development of amoxicillin suppositories and in vitro/ in

Development of amoxicillin suppositories

and in vitro/ in vivo evaluations

Trusha Jaimin Purohit

M Pharm

A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in Pharmaceutics, the University of Auckland, November 2021.

Dedication

To my family

i

Acknowledgements

There are several individuals whose endless support and sacrifices made this PhD

successful.

First of all, I would express my deepest gratitude towards my supervisors A/ Prof Zimei Wu

and Dr Sara Hanning for their continuous support both academically and financially. Their

perseverance, guidance and thoughtful suggestions always helped me realise and understand

the amazing principles of science involved in various aspects of my research. This thesis

would not have been possible without your mentorship and expertise. It was wonderful

growing as a PhD researcher under your observation.

I am grateful to the School of Pharmacy (SOPPBRF 2017) for their financial support in

carrying out this research, controlled release society - New Zealand chapter for their

conference travel award.

My sincere thanks to Andrew Brown and Louis Tessier-Varlet from the Vernon Jansen unit

for their help and support during the in vivo study. Thanks to Satya Amirapu for her expertise

and guidance for the histology study and Ratish Kurian for his help in carrying out elemental

analysis using scanning electron microscopy associated with energy disruptive X-ray

detector. I am thankful to Alec Asadov for his technical assistance in powdered X-ray

diffraction analysis.

I am in debt to my colleagues at the School of Pharmacy, particularly Tianjiao Geng, Yuki

Gao, Joy Reginald-Opara, Marvin Tang, Patrick Pan, Sanjukta Duarah and Mahima Bansal,

for their friendship and support throughout this fact-finding phase. Thanks to Danhui Li,

Stella Dawkins, Alvin Zhou, and Mike Goldthorpe for their technical expertise in the lab.

Above all, my deepest gratitude to my husband Jaimin and daughter Devanshi whose love,

encouragement and endurance continually restored my confidence and faith during the

challenging phases of my journey. Sincerest regards to my parents and in law, for their

blessings and immense support. Finally, a big shoutout to my siblings, friends and family

members for their prayers and well wishes.

ii

Abstract Background: Rectal drug delivery is a potential route of administration, especially for

children in low- and middle-income countries, where pneumonia is a leading cause of

childhood death. An inexpensive, accessible, stable rectal formulation suitable for an

outpatient setting could help reduce childhood mortality due to pneumonia. This research

aimed to develop amoxicillin suppositories using lipophilic and hydrophilic bases and to

investigate the mechanisms of absorption of amoxicillin from the rectum as well as

influencing factors such as salt form of drug, base, and drug-base interactions.

Methods: Hydrophilic suppository base, polyethylene glycol (PEG) 1500:PEG 4000 (70:30

w/w) and lipophilic suppository base, Suppocire® NA 15 (SNA 15) were selected with the

aid of differential scanning calorimetry (DSC). Following preformulation studies, a

validated high-performance liquid chromatographic (HPLC) method was developed and

applied to validate the extraction protocol developed by leveraging the solubility and

stability of amoxicillin. The developed HPLC method was then used for in vitro and in vivo

quantification of amoxicillin. Amoxicillin sodium (AS) 100 mg and 200 mg conventional

suppositories prepared in PEG or SNA 15 base were evaluated using standard

pharmacopoeial tests and solid-state characterisation, along with in vitro release and stability

over 3 months at 20 ± 0.2 °C. Absolute bioavailability (F) of AS from each suppository was

investigated in a rabbit model to determine the extent and mechanism of absorption of

amoxicillin along with irritation potential. PEG 1500 shells encapsulating amoxicillin

trihydrate (AMT) or AS loaded in SNA 15 core were used to develop a prototype of 250 mg

amoxicillin hollow-type (HT) suppository. F of AS vs AMT was predicted using an in vitro-

in vivo correlation model developed for the conventional AS suppositories.

iii

Results and discussion: A rapid, sensitive, stability-indicating isocratic HPLC method was

developed and validated and used for quantitation of amoxicillin. A five-fold increase in

solubility and two-fold increase in stability of amoxicillin was achieved by addition of

acetate buffer (0.1 M, pH 5.0) in acetonitrile, resulting in an extraction recovery of > 82%

for all the samples. The HPLC assay following extraction was found linear (R2 >0.9999)

over the range of 0.2 -20 µg/mL. Conventional AS suppositories complied with the

pharmacopoeial tests. Solid-state analysis revealed AS distributed as solid dispersion in PEG

or as an anhydrous crystalline dispersion in SNA 15 suppositories. In dissolution test,

melting and spreading of SNA 15 suppositories accounted for rapid drug release and

complete amoxicillin absorption (F close to 100%). AS up to 200 mg in SNA 15

suppositories was rectally absorbed via non-saturated transport, with a double peak PK

profile alongside minimal tissue irritation in rabbits. AS was more stable in SNA 15 than

PEG suppositories (91% vs 80% at 3 months). Shells prepared with PEG 4000 were found

brittle compared with PEG 1500 shells. Solid-state evaluation confirmed the distribution of

orthorhombic crystals of AMT and anhydrous crystals of AS in the SNA 15 core of HT

suppositories. Slow drug release from AMT HT suppositories (12% at 4 h) led to a predicted

F of < 1% compared with 79.4% for AS, corresponding to their solubilities. After three

months, PEG 1500 shells were found to protect the drug-loaded core from degradation, with

>98% amoxicillin remaining at 25 °C compared with the AS suppositories without shells

(90%). Further study is necessary to improve the stability of HT suppository above 37 °C.

Conclusion: This research is the first to determine the extent and mechanism of absorption

of amoxicillin following rectal administration, which could be affected by the

physicochemical properties of the drug salt form and type of formulation. This research

highlighted potential for the development of AS HT suppositories for children using

SNA 15 base with a PEG shell.

iv

Dissemination of Work Arising from This Thesis Peer-reviewed journal articles

Purohit TJ, Hanning SM, Wu Z. Advances in rectal drug delivery systems. Journal of

Pharmaceutical Development and Technology. 2018; 23(10):942-52

Purohit TJ, Wu Z, Hanning SM. Simple and reliable extraction and a validated high-

performance liquid chromatographic assay for quantification of amoxicillin from plasma.

Journal of Chromatography A. 2020;1611:460611

Purohit TJ, Hanning SM, Satya Amirapu, Wu Z. Rectal bioavailability of amoxicillin

sodium in rabbits: Effects of suppository base and drug dose. Journal of Controlled Release.

2021; 338:858-869

Conference presentations

Purohit TJ, Hanning SM, Wu Z. Preformulation studies for the development of a novel

suppository to deliver amoxicillin, QMB satellite drugs, devices and diagnostics meeting

2018. Queenstown, New Zealand. 26-27 August 2018. Abstract and poster.

Purohit TJ, Hanning SM, Wu Z. Solubility and stability analysis of amoxicillin in different

solvent systems used for quantitative analysis of amoxicillin, 46th annual meeting of

controlled release society, Valencia, Spain. 21-24 July 2019. Abstract and poster.

Purohit TJ, Hanning SM, Wu Z. Feasibility of amoxicillin suppositories: Storage stability

and rectal bioavailability, the 12th annual meeting of European paediatric formulation

initiative (EuPFI), Virtual, 09-10 September 2020. Poster, and oral presentations

v

Purohit TJ, Hanning SM, Wu Z. In vitro and in vivo evaluation of amoxicillin suppositories,

HealtheX 2020 - The University of Auckland, New Zealand. 18th September 2020. Oral

presentation

Purohit TJ, Hanning SM, Wu Z. Amoxicillin suppositories for children: Formulation

development and in vitro/in vivo evaluation, monthly webinar series, New Zealand local

chapter of controlled release society (NZCRS), virtual, 25 August 2021. Oral presentation

Awards

Travel award from New Zealand Controlled Release Society to attend 46th annual meeting

of Controlled Release Society, Valencia, Spain.

Best Poster Award from Professional Compounding Centres of America (PCCA) at the

12th annual meeting of European Paediatric Formulation Initiative (EuPFI).

vi

Table of Contents Acknowledgements ............................................................................................................... i

Abstract ................................................................................................................................ii

Dissemination of Work Arising from This Thesis ........................................................... iv

Table of Contents ................................................................................................................ vi

List of Tables ..................................................................................................................... xvi

List of Figures ................................................................................................................... xix

List of Abbreviations ..................................................................................................... xxiii

Co-Authorship Forms ..................................................................................................... xxv

Publication/Copyright Permissions ........................................................................... xxviii

Background and Thesis Structure ............................................................................. 1

1.1 Background .................................................................................................................... 2

1.2 Rectal suppositories ....................................................................................................... 2

Suppository bases ...................................................................................................... 3

Hydrophilic suppository bases .............................................................................. 3

Lipophilic suppository bases ................................................................................. 3

Preparation of suppositories ...................................................................................... 6

Hand rolling ........................................................................................................... 6

Compression moulding .......................................................................................... 6

Fusion moulding .................................................................................................... 6

1.3 Pneumonia – a leading cause of death in children ........................................................ 7

vii

1.4 Amoxicillin and its general properties .......................................................................... 8

Mechanism of action of amoxicillin .......................................................................... 9

Physicochemical properties of amoxicillin .............................................................. 11

Pharmacokinetics of amoxicillin ............................................................................. 12

1.5 Challenges in pneumonia treatment with existing amoxicillin formulations .............. 12

1.6 Ideal rectal amoxicillin formulation ............................................................................ 15

1.7 Hypothesis ................................................................................................................... 15

1.8 Thesis aims and objectives .......................................................................................... 16

1.9 Thesis structure ............................................................................................................ 16

Literature Review ...................................................................................................... 19

2.1 Introduction ................................................................................................................. 20

Rectal anatomy and scope of drug selection ............................................................ 21

Epidemiology of rectal dosage forms ...................................................................... 24

2.2 Literature search strategy ............................................................................................ 24

2.3 Traditional rectal dosage forms ................................................................................... 25

Solid rectal dosage forms ......................................................................................... 28

Suppositories ....................................................................................................... 28

Rectal capsules, tablets or powder for reconstitution .......................................... 28

Liquid rectal dosage forms ...................................................................................... 28

Semi-solid rectal dosage forms ................................................................................ 29

2.4 Novel rectal drug delivery systems and treatment opportunities ................................ 30

Hollow type suppository .......................................................................................... 30

Thermo-responsive liquid suppository (LS) ............................................................ 32

viii

Muco-adhesive gel ................................................................................................... 33

Micro and nanoparticles .......................................................................................... 33

Vesicular drug delivery systems .............................................................................. 35

2.5 In vitro and in vivo evaluation of RDDS ..................................................................... 35

In vitro release studies of RDDS ............................................................................. 36

In vitro, ex vivo and in vivo studies for prediction of absorption from RDDS ....... 40

2.6 Recent trends in the formulation of RDDS ................................................................. 41

2.7 Conclusion and future perspectives ............................................................................. 46

Development of Stability-Indicating HPLC Assay and Preformulation Studies . 48

3.1 Introduction ................................................................................................................. 49

3.2 Material and methods .................................................................................................. 50

Materials .................................................................................................................. 50

Selection of chromatographic parameters ............................................................... 51

Mobile phase ....................................................................................................... 51

Detection wavelength .......................................................................................... 51

HPLC method validation for in vitro quantification of amoxicillin ........................ 52

Chromatographic conditions ............................................................................... 52

Preparation of standard solutions for HPLC method validation ......................... 52

Validation of HPLC ............................................................................................. 52

3.2.3.3.1 Specificity ........................................................................................................ 52

3.2.3.3.2 Linearity .......................................................................................................... 53

3.2.3.3.3 Accuracy and precision ................................................................................... 53

3.2.3.3.4 Sensitivity ........................................................................................................ 53

Preliminary stability evaluation of amoxicillin ....................................................... 53

ix

pH-dependent stability evaluation of amoxicillin ............................................... 53

Forced degradation studies of amoxicillin........................................................... 54

Selection of suppository bases using differential scanning calorimetry (DSC) ...... 54

Calculations for batch formulation of amoxicillin suppositories ............................ 55

Suppository mould capacity ................................................................................ 55

Displacement value (DV) .................................................................................... 56

3.2.6.2.1 Preparation of blank suppositories .................................................................. 56

3.2.6.2.2 Preparation of amoxicillin containing suppositories ....................................... 56

Quantities required for a batch formulation ............................................................. 57

3.3 Results and discussion ................................................................................................. 57

Selection of chromatographic parameters ............................................................... 57

Mobile phase selection ........................................................................................ 57

Detection wavelength .......................................................................................... 59

Method validation .................................................................................................... 60

Specificity ............................................................................................................ 60

Linearity .............................................................................................................. 62

Accuracy and precision ....................................................................................... 62

Sensitivity ............................................................................................................ 63

Preliminary stability evaluation of amoxicillin ....................................................... 63

pH-dependent stability evaluation of amoxicillin ............................................... 63

Forced degradation studies of amoxicillin........................................................... 64

Selection of suppository bases using differential scanning calorimetry .................. 66

Suppository mould capacity .................................................................................... 69

Calculation of displacement value ........................................................................... 70

Calculations for preparation of a batch of suppositories ......................................... 70

x

3.4 Conclusions ................................................................................................................. 71

Development and Validation of a Stability-Indicating Extraction Protocol for

Quantification of Amoxicillin from Plasma ............................................................ 72

4.1 Introduction ................................................................................................................. 73

4.2 Materials and methods ................................................................................................. 75

Materials .................................................................................................................. 75

Chromatographic conditions .................................................................................... 75

Solubility and stability analysis of amoxicillin ....................................................... 76

Extraction protocol .................................................................................................. 77


Preparation of calibration and quality control (QC) standards in plasma ........... 78

Sensitivity ............................................................................................................ 78

Linearity .............................................................................................................. 79

Selectivity and specificity .................................................................................... 79


Post-extraction and sample storage stability ....................................................... 80

Pharmacokinetic study ............................................................................................. 80

Data analysis ............................................................................................................ 81

4.3 Results and discussion ................................................................................................. 81

Challenges in the extraction of amoxicillin during preliminary studies .................. 81

Solubility and stability analysis ............................................................................... 82

Solubility of amoxicillin ...................................................................................... 82

Stability of amoxicillin ........................................................................................ 83

Development and selection of extraction protocol .................................................. 85

xi

Extraction recovery.............................................................................................. 86


Sensitivity ............................................................................................................ 86

Linearity .............................................................................................................. 86

Selectivity and specificity .................................................................................... 86


Post-extraction and sample storage stability ....................................................... 88

Pharmacokinetic study ............................................................................................. 90

4.4 Conclusions ................................................................................................................. 91

Development and Evaluation of Conventional Amoxicillin Suppositories .......... 93

5.1 Introduction ................................................................................................................. 94

5.2 Materials and methods ................................................................................................. 96

Materials .................................................................................................................. 96

Preparation of suppositories .................................................................................... 97

Characterisation of suppositories ............................................................................. 97

Uniformity tests ................................................................................................... 98

Hardness and disintegration time ........................................................................ 98

Solid-state Characterisation of the suppositories ..................................................... 99

Drug-base interactions by differential scanning calorimetry (DSC) ................... 99

Powder x-ray diffraction ...................................................................................... 99

SEM/EDX microanalysis .................................................................................... 99

Intermolecular interactions by FTIR analysis ................................................... 100

In vitro release and kinetics ................................................................................... 100

Stability evaluation of the suppositories ................................................................ 101

xii

Absolute bioavailability study ............................................................................... 102

Drug administration ........................................................................................... 102

Plasma sample analysis ..................................................................................... 103

Pharmacokinetic data analysis ........................................................................... 103

Histological analysis .............................................................................................. 103

In vitro - in vivo correlation (IVIVC) .................................................................... 104

Statistical analysis .................................................................................................. 104

5.3 Results and discussion ............................................................................................... 104

Characterisation of suppositories ........................................................................... 104

Uniformity tests ................................................................................................. 105

Hardness and disintegration time evaluation ..................................................... 106

Solid-state Characterisation of the suppositories ................................................... 106

Drug-base interactions by DSC ......................................................................... 106

Powder X-ray diffraction ................................................................................... 107

SEM/EDX microanalysis .................................................................................. 108

Intermolecular interactions by FTIR analysis ................................................... 110

In vitro drug release and kinetics ........................................................................... 112

Stability evaluation of the suppositories ................................................................ 114

Absolute bioavailability study ............................................................................... 115

Histological analysis .............................................................................................. 119

In vitro - in vivo correlation (IVIVC) .................................................................... 121

5.4 Conclusions ............................................................................................................... 121

Formulation Development of Amoxicillin Hollow-Type Suppositories:

Comparison of Amoxicillin Sodium Salt and Trihydrate .................................... 123

xiii

6.1 Introduction ............................................................................................................... 124

6.2 Materials and methods ............................................................................................... 126

Materials ................................................................................................................ 126

Screening of suppository bases for preparation of shells ...................................... 127

Characterisation of the suppository shells ............................................................. 128

Weight variation ................................................................................................ 128

Disintegration time ............................................................................................ 128

Hardness and frangibility evaluation using texture analyser ............................. 128

Preparation of amoxicillin HT suppositories ......................................................... 129

Characterisation of amoxicillin containing HT suppositories ............................... 130


Hardness test of HT suppositories ..................................................................... 130

Solid-state characterisation .................................................................................... 131

Characterisation of the core using powder X-ray diffraction (XRD) ................ 131

Differential scanning calorimetry ...................................................................... 131

Fourier transform infrared spectroscopy ........................................................... 132

In vitro drug release and kinetics ........................................................................... 132

Prediction of absolute bioavailability (F) using IVIVC model.............................. 134

Stability of HT suppositories ................................................................................. 134

6.3 Results and discussion ............................................................................................... 135

Screening of suppository bases for preparation of shells ...................................... 135

Characterisation of the suppository shells ............................................................. 136

Weight variation ................................................................................................ 136

Disintegration time ............................................................................................ 136

Hardness and frangibility evaluation using texture analyser ............................. 136

xiv

Characterisation of amoxicillin HT suppositories ................................................. 138


Hardness test of HT suppositories ..................................................................... 139

Solid-state characterisation .................................................................................... 140

Characterisation of the drug loaded core using XRD ........................................ 140

Differential scanning calorimetry (DSC) .......................................................... 141

Fourier transform infrared spectroscopy ........................................................... 142

In vitro release analysis ......................................................................................... 143

Prediction of absolute bioavailability using IVIVC model ................................... 148

Stability of HT suppositories ................................................................................. 149

Visual evaluation ............................................................................................... 149

Change in hardness - Effect of amoxicillin form .............................................. 150

Intermolecular interactions by DSC and FTIR .................................................. 151

Content of amoxicillin remaining ...................................................................... 153

Other observations ............................................................................................. 153

6.4 Conclusions ............................................................................................................... 154

General Discussion and Future Directions ............................................................ 155

7.1 General discussion ..................................................................................................... 156

Selection of suppository bases: Preformulation studies ........................................ 157

Development of an extraction protocol for in vivo quantitation of amoxicillin .... 158

Development and solid-state characterisation of amoxicillin suppositories .......... 159

In vitro – in vivo evaluation of amoxicillin suppositories: Effects of base and dose

............................................................................................................................... 159

Development of HT amoxicillin suppositories ...................................................... 161

xv

7.2 Future directions ........................................................................................................ 162

PK study for HT suppositories .............................................................................. 163

Modification and development of HT suppository shells to improve drug stability

............................................................................................................................... 163

Automated process in preparation of the core ....................................................... 164

Translation of the bioavailability data in humans .................................................. 164

Gut microbiome evaluation ................................................................................... 165

Co-delivery of a β-lactamase inhibitor with amoxicillin ....................................... 165

7.3 Thesis conclusions ..................................................................................................... 166

Appendices ....................................................................................................................... 167

References......................................................................................................................... 184

xvi

List of Tables

Table 1.1 Different types of lipophilic suppository bases, their composition and

physical properties ..................................................................................... 5

Table 1.2 Summary of different types of amoxicillin formulations available on

the market. ............................................................................................... 13

Table 2.1 Molecular weight (Mol. Wt), Log P and pKa of the commonly used

APIs in the marketed rectal dosage forms obtained from the FDA

Orange Book ........................................................................................... 23

Table 2.2 Summary of marketed formulations available in the USA, UK, New

Zealand, and Australia. ............................................................................ 26

Table 2.3 Summary of novel rectal dosage forms and different mechanisms

involved in drug release from different type of dosage forms. ............... 38

Table 2.4 Summary of various excipients and their functions in the formulation

of the rectal dosage forms. ...................................................................... 43

Table 3.1 Effect of modulation of buffer to organic solvent ratio on the retention

time of amoxicillin. ................................................................................. 58

Table 3.2 Amoxicillin peak response evaluation using PDA at different

wavelengths. ............................................................................................ 59

Table 3.3 Accuracy and precision of the method for quantifying amoxicillin at

concentration levels of 3, 15 and 40 µg/mL. ........................................... 63

Table 3.4 Effect of stress conditions on degradation rate of AMT (equivalent to

200 µg/mL amoxicillin) after 24 hours. .................................................. 65

Table 3.5 Calculation of displacement value of AMT and AS equivalent to 100

mg amoxicillin for each type of suppository base ................................... 70

xvii

Table 3.6 Summary of calculated quantities of the drug and base for preparation

of a batch of 20 suppositories containing 100 mg amoxicillin. .............. 71

Table 4.1 Degradation kinetics of the solvent systems with or without acetate

buffer (0.1 M, pH 5.0). ............................................................................ 85

Table 4.2 Accuracy, precision and extraction recovery data for amoxicillin at

concentrations of 0.5, 4 and 16 µg/mL. .................................................. 89

Table 4.3 Auto sampler, freeze-and-thaw and long-term storage (- 20 °C)

stability of amoxicillin. ........................................................................... 89

Table 4.4 Pharmacokinetic parameters following IV administration of

amoxicillin at a dose of 53 mg/mL amoxicillin sodium solution

equivalent to 100 mg amoxicillin. ........................................................... 91

Table 5.1 Summary of the elements detected in PEG and SNA 15 suppositories

at 100 mg and 200 mg dose levels and chemical composition of

amoxicillin. ............................................................................................ 109

Table 5.2 Drug release kinetic modelling for the suppositories containing 106

mg of AS (equivalent to 100 mg of amoxicillin) prepared with PEG

base and SNA 15 base. .......................................................................... 113

Table 5.3 PK parameters of amoxicillin following rectal administration of

amoxicillin suppositories at two different dose levels compared with

intravenous (IV) injection (at a dose of 100 mg per rabbit) in rabbits. . 118

Table 6.1 Properties of components of the HT suppositories, individual and as

a system. ................................................................................................ 138

Table 6.2 Summary of release kinetics of amoxicillin from AS HT and AMT

HT suppositories. .................................................................................. 147

xviii

Table 6.3 Predicted bioavailability of AS and AMT in the HT suppositories

using the previously developed IVIVC model. ..................................... 148

Table 6.4 Effect of amoxicillin form, AS or AMT on the hardness of HT

suppositories following storage at 25 °C, 60% RH and at 37 °C for

three months. ......................................................................................... 150

xix

List of Figures

Figure 1.1 Pneumonia infection and comparison of healthy and mucus filled

alveoli. ....................................................................................................... 7

Figure 1.2 Bacterial cell wall synthesis and mechanism of action of amoxicillin. .. 10

Figure 1.3 Chemical structures of amoxicillin. ........................................................ 11

Figure 2.1 Schematic showing venous and lymphatic drainage from the rectum

and porto systemic shunting. ................................................................... 22

Figure 2.2 Schematic diagram representing longitudinal cross-section of HT

suppository. ............................................................................................. 31

Figure 3.1 Chromatograms showing complete resolution of AMT equivalent to

amoxicillin (200 µg/mL) from the degradation products (DP) generated

at different forced conditions (pH 2-10) after 24 hours. ......................... 61

Figure 3.2 Linearity curve of amoxicillin. ................................................................ 62

Figure 3.3 Stability of AMT equivalent to amoxicillin 40 µg/mL at pH 4, 5 and 6

for up to 24 days at (a) 2 - 8 °C and (b) ambient temperature. ............... 64

Figure 3.4 Proposed degradation products of amoxicillin in aqueous conditions

(pH 2-10). ................................................................................................ 65

Figure 3.5 Physical appearance of suppository bases and their DSC thermograms.

................................................................................................................. 67

Figure 3.6 DSC analysis of different ratios of PEG 1500:4000 and its effect on the

melting and solidification behaviour of the suppository base mixture. .. 69

Figure 4.1 Solubility of amoxicillin in different solvent systems (n=3) at 25 °C .... 83

xx

Figure 4.2 Degradation graphs for amoxicillin in two different solvent systems

(n=3), acetonitrile and plasma (95:5 v/v) and acetonitrile, plasma and

acetate buffer (90:5:5 v/v) ....................................................................... 84

Figure 4.3 Overlaid chromatograms of blank sample and a spiked sample at LLOQ

concentration (0.2 µg/mL). ..................................................................... 87

Figure 4.4 Chromatograms showing amoxicillin peak eluting at 6.2 ± 0.2 min ...... 90

Figure 4.5 Drug release profiles in rabbits (n=3) following IV amoxicillin

administration at a dose of 53 mg/mL amoxicillin sodium solution

equivalent to 100 mg amoxicillin. ........................................................... 90

Figure 5.1 Images of the freshly prepared amoxicillin suppositories: (a) PEG base

and (b) SNA 15 base. ............................................................................ 105

Figure 5.2 DSC Characterisation of the state of AS in suppositories composed of

PEG 1500/4000 (70:30 w/w) and SNA 15 bases. DSC thermograms of

AS, suppository base and AS containing (a) PEG 1500/PEG 4000 and

(b) SNA 15 suppositories. ..................................................................... 107

Figure 5.3 Comparison of XRD patterns of AS, suppository base alone, physical

mixture of AS and suppository bases and powdered samples of 100 mg

and 200 mg AS suppositories: (a) PEG suppositories and (b) SNA 15

suppositories. ......................................................................................... 108

Figure 5.4 SEM/EDX analysis of suppositories containing 100 mg AS in (a) PEG

(b) SNA 15; and 200 mg AS in (c) PEG (d) SNA 15. .......................... 109

Figure 5.5 Intermolecular interactions between AS and suppository bases

evaluated using FTIR, (a) PEG and (b) SNA 15. .................................. 111

Figure 5.6 In vitro drug release from (a) 100 mg and (b) 200 mg amoxicillin

suppositories made from PEG (●) and SNA 15 (■), respectively. Free

xxi

drug release (◊) 100 mg/5 mL solution from dialysis bag was used as

reference. ............................................................................................... 113

Figure 5.7 Amoxicillin plasma concentration (μg/mL) versus time profiles

following rectal administration of from PEG base and SNA 15

suppositories in rabbits at two dose levels, (a) 100 mg and (b) 200 mg

compared with intravenous injection at a dose of 100 mg/rabbit. ........ 117

Figure 5.8 Representative histological images of rectal tissues from non-treated

rabbits (control) and rabbits treated with PEG suppositories and SNA

15 suppositories containing amoxicillin (200 mg) to understand the

irritation to the rectal mucosa, at two magnifications: 4 X and 10 X. .. 120

Figure 5.9 Level C IVIVC by regression analysis of mean dissolution time (MDT)

vs (a) % F and (b) mean absorption time (MAT). ................................. 121

Figure 6.1 A schematic diagram of amoxicillin containing HT suppositories ....... 125

Figure 6.2 A Modified hollow-type suppository mould for the preparation of PEG

shells (a), and a schematic diagram showing the dimensions of the

modified suppository mould used to prepare the HT suppository shell

(b). ......................................................................................................... 127

Figure 6.3 Images of suppository shells prepared with (a) PEG 1500, (b) PEG

4000 and (c) PEG 1500:4000 mixed in a weight ratio of 80:20. .......... 135

Figure 6.4 Graphical representation of breakpoint evaluation by texture analyser

showing the difference in breakpoint of PEG 1500 shells (blue line) and

PEG 4000 shells (red line) (n=3 each). ................................................. 137

Figure 6.5 Images representing the components of HT suppositories .................... 139

Figure 6.6 Comparison of XRD patterns of AMT and AS, their physical mixtures

with SNA 15 and drug loaded core of the respective HT suppositories.140

xxii

Figure 6.7 Drug-base interactions shown by DSC analysis of (a) AS HT

suppositories and (b) AMT HT suppositories along with FTIR spectra

of (c) AS HT suppositories and (d) AMT HT suppositories. ................ 142

Figure 6.8 In vitro drug release from HT freshly prepared suppositories. AS HT

suppositories (green line) and AMT HT suppositories (red line). ........ 144

Figure 6.9 Time lapse up to 72 h showing drug release from HT suppositories kept

in a dialysis bag (MWCO 14000 Da) containing PBS, pH 7.4 at 37 °C146

Figure 6.10 Images showing different degrees of colour change in HT suppositories

stored at 25 °C, 60% RH after three months. ........................................ 149

Figure 6.11 Stability of amoxicillin HT suppositories evaluated with DSC (a and b)

and FTIR (c and d). ............................................................................... 152

xxiii

List of Abbreviations AMT Amoxicillin trihydrate AMT HT Amoxicillin trihydrate hollow-type ANOVA Analysis of variance APIs Active pharmaceutical ingredients AS Amoxicillin sodium salt AS HT Amoxicillin sodium hollow-type ATR Attenuated total reflection AUC Area under curve AUMC Area under the moment curve BCS Biopharmaceutics classification systems Cl Clearance DSC Differential scanning calorimetry DT Dispersible tablets DV Displacement value F Absolute bioavailability FTIR Fourier transform infrared GI Gastro intestinal H2O2 Hydrogen peroxide HCl Hydrochloric acid Hib Haemophilus influenzae type b HIV Human immunodeficiency Virus HPC Hydroxypropyl cellulose HPLC High-performance liquid chromatography HPMC Hydroxypropyl methylcellulose HT Hollow-type IBD Inflammatory bowel disease ICH International Council for Harmonisation IV Intravenous IVIVC In vitro-in vivo correlation LLE Liquid-liquid extraction LLOQ Lower limit of quantification LMICs Lower and middle income countries LOD Limit of detection LOQ Limit of quantitation

xxiv

LS Liquid suppositories MAT Mean absorption time MC Mould capacity MDT Mean dissolution time MIC Minimum inhibitory concentration Mol Wt Molecular weight MRT Mean residence time MWCO Molecular weight cut-off NAG N-acetyl glucosamine NAMA N-acetyl muramic acid NaOH Sodium hydroxide NBF Neutral buffered formalin PBPs Penicillin binding proteins PBS Phosphate buffer saline PDA Photo diode array PEG Poly ethylene glycol PK Pharmacokinetics PP Protein precipitation PVP Poly vinyl pyrollidone QC Quality control RDDS Rectal drug delivery systems RH Relative humidity RPM Rotations per minute RSD Relative standard deviation SD Standard deviation SEM/EDX Scanning electron microscopy conjugated with energy dispersive X-ray SLN Solid lipid nanoparticles SNA 15 Suppocire® NA 15 SPE Solid phase extraction SR-HT sustained release hollow-type UNICEF United Nations International Children's Emergency Fund Vd Volume of Distribution WHO World Health Organization

xxv

Co-Authorship Forms

xxviii

Publication/Copyright Permissions Chapter 2 – Literature review

Purohit TJ, Hanning SM, Wu Z. Advances in rectal drug delivery systems.

Pharmaceutical Development and Technology. 2018;23(10):942-952, DOI:

https://doi.org/10.1080/10837450.2018.1484766

Chapter 4 - Development and Validation of a Stability-Indicating Extraction Protocol

for In Vivo Quantification of Amoxicillin from Plasma

Purohit TJ, Wu Z, Hanning SM. Simple and reliable extraction and a validated high

performance liquid chromatographic assay for quantification of amoxicillin from plasma.

Journal of Chromatography A. 2020;1611:460611, DOI:

https://doi.org/10.1016/j.chroma.2019.460611

https://doi.org/10.1080/10837450.2018.1484766


xxix

Chapter 5 - Development and Evaluation of Conventional Amoxicillin Suppositories

Purohit TJ, Hanning SM, Amirapu S, Wu Z. Rectal bioavailability of amoxicillin sodium

in rabbits: Effects of suppository base and drug dose. Journal of Controlled Release.

2021;338:858-869, DOI: https://doi.org/10.1016/j.jconrel.2021.09.015

https://doi.org/10.1016/j.jconrel.2021.09.015

1

Background and Thesis Structure

Chapter 1

2

This Chapter provides a general overview of this thesis following a brief review of the

history of rectal drug delivery and its specific advantages over other drug delivery routes.

Aims, objectives and hypotheses to investigate rectal delivery of amoxicillin have been

discussed.

