6 ex vivo in vivo studyshodhganga.inflibnet.ac.in/bitstream/10603/10535/15/15_chapter6.pdf ·...

Chapter-6 Ex vivo / In vivo studies

Department of Pharmaceutical Sciences, M. M. University, Mullana 98

66..11 OOBBJJEECCTTIIVVEE

After successful formulation of MDTs by modified effervescent method, the selected

formulation of Cefixime (C4) was subjected to ex vivo and in vivo studies. Objective of this

study includes

a. Ex-Vivo Permeability Study: Study the drug permeability of Cefixime from

Formulation C4 in comparison to pure drug and evaluate the permeability

enhancement of Cefixime from the finalized formulation.

b. In-Vivo Study: Study the In vivo parameter of Cefixime in Formulation C4 in

comparison to pure drug after oral administration in animal and to perform

i) Pharmacodynamic studies: To evaluate the Pharmacodynamic effectiveness

of Cefixime in formulation C4 in comparison to pure drug

ii) Assessment of Pharmacokinetic Parameters: To determine the

pharmacokinetic parameters of Cefixime after oral administration of

formulation C4 in rabbit model.

66..22 MMAATTEERRIIAALLSS

a. Chemicals: Sodium Chloride, Sodium Citrate, Nutrient Agar (Qualikems, New

Delhi, India.), Acetonitrile HPLC grade, Methanol HPLC grade, Ammonium Acetate

HPLC grade (Ranbaxy Fine Chemicals Ltd, India) were purchased from the standard

concern. . All other chemicals used were of analytical grade.

b. Microbial Stain: Proteus Vulgaris (ATCC No. 13315), procured from Department of

Microbiology, M.M. Institute of Medical Science and Research, M.M. University,

Mullana, Haryana INDIA

c. Animals: 12 Albino male rabbits (approximately 1kg weight) obtained from

Departmental Animal House, M.M. College of Pharmacy, M.M. University, Mullana,

Haryana, INDIA and kept under standard laboratory conditions in 12 h light/dark

6. Ex vivo / In vivo studies


cycle at 25±2 °C. Animals were marked with picric acid solution (for easy

identification) and provided with pellet diet (Lipton, India) and water ad libitum.

66..33 EEQQUUIIPPMMEENNTTSS

Digital Balance (Shimadzu, Japan), Vortex shaker (Electro Lab, India), Water bath

incubator shaker (Tanco Pvt. Ltd., India), Vaccum Oven (Q5247, Navyug, India), UV

Spectrophotometer (UV 1800, Shimadzu, Japan), HPLC (LC 10AT SHIMADZU) were used.

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6.4.1 Protocol Approval

Protocol no. MMCP/IEC/10/18 approved by Institutional Animal Ethical Committee

(1355/ac/10/CPCSEA), M.M. College of Pharmacy, M.M. University, Mullana.

6.4.2 Methodology

Ex vivo permeability studies of Cefixime in formulation C4 were carried out using non-

everted gut sac technique. The small intestine of freshly sacrificed male albino rabbit was

removed by cutting across the upper end of the duodenum and the lower end of the ileum and

manually stripping the mesentery. The small intestine was washed out carefully with cold

normal oxygenated saline solution (0.9%w/v NaCl) using a syringe equipped with blunt end.

The clean intestinal tract was prepared into 6 ± 0.2 cm long sacs. Each sac was filled with

1ml of pure drug and formulation C4 (equivalent To 10 mg of Cefixime, suspended in 0.1N

HCl) via a blunt needle and the two sides of the intestine were tied tightly with thread. Each

non-everted intestinal sac was placed in a glass conical flask containing 50 ml physiological

solution (Tyrode solution). The entire system was maintained at 370C±0.50C in a shaking

water bath operating at 50 rpm and aerated with oxygen (10–15 bubble/min) using laboratory

aerator. From outside of the sac 1ml samples were withdrawn for 12 h and replaced with

fresh physiological solution. The samples were analyzed using UV–visible

spectrophotometer (UV 1800, Shimadzu, Japan). Amount released per unit area of sac and



percent drug permeation in the receptor compartment were calculated (Ruan et al., 2006 S ).

Studies were performed in six replicates.

