formulation and evaluation of enalapril maleate sustained...

International Journal of PHARMACEUTICAL AND BIOMEDICAL

RESEARCH

Research article

Formulation and evaluation of enalapril maleate sustained release matrix tablets

Somnath Sakore*, Bhaswat Chakraborty Cadila Pharmaceuticals Ltd, Research & Development, Survey no. 1389, Trasad Road, Dholka, Ahmedabad- 387 810, Gujarat, India

Received: 06 Dec 2012 / Revised: 30 Dec 2012 / Accepted: 10 Jan 2013 / Online publication: 31 Jan 2013

ABSTRACT

The aim of the present research work was to develop and evaluate the sustained release matrix tablets of enalapril maleate. The tablet was formulated using HPMC KM and HPMC K15 M polymers by wet granulation method. In vitro drug release study was carried out in simulated gastric fluid (0.1 N HCl) for the first 2h and in phosphate buffer (pH 6.8) for the next 3h following USP apparatus II paddle method. An independent model method, Lin Ju and Liaw’s similarity factor (ƒ2) were used to compare various dissolution profiles. In order to describe the enalapril maleate release kinetics from individual tablet formulations, the corresponding dissolution data were fitted in various kinetic dissolution models: zero order, first order, Higuchi, Korsmeyer Peppas and Hixon Crowell. The results indicated that the drug release characteristics from HPMC polymer matrices follow Higuchi square root time kinetics and the mechanism of drug release was both diffusion and erosion.

Key words: Oral dosage form, Controlled drug release, Half-life, HPMC KM, Release kinetics

1. INTRODUCTION

Oral dosage forms has long been the most popular and convenient route of drug delivery. Various types of modified release formulations have been developed to improve the patient compliance and also clinical efficacy of the drug. The sustained release oral dosage forms have been demonstrated to improve therapeutic efficacy by maintaining steady state drug plasma concentration.

Nonionic cellulose ethers and hydroxypropyl methyl cellulose (Hypromellose, HPMC) have been widely studied for their application in oral sustained release formulations [1]. Such hydrophilic polymers are most popular because of their flexibility to get a desirable drug release profile, cost effectiveness and broad regulatory acceptance [2]. HPMC has always been a first choice for formulation of hydrophilic matrix systems, because of providing robust mechanism, choice of viscosity grades, nonionic nature, consistent reproducible release profiles, cost effectiveness and

utilization of existing conventional equipment and methods [3]. HPMC most widely used as the gel forming agent in the formulations of solid, liquid, semisolid and controlled release dosage forms. The adjustment of the polymer concentration, the viscosity grades and the addition of different types and levels of excipients to the HPMC matrix can modify the drug release rates [4].

Drug release from dosage form like tablets capsules, and granules shows complex interaction between mechanisms like wetting, capillary penetration, swelling, disintegration diffusion, dissolution, erosion etc. These processes are mainly depends on type, quantity and properties of the drug and excipients as well as manufacturing processes. Polymer dissolution, erosion in solvent is an important area in drug delivery system, which allows for optimization of design and processing of drug dosage form as well as selection of suitable excipients. The ideal drug delivery system is one which provides the drug only when and where required and in minimum dose is required to give the desired therapeutic.

When the dosage form introduced into the solvent, swelling occur allowing increased mobility of drug and it diffuses out of polymer in the surrounding fluid. Various mathematical models can be applied to the dissolution profile

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Somnath Sakore and Bhaswat Chakraborty, Int J Pharm Biomed Res 2013, 4(1), 21-26

22

to describe the mechanism and kinetics of dissolution process, whereas, it is quite difficult to create mathematical equations due to different dissolution curves shows very different shapes.

The hypertensive patients are more prone to morning surge in blood pressure and hypertensive attacks during morning hours between 5 a.m. to 9 a.m. The development sustained release tablets of enalapril are expected to avoid acute overdose, and to prevent morning hypertension [5]. The other advantages of sustained release dosage forms are patient compliance, reduction of local and systemic side effects, minimization of peaks and valleys in drug blood levels [6].

Enalapril, an orally-active, long-acting, nonsulphydryl angiotensin-converting enzyme (ACE) inhibitor, is extensively hydrolyzed in vivo to enalapril at its bioactive form. Bio activation probably occurs in the liver. Metabolism beyond activation to enalapril is not observed in man. Administration with food does not affect the bioavailability of enalapril; excretion of enalapril and enalapril at is primarily renal. Enalapril reduces blood pressure in hypertensive patients by decreasing systemic vascular resistance. The blood pressure reduction is not accompanied by an increase in heart rate. Furthermore, cardiac output is slightly increased and cardiovascular reflexes are not impaired [7].

In the present work, enalapril maleate sustained release matrix system has been developed using HPMC KM and HPMC K15 M polymers by wet granulation method. In order to compare the dissolution profiles, Lin Ju and Liaw’s similarity factor (ƒ2) was used. In order to describe the release kinetics and the mechanism of drug release, dissolution data were fitted in various kinetic dissolution models: zero order, first order, Higuchi, Korsmeyer Peppas and Hixon Crowell.

2. MATERIALS AND METHODS

2.1. Chemicals and reagents

Enalapril maleate was a gift sample from Cadila Pharmaceuticals, Ahmedabad. All other chemicals used were of analytical reagent grades.

2.2 Preparation of tablets

Different tablets formulations were prepared by wet granulation technique. All powders were passed through 60 mesh. Required quantities of drug and polymers were mixed thoroughly, and sufficient quantity of isopropyl alcohol and methylene dichloride was added slowly as granulating fluid. Dummy granules were added to improve flow property of granules. The granules were passed through 22/44 mesh and dried at room temperature for 12h. Magnesium stearate was added as lubricant. Lactose was used as diluents, Starch paste is used as binder for granules. Finally were subjected to

compression. Prior to compression, the granules were evaluated for several tests. In all formulations, the amount of the active ingredient is equivalent to 20mg of enalapril maleate (Table 1).

Table 1 Formulation of HPMC K4M and K15 M based enalapril maleate sustained release matrices Formulation code

Enalapril maleate (mg)

HPMC K4M (mg)

HPMC K15M (mg)

F1 20 -- 15 F2 20 15 15 F3 20 20 15 F4 20 20 10 F5 20 15 15 F6 20 15 10 F7 20 12.5 12.5 F8 20 15 17.5 F9 20 10 15 F10 20 10 12.5 F11 20 15 20

2.3. Angle of repose

The angle of repose of granules was determined by the funnel method. The accurately weighed granules were taken in funnel. The height of the funnel was adjusted in such a way that the tip of the funnel just touched the apex of the heap of the granules. The granules were allowed to flow from the funnel on the surface. The diameter and height of the heap formed from the granules were measured. The angle of repose was calculated using following formula [8]: Tan Ѳ= h/r ………………………………………. Eqn.(1) Where, “h” is height of the heap and “r” is the radius of the heap of granules.

2.4. Carr’s compressibility index

The Carr’s compressibility Index was calculated from Bulk density and tapped density of the granules. A quantity of 2g of granules from each formulation, filled into a 10mL of measuring cylinder. Initial bulk volume was measured, and cylinder was allowed to tap from the height of 2.5cm. The tapped frequency was 25±2 per min to measure the tapped volume of the granules. The bulk density and tapped density were calculated by using the bulk volume and tapped volume. Carr’s compressibility index was calculated by using following formula [9]: Carr’s compressibility index (%) = [(Tapped density-Bulk density) X100]/Tapped density

……….. Eqn.(2)

2.5. Evaluation of tablets

The weight of tablets was evaluated on 20 tablets using an electronic balance. The flow properties were measured by Carr’s compressibility index, Friability was determined using


23

6 tablets in Roche friability tester at 25rpm. Hardness of the tablets was evaluated using an ERWEKA hardness tester (Erweka GmbH, Germany). The hardness of all the formulation was between 4-6kg/cm2.

2.6. In vitro dissolution studies

In vitro drug release studies from the prepared matrix tablets were conducted using USP type II apparatus at 37°C at 50rpm. Dissolution mediums used were 900mL of 1.0N HCl and phosphate buffer of pH 6.8. The release rates from matrix tablets were conducted in HCl solution (pH 1.2) for 2h and changed to phosphate buffer (pH 6.8) for further time periods. The samples were withdrawn at desired time periods from dissolution media and the same were replaced with fresh dissolution media of respective pH. The samples were analyzed by HPLC. The amounts of drug present in the samples were calculated with the help of appropriate calibration curves constructed from reference standards. Drug dissolved at specified time periods was plotted as percent release versus time curve.

2.7. Dependent-model method (Data analysis)

In order to describe the enalapril maleate release kinetics from individual tablet formulations, the corresponding dissolution data were fitted in various kinetic dissolution models: zero order, first order, Higuchi, Korsmeyer Peppas and Hixon Crowell. When these models are used and analyzed in the preparation, the rate constant obtained from these models is an apparent rate constant. The release of drugs from the matrix tablets can be analysed by release kinetic theories [10-14].

To study the kinetics of drug release from matrix system, the release data were fitted into Zero order as cumulative amount of drug release vs. time (Eqn.3), first order as log cumulative percentage of drug remaining vs. time (Eqn.4), Higuchi model as cumulative percent drug release vs. square root of time (Eqn.5), Hixon-Crowell cube root law as cube root of percent drug remaining vs time (Eqn.6). To describe the release behavior from the polymeric systems, data were fitted according to well known exponential Korsmeyer –Peppas equation as log cumulative percent drug release vs log of time equation (Eqn.7).

(i) Zero order kinetics Qt=K0t……………………………Eqn.(3) Where, Q= Amount of drug release in time t K0 = Zero order rate constant expressed in unit of concentration /time t = Release time (ii) First order kinetics Log Q=Log Q0-kt/2.303…………Eqn.(4) Where,

Q0= is the initial concentration of drug k= is the first order rate constant t =release time (iii) Higuchi kinetics Q=kt1/2………………………...…Eqn.(5) Where, k= Release rate constant t=release time, Hence the release rate is proportional to the reciprocal of the square root of time. (iv) Hixon Crowell cube root law Q 01/3- Q t1/3=K HC t………...……Eqn.(6) Q t= the amount of drug release at time t Q 0= initial amount of drug in tablets K HC= rate constant for Hixon Crowell. (v) Korsmeyer-Peppas First 60% in vitro release data was fitted in equation of Korsmeyer et al. to determine the release behavior from controlled release polymer matrix system. The equation is also called as power law, Mt /M∞ =Kt n …………………… Eqn.(7) Where, Mt = amount of drug released at time t M∞ = amount of drug released after infinite time Mt /M∞ = fraction solute release t = release time K = kinetic constant incorporating structural and geometric characteristics of the polymer system n = diffusional exponent that characterizes the mechanism of the release of traces.

The magnitude of the release exponent “n” indicates the release mechanism (i.e. Fickian diffusion, Non Fickian, supercase II release). For matrix tablets, values of n of near 0.5 indicates Fickian diffusion controlled drug release, and an n value of near 1.0 indicates erosion or relaxational control (case II relaxational release transport, non Fickian, zero order release) [15-16].

Values of n between 0.5 and 1 regarded as an indicator of both diffusion and erosion as overall release mechanism commonly called as anomalous release mechanism [17]. The values of n and k are inversely related. A very high k values may suggest a burst drug release from the matrix [18,19].

2.8. Independent-model method (Data analysis)

The dissolution profile was statistically analyzed by using dissolution similarity factor (f2), which was calculated by the following formula:

ƒ2 = 50 × log {[1 / (1 + (Σ (Rt - Tt) 2) / n)] 1/2 × 100}…Eqn.(8) Where, n=Number of dissolution time points.st that Rt=Reference dissolution value at time t


24

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0 2 4 6

Log % Drug Re

maining

Time (h)

F1

F2

F3

F4

F5

F6

F7

F8

F9

F10

F11

Tt = Test dissolution value at time t In vitro release profile of test formulation was compared

with the desired theoretical dissolution profile. The f2 value between 50-100 ensures sameness and equivalence between two dissolution profiles.

3. RESULTS AND DISCUSSION

3.1. Physical characteristics of granules and tablets

The granules of different formulations were evaluated for angle of repose, Carr’s compressibility index etc (Table 2). The results of Angle of repose and Carr’s compressibility Index (%) ranged from 22-27 and 16-24, respectively. Which showed that granules from all the formulations having good flow property. The hardness and percentage friability ranged from 4-6kg/cm2 and 0.64-0.81% respectively.

Table 2 Physical properties of enalapril maleate granules and tablets Formulation code Friability (%) Angle of repose Carr’s index F1 0.64 22 16 F2 0.72 24 20 F3 0.69 23 18 F4 0.67 25 19 F5 0.71 23 21 F6 0.63 28 24 F7 0.69 27 23 F8 0.66 23 17 F9 0.73 22 16 F10 0.63 25 15 F11 0.62 22 17

3.2. In vitro dissolution studies

Enalapril maleate sustained release tablets were prepared by using HPMC polymers. The release profiles of enalapril maleate sustained release tablets were plotted as Fig.1-5. The release rate of enalapril maleate mainly controlled by the hydration and swelling properties of HPMC which forms a gel layer that controls the water penetration and drug diffusion. The effect of polymer concentration on drug release could be clearly seen from the variation of the dissolution profiles. It was found that drug release from F1 and F2 composed of HPMC single polymer was no longer than 2.5h, and significantly higher drug release rate than other formulation which were prepared by using combination of HPMC K4M and HPMC K15 M. Formulations F3 to F11 shows different release rate profile up to 5h period. Formulation F3 and F8 shows less than 70% drug release in 2h. However other formulation shows more than 80% of drug release within first 2h, which is due to higher amount of drug release retarding polymers. The release rate decreased significantly and the drug release prolonged as the polymer concentration was increased. The release profiles were compared with target release profiles. The release profiles of

F1, F2, F3, F4, F5, F6, F7, and F8 were found to be out of the target release profile during first 1h. The drug release was comparatively slower than the target profile. This might be due to use of higher proportion of HPMCK4M to HPMCK15M polymers. However F9 and F10 showed the proper dissolution profiles which were required for target release profile of enalapril maleate for 5h.

Fig.1. Zero order release plot for enalapril maleate matrix tablets

Fig.2. First order release plot for enalapril maleate matrix tablets

Fig.3. Higuchi release plot for enalapril maleate matrix tablets


25

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

‐1.5 ‐1 ‐0.5 0

Log % Release

Log time

F1

F2

F3

F4

F5

F6

F7

F8

F9

F10

F11

Fig.4. Hixon Crowell release plot for enalapril maleate matrix tablets

Fig.5. Korsmeyer Peppas release plot for enalapril maleate matrix tablets The hydration rate of HPMC depends on the nature of the

substituent like hydroxypropyl group contents. HPMC K4M and HPMC K15M are having viscosity 4000cps and 15000cps respectively, which forms a strong viscous gel in contact with aqueous media, which controls the release rate of enalapril maleate. For formulation F3, F4, F8 containing highest amount of polymer shows more controlled release of drug in both pH 1.2 and distilled water. This may be owing to a more rigid complex formed by presence of higher proportion of HPMC K4M and HPMC K15M which helped in retaining the drug in matrix and did not allow rapid diffusion of drug from matrix.

The rate and amount of drug release were decreased with increasing the amount of HPMC polymers. This polymers ability to retard the drug release rate is depends on its viscosity. The increase in polymer content decreases the total porosity therefore drug release extended for prolonged period because decreased porosity have lower lateral area. The release rate decreased significantly and drug release retarded as the polymer proportion was increased.

The drug release became sustained with increasing HPMC concentration because of poorer wetability, slower hydration and formation of gelatinous layer. Another important factor is

viscosity of the polymers which is higher as the molecular weight polymer increases. If the viscosity of the polymer increases the gel layer viscosity also increases so that the gel layer becomes resistant to diffusion and erosion. The release rate therefore decreases. Different levels of methyl and hydroxypropoxy substitution resulted in intrinsically different hydration rates, which affected the performance of the polymer in the initial stages of tablet hydration.

When glassy polymer comes into contact with water or any other medium with which it is thermodynamically compatible, the solvent penetrates into the free spaces on the surface between macromolecular chains. When enough solvent has entered into the matrix, the glass transition temperature of the polymer drops to the level of the experimental temperature (which is usually 37°C). The presence of solvent in the glassy polymer causes stresses, which are then accommodated by an increase in the radius of the gyration and end to end distance of the polymer molecules. i.e., the polymer chains get solvated. The solvent molecules move into the glassy polymer matrix. The thickness of the swollen or rubbery region increases with time in the opposite direction. This phenomenon is a characteristic for that particular polymer/solvent system.

3.3. Influence of ionic strengths on drug release

The release rate of drug is known to be affected by changing the pH of dissolution medium although HPMC hydration and gel formation is not affected by changes in pH. Bravo et al [20], studied the effect of pH on diclofenac sodium release from HPMC matrices. Study showed the release rate of diclofenac sodium was extremely slow in acidic pH, since after 2h only 1% of drug was released and showed faster drug dissolution rates at pH 6.8.

Various attempts have been made to quantify the influence of the solution containing phosphate and chloride ions at different ionic strengths on dissolution rates from HPMC SR tablets. In this study effect of phosphate buffer on drug release from HPMC matrices have been studied. No significant changes in drug dissolution in buffer compared to water medium observed for enalapril maleate.

3.4. Release kinetics

The best fit with higher correlation coefficient was found with Higuchi model which indicates that the amount of drug release is proportional to the square root of total amount of drug in tablet and solubility of drug in polymer matrix and time. (Fig.3) The rate of release can be altered by increase or decrease in the drug solubility and concentration of drug in matrix system. The drug release mechanism based upon entrance of the surrounding medium into a polymer matrix where it dissolves and leaches out the soluble drug, leaving a shell of polymer and empty pores. Depletion zone moves to the centre of the tablet as the drug released. Since the boundary between the drug matrix and the drug depleted


26

matrix recedes with time and the thickness of the empty matrix through which drug diffuses also increases with time. As the drug passes out of a homogeneous matrix, the boundary of the drug moves to the inside by an infinitesimal distance.

Table 3 Release kinetics of enalapril maleate sustained release tablet formulations Formulation Zero

order First order

Hixon Crowell

Higuchi Korsmeyer Peppas

F1 0.803 0.962 0.549 0.957 0.988 F2 0.633 0.887 0.460 0.992 0.983 F3 0.895 0.979 0.583 0.976 0.994 F4 0.861 0.972 0.550 0.969 0.983 F5 0.835 0.969 0.478 0.969 0.989 F6 0.82 0.953 0.525 0.930 0.834 F7 0.798 0.947 0.583 0.986 0.976 F8 0.900 0.967 0.593 0.974 0.966 F9 0.800 0.964 0.484 0.984 0.995 F10 0.796 0.961 0.478 0.986 0.987 F11 0.842 0.970 0.549 0.989 0.987

To confirm the mechanism of drug release, dissolution

data was fitted into Korsmeyer Peppas equation (Fig.5). All formulations showed good linearity. The correlation coefficient was found to be 0.988-0.994 with diffusional coefficient n values between 0.552-0.839, which indicates both mechanisms as drug diffusion and erosion, so called anomalous diffusion. (Table 3) All formulations showed Mathematical modeling of drug release from dosage form has two major benefits: first, elucidation of the underlying transport mechanism and second the possibility to predict the resulting drug release kinetics as a function of the design of the formulation. Swellable matrix tablets are activated by water, and the drug release is controlled by the interaction among the water, the polymer, and the drug. Due to the water penetration, the gel layer is exposed to continuous changes in its structure and thickness.

Similarity factor f2 analysis is used to ensure the sameness and equivalence in drug release between two profiles. Formulation F9 showed the highest value of f2 i.e. 77.22 which confirms that the release of drug from prepared formulation is similar with desired target release profile.

4. CONCLUSIONS

The sustained release tablets of enalapril maleate were prepared successfully using HPMC polymer of different viscosity. According to in vitro release studies, the release rate was decreased with increasing viscosity and amount of polymer. The results of the study clearly demonstrated that HPMC matrix tablet formulation is an effective and promising drug delivery system for once daily administration of enalapril maleate.

The analysis of the release profiles in the light of distinct kinetic models (zero order, first order, Higuchi, Korsmeyer Peppas and Hixon Crowell) led to the conclusion that, the drug release characteristics from HPMC polymer matrices follows Higuchi square root time kinetics and the mechanism of drug release was both diffusion and erosion.

REFERENCES

[1] Rajabi-Siahboomi, A.R., Jordan, M.P., Eur Phar Rev 2000, 5, 21-23. [2] Lordi, N.G., Sustained release dosage form. In: The Theory and

Practice of Industrial Pharmacy, 3rd Edn., Lea and Febiger, Philadelphia, USA, 1986.

[3] Reddy, K.R., Mutalik, S., Reddy, S., AAPS Pharm Sci Tech 2003, 4, Article 61.

[4] Bravo, S.A., Lamas, O., Salomon, C.J., J Pharm Pharmaceutical Sciences 2002, 5, 213-219.

[5] Hermida, R.C., Ayala, D.E., Mojon, A., Farnandez, J.R., J Am Soc Nephrol 2011, 22, 2313-2321.

[6] Kiattisak, S., Yanee, P., Helmut, V., Siriporn, O., Sci Pharm 2007, 75, 147-163.

[7] Gomez, H.J., Cirillo, V.J., Irvin, J.D., Drugs 1985, 30, 13-24. [8] Cooper, J., Gunn, C., Tutorial Pharmacy. CBS Publishers and

Distributors, New Delhi, India 1986. [9] Martin, A., Physical Pharmacy, Lippincott Williams & Wilkins 2001.

[10] Hixon, A.W., Crowell, J.H., Ind Eng Chem 1931, 23, 923-931. [11] Higuchi. T., J Pharm Sci 1963, 52, 1145-1149. [12] Gibaldi, M., Feldman, S., J Pharm Sci 1967, 56, 1238-1242. [13] Korsmeyer, R.W., Gurny, R., Doelker, E.M., Buri, P., Peppas, N.A., Int

J Pharm 1983, 15, 25-35. [14] Costa, P., Sousa, Lobo, J.M., Eur J Pharm Sci 2001, 13, 123-133. [15] Ford, J.L., Mitchell, K., Rowe, P., Armstrong, D.J., Elliott, P.N.C.,

Rostron, C., Hogan, J.E., Int J Pharm 1991, 71, 95-104. [16] Wadher, K.J., Kakde, R.B., Umekar, M.J., Bri J Pharm Res 2011, 1, 29-

45. [17] Talukdar, M.M., Plaizier-vercammen, J., Drug Dev Ind Pharm 1993,

19, 1037-1046. [18] Marina, L., Ali, R., Rajabi-Siaboomi , J Pharm Sci 2004, 93, 2743-

2754. [19] Bhavani Boddeda, P.V., Kamala Kumari, Chowdary, K.P.R., Int J

Pharm Biomed Res 2012, 3, 44-48. [20] Bravo, S.A., Lamas, M.C., Salomon, C.J., J Pharm Sci 2002, 5, 213-

219.

Sakore, et al. Int J Pharm 2012; 2(3): 598-604 ISSN 2249-1848

www.pharmascholars.com 598

Research Article CODEN: IJPNL6

IN-VITRO AND IN-VIVO CORRELATION OF IMMEDIATE RELEASE ACYCLOVIR TABLET USING

WAGNER NELSON METHOD

Somnath Sakore* and Bhaswat Chakraborthy

Cadila Pharmaceuticals Ltd. Dholka, Ahmedabad, Gujrat. India

*Corresponding author e-mail: [email protected]

ABSTRACT

The purpose of this study was to establish In-Vitro and In-Vivo correlation of immediate release Acyclovir tablets of

800 mg. In vitro and in vivo studies are done on the test product as Acyclovir Tablet USP 800 mg (containing

Acyclovir 800 mg) of Cadila Pharmaceuticals Ltd., India versus Zovirax® Tablet 800 mg (containing Acyclovir 800

mg) of GlaxoSmithKline, USA. In vivo studies are done in 36 healthy, adult, human subjects under fasting

condition.In vitro dissolution study was done using USP apparatus II at 50 rpm in 0.1N HCL for 45 minutes. The in

vitro–in vivo correlation of Acyclovir shows R-squared value 0.9794 in excel work sheet, which depicts a successful

correlation between in vitro and in vivo Characteristic of the drug. In addition, %PE AUC and %PE Cmax was found to

be –4.604 and -11.19 respectively for each formulation. The present study shows a good correlation between in vivo

and in vitro PK profiles of the formulation used as the test drug in the study.

Keywords: Acyclovir 800 mg tablets, In Vitro Dissolution, In Vivo absorption, IVIVC.

INTRODUCTION

In vitro in vivo correlations play a key role in the

drug development and optimization of formulation is

an integral part of manufacturing and marketing

which is certainly a time consuming and expensive

process. In vitro-in vivo correlation (IVIVC)

demonstrates the direct relationships between in

vitro dissolution / release and in vivo absorption

profiles. The in vitro property generally is the rate or

amount of drug dissolution or release, while in vivo

response is plasma drug concentration or amount of

drug absorbed. [1]

The in vitro release data of a dosage form containing

the active substance serve as characteristic in vitro

property, while the in vivo performance is generally

represented by the time course of the plasma

concentration of the active substance. These In vitro

& In vivo data are then treated scientifically to

determine correlations. For oral dosage forms, the in

vitro release is usually measured and considered as

dissolution rate. The relationship between the in vitro

and in vivo characteristics can be expressed

mathematically by a linear or nonlinear correlation.

However, the plasma concentration cannot be directly

correlated to the in vitro release rate; it has to be

converted to the in vivo release or absorption data,

either by pharmacokinetic compartment model

analysis or by linear system analysis. Different

IVIVC model are used as a tool for formulation

development and evaluation of immediate and

extended release dosage forms for setting a

dissolution specification and as a surrogate for

bioequivalence testing. Practically, the purpose of

IVIVC is to use drug dissolution results from two or

more products to predict similarity or dissimilarity of

expected plasma drug concentration (profiles).

Before one considers relating in vitro results to in

vivo, one has to establish as to how one will establish

similarity or dissimilarity of in vivo response i.e.

plasma drug concentration profiles.

As a result, considerable effort goes into their

development and the main outcome is “the ability to

predict, accurately and precisely, expected

bioavailability characteristics for an extended release

(ER) drug product from dissolution profile

characteristics. [2,3]

The methodology of establishing

similarity or dissimilarity of plasma drug

concentrations profile is known as bioequivalence

testing. There are very well established guidances

International Journal of Pharmacy Journal Homepage: http://www.pharmascholars.com



and standards available for establishing

bioequivalence between drug profiles and products [4,

5]. There are four levels of IVIVC that have been

described in the FDA guidance, which include levels

A, B, C, and multiple C [6,7]

.

The concept of correlation level is based upon the

ability of the correlation to reflect the complete

plasma drug level-time profile which will result from

administration of the given dosage form. An IVIVC

Level A correlates the entire in vitro and in vivo

profiles has regulatory relevance. This level of

correlation is the highest category of correlation and

represents a point-to-point relationship between in

vitro dissolution rate and in vivo input rate of the

drug from the dosage form [8]

.