1.1 Background

Drug delivery through the distal end of the gastrointestinal (GI) tract, known as the rectum,

has been in use for centuries [1]. The use of retention rectal enemas was mentioned in

Babylonians and Assyrians, indicating extensive use of rectal route since about 200 B.C. [2,

3]. The historical use of enemas and suppositories have suggested that the rectal route was

used for therapeutic procedures and home remedies of infectious diseases [4, 5]. However,

it is not a commonly used route for drug administration in present times. The rectal route is

an alternative approach for drug administration with specific advantages over the oral or

parenteral drug delivery systems. Rectal drug delivery systems (RDDS) can be particularly

useful for paediatric, geriatric or comatose patients as there are no swallowability concerns

or needle phobia associated with them. It also minimises first-pass metabolism of the drug

and enables systemic drug delivery, unlike oral administration [6, 7]. Due to the small

rectum size in infants (3.0 x 6.0 cm for a 3-month-old and 3.5 x 7 cm for a 1-year-old) and

the tendency of enemas to induce strong peristaltic movements in the rectum, suppositories

are preferrable over enemas for children [2, 3, 8]. Therefore, rectal suppositories have been

studied in detail for this thesis.

1.2 Rectal suppositories

Rectal suppositories are a unit solid dosage form, which either melt or dissolve in body fluid

to release the therapeutic contents that are either dissolved or suspended in the suppository

base.

Chapter 1

3

Suppository bases

A suppository base should be non-toxic and non-irritant to the rectal mucosa and remain

solid at room temperature [9]. Suppository bases can be hydrophilic or lipophilic.

Hydrophilic suppository bases

Glycerogelatine and polyethylene glycol (PEG) are water soluble or water miscible

suppository bases that dissolve in body fluid to release the drug. Glycerogelatine base is

moisture sensitive and, therefore, requires a moisture-proof packaging system [10].

Glycerogelatine base is more suitable for vaginal preparations and requires preservatives in

the formulation to avoid microbial growth due to the presence of water [9, 11]. In contrast,

PEGs are most frequently used for suppository bases because they are cost-effective, freely

soluble in body fluid, stable at room temperature with high melting temperatures and do not

require preservatives [9, 12-15]. Unlike glycerogelatine base, PEGs shrink significantly

upon cooling. Therefore, they do not stick to the suppository mould during demoulding of

suppositories [9, 16]. Both low and high molecular weight PEGs are frequently used for the

preparation of suppositories. The increase in the length of PEG chain essentially increases

the firmness and reduces its solubility. Consequently, a mixture of both high and low

molecular weight PEGs is required to achieve optimal mechanical strength and drug release

from suppositories [17].

Lipophilic suppository bases

Lipophilic suppository bases melt at body temperature to release the drug. Cocoa butter is

the traditional, safe to use lipophilic suppository base [1, 9, 18]. It is a polymorphic

compound with relatively low melting point (30 °C to 35 °C) and can take a long time to

solidify if overheated [1, 9]. Currently, semi-synthetic lipophilic bases are also widely used

for the preparation of suppositories due to their ability to produce elegant and smooth

Chapter 1

4

suppositories compared with PEGs [19]. Synthetic triglycerides such as Witepsol®, Novata®,

Suppocire®, Fattibase®, Wecobee®, Hydro-kote® and Dehydag® are some examples of

commercialised semi-synthetic lipophilic bases derived from the esterification of

hydrogenated vegetable oils [10, 20]. Their composition and physical properties have been

summarised in Table 1.1. These suppository bases are available in many different grades

with slight variations in the compositions resulting in changes to the melting range of the

suppository base.

Chapter 1

5

Table 1.1 Different types of lipophilic suppository bases, their composition and physical

properties [21, 22]. NA – not available

Suppository

base

Composition Melting

range (°C)

Hydroxyl value

(mg KOH/g)

Dehydag® Hydrogenated fatty alcohols and

esters

3 - 39 NA

Fattibase® Triglycerides from palm, palm kernel

and coconut oil together with self-

emulsifying glycerol monostearate

and polyoxyl stearate

35.5 - 37 NA

Hydro-kote® Hydrogenated palm kernel oil and soy

lecithin

31 - 45 NA

Novata® Mixture of mono, bi and triglyceride

chains bonded to a glycerol molecule

via an ester bond.

32 - 44 5 - 40

Suppocire® Mixture of mono-bi and tri glyceride

esters of fatty acids (C10 – C18).

32 - 45 5-60

Wecobee® Hydrogenated palm kernel oil 31 - 43 NA

Witepsol® Mixture of mono (1-5%)-di (10-35%)

and tri glycerides (65-80%)

32 – 48 3 - 70

For this thesis, particular focus has been given on hydrophilic PEG and lipophilic

Suppocire® NA 15 (SNA 15) base.

Chapter 1

6

Preparation of suppositories

Suppositories can be prepared in different shapes and sizes using different techniques,

including hand rolling, compression moulding and fusion moulding.

Hand rolling

This is the traditional method for preparation of suppositories using cocoa butter. The

suppository base and the drug are triturated using mortar and pestle and rolled in a uniform

cylinder using a spatula. The cylinders are cut to a desirable size and used as a suppository.

This method does not involve heating of the cocoa butter and can be useful to incorporate

heat-labile drugs. Nonetheless, it is difficult to produce uniform suppositories using this

method [11, 16, 23].

Compression moulding

Compression moulding is also known as cold compression. This is a method where the drug

and the suppository base are triturated and mixed together, then compressed using a pre-

calibrated mould or die. This method produces more uniform suppositories than with hand

rolling, but air can become entrapped, causing weight variation in the suppositories and

possible oxidative degradation of the formulation [11, 16].

Fusion moulding

Fusion moulding is the most commonly used method for preparation of suppositories where

the drug is uniformly mixed in a melted suppository base. The mixture is then congealed

into the required size and shape of suppositories using an appropriate mould. This method

produces uniform suppositories provided the drug is stable at the temperature required to

melt the base [11, 16].

Chapter 1

7

1.3 Pneumonia – a leading cause of death in children

Pneumonia, an air-borne infectious disease affecting the respiratory system, has been

identified as the leading cause of death in children [24-26]. In 2015, the World Health

Organisation (WHO) reported that pneumonia was responsible for 16% of childhood deaths

globally and most of the victims were children under two years old [27, 28]. This means 100

children died every hour and approximately 2400 children every day [29]. Pneumonia is

characterised by the accumulation of mucus or pus in the tiny air pockets of lungs known as

alveoli. Accumulation of fluid in the lungs hinders the gaseous exchange of O2 and CO2 and

thus makes breathing difficult (Figure 1.1).

Figure 1.1 Pneumonia infection and comparison of healthy and mucus filled alveoli.

Image created using biorender.com (Bio Render, Toronto, Canada).

Chapter 1

8

The symptoms of pneumonia include fever, chills, cough, shortness of breath and chest pain

[27]. The symptoms worsen to hypoxia, lower chest indrawing, central cyanosis, nasal

flaring and oxygen saturation <90% in severe infections, which may lead to death [30].

Pneumonia can be caused by various pathogens, including bacteria (Streptococcus

pneumoniae), viruses (respiratory syncytial virus and Haemophilus influenzae type b (Hib))

and fungi (Pneumocystis jiroveci) [27]. Of these, bacterial pneumonia is most common.

Appropriate hygiene and vaccination are being promoted for prophylaxis of pneumonia.

Although pneumonia is treatable and vaccine preventable, it is most prevalent in resource

constrained low- and middle-income countries (LMICs), where treatment options may be

limited. To resolve this global issue, WHO and The United Nations International Children's

Emergency Fund (UNICEF) have jointly prepared an action plan involving protection,

prevention and treatment of pneumonia [27, 29].

Currently, WHO and UNICEF recommend amoxicillin as the first-line antibiotic for

outpatient paediatric pneumonia [31-34]. Children with fast breathing and no chest

indrawing pneumonia are recommended to receive oral amoxicillin up to 40 mg/kg/dose

twice daily for five days [33-36]. Other antibiotics such as benzylpenicillin, co-trimoxazole

and erythromycin have also been listed as alternatives for treatment, where amoxicillin is

not suitable or is unavailable [30, 33, 37].

1.4 Amoxicillin and its general properties

Amoxicillin is a broad spectrum, semi-synthetic, β-lactam amino-penicillin antibiotic

(Figure 1.2). Apart from pneumonia, amoxicillin is used for the treatment of various

infections, including otitis media, upper and lower respiratory tract infections, dental

abscesses and urinary tract infections.

Chapter 1

9

Mechanism of action of amoxicillin

Amoxicillin has shown bactericidal activity against Streptococcus pneumoniae at plasma

concentrations above 2 mg/L [38]. The five-membered β-lactam ring in the structure of

amoxicillin is responsible for its bactericidal activity [39]. Streptococcus pneumoniae are

gram-positive, extracellular pathogens. Their cell wall comprises peptidoglycans, which are

made up of cross-linked, linear chains of two amino sugars, N-acetyl glucosamine (NAG)

and N-acetyl muramic acid (NAMA) [40]. Penicillin binding proteins (PBPs) help to form

the tripeptide linkage between the short amino acid chains from each NAMA unit [41].

Amoxicillin inhibits PBPs (Figure 1.2) and interferes in the synthesis of peptidoglycan

chains, which are the building blocks of bacterial cell wall, and results in cell lysis [39].

Chapter 1

10

Figure 1.2 Bacterial cell wall synthesis and mechanism of action of amoxicillin. PBP - penicillin binding protein, NAMA – N acetyl muramic

acid, NAG – N – acetyl glucosamine.

Chapter 1

11

Physicochemical properties of amoxicillin

Amoxicillin is available as a trihydrate (molecular weight 419.4 g/mol) and sodium salt

(molecular weight 387.4 g/mol) [42]. It is insoluble in oils and very slightly soluble in

ethanol. The sodium salt is highly soluble in water (nearly 150 mg/ mL) [43, 44], while the

trihydrate form is slightly soluble in water (2.7 mg/mL) [45], but dissolves in dilute acid and

alkali solutions [46]. The chemical structure of amoxicillin includes an amino and carboxylic

acid group (Figure 1.3), which together are responsible for the amphoteric characteristics of

this drug, resulting in two pKa values: 3.2 (acid) and 11.7 (basic) [47]. In aqueous solutions

amoxicillin is stable for 14 days at 2-8 °C and for seven days at ambient temperature [48].

Figure 1.3 Chemical structures of amoxicillin. (2S,5R,6R)-6-[[(2R)-2-amino-2-(4-

hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-

carboxylic acid (a) and its sodium salt (b) and trihydrate (c). Formula: C16H19N3O5S,

C16H18N3O5SNa and C16H25N3O8S.3H2O. Molecular weights 365.4 g/mol, 387.4 g/mol,

and 419.450 g/mol, respectively [42].

Chapter 1

12

Pharmacokinetics of amoxicillin

Amoxicillin is well tolerated, acid resistant and rapidly absorbed from the GI tract.

Following oral administration, peak serum concentration is observed within 1-2 h [49, 50].

Unlike other penicillins, amoxicillin has low plasma protein binding, ranging from 17-20%.

Therefore, there is a high unbound drug concentration for antibacterial activity [51].

Amoxicillin is mainly excreted renally and the elimination half-life of amoxicillin is 1 – 1.5

hours following oral administration in humans [48-50].

1.5 Challenges in pneumonia treatment with existing amoxicillin formulations

There are several amoxicillin formulations available on the market (Table 1.2), including

capsules, tablets, powder for oral suspension, injections and dispersible tablets (DTs) [32].

Chapter 1

13

Table 1.2 Summary of different types of amoxicillin formulations available on the

market.

Type of

formulation Available strengths Some of the manufacturer(s)

Capsule 250, 500, 1000 mg Teva pharmaceuticals, US

Ranbaxy pharmaceuticals, US

Dispersible tablets* 125, 250 mg Micro Labs, India

Sandoz, Denmark

Injection 250, 500, 1000 mg powder for

reconstitution GlaxoSmithKline, India, UK

Oral drops 100 mg/mL - powder for

reconstitution

GlaxoSmithKline, US

Teva pharmaceuticals, US

Oral suspension* 125 mg/5 mL, 250 mg/5 mL –

powder for reconstitution

Par pharmaceuticals, US


Sachet 3 g GlaxoSmithKline, US

Tablet 500, 875 mg

Chewable 125, 200, 250, 400 mg


Ranbaxy pharmaceuticals, US

* Commonly used formulations for children in LMICs. All the formulations are available as amoxicillin trihydrate form except injections which contain the amoxicillin sodium salt.

Chapter 1

14

Despite amoxicillin tablets and capsules being commonly used oral formulations, they are

not useful for paediatric patients due to the bitter taste of antibiotic and poor swallowability

in children. Intravenous (IV) administration of amoxicillin is a quick and easy alternative

but requires a trained person for drug administration and often, hospitalisation. Furthermore,

needle phobia is associated with IV administration especially for paediatric patients. Liquid

amoxicillin formulations are the most commonly used child-friendly formulations and they

can be taste masked, but the instability of those formulations is an issue as amoxicillin

readily undergoes hydrolysis in presence of water and only 50% of its activity remains intact

after seven days at ambient temperature [48]. Amoxicillin 250 mg DTs are currently in use

as the cheapest alternative providing similar strength of dose as oral suspensions, but in a

compact form. Thus, DTs are a cost-effective alternative that ensure accurate dosing without

the need for a measuring device. Also, unlike liquid formulations, refrigeration is not

required. However, not all of these dosage forms are appropriate for use in resource

constrained countries [36]. For example, clean water may not be available to reconstitute the

powder into a suspension or to disperse the DTs. Additionally, refrigerators may not be

available for storage to ensure stability of these formulations. Although amoxicillin is well

absorbed via the oral route, it undergoes first-pass metabolism and prolonged oral treatment

with amoxicillin affects the healthy gut microflora leading to GIT disturbances and

associated adverse effects [52, 53].

In contrast, RDDS containing amoxicillin may offer an alternative treatment option as they

offer some specific advantages over oral and parenteral amoxicillin dosage forms and can

be prepared using inexpensive, readily available traditional excipients [1, 54]. Furthermore,

unlike oral amoxicillin formulations, they may also offer the advantage of reduced effect on

the gut microbiome by minimising first-pass metabolism. However, like all other drug

delivery systems, RDDS also have limitations. Solubility of drug in the small fluid volume

Chapter 1

15

(1-3 mL) of the rectum and the absence of carrier mediated transport in the rectum limits the

choice of drug molecule [6, 7]. Nevertheless, drug delivery through the rectal route have not

been explored extensively due to perceived cultural barriers to this route of administration

[1, 13, 14].

1.6 Ideal rectal amoxicillin formulation

Amoxicillin is a time-dependent antibiotic, meaning the bactericidal effect depends on the

length of time amoxicillin concentration is above the minimum inhibitory concentration

(MIC) [33, 55]. MIC is defined as the lowest concentration of the antibiotic required to

inhibit the visible growth of the microorganism following an overnight incubation [56].

Therefore, this research was focused on the development of rectal suppositories containing

amoxicillin for the treatment of pneumonia in children under five years, especially in LMICs

(Chapters 5 and 6). Due to the high pneumonia burden and unsuitability of the currently

available formulations, the ideal amoxicillin-containing rectal dosage form should be simple

to manufacture, easy to administer, stable at tropical climatic conditions and bioequivalent

to the oral dosage forms.

Many antibiotics have been investigated for rectal administration using suppositories as a

dosage form [12, 13, 57-59]. Ampicillin, a close analogue of amoxicillin has shown potential

for the development of rectal suppositories [60]. This may suggest that amoxicillin could

also be a suitable candidate for the development of rectal suppositories.

1.7 Hypothesis

In this PhD project, it was hypothesised that due to the small molecular size, amoxicillin

would be readily absorbed from the rectum, however, systemic absorption would be

controlled by its release (as free drug) and thus dominated by the physicochemical properties

of the drug as well as the suppository matrix. Furthermore, the stability of amoxicillin would

Chapter 1

16

be improved by loading the drug in an inert lipophilic base and covering it with a protective

suppository shell made with a suppository base having high melting point, in the form of a

hollow-type (HT) suppository.

1.8 Thesis aims and objectives

This research aimed to develop amoxicillin suppositories using lipophilic and hydrophilic

suppository bases and investigate the mechanism of drug release and absorption from the

rectum, along with their rectal tissue compatibility. Secondly, this research intended to

develop and evaluate a stable, readily available, preliminary prototype of amoxicillin

containing HT suppositories for the treatment of childhood pneumonia in the LMICs. The

specific objectives of this research were to:

1) Develop and validate reliable and sensitive high-performance liquid

chromatographic (HPLC) methods for quantification of amoxicillin suitable for both

in vitro (in Chapter 3) and in vivo studies (in Chapter 4). For these, the stability of

amoxicillin was studied, and methods for extraction from plasma were compared.

2) Design and characterise hydrophilic and lipophilic amoxicillin suppositories and

understand the in vitro release and bioavailability of amoxicillin along with in vitro-

in vivo correlation (IVIVC) and preliminary stability evaluation. (Chapter 5).

3) Develop a preliminary prototype of HT amoxicillin suppository to investigate the

stability improvement due to the PEG shell (Chapter 6).

1.9 Thesis structure

This thesis is written in compliance with the University of Auckland 2020 PhD statute

guidelines titled “Doctoral thesis policies and procedures”. Chapters 2 and 4 of this thesis

have been published in peer-reviewed journals, while Chapter 5 is under review in a peer-

reviewed journal. Chapters that are already published papers have been included without

Chapter 1

17

any changes except for the table and figure numbers and citations to maintain the

consistency in the formatting, citation style and terminology used throughout the thesis.

Where necessary, additional data have been added at the end of the chapters. The published

papers have been edited and included with necessary permission from the publishers. Each

chapter is structured as introduction, methods, results and discussion (combined), and

conclusion.

The background, aims and objective of this research and thesis structure have been

introduced in Chapter 1. A thorough literature review about RDDS has been included as

Chapter 2 [61], where a detailed review of rectal dosage forms, their characteristics,

advantages and disadvantages are presented. Traditional and novel RDDS, along with

techniques for in vitro and in vivo characterisation of RDDS have been summarised and

discussed.

Chapter 3 comprises preformulation studies and the development of a rapid, sensitive, and

stability-indicating HPLC method for the quantification of amoxicillin in in vitro studies.

Stress degradation studies and pH-dependent separation of amoxicillin were investigated to

satisfactorily resolve amoxicillin from its degradation products and without any interference

of the suppository base. For formulation development, different batch processing parameters

such as mould capacity (MC) and displacement value (DV) were also determined prior to

the formulation of a batch of suppositories.

In Chapter 4, the development and optimisation of an extraction protocol for quantification

of amoxicillin from rabbit plasma have been described [45]. An understanding of pH-

dependent solubility and stability of amoxicillin facilitated the development of a reliable and

reproducible protocol for the extraction of amoxicillin from plasma. The extraction protocol

was validated for quantification of amoxicillin from rabbit plasma using the previously

developed HPLC method (Chapter 3) and further applied for in vivo studies.

Chapter 1

18

Formulation and development of conventional amoxicillin sodium suppositories

(approximately 1 g each) are discussed in Chapter 5. The suppositories were prepared at

two dose levels (100 mg and 200 mg) from two types of suppository bases, a hydrophilic

(PEG) base mixture and lipophilic base (SNA 15). The effects of each base and doses were

evaluated based on sold-state characterisation, in vitro drug release, preliminary stability at

20 ± 0.2 °C and in vivo absorption. The mechanism and extent of absorption of amoxicillin

from the rectal mucosa were studied in a rabbit model and pharmacokinetic (PK) parameters

were calculated, which were then utilised to determine in vitro – in vivo correlation (IVIVC).

The irritation potential of two types of suppositories to the rectal mucosa was also evaluated

by histological examination. Findings of PK study indicated the potential for development

of a novel rectal dosage form containing amoxicillin which led to the chapter 6 studies.

In Chapter 6, to increase the stability of the drug, formulation development of HT

amoxicillin suppositories was carried out. The HT suppositories were formulated using two

forms of amoxicillin, sodium salt and amoxicillin trihydrate and characterised by solid-sate

analysis, in vitro drug release and absolute bioavailability were predicted from the

previously IVIVC model developed in Chapter 5. The suitability of the PEG shells to

improve the stability of HT suppositories was evaluated at 25 °C, 60% relative humidity and

37 °C. The AS HT suppositories were found to be a potential candidate for the age-

appropriate dosage form for rectal delivery of amoxicillin, provided further modulation in

the preparation technique is undertaken.

In Chapter 7, research findings and implications of this work have been discussed. Future

pathways and directions for further research and challenges associated with this research

project have been outlined.

19

Literature Review

This chapter is published in Journal of Pharmaceutical

Development and Technology.

Purohit TJ, Hanning SM, Wu Z. Advances in rectal drug delivery

systems. Pharm Dev Technol. 2018;23(10):942-952, DOI:

https://doi.org/10.1080/10837450.2018.1484766.

https://doi.org/10.1080/10837450.2018.1484766

Chapter 2

20

In this chapter, rectal drug delivery as an alternative to other conventional dosage form has

been discussed in detail. Traditional and advanced rectal drug delivery techniques have also

been described in detail.

2.1 Introduction

Drug delivery systems can be utilised to modulate therapeutic drug concentration depending

upon the disease and patient condition. While oral and parenteral routes remain the most

commonly used drug delivery routes, rectal delivery is also an effective approach, not only

for systematic treatment but also for localised drug delivery. The rectal route of drug

administration has been in use for centuries particularly for children, elderly and comatose

patients [1]. However, due to privacy concerns and cultural barriers, rectal drug

administration is neglected or avoided for the delivery of a therapeutically active moiety in

some countries [9, 62].

Despite these concerns, rectal drug administration is a choice for post-operative treatment

when patients are unconscious, vomiting or unable to swallow, or are reluctant towards

parenteral administration [7, 63]. There are several types of traditional rectal dosage forms

in existence such as suppositories, enemas, creams, ointments, gels, and rectal capsules [63,

64]. The rectal route of drug administration has the following advantages over the oral route:

1) Drug delivery to paediatric, geriatric, comatose or vomiting patients where

swallowing may be difficult [65-68];

2) Protection of enzymatically unstable drugs due to the absence of enzymes in the

rectum, such as insulin [69-71];

3) Minimal first-pass metabolism of the drug provided the suppository is administered

at an appropriate distance in the rectum [62, 72];

Chapter 2

21

4) Prevention of gastric mucosa being exposed to irritant drugs such as non-steroidal

anti-inflammatory drugs [73, 74];

5) Improved efficacy of localised treatments for some conditions, such as haemorrhoids

and constipation.

With the help of modern pharmaceutics and novel rectal drug delivery systems (RDDS),

higher bioavailability and controlled release of the drug have become possible. Integration

of various formulation techniques and basic physiological principles have resulted in the

development of some promising RDDS. Such systems could be modified to act either locally

or systematically and could be either immediate acting or extended release.

Rectal anatomy and scope of drug selection

Anatomically, the rectum forms the distal part of the large intestine. A normal adult human

rectum is about 12-15 cm long and deviates in three lateral curves: upper, middle and lower

[75]. It is considered a cylindrical organ that does not contain villi or microvilli on the

luminal surface and is mainly associated with water absorption and re-absorption from the

gastrointestinal (GI) contents. Average rectal surface area is reported to be around 200-

400 cm2 and the pH is 7.2 to 7.4, varying from patient to patient while the average rectal

fluid volume is 1-3 ml [7]. The rectum is structured with columnar epithelial cells along

with goblet cells that are responsible for mucous secretion. The mucous forms a layer

approximately 100 µm thick, that acts as a protectant to the rectal epithelia and is also a

barrier for drug absorption [1, 75].

Three haemorrhoidal veins drain the rectum: superficial, middle and inferior (Figure 2.1).

The superficial vein drains from the upper rectum into portal circulation while the middle

and inferior veins drain from the lower rectum directly into systemic venous circulation [75].

This porto-systemic shunting and lymphatic drainage of the rectum play a significant role in

Chapter 2

22

systemic absorption of lipophilic drugs [1, 6] and this is why the rectal route can be

considered a candidate for systemic drug delivery. The rectal route may minimise first-pass

metabolism of drugs, depending on the position of the formulation in the rectum. If the drug

reaches to the end of the colon, drug can get lost in the portal circulation.

Figure 2.1 Schematic showing venous and lymphatic drainage from the rectum and porto

systemic shunting.

Absorption of various drug compounds such as anticonvulsants, anti-inflammatory agents,

sedatives, laxatives, analgesics, anti-emetic and antibiotic drugs have been studied following

administration of different rectal dosage forms [6, 76]. The main absorption pathways from

the rectum are transcellular or paracellular; however, no carrier mediated transport has been

reported [6, 7]. Rectal degradation of drug has shown to be relatively lower than that from

the upper GI tract due to absence of enzymes in the rectum [77]. A summary of the active

pharmaceutical ingredients (APIs) used in marketed rectal dosage forms listed in the Orange

Book of the United States Food and Drug Administration (FDA) [78] and other available

marketed rectal dosage forms has been tabulated (Table 2.1). It is observed that the majority

Chapter 2

23

of APIs formulated for rectal administration have high log P values and are small molecules

(molecular weight <500 g/mol). This could be the reason for their rapid absorption through

rectal mucosa. However, like for oral administration, other factors such as rectal pH, drug

release, residence time, quantity of rectal fluid, contents of the rectum, motility of the rectal

wall and spreading of formulation in the rectal area play an important role in determination

of the rate and extent of absorption [6, 76].

Table 2.1 Molecular weight (Mol. Wt), Log P and pKa of the commonly used APIs in the

marketed rectal dosage forms obtained from the FDA Orange Book [78].

Name of API Physical properties

Mol. Wt (g/mol) Log P pKa

Budesonide 430.54 1.90 13.74

Diazepam 284.74 2.82 2.82

Hydrocortisone 362.76 1.61 1.61

Indomethacin 357.79 4.27 4.27

Mesalamine 153.14 1.20 1.20

Ondansetron 293.37 2.40 7.34 (pKa1), 15.39 (pKa2)

Prochlorperazine 373.94 4.88 4.88

Promethazine

hydrochloride 320.88 4.81 4.81

Bisacodyl 361.40 3.37 4.08

Acetaminophen

(Paracetamol) 151.17 0.46 9.38

Diclofenac sodium 296.15 4.15 4.51

Chapter 2

24

Epidemiology of rectal dosage forms

During the 18th century, rectal dosage forms were used for the management of constipation

or as a bowel stimulant to help patients expel their bowel contents [1, 2]. Early formulations

were made of some modified natural sources such as honey or animal fat, which was later

replaced by cocoa butter and then eventually other water miscible suppository bases such as

polyethylene glycol (PEG), glycero-gelatine bases and more recently, emulsification bases

such as triglycerides of vegetable oils [1]. Initially, suppository dosage forms were used as

an alternative to enemas and eventually various other rectal dosage forms such as creams,

ointments, capsules and tampons were introduced. Recently, an increase in the interest in

development of RDDS has been observed for the development of more controlled release

dosage forms than the conventional formulations.

The aim of this report is to review various RDDS and their applicability in the treatment of

various diseases. A particular focus is given to advances in the development of novel RDDS.

The mechanism of rectal absorption and epidemiology is also briefly discussed. Methods

for in vitro drug release and in vivo absorption are also briefly reviewed. Ultimately, the

review aims to provide insight to the flexibility of RDDS for inclusion of other

therapeutically active moieties and development of age specific formulations.

2.2 Literature search strategy

Articles from databases Scopus (1973 – 2017) and Medline (Ovid) (1969 – 2017) were

searched with the key terms “rectal dosage form”, “rectal drug delivery”, “rectal drug

administration”, “suppository”, ”enema”, and “rectal gel” to review traditional rectal dosage

forms. Articles for advances in rectal drug delivery were searched with the key terms

“hollow-type suppository”, “liquid suppository”, “liposomes”, “nanoparticles”, “hydrogel”

and “microspheres” using ‘AND’ as a Boolean operator between keywords for site specific

Chapter 2

25

delivery system and a keyword for each formulation type mentioned above. Also, a

combined search for all novel formulation types and “rectal drug administration” was

conducted using OR as a Boolean operator.

All articles that mentioned RDDS were selected based on abstract screening and duplicates

were removed. Review articles, book chapters, pharmacopoeias and regulatory websites

were referred to summarise different traditional rectal dosage forms.

Articles that described advances in rectal formulation development were shortlisted and 139

full-text articles were accessed. Research articles describing the development of novel

RDDS were included for this review. The cumulative literature review data was derived

from 38 articles: 11 articles related to hollow-type (HT) suppository development, 13

articles on thermo-responsive liquid suppositories and 14 articles based on micro- and nano-

structure containing RDDS. Articles describing the effect of formulation parameters were

also included in the review to make a scientific explanation for choice of excipient. The

formulation design and the mechanisms are the basis of classification as traditional rectal

dosage forms and novel RDDS in this paper.

2.3 Traditional rectal dosage forms

Lack of acceptance and patient preference towards the rectal route has meant that rectal drug

delivery has not been explored to the same extent as other routes of administration.

Nevertheless, traditional rectal dosage forms are already available in many countries. These

can be categorised into solid, liquid and semi-solid dosage forms, as illustrated with some

marketed products available in USA, UK, New Zealand, and Australia (Table 2.2).

Chapter 2

26

Table 2.2 Summary of marketed formulations available in the USA, UK, New Zealand, and Australia.

Category Dosage form Brand name APIs Strength(s) Indication

Solid Suppository MIGERGOT® Caffeine, Ergotamine

tartarate

100 mg Caffeine USP , 2 mg

Ergotamine tartrate USP

Migraine

CANASA® Mesalamine 1 g Ulcerative colitis and inflammatory

bowel disease

COMPRO® Prochlorperazine 25 mg Nausea and vomiting

ACEPHEN® Acetaminophen

(Paracetamol)

325, 650 mg Fever and pain relief

FEVERALL® Acetaminophen

(Paracetamol)

80, 120, 325 mg Fever and pain reliever for infants

ALVEDON® Paracetamol 60, 125, 250 mg Fever and pain relief

DULCOLAX® Bisacodyl 10 mg Constipation relief

Liquid Enema COLOCORT® Hydrocortisone 100 mg/60 mL Ulcerative colitis and inflammatory

bowel disease CORTENEMA®

Microenema MICROLAX® Sodium citrate/sodium

lauryl

sulfoacetate/glycerol

450 mg sodium citrate, 45 mg

sodium lauryl sulfoacetate, 3.125

mg sorbitol/ 5 mL,

Constipation relief

Chapter 2

27

Table 2.2 Summary of marketed formulations available in USA, UK, New Zealand, and Australia- continued.

Category Dosage form Brand name APIs Strength(s) Indication

Liquid MICOLETTE® *(see below)

Foam UCERIS® Budesonide 2 mg Ulcerative colitis

Suspension KIONEX® Sodium polystyrene

sulfonate

15 mg/60 mL Constipation relief

Semi-solid Gel DIASTAT®

ACUDIAL™

Diazepam 5 mg/mL Antiepileptic

Ointment RECTOGESIC® Glyceryl trinitrate 0.2% w/w Anal fissure, haemorrhoids

Medicinal

devices

Rectal tampon SURGISPON®

ANAL

Absorbable gelatine

sponge

First grade pure gelatine Wound healing and surgical

procedures

PERISTEEN®

ANAL PLUG

Absorbent sponge Soft and absorbent foam Prevention of faecal incontinence up

to 12 hrs

*:90 mg sodium citrate dihydrate, 9 mg sodium lauryl sulfoacetate, 887.5 mg/mL sorbitol solution (70%, non-crystallising).

Chapter 2

28

Solid rectal dosage forms

Solid rectal dosage forms are fixed dose formulations intended for delivery via rectal route

and include suppositories, capsules and tablets or powder for reconstitution.

Suppositories

Suppositories are solid single unit dosage forms that contain the active compound either

solubilised or suspended in a suppository base, which upon melting or solubilisation at

physiological conditions releases the API for either local or systemic action [79, 80].

Suppositories can be formulated using different suppository bases depending upon the

physicochemical properties of the drug and compatibility with the bases. The difference in

physicochemical properties of drug and suppository bases could be effectively used to

modify the release and absorption of the drug. Depending upon the site of administration

and treatment group, suppositories are formulated in different sizes. To avoid falling out, a

suppository should be inserted past the muscular sphincter, which equates to about half an

inch into the rectum for infants and 1 inch for children and adults [81]. Also, as described

above, location of suppository in the rectum determines the fate of the drug that whether it

will undergo first-pass metabolism after absorption or not [62, 82, 83].

Rectal capsules, tablets or powder for reconstitution

Rectal capsules have been used to improve absorption for certain drugs like lidocaine and

propranolol that are extensively metabolised by first-pass metabolism [84]. However, there

are limited marketed rectal capsules, or tablets or powder for reconstitution [9].

Liquid rectal dosage forms

Any solution, emulsion or suspension for rectal administration is referred to as an enema, or

micro-enema if administration volume is small such as 5 to 10 mL. These formulations

Chapter 2

29

contain either oil, glycerine or low molecular weight macrogol as vehicle [6].