66..44..33 Results and Discussion

Table 6.1 Parameters for Permeability studies by non everted gut sac method

Method Non - Everted Gut Sac method

Medium 50ml physiological Solution (Tyrode Solution)

Sample Taken 1 ml

Dilution Factor 10 times

Conc. Conversion factor (Abs-0.018)/0.049 mcg/ml

Theoretical Drug Tablet Formulation C4 equivalent to 10mg cefixime

Table 6.2 Cumulative % drug Permeated from formulation C4 in comparison to pure drug

Time

(hrs.)

Cumulative % Drug Permeated

Pure Drug Cefixime Formulation (C4)

0.25 7.04±0.9 10.51±1.2

0.5 9.73±1.2 10.72±1.1

1 14.89±2.1 20.92±1.5

1.5 18.05±1.9 35.52±2.1

2 21.07±2.4 37.44±3.2

4 27.86±3.2 53.18±3.6

6 33.92±3.3 69.52±4.3

8 43.22±3.1 80.76±3.5

10 57.99±4.3 99.87±3.8

S.D for six trails, P<0.005



Fig 6.1 Comparison of Cumulative % drug Permeated from formulation and pure drug

Selected formulation of Cefixime MDT (C4) was evaluated for Ex-vivo permeability

studies as per the procedure described in Methodology. Result (Table 6.2, figure 6.1) shows

that only <10% Cefixime was permeated through the intestinal membrane from formulation

as well as pure drug in 30 minutes and permeability increased after 1hours. This may be due

to the initiation of dissolution and absorption of Cefixime through intestinal membrane. The

permeability of Cefixime was linear from pure drug and only 57.99±4.3% drug was

permeated even after 10hours. In formulation C4, more than 53.18±3.6% of the drug was

absorbed within 4 hours (T50% = 3.5hrs and T90% = 9hrs) and found 99.87±3.8% permeation

within 10 hours of study. Thus, in formulation C4, the permeability of Cefixime was

enhanced in comparison to pure drug (T1/2 < 3.5hrs in formulation, T1/2 < 9hrs in pure drug).



66..55 IINN VVIIVVOO PPHHAARRMMAACCOODDYYNNAAMMIICC SSTTUUDDYY


Protocol no. MMCP/IEC/10/18 approved by Institutional Animal Ethical

Committee (1355/ac/10/CPCSEA), M.M. College of Pharmacy, M.M. University,

Mullana.

6.5.2 Methodology

Selection of Animal: - Six Healthy rabbits (1Kg approx.) were selected and subjected to physical examination.

Standard: - Pure Drug (suspension of 2mg equivalent Cefixime).

Test: - Cefixime Formulation C4 (2mg equivalent suspension)

Delivery method: - 1ml suspension was administered orally in each animal with feeding needle.

Sampling: - 0.5ml blood samples were withdrawn from marginal ear vein of animal at

regular time interval and immediately diluted with anticoagulant solution (3.2% w/v

Sodium Citrate). Samples were centrifuged and plasma was stored at -200C until assayed

for antimicrobial activity.

6.5.3 Evaluation: Antimicrobial study

Method: - Disc Plate Agar Diffusion method

Microbial stain: - Proteus Vulgaris (ATCC No. 13315)

Media: - Nutrient Agar Media

6.5.3.1 Reference Curve

Sterilized nutrient agar media (about 15ml) was poured in each sterilized petridish

and allowed to solidify. The microorganisms were inoculated aseptically by spread plate

method. The drug loaded disc (different concentrations of Cefixime loaded in 5mm



diameter disc) were made and placed in nutrient agar plate. The petridish were incubated

for 24 hours at 380C. The resultant growth inhibition zone was measured, and tabulated.

These results were drawn as a curve of the concentration verses zone diameter and range

of linearity was determined.

6.5.3.2 Sample assay

The same method was adopted to assess the antimicrobial activity of the antibiotic

in the animal plasma. Plasma samples were loaded in disc (5mm diameter) and placed on

freshly microbial spreaded nutrient agar plate, and incubated for 24 hours at 380C. The

zone of inhibition was recorded. The concentration of unknown samples was read from

the reference curve.

66..55..44 Result and Discussion

6.5.4.1 Reference Curve

The resultant growth inhibition zone in millimeter was measured by calibrated

scale and reference curve (table 6.3, Fig 6.2) was plotted against zone of inhibition (mm)

versus concentration of Cefixime (mcg/ml).

Table 6.3 Zone of Inhibition (mm) by Cefixime with concentration (mcg/ml)

Conc.

(mcg/ml) Zone of

Inhibition* (mm)

Conc.