The objective of IVIVC evaluation is to estimate the

magnitude of the error in predicting the in vivo

bioavailability results from in vitro dissolution data.

This objective should guide the choice and

interpretation of evaluation methods. Any appropriate

approach related to this objective may be used for

evaluation of predictabilityPrediction errors are

estimated for Cmax and AUC to determine the validity

of the correlation. Various approaches of are used to

estimate the magnitude of the error in predicting the

in vivo bioavailability results from in vitro

dissolution data [8,9]

.

It can be calculated by Prediction error that is the

error in prediction of in vivo property from in vitro

property of drug product. Depending on the intended

application of an IVIVC and the therapeutic index of

the drug, evaluation of prediction error internally

and/or externally may be appropriate. [5]

Acyclovir is a synthetic purine nucleoside analogue

with in vitro and in vivo inhibitory activity against

herpes simplex virus types 1 (HSV-1), 2 (HSV-2),

and varicella-zoster virus (VZV). The inhibitory

activity of Acyclovir is highly selective due to its

affinity for the enzyme thymidine kinase (TK)

encoded by HSV and VZV. This viral enzyme

converts Acyclovir into Acyclovir monophosphate, a

nucleotide analogue. The monophosphate is further

converted into diphosphate by cellular guanylate

kinase and into triphosphate by a number of cellular

enzymes. In vitro, Acyclovir triphosphate stops

replication of herpes viral DNA. The greater antiviral

activity of Acyclovir against HSV compared to VZV

is due to its more efficient phosphorylation by the

viral TK [10]

. Acyclovir pharmacokinetics has been

extensively investigated during the various phases of

clinical development. Most of the administered drug

is eliminated from the body unchanged, via the

kidneys by glomerular filtration and tubular

secretion. After intravenous dosing of patients with

normal renal function, 8 to 14% of the dose is

recovered in the urine as the metabolite 9-

carboxymethoxymethylguanine.

After oral administration, the bioavailability of

Acyclovir was approximately 20%. The plasma

elimination half life of acyclovir is 2.5 to 3.3 hr and

protein binding is 9%-33% [11]

.

MATERIAL AND METHODS

Materials: All materials used for analysis were of

analytical grade. The test product for In Vivo study

used Acyclovir Tablet USP 800 mg of Cadila

Pharmaceuticals Ltd., India reference product used

was Zovirax® Tablet 800 mg of GlaxoSmithKline.

In vitro Evaluation:The dissolution of Acyclovir

tablets was carried out using USP Type II Dissolution

apparatus for 45 minutes. The dissolution media used

was 900 ml 0.1 N HCL at 37ºC ± 0.5ºC and rotated

at a speed of 50 rpm. Analysis of the withdrawn

samples was carried out using UV spectrophotometer

at the maximum 254 nm against 0.1 N Hydrochloric

acid as a blank.

Dissolution procedure: Parameters of the instrument

were set as mentioned above and the medium was

degas prior to use. 900 ml of Dissolution media was

transferred into each of the six dissolution vessels

and apparatus was operated as per requirement. One

tablet was dropped into each of six different vessels

& dissolution apparatus was started immediately.

After 45 min solution was withdrawn, & fiiltered the

solution through whatmann filter paper No.1; discard

first 2-3 ml of the filtrate. One mL of the filtered

sample was diluted to 100 ml with dissolution

medium and mixed. (Use this solution as sample

preparation)

Procedure for analysis: Absorbance was measured

for the standard preparation and sample preparation

on a suitable spectrophotometer at the maximum

254nm against 0.1 N Hydrochloric acid as a blank.

Percentage of Acyclovir dissolved in 45 minutes in

individual tablets was calculated.

In vivo Absorption study: A randomized, open label,

two-treatment, two-period, two-sequence, two-way

crossover comparative bioavailability study of a

single oral dose of Acyclovir Tablet USP 800 mg

(containing Acyclovir 800 mg) of Cadila

Pharmaceuticals Ltd., India versus Zovirax® Tablet

800 mg (containing Acyclovir 800 mg) of



GlaxoSmithKline, USA in 36) healthy, adult, human

subjects under fasting condition.

Screen Procedure: During screening procedure

Demography data, standard physical examination

with Vital signs, Clinical laboratory tests on blood

and urine samples, Electrocardiogram (ECG) and

Chest X-ray were done.

Study Design; A randomized, open-label, two-

treatment, two-period, two-sequence, two-way

crossover, comparative bioavailability study, during

which subjects were administered a single dose of

test or reference product under fasting condition with

at least 7 days washout period between each

administration.

Sample Size: Minimum of 36 + (04 standby) healthy,

adult, subjects was enrolled to allow dosing in both

periods.

Administration: Single a single oral dose of test

(Acyclovir Tablet USP 800 mg) or reference product

( Zovirax® Tablet 800 mg) products were

administered along with 240 mL of drinking water

after an overnight fasting of at least 10 hours in each

Period.

Bio-analysis of Plasma sample: Samples were

analyzed for the quantification of Acyclovir in

plasma using Liquid Chromatography with Mass

Spectrometry (LCMS) procedures. Data analysis was

carried out using Win-Nonlin software to get the

Cmax, AUC 0-t, AUC 0- and kel.

Statistical Analysis: Statistical analysis was

performed on the pharmacokinetic parameters data

obtain from subjects (Completing both the periods)

using the SAS Statistical Software version 9.1.3

(SAS Institute INDIA Pvt. Ltd).

RESULTS AND DISCUSSION

In Vitro dissolution: The mean percent dissolved is

calculated on the basis of time and it showed that

within the first 15 min, 91.0% of Acyclovir drug and

within the first 20 min, 89 % of Zovirax had

dissolved. It was observed that 96.0 % of acyclovir

and Zovirax dissolved within 45 min.

Percent dissolved versus in vitro dissolution time (in

min) when plotted generates a dissolution profile

curve as shown below in table 1. The amount of drug

dissolved over a period of time for test & reference

formulation is given in the figure 1.

In vivo absorption: The fraction of drug absorbed

was calculated by Wagner Nelson method using

following equation.

0

T

0

A

TA

CdtK

CdtKCT

)X(

)X(

Above equation relates the cumulative amount of

drug absorbed after a certain time to the amount of

drug absorbed. The fraction of drug absorbed

calculated using Wagner Nelson method for test &

reference formulation is summarized in the Table 2.

The following figure 2 and figure 3 summarizes the

fraction of drug absorbed of the Acyclovir molecule

Test and reference respectively at given blood

sampling time points for test drug. It shows that at 1

hr almost 90% of the drug was absorbed for both test

& reference formulation.

Determination of intensity factor : Since the in

vitro dissolution data was available only for one hour

and hence a time scaling factor (i.e., intensity factor)

was calculated using Ratio of time of 90 % absorbed

and 90 % dissolved & Ratio of time of 50 %

absorbed and 50 % dissolved.

In vivo observed data: With the help of time

scaling factor the in vitro data was compared with In

vivo data. Table 3 summarizes fraction of drug

absorbed & percent drug absorbed vs. time data of

Zovirax 800 mg tablets obtained using Wagner

Nelson Method.

Development and evaluation of Level A IVIVC

Model: The in vitro data was taken based on minutes

and the in vivo absorption based on hours. It was

apparent that these two processes occurred over

different time scale. To make the time difference

between in vitro and in vivo data, uniform, a time

scaling factor (intensity factor) was calculated. The

intensity factor, I, obtained was 6.66. Thus by using

this factor we have converted the in vitro time points

to hours in order to match with the in vivo time

points. Time scaled normalization of in vitro and in

vivo data with intensity factor is given in figure 4.

Correlation calculation: The correlation graph

(Figure 5) was plotted as % absorbed verses %

dissolved and there exists an extremely good

correlation of formulation between in vitro and in

vivo data.



Prediction Error: IVIVC model predictability was

determined by the calculating percent Cmax and

AUC prediction errors. Prediction error as %PE,

AUC and %PE Cmax was found to be – 4.604 and -

11.19 respectively for each formulation.

An IVIVC should be evaluated to demonstrate that

predictability of in vivo performance of a drug

product from its in vitro dissolution characteristics is

maintained over a range of in vitro dissolution rates

and manufacturing process.

If in vitro dissolution is shown to be independent of

dissolution conditions such as PH, surfactants,

osmotic pressure, agitation intensity, a set of

dissolution data obtain from one formulation is

correlated with that of in vivo absorption data.

To demonstrate a correlation, fraction absorbed in

vivo should be plotted against fraction dissolved in

vitro. If this relationship becomes linear with a slope

of 1, then curves are super imposable, and there is a

1:1 relationship which is defined as point-to-point or

Level A correlation.

Regression analysis was also performed on Excel

spread sheet with the same in vitro and in vivo data.

R-squared value obtained from graph was 0.9794.

According to FDA guidelines and expertise, statistic

from Level A analysis is r, the correlation coefficient.

It’s square, i.e. R-squared, ranges from 0 to 1 and is a

measure of strength of relationship between fractions

absorbed against fraction dissolved. Often, results

with sufficient large R-squared (e.g., greater than 0.9)

yielded “a successful correlation”.

The in vitro–in vivo correlation of Acyclovir shows

R-squared value 0.9794 in excel work sheet, which

depicts a successful correlation between in vitro and

in vivo Characteristic of the drug.

In a linear correlation, in vitro dissolution and in vivo

input curves may be directly super imposable or may

be made to be super imposable by the use of

appropriate scaling factor (time corrections). Time

scaling factor should be the same for all formulations

and different time scales for each formulation

indicate absence of an IVIVC.

Percent prediction error (PE%) of 10% or less for

Cmax and AUC establishes the predictability of the

IVIVC. In addition, the PE% for each formulation

should not exceed 15%. Here %PEAUC and %PECmax

was found to be –4.604 and -11.19 respectively.

CONCLUSION

The present study shows a good correlation between

in vivo and in vitro PK profiles of the formulation

used as the test drug in the study. The in vitro–in vivo

correlation of Acyclovir shows R-squared

value

0.9794 in excel work sheet, which depicts a

successful correlation between in vitro and in vivo

Characteristic of the drug. In addition, %PE AUC and

%PE Cmax was found to be –4.604 and -11.19

respectively for each formulation. The concept of

correlation level is based upon the ability of the

correlation to reflect the complete plasma drug level-

time profile which will result from administration of

the given dosage form. An IVIVC Level A correlates

the entire in vitro and in vivo profiles has regulatory

relevance. This level of correlation is the highest

category of correlation and represents a point-to-

point relationship between in vitro dissolution rate

and in vivo input rate of the drug from the dosage

form.

TABLE 1: IN VITRO DISSOLUTION DATA OF ACYCLOVIR 800 MG TABLETS (TEST) AND ZOVIRAX 800

MG TABLETS (REFERENCE)

0.1 N HCl

Time (min) Acyclovir 800 mg (test)

Tablets

Zovirax 800mg (Reference)

tablets

0 0 0

10 86 74

15 91 81

20 94 89

30 95 94

45 96 96



TABLE 2: FRACTION OF DRUG ABSORBED FOR TEST & REFERENCE

Fraction of Drug Absorbed

Time (hr) Test Reference

0 0 0

0.333 0.208509 0.283505

0.667 0.600566 0.809927

1 0.902686 0.99656

1.333 1.086002 1.195828

1.667 1.187093 1.321714

2 1.13907 1.327762

2.5 1.173928 1.330732

3 1.257711 1.281141

4 1.137136 1.175827

5 1.067112 1.088926

6 0.956591 0.981582

8 0.886052 0.901321

10 0.851755 0.878418

12 0.835008 0.851137

14 0.841072 0.844374

16 0.852433 0.850673

24 0.876586 0.875232

TABLE 3: Fraction of Drug Absorbed

Time (hr) Fraction drug absorbed % Drug absorbed

0 0 0

0.333 0.283505 28.3505

0.667 0.809927 80.9927

1 0.99656 99.656

1.333 1.195828 119.5828

1.667 1.321714 132.1714

2 1.327762 132.7762

FIGURE 1: FRACTION OF DRUG DISSOLVED VS TIME GRAPH FOR ACYCLOVIR 800 MG TABLETS



% Drug absorbed vs Time Graph

0

20

40

60

80

100

0 0.5 1 1.5 2 2.5 3

Time

% Ab

sorb

ed

% F

FIGURE 2: FRACTION OF DRUG ABSORBED VS TIME GRAPH FOR ACYCLOVIR 800 MG TABLETS

(TEST)

% Drug absorbed vs Time Graph

0

20

40

60

80

100

0 0.5 1 1.5 2 2.5 3

Time

% Ab

sorb

ed

% F

FIGURE 3: FRACTION OF DRUG ABSORBED VS TIME GRAPH FOR ZOVIRAX 800 MG TABLETS

(REFERENCE)

Time scale normalization of IVIVC

0

20

40

60

80

100

0 0.5 1 1.5 2 2.5

Time (hr)

% Dr

ug A

bsor

bed o

r

Diss

olved

In vivo observed In vitro observed In vitro predicted

FIGURE 4: TIME SCALED NORMALIZATION OF IN VITRO AND IN VIVO DATA WITH INTENSITY

FACTOR

FIGURE 5: THE LINEAR REGRESSION PLOT OF % ABSORBED AND % DISSOLVED



REFERENCES

1. Leeson L. J. Drug Information Journal, 1995; 29: 903-915.

2. Gaynor C, Dunne A, Davis J., International Biometric Society,

http://ibc2008abstracts.tibs.org/Other/IBC2008_Paper_ref_466.pdf

3. Modi NB, Lam A, Lindemulder E, Wang B, Gupta SK. Biopharm Drug Dispos, 2000; 21:321-326

4. Uppoor VRS. J Control Rel. 2001; 72:127-132.

5. Chilukuri DM, Sunkara G. Drug Delivery Technology, 2003; 3:4

6. Sirisuth N, Eddington ND, Int. J. Generic Drugs, 2002; Part 3:250-258.

7. Qureshi SA , The Open Drug Delivery Journal, 2010; 4: 38-47.

8. Jaber E. J. Pharm Pharmaceut Sci, 2006; 9 (2): 169-189.

9. Jayaprakasam B, Seeram NP, Nairs MG. Cancer Lett, 2003; 189 (1): 11-6.

10. www.rxlist.com

11. http://www.drugs.com/pdr/ Zovirax Tablets.html.

International Standard Serial Number (ISSN): 2249-6793

37 Full Text Available On www.ijupls.com

International Journal of Universal Pharmacy and Life Sciences 2(1): January-February 2012

IINNTTEERRNNAATTIIOONNAALL JJOOUURRNNAALL OOFF UUNNIIVVEERRSSAALLPPHHAARRMMAACCYY AANNDD LLIIFFEE SSCCIIEENNCCEESS

Research Article……!!!

Received: 29-12-2011; Accepted: 31-12-2011

BIOEQUIVALENCE STUDY OF OMEPRAZOLE CAPSULE: IN VITRO IN VIVO

CORRELATIONSomnath Sakore*, Bhaswat Chakraborty

Cadila Pharmaceuticals Ltd. Dholka, Ahmedabad, Gujarat. India.

Keywords:

Deconvolution, In vitro In vivo Correlation,

Omeprazole capsule, Pharmacokinetics

parameters

For Correspondence:

Somnath Sakore

Cadila Pharmaceuticals Ltd. Dholka, Ahmedabad,

Gujarat, India

E-mail:[email protected]

ABSTRACT

The aim of the present study was to develop In vitro In vivo correlation

model for Omeprazole Capsule. In vivo study was designed as

randomized, open-level, balanced, two-treatment, two-sequence, two-

way cross over, single dose comparative Bioavailability study in

healthy, adult, male, human, Indian subjects under fasting condition.

Various pharmacokinetic parameters as AUC0-t, AUC0- Kel, Tmax,

Cmax were computed using non-compartmental model of Win-Nonlin

Professional softwar. In vitro and In vivo data was compared using

deconvolution method and found to be highly correlative. To

demonstrate the correlation; a plot was generated with the percentage

absorbed in vivo versus the percentage released in vitro at the same

time. The r-squared value obtained by deconvolution method was

0.9789 and correlation coefficient was 0.9894 supports the extremely

significant correlation.

Pharmaceutical Sciences



1. INTRODUCTION

Drug regulatory authorities must ensure that all pharmaceuticals products, including generic drug products, conform to the same standards of quality, efficacy and safety required of innovator drug products. Therefore, regulatory frameworks must be established to prove that the generic drug products are therapeutically equivalent and interchangeable with their associated innovator’s product. Such regulatory frameworks would necessitate the proof of bioequivalence. [1, 2]

In vitro–In vivo Correlation (IVIVC) plays a key role in pharmaceutical development of dosage forms. This tool hastens the drug development process and leads to improve the product quality. It is an integral part of the immediate release as well as modified release dosage forms development process. IVIVC is a tool used in quality control for scale up and post-approval changes e.g. to improve formulations or to change production processes & ultimately to reduce the number of human studies during development of new pharmaceuticals and also to support the biowaivers.[3,4] An in vivo in vitro correlation (IVIVC) is defined as a “predictive mathematical model describing the relationship between an in vitro property of an extended release dosage forms and a relevant in vivo response, e.g. plasma drug concentration or amount of drug absorbed”. The main objective of such a mathematical model is to use data collected in vitro to predict the in vivo response without having to conduct in vivo studies. [5,6]The in vitro release data of a dosage form containing the active substance serve as characteristic in vitro property, while the in vivo performance is generally represented by the time course of the plasma concentration of the active substance. These In vitro & In vivo data are then treated scientifically to determine correlations. For oral dosage forms, the in vitro release is usually measured and considered as dissolution rate. The relationship between the in vitro and in vivocharacteristics can be expressed mathematically by a linear or nonlinear correlation. However, the plasma concentration cannot be directly correlated to the in vitro release rate; it has to be converted to the in vivo release or absorption data, either by pharmacokinetic compartment model analysis or by linear system analysis.There are four levels of IVIVC that have been described in the FDA guidance, which include levels A, B, C, and multiple C. [7,8] Level A correlation reflect the complete plasma drug level-time profile which will result from administration of the given dosage form and has a regulatory relevance .This level of correlation is the highest category of correlation and represents a point-to-point relationship between in vitro dissolution rate and in vivo input rate of the drug from the dosage form.Omeprazole belongs to a new class of anti-secretory compounds, the substituted benzimidazoles, that do not exhibit anti-cholinergic or H2 histamine antagonistic properties, but that suppress gastric acid secretion by specific inhibition of the H+/K+ ATP’ase enzyme system at the secretory surface of the gastric parietal cell. Because this enzyme system is regarded as the acid



(proton) pump within the gastric mucosa, Omeprazole has been characterized as a gastric acid-pump inhibitor, in that it blocks the final step of acid production. This effect is dose-related and leads to inhibition of both basal and stimulated acid secretion irrespective of the stimulus. [9]After oral administration, the onset of the anti-secretory effect of omeprazole occurs within one hour, with the maximum effect occurring within two hours. Inhibition of secretion is about 50% of maximum at 24 hours and the duration of inhibition lasts up to 72 hours. The anti-secretory effect thus lasts far longer than would be expected from the very short (less than one hour) plasma half-life, apparently due to prolonged binding to the parietal H+/K+ ATP’ase enzyme. When the drug is discontinued, secretory activity returns gradually, over 3 to 5 days. The inhibitory effect of omeprazole on acid secretion increases with repeated once-daily dosing, reaching a plateau after four days. Results from numerous studies of the anti-secretory effect of multiple doses of 20 mg and 40 mg of omeprazole in normal volunteers and patients are shown below. The "max" value represents determinations at a time of maximum effect (2-6 hours after dosing), while "min" values are those 24 hours after the last dose of omeprazole.[9, 10]

Omeprazole Delayed-Release Capsules contain an enteric-coated granule formulation of omeprazole (because omeprazole is acid-labile), so that absorption of omeprazole begins only after the granules leave the stomach. Absolute bioavailability (compared to intravenousadministration) is about 30–40% at doses of 20-40 mg, due in large part to pre-systemic metabolism. In healthy subjects the plasma half-life is 0.5–1 hour, and the total body clearance is 500-600 mL/min. Protein binding is approximately 95%. The bioavailability of omeprazole increases slightly upon repeated administration of Delayed-Release Capsules.

2. MATERIALS AND METHODS:

2.1 Materials

All materials used for analysis are of analytical grade. The test product for In vivo study used was Omeprazole Capsules 20 mg (Omeprazole) manufactured by Cadila Pharmaceuticals Ltd, Dholka, India. Reference Product used was Losec Capsules 20 mg (Omeprazole) manufactured by Laboratorio Tau, S.A. Madrid, Spain.

2.2 In vitro Evaluation

The dissolution of Omeprazole Capsule was carried out using USP Type II Dissolution apparatus. Taking 900 ml of Dissolution medium into each of the six different vessels, oneOmeprazole capsule was dropped into each of the Vessels and the apparatus was operated for exactly two hours. At the end of two hours 0.1 N HCL was decanted and 900 ml of pH 6.8 Phosphate buffer was transferred to the vessels at 37ºC ± 0.5ºC and rotated at a speed of 100 rpm. The solution was withdrawn at an interval of 2 min for the first 20 min then at an interval of10 min for 1 hour and sink conditions were maintained by constantly replenishing with the same volume of fresh buffer solution. The sample is withdrawn from the zone midway between



the surface of the medium and top of the rotating basket not less than 1 cm from the vessel wall. The solution is then filtered through the what- man filter paper o.1 discarding the first few mL of the filtrate. Immediately 5 ml of this sample was taken to test tubes containing 1 mL of 0.25 N Sodium hydroxide.

Analysis of Omeprazole was performed using a validated Ultra Performance liquid chromatographic (UPLC) assay.

The sample preparation was injected into the Chromatograph. Then the sample was analyzed using Variable wavelength, UV Detector at 302 nm using the same buffer solution of pH 6.8 blank. The Chromatograms are recorded and the responses for the principle peak are was measured.

2.3 In vivo Absorption

2.3.1 Title:

A randomized, two-treatment, two-period, two-sequence, single-dose, two-way crossover comparative bioavailability study on Omeprazole 20 mg (Containing Omeprazole HCl 20 mg) Capsules (Cadila Pharmaceuticals Ltd, India) with Losec 20 mg (Containing Omeprazole HCl 20 mg) Capsules (Laboratorio Tau, S.A. Madrid, Spain) in 37 healthy, adult, male, human Indian subjects under fasting condition.

2.3.2 Screen Procedure

During screening procedure Demography data, standard physical examination with Vital signs, Clinical laboratory tests on blood and urine samples, Electrocardiogram (ECG) and Chest X-raywere done.

2.3.3 Study Design

The protocol was approved by the “Ethique”- Independent Ethics committee. The study was carried out in accordance with the principles of the Declaration of Helsinki and its amendments(World Medical Association, 2008) and the International Conference on Harmonisation Guideline for Good Clinical Practice (59th WMA General Assembly, Seoul, October 2008)

The study was a randomized, open-level, balanced, two-treatment, two-sequence, two-way cross over, single dose comparative Bioavailability study in healthy, adult, male, human, Indian subjects under fasting condition with at least 7 days wash-out period between each administration. Each subject was given one capsule of either product i.e. test or reference after an overnight fast for 10 hours with 200 ml of drinking water at room temperature. The subjects received each treatment once according to the randomization schedule.



2.3.4 Administration: Single dose of 20 mg capsule of Omeprazole (Test) or Losec 20 mg Capsules (Reference)products was administered along with 200 mL of drinking water after an over night fasting of at least 10 hours in each Period.

2.3.5 Study endpoints:The endpoints was 90% confidence intervals of the ratio of least-squares means of the

Pharmacokinetic parameters AUC0-t, AUC0- and Cmax of the Omeprazole 20 mg Capsules (Test) and Losec 20 mg Capsules (Reference) products formulation for bioequivalence assessment.

2.3.6 Blood Sampling:5 ml blood samples were taken from a cubical vein into heparinized tubes at the following time points: 0 hr prior to administration of dose and at 0.333, 0.667 ,1.0, 1.336, 1.667, 2, 2.333, 2.667, 3.0, 3.5, 4.0, 5.0, 7.0, 9.0, 12.0, 16.0, 24.0 hours volume of blood drawn including 10 mL for screening, 5 mL for post safety assessment and 18 mL of ‘ discarded ’ anti coagulant mixed blood prior to each sampling from 0.0 hours to 24.0 hour samples during both Periods, will not exceed 213.0 mL.

2.3.7 Bio-analysis of Plasma sample:The Plasma sample was then analyzed to find out the Omeprazole in plasma concentrations by a validated UPLC method at Cadila Pharmaceuticals Ltd. After receiving the plasma concentration from the Bio analytical lab we carried out data analysis in Win-Nonlin software to get the Cmax,

AUC 0-t, AUC 0- and Kel.

2.3.8 Statistical AnalysisStatistical analysis was performed on the data obtained from subjects (completing both the periods) using WinNonlin software. Linear regression analysis, regression correlation was performed in NCSS, WinNonlin and Microsoft Excel Sheet.

3. RESULTS AND DISCUSSION

3.1 Confidence IntervalConfidence Interval was calculated for log-transformed Cmax, AUC0-t and AUC0-inf. The confidence interval was expressed as a percentage relative to the LSM of the reference treatments. To be considered as bioequivalent the 90% confidence intervals of these parameters for Omeprazole should be in the interval 80-125% for the log- transformed data of AUC0-t and AUC0-∞. For Cmax confidence intervals should be in the interval of 75-133% for the log –transformed data.The 90% confidence Interval Omeprazole was calculated and it showed that the Cmax having lower limit (%) 76.30 and upper limit 97.40. The Lower limit (%) and the Upper limit (%) of AUC0-t was 89.22 and 117.54 respectively.

42

The lower and upper limit of 90% confidence interval for AUC0respectively. Thus we can say that the drug is bioequivalent as the data are within the ratio of 80125%.

3.2 In vitro Dissolution: The mean % dissolved is calculated on the basis of min, 90.5% of the Omeprazole drug had dissolved. As observed that 90.5% dissolved within the first 18 min, we separated the time points in the increments of 2 min for the first 20 min followed by the increment of 10 min till 60 min. It was observed that 94.7 % of Omeprazole dissolved within 60 min.

Percent dissolved versus in vitrocurve as shown below in Figure 1

Fig. 1. In vitro

The above figure shows that 93% of Omeprazole capsule dissolved in 20 min at pH 6.8.

The measure or units of time for achieve uniformity of time unit, carried out in WinNonlin software.

3.3 In vivo absorption: After Bioanalytical study is over data is worked on by biostatistics department where data analysis is performed in PK/PD/NCA Analysis Wizard of WinNonlin for determination of Cmax, Kel, AUC0-t and AUC0-∞.