Administration volume may vary from 2.5 mL up to few hundred mL and applicators can

be used to assist administration such as the Macy catheter®, a recently FDA approved

medical device for rectal administration of fluids [85]. Enemas are used for rectal

stimulation to initiate defaecation before operative procedures [86] and for localised

treatments of inflammatory bowel disease or ulcerative colitis [87, 88] or as a medium to

deliver contrast agent before radiographic examination. Despite micro-enemas being more

advantageous than large volume enemas, the packaging and transportation costs make them

a less favourable formulation than large volume enemas [9].

Semi-solid rectal dosage forms

Gels, ointments and creams are common rectal semi-solid dosage forms in which the API is

dispersed in either hydrophilic (e.g., PEG 1500 & 4000) or lipophilic base (e.g., cocoa butter,

Witepsol®, Suppocire®) along with excipients such as glycerine or Tween® 80 to enhance

absorption. The semi-solid mixture is packed in a collapsible tube with an applicator.

Selection of the base for any semi-solid preparation affects the drug release due to solubility

of the APIs in the bases [89, 90]. Current commercial applicators are either supplied

separately or pre-filled with the formulation such as Diastat® Acudial™ (Table 2.2). Semi-

solid preparations comprising anti-inflammatory drugs or anaesthetics either alone or in

combination are widely used for localised treatments. Diazepam has been successfully

formulated in a rectal gel, Diastat®, and found to be effectively delivered to systemic

circulation. This formulation is useful in the treatment of acute repetitive seizures as it

increases the convulsion-free time by 12 hours [65] and reduces the frequency [66] and

duration of seizures [91].

Chapter 2

30

Considerable formulation development has been undertaken to achieve sustained drug

release from semisolid rectal formulations, such as hydrogel or xerogel dosage forms during

the 1970s - 1990s [92-97]. Though hydrogels provide sustained drug release, the dose

delivered to the site of administration may differ due to loss of formulation on the surface

of the applicator [97].

2.4 Novel rectal drug delivery systems and treatment opportunities

Traditional RDDS have recently improved by modifying the formulation properties such as

spreadability. The spreading characteristics of the various dosage forms can be demonstrated

as liquid > semi-solid > solid dosage forms [6]. In addition, prolonged retention and

controlled drug release may help to improve bioavailability and offer better pharmacokinetic

profiles or local treatment effect. Novel RDDS have been designed to offer better control

over spreading, retention and/or release of the drug via different formulation strategies, as

discussed in detail below.

Hollow type suppository

Hollow type (HT) suppositories (Figure 2.2) came into existence in the 1980s and various

APIs have been studied for modulation of release characteristics from these formulations.

HT suppositories contain a hollow cavity that can accommodate either solid, liquid or gel

[98, 99]. A special kind of mould equipped with a rod-like adaptor is used to make a hollow

cavity in the suppository [100]. As two phases, HT suppositories can prevent the interaction

between drug molecule and suppository base by eliminating the co-heating step and thus

can accommodate thermolabile APIs in their hollow cavity [101]. The other advantage of

HT suppositories is that the drug can be incorporated in the shell as well as in the hollow

cavity, which can provide rapid drug release from the core followed by sustained release

from the shell [99, 101].

Chapter 2

31

Figure 2.2 Schematic diagram representing longitudinal cross-section of HT suppository.

HT suppositories have been developed and evaluated for the effect of various formulation

additives to enhance drug absorption such as α-cyclodextrin [102-104] and sodium

decanoate [105]. A study was carried out using a HT system for indomethacin delivery. A

self-emulsifying system consisted of unsaturated fatty acid and a non-ionic surfactant which

dissolved indomethacin was filled in the core and an increase of approximately 41% was

found in rectal absorption of indomethacin compared with the powder-filled HT suppository

[106]. Further, HT suppositories have been reported to be advantageous over conventional

suppositories for the treatment of some chronic diseases such as asthma [99].

Since 2009, sustained release hollow-type (SR-HT) suppositories have offered more

controlled release of API from the suppository bases aided by muco-adhesive polymers [1].

Various natural gums including xanthan gum and alginate derivatives alter the viscosity of

the rectal fluid, while mucoadhesion increases mean residence time (MRT) and prevents

spreading of the formulation towards the colorectal area [99, 101, 102]. One study suggested

that selection of a carrier or vehicle is as important as the formulation technique [107]. For

example, hydrophilic bases are often mildly irritant and result in the secretion of more

mucous in the rectum compared with lipophilic suppository bases. In addition, the base

contributes to the variation in drug release characteristics [108, 109].

Chapter 2

32

Thermo-responsive liquid suppository (LS)

Flexibility of administration and intention to reduce the irritation to rectal mucosa led to

development of so called ‘liquid suppositories’ (LS) [67, 73, 110-113]. Liquid suppositories

are thermo-responsive rectal gels and are also referred to as thermoresponsive liquid

suppositories because the base material used in the formulation is a thermosensitive polymer

that becomes gel at physiological temperature (37 oC). LS differ from other liquid rectal

dosage forms by means of composition; enemas or micro enemas are suspensions or

emulsions applied to induce bowel movement to expel the rectal contents, while liquid

suppository forms a gel at physiological temperature to release the drug slowly either for

localised or systematic effect.

The main advantage of liquid suppositories over conventional or solid suppositories is that

they are easy to administer as they remain liquid at lower temperatures and minimise the

feeling of a foreign body compared with solid suppositories [110]. Thermosensitive

polymers such as poloxamers or pluronics form a gel at physiological temperature, thus

prevent leakage and excessive spreading in the rectum [67, 73, 110]. Moreover, the

combination of muco-adhesive and thermosensitive polymers provide more sustained

release of the drug compared with the thermosensitive polymer alone [68, 110, 114, 115].

A similar study has been carried out for the development of collagen based rectal in situ gels

containing mesalamine for the treatment of ulcerative colitis. The liquid formulation

undergoes phase transition from sol to gel under pH and temperature change, thus provided

sustained drug release. Efficacy was demonstrated using a mice model [116]. Viscosity,

gelling time and temperature threshold are all crucial factors in the formulation of liquid

suppositories and should be controlled otherwise the suppositories may either leak out from

the rectum or difficult to administer [115, 117]. This issue can be resolved by using a muco-

adhesive polymer in the formulation.

Chapter 2

33

Muco-adhesive gel

Muco-adhesive polymers can help to prolong the drug release by increasing the residence

time of the formulation in the rectum. Various muco-adhesive polymers have been studied

for their improved retention effect on formulation and pharmacokinetic parameters. The

results suggested that sodium alginate gives a sustained release with higher bioavailability

and less irritation to the rectal mucosa compared with hydroxypropyl cellulose (HPC), poly

vinyl pyrollidone (PVP), carbopol and polycarbophil [118]. Gaudin et al. investigated a

Carbopol based muco-adhesive gel containing artesunate, an anti-malarial drug, potentially

used for paediatric patients. Apart from the muco-adhesiveness and sustained release

properties which may improve bioavailability, this two-compartment formulation with the

drug and polymer separated from the liquid vehicle until prior to use, was also reported to

improve the stability of artesunate compared with current formulations [119]. Modified

chitosan containing hydrogel loaded with sulfasalazine also showed sustained and localised

release of drug which resulted into less production of toxic metabolites of sulfasalazine

[120].

The formulation experts suggested that the effect of drug and excipients on overall strength

of gel, mucoadhesivity and viscosity should be taken into consideration. It has been reported

that either API, excipient or both may affect the formulation characteristics [74, 112, 114,

121].

Micro and nanoparticles

Several articles have been published related to micro- or nanoparticles for rectal delivery

to achieve site specific and controlled drug delivery to the colorectal area using RDDS [122-

124]. These particulate systems were incorporated either into a suppository base or a gelling

polymer as a vehicle, which was used as a vehicle and a release modifier, respectively.

Chapter 2

34

Recently, silver-coated glass beads were delivered rectally for the localised treatment of

inflammatory bowel disease. The metal coated carrier system was incorporated into a

suppository base and the results suggested that almost 50% of the drug was released within

half an hour following administration [125]. However, several problems associated with

incorporation of a particulate system were reported, such as settling of the glass-beads at the

bottom and clogging of the particulate system [125].

Formerly metoclopramide and diazepam have been effectively delivered using solid lipid

nanoparticles (SLN) and a conventional suppository base [126, 127]. These SLN

formulations showed sustained release of the drugs due to coating of the lipids, which retards

the drug release by forming a hydrophobic matrix.

A novel approach integrating nanotechnology and liquid suppository was adopted for

targeted delivery of anti-cancer drugs, where delivery of docetaxel-loaded nano micelles in

a muco-adhesive LS showed promising anti-tumour activity with fewer adverse effects than

intravenous and oral formulations. Moreover, the surfactant used for the preparation of

micelles, Tween 80, is itself a permeation enhancer and it also formed a eutectic mixture

with docetaxel resulting in increased solubility and absorption of the drug [115].

Another approach uses nanotechnology in combination with thermo-responsive polymers in

rectal delivery, coined “double-reverse thermosensitive drug delivery” [128, 129]. This

system involves dispersion of API-loaded solid lipid nanoparticles in the thermo-responsive

in situ gel. This system provides a sustained release as absorption of the API is limited by

dissolution and diffusion. Such a system has been studied mainly for site specific drug

delivery such as for local treatment of colorectal cancer.

Chapter 2

35

Vesicular drug delivery systems

Currently, liposomes and niosomes are widely used for targeted and transdermal drug

delivery [130, 131]. However, very little work has been done to explore the applicability of

liposomes or niosomes for rectal drug delivery.

Unlike liposomes, niosomes do not contain phospholipids in their composition and can be

formulated by hydration of surfactant mixture alone. They are cheap and chemically more

stable than liposomes. A group of scientists developed and optimised the formulation of pro-

niosomal gel for the localised treatment of haemorrhoids. They concluded that the

concentration of surfactant affects entrapment efficiency and drug release. For the said

experiment the optimised pro-niosomal gel provided immediate release for the fraction of

API that could be beneficial for the localised treatment of diseases such as haemorrhoids

[132]. Recently, another vesicular drug delivery system containing nanotransferosomes was

studied. Nanotransferosomes are ultra-flexible liposomes containing edge activators. The

edge activators provide significant flexibility and deformability to the transferosomes [133].

Optimum transferosome formulations were then incorporated into a pluronic-based

thermoreversible gel including hydroxypropyl methylcellulose (HPMC). The

pharmacokinetics study in rabbits showed a two-fold increase in bioavailability along with

a longer half-life as compared to the oral drug solution [133].

2.5 In vitro and in vivo evaluation of RDDS

The United States, British and European Pharmacopoeias describe various in vitro

characterisation tests for the evaluation of rectal dosage forms including uniformity of mass,

content uniformity, liquefaction time and disintegration time [134-136]. Other tests like

gelling time [110], gel strength [67, 74, 137, 138] and muco-adhesive strength [69, 118, 121,

137, 138] need to be carried out specifically for thermo-responsive and muco-adhesive

Chapter 2

36

liquid suppositories, as described in detail in previously published papers [112, 113, 137].

The present review focuses on the release studies in the following paragraphs.

Various methods have been explored for the determination of drug release and absorption

from rectal dosage forms. Studies for in vitro release and in vivo/ex vivo absorption with

different RDDS and their release characteristics (Table 2.3) are reviewed in the following

sections. In addition to this, irritancy, spreadability and retention have also been evaluated.

In vitro release studies of RDDS

Different dissolution methods have been developed to study in vitro drug release from

various RDDS. These include using USP Type I rotating basket apparatus, USP Type II

paddle apparatus and USP Type IV flow through cell apparatus [139, 140]. Although none

of these are designed specifically for RDDS, the USP Type IV apparatus has been used on

a commercial scale to measure drug release from suppositories. The equipment contains a

flow cell that is filled with the glass bead-bed, limiting the exposure of suppository to the

dissolution medium, as well as a simulated rectal media that reflects in-use conditions. This

method provides easy changeover of solvents and pH adjustments, and is easy to maintain

sink conditions [141]. In addition, the Japanese Pharmacopoeia apparatus 3 has a similar

design to the USP type IV flow through cell apparatus [142]. The flow of dissolution

medium can be adjusted between 4 and 16 mL per minute. To simulate the rectal

environment, use of either phosphate buffer solution ranging from pH 7.2-7.4 or water has

been reported previously [74, 121, 126, 138]. Compared with water as medium, use of buffer

would provide more consistent results due to the control of pH and ionic strength to simulate

the in vivo environment. Different values for volume of dissolution medium and rotations

per minute (RPM) ranging from 200 to 1000 mL and 25 to 700 RPM, respectively, have

been reported [73, 102, 106, 114, 119]. However, the anatomical location of the rectum at

Chapter 2

37

the distal part of the gastrointestinal tract shows less movement than the intestine which

indicates that a lower stirring speed would better mimic the rectal environment.

A method was developed whereby the formulations were placed directly on a metallic net

(instead of a semipermeable membrane) inside a plastic cylinder, before being immersed in

the dissolution medium [99, 100]. Use of USP basket type I apparatus without any

modification [101] and with modification (by introducing the formulation in a specialised

dialysis bag) has been reported [116, 119]. Similarly, dialysis membranes or dialysis bags

can also be used in the type II apparatus [68, 73, 132]. Since the rectum has only paracellular

and transcellular transport [6] and the cell epithelium acts as a semipermeable membrane,

the use of dialysis bags can help to mimic the rectal epithelium and predict in vivo drug

release. Some rectal dosage forms have also been analysed using a fabricated vertical Franz

diffusion cell, which offers an effective diffusion area to mimic the rectum [67, 133].

Table 2.3 below shows a general categorisation of novel RDDS, their release characteristics

and their possible mechanism of release.

Chapter 2

38

Table 2.3 Summary of novel rectal dosage forms and different mechanisms involved in drug release from different type of dosage forms.

Category of novel

rectal dosage form

Subtype of dosage

form

Category of API used Release

characteristics

Possible mechanism

of release

References

Liquid Mucoadhesive -

thermosensitive

Antiemetic, anticancer,

analgesic, antihypertensive,

antipyretic, antimalarial

Extended release Dissolution and

diffusion

[68, 69, 74, 110,

118, 121, 137,

138]

Mucoadhesive nano

micelles in

thermosensitive

polymer

Anticancer Extended release Diffusion [115, 117]

SLN embedded into

thermosensitive

polymer

Anticancer, antiepileptic Extended release Dissolution and

diffusion

[126, 128, 129]

Chapter 2

39

nanotransferosomes Muscle relaxant Initial burst release

followed by gradual

drug release

Diffusion [133]

Semi-solid Niosomal gel Bioflavonoid as

antihaemorrhoid

Initial burst release

followed by

gradual drug

release

Diffusion [132]

Solid HT Analgesic, antiemetic,

NSAIDS

Immediate release Suspension followed

by dissolution

[98, 101, 106]

Sustained release

hollow-type (SR-

HT)

Antiasthmatic, analgesic,

antiparathyroid

Immediate release

followed by

sustained release

Dissolution followed

by diffusion

[99, 103, 105]

Chapter 2

40

Similar to conventional suppositories, HT suppositories show drug release due to dissolution

of the API, but the immediate release may be due to dual loading of the drug in both shell

and cavity, except material filled in the cavity is for prolonged release. Drug release can also

be altered by addition of a permeability enhancer or by using a carrier system [106]. In

contrast, SR-HT suppositories and LS contain release retarding and muco-adhesive

polymers, respectively, which provide slower drug release for an extended time period. This

may be beneficial for targeted drug delivery and to help minimise side effects of some potent

drugs [110, 129]. Drug release from liquid suppositories was further supported by the

Higuchi model, which meant that drug release from the liquid suppositories was Fickian

diffusion [68, 73, 138]. However, a group of scientists found a mixed Fickian and non-

Fickian diffusion for a gel based LS, possibly owing to the erosion of the gel matrix that led

the burst drug release [74]. Studies have also shown Fickian diffusion as the predominant

mechanism of drug release for vesicular RDDS [122, 132].

In vitro, ex vivo and in vivo studies for prediction of absorption from RDDS

Several studies suggested that caco2 cell lines could be used to predict the rectal absorption

of drugs [143-145]. However, caco2 are intestinal cell lines and form tight junctions that

prevent paracellular diffusion and may offer carrier-mediated transport. As this is reportedly

absent in rectum, it may not provide a suitable representation of the rectal environment [143,

146-148]. While cell culture methods have reduced the number of animal studies required,

animal models remain more representative of human in vivo rectal drug absorption.

Various in vivo pre-clinical models have been used to investigate the rectal drug absorption

and pharmacokinetics. For research, rats, rabbits, dogs and occasionally pigs have been used

for evaluation of LS and HT suppositories [99, 110, 128]. Previously, ex vivo studies has

been performed using isolated cattle and rat rectum to understand drug permeability [132,

Chapter 2

41

133]. Among these animals, rabbits are preferable for the pharmacokinetic study involving

multiple sampling points because rabbits have a larger blood volume compared with rats

and mice. While beagle dogs have also been used as an effective model for testing

suppositories [71, 149, 150], the expense of this model may be prohibitive. Both dogs and

rabbits have similar rectal physiology to humans [73, 151, 152].

Apart from absorption, another important testing parameter for RDDS is tissue irritancy.

The irritation due to rectal dosage forms can be analysed by histological examination of the

rectum of various animals, including rabbits and rats [67, 74, 99, 110, 133]. Also, to reveal

the location of the suppository upon administration blue lake dye has been incorporated in

the formulation, which could be of particular interest for liquid suppositories [112, 114].

This study would also provide information about the spreading and residence time of the

dosage form in the rectum [118].

2.6 Recent trends in the formulation of RDDS

The present literature review has indicated an increase in the development of novel RDDS

in the recent years. Novel RDDS have been studied for sustained release of some potent

drugs such as anti-cancer drugs [115, 128].

Being able to deliver a biphasic system in a single dosage form, HT suppositories can be

considered for further formulation development. Liquid suppositories provide prolonged

drug release due to the presence of thermosensitive and muco-adhesive polymers, which

form a muco-adhesive gel upon administration resulting in increased mean residence time

(MRT) and bioavailability [68, 110, 121, 153]. Muco-adhesive and thermogelling properties

of liquid suppository allow for both localised and systemic delivery of different drugs. In

addition, micro- and nano- particulate systems suspended in the thermo-responsive carrier

provide both targeted and controlled drug delivery [99, 115, 117, 129].

Chapter 2

42

Although RDDS may be more suitable than oral dosage forms in certain conditions, the

bioavailability and stability issues with these dosage forms need to be addressed. Advances

in the formulation technology and materials have encouraged the innovation of RDDS.

Apart from the RDDS discussed above, various other formulation approaches such as solid

dispersions [154], starch hydrolyse [155] and other formulation additives can be used to

modify the drug release and absorption. Table 2.4 summarises various excipients used and

their functions in the formulation of RDDS.

Chapter 2

43

Table 2.4 Summary of various excipients and their functions in the formulation of the rectal dosage forms.

Type of

excipient

Name of excipient Function References

Natural

polymer

Collagen Biocompatible, helps to replenish GI mucosa [116]

Xanthan gum Biocompatible, extends release [102]

Sodium alginate Biodegradable and mucoadhesive, extends release [68, 99, 118]

Gellan gum Biodegradable, thermo-sensitive, extends release [138]

Semi-synthetic

polymer

Hydroxy propyl methyl

cellulose (HPMC)

Mucoadhesive, helps to retard the release [67, 73, 121, 133, 137]

Methyl cellulose Retards drug release [68, 73, 121]

Chapter 2

44

Hydroxypropyl cellulose

(HPC)

Retards drug release [137]

Synthetic

polymer

Polyacrylate and sodium

polyacrylate

Extends drug release [99]

Poly vinyl pyrrolidone Extends drug release [73, 118, 121, 137]

Polycarbophil Extends release, and increases bio adhesiveness [137]

Carbopol® Mucoadhesive, extends release [73, 118, 119, 121, 137,

138]

Poloxamers Thermo-sensitive, extends release [67, 68, 115, 117, 118, 121,

126, 128, 129]

Surfactant Tween 80 Reduces surface tension, enhances permeation and

bioavailability

[115, 126, 128]

Sodium taurocholate Increases permeation and bioavailability [117]

Chapter 2

45

Sodium laurate Enhances absorption [156]

Sodium caprate Enhances absorption [156]

Sodium salicylate Enhances absorption [69]

Polyoxyethlene-9-lauryl ether Enhances absorption, increases bioavailability [70]

Lipid Compritol® Extends drug release [126, 127]

Imwitor® Extends drug release [126]

Tricarpine and triethanolamine Extends drug release [128, 129]

Others Sodium chloride Controls gel strength and mucoadhesivness ,

modifies absorption

[113]

Taurine Enhances absorption, cytoprotective [156]

α- Cyclodextrin Enhances viscosity and permeability [102]

Chapter 2

46

Notably, the drug-excipient and excipient-excipient interactions should not be neglected.

These interactions can significantly affect the final properties of the formulation such as

melting point, gelling properties and muco-adhesiveness [113], which are the key properties

for RDDS. In developing thermo-responsive and muco-adhesive liquid suppositories, Choi

et al. studied the effects of dug-excipient and excipient-excipient interaction. They found

that addition of acetaminophen (paracetamol) increased gelling temperature and decreased

the gelling strength and mucoadhesivness. In contrast, addition of carbopol and

polycarbophil decreased gelation temperature [137].

2.7 Conclusion and future perspectives

The RDDS have a number of advantages. Various formulation approaches for either

immediate or controlled delivery of API have been investigated for the formulation of rectal

dosage forms. The present review shows an increase in the development of modified RDDS

to achieve controlled drug delivery either systemically or locally by minimising the toxic

effects of some potent drugs. A wide range of excipients can be used, and desired properties

for drug release, spreadability and rectal retention can be achieved by modulating the

concentration, thus, optimising therapeutic outcomes. A challenge is drug transport through

rectal mucosa; due to smaller rectal fluid volume and relatively different epithelial surface

compared with intestinal mucosa drug absorption may vary depending upon the

physicochemical properties of the API. RDDS have potential for outpatient paediatric

treatment of some diseases at earlier stages before they progress to severe conditions such

as pneumonia or malaria. Also, formulation modification for better absorption is an area for

future focus. Complexity, stability, scale-up opportunity, end-user compatibility and clinical

relevance of pre-clinical data also play an important role in the development of a rectal

dosage form. This review also found that due to lack of an established rectal cell culture

Chapter 2

47

model, the in vitro evaluation of RDDS is reliant on animal model studies, which can present

ethical dilemmas and may not simulate human physiology, resulting in varied efficacy in

humans. This review recommends further research to improve the stability and

bioavailability of the rectal dosage forms. Apart from this, development of a clinically

relevant cell culture models that can provide reliable interpretation of absorption profile of

the rectal dosage form would be a huge contribution in the field of RDDS. Thus, the rectal

route is a potential alternative to the oral and parenteral route and it needs to be explored to

unleash its hidden potential in the field of drug delivery.

48

Development of Stability-Indicating HPLC Assay and Preformulation Studies

Chapter 3

49

This chapter describes preformulation studies including HPLC method development for

in vitro quantification of amoxicillin. Also, the preliminary screening of suppository bases

and batch formulation parameters for the preparation of amoxicillin suppositories have been

discussed in detail.

3.1 Introduction

Preformulation studies are essential preliminary tests to determine the physicochemical

properties of a drug molecule prior to development of a formulation [157]. Preformulation

data can minimise time and costs involved in the development of a new formulation [158,

159]. Although amoxicillin is a successfully marketed drug molecule for oral and parenteral

applications, it is also known to be sensitive to hydrolysis, which may lead to loss of activity

due to alteration in the molecular structure [160-162].Amoxicillin has pH-dependent

stability and solubility in aqueous media, with minimum solubility and optimal stability

between pH 4 – 6 and it exists in its most stable zwitterionic form within this pH range [161-

163]. Moreover, temperature dependent oxidative degradation of amoxicillin has also been

reported [164]. Due to these reasons, development of an efficient chromatographic method

that can quantify amoxicillin in the presence of degradation products while maintaining its

stability was deemed necessary.

As mentioned in Chapter 1, fusion melting is the most frequently used process for

preparation of suppositories where the drug is either dissolved or suspended in the melted

suppository base, then the mixture is poured into the suppository mould under continuous

stirring. Furthermore, the preparation of suppositories involves several steps of phase

change due to heating and cooling of drug and suppository base mixture. Thermodynamic

changes during this process may make amoxicillin prone to hydrolytic and oxidative

degradation [161-164]. The crystalline structure of suppository bases, especially synthetic

Chapter 3

50

lipophilic suppository bases, may also change during the preparation of the suppositories

due to heating and recrystallisation during cooling. This may alter its properties such as

melting behaviour and spreading in the rectal mucosa [165-167]. Therefore, evaluation of

drug-suppository base compatibility prior to formulation is essential. Compatibility of

amoxicillin for the preparation of suppositories can be evaluated using differential scanning

calorimetry (DSC).

Preparation of suppositories involves melting a specific mass of suppository base, which

occupies a specific volume upon filling the mould cavities. Different bases and drugs have

different densities. Hence, it is important to determine mould capacity (MC) and

displacement value (DV) [168, 169]. MC is different for specific moulds and suppository

bases while DV is different for different drugs in different suppository bases [169].

Therefore, calculation of DV is important to ensure accurate and uniform dosing while

preparing a batch of suppositories [18, 169].

This chapter aimed to carry out preformulation studies including development and

validation of a stability-indicating HPLC assay for in vitro quantification of amoxicillin and

to facilitate the process of suppository development. To prepare amoxicillin suppositories

using the fusion moulding method, thermal behaviour of hydrophilic and lipophilic

suppository bases were also characterised to decide the formulation strategy. The DV of

amoxicillin in each suppository base was determined to calculate the quantities of materials

required for the preparation of suppositories.

3.2 Material and methods

Materials

Amoxicillin trihydrate (AMT) (purity ≥ 99%) and Amoxicillin sodium (AS), purity >87%

were purchased from Alfa Aesar by Thermo Fisher scientific (Haverhill, Massachusetts,

Chapter 3

51

USA). Polyethylene glycol (PEG) at different molecular weights 1500 and 4000 (PEG 1500

and PEG 4000) were purchased from Sigma Aldrich (St. Louis, Missouri, USA). Suppocire®

NA 15 (SNA 15) pellets were kindly donated by Gattefossé SAS, France. Acetonitrile and

methanol were HPLC grade. The Milli-Q water for HPLC was obtained from Millipak

(Millipore, 0.22 µm). Potassium dihydrogen phosphate, potassium hydroxide pellets and

hydrochloric acid, used in the preparation of the buffer, were of analytical grade and

purchased from either Merck or Thermo Fisher Scientific, New Zealand. The 30-well,

stainless-steel, 1 g suppository mould used to prepare the suppositories was purchased from

Heros® Ltd (Olomouc, Czech Republic).

Selection of chromatographic parameters

Mobile phase

Potassium phosphate buffer (pH 2.5-5.5) was selected due to its efficiency in

chromatographic separation of amoxicillin as part of the mobile phase based on previous

studies [170-172]. Mobile phases comprising phosphate buffer ranging from 60 – 95% v/v

along with either methanol or acetonitrile or both at different ratios were evaluated for their

effect on retention time and peak response and peak symmetry.

Detection wavelength

The peak spectra were analysed at wavelengths ranging from 190 - 400 nm with a special

focus on the peak response of amoxicillin between the ranges of 227 – 230 nm, 254, 270,

273 nm using a photo diode array (PDA) detector [51, 172-177]. The selected wavelength

was also evaluated for amoxicillin peak purity using PDA.

Chapter 3

52

HPLC method validation for in vitro quantification of amoxicillin

The HPLC method was validated according to the International Council for Harmonisation

of Technical Requirements for Pharmaceuticals for Human Use (ICH) quality guidelines

about analytical validation of pharmaceuticals for human use [178].

Chromatographic conditions

An isocratic HPLC method for separation was developed using an Agilent 1260 series HPLC

instrument (Agilent Technologies, Germany) The chromatographic separation was carried

out on a Luna Omega PS C18 (250 x 4.6 mm, 5 µm) column from Phenomenex, Auckland,

New Zealand. The mobile phase was selected as 50 mM phosphate buffer, pH 5.0, methanol

and acetonitrile in a ratio of 93:5:2 %v/v and the flow rate was kept at 1 mL/min. The

injection volume was 20 µL and the detection was carried out at 228 nm using PDA detector.

Preparation of standard solutions for HPLC method validation

AMT was weighed to prepare a 1 mg/mL amoxicillin stock solution. From the stock

solution, appropriate dilutions were made to prepare quadruplets of seven calibration

standards at concentrations 0.2, 0.5, 1, 5, 10, 25 and 50 µg/mL. Another set of triplicates

were prepared at concentrations 3, 15 and 40 µg/mL using a separate stock solution as

quality control (QC) samples to determine the intra-day and inter-day variation of the

developed method.

Validation of HPLC

3.2.3.3.1 Specificity

To determine the specificity of the method, chromatograms of amoxicillin solution subjected

to different forced degradation studies were compared to observe satisfactory resolution of

amoxicillin from its degradation products.

Chapter 3

53

3.2.3.3.2 Linearity

The developed method was evaluated for linearity between the range of 0.2 – 50 µg/mL by

plotting the values of concentration versus respective peak area [178].

3.2.3.3.3 Accuracy and precision

Accuracy indicates the closeness of the determined concentration to the true value. The

accuracy of the method was determined by repeated injections of the QC sample prepared

at different concentration levels.

Precision of a method indicates the closeness of repeated measurements. Precision of the

developed method was determined by multiple injections of the QC samples prepared at

three concentration levels at different times in a day and for three consecutive days.

3.2.3.3.4 Sensitivity

The sensitivity of the developed method was determined by evaluating the limit of detection

(LOD), which indicates the ability of the method to determine the lowest concentration of

the analyte in the sample, and limit of quantitation (LOQ), which indicates the ability to

quantify the lowest amount of an analyte in the sample.

Both LOD and LOQ were determined by evaluation of signal-to-noise ratio of serially

diluted injections of amoxicillin. An acceptable signal-to-noise ratio was considered

between 3 or 2:1 for LOD and 10:1 for LOQ [178].

Preliminary stability evaluation of amoxicillin

pH-dependent stability evaluation of amoxicillin

To evaluate the stability during sample analysis, duplicates of amoxicillin 40 µg/mL

solutions were prepared using phosphate buffer solutions at pH 4, 5 and 6 at 2 - 8 °C and

Chapter 3

54

ambient temperature. Stability of amoxicillin was evaluated for 24 days to understand the

pH-dependent stability of amoxicillin. Stability of amoxicillin up to 24 h at ambient

temperature was considered optimum for sample preparation and analysis. All samples were

protected from light during the stability analysis.

Forced degradation studies of amoxicillin

To determine the stability-indicating nature of the HPLC assay, amoxicillin was subjected

to different hydrolysis (pH 2 or 10), photolytic and oxidative stress conditions to generate

the degradation products [179-181]. Amoxicillin solutions were prepared at 200 µg/mL

concentration using 0.1 mM hydrochloric acid (HCl), 0.1 mM sodium hydroxide (NaOH)

water and 0.3% hydrogen peroxide (H2O2). Duplicates of the acidic, basic and water samples

were kept at 60 °C, while the other set of duplicates in water were exposed to UV light

ranging from 320-400 nm at 25 °C. Oxidative stress degradation was carried out at 25 °C

protected from light. To determine the specificity of the method, samples were withdrawn

at predetermined time intervals, appropriately diluted and subjected to HPLC analysis to

evaluate the ability of the method to specifically resolve amoxicillin from its degradation

products. The degradation was carried out for up to seven days or until 10 % degradation of

amoxicillin was observed, whichever was earlier.

Selection of suppository bases using differential scanning calorimetry (DSC)

Initially, different grades of hydrophilic and lipophilic suppository bases were considered

for the preparation of suppositories. Physicochemical properties of suppository bases and

literature evidence were referred for the selection of suppository bases. The suppository

bases were screened based on their melting and solidification behaviour. Selected

suppository bases including, PEG 1500, 4000 and SNA 15 were then evaluated for their

melting behaviour by thermal analysis using DSC.

Chapter 3

55

Different ratios of PEGs have been reported previously for the preparation of suppositories

[12, 14]. Therefore, to determine effects of varying the ratios of two different molecular

weight PEGs and to identify the optimum PEG base, PEG 1500:4000 ratios of 70:30, 80:20,

85:15 and 90:10 were evaluated using DSC. A precisely weighed quantity of about 5-7 mg

of each of the samples were subjected to a heat/cool/heat cycle from 0-70 °C at the rate of

5 °C/min with the nitrogen gas flowing at the rate of 20 mL/min to simulate both formulation

process and in vivo temperature changes.

Calculations for batch formulation of amoxicillin suppositories

Following the selection of optimum ratio of PEG 1500:4000 base mixture (70:30 w/w), MC,

DV and subsequent batch formulation parameters for the preparation of conventional

amoxicillin suppositories were determined for PEG base mixture and SNA 15 suppository

bases. For each type of suppositories, the quantity of each component was calculated with

20% extra to compensate for manufacturing loss including shrinkage of suppository base

during solidification.