(mcg/ml) Zone of

Inhibition* (mm)

2 8.00 12 23.00

4 10.00 14 27.00

6 14.00 16 31.00

8 17.00 18 34.00

10 20.00 20 38.00

* Average of three readings



Fig 6.2 Reference curve of Cefixime (Zone of Inhibition [mm] vs. concentration [mcg/ml])

From the observed reference curve (fig 6.2), linearity was observed over

concentration range of 2-20mcg/ml.


The plasma concentration of the orally administered Cefixime pure drug and

in formulation C4 (2mg equivalent) in animal were determined by zone of inhibition

(mm) as shown in figure 6.3A to 6.3F and shows that the Antimicrobial activity of

formulation C4 (ZOI= 14±1.0 in 2.5hrs.) was found more (table 6.3, fig 6.4) in

comparison to pure drug (ZOI= 11±1.0 in 2.5hrs.).



Fig 6.3 Observed Zone of Inhibition (mm) of Cefixime pure drug and formulation C4



Table 6.4 Observed zone of Inhibition (mm) with time (hrs) from animal study

Time (hrs.) Zone of Inhibition (mm)

Cefixime Formulation (C4)

0 5±0.0 5±0.0

0.75 8±0.5 9±0.5

1.5 10±1.0 12±1.0

2.5 11±1.0 14±1.0

4 9±1.0 12±1.0

6 7±0.5 10±1.0

8 6±0.5 8±0.5

10 5±0.0 6±0.0

mean S.D for six reading

Fig 6.4 Zone of Inhibition (mm) versus time plot of Cefixime pure drug and formulation C4



Table 6.5 Plasma concentration (Cp) with time (hrs) of Cefixime and Formulation (C4)

Time (hrs.) Mean Plasma Concentration (Cp)

Cefixime Formulation (C4)

0 0.00±0.00 0.00±0.00

0.75 7.55±1.26 10.06±1.26

1.5 12.58±2.52 17.61±2.52

2.5 15.09±2.52 22.64±2.52

4 10.06±2.52 17.61±2.52

6 5.03±1.26 12.58±2.52

8 2.52±1.26 7.55±1.26

10 0.00±0.00 2.52±0.00


Fig 6.5 Comparative plot of plasma concentration (Cp) with time (hrs) of Cefixime and

Formulation (C4) from antimicrobial study



Table 6.6 Pharmacokinetic parameters in animal after oral administration of Cefixime and

Formulation (C4)

Pharmacokinetic parameters Cefixime Formulation (C4)

Cmax (mcg/ml) 15.09±2.1 22.64±1.8

AUC0-∞(mcg.h/ml) 78.7±9.2 140.0±10.2

Biological Half Life T1/2 (h) 3.58±0.21 4.17±0.32

Tmax(h) 2.5 2.5

Kel (h-1) 0.1938±0.0221 0.1664±0.0143

The peak plasma concentration (15.09±2.1 mcg/ml and 22.64±1.8mcg/ml) were

found to be achieved in 2.5 hrs in animal from Cefixime in comparison to formulation (C4).

The absorption of Cefixime from formulation (C4) was more compared to the Cefixime pure

drug. Further follow up of the plasma concentration shows that in at least 2.5 hours Cefixime

attained peak plasma concentration in pure drug as well as in formulation.

The elimination half life of Cefixime in the animal was found to be 3.58±0.21 hrs.

and 4.17±0.32 respectively and clearance of Cefixime was found almost same in both cases

(0.1938±0.0221 h-1 and 0.1664±0.0143 h-1).

From Pharmacodynamic study, the antimicrobial activity of Cefixime was found to

be enhanced in formulation (C4) in comparison to the pure drug (>77.89% with Cmax

22.64±1.8mcg/ml in 2.5hrs.)



66..66 IINN VVIIVVOO PPHHAARRMMAACCOOKKIINNEETTIICC SSTTUUDDYY


Protocol no. MMCP/IEC/10/18 approved by Institutional Animal Ethical

Committee (1355/ac/10/CPCSEA), M.M. College of Pharmacy, M.M. University,

Mullana.

6.6.2 Methodology

Selection of Animal: - Six Healthy albino male rabbits (1kg approx.) were selected and

subjected to physical examination.

Standard: - Pure Drug (suspension of 2mg equivalent Cefixime).

Test: - Cefixime Formulation C4 (2mg equivalent suspension in distilled water)

Delivery method: - 1ml suspension was administered orally in each animal with feeding needle.