The Omeprazole molecule shows Cmax of 381.484 ± 48.737, elimination rate constant (Kel) of

0.564 ± 0.054, AUC0-t around 1306.932 ± 328.105 a

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upper limit of 90% confidence interval for AUC0-∞ was 96.84 and 122.60 respectively. Thus we can say that the drug is bioequivalent as the data are within the ratio of 80

The mean % dissolved is calculated on the basis of time and it showed that within the first 18 min, 90.5% of the Omeprazole drug had dissolved. As observed that 90.5% dissolved within the first 18 min, we separated the time points in the increments of 2 min for the first 20 min followed

10 min till 60 min. It was observed that 94.7 % of Omeprazole dissolved

in vitro dissolution time (in min) plotted generates a dissolution profile 1

In vitro Percent dissolved Vs. Time plot


The measure or units of time for in vitro and in vivo are minutes and hours, respectively. achieve uniformity of time unit, in vitro time unit was converted to hours and deconvolution was carried out in WinNonlin software.

After Bioanalytical study is over data is worked on by biostatistics department where data nalysis is performed in PK/PD/NCA Analysis Wizard of WinNonlin for determination of

∞.


t around 1306.932 ± 328.105 and AUC0- of 1622.962 ± 414.656.

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∞ was 96.84 and 122.60 respectively. Thus we can say that the drug is bioequivalent as the data are within the ratio of 80-

time and it showed that within the first 18 min, 90.5% of the Omeprazole drug had dissolved. As observed that 90.5% dissolved within the first 18 min, we separated the time points in the increments of 2 min for the first 20 min followed

10 min till 60 min. It was observed that 94.7 % of Omeprazole dissolved

dissolution time (in min) plotted generates a dissolution profile


are minutes and hours, respectively. So to time unit was converted to hours and deconvolution was

After Bioanalytical study is over data is worked on by biostatistics department where data nalysis is performed in PK/PD/NCA Analysis Wizard of WinNonlin for determination of


of 1622.962 ± 414.656.



Table 1 summarizes the Pharmacokinetic parameters of the Omeprazole molecule.

Cmax 381.484 ± 48.737Kel 0.564 ± 0.054

AUC0-t 1306.932 ± 328.105

AUC0- 1622.962 ± 414.656

Table 1. Summary of Pharmacokinetic parameters

Mean plasma concentration of the 37 subjects was determined. Mean plasma concentration was plotted against time to produce mean plasma concentration time graph. The lower limit of Quantification (LLQ) was established at 20.17 ng/mL for Omeprazole in Human Plasma.

3.3.1 Mean plasma concentration of test drug In vivo mean plasma concentration data were obtained up to 24 hrs, after administration of Omeprazole 20 mg capsule to 37 healthy human volunteers and was subjected to deconvolution analysis to obtain percentage in vivo absorbed profiles. Mean plasma concentration of test drug shown in Figure 2

Fig.2. Mean Plasma drug Concentration vs. Time

3.4 Development and evaluation of Level A IVIVC Model:A preliminary inspection of the % in vivo absorption time profile indicated that in vivoabsorption of Omeprazole occurred for 24 hrs for some subjects after dose administration. Since the in vitro dissolution data was available only for 1 hr, and in vivo absorption data for 24 hrs, a time scaling factor (i.e., intensity factor) was calculated using the formula:-

I = time for 50% absorption in vivo / time for 50% dissolution in vitro The obtained time scaling factor, I, was then used to compare the in vivo and in vitro data. From the in vivo deconvoluted profile we found that the 50% absorption was between 0.72 and 0.96 hrs. For accurate

Plasma concentration

1.0

10.0

100.0

1000.0

0 4 8 12 16 20 24

Time in Hours

Cp (n

g/ml)



determination of time points of 50% in vivo absorption we considered 0.84 hr, average of 0.72 and 0.96 hrs. In vitro 50% dissolution was observed at 0.32 hr. Thus by applying the above formula an intensity factor (I) = 2.625 was obtained.

Intensity factor was then applied to convert the time difference between in vitro and in vivo data from minutes to hours. In vitro–in vivo concentration was subsequently calculated for each transformed time points.The in vitro % dissolved and in vivo % absorbed data considered for correlation is given in table 2:

In vivo % absorbed

In vitro % dissolved

0 00.1225 0.234360.34226 0.41330.60627 0.571650.78383 0.707780.88136 0.806040.93489 0.890510.96427 0.951470.98039 0.99603

Table 2. In vivo % absorbed vs. In vitro % dissolved

3.4.1 Correlation calculationOn entering in vitro and in vivo data to the NCSS (statistical and power analysis software 2007) a Linear Regression plot section was obtained along with summary output. The output results are shown in Table 3 and the linear regression plot is given in figure 3

Parameters ValueIntercept -0.0522slope 1. 0923R-squared 0.9789correlation 0.9894

Table 3. In vitro–In vivo correlation parameters



Fig.3. The linear regression plot of % absorbed and % dissolved.

3.5 Summary statement:The equation of the straight line relating C1 in vivo absorbed at time t (%) and C2 in vitrodissolved at time t/I (%) was estimated as: C1 in vivo absorbed at time t (%) = (-0.0522) + (1.0923) C2 in vitro dissolved at time t/I (%) using the 9 observations in this dataset. The y-intercept, the estimated value of C1 in vivo absorbed at time t (%) when C2 in vitro dissolved at time t/I (%) was zero, was -0.0522 with a standard error of 0.0424. The slope, the estimated change in C1 in vivo absorbed at time t (%) per unit change in C2 in vitro dissolved at time t/I (%), was 1.0923 with a standard error of 0.0606. The value of R-Squared, the proportion of the variation in C1 in vivo absorbed at time t (%) that can be accounted for by variation in C2 in vitro dissolved at time t/I (%), was 0.9789. The correlation between C1 In vivo absorbed at time t (%) and C2 in vitro dissolved at time t/I (%) was 0.9894.

A significance test that the slope was zero resulted in a t-value of 18.0161. The significance level of this t-test was far less than 0.05. Since 0.00 < 0.05, the hypothesis that the slope was zero is rejected.

The estimated slope was 1.0923. The lower limit of the 95% confidence interval for the slope was 0.9490 and the upper limit was 1.2357. The estimated intercept was -0.0522. The lower limit of 95% confidence interval for the intercept was -0.1524 and the upper limit was 0.0480.

3.6 Calculation on Excel Sheet:On inputting the in vitro and in vivo data to the Excel sheet, a Linear Regression plot was obtained along with the summary output. The output results shows in Table 4:

Regression StatisticsCorrelation Coefficient 0.9893R-square 0.9788

Table 4. Regression summary

-0.2

0.2

0.5

0.9

1.2

0.0 0.3 0.5 0.8 1.0

C1 In vivo absorbed at time t (%) vs C2 Invitro dissolved at time t/I (%)

C2 Invitro dissolved at time t/I (%)

C1

In v

ivo

abso

rbed

at t

ime

t (%

)



The results obtained from excel spread sheet were reconfirmed using NCSS module/tool of Win stat software. Results obtained from both the methods were given in the following table 5:

Software Used R-squared Correlation CoefficientNCSS 0.9789 0.9894

Excel spread sheet 0.9788 0.9893

Table 5 Correlation of NCSS and Excel spread sheet results

In a linear correlation, in vitro dissolution and in vivo input curves may be directly super imposable or may be made to be super imposable by the use of appropriate scaling factor (time corrections). Time scaling factor should be the same for all formulations and different time scales for each formulation indicate absence of an IVIVC.

In cases where the dissolution rate depends on the experimental factors mentioned above the deconvoluted plasma concentration-time curves constructed following administration of batches of product with different dissolution rates (at least two formulations having significantly different behavior), are correlated with dissolution data obtained under the same dissolution condition. If there is no one-to-one correlation other levels of correlation can be evaluated.

The in vitro dissolution methodology should be able to adequately discriminate between the study formulations. Once a system with most suitable discrimination is developed, dissolution conditions should be the same for all formulations tested in the bio-study for development of the correlation.

During the early stages of correlation development, dissolution conditions may be altered to attempt to develop a one-to-one correlation between the in vitro dissolution profile and the in vivo dissolution profile.

An established correlation is valid only for a specific type of pharmaceutical dosage form (tablets, gelatin capsules, etc.) with a particular release mechanism (matrix, osmotic system, etc.) and particular main excipients and additives. The correlation is true and predictive only if modifications of this dosage form remain within certain limits, consistent with the release mechanism and excipients involved in it.

Extrapolation of IVIVC established in healthy subjects to patients has to be taken into account. Drugs are often taken just before, with or after meal. All these factors may increase variability. A posterior correlation might be established using the patients' data only to increase the knowledge of the drug.

The release rates, as measured by percent dissolved, for each formulation studied, should differ adequately (e.g., by 10%).



4. CONCLUSIONThe present study shows a good correlation between in vivo and in vitro PK profiles of the formulation used as the test drug in the study. In vitro and In vivo data was compared using deconvolution method and found to be highly correlative.

The r-squared value obtained by deconvolution method was 0.9789 the correlation coefficient value obtained was 0.9894 supports the extremely significant correlation.

In vitro-In vivo Correlation is scientifically accurate tool to predict and to design release pattern of a formulation in vivo. These can be utilized in designing of a generic formulation in order to make it bio-equivalent to the innovator. This implies that it can be effectively utilized for the formulation development of a bio-batch of generic formulation.

The established IVIVC was of level ‘A’ which confirms the accuracy of this in vitro model in simulating in vivo concentration. This type of correlation is important since it represents point-to-point relationship between in vitro dissolution and in vivo input rate of the drug from the dosage form. It was shown to be sensitive, effective and reliable in predicting in vivo performance of the drug formulation.

ACKNOWLEDGMENTWe gratefully acknowledge Mrs. Sangita Sakore & Mrs. Darshini Desai for their valuable inputsduring manuscript preparation.

REFERENCES

1. Leeson, L.J., Drug Information Journal 1995, 29, 903-915.2. Modi, N.B., Lam, A., Lindemulder, E., Wang, B., Gupta, S.K., Biopharm Drug Dispos

2001, 21,321-326. 3. Sirisuth, N., Eddington, D.N., Int J Generic Drugs 1999, 3(6).4. Chilukuri, D.M., Sunkara, G., Drug Delivery Technology 2003,3(4).5. http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulations6. Uppoor, V.R.S., J Control Rel 2001, 72,127-132.7. Jaber, E., J Pharm Pharmaceut Sci (www. cspsCanada.org) 2006, 9 (2),169-189.8. Qureshi, S.A., The Open Drug Delivery Journal 2010, 4, 38-47.9. Available from: URL: http://www.micromedex.com10. Available from: URL: http://www.rxlist.com

Research Article Open Access

Sakore and Chakraborty, J Bioequiv Availab 2011, S3http://dx.doi.org/10.4172/jbb.S3-001

Review Article Open Access

Bioequivalence & Bioavailability

J Bioequiv Availab ISSN:0975-0851 JBB, an open access journalBA/BE: LC-MS

Keywords: IVIVC definitions; Predictions; BCS classification; IVIVC Levels; Applications, Guidance

Abbreviations: IVIVC: In Vitro In Vivo correlation; FDA: Food and Drug Administration; AUC: Area Under Curve; MDT vitro: Mean in vitro Dissolution Time; MRT: Mean Residence Time; BCS: Biopharmaceutical Classification System

Introduction In vitro in vivo correlations (IVIVC) play a key role in the drug

development and optimization of formulation which is certainly a time consuming and expensive process. Formulation optimization requires alteration in formulation, composition, equipments, batch sizes and manufacturing process. If such types of one or more changes are applied to the formulation, the in vivo bioequivalence studies in human may required to be done to prove the similarity of the new formulation which will not only increase the burden of carrying out a number of bioequivalence studies but eventually increase the cost of the optimization process and ultimately marketing of the new formulation. To overcome these problems it is desirable to develop in vitro tests that reflect can bioavailability data. IVIVC can be used in the development of new pharmaceuticals to reduce the number of human studies during the formulation development. Thus, the main objective of an IVIVC is to serve as a surrogate for in vivo bioavailability and to support biowaivers.

IVIVC is a mathematical relationship between in vitro properties of a dosage form with its in vivo performance. The In vitro release data of a dosage form containing the active substance serve as characteristic in vitro property, while the In vivo performance is generally represented by the time course of the plasma concentration of the active substance. These In vitro & In vivo data are then treated scientifically to determine correlations. For oral dosage forms, the in vitro release is usually measured and considered as dissolution rate. The relationship between the in vitro and in vivo characteristics can be expressed mathematically by a linear or nonlinear correlation. However, the plasma concentration cannot be directly correlated to the in vitro release rate; it has to be converted to the in vivo release or absorption data, either by pharmacokinetic compartment model analysis or by linear system analysis [1].

IVIVC definitions

United state pharmacopoeia (USP) definition of IVIVC

The establishment of a rational relationship between a biological property, or a parameter derived from a biological property produced by a dosage form, and a physicochemical property or characteristic of the same dosage form [2].

Food and drug administration (FDA) definition of IVIVC

An In-vitro in-vivo correlation (IVIVC) has been defined by the Food and Drug Administration (FDA) as “a predictive mathematical model describing the relationship between an in-vitro property of a dosage form and an in-vivo response”.

Generally, the In vitro property is the rate or extent of drug dissolution or release while the In vivo response is the plasma drug concentration or amount of drug absorbed. Practically, the purpose of IVIVC is to use drug dissolution results from two or more products to predict similarity or dissimilarity of expected plasma drug concentration (profiles). Before one considers relating in vitro results to in vivo, one has to establish as to how one will establish similarity or dissimilarity of in vivo response i.e. plasma drug concentration profiles. The methodology of establishing similarity or dissimilarity of plasma drug concentrations profile is commonly known as bioequivalence testing. There are very well established guidances and standards available for establishing bioequivalence between drug profiles and products [3].

*Corresponding author: Somnath Sakore, Cadila Pharmaceuticals Ltd, Research & Development, 1389, Trasad Road, Dholka, Ahmedabad 387810, Gujarat, India, Tel: +91 9879112032; E-mail: [email protected]

Received October 06, 2011; Accepted December 31, 2011; Published January 02, 2012

Citation: Sakore S, Chakraborty B (2011) In Vitro–In Vivo Correlation (IVIVC): A Strategic Tool in Drug Development. J Bioequiv Availab S3. doi:10.4172/jbb.S3-001

Copyright: © 2011 Sakore S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

In Vitro–In Vivo Correlation (IVIVC) plays a key role in pharmaceutical development of dosage forms. This tool hastens the drug development process and leads to improve the product quality. It is an integral part of the immediate release as well as modified release dosage forms development process. IVIVC is a tool used in quality control for scale up and post-approval changes e.g. to improve formulations or to change production processes & ultimately to reduce the number of human studies during development of new pharmaceuticals and also to support the biowaivers. This article provides the information on the various guidances, evaluation, validation, BCS application in IVIVC, levels of IVIVC, applications of IVIVC in mapping, novel drug delivery systems and prediction of IVIVC from the dissolution profile characteristics of product.

In Vitro–In Vivo Correlation (IVIVC): A Strategic Tool in Drug DevelopmentSomnath Sakore* and Bhaswat Chakraborty

Cadila Pharmaceuticals Ltd, Research & Development, 1389, Trasad Road, Dholka, Ahmedabad 387810, Gujarat, India

http://dx.doi.org/10.4172/jbb.S3-001



Page 2 of 12


Purpose of IVIVCReduction of regulatory burden

IVIVC can be used as substitute for additional in vivo experiments, under certain conditions.

Optimization of formulation

The optimization of formulations may require changes in the composition, manufacturing process, equipment, and batch sizes. In order to prove the validity of a new formulation, which is bioequivalent with a target formulation, a considerable amount of efforts is required to study bioequivalence (BE) /bioavailability (BA).

Justification for “therapeutic’ product quality

IVIVC is often adequate for justification of therapeutically meaningful release specifications of the formulation.

Scale up post approval changes (Time and cost saving during the product development)

Validated IVIVC is also serves as justification for a biowaivers in filings of a Level 3 (or Type II in Europe) variation, either during scale-up or post approval, as well as for line extensions (e.g., different dosage strengths).

IVIVC as surrogate for in vivo bioequivalence and to support biowaivers (Time and cost saving)

The main purpose of an IVIVC model to utilize in vitro dissolution profiles as a surrogate for in vivo bioequivalence and to support biowaivers.

Levels of IvivcThere are four levels of IVIVC that have been described in the FDA

guidance, which include levels A, B, C, and multiple C [4]. The concept of correlation level is based upon the ability of the correlation to reflect the complete plasma drug level-time profile which will result from administration of the given dosage form.

Level A correlation

An IVIVC that correlates the entire in vitro and in vivo profiles has regulatory relevance and is called a Level A Correlation .This level of correlation is the highest category of correlation and represents a point-to-point relationship between in vitro dissolution rate and in vivo input rate of the drug from the dosage form [3,5].

Level A correlation is the most preferred to achieve; since it allows bio waiver for changes in manufacturing site, raw material suppliers, and minor changes in formulation. The purpose of Level A correlation is to define a direct relationship between in vivo data such that measurement of in vitro dissolution rate alone is sufficient to determine the biopharmaceutical rate of the dosage form.

Level B correlation

A level B IVIVC is based on the principles of statistical moment analysis. In this level of correlation, the mean in vitro dissolution time (MDT vitro) of the product is compared to either mean in vivo residence time (MRT) or the mean in vivo dissolution time (MDTvivo). MRT, MDTvitro and MDTvivo will be defined throughout the manuscript where appropriate [6]. A level B correlation does not uniquely reflect the actual in vivo plasma level curves, also in vitro data from such a

correlation could not be used to justify the extremes of quality control standards hence it is least useful for regulatory purposes [5].

Level C correlation

Level C correlation relates one dissolution time point (t50%, t90%, etc.) to one mean pharmacokinetic parameter such as AUC, tmax or Cmax. This is the weakest level of correlation as partial relationship between absorption and dissolution is established since it does not reflect the complete shape of plasma drug concentration time curve, which is the critical factor that defines the performance of a drug product.

Due to its obvious limitations, the usefulness of a Level C correlation is limited in predicting in vivo drug performance. In the early stages of formulation development Level C correlations can be useful when pilot formulations are being selected while waiver of an in vivo bioequivalance study (biowaiver) is generally not possible [5,6].

Multiple level C correlations

This level refers to the relationship between one or more pharmacokinetic parameters of interest (Cmax, AUC, or any other suitable parameters) and amount of drug dissolved at several time point of dissolution profile. Multiple point level C correlation may be used to justify a biowaivers provided that the correlation has been established over the entire dissolution profile with one or more pharmacokinetic parameters of interest. A multiple Level C correlation should be based on at least three dissolution time points covering the early, middle, and late stages of the dissolution profile. The development of a level A correlation is also likely, when multiple level C correlation is achieved at each time point at the same parameter such that the effect on the in vivo performance of any change in dissolution can be assessed [5,6].

Level D correlation

It is not a formal correlation but it is a semi quantitative (qualitative analysis) and rank order correlation and is not considered useful for regulatory purpose but can be serves as an aid in the development of a formulation or processing procedure [5,7] (Table 1).

IVIVC Models The relationship of observed drug concentration-time profiles

following administration of a tablet/capsule with drug dissolution and pharmacokinetics may be described graphically as shown in Figure 1.

It is generally assumed that absorption and dissolution have a

Level In vitro In vivo

A Dissolution curve Input (absorption) curves

B Statistical moments: meandissolution time (MDT)

Statistical moments: meanresidence time (MRT), mean absorption time (MAT), etc

C

Disintegration time, time to have 10%, 50%, 90% dissolved, dissolution rate,dissolution efficiency (DE)

Maximum observed concentration (Cmax), observed at time (Tmax), absorption constant (Ka), Time to have 10, 50, 90% absorbed, AUC(total or cumulative)

A: one-to-one relationship between in vitro and in vivo data, e.g., in vitro dissolution vs. in vivo absorptionB: correlation based on statistical moments, e.g., in vitro MDT vs. in vivo MRT or MATC: point-to-point relationship between a dissolution and a pharmacokinetic parameter, e.g., in vitro T50% vs. in vivo T max, Multiple C: relationship between one or several PK parameters and amount dissolved at several time points.

Table 1: Various parameters used in IVIVC depending on the level.



Page 3 of 12


linear relationship hence dissolution and absorption characteristics of a drug are commonly shown interchangeably. Thus from Figure 2, it is to be noted that one should be able to establish drug profiles with dissolution profiles combined with the pharmacokinetic characteristics of the drug as describe in the example above. This process of obtaining a drug profile from dissolution results is known as convolution. The opposite of this, i.e., obtaining or extracting a dissolution profile from a blood profile, is known as deconvolution Figure 2.

Convolution model

In the development of convolution model the drug concentration-time profiles obtained from dissolution results may be evaluated using criteria for in vivo bioavailability/ bioequivalence assessment, based on Cmax and AUC parameters.

In mathematical terminology, dissolution results become an input function and plasma concentrations (e.g. from IV) become a weighting factor or function resulting in an output function representing plasma concentrations for the solid oral product.

Implementation of convolution-based method involves the production of a user-written subroutine for the NONMEM

software

package, has shown that a convolution-based method based on that of O’Hara et al. [9] produces superior results. Using the NONMEM package, a nonlinear mixed effects model can be fitted to the data with a time-scale model linking the in vitro and in vivo components [10].

It has been demonstrates that the convolution based and differential equation based models can be mathematically equivalent [11]. Software has been developed which implements a differential equation based approach. This method utilises existing NONMEM libraries and is an accurate method of modeling which is far more straightforward for users to implement. This research shows that, when the system being modeled is linear, the use of differential equations will produce results that are practically identical to those obtained from the convolution method.

But is a task that can be time consuming and complex [12]. As a result, this methodology, despite its advantages over the deconvolution-based approach, is not in widespread use.

Mathematically we can write the convolution as:

0( ) ( ) ( ) ( ) ( )

tC t C t F t C F t dδ δ τ τ τ= = −∫ (1)

Where, C(t) = Plasma drug concentrations after oral dose

Cδ(t) = Plasma concentrations after an IV dose or a dose of oral solution

Upon taking the derivative of C(t) with respect to time:

0( ) ( ) ( ) (0) ( )

tC t C t F t C F dδ δ τ τ= + ∫

(2)

When Cδ(0) = 0

( ) * ( )C t F tδ (3)

Advantages of this approach relative to deconvolution-based IVIVC approaches include the following: The relationship between measured quantities (in vitro release and plasma drug concentrations) is modeled directly in a single stage rather than via an indirect two stage approach. The model directly predicts the plasma concentration time course. As a result the modeling focuses on the ability to predict measured quantities (not indirectly calculated quantities such as the cumulative amount absorbed). The results are more readily interpreted in terms of the effect of in vitro release on conventional bioequivalence metrics [5].

Deconvolution model

Deconvolution is a numerical method used to estimate the time course of drug input using a mathematical model based on the convolution integral.

The deconvolution technique requires the comparison of in vivo dissolution profile which can be obtained from the blood profiles with in vitro dissolution profiles. The observed fraction of the drug absorbed is estimated based on the Wagner-Nelson method. IV, IR or oral solution are attempted as the reference. Then, the pharmacokinetic parameters are estimated using a nonlinear regression tool or obtained from literatures reported previously. Based on the IVIVC model, the predicted fraction of the drug absorbed is calculated from the observed fraction of the drug dissolved. It is the most commonly cited and used method in the literature [10]. However this approach is conceptually difficult to use. For example: (1) Extracting in vivo dissolution data from a blood profile often requires elaborate mathematical and computing expertise. Fitting mathematical models are usually subjective in nature, and thus do not provide an unbiased approach in evaluating in vivo dissolution results/profiles. Even when in vivo dissolution curves are obtained there is no parameter available with associated statistical confidence and physiological relevance,

Figure 1: A typical drug concentration (in blood) – time profile reflecting the fate of a drug in the human body following an oral dose (tablet/capsule).

Cmax

Con

cent

ratio

n

Tmax Time

Figure 2: Schematic representation of deconvolution and convolution processes. Convolution is the process of combined effect of dissolution and elimination of drug in the body to reflect blood drug concentration-time profile (right to left). On the other hand, extracting dissolution profiles from blood drug concentration-time profile is known as the deconvolution process (left to right) [8].

Cmax

Con

c.

Tmax TimeBlood drug concentration

Deconvolution

Convolution

CO

NC

.%

DIS

SO

LVE

D

TimeElimination of drug

TimeDissoution profile



Page 4 of 12


which would be used to establish the similarity or dissimilarity of the curves [13]. A more serious limitation of this approach is that it often requires multiple products having potentially different in vivo release characteristics (slow, medium, fast). These products are then used to define experimental conditions (medium, apparatus etc.) for an appropriate dissolution test to reflect their in vivo behavior. This approach is more suited for method/apparatus development as release characteristics of test products are to be known (slow, medium, fast) rather product evaluation [14].

Differential equation based approach

Another approach, has been proposed is based on systems of differential equations [15]. The use of a differential equation based model could also allow for the possibility of accurately modelling non-linear systems and further investigation is being carried out into the case where the drug is eliminated by a nonlinear, saturable process. The convolution and deconvolution methods assume that the system being modelled is linear but, in practice, this is not always the case. Work to date has shown that the convolution-based method is superior, but when presented with nonlinear data even this approach will fail. It is expected that, in the nonlinear case, the use of a differential equation based method would lead to more accurate predictions of plasma concentration.

The incorporation of time-scaling in the PDx-IVIVC equation allows this parameter to be estimated directly from the in vivoand vitro release data. As a result, the predictability of an IVIVC model can be evaluated over the entire in vivo time course.Internal predictability of the IVIVC model was assessed using convolution.PDx-IVIVC Model Equation:

1 2 1 2

0, t 0.( )

( ), t 0.vivovitro

x ta a x b b t

≥= + − + ≥

(4)

For orally administered drugs, IVIVC is expected for highly permeable drugs, or drugs under dissolution rate-limiting conditions, which is supported by the Biopharmaceutical Classification System (BCS) [6,16]. For extended-release formulations following oral administration, modified BCS containing the three classes (high aqueous solubility, low aqueous solubility, and variable solubility) is proposed [17].

IVIVC Development Any well designed and scientifically sound approach would be

acceptable for establishment of an IVIVC. For the development and validation of a IVIVC model, two or three different formulations with different release rates, such as slow, medium, fast should be studied In vitro and In vivo [6].

A number of products with different release rates are usually manufactured by varying the primary rate controlling variable (e.g., the amount of excipient, or a property of the drug substance such as particle size) but within the same qualitative formulation. To develop a discriminative in vitro dissolution method, several method variables together with formulation variables are studied, e.g., different pH values, dissolution apparatuses and agitation speeds. Essentially at this stage a level A correlation is assumed and the formulation strategy is initiated with the objective of achieving the target in vitro profile.Development of a level A IVIVC model includes several steps.

In context of understanding the applications of IVIVR throughout

the product development cycle, it is useful to become familiar with the following terms as they relate to a typical product development cycle for oral extended-release product [5].

An assumed IVIVC is the one that provides the initial guidance and direction for the early formulation development activity. Thus, during step 1 and with a particular desired product, appropriate in vitro targets are established to meet the desired in vivo profile specification. This assumed model can be the subject of revision as prototype formulations are developed and characterized in vivo, with the results often leading to a further cycle of prototype formulation and In vivo characterization.