Suppository mould capacity

The MC of a specific suppository mould differs depending on the suppository base [168].

Therefore, mould calibration was carried out by filling the cavities of a 1 g suppository

mould with each melted suppository base. The cavities were filled with PEG 1500:4000

(70:30 w/w) base mixture or SNA 15 base, six cavities per base. The suppositories were

allowed to set for one hour and excessive base was trimmed off from the top of the mould.

Prepared suppositories were removed from the mould and weighed individually. The

average weight of each type of suppository was considered the MC for that particular base.

Chapter 3

56

Displacement value (DV)

DV was determined for both AMT and AS. DV of a drug is the amount by weight of a drug

displacing one part weight of the suppository base [168]. DV changes for different types of

suppository bases when a specific amount of drug is added to the suppository base [182].

To determine the DV, six suppositories of each type of suppository base were prepared with

and without drug by fusion moulding. The average weight of blank suppositories and

amoxicillin containing suppositories along with equivalent dose of AMT or AS per

suppository (D) were applied for the calculation of DV.

3.2.6.2.1 Preparation of blank suppositories

The amount of each type of suppository base required for preparation of n number of

suppositories was calculated using Eq 3.1.

𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 𝐴𝐴𝑜𝑜 𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 = 𝐴𝐴 × 𝑀𝑀𝑀𝑀 × 1.2 Eq 3.1

The calculated amount of suppository base was melted at 70 °C and poured into the mould.

The filled material was allowed to set for one hour. After solidification of suppositories,

excess base was scraped off, the suppositories were demoulded and their average weight (A)

was recorded and used for the calculation of DV.

3.2.6.2.2 Preparation of amoxicillin containing suppositories

Six 1 g suppositories containing 114.7 mg of AMT or 106 mg AS (equivalent to 100 mg

amoxicillin) were prepared from each type of suppository base. The amount of each type of

suppository base was calculated using Eq 3.1. The suppository base was melted at 70 °C and

the equivalent quantities of AMT or AS required to make six suppositories were added to it.

The drug-base were mixed thoroughly and poured into each mould cavity, with continuous

stirring to maintain dose uniformity, then allowed to set for one hour. Excess of the drug –

Chapter 3

57

base mixture was trimmed off and suppositories were removed from the mould and weighed

individually. The average weight of amoxicillin suppositories (B) was utilised in the

calculation of DV.

Quantities required for a batch formulation

For preparation of a batch of 20 suppositories from each base containing either AMT or AS

equivalent to 100 mg of amoxicillin, the quantities of drug and bases were calculated as

described below.

The total quantity of AMT or AS required to prepare n number of suppositories was

calculated from the equivalent amount of amoxicillin per suppository (D) as described in

Eq 3.2.

𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 𝐴𝐴𝑜𝑜 𝑑𝑑𝑑𝑑𝐴𝐴𝑑𝑑 = 𝐴𝐴 × 𝐷𝐷 × 1.2 Eq 3.2

Considering the DV of amoxicillin, Eq 3.3 was applied to calculated quantities of PEG and

SNA 15 to prepare a batch of n number of suppositories.

𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 𝐴𝐴𝑜𝑜 𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 = [(𝐴𝐴 × 𝑀𝑀𝑀𝑀) − (𝐴𝐴 × 𝐷𝐷 𝐷𝐷𝐷𝐷⁄ )] × 1.2 Eq 3.3

3.3 Results and discussion

Selection of chromatographic parameters

Mobile phase selection

To ensure maximum separation efficiency for reverse phase separation of amoxicillin,

selection of pH two units above or below pKa was essential [183]. Amongst all the buffer

systems, amoxicillin was found to be eluting most efficiently with phosphate buffer pH 5.0

(Figure 3.1) because of two pKa values of 3.2 and 11.7, as mentioned previously. To

Chapter 3

58

maintain stability of amoxicillin during the analysis at ambient temperature conditions,

50 mM phosphate buffer, pH 5.0 was selected. This was further confirmed in preliminary

stability studies (Section 3.3.3), which indicated amoxicillin was most stable at pH 5.0.

Modulation of buffer to organic solvent ratio altered the retention time of the peak where a

reduced proportion of buffer resulted in very early elution of amoxicillin within 2 - 3 min,

as shown in Table 3.1. Therefore, a high buffer proportion >90% v/v was used to optimise

the amoxicillin elution time between 6-8 min. An increase in the proportion of organic

solvent, particularly acetonitrile, resulted in increased peak response and reduced the

retention time. The flow rate of mobile phase was 1 mL/min based on existing literature [51,

170, 184-186].

Table 3.1 Effect of modulation of buffer to organic solvent ratio on the retention time of

amoxicillin.

Mobile phase composition (% v/v) Retention time

(± 0.2 min) 50 mM phosphate

buffer, pH 5.0 Methanol Acetonitrile

60 35 5 2.6

75 20 5 4.8

95 5 - 10.8

93 7 - 8.8

93 6 1 7.4

93 5 2 6.3

92 7 1 5.6

Chapter 3

59

Detection wavelength

From PDA spectra and peak response evaluation it was found that amoxicillin exhibited a

maximum response at 228 nm compared with the other reported wavelengths including 227,

230, 254, 270 nm [46, 51, 55, 175, 187]. The comparison of peak responses at different

wavelengths are compiled in Table 3.2.

Table 3.2 Amoxicillin peak response evaluation using PDA at different wavelengths.

Wavelength (nm) Peak response (mAU*S)

227 411.8

228 413.8

229 413.0

230 409.6

254 41.7

270 43.5

Thus, 228 nm was selected to detect the amoxicillin for optimal sensitivity of the HPLC

method. This was particularly important for detection of amoxicillin in the presence of a

complex matrix such as suppository bases or biological samples. Both acetonitrile and

methanol were used along with 50 mM phosphate buffer, pH 5.0 in a ratio of 2:5:93 % v/v

for chromatographic separation which eluted amoxicillin at 6.3 ± 0.2 minutes (n= 4) samples

at each standard concentration within the range.

Chapter 3

60

Method validation

Specificity

The developed method was found specific enough to satisfactorily resolve the degradation

products from amoxicillin as shown in Figure 3.1 [178]. Figure 3.1 (a), (b) and (c) represent

the hydrolysis of amoxicillin at 60 °C when kept in acid, base and water, respectively,

whereas Figure 3.1 (d) shows the degradation of amoxicillin solution (200 µg/mL) at 25 °C

exposed to UV light. Figure 3.1 (e) demonstrates the oxidative degradation of amoxicillin,

which was found instantaneous in that the initial chromatogram also showed degradation

products. Figure 3.1 (f) shows representative peak purity of amoxicillin determined at all

the degradation conditions.

Chapter 3

61

Figure 3.1 Chromatograms showing complete resolution of AMT equivalent to

amoxicillin (200 µg/mL) from the degradation products (DP) generated at different forced

conditions (pH 2-10) after 24 hours. (a) 0.1 mM HCl at 60 °C, (b) 0.1 mM NaOH at 60 °C,

(c) Water at 60 °C, (d) UV (320 –400 nm)at 25 °C, (e) 0.3% H2O2 at 25 °C, (f) Amoxicillin

peak showing peak purity within calculated threshold limit (representing all degradation

conditions)

Chapter 3

62

Linearity

Seven calibrators ranging from 0.2 -50 µg/mL showed a linear increase in the peak response

with an increase in concentration of amoxicillin (Figure 3.2) with an R2 value of >0.9999

[178]. This will facilitate the quantification of amoxicillin from suppositories following

appropriate dilution of samples.

Figure 3.2 Linearity curve of amoxicillin. Data represented as mean ± SD, n = 4.

Accuracy and precision

The developed method was found accurate and precise with the accuracy ranging from 97-

100% at all three QC concentrations (3, 15 and 40 µg/mL). The precision of the method was

also determined from the relative standard deviation (RSD) for each of the concentrations

found below 1% [178]. The results of accuracy, intra-day and inter-day evaluations have

been summarised in Table 3.3.

Chapter 3

63

Table 3.3 Accuracy and precision of the method for quantifying amoxicillin at

concentration levels of 3, 15 and 40 µg/mL. Data represented as mean ± SD (n=3). RSD:

relative standard deviation.

Concentration

(µg/mL)

Intra-day Inter-day

Accuracy

(% experimental)

Precision

(% RSD)

Accuracy

(% experimental)

Precision

(% RSD)

3 97.9 ± 0.6 0.6 98.0 ± 0.5 0.6

15 99.0 ± 0.2 0.2 99.2 ± 0.6 0.6

40 99.9 ± 0.2 0.2 100.1 ± 0.6 0.6

Sensitivity

The signal-to-noise ratio of the serially diluted samples of amoxicillin was determined to

find the LOD and LOQ of the developed method. The method was found sensitive to detect

amoxicillin concentration as low as 27 ng/mL (LOD), which was determined by a signal-to-

noise ratio above 3. The LOQ was found to be 85 ng/mL with the signal-to-noise ratio above

10 [178].

Preliminary stability evaluation of amoxicillin

pH-dependent stability evaluation of amoxicillin

From the preliminary evaluation, pH 5.0 was found to be optimum for sample analysis at

ambient conditions. After 72 hours, > 90% amoxicillin was found at pH 5.0 when kept at

2 – 8 °C, while it degraded rapidly at pH 4 and 6 (Figure 3.3). It has been reported that

amoxicillin exists in anion, cation, zwitterion or dianion form at different pH levels ranging

Chapter 3

64

from 1 - 10 and therefore, it has multiple pKa values [46, 161, 162]. It is most stable between

pH 4 – 6 [161].

Figure 3.3 Stability of AMT equivalent to amoxicillin 40 µg/mL at pH 4, 5 and 6 for up to

24 days at (a) 2 - 8 °C and (b) ambient temperature. Stability of amoxicillin up to 24 h at

ambient temperature was considered optimum for sample preparation. Quantity of

amoxicillin remaining was calculated as percentage of initial concentration of amoxicillin.

Data represented as the mean ± SD of duplicates.

Forced degradation studies of amoxicillin

During the forced degradation study, amoxicillin was subjected to different harsh conditions

ranging from pH 2-10. The pH of all degradation samples was found to reduce by 1-2 units

after 24 hours. This could be due to the hydrolysis of amoxicillin, leading to generation of

an acidic degradation product such as amoxicillin penicilloic or penilloic acid (Figure 3.4).

This was found to be consistent with a previous study showing formation of acidic

degradation products due to hydrolysis of amoxicillin [161, 188].

Chapter 3

65

Figure 3.4 Proposed degradation products of amoxicillin in aqueous conditions

(pH 2- 10).

Amoxicillin was found to be highly unstable under oxidative, acidic and alkaline stress

conditions, with 4.5%, 14.1% and 10% remaining after 24 hours, respectively. However,

90% of amoxicillin remained after 6 days exposure to UV light at 25 °C. The results of the

degradation study have been summarised in Table 3.4.

Table 3.4 Effect of stress conditions on degradation rate of AMT (equivalent to 200

µg/mL amoxicillin) after 24 hours. Data presented as percentage amoxicillin remaining

from an initial concentration.

Stress condition

Storage conditions

Amoxicillin remaining (%) pH

Sample 1 Sample 2 Mean Before After

Acidic 0.1 mM HCl, 60 °C

13.8 14.3 14.1 2.9 2.7

Alkaline 0.1 mM NaOH, 60 °C

12.2 7.8 10.0 9.2 7.9

Temperature 60 °C in water 63.4 69.7 66.5 5.5 3.6

UV (320-400 nm) 25 °C in water 94.3 94.0 94.1 5.3 4.9

Oxidation 0.3% H2O2, 25 °C 1.7 7.4 4.5 4.7 3.7

Chapter 3

66

Selection of suppository bases using differential scanning calorimetry

Selection of suppository base is an important step for the development of suppositories as

they influence the release of drug from the suppositories [9, 189]. Suppository bases having

a high melting point and ability to withstand higher temperature were deemed ideal for the

preparation of suppositories [9, 16, 17]. Based on the literature evidence and to withstand

the high temperatures suitable for low- and middle-income countries (LMICs), as discussed

in Chapter 1, PEG 1500 and 4000 were shortlisted as hydrophilic suppository bases for

further evaluation [9, 12-15]. Recent research showed compatibility of amoxicillin with a

lipophilic suppository base, SNA 15, demonstrating complete and rapid drug release [12].

Furthermore, the low hydroxyl value (5-15 mg KOH/g) of SNA 15 is considered

advantageous because amoxicillin is susceptible to hydrolysis [12, 90, 190]. Therefore, SNA

15 was chosen as the lipophilic suppository base.

The melting behaviour of the suppository bases was analysed by subjecting each sample to

a heat/cool/heat cycle using DSC. The first heat-cool cycle during DSC analysis was to

mimic the manufacturing process and the second heating run was to mimic in vivo melting

behaviour of the suppository. The physical appearance of each suppository base, along with

their melting behaviour, is summarised in Figure 3.5.

Chapter 3

67

Figure 3.5 Physical appearance of suppository bases and their DSC thermograms. (a)

PEG 1500, (b) PEG 4000, (c) SNA 15. Thermograms mimicking the thermal changes during

the manufacturing (blue line) and in vivo administration of suppositories (red line).

Chapter 3

68

Upon thermal analysis, SNA 15 base was found to be melting at 36 °C in the first run with

multiple endothermic peaks corresponding to its composition of a mixture of C10-18 mono,

di and triglyceride esters of fatty acids. However, in the second heating run the endothermic

peak corresponding to the melting point of SNA 15 reduced to 34 °C, which was within the

reference range supplied by the manufacturer, 32 – 36 °C [19] and suitable for rapid in vivo

melting and drug release [12, 166]. However, the exothermic peak corresponding to the

solidification of the base at 15 °C suggested that formulations prepared using SNA 15 base

require protection from hot environments, preferably at 2-8 °C. On the contrary, the

endothermic peaks corresponding to the melting points of PEG 1500 and 4000 were found

at 48 °C and 61 °C, respectively. Following the second heating run, PEG 4000 showed

multiple endothermic peaks within the range of 57-61 °C. However, upon cooling PEG 4000

base rapidly solidified resulting in a sharp exothermic peak at 43 °C while PEG 1500 started

solidifying at 37 °C with a broad exothermic peak indicating slower solidification of PEG

1500 base compared with PEG 4000.

Upon DSC analysis of different ratios of PEG 1500 and 4000 (Figure 3.6), the two PEG

grades merged into a single broad endothermic peak after the first heating run, indicating

mixing at a molecular level. Not surprisingly, increasing the ratio of PEG 4000 increased

the melting temperature range and solidification temperature range, as well as the hardness

of the base mixture. A mixture of low and high molecular weight PEGs has been reported

to achieve both the desired melting and mechanical strength of the suppositories [13, 58,

99]. Additionally, high molecular weight PEGs generate harder suppositories, which could

could improve the mechanical strength of the suppositories.

Chapter 3

69

Figure 3.6 DSC analysis of different ratios of PEG 1500:4000 and its effect on the melting

and solidification behaviour of the suppository base mixture.

Therefore, PEG 1500:4000 mixture at a ratio of 70:30 w/w was selected for the development

of suppositories. Furthermore, the selected ratio of PEG base mix showed the melting point

above 40 °C as well as solidification at higher temperature compared with the other ratios,

which would be ideal to withstand storage in warm conditions.

Suppository mould capacity

From the average weight of suppositories prepared using the 1 g suppository mould, the MC

was found to be 1.3 ± <0.01 g and 1.0 ± <0.01 g for PEG 1500:4000 (70:30 w/w) base

mixture and SNA 15 base, respectively. These values were used for further calculations.

Chapter 3

70

Calculation of displacement value

The DV was calculated for AMT and AS based on the average weights of blank and

medicated suppositories of prepared from PEG base mix and SNA 15 base. The detailed

calculation is summarised in Table 3.5.

Table 3.5 Calculation of displacement value of AMT and AS equivalent to 100 mg

amoxicillin for each type of suppository base i.e. PEG base mix and SNA 15. Wt. - Weight.

Calculation parameter AMT AS

PEG SNA 15 PEG SNA 15

Average wt. of blank suppository (A) (g) 1.271 1.017 1.271 1.017

Average wt. of medicated suppository (B) (g) 1.299 1.053 1.293 1.045

Wt. of drug per medicated suppository (D) (g) 0.115 0.115 0.106 0.106

Wt. of base per medicated suppository (C = B-D) (g) 1.184 0.937 1.185 0.939

Wt. of base displaced by amoxicillin (E = A – C) (g) 0.087 0.080 0.086 0.078

Drug displacement value (F = D/E) 1.322 1.438 1.233 1.359

Calculations for preparation of a batch of suppositories

The quantities of drug and suppository required for preparation of a batch of 20 suppositories

containing AMT or AS equivalent to 100 mg amoxicillin were calculated using Eq 3.2 and

Eq 3.3. The calculations have been summarised in Table 3.6 for AMT and AS containing

suppositories, respectively.

Chapter 3

71

Table 3.6 Summary of calculated quantities of the drug and base for preparation of a

batch of 20 suppositories containing 100 mg amoxicillin. The quantities of AMT or AS were

calculated equivalent to 100 mg of amoxicillin per suppository.

Quantities (g) AMT suppositories AS suppositories

PEG SNA 15 PEG SNA 15

Drug per suppository 0.115 0.115 0.106 0.106

Total Drug for 20 suppositories 2.75 2.75 2.54 2.54

Base per suppository 1.213 0.920 1.214 0.922

Total quantity of base for 20 suppositories 29.12 22.09 29.13 22.13

3.4 Conclusions

The preformulation studies were carried out to evaluate physicochemical properties of drug

and suppository bases to be utilised in further formulation development. Understanding of

pH-dependent stability of amoxicillin ensured development of a rapid, sensitive and

stability-indicating analytical method for in vitro estimation of amoxicillin in aqueous

samples. The same chromatographic conditions will be further utilised for validation of a

method for extraction of amoxicillin from rabbit plasma (Chapter 4). Additionally,

preliminary screening of SNA 15 bases indicated their suitability for rapid drug release

in vivo due to immiscibility of amoxicillin in oils as mentioned in Chapter 1, Section 1.4.2.

However, the physical stability of the suppository base needs to be improved to ensure

stability in tropical climatic conditions. On the contrary, higher melting points of PEG bases

suggests their physical stability in tropical climatic conditions. However, the melting point

optimisation by mixing PEG 1500 and 4000 is necessary for the design of amoxicillin

suppositories suitable for LMICs which would improve physical as well as mechanical

strength of the suppositories.

72

Development and Validation of a Stability-Indicating Extraction Protocol for

Quantification of Amoxicillin from Plasma

This chapter is published in Journal of Chromatography A.

Purohit TJ, Wu Z, Hanning SM. Simple and reliable extraction

and a validated high performance liquid chromatographic assay

for quantification of amoxicillin from plasma. J Chromatogr A.

2020;1611:460611, DOI: https://doi.org/10.1016/j.chroma.201

9.460611.



Chapter 4

73

This Chapter discusses development of an efficient, sensitive and repeatable extraction

protocol for quantitation of amoxicillin from rabbit plasma by understanding the pH-

dependent solubility and stability of amoxicillin in the extraction solvents, which was

validated using previously developed stability-indicating HPLC method.

4.1 Introduction

Extraction and quantitative analysis of a drug from biological samples such as plasma plays

an important role in pharmaceutical research. Plasma contains a variety of endogenous

substances including a large number of proteins, which need to be removed before

chromatographic drug analysis [163, 191]. Incomplete removal of proteins from plasma

samples leads to compromised column performance and low sensitivity of an analytical

method [191, 192]. It is a complex process involving exposure of a drug to a rigorous

separation process and a variety of solvents such as acids and organic solvents [193].

Different purification methods such as extraction (liquid-liquid or solid-phase) and protein

precipitation (PP) have been developed for the analysis of biological samples [194] with PP

being the most common and simplest amongst all. PP can be carried out by addition of either

organic solvents, acids, salts or metal ions. It mainly removes the proteins by lowering the

solubility of proteins in the plasma due to change in dielectric constant or isoelectric pH of

the proteins. Organic solvent mediated protein precipitation technique has been reported to

be the most common and efficient amongst all different methods [193]. However,

development of a specific purification method with high extraction efficiency is challenging

especially for chemically unstable molecules with low solubility, such as amoxicillin.

Amoxicillin is a broad spectrum β-lactam antibiotic used in the treatment of a variety of

infections. It is an amphoteric molecule having a log P value of 0.87 and pKa values of 3.2

and 11.7 [42]. It has a “U-shaped” pH-dependent solubility profile in aqueous media with

Chapter 4

74

lowest solubility between pH 4-6 [162]. In contrast, a pH-degradation profile with optimal

stability between pH 4-6 has been reported [162, 163]. As it degrades at extreme pH, acids

cannot be used to extract amoxicillin from biological samples [163]. Moreover, amoxicillin

readily undergoes methanolysis in presence of alcohols such as methanol [195, 196] and is

practically insoluble in acetonitrile, two of the most commonly used and efficient organic

solvents for protein precipitation [48].

There are numerous high-performance liquid chromatography (HPLC) methods available

for direct and indirect quantification of amoxicillin in biological matrix [163, 171, 172, 197-

199]. Previously described methods have involved solid phase extraction (SPE) [200],

liquid-liquid extraction (LLE) [172, 201] and PP [186, 202-205]. SPE and LLE shows

minimum matrix effect but they are often complex and time consuming [206] and the

extraction efficiency depends upon various parameters such as type of extraction cartridges,

solubility and partitioning in different solvents [191, 206]. In contrast, PP mediated

extraction is simple and quick, which makes it the most common method of extraction.

Furthermore, PP with an organic solvent is considered the most effective extraction method

for quantitative analysis of a drug with a low log P value from plasma samples [207].

Previously, quantification of amoxicillin from human plasma by external standard method

has been reported using methanol as an extraction solvent [208]. However, our preliminary

trials for extraction of amoxicillin by standard addition resulted in varying and low

extraction recovery, possibly due to poor stability. This necessitated pH-control by addition

of buffer to maintain solubility and stability of amoxicillin during the extraction process.

This led to development of a PP mediated protocol for extraction of amoxicillin from plasma

samples by understanding its solubility and stability in the extraction solvent(s) prior to

development of an extraction protocol.

Chapter 4

75

In this paper, the solubility and stability of amoxicillin in commonly used solvent systems

during extraction was first studied using a stability-indicating HPLC method. An optimal

extraction protocol for amoxicillin from plasma was developed in the selected solvent

system by leveraging its stability and solubility. The HPLC method for quantification of

amoxicillin from plasma was validated and applied in the analysis of a preliminary

pharmacokinetic study following intravenous (IV) drug administration to rabbits.

4.2 Materials and methods

Materials

Amoxicillin trihydrate (AMT) – European Pharmacopoeia standard was purchased from

Sigma Aldrich, New Zealand. Formic acid, sodium acetate, sodium/potassium dihydrogen

phosphate, and disodium hydrogen phosphate were of reagent grade and purchased from

Merck and ThermoFisher Scientific, New Zealand. Acetonitrile and methanol used in the

extraction process and liquid chromatography were HPLC grade. The Milli-Q water for

HPLC was obtained from Millipak (Millipore, 0.22 µm). Plasma was obtained from healthy

rabbits (The University of Auckland Animal Ethics Committee reference number 001663)

and for the pharmacokinetic study, New Zealand White rabbits weighing > 3 kg and 3-4

months old were used (The University of Auckland Animal Ethics Committee reference

number 002012).

Chromatographic conditions

An Agilent 1260 HPLC machine (Agilent Corporation, Germany) was used for sample

analysis. The samples were analysed using a Luna omega PS C18 100 Å, (250 x 4.6 mm, 5

µm) column from Phenomenex (Auckland, New Zealand). The mobile phase comprised 0.5

mM phosphate buffer (pH 5.0), methanol and acetonitrile (93:5:2 v/v) with the flow rate set

Chapter 4

76

at 1 mL/min, while the column temperature was maintained at 30 °C. The injection volume

was 20 µL. Detection was carried out at 228 nm using a photo diode array (PDA) detector.

The stability-indicating nature of the assay was validated with PDA using purity index

>99.9% under various forced degradation conditions (acidic, alkaline, UV and oxidative

stress) with the above-mentioned chromatographic conditions. This chromatographic

method was used in solubility and stability studies as well as plasma sample analysis

following extraction.

Solubility and stability analysis of amoxicillin

The solubility of AMT was determined in commonly used extraction solvents, with water

as control. Acetonitrile was mixed with 0.1% formic acid (pH 2.5), acetate buffer (0.1 M,

pH 5) and phosphate buffer (0.1 M, pH 7.4) to understand the effect of pH on solubility and

stability of amoxicillin. Water, acetonitrile, and acetonitrile mixed with either water,

methanol, acetate buffer (0.1 M, pH 5.0), or phosphate buffer (0.1 M, pH 7.4) at a ratio of

95:5 v/v were investigated. Rabbit plasma was also added to the binary systems (90:5:5).

Excess amount of AMT (>12 mg/mL) was added to each of the above solvents and the

samples were maintained on a shaking water-bath at 25 °C until equilibrium was achieved

(24 h). The solubility in different solvents was determined from the supernatants using a

stability-indicating HPLC method described above.

Systems showing solubility of amoxicillin above 120 µg/mL were further selected for

stability analysis, where the supernatant was split and stored, protected from light, at either

4 °C or ambient temperature (22 °C). Stability samples (10 µL) were taken at pre-determined

time intervals for up to 21 days or until 80% degradation of the drug was observed,

whichever came first. The degradation kinetics were further studied by application of a first

order kinetic model. Degradation rate constant (observed) (kobs), half-life (t1/2) and the time

Chapter 4

77

at which the drug retained 90% of its original concentration (t90) were determined using the

following equations Eq 4.1 and Eq 4.2:

𝐴𝐴1/2 = 0.693𝑘𝑘𝑜𝑜𝑜𝑜𝑜𝑜

Eq 4.1

𝐴𝐴90 = 0.105𝑘𝑘𝑜𝑜𝑜𝑜𝑜𝑜

Eq 4.2

Extraction protocol

To develop an optimised extraction protocol, initially various extraction solvent systems

were screened for efficient and repeatable extraction of amoxicillin from plasma by PP.

Solvent systems containing 100, 80, 70, 60, 50% acetonitrile in methanol, acidified methanol

or acetonitrile with either 0.1% formic acid [186, 205] or 10-20% perchloric acid [209, 210]

were evaluated for extraction efficiency. Following the solubility and stability studies,

extraction protocols were developed using the acetate buffer (0.1 M, pH 5.0) - acetonitrile

binary system (1:18) for extraction of amoxicillin from plasma samples. The protocol was

evaluated by applying one-step and two-step extraction processes, where the residue was

extracted with another 900 µL acetonitrile containing acetate buffer using the same process.

The extraction recoveries were determined using equation 4.3 below:

Recovery (%) =Peak area of the analyte extracted in plasma

Peak area of analyte in solution× 100 Eq 4.3

The samples were prepared as per the following method. Briefly, 50 µL of plasma was mixed

with and 900 µL acetonitrile and 50 µL of 0.1 M acetate buffer (pH 5.0). The mixture was

vortex mixed for 5 minutes at 4 °C at 2000 RPM and centrifuged at 13,400 RPM at 4 °C.

The supernatant was collected in another Eppendorf tube and evaporated at 40 °C with a

Chapter 4

78

gentle nitrogen stream. The residue was then reconstituted using 50 µL of mobile phase and

20 µL was injected for HPLC analysis.

The extraction recoveries of spiked quality control (QC) samples prepared at three different

concentration levels (0.5 µg/mL, 4 µg/mL and 16 µg/mL) were evaluated by injecting

triplicate samples of each concentration. The extraction recoveries were calculated using

Eq 4.3 above.

Method validation

Following extraction the method for plasma sample analysis was validated according to the

guidelines for validation of bioanalytical method [211].

Preparation of calibration and quality control (QC) standards in plasma

Different spiking solutions ranging from 2-200 µg/mL were prepared from a 1 mg/mL stock

solution of AMT in water. Blank rabbit plasma was spiked with the appropriate volume of

respective spiking solution to prepare eight calibration standards (0.2, 0.3, 0.4, 0.8, 2, 6, 12

and 20 µg/mL) for determination of linearity. Three different QC standards: 0.5 µg/mL,

4 µg/mL and 16 µg/mL, were spiked similarly as standards to determine accuracy and

precision of the method.

Sensitivity

The sensitivity of the method was determined in nine samples by measuring the analyte

response from blank samples and the lowest non-zero concentration. To meet the criteria the

analyte response from the lowest non-zero concentration should be ≥ 5 the response from

blank calibrator.

Chapter 4

79

Linearity

Linearity shows that the sample solutions are in a concentration range where analyte

absorption is linearly proportional to concentration. This was performed by analysing

freshly prepared spiked plasma samples (n=3) ranging from 0.2 – 20 µg/mL for three

consecutive days. A linearity standard curve was obtained by plotting the peak area against

concentration.

Selectivity and specificity

Blank plasma samples (n=6) were prepared at different time points were compared and

evaluated for interference of an endogenous compound during analysis to establish the

selectivity of the method. The specificity of the method was evaluated based on the

selectivity of the method and ability to detect the analyte of interest (± 20% LLOQ) from

the sample matrix. The chromatograms of blank samples and spiked samples (0.2 µg/mL;

n=3) were analysed and compared for determination of specificity of the method.


The accuracy (%A) of the method is the degree of closeness of the measured concentration

of the analyte to the true concentration of the analyte in QC samples. The accuracy 𝐴𝐴(%)

was determined by analysing nine QC samples at three different concentration levels, 0.5, 4

and 16 µg/mL, using Eq 4.4 mentioned below:

𝐴𝐴(%) = [ 𝑀𝑀𝑏𝑏𝑏𝑏𝑏𝑏𝐴𝐴𝑑𝑑𝑏𝑏𝑑𝑑 𝑐𝑐𝐴𝐴𝐴𝐴𝑐𝑐𝑏𝑏𝐴𝐴𝐴𝐴𝑑𝑑𝑏𝑏𝐴𝐴𝑐𝑐𝐴𝐴𝐴𝐴 𝑁𝑁𝐴𝐴𝐴𝐴𝑐𝑐𝐴𝐴𝑏𝑏𝑁𝑁 𝑐𝑐𝐴𝐴𝐴𝐴𝑐𝑐𝑏𝑏𝐴𝐴𝐴𝐴𝑑𝑑𝑏𝑏𝐴𝐴𝑐𝑐𝐴𝐴𝐴𝐴⁄ ] × 100 Eq 4.4

Precision is the degree of closeness between the results. Intra-day and inter-day precision

was determined by applying multiple injections of QC samples in a single day and for three

consecutive days, respectively, and calculating the relative standard deviation (RSD). The

Chapter 4

80

accuracy and the precision of the developed method was determined provided the % RSD

values remain below 15.

Post-extraction and sample storage stability

The stability of amoxicillin kept in auto sampler (without temperature control) was

determined by reinjecting amoxicillin samples prepared at two concentration levels, 0.5 and

16 µg/mL, in triplicate over a period of 72 h. Freeze-and-thaw stability of spiked plasma

samples at two concentrations (0.5 and 16 µg/mL) were studied in triplicate for stability of

amoxicillin over three freeze-and-thaw cycles (-20 °C to ambient temperature). Separate sets

of spiked plasma samples with the same concentrations were prepared to determine long-

term freezing (- 20 °C) stability for up to two months. The stabilities were determined

provided the accuracy at each concentration level remained within ± 15%.

Pharmacokinetic study

The analytical study to determine blood concentration of amoxicillin in rabbit plasma was

approved by The University of Auckland Animal Ethics Committee (reference number

002012). The study was carried out on New Zealand white rabbits (n=3). All the rabbits

were acclimatised for five days prior to the start of the study and were allowed free access

to food and water. The rabbits received an IV bolus of amoxicillin sodium solution

(53 mg/mL) at a total dose equivalent to 100 mg amoxicillin. Blood samples were collected

at 0, 0.25, 0.5, 1, 2, 4, 6 and 8 hours then centrifuged at 7800 RPM for 10 min at 4 °C.

Plasma was separated and stored at -20 °C until analysis. The samples were extracted and

analysed as per the method described under Section 4.2.4.

Chapter 4

81

Data analysis

Pharmacokinetic parameters were calculated using PKsolver 2.0, an add-in program for

pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel [212]. The

pharmacokinetic parameters: half-life (t1/2), area under the drug-concentration – time curve

(AUC), area under the moment curve (AUMC), volume of distribution (Vd) and clearance

(Cl) were determined using a non-compartmental pharmacokinetic model and all the results

were reported as mean ± standard deviation (SD). Statistical evaluation was carried out using

IBM SPSS statistics software (Version 25, IBM Corporation). An independent t-test was

used to evaluate the recoveries of one-step and two-step extraction protocol. Differences

were considered significant if p < 0.05.