Sampling: - 0.5ml blood samples were withdrawn from marginal ear vein of animal at

regular time interval and immediately diluted with anticoagulant solution (3.2% w/v

Sodium Citrate). Samples were centrifuged and plasma was stored at -200C until assayed

for antimicrobial activity.

6.6.3 Evaluation: HPLC Analysis

HPLC specifications

Instrument: - LC 10AT SHIMADZU with Wakosil II C18 250 x 4.6 mm column

Mobile phase: - Acetonitrile, methanol, 0.5% ammonium acetate buffer in ratio 44:16:40 v/v/v (pH 5.54)

Injection Vol.: - 100 µl

Flow rate : - 0.8 ml/min

Detector : - UV detector at 220nm



6.6.3.1 Linear Curve

HPLC method was developed for estimation of Cefixime using HPLC

(SHIMADZU LC 10AT) fitted with Rheodyne injector. The column (Wakosil II C18)

was eluted with the mobile phase (acetonitrile, methanol, 0.5% ammonium acetate

buffer in the ratio of 44:16:40 v/v/v) and retention time of cefixime was found.

Linearity of the HPLC method was determined by mathematical treatment of test

results obtained by analysis of samples with Cefixime concentrations across the

claimed range. Area was plotted graphically as a function of Cefixime concentration.


The plasma samples were treated with 12.5% tricloro-acetic acid (TCA) for

protein precipitation. The samples were eluted with mobile phase from Wakosil II C18

column and monitored in UV detector. Quantification was achieved by measurement

of the peak area and peak plasma concentration was determined from linear plot.

66..66..44 Result and Discussion

6.6.4.1 Linear Curve

Retention time of Cefixime was found to be 3.17 minutes.

Table 6.7 Peak area versus concentration of Cefixime in HPLC analysis

Sr. No. Concentration (mcg/ml) Peak Area*

1 5 67345

2 10 134054

3 15 200780

4 20 278194

5 30 426976

6 40 545821

*average of 3 reading



Fig 6.6 Linearity plot of Cefixime (peak area versus concentration [mcg/ml])

From the observed reference curve (fig 6.6), linearity was observed over

concentration range of 5-50mcg/ml.


Table 6.8 Level of plasma concentration (Cp) of Cefixime (mcg/ml) from rabbit after oral administration of pure drug and formulation (C4)

Time (min) Mean Plasma Concentration (Cp)

Pure Drug Formulation C4

0 0 0 0.75 5.45±0.8 9.98±0.6 1.5 10.67±1.6 15.23±1.8 2.5 14.64±1.9 19.21±2.1 4 11.67±1.4 16.87±1.5 6 6.34±1.2 10.13±1.1 8 2.67±1.1 5.23±0.8 10 1.3±0.7 2.45±0.5




Fig 6.7 Plasma concentration (Cp) of Cefixime versus time plot from animal plasma after oral administration of pure drug and formulation (C4)

Table 6.9 Pharmacokinetic parameter of Formulation C4 in comparison to pure drug after oral administration in rabbits

Pharmacokinetic parameters Pure Drug Formulation C4

Cmax (mcg/ml) 14.64±1.9 19.21±2.1

AUC0-∞(mcg.h/ml) 77.9±8.7 121.3±8.4

Biological Half Life T1/2 (h) 3.41±0.45 3.91±0.62

Tmax(h) 2.5 2.5

Kel (h-1) 0.2033±0.0341 0.1772±0.0114



The peak plasma concentration (Cp) from pure drug was found 14.64±1.9mcg/ml in

2.5 hours (table 6.9) whereas in formulation (C4), Cp was enhanced to 19.21±2.1mcg/ml

indicating more absorption of Cefixime from formulation (C4) was as compared to the

Cefixime pure drug.

From pharmacokinetic study (Fig 6.7, table 6.9), the elimination half life of Cefixime

in the animal was found to be 3.41±0.45 hours and 3.91±0.62 hours from pure drug and

formulation (C4) respectably and clearance of Cefixime was found almost same in both cases

(0.2033±0.0341 h-1 and 0.1772±0.0114 h-1). The AUC0-∞ of formulation was found

121.3±8.4 mcg h/ml in comparison to Cefixime pure drug (77.9±8.7 mcg h/ml), thus

bioavailability of the drug was found to enhanced by more than 50% in formulation (C4) in

comparison to the pure drug (Cmax = 19.21±2.1mcg/ml in 2.5hrs.).

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