Out of this product development cycle and In vivo characterization and, of course, extensive in vitro testing is often developed what can be referred to as retrospective IVIVC.

The defined formulation that meets the in vivo specification is employed for Stage 2. At this stage based on a greater understanding and appreciation of defined formulation and its characteristics, a prospective IVIVC is established through a well defined prospective IVIVC study [18,5].

Step 1

In the first step, the In vivo input profile of the drug from different formulations is calculated from drug concentrations in plasma (Figure 3). The target In vivo profile needs to be first established, based on, if possible, pharmacokinetic/pharmacodynamic models. Certainly, step 1 activity should culminate in a pilot PK study. This is typically a four or five-arm cross-over study. The size of this pilot pharmacokinetic study will vary depending on the inherent variability of the drug itself but typically range from 6 to 10 subjects [5]. The results of this pilot PK study provide the basis for establishing what has been referred to as a retrospective IVIVC .To separate drug input from drug distribution and elimination, model-dependent approaches, such as Wagner-Nelson and Loo-Riegelman, or model independent procedures, based on numerical deconvolution, may be utilised [19,20,21]. In step 1, the parameters that describe drug input rate, drug distribution and/or elimination are determined. In the model dependent approaches, the distribution and elimination rate constants describe pharmacokinetics after absorption. In the numerical deconvolution approach, the drug unit impulse response function describes distribution and elimination phases, respectively. The physicochemical characteristics of the drug substance itself, in relevance to formulation approach and dissolution at distal sites in the gastro-intestinal tract, need to be taken into account. Based on this information a priori in vitro methods are usually then developed and a theoretical in vitro target is established, which should achieve the desired absorption profile [5,18].

Step 2

By this phase of the development process, a defined formulation that meets the In vivo targets has been achieved. Extensive In vitro characterization is again performed across pH, media and apparatus, along with the consideration of results of stage 1. This leads to execution of a prospective IVIVC study. The IVIVC is developed and defined after an analysis of the result of that prospective in vivo study. It can often involved further in vitro method development in the context of the observed results, but clearly with the objective of establishing a definitive IVIVC. In this step, the relationship between



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Step1;

Step3;

Step 2: IVIVC

% absorbed

%Dissolved

Prospective

Time (hr)

Time (hr)

% Released

In vitro DissolutionRelease rate:

Release rate:

Release rate:

Release rate:

Fast

Fast

Fast

Fast

Medium

Medium

Medium

Medium

Slow

Slow

Slow

Slow

Assumed IVIVC

Retrospectiv IVIVC Defined formulation

DECONVOLUTION

IVIVC

IVIVC

In vivo absorption

Absorbed

Time

Comcentration

(mg/ml)

VALIDATION

Predicted Plasma Levels

Concentration

(ng/ml)

Time

observed plasma LeveiS

CONVOLUTION

Figure 3: Development of IVIVC with validation.

in vitro dissolution and the in vivo drug input profile is determined (Figure 3). Either a linear or nonlinear relationship may be found. In some cases, time-scaling of in vitro data must be used, because In vitro dissolution and In vivo input may follow the same kinetics but still have different time-scales [6,22]. The time-scaling factor should be the same for all formulations if an IVIVC at level A is sought. During the early stages of correlation development, dissolution conditions may be altered to attempt to develop a 1-to-1 correlation between the in vitro dissolution profile and the In vivo dissolution profile. This work should also result in the definitive in vitro method that has been shown to be correlated with in vivo performance and sensitive to the specific formulation variables.

Step 3

In this phase plasma drug concentration profiles are predicted and compared to the observed time courses for different formulations (Figure 3). To generate predicted time courses, the drug input profile is predicted based on In vitro dissolution data and the In vitro-In

vivo relationship generated in step 2. In the convolution process, the predicted drug input and parameters describing drug distribution and/or elimination phases are combined in order to get predicted time courses. This procedure, which includes steps 1-3, is called two-stage deconvolution. Alternatively, a drug input profile based on in vitro dissolution data can be solved together with parameters describing systemic pharmacokinetics, i.e. distribution and elimination. This approach is called direct convolution.

Different IVIVC model are used as a tool for formulation development and evaluation of immediate and extended release dosage forms for setting a dissolution specification and as a surrogate for bioequivalence testing.

As a result, considerable effort goes into their development and the main outcome is “the ability to predict, accurately and precisely, expected bioavailability characteristics for an extended release (ER) drug product from dissolution profile characteristics [10].



Page 6 of 12


Once the IVIVC is established and defined it can be then used to guide the final cycle of formulation and process optimization program statistically based experimental design studies looking at critical formulation and process variables. This information can also be used into the activities of scale-up, pivotal batch manufacture, and process validation culminating in registration, approval and subsequent post-approval scale-up and other changes. Thus rather than viewing the IVIVC as a single exercise at a given point in a development program, one should view it as a parallel development in itself starting at the initial assumed level and being built on and modified through experience and leading ultimately to a prospective IVIVC”.

Validation of IVIVC Model Evaluation of predictability of IVIVC

Prediction errors are estimated for Cmax and AUC to determine the validity of the correlation.

Various approaches of are used to estimate the magnitude of the error in predicting the in vivo bioavailability results from in vitro dissolution data.

Predictability of correlation

The objective of IVIVC evaluation is to estimate the magnitude of the error in predicting the in vivo bioavailability results from in vitro dissolution data. This objective should guide the choice and interpretation of evaluation methods. Any appropriate approach related to this objective may be used for evaluation of predictability [5,23]. It can be calculated by Prediction error that is the error in prediction of in vivo property from in vitro property of drug product (Figure 3).

Depending on the intended application of an IVIVC and the therapeutic index of the drug, evaluation of prediction error internally and/or externally may be appropriate [24].

Internal predictability

Evaluation of internal predictability is based on the initial data used to define the IVIVC model. Internal predictability is applied to IVIVC established using formulations with three or more release rates for non-narrow therapeutic index drugs exhibiting conclusive prediction error. If two formulations with different release rates are used to develop IVIVC, then the application of IVIVC would be limited to specified categories. The bioavailability (Cmax, tmax/AUC) of formulation that is used in development of IVIVC is predicted from its in vitro property using IVIVC. Comparison between predicted bioavailability and observed bioavailability is done and % P.E is calculated. According to FDA guidelines, the average absolute %P.E should be below 10% and %P.E for individual formulation should be below 15% for establishment of IVIVC.

Under these circumstances, for complete evaluation and subsequent full application of the IVIVC, prediction of error externally is recommended [23].

Acceptance criteria

According to FDA guidance

1) ≤15% for absolute prediction error (%P.E.) of each formulation.

2) ≤ 10% for mean absolute prediction error (%P.E.).

External predictability

Most important when using an IVIVC as a surrogate for bioequivalence is confidence that the IVIVC can predict in vivo performance of subsequent lots of the drug product. Therefore, it may be important to establish the external predictability of the IVIVC.

Evaluation of external predictability is based on additional test data sets [5]. External predictability evaluation is not necessary unless the drug is a narrow therapeutic index, or only two release rates were used to develop the IVIVC, or, if the internal predictability criteria are not met i.e. prediction error internally is inconclusive [4,23]. The predicted bioavailability is compared with known bioavailability and % P.E is calculated. The prediction error for external validation should be below 10% whereas prediction error between 10-20% indicates inconclusive predictability and need of further study using additional data set [24].

The % prediction error can be calculated by the following equation:

Prediction error

For Cmax

max max

max

( )%Prediction error (P.E.)= 100

C observed C predictedC observed

−× (5)

For AUC:(AUC AUC )%Prediction error (P.E.)= 100

AUC observed predicted

observed−

× (6)

Limitation of predictability metrics

Metrics used to evaluate the predictability is described simply the prediction error (%P.E.) for only two PK parameters i.e. Cmax and AUC. Emax predicted with IVIVC model represents the maximum of the mean plasma profiles but is compared with the mean Cmax observed calculated as the average of individual profile at different Tmax. But Tmax is not included in predictability metrics.

Factors to be Consider in Developing a CorrelationBiopharmaceutics classification system (BCS)

Biopharmaceutics Classification System (BCS) is a fundamental guideline for determining the conditions under which in-vitro, in-vivo correlations are expected [25]. It is also used as a tool for developing the in-vitro dissolution specification.

The classification is based on the drug dissolution and absorption model, which identifies the key parameters controlling drug absorption as a set of dimensionless numbers: the Absorption number, the Dissolution number and the Dose number [25-27].

The Absorption number is the ratio of the mean residence time to the absorption time.

2/ ( / ) / ( / )n res abs effA T T R L Q R Pπ= + (7)

The Dissolution number is a ratio of mean residence time to mean dissolution time given as equation 2

2 2 min0/ ( / ) / ( / 3 )n res diss sD T T R L Q pr DCπ= +

(8)

The Dose number is the mass divided by an uptake volume of 250 ml and the drug’s solubility as Equation 3

min/ ( / )o o sD Dose V C=

(9)

The mean residence time here is the average of the residence time



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in the stomach, small intestine and the colon. The fraction of dose absorbed then can be predicted based on these three parameters. For example, Absorption number 10 means that the permeation across the intestinal membrane is 10 times faster than the transit through the small intestine indicating 100% drug absorbed.

In the BCS, a drug is classified in one of four classes based solely on its solubility and intestinal permeability [27].

A biopharmaceutic drug classification scheme for correlating in vitro drug product dissolution and in vivo bioavailability is proposed based on recognizing that drug dissolution and gastrointestinal permeability are the fundamental parameters controlling rate and extent of drug absorption. This classification system was devised by Amidon et al. [27].

The drugs are divided into high/low-solubility and permeability classes. Currently, BCS guidelines are provided by USFDA, WHO, and EMEA (European Medicines Academy)

Class I: HIGH solubility / High permeability,

Class II: LOW solubility / High permeability,

Class III: HIGH solubility / LOW permeability,

Class IV: LOW solubility / LOW permeability.

Class I: High solubility- high permeability drugs

In case of class I , drugs (such as metoprolol) is well absorbed (though its systemic availability may be low due to first pass extraction/ metabolism) and the rate limiting step to drug absorption is drug dissolution or gastric emptying if dissolution is very rapid. The dissolution specification immediate release (IR) dosage forms of perhaps 85% dissolved in less than 15 min. May insure bioequivalence. To insure bioavailability for this case, the dissolution profile must be well defined and reproducible. [5,27].

Class II: Low solubility- high permeability drugs

This is the class of drugs (such as phenytoin) for which the dissolution profile must be most clearly defined and reproducible. More precisely this is the case where absorption number, (An) is high and Dissolution number (Dn) is low. Drug dissolution in vivo is then the rate controlling step in drug absorption and absorption is usually slower than for class I [28-31].

Class III: High solubility-low permeability drugs

For this class of drugs (such as cimetidine) Permeability is the rate controlling step in drug absorption. While the dissolution profile must be well defined, the simplification in dissolution specification as in Class I is applicable for immediate release dosage forms where drug input to the intestine is gastric emptying rate controlled.. Both rate and extent of drug absorption may be highly variable for this class of drugs, but id dissolution is fast i.e. 85% dissolved in less than 15 min, this variation will be due to the variable gastrointestinal transit, luminal contents , and membrane permeability rather than dosage form factors [5].

Class IV: Low solubility-low permeability drugs

This class of drugs present significant problems for effective oral delivery. The number of drugs that fall in this class will depend on the precise limits used from the permeability and solubility classification.

Applications

This concept underlying the BCS published finally led to introducing the possibility of waiving in vivo bioequivalence (BE) studies in favor of specific comparative in vitro testing to conclude BE of oral immediate release (IR) products with systemic actions [32].

In terms of BE, it is assumed that highly permeable, highly soluble drugs housed in rapidly dissolving drug products will be bioequivalent and that, unless major changes are made to the formulation, dissolution data can be used as a surrogate for pharmacokinetic data to demonstrate BE of two drug products. The BCS thus enables manufacturers to reduce the cost of approving scale-up and post approval changes to certain oral drug products without compromising public safety interests [33].

It is a drug-development tool that allows estimation of the contributions of three major factors, dissolution, solubility and intestinal permeability that affect oral drug absorption from IR solid oral dosage forms. It was first introduced into regulatory decision-making process in the guidance document on immediate release solid oral dosage forms: Scale-up and post approval changes [2]. BCS system is an indicator of developing a predictive IVIVC and also examined the importance of drug dissolution and permeability on IVIVC validity (Table 2).

The establishment of correlation needs, as described in the FDA or USP definitions, to use various parameters summarized in following table: Waiver of in vivo BE studies

Class Solubility Permeability IVIVC correlation for IR Products

I High HighIVIVC correlation if dissolution rate is slower than gastric emptying rate, otherwise limited or no correlation

II Low HighIVIVC correlation expected if in in vitro dissolution rate is similar to in vivo dissolution rate , unless dose is very high

III High LowAbsorption [permeability] is rate determining and limited or no IVIV correlation with dissolution rate.

IV Low Low Limited or no IVIV correlation expected.

Table 2: IVIV correlation expectation for immediate release product based on biopharmaceutic class.

Biopharmaceutics Drug Classificationfor Extended Release Drug Products **

Class Solubility Permeability IVIVC

IA High & Site Independent High & Site Independent IVIVC Level A

expected

IB High & Site Independent

Dependent on site & Narrow Absorption Window

IVIVC Level C expected

IIaLow & Site Independent High & Site Independent IVIVC Level A

expected

IIbLow & Site Independent Dependent on site & Narrow

Absorption WindowLittle or no IVIVC

Va: Acidic Variable Variable

Little or no IVIVC

Vb: basic Variable Variable IVIVC Level A

expected.

Table 3: Biopharmaceutics Drug Classification for Extended Release Drug Products.



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based on BCS Recommended for a solid oral Test product that exhibit rapid (85% in 30 min) and similar in vitro dissolution under specified conditions to an approved Reference product when the following conditions are satisfied (Table 3,4):

• Products are pharmaceutical equivalent

• Drug substance is highly soluble and highly permeable and is not considered have a narrow therapeutic range

• Excipients used are not likely to effect drug absorption

In vitro dissolution

Dissolution plays the important role in the formulation development as an obvious stage in IVIVC development when the dissolution is not influenced by factors such as pH, surfactants, osmotic pressure, mixing intensity, enzyme, ionic strength. Drug absorption from a solid dosage form following oral administration depends on the release of the drug substance from the drug product, the dissolution or solubilization of the drug under physiological conditions, and the permeability across the gastrointestinal tract. The purpose of the in-vitro dissolution studies in the early stage of drug development is to select the optimum formulation, evaluate the active ingredient and excipients, and assess any minor changes for drug products. During the early stages of correlation development, dissolution conditions may be altered to attempt to develop a one-to-one correlation between the in vitro dissolution profile and the in vivo dissolution profile [5]. For the IVIVC perspective, dissolution is proposed to be a surrogate of drug bioavailability. Thus, dissolution standard may be necessary for the in-vivo waiver [26]. The dissolution methodology, which is able to discriminate between the study formulations and which best, reflects the in vivo behavior would be selected. Once a discriminating system is developed, dissolution conditions should be the same for all formulations tested in the biostudy for development of the correlation and should be fixed before further steps towards correlation evaluation are undertaken [34]. The types of dissolution apparatus used as per USP recommended in the FDA guidance especially, for modified release dosage form are specified by the USP and are:

[1] Rotating basket,

[2] Paddle method,

[3] Reciprocating cylinder,

[4] Flow through cell,

Other dissolution methodologies may be used, however, the first four are preferred, especially the basket and paddle. But primarily it is

recommended to start with the basket or paddle method prior to using the others [26].

The in vitro dissolution release of a formulation can be modified to facilitate the correlation development. Changing dissolution testing conditions such as stirring speed, choice of apparatus, pH of the medium, and temperature may alter the dissolution profile.

As previously described, appropriate dissolution testing conditions should be selected so that the formulation behaves in the same manner as the in vivo dissolution.

For an appropriate dissolution test, in general and in particular for developing IVIVC, one requires to conduct the test selecting experimental conditions to simulate an in vivo environment as closely as possible. Commonly the following experimental conditions should be considered in this regard.

A common dissolution medium is dearated water, simulated gastric fluid (pH 1.2), or intestinal fluid (pH 6.8 or 7.4) without enzyme, and buffers with a pH range of 4.5 to 7.5 and be maintained at 37°C. For sparingly water-soluble drugs, use of surfactants in the dissolution medium is recommended [34,35]. A simple aqueous dissolution media is also recommended for BCS Class I drug as this type of drug exhibits lack of influence of dissolution medium properties [5,36]. Water and simulated gastric fluid then are the default mediums for most of the Class I drugs. A typical medium volume is 500 to 1000 ml.

1. Frequent samples (8-10) should be withdrawn to obtain a smooth dissolution profile leading to complete dissolution within the dosing interval of the test product in humans.

2. The normal test duration for immediate release is 15 to 60 minutes with a single time point. For example, BCS class I recommend 15 minutes. Additionally, two time points may be required for the BCS class II at 15 minutes and the other time at which 85% of the drug is dissolved [36].

3. In contrast, in vitro dissolution tests for a modified release dosage form require at least three time points to characterize the drug release. The first sampling time (1-2 hours or 20- 30% drug release) is chosen to check dose-dumping potential. The intermediate time point has to be around 50% drug release in order to define the in vitro release profile.

4. The dissolution medium should not be de-aerated. Preference should be given that the medium be equilibrated at 37°C with dissolved air/gasses, particularly for IVIVC studies.

5. An apparatus should be selected to have an appropriate

BCS Class Examples Drug delivery technology

Class I Metoprolol, Diltizem, Verapamil, Propranolol, Acyclovir, Atropine, verapamil.

Macrocap, Micropump, MODAS (Multiporous oral drug absorption system), SCOT (Single composition osmotic tablet system), and SPDS (Stabilized pellet delivery system) [28,30,31].

Class IIPhenytoin, Danazole, Ketokonazole, Mefenamic acid, Tacrolimus, Piroxicam, griseofulvine, Warfarin,

Micronization, stabilization of high-energy states (including lyophilized fast-melt systems), use of surfactants, emulsion or microemulsion systems, solid dispersion and use of complexing agent such as cyclodextrins.e.g nanosuspension and nanocrystals are treated as hopeful means of increasing solubility and BA of poorly water-soluble active ingredients [28,30,31].

Class III Cimetidine, Neomycin, ranitidine, Amoxycillin, Oral vaccine system, Gastric retention system, High-Frequency Capsule and Telemetric Capsule [28,30,31].

Class IV Cyclosporin A, Furosemide, Ritonavir, Saquinavir andTaxol.

The class IV drugs present a major challange for the development of drug delivery systems due to their poor solubility and permeability characteristics. These are administered by parenteral route with the formulation containing solubility enhancers [ 28,30,31].

Table 4: BCS class and drug delivery technology.



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mechanism to provide thorough but gentle mixing and stirring for an efficient product/medium interaction. Use of sinkers may be avoided as these often alter the dissolution characteristics of the test products. Paddle and basket apparatuses are known for their inefficient stirring and mixing, thus their use should be critically evaluated before use for IVIVC studies.

6. The last time point is to define essentially complete drug release. The dissolution limit should be at least 80% drug release. Further justification as well as 24 hours test duration are required if the percent drug release is less than 80 [34,37].

7. If the dissolution results are not as expected, then the product/formulation should be modified to obtain the desired/expected release characteristics of the product. However, altering experimental conditions such as medium, apparatus, rpm etc. should be avoided as these are generally linked to GI physiology which remains the same for test to test or product to product. Obtaining dissolution results by altering testing (experimental) conditions may void the test for IVIVC purposes.

Once the discriminatory system is established, dissolution testing conditions should be fixed for all formulations tested for development of the correlation [6]. A dissolution profile of percentage or fraction of drug dissolved versus time then can be determined.

Comparison between dissolution profiles could be achieved using a difference factor (f1) and a similarity factor (f2) which originates from simple model independent approach. The difference factor calculates the percent difference between the two curves at each time point and is a measurement of the relative error between the two curves:

11 1

/ *100n n

t t tt r

f R T R= =

= − ∑ ∑ (10)

Where, n is the number of time points, Rt is the dissolution value of the reference batch at time t, and Tt is the dissolution value of the test batch at time t. The similarity factor is a logarithmic reciprocal square root transformation of the sum squared error and is a measurement of the similarity in the percent dissolution between the two curves

0.5

21

50* log 1 (1 / ) *100n

t tt

f n R T−

2

=

= + ( − ) ∑ (11)

Generally, f1 values up to 15 (0-15) and f2 values greater than 50 (50-100) ensure sameness or equivalence of the two curves. The mean in vitro dissolution time (MDTvitro) is the mean time for the drug to dissolve under in vitro dissolution conditions. This is calculated using the equation 6:

0( ( )) /vitroMDT M M t dt M

∞

∞ ∞= −∫ (12)

For the IVIVC development, the dissolution profiles of at least 12 individual dosage units from each lot should be determined. The coefficient of variation (CV) for mean dissolution profiles of a single batch should be less than 10%. Since dissolution apparatuses tend to become less discriminative when operated at faster speeds, lower stirring speeds should be evaluated and an appropriate speed chosen in accordance with the test data. Using the basket method the common agitation is 50-100 rpm; with the paddle method, it is 50-75 rpm and 25 rpm for suspension [5].

In vivo absorption (Bioavailability studies)

The FDA requires in vivo bioavailability studies to be conducted

for a New Drug Application (NDA). A bioavailability study should be performed to characterize the plasma concentration versus time profile for each of the formulation. These studies for the development of IVIVC should be performed in young healthy male adult volunteers under some restrictive conditions such as fasting, non-smoking, and no intake of other medications. In prior acceptable data sets, the number of subjects has ranged from 6 to 36. Although crossover studies are preferred, parallel studies or cross-study analyses may be acceptable. The latter may involve normalization with a common reference treatment. The drug is usually given in a crossover fashion with a washout period of at least five half-lives.

The bioavailability study can be assessed via plasma or urine data using the following parameters: (I) area under the plasma time curve (AUC), or the cumulative amount of drug excreted in urine (Du), (II) maximum concentration (Cmax), or rate of drug excretion in urine (dDu/dt), and (III) a time of maximum concentration (Tmax).

Several approaches can be used for determining the In vivo absorption. Wagner-Nelson, Loo-Riegelman, and numerical deconvolution are such methods [2,37]. Wagner Nelson and Loo-Riegelman are both model dependent methods in which the former is used for a one-compartment model and the latter is for multi-compartment system.

The Wagner Nelson method is less complicated than the Loo-Riegelman as there is no requirement for intravenous data. However, misinterpretation on the terminal phase of the plasma profile may be possible in the occurrence of a flip flop phenomenon in which the rate of absorption is slower than the rate of elimination.

Application of An IVIVCApplication in drug delivery system

Various rate controlling technologies are used as the basis for Modified release dosage forms e.g. Diffusion-dissolution, matrix retardation, osmosis, etc. to control, and prolong the release of drugs, for the administration by oral or parenteral route [24,38].

The novel drug delivery systems have been developed such as OROS, liposomes, niosomes, pharmacosomes, microspheres, nanoparticles, implants, in situ gelling system, organogels, transdermal drug delivery systems, parenteral depots, etc. as a substitute for conventional dosage forms. The obvious objective of these dosage forms is to achieve zero-order, long term, pulsatile, or “on demand” delivery. Major applications of IVIVC related to oral drug delivery and a few issues related to the development of IVIVC models for parenteral drug delivery are addressed herewith [39].

In early stages of drug delivery technology development

The most crucial stage in the drug development is drug candidate selection. Such selection is primarily based on the drug “developability” criteria, which include physicochemical properties of the drug and the results obtained from preformulation, preliminary studies involving several in vitro systems and in vivo animal models, which address efficacy and toxicity issues [24,40]. During this stage, IVIVC (exploring the relationship between in vitro and in vivo properties) of the drug in animal models provide an idea about the feasibility of the drug delivery system for a given drug candidates. In such correlations, study designs including study of more than one formulation of the modified-release



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dosage forms and a rank order of release (fast/slow) of the formulations should be incorporated. Even though the formulations and methods used at this stage are not optimal, they promise better design and development efforts in the future.

Formulation assessment: In vitro dissolution

A suitable dissolution method that is capable of distinguishing the performance of formulations with different release rates in vitro and in vivo is an important tool in product development. Depending on the nature of the correlation, further changes to the dissolution method can be made. When the discriminatory in vitro method is validated, further formulation development can be relied on the in vitro dissolution only.

Dissolution specifications

Modified-release dosage forms typically require dissolution testing over multiple time points, and IVIVC plays an important role in setting these specifications [24,39]. Specification time points are usually chosen in the early, middle, and late stages of the dissolution profiles. In the absence of an IVIVC, the range of the dissolution specification rarely exceeds 10% of the dissolution of the pivotal clinical batch. However, in the presence of IVIVC, wider specifications may be applicable based on the predicted concentration-time profiles of test batches being bioequivalent to the reference batch.

The process of setting dissolution specifications in the presence of an IVIVC starts by obtaining the reference (pivotal clinical batch) dissolution profile. The dissolution of batches with different dissolution properties (slowest and fastest batches included) should be used along with the IVIVC model, and prediction of the concentration time profiles should be made using an appropriate convolution method. Specifications should optimally be established such that all batches with dissolution profiles between the fastest and slowest batches are bioequivalent and less optimally bioequivalent to the reference batch. The above exercise in achieving the widest possible dissolution specification allows majority of batches to pass and is possible only if a valid Level A model is available [24].

Future biowaivers

Frequently, drug development requires changes in formulations due to a variety of reasons, such as unexpected problems in stability, development, availability of better materials, better processing results, etc. Having an established IVIVC can help avoid bioequivalence studies by using the dissolution profile from the changed formulation, and subsequently predicting the in vivo concentration-time profile [24,41].

This predicted profile could act as a surrogate of the in vivo bioequivalence study. This has enormous cost-saving benefit in the form of reduced drug development spending and speedy implementation of post-approval changes. The nature of post-approval changes could range from minor (such as a change in non release-controlling excipient) to major (such as site change, equipment change, or change in method of manufacture, etc) [24,42].

IVIVC - Parenteral drug delivery

IVIVC can be developed and applied to parenteral dosage forms, such as controlled-release particulate systems, depot system, implants, etc, that are either injected or implanted. However, there are relatively fewer successes in the development of IVIVC for such dosage forms, which could be due to several reasons, a few of which are discussed

further. Sophisticated modeling techniques are needed to correlate the in vitro and in vivo data, in case of burst release which is unpredictable and unavoidable [24,43].

Potent Drugs & Chronic Therapy - In general, several parenteral drug delivery systems are developed for potent drugs (eg, hormones, growth factors, antibiotics, etc) and for long-term delivery (anywhere from a day to a few weeks to months). In such instances, to establish a good IVIVC model, the drug concentrations should be monitored in the tissue fluids at the site of administration by techniques such as microdialysis, and then the correlation should be established to the in vitro release.