Challenges in the extraction of amoxicillin during preliminary studies

Extraction and PP with the most commonly used organic solvents, acetonitrile and methanol,

either alone or in combination did not give acceptable extraction recovery of amoxicillin.

Extraction with methanol resulted in extremely high recoveries ranging from 140-180% and

further resulted in compromised column performance. The cause for this high extraction

recovery may be related to evaporation of methanol due to low boiling point or methanolysis

of amoxicillin, although this is yet to be fully explored. It was observed that acetonitrile was

a better precipitating agent as compared to methanol, which is similar to the findings by

Polson et al [193]. Therefore, an acetonitrile mediated PP approach was employed for

extraction trials.

One and two-step extraction with acetonitrile resulted in low and varying extraction

recoveries ranging from 10-40%. This could be due to limited solubility of amoxicillin in

acetonitrile, which is in agreement with the findings by Bhattacharya [48] that amoxicillin

Chapter 4

82

is practically insoluble in acetonitrile. Lindegardh et al. reported an improvement in

extraction recovery using a plasma to acetonitrile ratio greater than 1:12 [200], however, our

trials with plasma to acetonitrile ratios 1:12 and 1:15 contradicted this. We found that

increasing the quantity of acetonitrile did not improve the extraction recoveries, due to the

limited solubility of amoxicillin in acetonitrile. This was later confirmed by the solubility

and stability analysis (Section 4.3.2).

pH change mediated PP was also carried out by adding smaller proportion of either 0.1%

formic acid or 10% perchloric acid. It was observed that PP was better with formic acid than

with perchloric acid. Upon drying, the supernatant collected from the perchloric acid treated

samples resulted in dense, cloudy precipitates, indicating a higher amount of dissolved

proteins. This rendered the samples inappropriate for analysis. On the other hand, formic

acid samples resulted in low recoveries, possibly due to pH-dependent stability of

amoxicillin. Thus, variable extraction recoveries and non-repeatability in the results with

different sample purification approaches provided the basis to carry out preliminary analysis

of solubility and stability of amoxicillin in commonly used solvent systems in order to

develop a repeatable and reliable extraction protocol.

Solubility and stability analysis

Solubility of amoxicillin

The solubility equilibrium was established within 24 hours in all solvent systems

(Figure 4.1). There was no further increase in drug concentration in the supernatants found

at 48 h. The solubility of amoxicillin in water was found to be 2.7 mg/mL at ambient

temperature which was close to the reported values [42]. It has been reported that amoxicillin

is practically insoluble in acetonitrile [48]. This was supported by the low solubility of

amoxicillin (below 70 µg/mL) in acetonitrile alone or in binary systems along with either

Chapter 4

83

methanol, water or formic acid. Solubility in phosphate buffer (0.1 M, pH 7.4) was found

slightly above 100 µg/mL. Addition of acetate buffer (0.1 M, pH 5.0) resulted in a 16- and

5-fold increase in solubility of amoxicillin in the acetonitrile alone and along with plasma,

respectively.

Figure 4.1 Solubility of amoxicillin in different solvent systems (n=3) at 25 °C : Water

(●), Acetonitrile (ACN) (■), ACN+ Methanol+ Water (▲), ACN+ Water (▼), ACN+

Acetate buffer, 0.1 M, pH 5.0 (), ACN+ Phosphate buffer, 0.1 M, pH 7.4 (), ACN+

Formic acid, pH 2.5 ( ), ACN+ Plasma (), ACN+ Acetate buffer, 0.1 M, pH 5.0+ Plasma

().

Stability of amoxicillin

Solvent systems containing water, acetonitrile with acetate buffer (0.1 M, pH 5.0) with or

without plasma and acetonitrile with plasma qualified for stability analysis. Amoxicillin was

found more stable at 4 °C than 22 °C in all solvent systems. Following storage at 4 °C for

24 hours, 87% and 78% amoxicillin was found as such in the solvent systems containing

acetonitrile and plasma with or without acetate buffer, respectively. A near two-fold increase

in stability was observed due to addition of acetate buffer to the binary system. Figure 4.2

Chapter 4

84

shows the degradation of amoxicillin in solvent systems containing acetate buffer with or

without plasma.

Figure 4.2 Degradation graphs for amoxicillin in two different solvent systems (n=3),

acetonitrile and plasma (95:5 v/v) and acetonitrile, plasma and acetate buffer (90:5:5 v/v)

: at 4 °C (a), 22 °C (b) and first-order degradation graphs at 4 °C (c) and 22 °C (d).

The results of linear regression analysis for first-order degradation kinetic model for both

the systems at 4 °C (Table 4.1) shows the effect of acetate buffer (pH 5.0) on drug stability.

Amoxicillin was found stable for about 2 days in the ternary solvent system containing

acetate buffer. This also justified the sample stability for the maximum analysis time, 12

hours a day.

Chapter 4

85

Table 4.1 Degradation kinetics of the solvent systems with or without acetate buffer (0.1

M, pH 5.0). All the samples were kept at 4 °C.

Solvent system R2 kobs (10-2 d-1) t1/2 (day) t90 (day)

Acetonitrile: plasma (without buffer) (95:5 v/v) 0.9785 8.412 8.2 1.2

Acetonitrile: plasma: acetate buffer (90:5:5 v/v) 0.9864 4.925 14.1 2.1

Thus, it was confirmed that, addition of acetate buffer improved both solubility and stability

of amoxicillin. Therefore, the protocol for extraction of amoxicillin from plasma was

developed using acetate buffer (0.1 M, pH 5.0) with acetonitrile.

Development and selection of extraction protocol

A plasma to acetonitrile ratio under 1:2 has been reported to be sufficient for protein removal

from plasma [193]. Addition of buffer may dissolve some of the plasma proteins; therefore,

a ratio of organic to inorganic solvents of 1:9 was selected to avoid accumulation of any

endogenous proteins and to improve the column life and performance. The extraction

protocol was developed with the solvent system containing acetonitrile, acetate buffer

(0.1 M, pH 5.0) and plasma in a ratio of 90:5:5, respectively.

Both the one-step and two-step extraction protocols were found repeatable, with extraction

recoveries of amoxicillin from plasma samples ranging between 80 and 88%. At the

concentration range of 1 µg/mL to 20 µg/mL, one-step and two-step extraction protocols

resulted in extraction recoveries of 81-83.6% and 82.9-87% respectively. When comparing

extraction recoveries, no significant improvement in extraction recovery was achieved using

the two-step protocol compared with the one-step extraction protocol (p = 0.834).

Furthermore, the two-step process was more time consuming and complex compared with

Chapter 4

86

the one-step extraction protocol. Therefore, a one-step extraction was selected for sample

preparation and method validation.

Extraction recovery

The extraction recovery was found between 82% and 89% for all samples. The extraction

recovery of QC samples at 0.5 µg/mL, 4 µg/mL and 16 µg/mL were found to be 84.6%,

83.5% and 82.6%, respectively. The results of extraction recovery have been summarised in

Table 4.2.

Method validation

The developed method was found to be simple, rapid, sensitive and reproducible with

amoxicillin eluting at 6.2 ± 0.2 min. The results of various validation parameters are

discussed below.

Sensitivity

The sensitivity of the method was determined with a concentration as low as 0.2 µg/mL

giving a response that was five times higher than the blank sample. Therefore, this was

considered the LLOQ for this method.

Linearity

The linear regression equation (n=3) for amoxicillin was linear in the concentration range

of 0.2 to 20 µg/mL in rabbit plasma. The R2 value was 0.9999 ± 0.0002 for all linearity

curves.

Selectivity and specificity

The developed method was found selective for amoxicillin in the presence of other

endogenous compounds. Analysis of multiple blank samples showed that there was no

Chapter 4

87

interference from the endogenous compounds in the sample matrix. Figure 4.3 shows

comparison of the chromatograms of blank plasma and lowest standard concentration,

indicating the specificity and sensitivity to amoxicillin of the developed method.

Figure 4.3 Overlaid chromatograms of blank sample and a spiked sample at LLOQ

concentration (0.2 µg/mL). Inset highlights the difference between blank and spiked sample

at LLOQ concentration (0.2 µg/mL).


The method was found accurate and precise at three different concentration levels. The

accuracy was found between the ranges of 97-115%. The percentage bias was found to be

below 6, which is below 15% for both inter-day and intra-day samples. This indicates that

the method is repeatable and reproducible. The results of accuracy and precision have been

summarised in Table 4.2.

Chapter 4

88

Post-extraction and sample storage stability

Stability of amoxicillin was determined on two aspects, post-extraction and freeze-and-thaw

to mimic the conditions during sample analysis, handling and storage. Results of the stability

study have been reported in Table 4.3. Amoxicillin was found stable for up to three days

post-extraction when kept in the auto sampler. Amoxicillin was found stable at both the

concentrations, 0.5 and 16 µg/mL, for up to 72 h. Only 2-3% of amoxicillin was degraded

from the initial concentration at both concentration levels. This indicated post-extraction

stability of amoxicillin in all the samples until analysis of the final sample in the sequence,

despite the temperature not being controlled in the auto sampler.

Amoxicillin was found stable for three freeze-and-thaw cycles (-20 °C to ambient

temperature). At the end of three freeze-and-thaw cycles only 2 - 5% amoxicillin was

degraded from the samples containing 0.5 and 16 µg/mL amoxicillin. In contrast,

amoxicillin was found unstable upon long-term freezing at -20 °C. At the end of two months,

the plasma samples spiked with amoxicillin showed degradation of amoxicillin, with 58.3%

and 48.9% remaining in the samples spiked with 0.5 and 16 µg/mL amoxicillin, respectively.

Amoxicillin has been reported stable for one month when kept at -80 °C [170]. However, it

is advisable to analyse clinical study samples within short period of time from collection, to

quantify amoxicillin as accurately as possible.

Chapter 4

89

Table 4.2 Accuracy, precision and extraction recovery data for amoxicillin at concentrations of 0.5, 4 and 16 µg/mL. All the values are

expressed as a mean percent (%). RSD – Relative standard deviation.

Concentration

(µg/mL)

Intra-day (n=3) Inter-day (n=9) Extraction recovery (n=3)

Accuracy

(% Nominal)

Precision

(% RSD)

Accuracy

(% Nominal)

Precision

(% RSD) Mean (%) RSD (%)

0.5 113.6 1.4 108.3 5.3 84.6 2.3

4 99.9 2.8 97.9 2.6 83.5 3.2

16 99.7 1.5 98.7 2.1 82.6 2.2

Table 4.3 Auto sampler, freeze-and-thaw and long-term storage (- 20 °C) stability of amoxicillin. *RSD (relative standard deviation).

Concentration

(µg/mL)

Auto sampler, 72 h (n=3) Three cycles, freeze-and-thaw (n=3) 2 months, storage at -20 °C (n=3)

Drug remaining (%) RSD (%) Drug remaining (%) RSD (%) Drug remaining (%) RSD (%)

0.5 99.5 6.0 98.4 6.2 58.3 6.3

16 98.3 8.0 94.9 3.7 48.9 2.1

Chapter 4

90

Pharmacokinetic study

The method was successfully applied in the determination of concentration of amoxicillin

in rabbit plasma following IV amoxicillin administration. Figure 4.4 shows chromatograms

of the samples prepared from spiked blank plasma and pharmacokinetic study sample.

Figure 4.4 Chromatograms showing amoxicillin peak eluting at 6.2 ± 0.2 min; spiked

sample, 12 µg/mL (a) and a pharmacokinetic study sample (b).

Figure 4.5 Drug release profiles in rabbits (n=3) following IV amoxicillin administration

at a dose of 53 mg/mL amoxicillin sodium solution equivalent to 100 mg amoxicillin.

Chapter 4

91

The drug release study following IV administration of amoxicillin was carried out and the

release profile (n =3, each time point) is shown in Figure 4.5 and the pharmacokinetic

parameters are summarised in Table 4.4.

Table 4.4 Pharmacokinetic parameters following IV administration of amoxicillin at a

dose of 53 mg/mL amoxicillin sodium solution equivalent to 100 mg amoxicillin. Data are

expressed as mean ± SD for n=3 rabbits.

Dosing t1/2 (h) AUC 0-∞ (μg/

mL*h)

AUMC

(μg/mL*h2)

Vd (L) Cl (L/h)

IV amoxicillin 0.62 ± 0.04 20.16 ± 4.69 12.94 ± 5.13 4.56 ± 0.79 5.14 ± 1.15

Table 4.4 shows that, following IV administration, amoxicillin was cleared rapidly with a

t1/2 of 0.6 h (35 – 40 minutes). This is close to the reported t1/2 values of 1 h in humans [213].

However, the values of Vd and Cl were found noticeably lower than those in human treated

with 500 mg IV amoxicillin, 20.2 L and 13.3 L/h, respectively [213].

4.4 Conclusions

Quantitative analysis of a drug from plasma samples usually involves protein removal and

maximum extraction of the drug using a suitable extraction protocol with the consideration

of drug stability. In this research, the solubility and stability analysis of amoxicillin was

carried out in commonly used solvents by a stability-indicating HPLC assay. Addition of a

small amount of acetate buffer (0.1 M, pH 5.0) improved the solubility as well as stability

of amoxicillin. The synergy of organic solvent and pH-control was further utilised in the

development of a suitable extraction protocol for extraction of amoxicillin from plasma. A

simple, rapid, one-step and easy to follow sample purification procedure by PP was

developed for extraction of amoxicillin from plasma before subjecting the samples to HPLC

Chapter 4

92

for quantitative analysis. The HPLC method for quantification of amoxicillin was found to

be simple and repeatable with the stability-indicating nature. Amoxicillin was found stable

for up to 3 days post-extraction in the auto sampler without temperature control. However,

the stability was compromised when the samples were kept at -20 °C for two months with

nearly 50% of amoxicillin was found degraded. The method was successfully applied for

the quantitative analysis of amoxicillin from a pharmacokinetic study in rabbits. This

research highlighted the importance of understanding the physicochemical properties,

particularly pH-dependent solubility and stability, which is frequently overlooked for many

drugs.

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93

Development and Evaluation of Conventional Amoxicillin Suppositories

This chapter is published in Journal of Controlled Release.

Purohit TJ, Hanning SM, Amirapu S, Wu Z. Rectal bioavailability

of amoxicillin sodium in rabbits: Effects of suppository base and

drug dose. Journal of Controlled Release. 2021;338:858-869, DOI:



Chapter 5

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This Chapter describes formulation development and in vitro-in vivo evaluation of

conventional amoxicillin sodium suppositories to determine the extent and mechanism of

absorption of amoxicillin from the rectum. Along with that, preliminary stability and rectal

tissue irritancy evaluation have also been discussed in detail.

5.1 Introduction

Amoxicillin is a broad-spectrum β-lactam antibiotic commonly used to treat bacterial

infections. It is available in various dosage forms such as tablets, capsules, suspensions and

injections [29, 214]. Oral dosage forms are the most popular and convenient formulations,

but they are not suitable when the patient is vomiting or having swallowing problems [67,

215]. In such conditions, rectal dosage forms, particularly suppositories, may be a suitable

alternative to oral drug administration [1, 61].

Drug delivery through the rectal route has specific advantages, such as enabling systemic

drug delivery via portal drainage from the middle and inferior rectal vein and minimizing

first-pass metabolism, depending on how it is inserted [61]. There are also limitations of this

route, such as patient barriers related to negative perceptions of this route of administration.

Further, high aqueous solubility and small molecular size of the drug are critical for

absorption due to low fluid volume (1-3 mL) in the rectum [1, 6, 7, 216]. While novel rectal

dosage forms have been developed, such as hollow-type suppositories and rectal in-situ gels,

traditional suppositories remain the most common rectal drug delivery system [61, 62, 72,

217].

Rectal suppositories have been investigated for different categories of drugs, including those

for management of pain and fever [218-220], diabetes [70, 71, 221] and inflammatory bowel

disease [120, 125]. Various classes of antibiotics have also been studied for delivery via the

rectal route [12-14, 58, 59, 190, 222, 223] and have shown potential to be delivered with

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suppositories. For example, β-lactam suppositories have been reported to produce

satisfactory pharmacokinetic profiles in healthy volunteers [59, 223]. Therefore,

development of rectal delivery systems of amoxicillin could be beneficial for patients having

swallowability concerns, for example, paediatric or elderly patients. To date, few studies

have attempted to develop amoxicillin suppositories. Webster et al. compared pre and post

storage release properties of amoxicillin trihydrate from different suppository bases [190].

A recent study reported preparation and in vitro Characterisation of amoxicillin trihydrate

suppositories using hydrophilic polyethylene glycol (PEG) base and lipophilic Suppocire

NA 15 (SNA 15) base, respectively, and compared their antimicrobial efficacy and irritation

potential on a slug mucosal irritation assay model [12]. To the best of our knowledge, there

have been no reports of in vivo assessment of the rectal bioavailability of amoxicillin. Oral

amoxicillin (as trihydrate) formulations at a dose below 875 mg show almost complete

absorption and can be classified according to the Biopharmaceutics Classification System

(BCS) as class I (high solubility and high permeability). However, at increased dose levels

above 1000 mg, amoxicillin trihydrate could be BCS class II (solubility limited) or even

BCS class IV (both solubility and permeability limited) [224]. Indeed, studies have

suggested a non-linear relationship between bioavailability and the oral dose, indicating

zero-order absorption of amoxicillin at dose levels above 875 mg [225, 226]. This means

that in vivo absorption of amoxicillin can be improved by increasing the solubility of

amoxicillin.

An earlier study on human volunteers suggested a site-specific or carrier-mediated

absorption of amoxicillin, where a higher fraction of amoxicillin was found to be absorbed

from the duodenum and jejunum compared with minimal absorption from the colon [227].

In contrast, another research group found no evidence of carrier mediated transport in the

rectum and only paracellular and transcellular diffusion have been reported as the transport

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mechanism [6]. Therefore, more evidence is needed to shed light on the mechanism of

amoxicillin absorption from the rectal mucosa.

This research aimed to develop and compare amoxicillin suppositories with a hydrophilic

base or a lipophilic base, focusing on the mechanisms involved in the in vitro release and in

vivo rectal absorption of amoxicillin. Hydrophilic bases may dissolve in body fluid, whereas

lipophilic bases with a melting point slightly lower than the body temperature can melt in

the rectum, so the properties of suppositories prepared using the two bases are expected to

be different. Amoxicillin sodium (AS) was used to overcome the solubility limitation of

amoxicillin trihydrate (150 mg/mL [44] vs 2.7 mg/mL [45] in water at 25 °C). The

suppositories were evaluated using standard pharmacopoeial tests. Drug-base interaction,

drug distribution in suppository bases, stability and mechanism of drug release and kinetics

were characterised. Pharmacokinetics (PK) of both types of suppositories formulated at two

dose levels (100 mg and 200 mg) were assessed in rabbits to understand the mechanism of

rectal absorption of amoxicillin. The absolute bioavailabilities (F) were also estimated by

comparing the PK profiles with intravenous (IV) administration of drug solution. In

addition, the rectal irritation potential of the suppositories was investigated by histological

analysis at the end of the PK study.


Materials

Antibiotic AS was purchased from Alfa Aesar by ThermoFisher Scientific (Massachusetts,

USA). PEG 1500, PEG 4000 and dialysis bags with molecular weight cut-off (MWCO)

14,000 Da were purchased from Sigma Aldrich (St. Louis, Missouri, USA). SNA 15 pellets,

a mixture of mono-, bi- and tri-glyceride esters of fatty acids (C10-C18), were kindly

donated by Gattefossé SAS, France. Acetonitrile and methanol used in the extraction process

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and chromatography were high-performance liquid chromatography (HPLC) grade. The

Milli-Q water for HPLC was obtained from Millipak (Millipore, 0.22 µm). Acetic acid,

sodium acetate, sodium/potassium dihydrogen phosphate, and disodium hydrogen

phosphate were of reagent grade and purchased from either Merck or Thermo Fisher

Scientific, New Zealand. To prepare suppositories, a Stainless-steel 30 well, 1 g suppository

mould was purchased from Heros® Ltd (Olomouc, Czech Republic).

Preparation of suppositories

Two types of suppository bases, hydrophilic (a mixture of PEG 1500:4000, 70:30 w/w) and

lipophilic (SNA 15), were selected to prepare AS suppositories. The displacement values

(DV) of amoxicillin were found to be 1.23 and 1.36 for PEG and SNA 15 bases, respectively.

Both types of suppositories were prepared by the fusion moulding technique. Briefly, PEG

base mixture or SNA 15 base were melted at 80 °C and 50 °C, respectively, on a heating

plate. AS 106 mg or 212 mg (equivalent to 100 and 200 mg amoxicillin, respectively) was

added to each base and mixed thoroughly to ensure uniform dispersion of AS. The mixture

was then poured into a 1 g suppository mould. After 1 h standing at ambient temperature,

the suppositories were trimmed, de-moulded and stored in an air-tight container at ambient

temperature, protected from light before further use.

Characterisation of suppositories

All suppositories were visually observed for integrity. Following that, uniformity of weight,

uniformity of content, hardness and disintegration time were determined, as described in the

British Pharmacopoeia (BP) [228].

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Uniformity tests

To evaluate the uniformity of weight, ten suppositories from each type of formulation were

randomly selected and weighed. Uniformity of weight was determined based on the

acceptance criteria described in the BP, where none of the suppositories should be weighing

± 5% of the average weight of the suppositories [228].

To determine content uniformity, three suppositories from each batch were randomly

selected. Each 100 mg or 200 mg suppository was added to a separate beaker containing

100 mL or 200 mL of distilled water at 37 °C. The contents of the beaker were stirred at 300

RPM with a magnetic stirrer for 30 min. From each beaker, 1 mL sample was withdrawn

and diluted using the mobile phase and analysed using a previously validated stability-

indicating HPLC method [45].

Hardness and disintegration time

For selected suppository formulations, three samples from each type were randomly selected

and analysed for hardness using a Monsanto hardness tester. Each suppository was kept in

an upright position between two plungers of the hardness tester and pressure was applied

until the suppository deformed or snapped.

Both hydrophilic and lipophilic suppositories (n = 3) were subjected to the disintegration

test described in the BP for oral tablets and capsules [228]. The disintegration test was

carried out on a basket-rack disintegration test assembly Erweka ZT 320 (Erweka GmbH,

Germany). Milli-Q water (37 ± 2 °C) was used as an immersion fluid and the basket was

immersed in the fluid at a constant frequency rate between 29 and 32 cycles per min. At the

end of 30 min for SNA 15 and 60 min for PEG suppositories, the basket was lifted from the

apparatus to observe the remaining samples.

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Solid-state Characterisation of the suppositories

Drug-base interactions by differential scanning calorimetry (DSC)

To determine any drug-base interactions as well as the physical state of the drug in

suppositories, AS, PEG and SNA 15 bases and the medicated suppositories were analysed

using a differential scanning calorimeter (TA Q 2000, TA instruments, New Castle, USA)

equipped with TA-Q Universal Analysis software. The samples were evaluated for any

noticeable change in melting behaviour and drug-excipient interactions. Approximately

5 - 7 mg of precisely weighed solid samples were placed in an aluminium pan and

hermetically sealed. The samples were heated at the rate of 5 °C/min from 0 to 250 °C with

the nitrogen gas flowing at 20 mL/min.

Powder x-ray diffraction

To identify crystalline or amorphous nature of AS in different suppositories, powdered drug

samples as supplied, SNA 15 base, physical mixtures of AS and base (1:4 w/w) and crushed

samples of 100 mg and 200 mg suppositories were analysed using an Ultima IV X-ray

diffractometer (Rigaku Corporation, Tokyo, Japan). The diffractometer was operated in

continuous rotation scan mode at a generator tension of 40 kV and 30 mA using copper Kα

radiation (1.54056 Å) within the range of 5° < 2θ < 45° and the with a fixed step size of

0.02°, 2θ every 20s at 25 °C.

SEM/EDX microanalysis

The drug distribution in the microstructures of each type of suppositories at two dose levels

was further evaluated by elemental analysis using a scanning electron microscope equipped

with energy dispersive X-ray spectrometer (SEM/EDX) (Hitachi TM 3030 plus, Hitachi

Ltd., Tokyo, Japan). A silicon drift detector (Quantax 70, Bruker, Billerica, MA, USA) was

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used to determine the surface elements of materials by detecting electron beam mediated

characteristic atomic X-rays. The suppositories were crushed and samples were spread on

the two-way tape attached to a Stainless-steel stud. The analysis was carried out at 500 X

and 1800 X magnification, 15 KV current with a working distance of 8.3 ± 0.3 mm and spot

diameter of 174 µm.

Intermolecular interactions by FTIR analysis

Intermolecular interactions between drug and suppository base were further evaluated using

Fourier transfer infrared (FTIR) spectroscopy. Samples from each suppository type at both

100 mg and 200 mg dose levels were analysed with a Bruker Tensor 37 Attenuated total

reflection FTIR spectrometer (Billerica, MA, USA) using a diamond crystal. The FTIR

spectra were generated from an average of 32 sample scans following an automatic

background subtraction within the range of 400 - 4000 cm-1 with a resolution of 2 cm-1.

In vitro release and kinetics

In vitro drug release was carried out using a United States Pharmacopoeia (USP) type II

dissolution apparatus with 250 mL phosphate buffer saline (PBS, pH 7.4 – reflective of

rectal pH) maintained at 37 °C as the dissolution medium. Given the high aqueous solubility

of AS, the release study was operated under sink conditions at both dose levels. The stirring

speed was set at 50 RPM. Freshly prepared suppositories were randomly selected (n = 3

from each type). Each suppository was kept in a dialysis bag (MWCO 14000 Da) and

immersed in the dissolution medium. Dialysis bags (n = 3) containing 5 mL of free drug

solution (20 mg/mL) were set up under the same conditions and used as reference. At each

time interval, 1 mL sample was withdrawn and filtered using a 0.22 µm nylon filter and

replaced with the same volume of fresh PBS. Samples were diluted using the mobile phase

and analysed using a stability-indicating HPLC method [45]. The mean dissolution time

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(MDT), defined as time to reach 50% of drug release, and the similarity factor (f2) of

different release profiles were calculated as per Eqs 5.1 and 5.2, respectively, using

Microsoft Excel add-in DDSolver [229].

𝑀𝑀𝐷𝐷𝑀𝑀 = � 𝐴𝐴�̅�𝑖. ∆𝑀𝑀𝑖𝑖

𝑛𝑛

𝑖𝑖=1� ∆𝑀𝑀𝑖𝑖

𝑛𝑛

𝑖𝑖=1� Eq 5.1

Where n indicates the number of sampling time points; ti is ith time point; 𝐴𝐴�̅�𝑖is time at the

middle point between i and i–1; ∆Mi is the additional amount of drug dissolved between i

and i–1 [229].

f2 = 50 log{[ 1 + (1/m) � |Rj − Tj|2]−0.5

m

j=1

× 100 Eq 5.2

Where m represents total time points and (Tj and Rj) is the difference in % release between

the two types of suppositories for comparison.

The effects of each base as well as drug dose on MDT and overall drug release profiles were

evaluated. According to the U.S. Food and Drug Administration guideline, an f2 value in the

range 50 - 100 indicates similar drug release profiles, whereas values below 50 indicate

dissimilarity in drug release [230].

In addition, to understand the mechanism of drug release, the release profiles were fit into

different kinetic models, including zero order, first order, Higuchi, Hixon Crowell and

Korsmeyer Peppas using GraphPad Prism 8.2.1 software (GraphPad Inc, San Diego, USA).

The correlation coefficient (R2) was used as a measure of goodness of fit to determine the

best model to describe the release kinetics.

Stability evaluation of the suppositories

The physical and chemical stability of the suppositories were monitored over a period of

three months. Each suppository was wrapped individually in aluminium foil and stored

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102

protected from light in an air-tight container kept at 20 ± 0.2 °C. At the end of three months,

changes in the physical appearance of the suppositories were evaluated visually and the

content of amoxicillin remaining analysed by HPLC.

Absolute bioavailability study

New Zealand White - Californian crossbreed rabbits of either gender weighing 3.23 ± 0.19

kg were used following at least 5 days of acclimation. Ethical approval was obtained from

The University of Auckland Animal Ethics Committee (reference number 002012) to

conduct the in vivo experiments. Animals were handled and monitored according to the

standards for care and management of experimental animals of New Zealand.

Drug administration

The rabbits were randomly divided into three groups (n = 6). One group received an IV bolus

of 2 mL AS solution (53 mg/mL) equivalent to 100 mg amoxicillin and the other two groups

received either hydrophilic or lipophilic suppositories. A crossover design with two

treatment levels and a 14-day washout period between each treatment was adopted for the

suppository groups. The oral amoxicillin dose for children ranges from 15 to 40 mg/kg/dose

given two or three times a day with a maximum daily dose of 100 mg/kg or 4 g [33, 231-

233]. This is equivalent to a dose of 27.4 - 73.2 mg/kg in rabbits [234, 235]. There is a lack

of data about the rectal absorption of amoxicillin in rabbits. However, given the penicillin

class has a wide therapeutic window with a known short half-life [224], amoxicillin doses

100 mg and 200 mg were selected for this study. Each rabbit received a low dose (100 mg)

suppository in the first period and a higher dose (200 mg) suppository of the same base in

the second period. Following administration, blood samples (1 mL) were collected at time

0 (before administration), 0.25, 0.5, 1, 2, 4, 6 and 8 h and centrifuged at 7142 g for 10 min

at 4 °C to obtain plasma samples.

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Plasma sample analysis

Amoxicillin was extracted from plasma samples as per a previously developed protein-

precipitation method [45]. Briefly, 50 µL of the plasma sample was mixed with 900 µL

acetonitrile and 50 µL of 0.1 M acetate buffer (pH 5.0). The mixture was vortexed and

centrifuged at 4 °C. The supernatant was evaporated to dryness at 40 °C with a gentle

nitrogen stream. The residue was then reconstituted using 50 µL of mobile phase and

analysed using HPLC. The method leveraged the solubility and stability of amoxicillin, both

were pH-depended. The extraction recovery was 82 - 89%, and the assay was linear from

0.2 - 20 µg/mL with a limit of quantification (LoQ) of 0.2 µg/mL in plasma.

Pharmacokinetic data analysis

The PK parameters, including the area under the curve (AUC), elimination half-life (t1/2),

mean residence time (MRT), volume of distribution (Vd) and clearance (Cl) were determined

by formulating the non-compartmental PK model using Microsoft-Excel [236]. The extent

of absorption was analysed by comparing the AUC values of the suppositories at each dose

level. The absolute bioavailability (F) was calculated using Eq 5.3.

F (%)=� AUC0-∞(rectal) ×DoseIV AUC0-∞ (IV) × Doserectal⁄ �×100 Eq 5.3

In addition, the mean absorption time (MAT) from each suppository was calculated as the

difference in MRT values of amoxicillin from each type of suppository and IV treatment.

Histological analysis

At the end of PK study, rabbits were euthanised and rectal tissue was harvested and fixed in

10% v/v neutral buffered formalin for 24 hours before being transferred to 70% v/v ethanol

and stored at 4 °C. The fixed tissues were paraffin embedded and 5 µm thick sections were

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cut. The sections were subsequently stained with haematoxylin and eosin and observed

under a light microscope for imaging and analysis.

In vitro - in vivo correlation (IVIVC)

To understand whether drug release from suppositories was the predominant factor to

determine rectal bioavailability, a Level C IVIVC was attempted based on the in vitro release

parameter, MDT and PK parameters (F and MRT) [237]. Regression analysis was performed

using GraphPad Prism 8.2.1 software (GraphPad Inc, San Diego, USA).

Statistical analysis

All the results were reported as mean ± standard deviation (SD). An unpaired t-test was

employed to analyse the parameters arising from the double peak phenomenon in the

absorption of amoxicillin from two suppository bases. A two-way analysis of variance

(ANOVA) was conducted, followed by Tukey’s multiple comparison tests, to determine the

effect of suppository base on absorption of amoxicillin using GraphPad Prism 8.2.1 software

(GraphPad Inc, San Diego, USA). Differences were considered significant if p < 0.05.


Characterisation of suppositories

All suppositories were white to slightly off-white in colour and had a smooth and even

surface without any visible cracks (Figure 5.1).

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Figure 5.1 Images of the freshly prepared amoxicillin suppositories: (a) PEG base and

(b) SNA 15 base. Dimensions: Diameter 0.8 cm and length 2.5 cm.

Uniformity tests

Both types of suppositories (n = 10) were found uniform in weight with an average weight

of 1.28 ± <0.01 g for PEG base and 1.05 ± <0.01 g for SNA 15 suppositories, respectively,

for those containing 100 mg amoxicillin. The 200 mg amoxicillin suppositories weighed

1.31 ± 0.01 g and 1.09 ± <0.01 g for PEG and SNA 15 suppositories, respectively. None of

the suppositories deviated by more than 5% of the average weight as per the BP standard

[228].

The content of amoxicillin was found to be 92.8 ± 0.8% and 106.3 ± 15.2% in PEG and

SNA 15 suppositories containing 100 mg amoxicillin, respectively. Increasing the dose did

not change uniformity as the corresponding values were 95.6 ± 1.2% and 104.0 ± 4.2% for

the 200 mg suppositories. As per BP 2020 [228], drug content in every individual

suppository should be within the range of 75 – 125% of the specified dose. This indicated

that the fusion moulding method developed in this study was appropriate in obtaining

uniform distribution of amoxicillin in both suppository bases.

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Hardness and disintegration time evaluation

Both types of suppositories at both dose levels were found physically robust and the break

point of each type of suppository was found to be above 2 kg. This indicated the feasibility

of suppositories to withstand the forces that may be applied during transport or storage.

In water, the PEG suppositories disintegrated and dissolved within 25 - 30 min, while the

lipophilic suppositories melted within 10 - 15 min. These timeframes were within those

specified in the BP for the disintegration time of hydrophilic and lipophilic suppositories

[228].

Solid-state Characterisation of the suppositories

Drug-base interactions by DSC

As shown in the DSC thermograms (Figure 5.2), an endothermic peak with a small enthalpy

change of 1.9 w/g appeared at 132.6 °C in the AS samples, followed by an exothermic peak

at 223.7 °C.

In the physical mixture of PEG 1500 and PEG 4000, two distinct endothermic peaks

appeared at 47.6 °C and 57 °C, corresponding to the individual melting points as semi-

crystalline polymers [238, 239]. As a result of heating at 80 °C during suppository

preparation, the two endothermic peaks merged at 55.4 °C, indicating the two PEG polymers

were thoroughly mixed in the suppository base while maintaining their semi-crystalline

nature. Notably, no drug peaks were observed, suggesting that AS was primarily distributed

as a solid dispersion in the PEG suppositories (Figure 5.2 a). PEG polymers are widely used

as drug carriers to form solid dispersions, resulting in reduction or disappearance of the peak

corresponding to the melting point of the drug, primarily due to conversion of the drug to its

amorphous form [240-242].

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107

With SNA 15 base, there were either multiple endothermic peaks or a large band observed

between 32 and 36 °C (Figure 5.2 b), reflecting the composition of SNA 15 as a mixture of

C10-C18 mono-, di- and tri-glyceride esters of fatty acids [12, 19]. Both endothermic and

exothermic peaks of AS were observed in SNA 15 suppositories, albeit with some shift,

suggesting the presence of AS particles in the SNA 15 base. No interference was observed

in the melting points of either PEG base or SNA 15 base due to the addition of AS.

Figure 5.2 DSC Characterisation of the state of AS in suppositories composed of PEG

1500/4000 (70:30 w/w) and SNA 15 bases. DSC thermograms of AS, suppository base and

AS containing (a) PEG 1500/PEG 4000 and (b) SNA 15 suppositories. All the samples were

heated at the rate of 5 °C/min from 0 to 250 °C.

Powder X-ray diffraction

As shown in Figure 5.3 a, the XRD patterns of AS powder showed anhydrous crystal peaks

between 2θ of 5°– 45°, consistent with a previous report [243]. These peaks were suppressed

in the AS - PEG physical mixture and low-dose PEG suppository samples. Only some peaks

around 2θ of 18°, corresponding to AS, were observed with much lowered intensity in the

high dose PEG suppository sample. The two significant peaks reflecting the semi-crystalline

nature of PEG remained unchanged at 2θ of 20.12° and 23.34° at both dose levels. Consistent

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with the DSC results, these results again indicated AS was primarily present as a solid

dispersion in the PEG base. In contrast, the diffractograms of AS-SNA 15 physical mixture

and SNA 15 suppositories at both dose levels indicated the presence of AS in crystalline

form as confirmed by the peaks near 2θ 9.38° and 10.52° (Figure 5.3 b).

Figure 5.3 Comparison of XRD patterns of AS, suppository base alone, physical mixture

of AS and suppository bases and powdered samples of 100 mg and 200 mg AS suppositories:

(a) PEG suppositories and (b) SNA 15 suppositories. Dotted vertical lines indicate sample

peaks.

SEM/EDX microanalysis

The SEM/EDX analysis provided surface information on the elements (carbon, oxygen and

sulfur) and their distribution in the microstructure of the suppositories (Figure 5.4;

Table 5.1). The sodium (Na) element from AS was not detectable in any of the

suppositories. Instead, trace level of the sulfur (S) element, which belongs explicitly to AS

(C16H19N3O5SNa), was detected in all suppository samples except the 100 mg SNA 15

suppository.

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109

Figure 5.4 SEM/EDX analysis of suppositories containing 100 mg AS in (a) PEG (b) SNA

15; and 200 mg AS in (c) PEG (d) SNA 15. Overall, the SNA 15 suppositories show more

carbon element (red) and less oxygen (green) compared with PEG suppositories, as

summarised in Table 5.1. The scale bar =20 µm at 1800 X (left panel) and 60 µm at 500 X

for the other panels. ND: Not detected.

Table 5.1 Summary of the elements detected in PEG and SNA 15 suppositories at 100 mg

and 200 mg dose levels and chemical composition of amoxicillin. ND: Not detected.

Type of suppository Detected elements (wt. %)

C O S

PEG 100 mg 60.1 39.4 0.5

SNA 15 100 mg 80.8 19.2 ND

PEG 200 mg 59.7 38.4 1.2

SNA 15 200 mg 80.5 18.9 0.6

As shown in Table 5.1, the quantities of S detected on the surface of PEG suppositories

increased approximately linearly with the dose of AS, indicating uniformity of S element

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110

distribution. The elemental contribution of C:O:S in AS (C16H19N3O5SNa) alone was 49.6%,

20.7% and 8.3% respectively [243]. The experimentally determined higher ratios of O or C

in the suppositories were contributed from the respective suppository bases PEG

(CnH4n+2On+1) or SNA 15 (a mixture of C10-C18 mono-, di- and tri-glyceride esters of fatty

acids).

Theoretically, the detection of S element (representing AS) in the suppositories should be

dose-dependent irrespective of the base type if AS were dispersed homogeneously. Indeed,

the quantity of S in 200 mg PEG suppositories was twice that in 100 mg PEG suppositories

and S element was found to be distributed uniformly in the images (Figure 5.4 left panel,

1800 X). However, in 100 mg SNA 15 suppositories, S was undetectable in the area of

interest, despite the same quantity of drug as in the PEG suppositories.

Intermolecular interactions by FTIR analysis

The FTIR spectra of AS powder (Figure 5.5) produced its characteristic peaks (I-V), C = O

stretching of β-lactam at 1770 cm-1 (Peak I), amide I, C=O stretching peak at 1693 cm-1

(Peak II) and symmetric stretching of carboxylate (Peak III) at 1593 cm-1, that were found

consistent with the reported values for AS [35, 244]. The other peak at 1390 cm- 1

corresponded to COO symmetric stretch and phenol OH combination (Peak IV), whereas

fused thiazolidine ring stretch peak was detected at 1310 cm-1 (Peak V).

The FTIR spectra of PEG base (Figure 5.5 a) showed a broad band at 3296 cm-1 due to the

presence of a large number of OH groups. A characteristic peak at 2882 cm-1 due to C-H

stretching and intramolecular hydrogen bonding was observed in PEG base mix and the

suppository samples at both dose levels. The other peaks in the fingerprint region above

1100 cm-1 were because of C-O-C stretching in PEG base, consistent with the previous

report [245-247].

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111

On the contrary, SNA 15 base and suppositories exhibited characteristic C-H stretching

peaks at 2915 cm-1 and 2850 cm-1, corresponding to the asymmetric and symmetric

vibrations of CH2 groups of the triglyceride chain as well as ester peaks at 1740 cm- 1 and

1728 cm- 1 corresponding to the C=O stretching vibrations [248].

The characteristic drug peaks I - V were found at reduced intensities in both PEG and

SNA 15 suppositories (Figure 5.5, dotted box), possibly due to the dilution effect (100 mg

or 200 mg AS in >1 g of base). No peak shift or broadening was observed in the FTIR spectra

of any suppositories, which indicated absence of intermolecular interactions between AS

and the suppository bases.

Figure 5.5 Intermolecular interactions between AS and suppository bases evaluated using

FTIR, (a) PEG and (b) SNA 15. The boxed areas show FTIR spectra with increased

resolution between 1300 and 1800 cm-1 showing reduced β-lactam signals in PEG and

merged signals in SNA 15 suppositories. Where, I - V indicates characteristic peaks of AS

at 1770, 1693, 1593, 1390 and 1310 cm- 1, respectively.

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112

In vitro drug release and kinetics

SNA 15 suppositories melted and spread quickly, exhibiting significantly rapid drug release

profiles compared with the PEG suppositories at both dose levels (Figure 5.6; f2 = 21.5 and

26.2, both <50). This was consistent with observations during the in vitro disintegration test.

Previously, Webster et al. reported rapid in vitro release of amoxicillin trihydrate from the

lipophilic bases compared with the hydrophilic base [190].

The AS release rate from SNA 15 suppositories at both dose levels was almost similar to

free drug solution (20 mg/mL) (f2 = 50.8 and 57.2, both >50). The PEG suppositories

displayed a 65 - 86 min longer MDT compared with SNA 15 suppositories. Doubling the

drug dose from 100 mg to 200 mg significantly retarded the drug release from SNA 15

suppositories (f2 = 42.3) with a 29 min increase in MDT. In contrast, doubling the drug dose

from 100 mg to 200 mg did not significantly change the release profile of PEG suppositories

(f2 = 67) and led to only an 8 min delay in the MDT. This could be attributed to SNA 15

suppositories melting in the dialysis bags in dissolution medium at 37 °C (consistent with

the DSC results). This allowed spreading in the semi-permeable dialysis bag, resulting in

relatively faster drug release due to the larger surface area. Even though AS was primarily

dispersed as drug particles, its high solubility led to rapid dissolution.

PEG is a hydrophilic base and, unlike SNA 15 base, does not melt at 37 °C, as evidenced in

the disintegration test. The relatively slow drug release from the PEG suppository was

mainly attributed to the slow erosion of the water-soluble matrix. Thus, the drug release only

relied on the dissolution of AS from the surface of suppositories, which decreased over time.

The hydrophilic nature of the drug (log P 0.87 [42]) could also contribute to the delayed

release of amoxicillin from the PEG base.

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Figure 5.6 In vitro drug release from (a) 100 mg and (b) 200 mg amoxicillin suppositories

made from PEG (●) and SNA 15 (■), respectively. Free drug release (◊) 100 mg/5 mL

solution from dialysis bag was used as reference. Data are mean ± SD (n=3). Mean

dissolution time (MDT): time to reach 50% drug release.

Table 5.2 Drug release kinetic modelling for the suppositories containing 106 mg of AS

(equivalent to 100 mg of amoxicillin) prepared with PEG base and SNA 15 base.

Model PEG SNA 15

R2 Slope intercept R2 Slope intercept

Zero order 0.8645 15.63 9.378 0.4528 11.77 54.12

First order 0.9811 -0.003 2.025 0.9940 -0.021 2.058

Higuchi 0.9346 44.21 12.52 0.7195 40.36 28.84

Hixon Crowell 0.9518 0.452 0.048 0.7318 1.041 1.513

Korsmeyer Peppas* 0.9560 0.8603 0.194 0.9256 0.962 0.254

*n = 0.8603 and 0.9621 for PEG and SNA 15 suppositories, respectively.

Drug release data were fitted into different mathematical models, which have been

summarised in Table 5.2. Both types of suppositories approximately followed first-order

drug release (R2 > 0.98), which indicated a concentration dependent drug release [249].

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According to the Korsmeyer-Peppas model, both suppositories followed non-Fickian

diffusion, with the n value greater than 0.45. However, the curve fitting was less significant

(R2 = 0.92 - 0.95). This could be possibly due to the heterogeneous shape of the suppositories

and changes in the surface area for drug release, which reduced over the period for PEG

suppositories and increased for SNA 15 suppositories.

Stability evaluation of the suppositories

After three months at 20 ± 0.2 °C, no colour change was observed in the SNA 15

suppositories. The PEG suppositories, however, turned yellowish, indicating possible drug

degradation. This was likely either due to the hygroscopic nature of PEG [250] or presence

of reactive impurities such as peroxides [251]. This might have led to hydrolytic or oxidative

degradation of amoxicillin [252] and imparted yellowish colour to the suppositories.

Consistently, HPLC analysis confirmed that the content of amoxicillin in PEG 100 mg and

200 mg suppositories after three months was found to be 78.1 ± 0.3% and 79.2 ± 1.4% of

the initial amount, respectively. In contrast, little degradation occurred to the 100 mg SNA

15 suppositories with 91.7 ± 0.2% AS remaining. This increased to 104.0 ± 3.3% as the dose

increased to 200 mg. All the solid-state characterisation experiments suggested AS was

distributed as a particulate suspension in SNA 15 suppositories. This solid-state of

amoxicillin should be more resistant to hydrolysis than the well-dispersed drug in PEG,

which has tendency to draw moisture from humid environment. In general, chemicals

including amoxicillin are more stable in their solid form than in solutions [35, 243]. Stability

was further increased as more AS particles existing in the 200 mg SNA 15 suppositories.

While suppositories are traditionally stored under refrigerated conditions (2 – 8 °C), stability

at ambient temperatures was investigated in this study. A potential application for

amoxicillin suppositories is in the treatment of pneumonia in children in low- and middle-

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income countries, where refrigeration may not be possible. Therefore, stability at

temperatures greater than 20 °C would be required in these settings. Further improvement

in the physical and chemical stability of amoxicillin suppositories may be achieved by

encapsulating the suppository in a shell [12] that has a higher melting point. Formulating

these hollow type suppositories could protect AS in the core from moisture and oxygen and

overcome the limitations of the low melting point of SNA 15. This would also require

investigation into stability at higher temperatures and greater humidity levels.

Absolute bioavailability study

Figure 5.7 shows the PK profiles following rectal administration of each type of

suppositories to rabbits at two dose levels (100 mg and 200 mg), in comparison with an IV

bolus injection of AS solution equivalent to 100 mg of amoxicillin per rabbit. The non-

compartmental PK parameters have been summarised in Table 5.3. To date, no data has been

published on the PK of amoxicillin in rabbits, other than in turkeys and chickens [253], or

dogs, pigs and goats [254]. Interestingly, the AUC0-∞ values following IV administration to

rabbits (approximately 30 mg/kg) in this study were similar to those found in turkeys and

chickens at the dose of 20 mg/kg [253].

SNA 15 suppositories provided faster and almost complete absorption of AS than PEG

suppositories. Interestingly, a double-peak phenomenon in the drug concentration-time

profiles was observed following administration of the SNA 15 suppositories at both dose

levels, Tmax1 at 15 min and Tmax2 at 3 h. A similar but less prominent trend was noticed for

PEG suppositories. The differences in the Cmax2 of SNA 15 suppositories were found about

1.7-fold higher than PEG suppositories at the corresponding dose (p = 0.017 and p = 0.026

at 100 mg and 200 mg, respectively). Amoxicillin is a time-dependent antibiotic and the

time spent above the minimum inhibitory concentration (MIC) is essential for its efficacy.

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Compared with IV administration, the AS suppositories produced prolonged (> 6 h) and

steady concentration profiles. With sufficient drug dose, drug concentrations could be

consistently above the MIC to ensure therapeutic effectiveness, as illustrated in Figure 7

using a Streptococcus Pneumoniae infection as an example. This study evaluated PK

following single dose administration. However, a five-to-seven-day dosing schedule with

multiple administration is more likely in the clinical settings.

Upon rectal administration, the increased surface area due to initial melting and spreading

of SNA 15 suppositories resulted in large tissue exposure. A similar pattern was also

observed during in vitro drug release. Rapid dissolution of the AS particles present on the

surface of the suppositories allowed rapid absorption of amoxicillin within 15 min, resulting

in the first peak shown in the PK profiles. The presence of AS particles within the SNA 15

base then required further transport through the lipophilic base to the absorption site prior to

dissolution and absorption, which likely explains the second peak observed at 3 h in SNA 15

suppositories at both doses. Of note, the F of amoxicillin at both dose levels from SNA 15

suppositories was found close to 100%.

In contrast, a less distinct double peak was observed in PEG suppositories compared with

the SNA 15 suppositories. This could be attributed to an erosion-controlled release

mechanism within a limited rectal area, even though the drug was predominantly distributed

as a solid dispersion in the PEG base. Furthermore, small volume of rectal fluid (1 - 3 mL)

resulted in slow drug absorption from PEG suppositories. The slower in vivo release from

the PEG base was reflected in a noticeable 1 – 1.5 h shorter MRT and MAT in the PEG

suppositories than the SNA 15 suppositories. The retarded drug release resulted in

incomplete absorption alongside significant variation in F (59.2 ± 23.6%) of PEG

suppositories. Increasing the dose of water-soluble AS in the PEG suppositories promoted

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drug release leading to a longer MAT (4.7 h vs 3.7 h) alongside a 1.3-fold increase (p < 0.01)

in F (77.3 ± 16.7%).

It has been reported that amoxicillin has non-linear, dose-dependent absorption when given

orally, so more frequent small doses seem to be more effective than less frequent and larger

doses [253, 255]. Further, saturated transport of amoxicillin through the small intestine of

rats has been reported [255-257]. The present study demonstrated non-saturated absorption

of amoxicillin through the rectal mucosa, indicating that drug transport most likely occurred

by passive diffusion via transcellular and paracellular transport within the tested dose range.

This finding corresponded to the previous reports highlighting an absence of active transport

in the rectum [6, 7]. These findings suggested that AS can be classified as BCS Class I with

no dissolution or permeability limiting steps. However, the permeability of AS through the

rectum could be further clarified in future using an ex vivo rectal membrane [258]. In

addition, further investigation is necessary at higher dose levels (> 200 mg) and with

multiple dose administration to confirm non-saturated transport of amoxicillin from the

rectum.

Figure 5.7 Amoxicillin plasma concentration (μg/mL) versus time profiles following

rectal administration of from PEG base and SNA 15 suppositories in rabbits at two dose

levels, (a) 100 mg and (b) 200 mg compared with intravenous injection at a dose of 100

mg/rabbit. All data are presented as mean ± SD (n=5). The minimum inhibitory

concentration (MIC) of amoxicillin for the treatment of S. Pneumoniae (2 μg/ mL) [38] is

shown for reference.

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Table 5.3 PK parameters of amoxicillin following rectal administration of amoxicillin suppositories at two different dose levels compared with

intravenous (IV) injection (at a dose of 100 mg per rabbit) in rabbits. Data are Mean ± SD; n=5 rabbits.

PK

Parameters

100 mg/rabbit 200 mg/rabbit

IV amoxicillin solution PEG suppository SNA 15 suppository PEG suppository SNA 15 suppository

AUC 0-8h (μg/mL h) 16.90 ± 5.58 8.38 ± 4.02 16.16 ± 1.73 23.67 ± 4.99 32.76 ± 4.98**

AUC0-∞ (μg/mL*h2) 17.20 ± 5.76 9.45 ± 3.76 20.37 ± 6.80 25.43 ± 5.51 33.88 ± 5.10

Tmax1 (h) - 0.65 ± 0.49 0.25 ± 0.00 0.75 ± 0.50 0.25 ± 0.00

Tmax2 (h) - 2.70 ± 0.97 3.00 ± 0.00 2.80 ± 0.84 3.20 ± 0.45

Cmax1 (μg/mL) 44.72 ± 34.53 1.93 ± 1.46 3.92 ± 1.19* 5.11 ± 2.77 8.72 ± 3.66

Cmax2 (μg/mL) - 2.36 ± 1.26 4.39 ± 0.85* 6.45 ± 1.55 10.96 ± 3.37*

t1/2 (h) 0.80 ± 0.25 2.21 ± 0.69 2.21 ± 1.93 1.78 ± 0.57 1.20 ± 0.17

MRT (h) 1.24 ± 0.57 4.93 ± 1.04 6.44 ± 0.83 5.61 ± 0.73 6.30 ± 1.26

MAT (h) - 3.69 ± 1.04 5.20 ± 0.83 4.37 ± 0.73 5.06 ± 1.26

F (%) 100 59.2 ± 23.6 111.4 ± 37.2* 77.3 ± 16.7 94.8 ± 14.3

Vd (L) 7.0 ± 1.6 44.2 ± 34.4 13.9 ± 8.0 20.6 ± 6.3 10.5 ± 2.6

Cl (L/h) 6.42 ± 2.32 12.45 ± 6.10 5.26 ± 1.34 8.24 ± 2.19 6.01 ± 0.93 Significant differences in the drug absorption between PEG and SNA 15 suppositories determined using two-way ANOVA, where * means p<0.05 and ** means p<0.01. “-” means not applicable.

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Histological analysis

The stained rectal tissues were observed under a light microscope to determine signs of

irritation to the rectal mucosa caused by the suppositories (Figure 5.8). The rectal tissues

from the control group showed the microstructures of the intact epithelium (IE) and lamina

propria (LP). The rectal tissue in the SNA 15 group showed minimal signs of irritation to

the mucosa. The epithelial architecture was intact except for dilated blood vessels (DBV)

with extravasation of blood compared with the control group.

In contrast, the rectal tissues collected from the PEG group showed apparent signs of

disrupted architecture (DA) of the rectal epithelium and tissue irritation, including loss of

goblet cells in the mucosa, oedematous epithelium (OE) and congestion (C). Furthermore,

the depth of glands was found to be reduced compared with the control tissue. This was

consistent with previous findings of the irritation potential of PEG bases [14, 259-261], as

hydrophilic PEG draws water from surrounding tissues causing irritation to the rectal

mucosa [252]. Irritation to mucosa due to PEG has recently been estimated using a slug

mucosal irritation potential assay, where the total mucus production was found to be 7.1%

and 10.2% of its body weight following exposure to PEG base alone and PEG with

amoxicillin trihydrate, respectively, suggesting mild irritation [12].

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Figure 5.8 Representative histological images of rectal tissues from non-treated rabbits (control) and rabbits treated with PEG suppositories and

SNA 15 suppositories containing amoxicillin (200 mg) to understand the irritation to the rectal mucosa, at two magnifications: 4 X and 10 X. Tissue

samples were taken 24 h following administration of suppositories. Where, C- Congestion, LP- Lamina propria, IE- intact epithelium, OE-

Oedematous epithelium, DBV- Dilated blood vessels, NA- Normal architecture, DA -Disrupted architecture, MA- Maintained architecture.

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In vitro - in vivo correlation (IVIVC)

Depending upon the formulation development, an IVIVC Level C was developed and

evaluated from the regression analysis (Figure 5.9). There was a clear trend of linearity

between MDT and F or MAT (p < 0.01 and p < 0.05), implying that a slow release would

result in poor absorption and shorter MAT. The correlations were found statistically

significant despite the low goodness of fit (R2 = 0.3686 and 0.2402) due to large individual

variations. The delayed release beyond 2 h from PEG suppositories was also reflected by

comparatively short MRT and incomplete absorption from the rectum. Notably, the F of the

four suppositories were also strongly correlated to their MAT, p = 0.021, indicating the

importance of rapid in vivo drug release in quick absorption of AS from the rectum.

Figure 5.9 Level C IVIVC by regression analysis of mean dissolution time (MDT) vs (a)

% F and (b) mean absorption time (MAT).

5.4 Conclusions

This study demonstrated different in vitro and in vivo behaviour of amoxicillin suppositories

formulated using two types of suppository bases, hydrophilic PEG (1500/4000) and

lipophilic SNA 15. SNA 15 suppositories provided rapid drug release and complete

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absorption, possibly aided by spreading due to the melting point (32 - 36 °C), which is lower

than the rectal temperature, as well as rapid dissolution of AS crystals. The delayed drug

release from PEG suppositories, dominated by slow erosion of the PEG base, resulted in

slow and incomplete absorption. In addition, SNA 15 suppositories exhibited better tissue

compatibility in the rectum than the PEG suppositories. AS existed predominantly in solid-

state as a suspension in SNA 15 base, therefore, was more chemically stable in SNA 15

suppositories compared with PEG suppositories. Altogether, this research highlighted

SNA 15 suppositories as a promising dosage form for rectal delivery of AS.

123

Formulation Development of Amoxicillin Hollow-Type Suppositories: Comparison of

Amoxicillin Sodium Salt and Trihydrate

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124

In this Chapter, the development and characterisation of amoxicillin containing hollow-type

(HT) suppositories have been discussed, focusing on stability improvement. The effect of

two salt forms of amoxicillin on in vitro drug release and bioavailability prediction have

been discussed, along with stability evaluation.

6.1 Introduction

As discussed in Chapter 1, pneumonia is an infectious respiratory disease. It has been

identified as the leading infectious cause of global childhood mortality [24, 26, 33, 262],

most commonly in South East Asia and Sub-Saharan Africa [33]. Currently, amoxicillin

dispersible tablets (DTs) are indicated as the first-line treatment in these regions, but there

are limitations to DTs as well as other available oral and parenteral formulations, especially

for paediatric patients [34]. Recently, minimally invasive hydrogel forming microneedle

arrays were investigated for transdermal delivery of amoxicillin in paediatric patients. This

study reported the instability of amoxicillin in microneedle arrays due to amoxicillin’s

sensitivity to moisture and temperature [263]. Furthermore, the higher manufacturing costs

of such technologies mean that they are not an ideal treatment option particularly in low-

and middle-income countries (LMICs). All these limitations can be overcome by rectal drug

delivery system containing amoxicillin.

Pharmacokinetics (PK) studies in Chapter 5 showed that amoxicillin sodium (AS) was

absorbed via non-saturated transport from the rectum. This indicated that rectal amoxicillin

delivery has the potential to be an alternative to the oral and parenteral dosage forms and

can be used for paediatric patients to treat a variety of diseases [12, 115, 120, 264, 265]. In

Chapter 5, two types of amoxicillin suppositories made with either polyethylene glycol

(PEG) 1500:4000 (70:30 w/w) or Suppocire NA 15 (SNA 15) were developed and

characterised in vitro. The absolute bioavailability study using a rabbit model indicated a

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rapid release and almost complete absorption of amoxicillin alongside better tissue tolerance

from SNA 15 suppositories than PEG suppositories at both dose levels (100 mg and

200 mg). Furthermore, three months storage at ambient conditions resulted in colour change

of PEG suppositories compared with no colour change in SNA 15 suppositories, consistent

with the HPLC analysis, which showed <80% amoxicillin remaining in PEG suppositories

compared with 91.7 ± 0.2% % and 104.0 ± 3.3% amoxicillin remaining in the low dose and

high dose SNA 15 suppositories, respectively. Thus, SNA 15 was considered a more suitable

suppository base for rectal delivery of amoxicillin than PEG.

However, the low melting point of the SNA 15 base, 30-36 °C [19] could potentially result

in physical instability of the suppositories at high temperatures such as the tropical climate

of many LMICs. Due to this, SNA 15 suppositories require cold chain transport, which

increases costs and barriers to accessibility and availability, particularly in LMICs [8]. This

may limit the use of SNA 15 suppositories for out-patient treatment of pneumonia in LMICs,

where the temperature is high. The literature review in Chapter 2 discussed hollow-type

(HT) suppositories, which can accommodate solid, liquid or semi-solid in the hollow core.

This approach could help improve the physical stability by encapsulating the amoxicillin

loaded SNA 15 core into a hollow shell that can withstand high temperature (Figure 6.1).

Figure 6.1 A schematic diagram of amoxicillin containing HT suppositories, where the

drug was loaded only in the SNA 15 core.

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It was hypothesised that using a shell prepared from PEG base with a high melting point, at

least above 40 °C, would improve the physical stability of drug loaded SNA 15 core as well

as enable rapid amoxicillin release due to quick dissolution in the body fluid due to its

narrow thickness. Furthermore, the addition of water soluble polymers along with PEG

would improve strength and flexibility of the PEG shells. It was also hypothesised that the

different solubilities of AS and amoxicillin trihydrate (AMT) would impose their effect on

the drug release and bioavailability.

Therefore, to further improve the stability of amoxicillin, this study aimed to develop

250 mg amoxicillin containing HT suppositories that would be inexpensive and easy to

produce using traditional suppository excipients. Following the experimental evidence

indicating absorption of AS from the rectum, AMT which is a more stable form of

amoxicillin than AS [35] was also evaluated as it has been previously reported in amoxicillin

suppository development [12, 190]. For this, HT suppository shells using the previously

selected PEG bases and PEG mixed with water soluble polymers polyvinyl pyrrolidone

(PVP) or polyvinyl alcohol (PVA) were developed and screened. The developed HT

suppositories were characterised by standard pharmacopoeial tests, solid-state analysis, in

vitro release and stability evaluation at 25 °C, 60% relative humidity (RH) and 37 °C. Also,

the bioavailability of AS and AMT was predicted from the previously established in vitro in

vivo correlation (IVIVC) model in Chapter 5.


Materials

AS, purity >87% and AMT, purity >99% were purchased from Alfa Aesar by Thermo Fisher

scientific (Haverhill, Massachusetts, USA). PEG 1500 and 4000 and dialysis bags having a

molecular weight cut-off (MWCO) 14000 Da were purchased from Sigma Aldrich (St.

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127

Louis, Missouri, USA). SNA 15 pellets were kindly donated by Gattefossé SAS, France.

The chemicals used in the preparation of buffer medium: acetic acid, sodium acetate, sodium

chloride, sodium/potassium dihydrogen phosphate, and disodium hydrogen phosphate, were

of reagent grade and purchased from either Merck or Thermo Fisher Scientific(Auckland,

New Zealand). Acetonitrile and methanol used in the chromatographic analysis were of

high-performance liquid chromatography (HPLC) grade. Milli-Q water was obtained from

a Millipak water dispenser (Millipore, 0.22 µm).

Screening of suppository bases for preparation of shells

Previously selected PEG 1500 and PEG 4000 bases were used to prepare the suppository

shells. Mixtures of PEG 1500 and 4000 in a ratio of 70:30, 80:20, and each type of PEG

alone were trialled to prepare the suppository shells. The shells were prepared using a

modified 1 g suppository mould shown in Figure 6.2. The stainless-steel pins were designed

to prepare the shells with a uniform thickness of 1 mm. The shell thickness was selected to

accommodate the required dose of amoxicillin (250 mg) and to minimise the irritation to the

rectal mucosa. Two polymers PVP- Kollidone® 12 PF and PVA were added (2% w/w) to

PEG 4000 for the preparation of shells to improve the shell hardness and flexibility.

Figure 6.2 A Modified hollow-type suppository mould for the preparation of PEG shells

(a), and a schematic diagram showing the dimensions of the modified suppository mould

used to prepare the HT suppository shell (b).

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Characterisation of the suppository shells

Following the preliminary screening, suppository shells prepared with PEG 1500

(100% w/w) or PEG 4000 (100% w/w) only were evaluated for weight variation,

disintegration time and hardness test using texture analyser.

Weight variation

Suppository shells prepared using PEG 4000 or PEG 1500, n=10, each was weighed

individually. The weight variation within ± 5% of the average weight was considered

uniform [228].

Disintegration time

A standard disintegration test was conducted using an Erweka basket-rack ZT 320

disintegration test assembly (Erweka GmbH, Germany). Individual suppository shells were

placed in each tube of the disintegration apparatus containing a beaker filled with 900 mL

of Milli-Q water at 37 ± 2 °C. The assembly was immersed in the disintegration fluid and

set at a constant frequency of 30 ± 1 cycles per minute. The suppository shells were visually

observed for the signs of complete disintegration of the suppository shells for up to 60 min

or the time when all the shells were dissolved, whichever was earlier, and the time was

recorded. According to the British Pharmacopoeia, the suppositories containing PEG base

should dissolve within 60 minutes [228].

Hardness and frangibility evaluation using texture analyser

The breakpoint of suppository shells made of PEG 1500 or 4000 (n=3 each) was measured

using a Texture Analyser equipped with Texture Plus 32 software for data analysis (TA.XT

Plus, Texture Systems). The TA system was calibrated with 2 kg cell load and 5 mm probe.

The suppository shells were kept vertical in the axial direction. The return to original

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129

position method parameters were set, where the 2 mm probe travelled at a speed of 1 mm

per second up to 6 mm distance once contact with the suppository shell was made. The 6 mm

travel distance covered the thickness of the apex of the suppository shell. The force (kg) at

which the suppository shell broke, - peak of the force-time curve, was recorded as hardness

of the suppository shells [266], while the time required to break each type suppository shells

represented their elasticity.

Preparation of amoxicillin HT suppositories

Amoxicillin HT suppositories were prepared by assembling the PEG 1500 shell and drug-

loaded SNA 15 core together using a modified 1 g suppository mould. As shown earlier

(Figure 6.2), PEG 1500 shells were prepared by the fusion moulding technique using

stainless-steel pins. Melted PEG base was poured into the cavity of the suppository mould.