Biowaivers

Validated IVIVC is applicable to serve as justification for a biowaiver in filings of a Level 3 (or Type II in Europe) variation, either during scale-up or post approval, as well as for line extensions (e.g., different dosage strengths). A biowaiver will only be granted if the prediction of the in vivo performance of the product with the modified in vitro release rate remains bioequivalent with the originally tested product (i.e., the new dissolution rate remains within the IVIVC based biorelevant corridor).

The FDA guidance outlines five categories of biowaivers: 1) biowaivers without an IVIVC, 2) biowaivers using an IVIVC: non-narrow therapeutic index drugs, 3) biowaivers using an

IVIVC: narrow therapeutic index drugs, 4) biowaivers when in vitro dissolution is independent of dissolution test conditions and 5) situations for which an IVIVC is not recommended for biowaivers Biowaivers may be granted for manufacturing site changes, equipment changes, manufacturing process changes, and formulation composition changes according to a predictive and reliable IVIVC. The changes may range from minor changes that are not significant to alter product performance to major ones where an IVIVC is not sufficient to justify the change for regulatory decision [4,24].

Establishment of dissolution specifications

It is relatively easy to establish a multipoint dissolution specification for modified-release dosage forms .The dissolution behavior of the biobatch maybe used to define the amount to be released at each time point. However, the difficulty arises in the variation to be allowed around each time point [37]. The FDA guidance describes the procedures of setting dissolution specifications in cases of level A, multiple levels C, and level C correlation and where there is no IVIV correlation. Once an IVIVC developed, IVIVC should be used to set specifications in such a way that the fastest and lowest release rates allowed by the upper and lower dissolution specifications result in a maximum difference of 20% in the predicted Cmax and AUC. Predicted plasma concentration and consequent AUC and Cmax could be calculated using convolution or any other appropriate modeling techniques [24]. In the case of multiple level C correlation, the last time point should be the time point where at least 80% of drug has dissolved. For level C correlation, reasonable deviations from ±10 % may be acceptable if the range at any time point does not exceed 25%. When there is no IVIVC, the tolerance limits may be derived from the spread of in vitro dissolution data of batches with demonstrated acceptable in vivo performance (biobatch) or by demonstrating bioequivalence between batches at the proposed upper and lower limit of the dissolution range (the so called side batch



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concept). Variability in release at each time point is recommended not to exceed a total numerical difference of ±10% (a total of 20%) or less of the labeled claim. In certain cases, deviations from this criterion can be acceptable up to a maximum range of 25%. Beyond this range, the specification should be supported by bioequivalence studies [43].

Mapping

Mapping is a process which relates Critical Manufacturing Variables (CMV), including formulation, processes, and equipment variables that can significantly affect drug release from the product. The mapping process defines boundaries of in vitro dissolution profiles on the basis of acceptable bioequivalency criteria. The optimum goal is to develop product specifications that will ensure bioequivalence of future batches prepared within the limits of acceptable dissolution specifications. Dissolution specifications based on mapping would increase the credibility of dissolution as a bioequivalency surrogate marker and will provide continuous assurance and predictability of the product performance [5].

Some Limitations in the IVIVC Arising from the In Vivo Data

Could easily be understood:

1. More than one dosage form is needed and if possible intravenous or solution is essential to calculate deconvolution.

2. Pharmacokinetics and absorption of the drug should be ‘‘linear.’’ If the pharmacokinetic processes are dependent on the fraction of dose reaching the systemic blood flow (or of the dose administered) or on the rate of absorption, comparison between formulation and simulation cannot be made. This non-linearity may be owing to saturable absorption processes (active absorption), induction or inhibition of the metabolism, the first past effect, which is rate/absorption dependent, etc. Those points must be studied before any attempt to establish an IVIVC.

3. Absorption should not be the limiting factor, if the solubility is not the limiting factor in comparison to the drug release, an IVIVC may be attempted. The release must depend on the formulation, and must be the slowest phenomenon vs. dissolution and absorption.

ConclusionsThe pharmaceutical industry has been striving to find a ways to

saving precious resources in relevance to the budgets and increasing cost of drug development. IVIVC is a tool applied in various areas and stages of drug development to find a place in the regulatory bodies around the world. IVIVC can serve as surrogate for in vivo bioavailability and to support biowaivers also allows setting the dissolution specification and methods. The substitute of expensive clinical trials with the use of IVIVC is perhaps the most important feature of IVIVC. From the regulatory point of view IVIVC can assist certain scale-up and post-approval changes. IVIVC principles have been mostly applied to oral products, there exists a need to develop methodologies and standards for non-oral delivery systems, to develope more meaningful dissolution and permeation methods. Acknowledgement

The authors gratefully acknowledge Mrs. Sangita Sakore (Regulatory affairs, Formulation & Development, MKPPL, Pune, India) for her contribution.

References

1. Leeson LJ (1995) In vitro/ In vivo correlations. Drug Info J 29: 903-915.

2. US Department of Health and Human Services (1995) Guidance for industry: Immediate release solid oral dosage forms scale-up and postapproval changes: chemistry, manufacturing, and controls, in vitro dissolution testing, and in vivo bioequivalence documentation. Center for drug Evaluation and Research.

3. Center for Drug Evaluation, USFDA (1995) Guidance for Industry: Immediate Release Solid Oral Dosage Forms: Scale Up and Post Approval Changes.

4. Uppoor VRS (2001) Regulatory perspectives on in vitro (dissolution)/in vivo (bioavailability) correlations. J Control Rel 72: 127-132.

5. Jaber E (2006) In vitro - In vivo Correlation: From Theory to Applications. J Pharm Pharmaceut Sci 9: 169-189.

6. Food and Drug Administration (1997) Guidance for industry: extended release oral dosage forms: Development, evaluation, and application of in vitro/in vivo correlations.

7. Sirisuth N, Eddington ND (2002) In vitro in vivo correlations, systemic methods for the development and validation of an IVIVC metoprolol and naproxen drug examples. Int J Generic Drugs 3: 250-258.

8. Qureshi SA (2010) In Vitro-In Vivo Correlation (IVIVC) and Determining Drug Concentrations in Blood from Dissolution Testing - A Simple and Practical Approach. Open Drug Delivery J 4: 38-47.

9. O’Hara T, Hayes S, Davis J, Devane J, Smart T, et al. (2001) In vivo-In Vitro Correlation (IVIVC) Modeling Incorporating a Convolution Step. J Pharmacokinet Pharmacodyn 28: 277-298.

10. Gaynor C, Dunne A, Davis J (2008) A Differential Equations Approach to In Vitro – In Vivo Correlation Modelling. International Biometric Conference, Dublin.

11. Dunne A (2007) Approaches to developing IVIVC models, Ch 5. In Chilukuri, Sunkara and Young (Eds), Pharmaceutical Product Development: In Vitro – In vivo Correlation. Taylor and Francis, New York.

12. Beal SL, Sheiner LB (1992) NONMEM User’s Guides, NONMEM Project Group, University of California, San Francisco.

13. Meyer MC, Straughn AB, Mhatre RM, Shah VP ,Williams RL (1998) Lack of in vitro / in vivo correlations of 50 mg and 250 mg primidone tablets. Pharm Res 15: 1085-1089.

14. Food and Drug Administration. Rockville, MD Abdou, HM (1989) Dissolution Bioavailability and Bioequivalence. Easton Pennsylvania: Mack Printing.

15. Buchwald P (2003) Direct, differential-equation-based in-vitro-in-vivo correlation (IVIVC) method. J Pharm Pharmacol 55: 495-504.

16. Dressman JB, Amidon GL, Reppas C, Shah VP (1998) Dissolution testing as a prognostic tool for oral drug absorption: immediate release dosage forms. Pharm Res 15: 11-22.

17. Corrigan OI (1997) The biopharmaceutic drug classification and drugs administered in extended release (ER) formulations. Adv Exp Med Biol 423: 111-128.

18. Yu H, Joves R (2004) The STATs of cancer--new molecular targets come of age. Nat Rev Cancer 4: 97-105.

19. Wagner JG, Nelson E (1964) Kinetic analysis of blood levels and urinary excretion in the absorptive phase after single doses of drug. J Pharm Sci 53: 1392-1403.

20. Loo JC, Riegelman S (1968) New method for calculating the intrinsic absorption rate of drugs. J Pharm Sci 57: 918-928.

21. Cutler DJ (1978) Linear system analysis in pharmacokinetics. Pharmacokinet Biopharm 6: 265-282.

22. Brockmeier D, Voegele D, von Hattingberg HM (1983) In vitro-in vivo correlation, a time scaling problem? Basic techniques for testing equivalence. Arzneimittelforschung 33: 598-601.

23. Jayaprakasam B, Seeram NP, Nairs MG (2003) Anticancer and antiinflammatory activities of cucurbitacins from Cucurbita andreana. Cancer Lett 189: 11-16.


http://cat.inist.fr/?aModele=afficheN&cpsidt=3633089

http://www.ncbi.nlm.nih.gov/pubmed/11389991




http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulations



http://benthamscience.com/open/toddj/articles/V004/SI0001TODDJ/38TODDJ.pdf



http://ibc2008abstracts.tibs.org/Other%5CIBC2008_Paper_ref_466.pdf





























Page 12 of 12


24. Chilukuri DM, Sunkara G (2003) IVIVC: An Important Tool in the Development of Drug Delivery Systems. Drug Deliv Technol 3: 4.

25. Young D, Devane JG, Butler J (1997) In Vitro-In Vivo Correlations. New York: Plenum press.

26. Amidon GL, Robinson JR, Williams RL (1997) Scientific Foundations for Regulating Drug Product Quality. American Association of Pharmaceutical Scientists. Alexandria, Virginia: AAPS Press.

27. Amidon GL, Lennernas H, Shah VP, Crison JR (1995) A theoretical basis for a biopharmaceutic drug classification: The correlation of in vitro drug product dissolution and in vivo bioavailabilty. Pharm Res 12: 413-419.

28. Gohel MC, Mehta NR (2005) An audit of recent inputs on biopharmaceutical classification system. Pharmaceut Rev.

29. Yu LX, Amidon GL, Polli JE, Zhao H, Mehta MU, et al. (2002) Biopharmaceutics classification system: The scientific basis for biowaiver extension. Pharm Res 19: 921-925.

30. Wilding I (1999) Evolution of the Biopharmaceutics Classification System (BCS) to Modified Release (MR) formulations: What do we need to consider? Eur J Pharm Sci 8: 157-159.

31. Devane J (1998) Oral drug delivery technology: Addressing the solubility/permeability paradigm. Pharmaceut Technol 22: 68-80.

32. Helga M (2002) The Biopharmaceutical Classification System (BCS) and its usage. Drugs Made in Germany 45: 63-65.

33. Dressman J, Butler J, Hempenstall J, Reppas C (2001) The BCS: Where do we go from here? Pharmaceut Technol 25: 68-76.

34. Sievert B, Siewert M (1998) Dissolution tests for ER products. Dissolut Technol 5.

35. Shargel L, Yu ABC (1993) Applied Biopharmaceutics and Pharmacokinetics. East Norwark, Connecticut: Appleton & Lange.

36. Galia E, Nicolaides E, Horter D, Lobenberg R, Reppas C, et al. (1998) Evaluation of various dissolution media for predicting in vivo performance of class I and class II drugs. Pharm Res 15: 698-705.

37. Abdou HM (1989) Dissolution, Bioavailability and Bioequivalence. Easton, Pennsylvania: Mack Printing.

38. Strickley RG (1999) Parenteral formulations of small molecules therapeutics marketed in the United States Part I, PDA. J Pharm Sci Tech 53: 324-349.

39. Modi NB, Lam A, Lindemulder E, Wang B, Gupta SK (2000) Application of in vitro-in vivo correlation (IVIVC) in setting formulation release specifications. Biopharm Drug Dispos 21: 321-326.

40. Venkatesh S, Lipper RA (2000) Role of the development scientist in compound lead selection and optimisation. J Pharm Sci 89: 145-154.

41. Hwang SS, Gorsline JJ, Louie J, Dye D, Guinta D, et al. (1995) in vitro and in vivo evaluation of a once-daily controlled release pseudoephedrine product. J Clin Pharmacol 35: 259-267.

42. FDA (1997) Guidance for Industry: SUPAC-MR: Modified release solid oral dosage forms: scale-up and post-approval changes: chemistry, manufacturing and controls, in vitro dissolution testing, and in vivo bioequivalence documentation.

43. Young D, Chilukuri D, Becker R, Bigora S, Farrell C, et al. (2002) Approaches to developing a Level-A IVIVC for injectable dosage forms. AAPS Pharm Sci 4: M1357.

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Research Article

BIOWAIVER MONOGRAPH FOR IMMEDIATE RELEASE SOLID ORAL DOSAGE FORMS: OFLOXACIN

SOMNATH SAKORE*, SUNIL CHOUDHARI, BHASWAT CHAKRABORTY

1,2Cadila Pharmaceuticals Limited, 1389, Trasad Road, Dholka387810, Ahmedabad, Gujarat. India Email: [email protected]

Received: 23 July 2010, Revised and Accepted: 23 August 2010

ABSTRACT

Literature and experimental data relevant to the decision to allow a waiver of in vivo bioequivalence testing for the approval of immediate release (IR) solid oral dosage forms containing ofloxacin have been reviewed. According to the current Biopharmaceutics Classification System (BCS), ofloxacin should be assigned to Class I. Therapeutic use, therapeutic index, pharmacokinetic properties, reported BE/bioavailability (BA) and data related to the possibility of excipient interactions studies of Ofloxacin were also taken into consideration in order to ascertain whether a biowaiver can be recommended. Ofloxacin seems not to be critical with respect to a risk for bioinequivalence, as no examples of bioinequivalence have been identified. However; if (a) the test product contains only excipients in their usual amounts present in ofloxacin solid oral IR drug products approved in ICH or associated countries, for instance as presented in this article; and (b) the comparator and the test product both are very rapidly dissolving a biowaiver for IR ofloxacin solid oral drug products is considered justified for all tablet strengths.

Keywords: Absorption, Biopharmaceutics Classification System (BCS), Ofloxacin, Permeability, Solubility.

INTRODUCTION

A biowaiver monograph of ofloxacin based on literature data together with some additional experimental data has been presented here. A biowaiver implies that bioequivalence (BE) assessment studies would be waived for marketing authorizations (MA) by Health Authorities for a new tablet or capsules, or a new formulation of an existing immediate release (IR) dosage form, and hence the product is considered bioequivalent to its reference product, without carrying out a bioequivalence (BE) study. The risks of waiving in vivo BE testing for the approval of new and/or reformulated immediate release (IR) solid oral dosage forms containing ofloxacin, including both reformulated products and new multi source products, are evaluated in consideration of their biopharmaceutical and clinical properties. The scientific basis for a waiver request for ofloxacin tablets has been developed according to Biopharmaceutical Classification System (BCS)1 .

The BCS states that three major factors govern the rate and extent of drug absorption of IR solid oral dosage forms: dissolution rate, solubility and intestinal permeability. For IR dosage forms containing active pharmaceutical ingredients (APIs) showing high solubility, high intestinal permeability, and rapid dissolution, a waiver from performing BE studies (biowaiver) can be scientifically justified. In the regulatory domain this is adopted by both the US FDA and the European CPMP in their guidances for industry, Waiver of In Vivo Bioavailability (BA) and BE Studies for Immediate‐Release Solid Oral Dosage Forms Based on a BCS 2 and the Note for Guidance on the Investigation of BA and BE 3 respectively, have been together referred to as the Guidances in this article. In particular, the US FDA document describes in detail the data that are necessary for a successful application for a biowaiver. To explore the scope and the possibilities of gathering BCS related data from scientific literature, and in order to set up such BCS‐monographs, a literature search was carried out on ofloxacin.

The aim of this monograph is to evaluate all pertinent data available from literature sources for ofloxacin to assess the risks associated with a biowaiver. For these purposes risk is defined as the probability of an incorrect biowaiver decision as well as the consequences of an incorrect biowaiver decision in terms of public health and individual patient risk. On the basis of these considerations, a recommendation can be made as to whether a biowaiver is advisable or not for ofloxacin solid oral dosage forms. This systematic approach to recommend or advice against a

biowaiver decisions is referred to in recently published World Health Organization (WHO) Guideline.

General characteristics of Ofloxacin

Chemical, Pharmaceutical and Pharmacokinetic BCS‐related information on ofloxacin was obtained by means of a literature search. The following data‐fields were defined in order to standardize the dataset: indication, solubility, dissolution, poly‐morphism, partition coefficient, pKa, available dose, permeability, stereospecificity, pharmacokinetic properties. Literature data was accessed from PubMed, PubChem, Medicines Complete, WHO search engine, WHOLIS, the BIAM, 16 ROTE LISTE, and VIDAL databases. Key words used for searching were: Ofloxacin, bioequivalence, bioavailability, biowaiver, solubility, permeability, dissolution, excipient, toxicity, polymorphism, and pharmacokinetics.

Nomenclature

Ofloxacin (INN) It’s chemical name is (±)‐9‐fluoro‐2,3‐dihydro‐3‐methyl‐10‐(4‐methyl‐1‐piperazinyl)‐7‐oxo‐7H‐pyrido[1,2,3‐de]‐1,4‐benzoxazine‐6‐carboxylic acid. 4

Its structure is shown in below Fig 1.

N

O

N

O

COOH

CH3

NH3C

F

Fig. 1: Structure of ofloxacin

Therapeutic indications

Ofloxacin is a new fluoroquinolone with a spectrum of activity similar to other fluoroquinolones with activity which includes Chlamydia trachomatis, Mycobacterium spp., Mycoplasma spp. and Legionella pneumophila. Ofloxacin may be less susceptible to the development of resistance from Staphylococcus aureus commonly seen with currently available fluoroquinolones5.

It is also used in chlamydial infections including nongonococcal urethritis in treating mycobacterial infections such as leprosy. Ofloxacin tablets are indicated for the treatment of adults with mild to moderate infections (unless otherwise indicated) caused by susceptible strains of the designated microorganisms in the infections listed below 6.

International Journal of Pharmacy and Pharmaceutical Sciences

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Sakore et al.

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• Acute bacterial exacerbations of chronic bronchitis due to Haemophilus influenzae or Streptococcus pneumoniae.

• Community‐acquired Pneumonia due to Haemophilus influenzae or Streptococcus pneumoniae.

• Uncomplicated skin and skin structure infections due to methicillin‐susceptible Staphylococcus aureus, Streptococcus pyogenes, or Proteus mirabilis.Acute, uncomplicated urethral and cervical gonorrhea due to Neisseria gonorrhoeae.

• Nongonococcal urethritis and cervicitis due to Chlamydia trachomatis.

• Mixed Infections of the urethra and cervix due to Chlamydia trachomatis and Neisseria gonorrhoeae.

• Acute pelvic inflammatory disease (including severe infection) due to Chlamydia trachomatis and/or Neisseria gonorrhoeae.

• Uncomplicated cystitis due to Citrobacter diversus, Enterobacter aerogenes, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, or Pseudomonas aeruginosa.

• Complicated urinary tract infections due to Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Citrobacter diversus, or Pseudomonas aeruginosa.

• Prostatitis due to Escherichia coli (http://www.rxlist.com/floxin‐drug.htm)

Therapeutic index and toxicity

An adult oral or intravenous dose ranges from 200 mg daily to 400 mg twice daily depending on the severity and the nature of the infection. Oral doses up to 400 mg may be given as a single dose, preferably in the morning. For intravenous use a 0.2% solution is infused over 30 minutes or a 0.4% solution over 60 minutes. A dose of 400 mg daily or intermittently by mouth has been recommended by WHO as part of alternative multidrug therapy regime for leprosy 7.

The following is a compilation of the data for ofloxacin based on clinical experience with both the oral and intravenous formulations. The incidence of drug‐related adverse reactions in patients during Phase 2 and 3 clinical trials was 11%. Among patients receiving multiple‐dose therapy, 4% discontinued ofloxacin due to adverse experiences. In clinical trials, the following events were considered likely to be drug‐related in patients receiving multiple doses of ofloxacin: nausea 3%, insomnia 3%, headache 1%, dizziness 1%, diarrhea 1%, vomiting 1%, rash 1%, pruritus 1%, external genital pruritus in women 1%, vaginitis 1%, dysgeusia 1%. Information on overdosage with ofloxacin is limited. One incident of accidental overdosage has been reported. In this case, an adult female received 3 grams of ofloxacin intravenously over 45 minutes. A blood sample obtained 15 minutes after the completion of the infusion revealed an ofloxacin level of 39.3 μg/mL. In 7 h, the level had fallen to 16.2 μg/mL, and by 24 h to 2.7 μg/mL. During the infusion, the patient developed drowsiness, nausea, dizziness, hot and cold flushes, subjective facial swelling and numbness, slurring of speech, and mild to moderate disorientation. All complaints except the dizziness subsided within 1 h after discontinuation of the infusion. The dizziness, most bothersome while standing, resolved in approximately 9 h. Laboratory testing reportedly revealed no clinically significant changes in routine parameters in these patient. Thus, ofloxacin does not have a narrow therapeutic index 6.

Chemical properties

Stereoisomers and polymorphs

Ofloxacin chemically a fluorinated corboxyquinolone, and it’s the racemate 6 Polymorphic forms have not been reported in the literature.

Partition coefficient (logP)

The n‐octanol/water partition coefficient (log P) of ofloxacin was reported as ‐0.48 8.

pKa

Ofloxacin is an amphoteric drug with two protonation sites9,10. Its pKa are 6.05 for the carboxylic group and 8.22 for the piperazine nitrogen given in figure 2.

N

O

N

O

COOH

CH3

NH3C

F

pKa = 6.0

pKa = 8.0

Fig. 2: The molecular structure of ofloxacin with the approximate pKa values.

Solubility

Ofloxacin is freely soluble in acetic acid, slightly soluble in water, methanol, ethanol or acetone8.

Dosage form strengths

The WHO Essential Medicines List (EML) lists Ofloxacin Tablet strengths from 200 to 400 mg (WHO drug information, 2007) Ofloxacin tablet dosage form existing in different countries through out world on different brand names by different marketing authorization7 . In the United States, NDA exists for strengths in the range of 200 to 400 mg3 .

Pharmacokinetic properties

Permeability and absorption

One of the permeability studies for ofloxacin drug substance was carried out with Caco‐2 assay method. This cell culture model was previously evaluated and determined to be a suitable method according to the BCS Guidance as it demonstrated a rank‐order correlation between in vitro permeability and human extent of absorption for the model drugs, with a clear segregation between high and low permeability drug substances11. Based on the previous reports of human absolute bioavailability, it was expected that ofloxacin would be classified as a highly permeable (HP) drug12,13 . Also, in the Caco‐2 permeability assays, ofloxacin was classified as HP drugs. Thus, the in vitro results matched human in vivo data based on absolute bioavailability. In addition, there was evidence that ofloxacin underwent some active transport as its permeability apparent values decreased with concentration 14 .

Following oral administration, there is rapid and extensive oral absorption from the gastrointestinal tract achieving peak serum concentration within 1 – 3 h and levels in excess of 100 g/ml in the urine and bladder 8.

The pharmacokinetics of ofloxacin are characterised by almost complete bioavailability (95 to 100%), peak serum concentrations in the range of 2 to 3 mg/L after a 400mg oral dose and an average half‐life of 5 to 8h. Ofloxacin is rapidly and well absorbed from the gastrointestinal tract5.Oral bioavailability is almost 100% and a peak plasma concentration of 3 to 4 μg/mLis achieved within 1 to 2 hours after a dose of 400 mg by mouth. Absorption may be delayed by the presence of food, but the extent of absorption is not substantially affected 7 .

Distribution

About 25% of ofloxacin is bound to plasma proteins. Ofloxacin is widely distributed in body fluids, including the CSF, and tissue penetration is good. It crosses the placenta and is distributed into breast milk. Relatively high concentration is achieved in bile 7 .

Metabolism and excretion

There is limited metabolism to desmethyl and N‐oxide metabolites; desmethylofloxacin has moderate antibacterial activity. Ofloxacin is eliminated mainly by the kidneys. Excretion is by tubular secretion and glomerular filtration and 75 to 80% of a dose is excreted unchanged in the urine over 24 to 48 hours, resulting in high urinary concentrations. Less than 5% is excreted in the urine as metabolites. From 4 to 8% of a dose may be excreted in the faeces. Only small

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amount of ofloxacin are removed by Dialysis 7 . In comparison with other available quinolones, elimination is more highly dependent on renal clearance, which may lead to more frequent dosage adjustments in patients with impaired renal function6.

Food and excipients interaction

Ofloxacin interacts with multivalent cation‐containing products, such as aluminum‐ or magnesium‐containing antacids and products containing calcium, iron, or zinc. Concomitant use invariably results in marked reduction of oral absorption of this antimicrobial. The mechanism of this interaction is formation of insoluble chelation complexes in the gastrointestinal tract that inhibit drug absorption 15,16 .

Multivitamin preparations that contain minerals should be avoided. Similar adverse effects on fluoroquinolone absorption were observed with concomitant administration of ferrous sulfate (iron), with decreases in bioavailability of the antibiotic of 19‐66 %.17,18 .

Although it is usually recommended that concomitant intake of calcium‐rich foods (e.g., milk) be avoided because of the potential for chelation effects, the actual influence of dairy products on fluoroquinolone absorption varies19,20 . Milk did not alter the rate or extent of absorption of ofloxacin or its elimination 21 .

Sucralfate significantly interferes with oral absorption of fluoroquinolones. It decreased the bioavailability of these drugs by up to 98% when given within 2 hours of antibiotic administration. The mechanism of this interaction has been attributed to both the aluminum content of the sucralfate salt and direct binding of the fluoroquinolone by the sucralfate itself 22,23. The degree to which fluoroquinolones are absorbed is not significantly affected by food. Studies involving ciprofloxacin, levofloxacin, gatifloxacin, and moxifloxacin consistently reported alterations in drug absorption rates without change in the extent of absorption 24.

When fluoroquinolones are administered with food, peak concentration times are usually slightly delayed, and maximum

plasma concentrations (C max) are decreased 8‐16%. The area under the plasma concentration versus time curve (AUC) is invariably unchanged and alterations in absorption rates are considered to be clinically insignificant21.

Dosage form performance

Bioavailability and bioequivalence

Bioavailability of oral and intravenous ofloxacin was investigated after the administration of multiple doses of 400 mg every 12 h to 20 healthy male volunteers in a randomized, crossover, open‐label study. Ofloxacin concentrations in plasma were evaluated after 4 days of oral or intravenous (1‐h infusion) dosing with a 3‐day wash‐out period between regimens. As expected, delivery to the systemic circulation took slightly longer after the oral dosing (time to maximum concentration of drug in serum of 1.7 h) relative to the 1‐h intravenous infusion, but the systemic availabilities of ofloxacin by the two routes of administration were equivalent (area under the concentration‐time curve from 0 to 12 h ratio of 95%)25.