A lubricated stainless-steel pin was placed immediately into the mould cavity and the PEG

shell was allowed to solidify. The solidified shells were then carefully separated from the

pins and stored in an airtight container.

The core was prepared by the warm compression method using a stainless-steel bespoke

mould prepared in-house. AS or AMT, equivalent to 250 mg amoxicillin, were weighed

individually and physically mixed with 200 mg of crushed SNA 15 base for each unit of

suppository. Excess mixture (2%) was prepared to compensate for any formulation loss

during the manufacturing process. The mixture was simultaneously compressed and filled

in a cavity of pre warmed (35 °C) stainless-steel mould. The compressed cores were left to

solidify at room temperature for 1 hour. The compressed cores were then demoulded and

transferred inside the hollow cavity of the PEG shells. The open end of the shell was then

sealed with melted PEG 1500 base. Excess PEG base was scraped off from the seal and the

HT suppositories were demoulded.

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Characterisation of amoxicillin containing HT suppositories

All suppositories were visually observed for uniformity of the shape, size, colour and

evaluated for other pharmacopoeial tests according to the British Pharmacopoeia [228].

Uniformity tests

Ten individual units from a batch of both amoxicillin sodium hollow-type (AS HT) and

amoxicillin trihydrate hollow-type (AMT HT) suppositories were selected randomly and

weighed. The weights of individual suppositories were recorded and the uniformity was

determined as per the acceptance criteria mentioned in the British Pharmacopoeia where

none of the individual units should deviate by 5% of the average weight of suppositories

[228].

To determine content uniformity, three individuals from each type of HT suppositories were

selected and kept in separate beakers containing 200 mL milli-Q water at 37 °C. The contents

of the beaker were stirred at 300 RPM using a magnetic stirrer for 45 min and 1 mL sample

was withdrawn and filtered using 0.22 µm syringe filter. The samples were appropriately

diluted and quantified using a stability-indicating HPLC method described in Chapter 3.

Hardness test of HT suppositories

The hardness of the HT suppositories was evaluated using the Erweka hardness tester

(Erweka GmbH, Germany). Three suppositories from each batch were placed such that the

force of the moving plunger was applied on the longitudinal axis of the suppository. The

force (kg) when the HT suppository broke or cracked was noted as the hardness of the

suppository.

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Solid-state characterisation

To determine any drug-base interactions, individual samples of each type of HT

suppositories were characterised by powder x-ray diffraction (XRD) technique, differential

scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy.

Characterisation of the core using powder X-ray diffraction (XRD)

As the drug was loaded in the SNA 15 core, crushed samples of SNA 15 base, AS, AMT,

and the drug loaded core of AS HT and AMT HT suppositories were compared by XRD

using the Ultima IV X-ray diffractometer (Rigaku Corporation, Tokyo, Japan). The samples

were analysed using Cu Kα radiation (1.54056 Å) at a scattering angle of 5°<2θ<45° in a

continuous rotation scan mode with a step size of 0.02θ every 20s. The diffractometer was

operated at a generator tension of 40 KV and current of 30 mA. Diffraction line intensity

versus 2θ were plotted and analysed for changes in drug peaks in the presence of SNA 15

base.

Differential scanning calorimetry

Thermal analysis of individual samples of PEG shell, unmedicated SNA 15 base, AS, AMT,

and the HT suppositories of each type were investigated using a differential scanning

calorimeter (TA Q2000, TA instruments, New Castle, USA) equipped with TA-Q Universal

Analysis software. Approximately 5-7 mg sample was precisely weighed and heated from

0 – 250 °C at a rate of 5 °C/min with nitrogen gas flowing at 20 mL/min. The samples were

evaluated for any interference in melting behaviour of the HT suppository shell and the drug

loaded core.

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Fourier transform infrared spectroscopy

To determine whether there were any drug-excipient interactions, an FTIR spectrometer

(Bruker, United States) equipped with a diamond crystal (Bruker Tensor 37, attenuated total

reflection - ATR) was employed. A small amount of sample, enough to cover the crystal

surface, was taken from each batch of suppositories and analysed using FTIR spectroscopy.

The samples were kept directly on the surface of the diamond crystal and were examined

from an average of 32 scans and automatic background subtraction between the range of

400-4000 cm-1 and resolution of 2 cm-1.

In vitro drug release and kinetics

In vitro drug release of HT suppositories was determined using USP type II paddle type

apparatus (Hansen Research SR 8 Plus, Hanson Research, Chatsworth, USA). Phosphate

buffer saline (PBS) pH 7.4 kept at 37 °C was used as dissolution medium to mimic the rectal

pH. Each of the AS HT and AMT HT suppositories were kept in a dialysis bag (MWCO

14000 Da) in 500 mL of PBS (n=3). The contents of the vessel were stirred at 50 RPM and

1 mL sample was withdrawn at 0, 15, 30 and 45 min, then 1, 1.5, 2, 2.5, 3, 4, 5, 7, 9, 24, 27,

30, 48, 52, 60 and 72 hours. The samples were diluted as required and 20 µL was injected

for quantitative analysis of amoxicillin using a previously developed stability-indicating

HPLC method, as described in Chapter 3.

The release profiles were compared and the time to release 50% of drug from the HT

suppositories and similarity factor (f2) were calculated using DDSolver, a Microsoft Excel

add-in programme using Eq 5.1 and 5.2, as mentioned in Chapter 5 [229]. According to the

United States Pharmacopeia, an f2 value between 50-100 indicates similarity in drug release

from different formulations, while a value below 50 indicates dissimilar drug release from

two formulations [230].

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The release profiles were then fitted to different mathematical models to determine the

mechanism involved in the in vitro drug release. Different mathematical models such as first

order (Eq 6.1), Hixon Crowell (Eq 6.2), Higuchi (Eq 6.3) and Korsmeyer-Peppas (Eq 6.4)

were fitted to the drug release profiles using GraphPad prism software 8.2.1 (GraphPad

software, Inc) to understand the impact of drug solubility on release.

𝐿𝐿𝐴𝐴𝑑𝑑𝑀𝑀 = 𝐿𝐿𝐴𝐴𝑑𝑑𝑀𝑀0 − 𝑘𝑘𝐴𝐴/2.303 Eq 6.1

Where, C0 is the initial drug concentration found in the freshly prepared suppositories, C is

the drug concentration at time t and k is the first-order rate constant.

𝑄𝑄 01/3 − 𝑄𝑄𝑡𝑡

1/3 = 𝐾𝐾𝐻𝐻𝐻𝐻𝐴𝐴 Eq 6.2

Where, Qt is the amount of drug released at time t while Q0 indicates amount of drug in the

suppository at the time 0 and KHC is the Hixon Crowell rate constant.

𝑄𝑄 = 𝐾𝐾𝐻𝐻√𝐴𝐴 Eq 6.3

Where, Q means the amount of drug released at time t and KH is the Higuchi rate constant.

𝑀𝑀𝑡𝑡 𝑀𝑀∞⁄ = 𝐾𝐾𝐴𝐴𝑛𝑛 Eq 6.4

Where, Mt/M∞ indicates the fraction of drug released at time t, K is the rate constant and n

is the release exponent. The value of n indicates different release mechanisms. The n value

of 0.45 indicates Fickian diffusion while 0.45 < n < 0.89 indicates drug release via non-

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Fickian diffusion [267]. The drug release can be considered as case II transport in case the

value of n ≥ 0.89.

Prediction of absolute bioavailability (F) using IVIVC model

The previously developed IVIVC model described in Chapter 5 was used to predict the

bioavailability of the HT suppositories. The Level C correlation equation (Eq 6.5) derived

from the plot of F vs mean dissolution time (MDT) was utilised for the calculation of

bioavailability.

𝑦𝑦 = −0.4550𝑥𝑥 + 126.7 Eq 6.5

Where, y indicates the F (%) and x is the MDT (min) of HT suppositories.

Stability of HT suppositories

All the suppositories were individually wrapped in aluminium foil, stored in an airtight

container and evaluated for evaluated for physical and chemical stability. Both AMT HT

and AS HT suppositories (n=12 each) were stored at 25 °C and 60% RH for three months

or until >10% degradation of amoxicillin was observed. A separate set of AMT and AS HT

suppositories (n=4, each) were stored at 37 °C to evaluate the storage stability of the

suppositories. At the end of three months, suppositories were observed physically for any

signs of melting or colour change, hardness and amoxicillin content as per the methods

described previously. Finally, the suppositories were also evaluated by DSC and FTIR for

any signs of degradation and possible change in the intermolecular interaction compared

with the freshly prepared samples.

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Screening of suppository bases for preparation of shells

The suppository shells made with different mixtures of PEG 1500 and 4000 were found

brittle and cracked (Figure 6.3), likely due to crystallinity differences between the low and

high molecular weight PEGs [268]. PEG 1500 solidifies slowly, starting at 37 °C compared

with PEG 4000 which rapidly solidifies at 43 °C, respectively, this solidification rate

difference could lead to rapid shrinkage of PEG and generate cracks on the shell due to its

narrow thickness of 1 mm.

Figure 6.3 Images of suppository shells prepared with (a) PEG 1500, (b) PEG 4000 and

(c) PEG 1500:4000 mixed in a weight ratio of 80:20.

The shells prepared using PEG 4000 alone were also found brittle and cracked, likely

because of the higher solidification point. Furthermore, plasticity of PEG decreases as the

chain length increases resulting into brittle and cracked suppository shells. Consequently,

incorporation of 2% w/w of PVP or PVA did not improve the strength or plasticity of PEG

4000 shells. This is consistent with recent findings which found that a ratio of PEG >20%

in a PVP-PEG mixture resulted in cracked and brittle scaffolds [268, 269]. Therefore,

suppository shells made with only PEG 4000 or PEG 1500 (100% w/w) were further

evaluated.

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Characterisation of the suppository shells

Weight variation

Both type of suppository shells were found uniform in weight. The average weight variation

of the shells made with PEG 1500 and PEG 4000 was found to be 0.63 ± <0.01 g and

0.62 ± <0.01 g, respectively. No samples deviated by more than 5% of the average weight

of the shells.

Disintegration time

Both types of suppository shell completely dissolved in water within 7 - 10 min, which was

unsurprising due to the narrow 1 mm thickness of each shell as well as high water solubility

of PEG base. This suggested rapid removal of the PEG shell and consequent exposure of

amoxicillin loaded core upon administration.

Hardness and frangibility evaluation using texture analyser

The force-time curve (Figure 6.4) indicated the mean break point of shells was 2.35 ±

0.79 kg and 1.47 ± 0.23 kg for PEG 1500 and PEG 4000, respectively. As per United States

Pharmacopoeia guidelines, the hardness of the suppository should be above 2 kg [135].

Therefore, only PEG 1500 shells were found compliant, indicating their suitability to

withstand the mechanical forces during transportation and drug administration.

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137

Figure 6.4 Graphical representation of breakpoint evaluation by texture analyser

showing the difference in breakpoint of PEG 1500 shells (blue line) and PEG 4000 shells

(red line) (n=3 each).

In addition to the greater force required to break PEG 1500 shells compared with PEG 4000

shells, the time required to break the PEG 1500 shell was also longer. The PEG 1500 shell

broke at 0.74 ± 0.06 sec while the same was found at 0.2 ± 0.01 sec for PEG 4000 shells.

PEG 4000 generated brittle suppository shells as seen by sharp decline in the applied force,

while PEG 1500 generated a comparatively elastic curve. This is consistent with previous

reports, which suggested increased plasticity with lower molecular weight PEGs [250, 270].

Furthermore, the plasticity of PEGs decreases with an increase in chain length [250]. This

could explain why PEG 4000 produced brittle and fragile shells, especially at a thickness of

1 mm. On the contrary, PEG 1500 shells were found elastic, generating a broad time-force

curve compared with PEG 4000 shells. Thus, suppository shells made of only PEG 1500,

melting point > 45 °C, were further used for the preparation of the drug loaded suppositories.

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Characterisation of amoxicillin HT suppositories

Following the screening of the suppository shells, HT suppositories were prepared with AS

or AMT. Table 6.1 summarises the composition of each HT suppository.

Table 6.1 Properties of components of the HT suppositories, individual and as a system.

AS HT AMT HT Property

Shell PEG 1500 PEG 1500 Rapidly dissolves in the

rectal fluid enabling

immediate drug release.

Core AS equivalent to

amoxicillin in SNA 15

AMT equivalent to

amoxicillin in SNA 15

Lipophilic core melts and

spreads into the rectum

releasing amoxicillin.

Hypothesis Rapid drug release due

to high solubility of

amorphous AS, 150

mg/mL [44]

Delayed drug release

due to low solubility of

crystalline AMT. 2.7

mg/mL [45].

All suppositories loaded with AS or AMT core were found uniform in size, shape, and

slightly off-white in colour (Figure 6.5).

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139

Figure 6.5 Images representing the components of HT suppositories: (a) separate shell

and drug loaded core, (b) HT suppositories. (c) schematic diagram of HT suppository along

with the dimensions.

Uniformity tests

Both AS HT and AMT HT suppositories were found uniform in weight with an average

weight of 1.17 ± <0.01 g and 1.18 ± <0.01 g, respectively. None of the suppository weights

deviated by more than 5 % of the average weight of the suppositories [228].

The average content of amoxicillin in 250 mg AS HT and AMT HT suppositories was found

to be 99 ± 2.4 % and 109.5 ± 1.9%, respectively. The content of both formulations were

found compliant with the content uniformity criteria of the British Pharmacopoeia,

according to which the content of an individual suppository should be within 75 and 125 %

of the average content [228].

Hardness test of HT suppositories

The suppositories were tested for hardness along the longitudinal axis to simulate the

pressure applied during the administration as well as during transportation of the

suppositories. The hardness of AMT HT suppositories was found slightly higher

(3.7 ± 1.0 kg) compared with the hardness of AS HT suppositories (3.1 ± 0.3 kg). The

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140

hardness of both types of HT suppositories above 2 kg indicated that the HT suppositories

were able to withstand the forces that may apply during the administration as well as

transportation [135].

Solid-state characterisation

Characterisation of the drug loaded core using XRD

The XRD patterns indicated crystallinity of AMT and AS drug powders (Figure 6.6).

However, the diffraction patterns were found different for AMT and AS drug powders

corresponding to their crystal structures; orthorhombic shaped structures and irregular

clusters of crystals of anhydrous AS, respectively [243, 271]. The analysis of physical

mixture and drug loaded core resulted in XRD patterns of a partially amorphous mixture

likely due to the presence of crystalline AMT or AS in amorphous SNA 15 matrix. Similar

XRD patterns were observed from the samples of AS conventional suppositories prepared

using SNA 15 base (Chapter 5).

Figure 6.6 Comparison of XRD patterns of AMT and AS, their physical mixtures with SNA

15 and drug loaded core of the respective HT suppositories.

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141

Differential scanning calorimetry (DSC)

The results of thermal evaluation of individual samples as well as the HT suppositories using

DSC have been summarised in Figure 6.7.

Both AS and AMT were found to be degrading above 230 °C, as seen from the thermograms,

consistent with the previous reports [12, 35]. In AS sample, a broad and small endothermic

peak was observed at 132.6 °C representing its melting, which was close to the value

supplied by the manufacturer as 130 °C [272], followed by an exothermic peak at 223.7 °C.

Such atypical small endothermic melting peaks are indicative of anhydrous crystals. On the

contrary, orthorhombic AMT crystals in the drug powder showed a sharp endothermic peak

due to its melting at 156.7 °C followed by multiple noise peaks indicating degradation of

AMT. The samples of PEG 1500 shells showed an endothermic peak at 47 °C, corresponding

to the melting point of PEG 1500 whereas SNA 15 showed multiple endothermic peaks

within the range of 33-36 °C corresponding to its melting point at 33.6 °C [19].

DSC analysis indicated no change in the melting behaviour of SNA 15 base due to the

presence of amoxicillin (Figure 6.7 a and b). Despite AS being distributed as a particulate

solid suspension in the SNA 15 matrix, the AS peak was not detected in AS HT suppositories

unlike the conventional suppositories. This could be due to relatively smaller proportion of

AS in the DSC samples, as seen from a small endothermic peak corresponding to SNA 15

melting and lower enthalpy of melting of AS.

In contrast, AMT HT suppositories showed multiple peaks within the range of 135 -158 °C

with a maximum at 138 °C likely due to crystalline rearrangement of the AMT molecules

present in the SNA 15 base. The samples were further analysed using FTIR spectroscopy

for any signs of drug-base interactions.

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142

Figure 6.7 Drug-base interactions shown by DSC analysis of (a) AS HT suppositories and

(b) AMT HT suppositories along with FTIR spectra of (c) AS HT suppositories and (d) AMT

HT suppositories. All the samples were heated from 0- 250 °C at the rate of 5 °C/ min.

Fourier transform infrared spectroscopy

Corresponding to the DSC analysis, qualitative analysis by FTIR also showed absence of

intermolecular interactions between the drug and SNA 15 base, shown in Figure 6.7 c and d.

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143

Both AS HT and AMT HT suppositories showed strong characteristic peaks at 1770 and

1772 cm-1 (C=O stretch of the β-lactam ring), 1685, 1693 cm-1 (amide I, C=O stretch), 1391,

1377 cm -1 (gem-dimethyl CH deformation and phenol OH combination band ) and at 1312,

1308 cm-1 (fused thiazolidine β-lactam stretch and preliminary amide, C=O stretch),

corresponding to the reported values [35].

In the FTIR spectra of AS HT suppositories, the characteristic β-lactam C=O stretching peak

at 1770 cm-1 were found mixed with the characteristic ester C=O stretch peak of SNA 15

base at 1740 cm-1. This was in line with the previous findings reported in Chapter 5, where

amoxicillin showed combined peaks but no interaction with the SNA 15 base possibly

resulting in improved stability of amoxicillin suppositories. In the contrast, AMT HT

suppositories showed β-lactam C=O stretching peak at 1772 cm-1 as well as amide I C=O

stretching peak at 1693 cm –1. The peaks in the fingerprint region were found merged with

the peaks of the PEG 1500 or SNA 15 bases used in the preparation of shell and core of both

types of HT suppositories.

In vitro release analysis

Dissimilar drug release profiles (Figure 6.8) were observed from the suppositories when AS

was replaced with AMT, with a similarity factor f2 of 18.1, much smaller than the minimum

threshold of 50 for being similar [230]. The two types of suppository had a large difference

(25.6 h) in MDT, which was found to be 1.7 h for AS HT and 27.3 h for AMT HT

suppositories.

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Figure 6.8 In vitro drug release from HT freshly prepared suppositories. AS HT

suppositories (green line) and AMT HT suppositories (red line). Data presented as mean ±

SD of n =3 suppositories. Dotted line in represents the MDT. Inset graph shows difference

in drug release within 4 h.

A <10 min lag time was observed in the AS release from HT suppositories due to the

presence of the shell which may cause delay in in vivo absorption, particularly when the

in vivo fluid volume is minimal (1 – 3 mL) . Despite existence of AS and AMT as a solid

suspension in SNA 15 matrix, nearly 80% of amoxicillin released in 4 h from AS HT

suppositories whereas only 12% drug was released from AMT HT suppositories over the

same period (Figure 6.8 inset). The high aqueous solubility of AS (150 mg/mL in water at

25 °C [44]) resulted in rapid dissolution particles and release of the drug (Noyes-Whitney

theory). Additionally, melting and spreading of SNA 15 base could have increased the

surface area for particulate dissolution. However, this was not the case for AMT HT

suppositories. The orthorhombic crystalline arrangement of AMT, determined by XRD,

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145

might have contributed to the significantly slow drug dissolution and release. The highly

organised and stable crystalline lattice of AMT limits the loss of energy by solubilisation

[35] compared with the anhydrous AS crystals which can also observed from the time-lapse

images during the dissolution study (Figure 6.9).

Furthermore, the delayed release from AMT HT suppositories could be associated with the

formation of hard suppositories due to the trihydrate form of the drug, which was also

reflected in the hardness test [273, 274].

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146

Figure 6.9 Time lapse up to 72 h showing drug release from HT suppositories kept in a dialysis bag (MWCO 14000 Da) containing PBS, pH

7.4 at 37 °C (a) AS HT suppositories and (b) AMT HT suppositories.

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147

The release profile of AMT HT suppositories fitted the Hixon Crowell model whereas AS

HT suppositories fitted the first-order release model, as summarised in Table 6.2. The Hixon

Crowell model is appropriate for drug delivery systems where drug release depends on the

velocity of dissolution and not the diffusion [275]. This was found consistent with the drug

release profiles from AMT HT suppositories, where the drug release was attributed to the

velocity of dissolution rather than diffusion from the dialysis bag. The n values of the

Korsmeyer-Peppas model suggested anomalous drug transport from AS HT suppositories,

indicating simultaneous dissolution of drug from the melted base and diffusion from the

dialysis bag [249, 267], whereas Fickian transport was observed from AMT HT

suppositories due to low solubility of AMT and subsequently slower dissolution and

diffusion of the drug.

Table 6.2 Summary of release kinetics of amoxicillin from AS HT and AMT HT

suppositories.

Model AMT HT AS HT

R2 Slope intercept R2 Slope intercept

First-order 0.6869 -0.0255 2.0870 0.9930** -0.2533 2.0890

Hixon Crowell 0.9336** 0.0458 0.0145 0.9748 0.4629 0.1072

Higuchi 0.9804 12.4800 8.5010 0.9405 41.5000 7.7850

Korsmeyer

Peppas* 0.9519 1.0200 0.2977 0.6049 1.0610 1.2080

*n = 0.2977 and 1.2080 for AMT HT and AS HT suppositories, respectively. ** the best model to describe the release kinetics

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Prediction of absolute bioavailability using IVIVC model

The F of AS HT and AMT HT suppositories were predicted based on the MDT values using

Eq 6.5. The F (%) values have been summarised in Table 6.3.

Table 6.3 Predicted bioavailability of AS and AMT in the HT suppositories using the

previously developed IVIVC model. Data represented as mean ± SD, n =3.

Amoxicillin form MDT (min) F (%) predicted

AS 104.0 ± 3.5 79.4 ± 1.6

AMT 1636.0 ± 297.2 < 1

The predicted F values of AS were found consistent with the immediate in vitro release of

amoxicillin from AS HT suppositories. The high solubility of AS was expected to facilitate

rapid drug release in vivo despite a small rectal fluid volume (1 - 3 mL). In contrast, the poor

solubility of AMT delayed drug release and absorption. Therefore, AS was considered to be

an appropriate salt form for the rectal administration of amoxicillin via HT suppositories.

Initially, bioavailability was planned to be evaluated using the developed rabbit model

described in Chapter 5. However, a national shortage of rabbits combined with disruptions

in global logistics and supply resulting from the COVID- 19 pandemic have meant that we

have been unable to procure rabbits in New Zealand. Therefore, after 8-months of waiting,

this study utilised the previously developed IVIVC model (Chapter 5, Section 5.3.7) to

predict the F of HT suppositories. The IVIVC model was established with few data points,

therefore, generalisability may be limited. The R2 values for the model were low, though the

correlation was statistically significant. Furthermore, the lack of ideal sink conditions during

the in vitro release study of AMT HT hollow type suppositories as AMT has low aqueous

solubility (mg/mL) could have led to false prediction of low F. It would be of great interest

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149

in future research to investigate the bioavailability of the HT suppositories using the rabbit

model, which would also further validate the IVIVC established in this thesis.

Stability of HT suppositories

Visual evaluation

Upon physical observation of the suppositories after three months, no signs of melting were

noticed in the suppositories stored at 25 °C and 60% RH, whereas the PEG shell of the

suppositories stored at 37 °C had softened. However, they regained their hardness within

minutes when exposed to ambient temperature. A slight colour change was observed at the

top of the AS HT suppositories stored at 25 °C and 60% RH, whereas the inner core showed

no colour change (Figure 6.10). In contrast, the surface of AMT HT suppository core turned

yellowish in colour. However, no colour change was observed inside the core upon breaking.

Figure 6.10 Images showing different degrees of colour change in HT suppositories stored

at 25 °C, 60% RH after three months. (a) AS HT and (b) AMT HT suppositories (c) An image

showing colour change only at the top end of the HT suppositories. Red arrows indicate the

location of colour change in the HT suppositories.

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150

All HT suppositories stored at 37 °C showed colour change at the end of one month. The

colour change and possible hydrolysis of drug present on the outer surface of the core could

be due to the hygroscopic nature of PEG [239, 268].

In addition, heat entrapment within the shell at the time of sealing open end of the

suppositories as a part of manual assembling core and shell, may also have led to degradation

of amoxicillin present on the surface of the core.

Change in hardness - Effect of amoxicillin form

After storage at different conditions for three months, the hardness of both AS HT and AMT

HT suppositories increased, as summarised in Table 6.4.

Table 6.4 Effect of amoxicillin form, AS or AMT on the hardness of HT suppositories

following storage at 25 °C, 60% RH and at 37 °C for three months. Data represented as

mean ± SD; n = 3 HT suppositories.

Amoxicillin form Storage condition Hardness (kg)

Before After

AS HT

25 °C, 60% RH

3.1 ± 0.3

4.4 ± 1.5

37 °C 3.8 ± 0.3

AMT HT

25 °C, 60% RH

3.7 ± 0.4

4.8 ± 1.7

37 °C 3.8 ± 0.4

After storage for three months at 25 °C, 60% RH, an increase in hardness by 1.3 kg and

1.1 kg was observed in AS HT and AMT HT suppositories, respectively. However, only AS

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151

HT suppositories showed an increase in hardness when stored at 37 °C. The change in

hardness was more pronounced due to 60% RH at lower temperature. The increase in

hardness of conventional suppositories is possibly due to rancidity of the lipophilic base

upon storage, a phenomenon that has been reported previously [190].

The presence of hydrates in the formulation have also been reported to affect the mechanical

strength of the pharmaceutical formulation upon storage [273, 274, 276, 277]. It was thought

that hardness over time would not change and thus, would improve with the newly

developed HT suppository formulation, where the PEG shell would protect the hardening

and rancidity of lipophilic SNA 15 core. However, this was not the case and further

improvements in suppository shell preparation and packaging is required to reduce changes

in hardness over time.

Intermolecular interactions by DSC and FTIR

No signs of change in thermal properties of the suppositories were observed (Figure 6.11 a

and b), despite the noticeable colour change over time. Both AS HT and AMT HT

suppository samples showed endothermic peaks corresponding to the melting of PEG shell

and SNA 15 base at 47 °C and 33.7 °C, respectively, consistent with the experimentally

determined values in section 3.3.4. AS HT suppositories stored at 25 °C, 60 % RH showed

no signs of any endothermic peak corresponding to AS. However, an unknown sharp blip at

about 100 °C in the baseline was observed in the thermogram of AS HT suppositories stored

at 37 °C. Similarly, the endothermic peak corresponding to AMT melting changed to a band

of multiple peaks between 135 – 159 °C with a peak at 138 °C, which remained consistent

in the suppositories stored at both the storage conditions.

In line with DSC findings, no intermolecular interactions were observed from the FTIR

spectra (Figures 6.11 c and d). Upon FTIR analysis, the characteristic β - lactam peak

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152

showing C=O stretching was found consistent at 1770 and 1772 cm-1 in AS HT and AMT

HT suppository samples, as observed at the time of preparation. Similarly, the other

characteristic peaks were also found consistent as observed in the freshly prepared HT

suppository samples.

Figure 6.11 Stability of amoxicillin HT suppositories evaluated with DSC (a and b) and

FTIR (c and d). DSC analysis of (a) AS -and (b) AMT HT suppositories. The HT

suppositories were kept at 25 °C, 60% RH and 37 °C. The samples were heated from 0-

250 °C at the rate of 5 °C/min. Intermolecular interaction analysis of (c) AS - and (d) AMT

HT suppositories using FTIR. Dotted line indicates β-lactam peak at 1772 cm-1 .

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153

Content of amoxicillin remaining

At the end of three months, > 98% amoxicillin was found remaining in the HT suppositories

kept at 25 °, 60% RH, indicating stability of the formulation as per the chemical stability

guidelines of United States Pharmacopoeia [278]. Despite the observed colour change in the

HT suppositories stored at 25 °C and 60% RH, an average of 98.3 ± 1.7% and 102.1 ± 1.8%

amoxicillin was found remaining after three months in AS - and AMT HT suppositories,

respectively. This indicated that the PEG shells were successful to improve the stability of

amoxicillin compared with the conventional suppositories, where 91.7 ± 0.2% AS was

remaining after three months storage at 20 ± 0.2 °C.

In contrast, only AMT HT suppositories were found stable with 100 ± 0.8% compared with

79.2 ± 8.8% amoxicillin remaining in AS HT suppositories following storage at 37 °C for

one month.

Other observations

It was observed that PEG 1500 started to lose its firmness around 36-37 °C, despite its

melting point at about 47 °C. As PEG 1500 is a waxy polymer, it starts softening before

complete melting of the base. It melts within a range of 44-49 °C [239]. Long exposure at

37 °C may have produced molecular changes in the PEG chain resulting in softening of PEG

1500 shell [279], which exposed the surface of the drug-containing core to high temperature

and moisture during the stability study at 37 °C. Although the PEG shells softened at 37 °C,

no direct relation of glass transition temperature to structural firmness was observed [280].

This is a limitation of PEG 1500 shells in protecting the drug loaded core. Furthermore,

during content analysis after three months, the time to completely dissolve AMT HT

suppositories was longer than the same for AS HT suppositories. This could lead to delayed

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154

drug release from the suppositories in vivo. This observation was found consistent with the

increased hardness after the storage.

Apart from this, amoxicillin content was found above 98% in both AS HT and AMT HT

suppositories stored at 25 °C, 60% RH, the shells were found unsuitable to prevent colour

change and surface hydrolysis of the drug particles. Therefore, further improvement in

preparation of shells and packaging of the suppositories is required to improve the stability

of the HT suppositories.

6.4 Conclusions

HT suppositories containing different forms of amoxicillin, AMT and AS were developed

by loading them in SNA 15 core which was encapsulated within the PEG shell. The HT

suppositories were found uniform in weight with an average content of 99 ± 2.4 % (AS) and

109.5 ± 1.9% (AMT). Thermal analysis and FTIR spectroscopy indicated no change in

melting behaviour of the suppository bases and absence of intermolecular interaction. The

minimal thickness of PEG shells promoted fast exposure of the drug loaded core to the

release medium. However, only AS HT suppositories showed rapid and complete release

within 7 h resulting in predicted F value of 79.4 ± 1.6%. The PEG shells were found to

protect the amoxicillin loaded core when stored at 25 °C, 60% RH, with > 98% amoxicillin

remaining in both type of HT suppositories after three months. However, the protection of

the PEG shells was not sufficient at 37 °C due to degradation of amoxicillin in both types of

HT suppositories. All these evaluations indicated the potential in development of

amoxicillin HT suppositories using highly soluble AS.

155

General Discussion and Future Directions

156

7.1 General discussion

The rectal route has several advantages for drug delivery, especially in children, but has not

been extensively explored. This non-invasive drug delivery approach with minimal first-

pass metabolism makes the rectal drug delivery systems (RDDS) an alternative to parenteral

and oral formulations. Furthermore, rich lymphatic supply and porto-systemic shunting

enable systemic delivery of the drugs from the rectum, as discussed in Chapter 2. However,

this route has not been explored extensively for drug delivery because of perceived barriers

and patient hesitation due to the unconventional site of administration [1, 61]. RDDS, most

commonly suppositories, can be prepared using low-cost, readily available excipients and

modified by varying the drug - excipient ratio to improve the rectal retention or drug release.

Amoxicillin is used as the first-line antibiotic for the treatment of paediatric pneumonia in

low- and middle-income countries (LMICs). However, only one-third of paediatric

pneumonia cases receive antibiotic treatment [32], despite the availability of several child-

friendly formulations such as dispersible tablets and oral suspensions. This may be because,

these formulations are often unsuitable due to limited stability [281] and possible

swallowing difficulty. Until now, rectal amoxicillin formulations have not been investigated

despite considerable advantages of rectal drug delivery in paediatric patients. Recent

literature suggests a resurgence in exploring drug delivery through the rectum using novel

RDDS that can also enable antibiotic delivery.

This PhD research provided important fundamental understanding in future exploration for

development of an inexpensive, uniform, stable and age-appropriate amoxicillin

suppositories. Both conventional and hollow-type (HT) amoxicillin suppositories were

developed and characterised to understand the bioavailability and mechanism of absorption

157

of amoxicillin in different chemical forms, amoxicillin sodium salt (AS) and amoxicillin

trihydrate (AMT), along with their tissue tolerance following rectal administration.

Two types of suppository bases were studied, hydrophilic polyethylene glycol (PEG) and

lipophilic Suppocire® NA 15 (SNA 15). It was hypothesised that PEG would dissolve in

rectal fluid while SNA 15 would melt in vivo upon administration to release the drug.

Different solubilities of AS (150 mg/mL [44]) and AMT (2.7 mg/mL [45]) were expected

to impose their effect on drug release and absorption due to their different crystallinities. AS

was expected to release and be absorbed rapidly (BCS class I) due to its high solubility

compared with AMT. The key findings of this thesis are discussed below.

Selection of suppository bases: Preformulation studies

In Chapter 3, preformulation studies required for the development and evaluation of

suppositories were carried out. For the preparation of suppositories, PEG 1500, PEG 4000

and SNA 15 base were selected following a literature review [9, 12-15] and thermal

evaluation by differential scanning calorimetry (DSC). The SNA 15 base was selected based

on the low hydroxyl value (5 – 15 mg KOH/g) and ability to melt below body temperature,

30-36 °C [12], while PEG 1500 and 4000 were selected due to their high melting points,

48 °C and 61 °C, respectively, without any noticeable change in the second heating cycle.

To improve the solid-state stability and mechanical strength of the suppository PEG 1500

and PEG 4000 were mixed in a ratio of 70:30 w/w was selected to maintain the physical

stability of the PEG base, which was then used for the determination of mould capacity and

displacement values of AMT and AS.

Along with this, a simple, rapid high-performance liquid chromatography (HPLC) method

was developed and validated, which was then applied for in vitro quantitation of amoxicillin.

The method was found linear (R2 >0.9999), accurate and precise, sensitive and stability-

158

indicting. Amoxicillin has pH-dependent solubility and stability [162]. Upon stability

evaluation within the narrow range of pH 4 - 6, amoxicillin was found most stable at pH 5.0

due to the presence of amoxicillin in its zwitterionic form [161]. This finding was then

further extended to the development of an extraction protocol to quantify amoxicillin from

plasma (Chapter 4). All these preformulation studies were essential to ensure the

reproducible preparation of the suppositories having a uniform distribution of amoxicillin

as well as for rapid and repeatable quantitation of amoxicillin from the suppositories. The

findings and developed analytical process were further employed in the formulation

development and characterisation of conventional amoxicillin suppositories (Chapter 5).

Development of an extraction protocol for in vivo quantitation of amoxicillin

To ensure complete extraction and stability of amoxicillin, solubility and stability evaluation

in the commonly used extraction solvents was carried out in Chapter 4. The addition of a

small volume (50 µL) of 0.1 M acetate buffer pH 5.0 resulted in a five-fold increase in

solubility and a two-fold increase in the stability of amoxicillin [45]. Therefore, a single-

step extraction protocol was developed using the acetate buffer (0.1 M, pH 5.0) - acetonitrile

binary system (1:18) for the extraction of amoxicillin from plasma samples and validated

within the range of 0.2 - 20 µg/mL with extraction recovery >80% for all the samples.

Amoxicillin was found stable for up to 72 h post-extraction reflecting its stability at pH 5.0.

The extraction protocol developed using pH control was then employed to estimate

amoxicillin from plasma samples in the pharmacokinetics (PK) study (Chapter 5).

The criticality associated with the pH-dependent solubility and stability of amoxicillin was

a major challenge. An appropriate understanding of physicochemical properties and pH-

dependent solubility and stability of amoxicillin ensured efficient and reproducible

extraction of amoxicillin from plasma samples.

159

Development and solid-state characterisation of amoxicillin suppositories

Conventional AS suppositories 100 and 200 mg were prepared and evaluated in vitro and

in vivo in Chapter 5. Thorough mixing of drug and suppository base during the preparation

of suppositories ensured uniform distribution of AS in the suppositories as determined by

the weight and content uniformity tests. The solid-state characterisation using powder X-ray

diffraction (XRD) and DSC indicated distribution of AS as anhydrous crystals in SNA 15

whereas it existed as a solid dispersion in PEG suppositories, which did not interfere in the

melting behaviour of the PEG base (47 °C) and SNA 15 base (30-36 °C). This was further

confirmed by the absence of intermolecular interactions determined by Fourier transform

infrared (FTIR) spectroscopy. Similarly, scanning electron microscopy coupled with energy

disruptive x-ray analysis also suggested the distribution of AS as a particulate suspension in

both types of suppository bases, evidenced by the detection of sulfur (S) element.

The major challenge during the preparation of the suppositories was to ensure uniform drug

distribution in the suppository bases to avoid sedimentation of AS at the tip of the

suppository mould. Preformulation studies of suppository bases helped identify an

appropriate temperature to pour the drug-base mixture in the suppository mould near their

congelation point to avoid sedimentation of the drug at the bottom of the suppository mould.

In vitro – in vivo evaluation of amoxicillin suppositories: Effects of base and dose

The difference in disintegration time was reflected in the in vitro drug release from both

types of suppositories, n=3 (Figure 5.6) at 100 mg and 200 mg, f2 values of 21.5 and 26.2,

respectively. The slow drug release from PEG suppositories was controlled by erosion,

whereas rapid release from SNA 15 suppositories was due to the increased surface area aided

by melting and spreading of the base. The presence of solid drug particles in both types of

suppositories was also reflected on the mean dissolution time (MDT) with the increase in

160

dose from 100 mg to 200 mg. AS present as solid suspension was found stable in SNA 15

suppositories compared with PEG suppositories with >91% AS remaining after three

months at both dose levels. In contrast, the tendency of PEG to draw moisture from the

surroundings [252] led to the hydrolysis of amoxicillin in PEG suppositories.

To date, there are no reports of rectal bioavailability of amoxicillin. This study showed that

AS was almost completely absorbed from the SNA 15 suppositories at both dose levels with

better tissue tolerance. On the contrary, slow drug release from PEG suppositories showed

incomplete absorption which increased by 1.3 fold (77.3%) with an increase in the dose.

Both suppositories exhibited a double peak phenomenon (Figure 5.7) with significantly

different Cmax1 at 15 min in 100 mg suppositories (p = 0.046) and Cmax2 at 3 h in the 100 mg

and 200 mg suppositories (p = 0.017 and 0.026, respectively), confirming the presence of

drug particles in both suppositories. The linear increase in the area under the curve of both

the suppositories with dose indicated non-saturated transport of AS from the rectum up to

200 mg dose per suppository, which is reportedly the only mode of drug transport in the

rectum [1, 6]. Unlike clinical practice, this PK study only evaluated single dose

administration, therefore, more evidence showing amoxicillin absorption at higher dose

levels and multiple administration at set dose intervals is required. The values of absolute

bioavailability (F) were then successfully correlated with the MDT values (p<0.01) and were

further used in Chapter 6 to predict F of AMT and AS from HT suppositories.

The PK study was a significant challenge in this research. Although other drugs belonging

to the β-lactam category showed promising PK in human trials [59, 223], the absorption of

amoxicillin from the rectum cannot be predicted without experimental determination.

Furthermore, amoxicillin has been reported to be absorbed via carrier-mediated transport

and passive diffusion from the small intestine [227]. However, carried-mediated transport is

reportedly absent in the rectum [1, 6, 61]. Besides this, oral amoxicillin causes severe gastro-

161

intestinal upset in the rabbits, which can cause death [282, 283]. Therefore, only absolute

bioavailability of amoxicillin was determined using IV amoxicillin as reference.

Development of HT amoxicillin suppositories

Based on the stability findings of the conventional suppositories, HT amoxicillin

suppositories were prepared in Chapter 6 to improve the physical stability of the SNA 15

base at a higher temperature. The HT suppository shells prepared with the PEG 1500 or

4000 mixed in any proportion or in the presence of the polymers resulted in cracked and

broken suppository shells because of the low thickness (1 mm) of the suppository shell and

different crystallinity of the PEGs as reported previously [268]. PEG 1500 shells were

selected due to their elasticity and higher breakpoint (Figure 6.4) than PEG 4000 shells.

Orthorhombic AMT crystals and anhydrous AS crystals were observed uniformly

distributed in cores of AMT HT and AS HT suppositories, which was determined by XRD

and DSC evaluation. The highly ordered arrangements of the orthorhombic crystals of AMT

delayed drug release from AMT HT suppositories resulting in F <1% prediction compared

with 79.4% for AS HT suppositories. However, further validation of the IVIVC model is

necessary.

The PEG shells were explicitly evaluated for their efficiency in improving the stability of

the HT suppositories. Both AS HT and AMT HT suppositories showed colour change of the

core (Figure 6.10) with more prominent effects at 37 °C, possibly due to the entrapment of

heat during the process of sealing the open end of the suppositories resulting in hydrolysis

of amoxicillin molecules. Despite having a high melting point of 47 °C, PEG 1500 shells

started losing their integrity when stored at 37 °C, exposing drug loaded core to temperature

and moisture and resulted in the degradation of amoxicillin in the HT suppositories. When

stored at 25 °C, 60% relative humidity, the PEG shells were suitable to protect the drug

162

loaded core of HT suppositories with >98% amoxicillin remaining in both types of

suppositories. However, further modifications in the preparation of shells are necessary to

improve their stability at temperatures greater than 37 °C.

The development of both HT suppository shell and core was challenging, as it involved

many trial and errors during the modification of suppository mould. Different types of

modified moulds and stainless-steel pins were trialled to generate suppository shells with

various thicknesses. A bespoke stainless-steel mould was manufactured in-house according

to the dimensions of the hollow cavity of the shell, which was then utilised for the

preparation of the core. Although the method of preparation of shell and core was

reproducible, it was slow and time-consuming. Therefore, further improvement and

automation in the core preparation process is recommended.

7.2 Future directions

These days, different novel RDDS are being investigated for localised and systemic drug

delivery of different drugs considering the specific advantages of the rectal route [20, 125,

218, 264, 284-287]. However, the translation of lab based research to commercialisation of

the formulation is limited. Despite the potential for systemic drug delivery, the rectal drug

delivery is challenging due to the limited rectal fluid and absence of carrier mediated

transport [1, 7, 61]. BCS class I drugs are the most suitable candidates for rectal drug

delivery, which was observed with the slow drug release and negligible bioavailability of

AMT in HT suppositories. Therefore, suitability of processing techniques and drug-

excipient compatibility is a crucial factor in formulation development, which was explored

in this thesis.

Some potential strategies could be applied in further formulation development of the HT

suppositories to improve their stability and rapid prototyping. This could ultimately lead to

163

commercial translation of the developed formulation. Some of these strategies are briefly

explained in the following sections.

PK study for HT suppositories

This research intended to determine the PK of AMT HT and AS HT suppositories using the

rabbit model developed in Chapter 5. However, this study was unable to be completed

because the rabbits required for this experiment are yet to arrive in New Zealand due to the

disruption in supply chain and logistics due to the COVID – 19 pandemic. This would be a

priority for future research, not only to investigate the bioavailability of the new formulation

but also to further validate the IVIVC model established in Chapter 5

It was expected that the different solubilities of AMT and AS would impact on in vivo release

and absorption of amoxicillin. Although the predicted F showed apparent differences

between the absorption of AMT and AS, PK evaluation in an animal model is necessary to

verify this finding. Furthermore, translation from animals to human could be largely

depended on the animal species.

Modification and development of HT suppository shells to improve drug stability

Alternative water soluble excipients that are stable above 40 °C should be evaluated in future

studies for the preparation of the HT suppository shells. Recent research by Tagami et al.

[287] used highly water soluble polyvinyl alcohol (PVA) filament to prepare suppository

shells. Such approaches have the potential to improve the physical stability of the

suppository shells at higher temperatures, which should be evaluated.

Furthermore, the current HT suppository preparation process involves manual preparation

of shells which is tiring and time consuming. An automated approach in the formulation of

the suppositories such as three-dimensional (3D) printing has been reported to eventually

remove the traditional approach of “one size fits all” [288-291]. Such approaches involving

164

3D printer compatible polymers can enable rapid prototyping using different polymers to

prepare and evaluate the HT suppository shells, provided the formulation parameters and

excipients are compatible with the drug.

Automated process in preparation of the core

The current preparation of core is a manual, tiring, and time-consuming process and also

involves addition of excess materials to compensate the material loss during the

manufacturing process. Therefore, novel vacuum compression technique that is reported to

be efficient, lossless and reproducible [292, 293] should be explored for the preparation of

a core having desired shape, size, and thickness in a short time [292-294]. Thus, inclusion

of automated, cost-effective and time savvy techniques would contribute to faster translation

of laboratory-based research to large scale manufacturing.

Translation of the bioavailability data in humans

Based on the anorectal structural similarities between rabbits and humans [151, 152], a

rabbit model was selected for the PK studies (Chapter 5). Rabbits are convenient, cost-

effective, and easy to handle for lab-scale analysis. However, oral amoxicillin has been

reported to cause severe diarrhoea leading to loss of life in rabbits [282, 283]. Therefore,

absolute bioavailability was estimated using IV amoxicillin solution as a reference. During

the study, rectal administration of amoxicillin suppositories did not produce any adverse

effects in rabbits for up to two weeks.

Given the extensive use of oral amoxicillin in clinical settings, the ultimate goal would be

to evaluate the bioavailability of amoxicillin following suppository administration in

humans. This would give a better understanding of the efficacy and tolerance of amoxicillin

following rectal administration in humans. However, before proceeding to the clinical trials,

165

further formulation development and pre-clinical evaluation, preferably in a large animal

model that can tolerate oral amoxicillin such as dog, sheep or pig [254], are required.

Gut microbiome evaluation

Although amoxicillin is well absorbed via the oral route, it undergoes first-pass metabolism

and prolonged oral treatment with amoxicillin affects resident gut microflora, leading to GIT

disturbances and associated adverse effects [52, 53]. An important avenue for future

research would be a comparison of oral vs rectal amoxicillin on gut microflora, to better

understand the effect on the gut microbiome before and after treatment.

Immune development, resistance to pathogen colonisation and food metabolism are vital

functions of the gut microbiome, which upon disruption, can cause and exacerbate a variety

of diseases [295]. Intravenous antibiotics have been reported to have less effect on the

microbiome than oral antibiotics [296]. It would be an added advantage of the rectal route

if rectal antibiotic administration had similar effects on the gut microbiome as intravenous

administration. This would be an important finding that may interest potential stakeholders

for commercial research that can translate to more widespread clinical application.

Co-delivery of a β-lactamase inhibitor with amoxicillin

Due to the lack of experimental evidence of rectal absorption of amoxicillin, the PK of

amoxicillin containing suppositories were evaluated in Chapter 5. However, given the

extended spectrum of activity of co-administered β-lactamase inhibitors such as clavulanic

acid with amoxicillin, a systematic approach involving preformulation development and in

vitro-in vivo characterisation should be explored for the development of HT suppositories.

Such approaches could extend the applicability of the developed formulation to address

additional clinical needs. Further development of such formulations might be of interest to

potential stakeholders interested in commercialisation.

166

7.3 Thesis conclusions

This PhD research provided the basis for systemic delivery of amoxicillin via the rectum by

comparing the effects of suppository bases and drug forms This is the first in vivo study to

demonstrate the extent and mechanism of rectal absorption of amoxicillin.

This research demonstrated that the formulation development of AS suppositories in the

SNA 15 base exhibited the potential to be extended to the clinical settings via an appropriate

channel of commercialisation, albeit with further improvements.

The key findings of this research are:

1) Following rectal administration, the release and absorption of amoxicillin in

hydrophilic suppositories was controlled by erosion and dissolution of PEG base,

whereas in lipophilic suppositories it was controlled by melting and spreading from

SNA 15 base.

2) Non-saturated transport of amoxicillin through the rectum was observed in rabbits

up to a dose level of 200 mg per suppository (equivalent child dose 24 mg/kg).

3) Rapid and complete absorption of amoxicillin was possible from the lipophilic SNA

15 base aided by the high solubility of AS and melting and spreading of SNA 15

base.

4) The PEG 1500 shell improved stability of amoxicillin at 25 °C, 60% relative

humidity. However, further modification in shell composition is required to improve

the physical stability of the suppositories at higher temperatures (> 37 °C).

167

Appendices

Appendices

168

Appendix 1 Animal ethics approval for PK study

A 1.1 Animal ethics approval letter from the animal ethics committee of The University of

Auckland to carry out PK study of amoxicillin suppositories on rabbits.

Appendices

169

A 1.2 Animal welfare monitoring sheet used for the monitoring of rabbits during PK study

Animal/Species # NZ – White rabbits Study group: Date:

Pre-study Bodyweight: Investigator: _____________AEC #: 002012

Cage #: Animal #:

Date

Day

Time PRE

PAIN/DISTRESS SCORE 0 = not present, 1 = moderately present, 2 = obviously present

Orbital tightening

Cheek flattening

Flattening of cheek

Nostril shape

Whiskers change & position

Ear shape & position

Diarrhoea

Suspected dehydration

Eye wetting/squinting

Sudden aggression

Loud tooth grinding/crying

Tremor

Total pain/distress score1*

OTHER OBSERVATIONS

Rate of breathing

Type of breathing2

Drug administration site3

Catheter site3

Body wt (kg)4

% body wt change4*

FOOD AND WATER

Wt of food (g)

Food intake today (g)

Wt of water bottle (g)

Water consumed today (ml)

EXAMINATION ON DAY OF INTERVENTION

Rectal temperature (oC)5

Excrement Mass (g)

Time

ANY OTHER OBSERVATIONS (include date/time)

Other observations (include date/time):

Signature

Appendices

170

Scoring details: 1 A total pain/distress score of more than 4 requires continual monitoring. A score of greater than 7 persistently for 30 minutes is a humane endpoint. 2 Breathing R = rapid, S = shallow, L = laboured, N = normal 3 Drug administration site check – score 0 = no signs of infection/reaction, 1 = slight redness, 2 = swelling/severe redness or colour change, puss (notify AWO if score = 2) 4 Body weight to be measured immediately before and 24 hours after drug administration, then twice a day during the washout period.

5 Rectal temperature only needs to be determined immediately prior to drug administration

on the days of experiment only.

See https://www.nc3rs.org.uk/sites/default/files/documents/RbtGS%20Manual.pdf for further details

https://www.nc3rs.org.uk/sites/default/files/documents/RbtGS%20Manual.pdf

Appendices

171

Appendix 2 Graphical abstract summarising the finding of Chapter 5

Appendices

172

Appendix 3 Similarity factor (f2) calculation for conventional amoxicillin suppositories

A 3.1 Calculation of f2 value between the drug release from PEG and SNA 15 suppositories

containing AS equivalent to 100 mg amoxicillin.

Time (min) PEG - 1 F(%) PEG - 2 F(%) PEG -3 F(%) Mean SD RSD(%)

0 0.00 0.00 0.00 0.00 0.00 5 2.20 1.60 1.30 1.70 0.46 26.96

15 7.10 5.50 4.50 5.70 1.31 23.01

30 13.50 11.60 9.30 11.47 2.10 18.34

45 22.10 17.20 14.40 17.90 3.90 21.77

60 27.60 21.40 18.70 22.57 4.56 20.22

90 38.90 33.30 30.60 34.27 4.23 12.35

120 50.20 59.40 75.90 61.83 13.02 21.06

150 57.90 71.50 87.10 72.17 14.61 20.25

180 65.00 85.50 96.30 82.27 15.90 19.33

240 76.00 95.10 98.10 89.73 11.99 13.36

300 84.60 99.20 103.80 95.87 10.02 10.46

420 94.80 103.30 104.70 100.93 5.36 5.31

Time (min) SNA 15 – 1 F(%)

SNA 15 – 2 F(%)

SNA 15 – 3 F(%) Mean SD RSD(%)

0 0.00 0.00 0.00 0.00 0.00 5 11.80 11.50 9.50 10.93 1.25 11.44

15 33.50 42.80 39.70 38.67 4.74 12.25

30 64.40 74.30 64.30 67.67 5.74 8.49

45 78.10 87.70 77.80 81.20 5.63 6.93

60 82.60 90.60 87.20 86.80 4.01 4.63

90 90.70 95.20 92.60 92.83 2.26 2.43

120 95.30 97.50 94.10 95.63 1.72 1.80

150 98.90 98.90 95.20 97.67 2.14 2.19

180 96.90 96.10 98.30 97.10 1.11 1.15

240 98.50 97.30 99.80 98.53 1.25 1.27

300 101.50 99.50 101.20 100.73 1.08 1.07

420 102.40 101.20 101.20 101.60 0.69 0.68

Appendices

173

Similarity factor: f2 f2 PEG - 1 PEG - 2 PEG - 3

SNA 15 - 1 21.58 22.33 22.56 SNA 15 - 2 19.23 19.61 19.71 SNA 15 - 3 21.14 21.77 21.88

Overall Statistics

Mean PEG vs Individual SNA 15 Mean PEG vs Mean SNA 15

Mean SE

f2 21.47 0.83 21.45 Is f2 ∈[50,100] between Mean PEG and Mean SNA 15 No

Similarity of PEG and SNA 15 Reject Where, F denotes cumulative release rate (%); f2 - similarity factor; SD - Standard Deviation; SE Standard error; RSD – Relative standard deviation.

Appendices

174

A 3.2 Calculation of f2 value between the drug release from PEG and SNA 15 suppositories

containing AS equivalent to 200 mg amoxicillin.

Time PEG -1 PEG - 2 PEG - 3

(min) F(%) F(%) F(%) Mean SD RSD(%)

0 0.00 0.00 0.00 0.00 0.00 5 2.60 2.90 19.10 8.20 9.44 115.13

15 8.40 5.30 6.60 6.77 1.56 23.01

30 10.60 11.10 10.60 10.77 0.29 2.68

45 14.80 15.30 14.10 14.73 0.60 4.09

60 22.80 23.30 18.90 21.67 2.41 11.12

90 29.90 30.90 29.80 30.20 0.61 2.01

120 39.30 41.20 74.00 51.50 19.51 37.88

150 48.80 49.60 81.30 59.90 18.54 30.95

180 72.60 59.10 86.20 72.63 13.55 18.66

240 81.80 88.00 90.30 86.70 4.40 5.07

300 94.50 93.80 91.90 93.40 1.35 1.44

420 96.80 98.60 96.00 97.13 1.33 1.37

Time SNA 15 - 1 SNA 15 - 2 SNA 15 - 3


0 0.00 0.00 0.00 0.00 0.00 5 7.20 6.90 16.60 10.23 5.52 53.90

15 20.60 21.20 30.00 23.93 5.26 21.99

30 37.20 36.60 46.60 40.13 5.61 13.97

45 52.30 51.30 65.00 56.20 7.64 13.59

60 66.60 60.60 77.60 68.27 8.62 12.63

90 81.00 74.40 87.00 80.80 6.30 7.80

120 96.30 83.90 96.00 92.07 7.07 7.68

150 95.10 88.50 97.80 93.80 4.78 5.10

180 100.70 91.60 100.30 97.53 5.14 5.27

240 105.70 102.30 110.00 106.00 3.86 3.64

300 104.90 100.20 106.70 103.93 3.36 3.23

420 107.70 104.60 110.20 107.50 2.81 2.61

Appendices

175

Similarity factor: f2 f2 PEG - 1 PEG - 2 PEG - 3

SNA 15 - 1 24.35 24.15 29.17 SNA 15 - 2 27.79 27.65 32.31 SNA 15 -3 21.31 21.20 25.05

Overall Statistics

Mean PEG vs Individual SNA 15 Mean PEG vs Mean SNA 15

Mean SE

f2 26.34 2.04 26.21 Is f2 ∈[50,100] between Mean PEG and Mean SNA 15 No

Similarity of PEG and SNA 15 Reject Where, F denotes cumulative release rate (%); f2 - similarity factor; SD - Standard Deviation; SE Standard error; RSD – Relative standard deviation.

Appendices

176

A 3.3 Cumulative drug release (F %) that were used for calculation of similarity factor (f2)

between the drug release from AS containing PEG suppositories prepared at two dose

levels; 100 mg and 200 mg.

Time 100 mg - 1 100 mg - 2 100 mg - 3


0 0.00 0.00 0.00 0.00 0.00 5 2.20 1.60 1.30 1.70 0.46 26.96

15 7.10 5.50 4.50 5.70 1.31 23.01

30 13.50 11.60 9.30 11.47 2.10 18.34

45 22.10 17.20 14.40 17.90 3.90 21.77

60 27.60 21.40 18.70 22.57 4.56 20.22

90 38.90 33.30 30.60 34.27 4.23 12.35

120 50.20 59.40 75.90 61.83 13.02 21.06

150 57.90 71.50 87.10 72.17 14.61 20.25

180 65.00 85.50 96.30 82.27 15.90 19.33

240 76.00 95.10 98.10 89.73 11.99 13.36

300 84.60 99.20 103.80 95.87 10.02 10.46

420 94.80 103.30 104.70 100.93 5.36 5.31

Time 200 mg - 1 200 mg - 2 200 mg - 3


0 0.00 0.00 0.00 0.00 0.00 5 2.70 3.00 19.90 8.53 9.84 115.37

15 8.80 5.50 6.90 7.07 1.66 23.44

30 11.10 11.70 11.10 11.30 0.35 3.07

45 15.50 16.00 14.80 15.43 0.60 3.91

60 23.80 24.40 19.80 22.67 2.50 11.03

90 31.30 32.30 31.10 31.57 0.64 2.04

120 41.10 43.10 77.40 53.87 20.40 37.88

150 51.10 51.90 85.00 62.67 19.35 30.87

180 75.90 61.80 90.20 75.97 14.20 18.69

240 85.50 92.00 94.40 90.63 4.60 5.08

300 98.80 98.10 96.10 97.67 1.40 1.43

420 101.20 103.20 100.40 101.60 1.44 1.42

Appendices

177

Similarity factor: f2 f2 100 mg - 1 100 mg - 2 100 mg - 3

200 mg - 1 56.44 53.09 40.28 200 mg - 2 56.48 50.44 38.83 200 mg - 3 40.50 53.87 60.27

Overall Statistics

Mean 100 mg vs Individual 200 mg Mean 100 mg vs Mean 200 mg

Mean SE

f2 52.72 1.09 66.95 Is f2 ∈[50,100] between Mean 100 mg and Mean 200 mg Yes

Similarity of 100 mg and 200 mg Accept Where, F denotes cumulative release rate (%); f2 - similarity factor; SD - Standard Deviation; SE Standard error; RSD – Relative standard deviation.

Appendices

178

A 3.4 Cumulative drug release (F %) that were used for calculation of similarity factor (f2)

between the drug release from AS containing SNA 15 suppositories prepared at two dose

levels 100 mg and 200 mg.

Time 100 mg - 1 100 mg - 2 100 mg - 3


0 0.00 0.00 0.00 0.00 0.00 5 11.80 11.50 9.50 10.93 1.25 11.44

15 33.50 42.80 39.70 38.67 4.74 12.25

30 64.40 74.30 64.30 67.67 5.74 8.49

45 78.10 87.70 77.80 81.20 5.63 6.93

60 82.60 90.60 87.20 86.80 4.01 4.63

90 90.70 95.20 92.60 92.83 2.26 2.43

120 95.30 97.50 94.10 95.63 1.72 1.80

150 98.90 98.90 95.20 97.67 2.14 2.19

180 96.90 96.10 98.30 97.10 1.11 1.15

240 98.50 97.30 99.80 98.53 1.25 1.27

300 101.50 99.50 101.20 100.73 1.08 1.07

420 102.40 101.20 101.20 101.60 0.69 0.68

Time 200 mg - 1 200 mg - 2 200 mg - 3


0 0.00 0.00 0.00 0.00 0.00 5 6.90 6.60 16.00 9.83 5.34 54.33

15 19.80 20.40 28.80 23.00 5.03 21.88

30 35.80 35.20 44.80 38.60 5.38 13.93

45 50.30 49.40 62.60 54.10 7.37 13.63

60 64.00 58.30 74.60 65.63 8.27 12.60

90 77.90 71.50 83.70 77.70 6.10 7.85

120 92.60 80.70 92.30 88.53 6.79 7.66

150 91.50 85.10 94.10 90.23 4.63 5.13

180 96.90 88.10 96.50 93.83 4.97 5.30

240 101.60 98.40 105.80 101.93 3.71 3.64

300 100.90 96.40 102.60 99.97 3.20 3.20

420 103.60 100.60 106.00 103.40 2.71 2.62

Appendices

179

Similarity factor: f2 f2 100 mg - 1 100 mg - 2 100 mg - 3

200 mg - 1 43.35 36.38 41.80 200 mg - 2 39.70 33.98 38.53 200 mg - 3 54.12 43.93 51.89

Overall Statistics

Mean 100 mg vs Individual 200 mg Mean 100 mg vs Mean 200 mg

Mean SE

f2 42.46 3.72 42.28 Is f2 ∈[50,100] between Mean 100 and Mean 200 No

Similarity of 100 mg and 200 mg Reject Where, F denotes cumulative release rate (%); f2 - similarity factor; SD - Standard Deviation; SE Standard error; RSD – Relative standard deviation.

Appendices

180

Appendix 4 Summary of PK profiles of individual rabbits treated with conventional amoxicillin suppositories.

A 4.1 PK profiles of individual rabbits treated with conventional PEG suppositories prepared at two dose levels 100 mg and 200 mg. The minimum

inhibitory concentration (MIC) of amoxicillin for the treatment of S. Pneumoniae (2 μg/ mL) [38] is shown for reference.

Appendices

181

A 4.2 PK profiles of individual rabbits treated with conventional SNA 15 suppositories prepared at two dose levels 100 mg and 200 mg. The

minimum inhibitory concentration (MIC) of amoxicillin for the treatment of S. Pneumoniae (2 μg/ mL) [38] is shown for reference.

Appendices

182

Appendix 5 Similarity factor (f2) calculation for HT amoxicillin suppositories

A 5.1 Calculation of f2 value between the drug release from AMT HT and AS HT

suppositories containing AMT and AS equivalent to 250 mg amoxicillin, respectively.

Time AS HT - 1 AS HT - 2 AS HT - 3


0 0.00 0.00 0.00 0.00 0.00 0.08 0.30 0.90 0.50 0.57 0.31 53.91

0.25 2.00 4.40 3.20 3.20 1.20 37.50

0.5 7.70 11.00 12.50 10.40 2.46 23.61

0.75 17.70 21.20 23.90 20.93 3.11 14.85

1 26.50 30.90 32.80 30.07 3.23 10.75

1.5 41.50 45.20 47.80 44.83 3.17 7.06

2 56.30 58.30 62.80 59.13 3.33 5.63

2.5 64.40 66.10 71.40 67.30 3.65 5.43

3 72.20 75.40 77.40 75.00 2.62 3.50

4 83.60 87.40 88.20 86.40 2.46 2.84

5 90.50 92.90 92.40 91.93 1.27 1.38

7 96.60 98.50 99.40 98.17 1.43 1.46

9 98.90 98.90 100.60 99.47 0.98 0.99

24 93.80 93.50 96.70 94.67 1.77 1.87

28 93.50 93.10 97.40 94.67 2.38 2.51

31 95.40 95.90 98.30 96.53 1.55 1.61

48 92.40 91.40 105.50 96.43 7.87 8.16

52 94.20 90.00 97.60 93.93 3.81 4.05

60 95.60 93.10 99.00 95.90 2.96 3.09

72 94.50 100.60 98.40 97.83 3.09 3.16 Time AMT HT - 1 AMT HT - 2 AMT HT - 3


0 0.00 0.00 0.00 0.00 0.00 0.08 0.00 0.00 0.00 0.00 0.00 0.25 0.20 0.20 0.30 0.23 0.06 24.74

0.5 0.70 0.70 0.60 0.67 0.06 8.66

0.75 1.50 1.70 1.00 1.40 0.36 25.75

1 2.40 2.70 1.70 2.27 0.51 22.64

1.5 3.70 4.00 3.20 3.63 0.40 11.12

Appendices

183

2 5.40 5.70 4.80 5.30 0.46 8.65

2.5 6.80 7.60 6.60 7.00 0.53 7.56

3 8.70 9.00 8.50 8.73 0.25 2.88

4 11.90 11.20 12.80 11.97 0.80 6.70

5 15.60 15.50 22.10 17.73 3.78 21.33

7 21.20 21.00 33.10 25.10 6.93 27.61

9 27.20 25.50 41.80 31.50 8.96 28.45

24 41.90 54.90 63.00 53.27 10.64 19.98

28 48.10 62.30 69.30 59.90 10.80 18.03

31 54.90 66.80 74.30 65.33 9.78 14.97

48 63.40 71.80 80.90 72.03 8.75 12.15

52 78.90 83.90 87.70 83.50 4.41 5.29

60 84.20 87.80 89.30 87.10 2.62 3.01

72 100.80 100.10 98.90 99.93 0.96 0.96 Similarity factor: f2

f2 AS HT - 1 AS HT - 2 AS HT - 3 AMT HT - 1 17.60 16.97 15.89 AMT HT - 2 18.57 17.86 16.83 AMT HT - 3 20.53 19.69 18.64

Overall Statistics

Mean AS HT vs Individual AMT HT Mean AS HT vs Mean AMT HT

Mean SE

f2 18.07 0.82 18.12 Is f2 ∈[50,100] between Mean AS HT and Mean AMT HT No

Similarity of AS HT and AMT HT Reject Where, F denotes cumulative release rate (%); f2 - similarity factor; SD - Standard Deviation; SE Standard error; RSD – Relative standard deviation.

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