Excipients

Ofloxacin interact with multivalent cations present in fillers, binders and lubricants. Table 2 shows the excipients present in ofloxacin IR solid drug products in US market. It can be inferred that these drug products successfully passed an in vivo BE study. In Table 2 the amounts of various excipients found in single API ofloxacin products, along with the ranges specified by the FDA for oral drug products in general, are given 3 .

Excipients present in IR ofloxacin tablets with US MA are summarized in Table 3. In vivo comparisons of different formulations were not reported. Therapeutic inequivalence between brand‐name drug products and FDA‐approved generic drug products has not been reported and there have been no reports of bioinequivalence of IR tablets with an approval in India.

Table1: Excipients present in Ofloxacin IR solid oral drug with a Marketing Authorization United States (IIG Limits)

Excipients Max. amount present in solid oral dosage forms with a MA in the USA (mg) Lactose Anhydrous 735.20 Modified corn starch 433.32 Hydroxy propyl cellulose 46.00 Hypromellose 54.00Magnesium stearate 400.74Polyethylene Glycol 0.12 POLYSORBATE 80 21.25Sodium starch Glycolate 876.00Purified Talc 91.20 Titanium dioxide 27.00 Different countries having ofloxacin with following brand names:

Germany: Floxacil, Gyroflox, Oflo, Oflodura, Oflohexal, Oflox, Ofloxbeta, Tanvid, Uro Tanvid.

Finland : Excin,Tanvid

Netherlands: Tanvid, Trafloxal.

USA: Floxin.

Details of excipients used in the Ofloxacin tablet formulation from different countries were not available for studying.

Dissolution

The present biowaiver criteria state that, in addition to similarity of dissolution profiles, the test and the comparator drug product

should both be rapidly dissolving , which is defined as: not less than 85% of API releases within 30 min employing the dissolution conditions decribed therein 2,3 The Office of Generic Drugs (OGD), USFDA recommended dissolution medium is 0. 1 N HCl of 900ml, using USP‐II apparatus at 100 rpm with time points 10,20,30 & 45 minutes and specification for ofloxacin tablets is: not less than 80% (Q) should be dissolved in 45 mins.

MATERIALS AND METHODS

Solubility

The solubility of Ofloxacin as a function of pH is shown in Table 2. The solubility of ofloxacin was determined and all the solibility tests were conducted in triplicate. The media used were water media of various pH as 1.2, 4.5, 5.0, 6.0, 6.8, 7.2, 7.5, 8.2, and pH 9.2 and the temperatures were maintained at 37±0.5 ◦C.

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Table 2: Experimental solubility data (mg/ml) for ofloxacin and the corresponding dose/solubility (d/s) ratios for highest tablet strengths

S No pH pH after adittion of ofloxacin

Calculated solubility in mg/ml

Dose/solubility (D/S) ratios for highest tablet strengths (mg)

200 300 400 1 1.2 1.94 37.09 5.30 8.09 10.812 Water 7.15 2.66 75.19 112.81 150.38 3 4.5 5.20 14.62 13.69 20.53 27.38 4 5.05 6.0 16.49 30.82 46.22 61.635 6.0 6.32 5.04 39.68 59.52 79.376 6.8 6.80 4.57 43.76 65.64 87.53 7 7.2 7.15 2.85 70.18 105.26 140.35 8 7.5 7.30 2.80 71.43 107.14 142.869 8.2 7.87 3.64 54.95 82.42 109.9010 9.2 8.85 4.32 46.30 69.44 92.60

Formulation dissolution study

The release rate of Ofloxacin was determined using United States Pharmacopoeia (USP) Dissolution Testing Apparatus II (Paddle method). The dissolution test was performed in 900 ml of 0.1N HCl, in acetate buffer of pH 4.5 and simulated intestinal fluid pH 6.8, at 37 ± 0.5°C and 50 rpm. A sample (5 ml) of the solution was withdrawn

from the dissolution apparatus at intervals of 10, 20, 30 and 45 minutes and the samples were replaced with fresh dissolution medium. The samples were diluted to a suitable concentration with dissolution media. Absorbance of these solutions was measured at 294nm using a UV/Visible spectrophotometer. Cumulative percentage drug release was calculated using an equation obtained from a standard curve shown in table 3.

Table 3: Dissolution profiles of formulations of 200 mg, 300 mg and 400 mg of Ofloxacin

The present biowaiver criteria state that, in addition to similarity of dissolution profiles, the test and the comparator drug product should both be ‘‘rapidly dissolving,’’ which is defined as not less than 85% of API releases within 30 minutes, employing the dissolution conditions described therein. The same dissolution method was employed for evaluating randomly selected IR Ofloxacin drug products having a marketing authorization in USA and in India and we found that both formulations showing above 85% drug release within 30 minutes. Thus, it was found that the Ofloxacin drug products exhibited rapidly dissolving characteristics within the BCS limits.

DISCUSSION

Solubility

The USFDA defines ‘‘highly soluble drugs’’ exhibiting a dose/solubility (D/S) of <250 ml over the pH range 1–7.5, (3)(CDER, Guidance for Industry, 2000) while the EU and the recently revised WHO Guidelines limit the requirements to the pH range of 1–6.8. It was recently suggested that the USFDA should also redefine the solubility boundaries for BCS Class I (i.e high solubility and high permeability) to pH 1.2–6.8 (http://www.fda.gov/cder/foi/label/2007). At 250C, all tablet strengths conform to the criterion of 250 mL for the dose/solubility ratio at pH 6.8 and below (Table 1). At pH 7.5, the WHO recommended dose comply with this dose/solubility ratio criterion but higher tablet strengths do not. However, these data refer to 250C, not at 370C, as required by the Guidances3.

The solubility values found in the literature were not assessed under conditions specified for the Biopharmaceutics Classification System

(BCS) classification .Therefore new solubility determinations were carried out by us. The minimum solubility of Ofloxacin was about 2.66 mg/ mL. The corresponding dose/solubility (D/S) ratio, calculated for the highest commercially available tablet strength on the US market and on the WHO EML, was 150.38 mL or lower in the relevant pH range (Table 1). An API is ‘‘highly soluble’’ if its D/S ratio is below 250 mL 3. ( WHO, Proposal to waive in vivo bioequivalence requirements, 2006). Thus, ofloxacin can be regarded as ‘‘highly soluble”.

Permeability

According to BCS, a drug showing high solubility and high permeability is considered as Class‐1 drug. Ofloxacin is rapidly and well absorbed from the gastrointestinal tract. Oral bioavailability is almost 100% and a peak plasma concentration of 3 to 5 μg/mL is achieved 1 to 2 hours after a dose of 400 mg by mouth. Absorption may be delayed by the presence of food, but the extent of absorption is not substantially affected. The plasma half life ranges from 4 to 7 h in renal impairment values of 15 to 60 h have been reported. About 25% is bound to plasma proteins. Ofloxacin is widely distributed in body fluids, including the CSF and tissue penetration is good. It crosses the placenta and is distributed into breast milk. It also appears in bile.

There is limited metabolism to desmethyl and N‐oxide metabolites: demethylofloxacin has moderate antibacterial activity. Ofloxacin is eliminated mainly by the kidneys. Excretion is by tubular secretion and glomerular filtration and 65% to 80% of a dose is excreted unchanged in the urine over 24 to 48 h resulting in high urinary concentrations. Less than 5% is excreted in the urine as metabolites.

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From 4 to 8% of a dose may be excreted in the faces (USFDA Drug approved information26, 27.

Ofloxacin is an amphoteric drug with two protonation sites. Its pKas are 6.05 for the carboxylic group and 8.22 for the piperazine nitrogen 28. The log P value (octanol/water partition coefficient) is 0.33 29. At blood pH, 87% of the drug is in the zwitterionic neutral form HQ +/‐ 28 .This form of the molecule is the most hydrophobic and can readily diffuse through membrane lipids 30 . Fluoroquinolones could have a common transporter in the intestine and, according to their affinities, compete with each other for binding when coadministered 31. investigated the effect of P‐glycoprotein blockers on intestinal ofloxacin elimination in rats. P‐glycoprotein is an energy‐dependent drug‐efflux system located at several sites, particularly at the plasma apical membrane of intestinal cells 31, 32. This transport molecule seems to protect intestinal cells from plant alkaloids and other cytotoxic hydrophobic compounds 33. The stereoselectivity and saturability of intestinal ofloxacin secretion in vivo 34.

Ofloxacin is a moderately lipophilic quinolone with an octanol‐water partition coefficient of 0.41 at pH 7.0, 0.33 at pH 7.2, and 0.28 at pH 7.3 35 ,36.After administration of ofloxacin 200mg I.V. Infusion the ratios OF AUCCSF/AUCs (csf: cerebro spinal fluid & s: serum) and AUCCSF 0‐∞/AUCs0‐∞ there was a high level of penetration of ofloxacin into CSF (0.59 to 0.81 and 0.53 to 0.79, respectively 37.

Ofloxacin is excellently absorbed and has a biological half‐life of 3 to 3.5hr, high volume of distribution, predominant renal elimination, and only limited biotransformation38 . In vitro Caco‐2 assay results concluded that ofloxacin was classified as high permeable drug 39. In TC7 cells, ofloxacin displayed concentration‐dependent permeability and was actively absorbed 40.

BCS classification

Ofloxacin is ‘‘highly soluble.’’Data on its oral absorption and permeability are not fully conclusive but suggest this API to be a BCS Class III drug, with permeability properties approaching the border to BCS Class I. It should be noted that the cut‐off for ‘‘highly permeable’’ varies with regulatory authority. The FDA sets a limit for the fraction of dose absorbed of not less than 90%, the EMEA requires ‘‘high permeability’’ but does not define a limit for the fraction of dose absorbed and the WHO requires not less than 85% fraction of dose absorbed. Up to now, the FDA does not accept biowaivers for BCS Class III APIs, which would exclude ofloxacin from biowaiving. On the other hand, the recently revised WHO guidance extended the possibility of a biowaiver approval to BCS Class III APIs under certain conditions. Therefore ofloxacin is a candidate for biowaiver according to the WHO guidance.

Surrogate techniques for in vivo bioequivalence testing

Ofloxacin is ‘‘highly soluble’’ and the pure drug shows ‘‘very rapid dissolution’’. Furthermore bioinequivalence of ofloxacin formulations was reported neither in vivo nor in vitro and is unlikely to occur for this very soluble API. Hence, the stricter dissolution methodology for biowaiving of BCS Class III drugs according the WHO Guidance, that is, ‘‘very rapid dissolution’’ over the pH range of 1.2–6.8, should be capable of detecting poor quality of formulations. A caveat to the use of dissolution tests as surrogates for in vivo BE testing is that in vitro dissolution tests are not able to detect excipient influences on permeability and/or GI transit time which may cause bioinequivalence 3 .

Risks of bioinequivalence caused by excipients and/or manufacturing

Since no report of a bioinequivalent drug product has appeared in the accessible literature, the risk of bioinequivalence of ofloxacin IR dosage forms seems to be low. The risk of bioinequivalence caused by an excipient interaction is further reduced if the test product contains only excipients present in drug products having a MA in an ICH or associated country. The excipients present in a number of European countries are listed in Table 1. Patient risks associated with bioinequivalence of ofloxacin IR dosage forms can lead to decreased antibiotic efficacy. However, the risk of bioinequivalence

of ofloxacin IR dosage forms appears to be relatively low, especially if the test product is formulated only with excipients shown in Table 2 and complies with the criteria for ‘‘very rapidly dissolving.’’

ACKNOWLEDGEMENT

The authors gratefully acknowledge Mrs. Nilam Patel (Formulation & Development, Cadila Pharmaceuticals Ltd, Ahmedabad, India) for her contribution.

REFERENCES

1. Amidon GL, Lennerna SH. Shah VP, Crison JR. A theoretical basis for a Biopharmaceutics Drug Classification: The correlation of in vitro drug product dissolution and in vivo bioavailability. Pharm Res 1995; 12: 413–420.

2. Committee for Proprietary Medicinal Products (CPMP) London. The European Agency for the evaluation of Medicinal products, Evaluation of medicines for human use.. Note for guidance on the investigation of bioavailability and bioequivalence. Available from URL: 2001. http://www.emea.eu.int/pdfs/human/ewp/140198en.pdf.

3. U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER). Guidance for industry: Waiver of in vivo bioavailability and bioequivalence studies for immediate‐release solid oral dosage forms based on a Biopharmaceutics Classification System. 2000.

4. United States Pharmacopoeias, United States Phamacopoeial Convention. INC. Twin Brook Parkway. Rockville. 2004; p1355.

5. Lamp KC, Bailey EM, Rybak MJ. Ofloxacin clinical pharmacokinetics. Clin Pharmacokinet 1992; 22(1):32‐46.

6. U.S. Department of Health and Human services Food and Drug Administration, Center for Drug Evaluation and Research, 1998. Freedom of Information. Available from: http://www.fda.gov/cder/foi/label/2007/019735s058lbl.pdf

7. Parfitt K. Martindale, The Complete Drug Reference. Edited by The Pharmaceutical Press, London, 1999; 32nded.

8. Henry AO, Ikhuoria MA. Analytical profile of the fluoroquinolone antibacterials I Ofloxacin. African J Biotech 2008; 7: 670‐680.

9. Barbosa J, Berges R, Sanz‐Nebot V. Retention behaviour of quinolone derivatives in high‐performance liquid chromatography. Effect of pH and evaluation of ionization constants. J.Chromatogr A 1998 ;823: 411‐422.

10. Fabre D, Bressolle F, Kinowski JM, Bouvet O, Paganin F, Galtier M. A reproducible, simple and sensitive HPLC assay for determination of ofloxacin in plasma and lung tissue. Application in pharmacokinetic studies. J Pharm Biomed Ana 1994;12: 1463‐1469.

11. Volpe DA, Ciavarella AB, Asafu‐Adjaye EB, Ellison CD, Faustino PJ, Yu LX. Method suitability of a Caco‐2 cell model for drug permeability classification. AAPS Pharm Sci 2001;3: (S1).

12. Pickerill KE, Paladino JA, Schentag JJ. Comparison of the fluoro‐quinolones based on pharmacokinetic and pharmacodynamic parame‐ters. Pharmacother 2000; 20: 417‐428.

13. Bergan, T.; Pharmacokinetics of fluorinated quinolones. In: Andri‐ole VT, ed. The Quinolones. London, UK: Academic Press. 1988. 119‐152.

14. Castillo‐garit J A, Marrero‐ponce Y, Torrens F, Garcia‐domenech R. Predicting Caco‐2 Cell Permeability Using Atom‐Based Stochastic and Non‐Stochastic Linear Indices. J Pharm Sci 2008; 97: 1946‐1976.

15. Akerle JO, Okhamafe AO. Influence of oral co‐administered metallic drugs on ofloxacin pharmacokinetics. J Antimicrob Chemother 1991; 28: 87‐94.

16. Shimada J, Shiba K, Oguma T, Miwa H, Yoshimura Y, Nishikawa T, et al. Effect of antacid on absorption of the quinolone lomefloxacin. Antimicrob Agents Chemother 1992;36: 1219‐1224.

17. Cabarga MM, Navarro AS, Gandarillas CL, Dominguez‐Gil A. Effects of two cations on gastrointestinal absorption of ofloxacin. Antimicrob Agents Chemother. 1991; 35: 2102‐2105.

18. Polk RE, Healy DP, Sahai J, Drwal L, Racht E. Effect of ferrous sulfate and multivitamins with zinc on absorption of

160

Sakore et al.


3

ciprofloxacin in normal volunteers. Antimicrob Agents Chemother 1989;33: 1841‐1844.

19. Flor SD, Guay RP, Opsahl JA, Tack K, Matzke GR. Effects of magnesium‐aluminum hydroxide and calcium carbonate antacids on bioavailability of ofloxacin. Antimicrob Agents Chemother 1990; 34: 2436‐2438.

20. Verho, M., Malerczyk, V., Dagrosa, E., Korn, A. The effect' of food on the pharmacokinetics of ofloxacin. Curr Med Res Opin 1986; 10: 166‐171.

21. Dudley MN, Marchbanks C R, Flor SC , Beals B. The effect of food or milk on the absorption kinetics of ofloxacin. Eur J Clinical Pharmacol 1991; 41: 569‐571

22. Garrelts JC, Godley PJ, Peterie JD, Gerlach EH, Yakshe CC. Sucralfate significantly reduces ciprofloxacin concentrations in serum. Antimicrob Agents Chemother 1990;34: 9341‐9343.

23. Van S, Nix DE, Wiltop JH, Love JH, Spivey JM, Goldstein HR. Combined use of ciprofloxacin and sucralfate. The Annals Of Pharmacotherapy 1991;25: 578‐582.

24. Bruce AM,. Effect of Enteral Feeding with Ensure on Oral Bioavailabilities of Ofloxacin and Ciprofloxacin. Antimicrob Agents Chemother 1994; 38: 2101‐2105.

25. Flor SC. Bioequivalence of oral and intravenous ofloxacin after multiple –dose administration to healthy male volunteers. Antimicrob Agents Chemother 1993;37: 1468‐1472.

26. Lamp KC. Ofloxacin clinical pharmacokinetics. Clin Pharmacokinet 1992;22: 32‐46.

27. McMullin CM. The pharmacokinetics of once‐daily oral 400 mg ofloxacin in patients with peritonitis complicating continuous ambulatory peritoneal dialysis. J Antimicrob Chemother 1997;39:29‐31.

28. Furet YX, Deshusses J, Peche`re JC. Transport of pefloxacin across the bacterial cytoplasmic membrane in quinolone‐susceptible Staphylococcus aureus. Antimicrob Agents Chemother 1992;36: 2506–2511.

29. Hirai K, Aoyama H, Suzue S, Irikura T, Lyobe S, Mitsuhashi S. Isolation and characterization of norfloxacinresistant mutants of Escherichia coli K‐12. Antimicrob Agents Chemother 1986;30: 248‐253.

30. Gruber GU. Jaehde SF, Relationships between the chemical structure and the HPLC capacity factor of gyrase inhibitors and their metabolites in different methanol‐water eluents. Fresenius Z Anal. Chem 1988;330: 388–389.

31. Croop JM, Raymond M, Haber D, Devault RJ, Arceci PG, Houseman DE. The three multidrug resistance genes are expressed in a tissue‐specific manner in normal human tissues. Mol Cell Biol 1989;9: 1346–1350.

32. Thiebaut F, Tsuruo T, Hamada H, Gottesman MM, Pastan I, Willingham M C. Cellular localization of the multidrug‐resistance gene product P‐glycoprotein in normal human tissues. Proc Natl Acad Sci U. S. A. 1987; 84: 7735–7738.

33. Ince P, Elliott K, Appleton DR., Moorghen M, Finney KJ, Sunter JP, et al Modulation by verapamil of vincristine pharmacokinetics and sensitivity to metaphase arrest of the normal rat colon in organ culture. Biochem Pharmacol 1991;41: 1217–1225.

34. Lyndia R. intestinal elimination of ofloxacin enantiomers in the rat:evidence of a carrier‐mediated process. Antimicrob Agent Chemother 1996; 40: 2126–2130.

35. Ashby J, Piddock LJV, Wise R. An investigation of the hydrophobicity of the quinolones. J Antimicrob Chemother 1985;16: 805‐808.

36. Takacs NK, Jozan M, Hermecz I, Szasz G. Lipophilicity of antibacterial fluoroquinolones. Int J Pharm 1992;79: 89‐96.

37. Nau R. Pharmacokinetics of ofloxacin and its metabolites in cerebrospinal fluid after a single intravenous infusion of 400 mgs of ofloxacin. Antimicrob Agents Chemother 1994;38: 1849‐1853.

38. Hartmut I, Pharmacokinetics of ofloxacin after parenteral and oral administration. Antimicrob Agents Chemother 1987;31:1338‐1342.

39. Donna A, Volpe E. Permeability Classification of Representative Fluoroquinolones by a Cell Culture Method. AAPS Pharm Sci 2004; 6(2): Article 13.

40. Awadallah B, Wahl MA. Transport of ofloxacin enantiomers in the Caco‐2‐TC7 cell line. AAPS Pharm Sci 2002; 4 (S1).

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About SIPS Saraswati Institute of Pharmaceutical Sciences was established in 2006 under the auspices of Shree Saraswati Education Sansthan. The institute is situated in a green and clean environment at Dhanap, 10 kms away from Gandhinagar. The college is affiliated to Gujarat Technological University, approved by AICTE & PCI. The college possesses a very good infrastructure with highly equipped laboratories and state of the art equipments. SIPS conducts following courses :

• B.Pharm • M.Pharm (Pharmaceutics) • M.Pharm(Quality Assurance)

About Seminar A drug molecule has to pass through various phases before reaching market and all the disciplines of pharmaceutical sciences play a vital role in this process. Looking at the increasing number of biological targets identified for various diseases it is the need of the hour to not only have efficient and fast methods of chemical syntheses but also to have man power with good analytical skills along with a good working knowledge of specific screening techniques. Researchers have to keep themselves abreast of recent developments in the area of drug discovery and development. We have planned to have eminent speakers with ability to appeal to a multidisciplinary audience. Such symposiums are an essential part of an academic curriculum for motivating the young minds towards scientific research.

Objectives To bring together academicians and industrialists of multi-disciplinary areas for deliberations on recent research methodologies.

Symposium Schedule

24 - 9 - 2011 Registration and Inauguration 9:30-10:15 am Plenary Session Dr. M.R. Yadav 10:30-11:30 am Professor & Head, Department of Pharmacy , The Maharja Sayajirao Univ. of Baroda, Vadodara Title: Application of Spectrophotometric

Techniques in Pharmacy Field Dr. I.S. Anand 11:30-12:30 pm Professor and Head, Dept. of Pharmacology, Shree Sarvajanik Pharm. College, Mehsana. Title : Overview of Drug Discovery & Development Dr. ShitalKumar Zambad 12:30-1:30 pm AGM Department of Pharmacology , Torrent Research Centre. Gandhinagar. Title: GLP 1 Secretagogues: Advancing therapy for Type 2 Diabetes Lunch Break Dr. M.T. Chabbaria 2:30-3:30 pm Associate Professor, L.M. College of Pharmacy, Ahmedabad Title : Drug Design‐Serendipity to Success Mr. Somnath Sakore 3:30-4:30 pm Assistant Manager –CRO Cadila Pharmaceuticals Ltd. Ahmedabad Title: In Vitro In Vivo Co‐relations ‐ A tool in Drug Development

Valedictory

Symposium on

“Novel Technologies to Expedite Drug Discovery and Development”

Sept-24th, 2011

Organized by

Saraswati Institute of Pharmaceutical Sciences

At & Post Dhanap, Ta. & Dist. Gandhinagar: 382355

Tele.: 079-232731 01/ 31/51 Fax :079-23273111

E-mail: [email protected] URL : www.sips.org.in

In-Vitro-In-Vivo Correlation (IVIVC): A Tool In drug Development

Mr. Somnath SakoreCadila Pharmaceuticals Ltd

Symposium on Novel Technologies to Expedite Drug Discovery and

Development

Outline• Definition of IVIVC • Purpose of IVIVC• Levels of IVIVC• In vitro data • In vivo data• IVIVC models• IVIVC development• Predictability • IVIVC in drug development of extended release products• Issues• Factors to be consider for correlation development • Conclusion

ISSN 2229-5054 International Journal of Drug Formulation & Research Nov-Dec. 2010, Vol. 1 (iii) 324-348

Available online on www.ordonearresearchlibrary.org

324

International Journal of Drug Formulation & Research

LONG ACTING IN SITU GELLING VEHICLE FOR PHTHALMIC DELIVERY

Somnath Sakore1*, Sangita Kute2, Avinash Tekade2 , Bhaswat Chakraborty1,

1* - Cadila Pharmaceuticals Pvt.Ltd. 1389, Trasad road, Dholaka- 387810 Ahmedabad,

Gujrat, India.

2. Department of Pharmaceutics & QA,, R.C. Patel Institute of Pharmaceutical Education & Research, Shirpur- 425405, Dist: Dhule (M.S.), India.

Abstract

Objective of present study was to develop and characterize Pluronic F-127 (PF-127)/ Hydroxypropyl

methylcellulose (HPMC K4M) based in situ gelling vehicle for ophthalmic delivery of Ketorolac

tromethamine (KT). Various combinations of pluronic and HPMC K4M were studied to optimize the

system. The system was evaluated for various in vitro studies and in vivo ocular irritation study. The drug

release from optimized batch provides sustained release of KT over 6 h periods and showed excellent

ocular tolerance. The overall results of this study supports that the Pluronic/HPMC based vehicle could

be used for development of long acting ophthalmic drug delivery system.

Key words

Ocular In situ gelling system, Ketorolac tromethamine, Sustained release, Pluronic F-127, Hydroxypropyl

methylcellulose



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Introduction

The poor uptake of many ophthalmic drugs is mainly due to the rapid elimination process by tear

turnover. Another reason for the low bioavailability is the slow diffusion of water soluble drug [1]. A

large proportion of the topically applied drug in the form of solution is immediately diluted in the tear

film and excess fluid spills over the lid margin and the remainder is rapidly drained in to the

nasolachrymal duct. A proportion of the drug is not available for therapeutic action since it binds to the

surrounding extra orbital tissues. Due to these reasons corneal contact time is only about 1-2 min in

humans and an ocular bioavailability less than 10% [2, 3]. Nasolachrymal drainage also may cause

systemic toxicity of drugs. A high frequency of eye drops instillation is associated with patient non-

compliance. Inclusion of excess drug in the formulation in an attempt to overcome bioavailability

problems is potentially dangerous if the drug solution drained from the eye is systemically absorbed

from the nasolachrymal duct [8]. Due to poor ocular bioavailability, many ophthalmic drugs are applied

in high concentration which causes ocular and systemic side effects[4] .

A prolonged residence time would result in higher absorption and for some drugs a prolonged duration

of their therapeutic effects [5] .

Inserts can provide almost unlimited contact time and hold constant drug level during this time. On the

other hand, they may be expelled from the eye and can be difficult to put in and take out by older

patients.



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Use of mucoadhesive polymers is one of the suitable ways to increase the contact time and ultimately

improved bioavailability. The most common method to increase the residence of drug in the cul de sac is

enhancing the viscosity using polymers and by using suspension, ointments which shows longer

residence of drug in the eye better than solutions. But they may cause blurred vision and are usually

recommended at bed time (Robinson et al. 1995). Gel system is one of the better options for

conventional formulation but these are also difficult to administer for some patients [5] .

Limitations of all the conventional dosage form can be overcome by using in situ gelling systems, which

are conveniently instilled into the eye as a drop, which upon exposure to physiological conditions will

shift to the gel phase. This will increase the ocular residence time and ocular bioavailability.

The sol-gel transition occurs as a result of a chemical / physical change induced by the physiological

environment. The transition could be induced by a shift in the pH as for cellulose acetate phthalate [6] ,

polycarbophil [7] and carbopol .[8,9].

Temperature induced gelation was observed with poloxamer 407[2,9] . and xyloglucan [10]. Cation

induced gelation was other mechanism observed with gelrite [11] and alginic acid [12, 13] .

A wide range of synthetic and natural polymeric hydrogels have been widely used for drug delivery

carriers, tissue engineering templates and medical devices. Amongst them stimuli sensitive synthetic

hydrogels have been paid much attention for their unique properties such as thermo-sensitivity [14,15] .

Due to the unique thermo reversible gelation properties, PF-127 became one of the most extensively

investigated temperature responsive materials[16] . PF-127 consist of polyoxy ethylene units (70%) and

polyoxy propylene (30%) [17]. The sol-gel transition is induced by an increase in temperature, but

depends on the concentration of the additives such as salts and polymers [5, 18] .The phenomenon of

thermo gelling is characterized by a sol-gel transition temperature. Below this temperature, the sample

is fluid allowing a comfortable and precise delivery and above this transition temperature, the solution



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becomes gel according to the increment of local temperature [2]. The thermal gelation results from the

interaction between the different molecules of PF-127. The increment of the temperature modifies the

hydration spheres around the hydrophobic units which in turn induces higher interactions between

different units [2, 19].

At low temperature in an aqueous solution, a hydration layer surrounds PF-127 molecules. However, the

hydrophilic chains of the co-polymer become desolvated as the temperature get increased, because of

the breakage of the hydrogen bonds that had been formed between the solvent and these hydrophilic

chains. This phenomenon favors hydrophobic interactions among the polyoxy propylene domains and

results into gel formation. Hydroxyl groups become more accessible because of hydration [20]. It is

thought that the gel is miceller in nature. A liquid miceller phase is stable at low temperature but

transforms into the cubic structure by increasing the temperature. Above the critical miceller

temperature concentrated pluronic copolymers self assembled and hexagonal packed cylinder is formed

which is partly due to lower critical solution temperature (LCST).

However, when the physically formed Pluronic copolymer gel is exposed to a large volume of aqueous

solution, rapid dilution of the copolymer concentration occurs with immediate losing its gel structure

and integrity [21]. PF-127 gels containing various drugs have been used for treating patients with ocular

condition [2, 9, 22] .

At a concentration of 18% (w/w) or higher in aqueous solution, PF-127 is transformed from a low

viscosity solution to a gel under the ambient temperature. But this lower concentration solution will lose

the gelation ability after dilution by lachrymal fluid. Thus, 25% (w/w) PF-127 should be used to ensure

the completion of the phase transition under the physiological condition [9].

Objective of the present work was to formulate the in situ gelling system for sustained ocular delivery of

KT using PF-127 as thermosensitive polymer and HPMC K4M as viscosity enhancing agent to sustain the



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release of KT. KT is a non-steroidal anti-inflammatory drug (NSAID), which has potent analgesic and anti-

inflammatory activity due to prostaglandin related inhibitory effect of drug. KT is effective in inhibiting

postoperative inflammation of eyes. It is also effective in reducing conjunctivitis with no alteration of

corneal opacity. It does not facilitate herpes simplex, bacterial or fungal infection of the eye.

PF-127 alone has been widely explored by many researchers, therefore, in the present study, various

combinations of PF-127 and HPMC K4M were prepared to identify the suitable vehicle for in situ gelling

delivery of Ketorolac tromethamine.

Materials and Methods

Materials

The drug ketorolac tromethamine was obtained as a gift sample from Cadila pharmaceuticals,

Ahmedabad, India. Pluronic F 127 and HPMC K4M was a kind gift from Sigma Aldrich Chemie, Germany

and Colorcon, USA respectively. Mucin Type III was also supplied as a gift sample by Sigma Aldrich

Chemie, Gmbh, Germany All other materials, including those obtained from standard commercial

suppliers, were of pharmaceutical grade.

Methods

Preparation of PF-127/HPMC formulations

The formulations were prepared using cold method [2]. Drug and isotonicity adjusting agent were

dissolved in distilled de-ionized water and kept in refrigerator. After cooling, required quantity of PF-127

was added and kept at 4°C with periodical stirring to ensure complete dissolution. Solution of PF-127 in

the concentrations of 15, 18 and 25% were prepared and denoted as PF-15, PF-18 and PF-25

respectively.



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Formulation containing PF-127 and viscosity enhancing agent were also prepared. HPMC of different

grades were used as a viscosity enhancing agent. In this case, required amount of viscosity enhancing

agent was dissolved in hot water with continuous stirring for complete dissolution of HPMC. The drug

and isotonicity adjusting agents were then added. The resulting polymeric solutions were kept in

refrigerator. After cooling, the required amount of PF-127 were added in HPMC solutions and kept in

refrigerator at 4°C for complete dissolution of PF-127. The pH of all the formulations was measured and

found in the range of 6.5-7.2. The concentration of isotonicity adjusting agents that rendered the

formulation isotonic with eye fluid was calculated by sodium chloride equivalent method. Benzalkonium

chloride was added as a preservative.

In order to identify the composition suitable for use as in situ gelling, aqueous solutions of PF-127 and

HPMC with different concentration and grades were prepared and evaluated for gelling capacity and

transparency at physiological conditions. The formulations were then subjected to terminal sterilization

by autoclaving at 121°C and 15 psig for 20 min.

The gelling capacity was measured by visual method. 100 µl samples was placed in a vial containing 2 ml

of artificial tear fluid freshly prepared and equilibrated at 35°C and then visually assessing the gel

formation and noting the time taken for gel formation [9]. The composition of artificial tear fluid was

sodium chloride 0.670 g, sodium bicarbonate 0.200 g, calcium chloride 0.008 g, and purified water

q.s.100g [23] .

In Vitro Evaluation

Measurement of gelation temperature

i) Sample solution (10 ml) and magnetic bead were put into a transparent vial that was placed on a

magnetic stirrer. Thermometer was immersed in the sample solution and solution was heated with



330

continuous stirring at 100 rpm. The temperature was determined as a gelation temperature at which

the magnetic bead stopped moving due to gelation. Each sample measured was at least in triplicate [9] .

ii) The gelation temperature of poloxamer/ HPMC was measured in triplicate by vertical method. 1 ml of

poloxamer / HPMC solution was added into a 5 ml vial and kept into the incubator. The gelation was

measured by a visual inspection [24] .

In vitro drug release study

In vitro release of Ketorolac tromethamine was carried out by adding 1 ml of Ketorolac tromethamine

(0.5%) loaded poloxamer/HPMC solution into 5ml vials. The vials were then kept for gelation at 35±0.5°C

in an incubator. After gelation the above vials were filled with 2 ml of dissolution media as simulated

tear fluid maintained at same temperature. The temperature and stirring rate was maintained as

35±0.5°C and 50 rpm, respectively. Aliquots of 1 ml were withdrawn from release medium at specific

time interval and replaced by an equal volume of fresh medium at each sampling time. Aliquots were

diluted with simulated tear fluid and the amount of drug released was determined by using UV

spectrophotometer at 322.5 nm [24] .

Rheological studies

The rheological properties were determined using Brookfield viscometer (RVDV-E, Middleboro, USA).

The viscosity was measured at different rpm. In order to simulate the physiological disposition of gels

more literally, the polymer solution were diluted by simulated tear fluid (STF) in a ratio of 40:7 [25] and

temperature was maintained at 35±0.5°C.

In vitro mucoadhesion Study (Modified Balance Method)

The mucoadhesive properties of poloxamer/ HPMC in situ gelling system was studied by using mucin

pellet with modified balance method [26]. Mucin discs were prepared by using IR press with 13 mm die



331

and a compression force of 10 tones, applied for 30 s. The experimental techniques used for

determining the bioadhesive force has been derived from a previously published method [9,27]. The

experimental setup is presented in Fig. 1.

The Teflon cylinder (A) was suspended by means of a thin copper wire (E) to the left of the balance

.The mucin pellet (G) was then fixed to the Teflon cylinder. The balance was then leveled and balanced.

One ml of polymer solution(C) was added onto the sample cell, which was placed on a height-adjustable

pan (B). The height of the pan (B) was adjusted quickly to make the polymer solution just come into

contact with the mucin pellet. Then, the balance was disturbed with a weight (5g) put onto the left end

of the balance bar (F) so that the contact was made with the force of the Teflon cylinder (C) (5g). After

10 min. contact, the weight was removed, so that the balance (A) could regain balance. Then, the weight

was put into the balance pan (F). The weight in the pan (F) kept increasing until the gel and the mucin

pellet were detached. Bioadhesive force, the detachment stress (N) was determined from the minimal

weights that detached the gel and the mucin pellet. The mucin pellets were changed for each

measurement. To evaluate the changes in bioadhesive force after instillation and mixing with the tear

fluid, the bioadhesive force measurements were taken at 35±0.5ºC, pH 7.4 and after diluting the

formulations with STF.

Accelerated Stability Testing

The stability study of formulated in situ gelling system was carried out according to ICH

guidelines. A sufficient quantity of sol to gel system in amber colored bottle was kept in stability

chamber (Remi Motors, Mumbai, India) at 40 ± 0.5°C, 75% RH, at room temperature and 2-8°C

for three months. The effects of temperature and time on the formulation properties were

evaluated for assessing the stability of the prepared formulations. The different parameters that

were studied are gelation temperature, physical appearance, drug content and in vitro drug



332

release.

Ocular Irritation Study

Ocular irritation studies were performed according to the Draize technique [28]. New Zealand albino

rabbits weighing 1.5 kg were used for the test. The polymeric solution was instilled into the rabbit’s eyes

five times a day for 7 days. The rabbits were observed periodically for conjunctiva, iris, corneal changes

and irritation. Evaluation was carried out according to the Draize technique.

Results and Discussion

Optimization of in situ gelling vehicle

It has been found that the viscosity of pluronic /HPMC solution was significantly greater than that of

individual carbopol and pluronic solution at each rpm (shear rate). This observation can be explained by

the formation of crosslinking between two polymers. The above results and inferences drawn conclude

that the gel strength of the polymer solution in the physiological condition can be enhanced

significantly. By combining the two individual solutions, it was observed that sample preparation 1.5%

HPMC and 13% pluronic solution could flow freely at non physiological condition. Thus, without

increasing the concentration of individual polymer solution this mixed vehicle may be administered into

the eye as drop and forms strong gel following the sol to gel transition. The results suggest that the

combined polymer solution may have more strength to withstand the low shear forces likely to be

encountered in the cul de sac of the eye as well to prolong the residence time of the drug in the eye.

It was observed that the pluronic solution with concentration 15, 18 and 25% w/w forms the gel at non

physiological condition, does not observed free flowing properties at non physiological conditions. From

the results, it was found that the thermosensitivity of the pluronic gel is highly concentration

dependent. It was found that the pluronic concentration 13% w/w, the solution exhibited sol (liquid)

state at both non physiological and physiological condition. These observations suggest that the



333

concentration of 14%w/w exhibits liquid at non-physiological condition and gels at physiological

condition. Thus, the optimum concentration used as in situ gel system study as 13-15% w/w of PF-127

and 1-1.5% w/w of HPMC concentration. Optimum concentration of HPMC K4M and PF-127 was found

to be 1% and 13% w/w respectively for in situ gelling system at non physiological condition. (Table-1)

Gelation of HPMC solutions is primarily caused by the hydrophobic interaction between molecules

containing methoxy substitution. At low temperatures, the macromolecules are hydrated, and there is

little polymer–polymer interaction other than simple entanglement. As the temperature is raised, the

polymers gradually lose their water of hydration, which is reflected by a decline in relative viscosity.

Eventually, when sufficient but not complete dehydration of the polymer occurs, polymer–polymer

associations take place, and the system approaches an infinite network structure, as reflected

experimentally by a sharp rise in relative viscosity [29].

Effect of PF-127 concentration on gelation temperature

Temperature induced gelation was not observed for PF-127 below 12% w/w. Higher PF-127

concentration lead to faster gelation and lower the gelation temperature (Fig.2).

The phase transition temperature increases with decreasing concentration and all preparation had a

transition temperature below 35°C. Pluronics being nonionic, polyoxyethylene-polyoxypropylene-

polyoxyethylene triblock copolymers, aggregate into micelles at 34°C. Micellization is due to the

dehydration of the polymer blocks with temperature. It has been shown that at higher PF-127

concentrations the gel is more entangled. The gel formation is a result of micellar enlargements and

packing. As a result of these micelle entanglements, they cannot separate easily from each other, which

accounts for the rigidity and high viscosity of gel containing high concentrations of pluronic (Fig. 3) [ 2,

30,31].



334

Evaluation of formulations

The clarity, drug content, pH of the formulations underwent rapid phase transition in to gel at

35°C. There observed no effect of terminal sterilization by autoclaving on the gelling capacity. The

haziness that was observed after autoclaving (due to precipitation of HPMC at elevated temperature)

was found to be disappeared and it regained its original clarity after cooling.

Rheological studies

The formulation without drug exhibited pseudoplastic behavior as decrease in viscosity was observed

with increased rpm. Addition of drug is not affecting the rheological behavior. After dilution with

artificial lachrymal fluid at 35±0.5°C, the viscosity of the formulation significantly increased. HPMC

increases the viscosity of the preparation and decreases the amount of pluronic required for gelation.

HPMC of high molecular weight dissolves in water and yield much more viscous solutions compared

with 25% w/w PF-127 gel without additives (Fig. 4).

This might improve the patient compliance. The administration of ophthalmic preparation should have

as little effect as possible on the pseudoplastic character of the precorneal film [23, 32] , since the ocular

shear rate is very high, ranging from 0.03s-1 during inter-blinking to 4250-28500 s during blinking [33].

Viscoelastic fluids with a viscosity that is high under low shear rate and low under high shear rate

conditions are often preferred.

In vitro drug release

The release of 0.5% KT from gel formulations containing 15, 18 and 25% of PF-127 was studied at

35±0.5°C (Fig.5).



335

The results indicated that as the concentration of PF-127 increased, the amount of the drug released

decreased. The reason behind this is the structure of the gel which functioned as an increasingly

resistant barrier to drug release as the concentration of PF-127 increased. The mechanism for such

enhanced resistance may be due to reduction in the number and dimension of water channels and to

the increase in the number and size of micelles within the gel structure [34]. As the concentration

increases, the inter-micellar distance becomes shorter, leads to greater numbers of cross-links between

neighboring micelles leading to higher viscosity and lower rate of drug release [35, 36]. These results are

in agreement with those reported by El-Kamel (2002).

The release profiles of KT are depicted in Fig.6.

For the formulation (F1) containing 25% w/w PF-127, about 15.5% of KT was released into the medium

after 30min, and then release rate became slower, where 83% of the drug was released after 6 h. The

formulation F3 containing 13% w/w PF-127 and HPMC 1.5% had a sustained release trend as compare to

plain 15 % PF-127, which may be due to the HPMC which retards the drug release and gives more

sustained release pattern, at low concentration of PF-127. It releases almost 95% of the drug after 6 h.

In case of formulation F4, only 13% of drug was released into the medium after 30 min and

approximately 89.7 % after 6 h. Formulation F4 follows similar pattern of sustained release as that of F1

containing high concentration of PF-127. The formulation F7 had a similar release trend as that of

formulation F3 and releases about 35.7% in 30 min and about 98.9 % after 6 h (Fig. 6). The results

demonstrated that the formulation F4 had relatively better sustained release effect and can be used as

an ophthalmic sustained release drug delivery system.

Mucoadhesion study



336

In this investigation, mucoadhesive force means the force required to detach the gel from the mucin. The

mucoadhesion capacity (Fig.7) of F4 was found to be 3.7 folds more than PF 18 solution and about 24

folds more than that of F1. Addition of HPMC increases the mucoadhesive force of polymer solution.

Ocular tolerance

The result of ocular irritation studies indicates that the PF-127 / HPMC containing KT solution does not

present any local irritation after topical application and shows excellent ocular tolerance. No ocular

damage or abnormal clinical signs to the cornea, iris, or conjunctivae were observed. Average score was

found to be 0 (Table 2).

Stability studies

Stability study was carried out on formulation F4 for 1 month at 45ºC and 75% relative humidity (RH).

The formulation was found to be clear with no change in pH, drug content (97-99%), viscosity and in

vitro gelling capacity.

Conclusion

In conclusion, PF-127 formulations of KT can be used as liquid drops for instillation into the eye, which

upon exposure to physiological condition will shift to the gel phase. This formulation also showed

excellent ocular tolerance without any irritation. On the basis of in vitro results, KT formulation

containing 1.5% HPMC and low concentration of PF-127 (14%) showed potential for use as ocular

delivery system with improved bioavailability. All the results support that the solution of pluronic and

HPMC in combination can be a promising in situ gelling vehicle for ophthalmic delivery.



337

References

1. Edsman, K., Carlfors, J., Harju, K., Int.J.Pharm. 1996, 137, 233-241.

2. El-Kamel, A.H., Int.J.Pharm. 2002,241, 47-55.

3. Lee, VHL., In: Mitra, A.K. (Ed.), Ophthalmic Drug Delivery Systems, New York, Marcel

Dekker Inc. 1993, 59–81).

4. Urtti, A., Pipkin, J.D., Rork, G. , Reta, A.J., Int. J. Pharm. 1990 61, 235–240.

5. Edsman, K., Carlfors, J. ,Petersson, R., Eur. J. Pharm. Sci. 1998,6, 105–112.

6. Gurny. R., Boye , T., Ibrahim, H., J. Control. Release. 1985, 2, 353-361.

7. Middleton, D.L. , Robinson, J.R., STP Pharm. Sci. 1991, 1, 200-206.

8. Srividya, B., Cardoza, R.M., Amin, P.D., J. Control. Release. 2001,73 , 205–211.

9. Qi, H., Chen, W., Huang, C., Li, L., Chen, C., Li, W. , Wu, C., Int. J. Pharm. 2007,337,

178–187.

10. Mayazaki, S., Suzuki, S., Kawasaki, N., Endo, K., Takahashi, A., Attwood, D., Int. J.

Pharm. 2001, 229, 29-36.

11. Carlfors, J., Edsman, K., Petersson, R. , Jornving, K., Eur. J. Pharm. Sci. 1998, 6, 113-

119.

12. Sechoy, O., Tissie, G., Sebastian, C., Maurin, F., Driot, J.Y., Int. J. Pharm.2000, 207,

109-116.

13. Cohen, S., Lobel, E., Trevgoda, A., Peled, Y., J. Control Release.1997, 44,201-208.

14. Jeong, B.M., Kim, S.W., Bae, Y.H., Adv. Drug Deliver. Rev. 2002, 54, 37–51.

15. Drury, J.L., Mooney, D.J., Biomaterials.2003, 24, 437–51.

16. Wen-Di-ma, Hui, X., Chau, W., Shu, F. N. , Pan, W., Int. J. Pharm.2007, 350, 247-256.

17. Lin, H.R. , Sung, K.C., J. Control. Release.2000, 69, 379–388.



338

18. Gilbert, J.C., Hadgraft, J., Bye, A. , Brookes, L.G., Int. J. Pharm. 1986, 32, 223-228.

19. Dumortier, G., Grossiod, J.L., Zuber, M., Couarraze, G., Chaumeil, J.C., Drug Dev. Ind.

Pharm. 1991, 17, 1255– 1265.

20. Escobar-Chávez, J.J., López-Cervantes, M., Naïk, A., Kalia, Y.N., Quintanar-Guerrero,

D., Ganem-Quintanar, A., J. Pharm. Pharmaceut. Sci. 2006, 9, 339-358.

21. Chun, K.W., Lee, J.B., Kim, S.H., Park, T.G., Biomaterials.2005, 26, 3319–3326.

22. Mengi, S.A., Deshpande. S.G., J. Pharm. Pharmacol. 1995, 47 ,997–1001.

23. Liu, Z., Li, J., Nie, S., Liu, H., Ding, P. , Pan, W., Int. J. Pharm.2006, 315, 12–17.

24. Cho, K.Y., Chuno, T.W., Kim, B.C., Kim, M.K., Lee, J.H., Wee, W.R., Cho, C.S., Int. J.

Pharm. 2003,260, 83–91.

25. Wei, G., Xu, H., Ding, P.T., Li, S.M.,Zheng, J.M., J. Control. Release.2002, 83, 65–74.

26. Jones. D.S., David, W. A. , Brown, A.F., Int. J.Pharm. 1997,151, 223-233.

27. Koffi, A.A., Agnely, F., Ponchel, G., Grossiord, J.L., Eur. J. Pharm. Sci. 2006, 27, 328-

335.

28. Draize, J.H., Woodard, G., Calvery, H.O., J. Pharmacol. Exp. Ther. 1944,82, 377-390.

29. Eve, R., Jean-Christophe, L., Eur. J. Pharm. Biopharm.2004, 58, 409–426.

30. Cabana, A., Ait-Kadi, A., Juhasz, J., J. Colloid Interf. Sci.1997, 190, 307–312.

31. Jain, N., Aswal, V., Goyal, P., Bahadur, P., J. Phys. Chem.1998, 102, 8452–8460.

32. Bothner, H., Waaler, T., Wik, O., Drug Dev. Ind. Pharm. 1990, 16, 755-768.

33. Kumar, S., Himmelstein, K.J., J. Pharm. Sci.1995, 84, 344–348.



339

34. Schmolka, I.R., In: Tarcha PJ (Ed.), Polymers for Controlled Drug Delivery CRC Press,

Boca Raton, FL. 1991 , 189–214.

35. Bharadwaj, R., Blanchard, J., J. Pharm. Sci. 1996, 85, 915–918.

36. Alexandridis, P., Hatton, T. A., Colloid Surf.1995, 96, 1–46.



340

Tables and Figure – Captions and legends

Batch code Pluronic127F

(%)

HPMC E50 LV

(%)

HPMC K4M

(%)

Gelling

capacity a

F1 25 _ _ +++

F2 15 2 _ +++ (*)

F3 13 1.5 _ ++

F4 14 1.5 _ +++

F5 12 _ 1 +

F6 13 _ 1 +++

F7 15 1 _ +++

F8 12 2 _ +

(*): The polymer solution form stiff gel at room temperature only.

a + weak gelation;

+ + gelation immediate, remain for few hours;

+ + + gelation immediate, remain for extended period



341

Table 1 – Gelation Studies of Different Formulations

Formulation

Code

Eye Irritation Score at different time (h)

½

Cj I Co

1

Cj I Co

2

Cj I Co

4

Cj I Co

24

Cj I Co

48

Cj I Co

72

Cj I Co

F4 (Blank)

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

F4

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

Cj – Conjunctiva , I – Iris , Co – Cornea.

Average score = 0



342

Table 2- Ocular irritation test

Fig 1 - Mucoadhesive force measurement device – Modified balance- A- Teflon cylinder,

B- Height adjustment pan, C- Gel, D- Sample cell, E- Thin copper wire, F- Pan,

G- Mucin pellet



343

Fig2- Effect of different concentration of PF-127 on phase transition



344

Fig. 3- Behavior of PF-127 /HPMC solution at different temperature



345

Fig .4 - Effect of temperature on viscosity after dilution using simulated tear fluid (STF)



346

Fig. 5 - Release profile of KT from gel formulations containing various concentrations of

PF-127



347

Fig. 6 - In vitro release studies of Pluronic / HPMC formulations



348

Fig. 7- Mucoadhesive strength of different formulations

Sangita D.kute* et al. /International Journal Of Pharmacy&Technology

IJPT | June 2010 | Vol. 2 | Issue No.2 | 118-145 Page 118

IJPT ISSN: 0975-766X Available Online through Review Article

www.ijptonline.com FORMULATION APPROACHES IN OCULAR DRUG DELIVERY SYSTEM

Sangita D.kute*, Somnath C. Sakore1, Bhaswat S. Chakraborty2

SCSSS’s College of Pharmacy, Shirur-412210, Dist: Pune, Maharashtra (India) 1,2 Cadila Pharmaceuticals Limited,1389, Trasad road, Dholka- 387810 ,Ahmedabad, Gujrat (India)

Email: [email protected]

Received on 29-03-2010 Accepted on 22-04-2010

Abstract:

The bioavailability of conventional ophthalmic solutions is very poor due to efficient

protective mechanisms of the eye, blinking, reflex lachrymation and drainage which remove

rapidly various foreign substances including drug from the surface of the eye. Frequent instillation

of drug solution is necessary to maintain a therapeutic drug level in the tear or at the site of action

but the frequent use of highly concentrated solution may induce toxic side effects due to systemic

absorption of drug through nasolachrymal drainage. In recent years there has been significant

efforts directed towards the development of new systems for ophthalmic drug delivery. This

review focuses on recent literature regarding mucoadhesive systems, vesicular systems, semisolid

hydrogel and in situ gelling system. Moreover, attempt has been made to explore the applicability

of numerous polymers for ocular drug delivery system and also includes a detailed account on

various recent strategies that are developed and under development stage so far.

Key words: Colloidal system; Hydrogel; In situ gel ;Iontophoresis; Mucoadhesive system;; Ocular

inserts;.

1. Introduction

Many regions of the eye are relatively inaccessible to systematically administered drugs and

as a result, topical drug delivery remains the preferred route in most cases. Drug may be delivered

to treat the precorneal region for such infections as conjunctivitis and blepharitis, or to provide

intraocular treatment via the cornea for diseases such as glaucoma and uveitis [1]. Various



approaches that have been attempted to increase the bioavailability and the duration of therapeutic

action of ocular drugs can be divided into two categories. The first is based on the use of the drug

delivery systems, which provide the controlled and continuous delivery of ophthalmic drugs. The

second involves, maximizing corneal drug absorption and minimizing precorneal drug loss.

The bioavailability of ophthalmic drug is however, very poor due to efficient protective

mechanisms of the eye, blinking, baseline and reflex lachrymation and drainage remove rapidly

foreign substances, including drug, from the surface of the eye as shown in Fig.1. Moreover, the

anatomy, physiology and the barrier function of the cornea compromise the rapid absorption of

drug [2]. Frequent instillation of the eye drop is necessary to maintain a therapeutic drug level in

the tear film or at the site of action but the frequent use of highly concentrated solution may induce

toxic side effects [3].

Moreover, nasolachrymal drainage is also a major route to enter the circulatory system for

drugs that applied through topical administration. For potent drugs, the systemic exposure through



nasolachrymal drainage after topical administration can be sufficiently high to cause systemic

toxicity [4].

To enhance the amount of the active substances reaching the target tissue or exerting a

local effect in the cul de sac, the residence time of drug in the tear should be lengthened.

Moreover, once a day formulation should improve patient compliance. Recently, controlled and

sustained drug delivery has become the standard in modern pharmaceutical design and an intensive

research has been undertaken in achieving reliable, safety and feffective product. [5]. Numerous

strategies were developed to increase the bioavailability of ophthalmic drugs by prolonging the

contact time between the drug and cornea/ conjunctival epithelium. The use of a water soluble

polymer to enhance the contact time and possibly also the penetration of the drug was first

proposed by Swan [6].

Viscous semisolid preparations such as gels and ointments, proved a sustained contact with

the eyes but they cause a sticky sensation, blurred vision and induce reflex blinking due to

discomfort or even irritation [7]. Films, erodible and nonerodible inserts, rods and shields are the

most versatile drug delivery systems aimed at remaining for a long period of time in the front of

the eye. These systems sustained and control drug release and thus avoid pulsed entry.

Another approach has been the application of in situ gelling system or phase transition

system [8,9,10]. A further approach to optimize the ocular dosage forms was the implementation

of the mucoadhesive concept which was successful in buccal and oral application.

2. Ophthalmic disorders

Conditions treated by the topical application of the drugs include:

Glaucoma: The build up of pressure in the anterior and posterior chambers of the choroid layer that

occurs when the aqueous humour fails to drain properly.

Conjunctivitis: Inflammation of the conjunctiva which may be caused by bacterial and viral

infection, pollen and other allergens, smoke and pollutants.

Dry eye syndrome: An inadequate wetting of the ocular surface.



Keratitis: Inflammation to cornea, caused by bacteria, viral, or fungal infection.

Iritis (anterior uveitis): Commonly has an acute onset with the patient suffering pain and

inflammation of the eye.

Other conditions include the ophthalmic complications of Rosacea, blepharitis (inflammation of

the lid margins), chalazia (Meibomian cysts of the eyelid), and corneal ulcer.

3. Ophthalmic drug delivery

The most common method of ocular drug delivery is the instillation of drops into the lower

cul-de-sac. Eye drops provide pulsed entry of the drug followed by rapid decline in drug

concentration, approximate to first order kinetics [11]. This form also have disadvantages; the very

short time the solution stays at the eye surface, its poor bioavailability (a major portion i.e. 75% is

lost via nasolacrimal drainage), the instability of the dissolved drug, and the necessity of using

preservatives. The physiological factors attributing to poor bioavailability of eye drops shown in

figure 1.

Due to low viscosity such drops are usually drained quickly, aided by blinking reflex, and

the precorneal region returns to the normal resident of around 7µl.The retention of a solution in the

eye is influenced by viscosity, hydrogen ion concentration, the osmolality and the instilled volume.

To enhance the bioavailability and corneal contact time the polymers are added to ophthalmic

solution and suspensions to increase the viscosity of the vehicle. It has been reported that an

increase in the corneal penetration of a drug is at a maximum if the viscosity of the eye drop

solution is about 15 to 150 mPa s (cp). Any increase in viscosity would have less effect on the

drainage rate and tear film thickness and has been implicated with interference of vision and

resisting movement of the eye lid Extensive work has been done to prolong ocular retention of

drugs in the solution state by enhancing the viscosity or altering the pH of the solution [4-6].

3.1 Mucoadhesive polymers

The threshold required for successful mucoadhesion is a molecular weight of at least

100,000 Da. Excessive crosslinking in the polymer, however, decreases the chain length available



for interfacial penetration. Also excessive formation of interchain physical entanglement and

hydrogen bonding within the polymer itself can lead to confirmation hindering polymer diffusion

into the mucus layer [12,13]. Many high molecular weight polymers with different functional

groups (such as carboxyl, hydroxyl, amino and sulphate) capable of forming hydrogen bonds and

not crossing the biological membranes have been screened as a possible excipient in ocular

delivery system [14]. Various viscosifying polymers screened for ocular mucoadhesive capacity

are given in Table 1.

Table: 1

Viscosifying polymers screened for ocular mucoadhesive capacity.

Polymers Charge Mucoadhesive capacity Poly (acrylic acid) neutralized A +++

Carbomer (neutralized) A +++

Hyaluronan A +++

Chitosan C ++

Sodium carboxymethyl cellulose A ++ (+)

Sodium alginate A ++

Pectin A ++ (+)

Xantan gum A +

Xyloglucan A +

Scleraglucan A +

Poloxamer NI + (+)

Charge- A: anionic, C: cationic, NI: nonionic

Mucoadhesive capacity- + + +: excellent, + +: good, +: poor / absent



4.Hydrogel, In situ gelling system

Aqueous gel (hydrogel) consists of high molecular weight, hydrophilic, crosslinked

polymers or co-polymers that form a three- dimensional network in water. These gels have been

shown to combine significantly longer residence times in the cul-de-sac with increased drug

bioavailability [11]. Kim et al define the hydrogels as polymers which have the ability to swell in

water or aqueous solvents, and induce a liquid-gel transition [15]. The efficacy of ophthalmic

hydrogel is mostly based on an increase of ocular residence time via enhanced viscosity and

mucoadhesion properties. In particular, in situ gelling systems improve bioavailability and

decrease the side effects induced by the systemic absorption of topically applied ophthalmic drugs

[16]. Typical gelling agents include cellulose derivatives, polyvinyl alcohol, hyaluronic acid and

carbomer. In situ gels are promising ocular drug delivery systems since they are conveniently

dropped into the eye as a liquid where after they undergo a transition into a gel as a result of

special physical / chemical changes (for example pH, temperature, and a specific ion) in their

environment; in this case a cul-de-sac [17]. Due to their elastic properties hydrogels resist ocular

drainage leading to longer contact times. Hydrogel is the mot common method of improving the

ocular availability of drugs to increase precorneal residence time.

Qi et al. developed a thermosensitive in situ gelling and mucoadhesive ophthalmic drug delivery

system containing puerarin based on poloxamer analogs and carbopol. The incorporation of

carbopol 1342P NF not only did not affect the pseudoplastic behavior with hysteresis of the

poloxamer analogs solution and leads to a higher shear stress at each shear rate, but also enhanced

the mucoadhesive force significantly and sustained the drug release over a period of 8 h [17].

Kamel et al. developed a pluronic F 127 based formulations of timolol maleate (TM) aimed at

enhancing its ocular bioavailability. In vivo study showed that the ocular bioavailability of TM,

measured in albino rabbits, increased by 2.5 and 2.4 fold for PF127 gel formulation compared with

0.5% TM aqueous solution [18]. The mixture of 0.3% carbopol and 14% pluronic solutions



showed a significant enhancement in gel strength in the physiological condition. The pilocarpine

release was extended upto 6h by using this system [19].

Miyazaki et al. were developed a thermoreversible gel formed in situ by aqueous solution

of an enzyme degraded xyloglucan polysaccharide for sustained release vehicle for the ocular

delivery of pilocarpine hydrochloride. [20].

Grafting of poloxamer onto the hyaluronic acid for in situ gelling ophthalmic drug delivery

system for ciprofloxacin was reported by Cho et al [21]. Yanxia et al. investigated a novel

thermosensitive copolymer (poly 9 N- isopropylacrylamide) –chitosan (PNIPAAm- Cs) for its

thermosensitive in situ gel forming properties and potential utilization for ocular drug delivery for

timolol maleate over a period of 12 h [22].

Edsman et al evaluated the rheological properties of deacetylated gellan gum (Gelrite®) and

the effect of different ions in tear fluid (Na+, K+, Ca2+) on the gel strength [23]. Pandit et al

developed the in situ gelling system for Indomethacin by using ion sensitive sodium alginate. The

release of indomethacin was extended upto 8 h, and [24].

Mourice and Srinivas found a two fold increase in the permeation of fluoroscein in humans

by using gellan gum compared to an isotonic buffer solution [25]. Pan et al. developed ophthalmic

system of gatifloxacin using alginate (Kelton®) in combination with HPMC (methocel E50LV)

which acted as a viscosity enhancing agents. In vivo precorneal retention studies indicated that the

alginate / HPMC solution retained the drug better than the alginate or HPMC alone [26].

The pH triggered in situ gel of antibacterial agent; ofloxacin for ophthalmic delivery was

developed by Srividya et al. Polyacrylic acid (Carbopol® 940) [27]. Polycarbophil based pH

triggered in situ gelling system was reported. Polycarbophil is insoluble in water, but its high

swelling capacity in a neutral medium permits the entanglement of the polymer chains with the

mucus layer [28].

Lindell and Engstrom reported an in situ thermogelling system consisting of ethyl

(hydroxyethyl) cellulose and a charged surfactant releasing slowly timolol maleate [29].



Pluronic F-127-g-poly (acrylic acid) copolymer based in situ gelling vehicle was found to

have prolonged precoeneal residence time and improved ocular bioavailability. The studies

indicated that the drug release rates decreased as acrylic acid / pluronic molar ratio and copolymer

solution concentration increased [30]. Sol-to-gel system of ciprofloxacin hydrochloride was

prepared by utilizing the phase transition properties of hydroxyl propylmethyl cellulose K15M and

carbopol 934 [31]. Lui et al developed Aginate / HPMC based system for long acting delivery of

gatifloxacin [32].

5. Colloidal system

5.1 Microspheres and Nanparticle

The rational for the development of various particulate systems for the delivery of

ophthalmic drugs was based on possible entrapment of the articles in the ocular mucous layer and

the interaction of bioadhesive polymer chain with mucins including a prolonged residence and

slow drainage. Furthermore controlled drug release and enhanced absorption or even endocytosis

in the case of nanoparticles should improve bioavailability [33].

The first particular colloidal carrier system developed was Piloplex ®, consisting of

pilocarpine sonically bound to poly (methyl) methacrylate-co-acrylic acid nanoparticles [33].

According to Klein et al., twice daily instillation of pilocarpine in glaucoma patients are as

effective as 3 to 6 instillation of conventional pilocarpine eye drop per day [34]. Santos et al.

designed the microsphere for sustained delivery and enhanced intracellular penetration for ocular

administration of antisense oligonucleotides. Nanosized complexes of antisense TGF-²2

phosphorothioate oligonucleotides (PS-ODN) with polyethylnimine (PEI) and naked POS-ODN

were encapsulated into poly (lactide-co- glycolide) microsphere prepared by the double emulsion

evaporation method [35]. Gaini et al. formulated poly (lactide-co-glycolide) microsphere as a

carrier for the topical ocular delivery of peptide drug vancomycin with high and prolonged

vancomycin concentration and increased AUC values (Two fold) with respect to an aqueous

solution of the drug. [36].



The formulation developed of rhVEFG in poly (D, L-lactide–co-glycolide) (PLG)

microsphere that provide a continuous local delivery of intact protein. [37]. Sorin et al. developed

a new ophthalmic delivery system, pilocarpine loaded proteinaceous (gelatin albumin)

microspheres for better ocular bioavailability [38].

Various mucoadhesive polymers have been tried to prepare the microspheres and

nanoparticles to increase the total bioavailability and effectiveness of the dosage form like

Acrylate [39]. The hydrophilic Polyalkyl- (Cyanoacrylates) (PACA) and polyalkyl methacrylate

were most commonly used for the preparation of drug carriers in the size range 200-500nm, for

sustained drug release and prolonged therapeutic effect [40]. Zimmer et al. showed that poly

(butylcyanoacrylate) nanoparticles were taken up in the first cell layers of the cornea and

conjunctiva by endocytosis or due to lysis of the cell membrane resulting from the degradation of

the product. [41]. Pignatello et al. employed copolymers of poly (ethylacrylate), poly

(methylmethacrylate) and poly (chloromethyl-aminomethyl methacrylate containing quaternary

groups (4.5-6.8% and 8.8 -12%) for Eudragit® RS and RL respectively [42-44].

Gupta et al. developed polymeric micelles made of a copolymer of N-isopropylacrylamide,

vinyl pyrrolidone and acrylic acid cross-linked with N, N’- methylene bis-acrylamide, in which the

water insoluble drug ketorolac (free acid) was entrapped [45].

Hsiue et al. investigated the use of the thermosensitive polymer poly-n –

isopropylacrylamide (PNIPAAm) in controlled release delivery system for glaucoma therapy [46].

Micro and nanoparticles made of poly (D, L.-lactide-co-glycolide) (PLGA) were investigated for

topical application. [47].

Ginnavola et al changed the surface properties of the PLA nanoparticles loaded with

acyclovir by the incorporation of pegylated 1,2 distearyl-3 phosphotidyl ethanolamine (DSPE-

PEA) [48] instead of coating the external surface as in case of PACA nanoparticles [49]. Gelatin

nanoparticles encapsulating pilocarpine HCl or hydrocortisone as model drug were prepared using

desolvation method by J.Vandervoot and A. Ludwig. [50].



Hyaluronan and its chemical derivatives were also employed to prepare micro and

nanoparticles. The release of methyl prednisolone from particles consisting of hyaluronic acid

esters has been evaluated in vitro and in vivo on rabbits by kyyronen et al. [51].

Chitosan is an interesting polymer to formulate micro and nanoparticles due to mucoadhesive and

permeability enhancing properties and its biodegradability by lysozyme [52]. Genta et al. have

been compared the acyclovir loaded chitosan microsphere with aqueous suspension, an increase of

about 4 fold in aqueous humour concentration of acyclovir after a single instillation of acyclovir

loaded chitosan microsphere [53]. Chitosan microspheres and nanoparticles have a higher

precorneal retention than chitosan solution, and depending on the size the nanoparticles may enter

the corneal epithelium to a certain depth by a paracellular or transcellular pathway [54].

Nanosystem having surface aggregated chitosan or polyethylene glycol was found relatively stable

and also efficient at overcoming mucosal barrier. Chitosan interacts with mucin as shown in figure 2.

Figure: 2

(a)

b)



Fig 2 a) structure of chitosan, a biopolymer obtained hydrolytically from chitin of crustacean shell

b) Interaction between chitosan coated nanoparicles and sialic acid presesnt in the mucin layer is expected to improve bioavailability.

Polysaccharide like pectin used for the preperation of piroxicam nanoparticles with 205 fold

increased drug absorption compare to eye drop solution[55].

Various in vivo studies in rabbits reported the prolonged effect of drugs (Pilocarpine,

Piroxicam) incorporated in albumin particles compared to commercial preparations or aqueous and

viscous [56,57]. Zimmer et al. and Marta Mmerodio et al. Developed the albumin loaded

colloidal system for pilocarpine and gancyclovir respectively. [58,59].

Solid lipid nanoparticles observed with longer retention time on the corneal surface and in

the cul-de-sac is probably due to their small size. The nanoparticles are presumably entrapped and

retained in the mucus layer. Cavalli et al. evaluated the use of solid lipid nanoparticles (SLV) as

carrier for tobramycin [60]. Compared to commercial eye drops, the tobramycin –loaded SLN

produced a significantly higher bioavailability with no any ocular irritation.

Imprinted polymers are increasingly considered for biomedical applications, including drug

delivery. Feedback regulated drug delivery may be achievable by a combination of imprinted,

stimuli sensitive materials that would allow high drug-loading capacity molecularly imprinted

polymers (MIP’s) to respond to external stimuli (slight changes in pH, temperature, ionic strength,

concentration of biomolecules, or presence of specific receptors, and / or ligands) and modulate the

affinity of the network for the target molecules (bioactives), thus providing a regulatory capability

for the release process as in figure. 3.

Figure: 3



Fig-3: Schematic representation of targeted drug delivery and intelligent active release with a molecularly imprinted carrier

Colloidal nanosystem based on biodegradable polymeric materials that combine the

capabilities of stimulus response and molecular recognition promises significant improvement in

the ocular delivery of therapeutic agents. The formulation of biodegradable polymers as colloidal

system holds significant promise for ophthalmic drug delivery. Additionaly, surface modified

nanoparticulate carriers may use to accommodate a variety of actives [61] as in figure 4.

Figure 4:

Fig 4- Nanoparticles

a) Lipophilic nanoparticles

(b, c) Ionic nanoparticles nanoparticles

(d) Amphiphilic nanoparticles



6.Vesicular system

Vesicular systems not only helps in providing prolonged and controlled action at the corneal

surface but also helps in providing controlled ocular delivery by preventing the metabolism of the

drug from the enzymes present at the tear/corneal epithelial surface. Moreover, vesicles offer a

promising avenue to fulfill the need for an ophthalmic drug delivery system that has the

convenience of a drop, but will localize and maintain drug activity at its site of action. The rate of

drug penetration depends not only on the physicochemical properties of the drug itself. [62].

Vesicular drug delivery systems used in ophthalmics broadly include liposomes and niosomes

[63]. Liposomes are the microscopic vesicles composed of one or more concentric lipid bilayers,

separated by water or aqueous buffer compartments with a diameter ranging from 80 nm to 10µ m.

Liposomes (also called phospholipid vesicles) were first described by Bengham A. D. [64]. Such

vesicles (Fig. 5) composed of one or more phospholipid bilayer membranes can entrap both

hydrophilic and hydrophobic drugs, depending on the nature of the drug and hence, it is possible to

apply water-insoluble drugs in liquid dosage form. According to their size, liposomes are known as

either small unilamellar vesicles (SUV) (10–100 nm) or large unilamellar vesicles (LUV) (100–

3000 nm). If more than one bilayers are present, then they are referred to as multilamellar vesicles

(MLV).

Figure: 5

Fig-5 Basic structure of vesicular system



Liposomes can act as carriers for a wide variety of drug molecules, proteins, nucleotides, and

even plasmid endowing them with a great potential for their application in ophthalmics [65].

Another potential advantage of liposomes is their ability to come in an intimate contact with the

corneal and conjunctival surfaces, thereby, increasing the probability of ocular drug absorption

[66]. This ability is especially desirable for drugs that are poorly absorbed, for example, the drugs

with low partition coefficient, poor solubility or those with medium to high molecular weight [66]

and enzymes, like cholinesterase [67].

Positively charged liposomes on the other hand were reported to exhibit a prolonged

precorneal retention, than neutral and negatively charged liposomes because of electrostatic

interaction with the negatively charged corneal epithelium. It is proposed that these liposomes bind

intimately on the eye surface, increase the residence time and thus drug absorption [68].

Liposomes were used by Dean et al. [69] for nuclear targeting of plasmid DNA in human

corneal cells. Similarly, liposomes were used for intravitreal administration of oligonucleotides for

the treatment of ocular viral infections, like Herpes simplex virus or Cytomegalovirus (CMV) [70].

In order to enhance adherence to the corneal, conjuctival surface, dispersion of the liposomes

in mucoadhesive gels or coating the liposomes with mucoadhesive gel or wetting the liposomes

with mucoadhesive polymers was proposed [71]. Several mucoadhesive polymers were employed

which includes poly (acrylic acid) (PAA), hyaluronic acid (HA), chitosan and poloxamers [72,

73,74, 75]

The positively charged liposomes are more effective than negatively charged and neutral

liposomes [68].

Dendrimers

Synthetic, spherical, macromolecules named after their characteristic tree like or dendritic

branching around a central core, which possess unique properties (multivalency, globular

architecture and well defined molecular weight) that makes them a new scaffolds for drug delivery,

especially if formulated as micelles may prove effective vehicles for ocular drug delivery [61].



Vandamme developed poly (amidoamine) (PAMAM) dendrimers for controlled ocular drug

delivery of pilocarpine and tropicamide [76]. Devarakonda designed Polyamidoamine (PAMAM)

Dendrimers for Water-Insoluble Nifedipine [77]. Marano et al have have performed a long-term

study into the use of a lipophilic amino-acid dendrimer to deliver an anti-vascular endothelial

growth factor (VEGF) oligonucleotide (ODN-1) into the eyes of rats and inhibit laser-induced

choroidal neovascularization (CNV) [78]. The polymer micelle is a particle fundamentally formed

with a hydrophilic polymer chain as a shell and a hydrophobic polymer chain as core. The drug

delivery system of the invention can be effectively applied to photodyanamic therapies, when a

photosensitive drug used as a drug and can be subjected to therapy for age related macular

degeneration through occluding choroidal new vessels [79].

7. Solid dosage forms Ocular inserts

Films, erodible and nonerodible inserts, rods shields are the most logical delivery systems

aimed at remaining for a long period of time in the front of the eye, listed in table 2 [86-94].

Table2 various ocular inserts: Name Description SODI (soluble ocular drug insert) [86] of a soluble acrylamide, N-

Small oval wafer, composed Co-polymer consisting of vinyl pyrrolidone and ethyl softens on insertion

NODS(New or novel ophthalmic delivery system)[87]

Medicated solid polyvilnyl alcohol flag that is attached to a paper covered handle, an application flag detaches and gradually dissolves, releasing the drug.

Collagen shields [88] Erodible discs composed of crosslinked porcine scleral collagen. Ocusert [89] Flat, flexible elliptical insoluble device consisting of two layers enclosing a

reservoir, used commercially to deliver pilocarpine for 7 days. (OTS) Minidiscs or ocular therapeutic system [90]

4-5 mm diameter contoured either hydrophilic or hydrophobic disc.

Lacrisert [90] Rod shaped device made from hydroxy propyl used in the treatment of dry eye syndrome as an alternative to artificial tears.

Ophthalmic inserts [91] A cylindrical device containing mixtures of silicone elastomer and sodium chloride as a release modifier with a stable polyacrylic acid (PAA) interpenetrating polymer network grafted on to surface

Bioadhesive ophthalmic drug insert ( BODI) [92]

Adhesive rods based on mixtures of hydroxypropyl hydroxypropyl cellulose ethyl cellulose, polyacrylic acid, cellulose acetate phthalate.

Dry drop [93] A preservative free drop of hydrophilic polymer solution (hydroxyl propyl methyl cellulose) that is freeze dried on the tip of a soft hydrophobic carrier strip, immediately hydrates in the tear film.

Gelfoam [94] Slabs of Gelfoam Impregnating with mixtures of drug and cetyl ester wax in chloroform.



However, the inserts were not well tolerated or accepted by patients due to difficulties

encountered in the application, psychological factors and possible interference with vision [81].

Various researchers have developed and performed In vivo studies solid inserts by using various

polymer combinations [82, 83, 84, 85]

New devices for sustained release are being developed that allow the maintenance of a more

constant level of these compound in the posterior segment, for example- sustained release

flucinolone acetonide intravitrial implants (Retisert), significantly reduced uveitis recurrences,

improved visual acuity and decreased the need for adjunctive therapy in the patient population

studies [95]. Similarly biodegradable polymeric scleral plug that delivered sustained release of

tacrolimus (FK 506) in a rabbit model of experimental uveitis [96]. A biodegradable, deep scleral

lamear cyclosporine, a device, which was placed near to or in contact levels of drug to the uveal

tissues in the equine eyes [97].

8. Iontophoresis

Iontophoresis ia advanced non-invasive technique for ocular drug delivery. [98]

Transcorneal iontophoresis is iused to drive charged drug by an electric current into the cornea. In

a pseudomonas model model of bacterial keratitis, as well as in pharmacokinetic studies, ocular

iontophoresis of gentamycin, tobramycin, or ciprofloxacin was superior to topical ocular drops for

reducing pseudomonas in the cornea. This method was shown to be safe and nontoxic to the rabbit

corneal epithelium [99].

Molokhia et al. studied the transcorneal iontophoretic delivery and compared to passive

delivery and intravitrial injection using nuclear magnetic resonance imaging (MRI) and employed

the MRI to investgate the factors affecting transcorneal iontophoretic delivery [100]. Extending the

duration of iontophoresis at this site allowed the drug to be delivered into the vitreous more deeply

and to the greater extent than when the application site was at the back of the eye near the fornix.

The results showed that electrode placement was an important factor in transscleral iontophoresis



and the ciliary body (pars plana) was determined to be the pathway of least resistance for

iontophoresis transport.

The major causes of blindness in the United State and Europe are age related macular

degeneration (AMD) and diabetic retinopathy (DR). Posterior uvietis and retinitis secondary to

glaucoma also contribute considerably to loss of vision. These conditions affect tissues at the back

of the eye, where drug treatment is difficult to administer. Current method for ocular delivery have

limitations, invasive methods are inherently risky due to the potential for bleeding, infection,

retinal detachment and other local injuries. To meet these needs Iomed, Inc. is developing a novel

ocular iontophoresis system (OcuPhorTM) to deliver drugs safely and noninvasively to the back of

the eye [101].

The OcuPhorTM system consists of drug application dispersive electrode and an electronic

iontophoresis dose controller. A hydrogel pad to absorb the drug formulation and a small flexible

wire to connect the conductive element to the dose controller. The drug pad is hydrated with drug

solution immediately prior to use and the applicator is placed on the sclera of the eye under the

lower eye lid. Preliminary clinical studies in human volunteers have shown that the OcuPhor TM

system is well tolerated over a wide range of both positive and negative polarity current and does

not produce any ophthalmic changes as measured by a series of standard tests.[102]

Iomed used a model anionic drug, diclofenac to investigate the interstudy and intrastudy

reproducibility of transscleral iontophoresis to rabbit eyes. Significant amount of diclofenac was

found in retina / choroid tissues on average iontophoresis resulted in approximately a 16-fold

increase of diclofenac concentration in retina choroid as compared to passive no current control.

Relatively small amount of drug were delivered systemically, indicating predominantly local

delivery to the eye, with the transscleral Iontophoresis [101].

9. Recent developments in ocular drug delivery system

Challenges for effective front of the eye (FOTE) drug delivery include somehow

minimizing the use of preservatives in the drug solution being applied, and avoiding excess eye



drop solution being drained through the nasolachrymal duct with potential systemic absorption into

the circulatory system.

Currently available devices for improving FOTE drug delivery using eye drops include the

Visine pure tears single drop dispenser, which contains no preservatives. The Pfizer Xal-ease

FOTE drop delivery device which encloses a traditional eye drop bottle and the Autosqueeze and

Autodrop devices developed in the UK, with Royal national Institute for the Blind, which clip into

bottles of the eye drop.

Recent innovations in FOTE drug delivery devices include the Eye-Instill produced by

Med-Instill Inc., which have one way valve to ensure multiple dosings of sterile, preservative free

drug solution and the OptiMyst device, which dispenses medication as a mist rather than as a drop.

The latter provides much less medication per dose, below blink and lachrymation thresholds.

The VersiDoser TM drug delivery system under development by Mystic pharmacueticals,

Inc., holds the near term potential for setting new slandard for effective FOTE drug delivery. The

VersiDoser platform utilizes the pack with Novel multidose delivery device that dispenses the drug

into the eye in a predictable manner irrespective of the orientation of the device and the eye. These

devices are capable of the self administered precision dosing in the 12-15µl range and provide

automatic dose counters. Preservative free packaging and ergonomic design will significantly

enhance compliance, ease of use and therapeutic benefits for elderly and padiatric patients [103].

10. Conclusion

Formulating a drug delivery system for the sensitive organ like eye always remains a

challenge for the formulation chemist. Though intensive work has been done on this drug delivery

system still there is no such formulation which increases patient compliance and reduces side

effects due to systemic absorption of drug substances. Number of new approaches is being used

but still need to work on this important drug delivery system.



References

1. C.A.Le-Bourlais, A.L.Treupel, C.T. Rhodes, P.A.Sado, R.Leverge, New- ophthalmic drug

delivery system, Drug Devel. Ind. Pharm. 21 (1998) 19-59.

2. V.H.L. Lee, J.R. Robinson, Review: topical ocular drug delivery: recent development and

future challenges, J.Ocul.Pharmacol. 2 (1986) 67-108

3. A.Topalkara, C.Guler, D.S.Arici, M.K.Arici, Adverse effects of topical antiglaucoma drugs on

the ocular surface, Clin.Exp.Ophthalmol. 28(2000) 113-117.

4. A.Urtti, L.Salminen, Minimizing systemic absorption of topically administered ophthalmic

drugs, Surv.Ophthalmol. 37(1993) 435-456.

5. Y.W.Chien, Ocular drug delivery and delivery systems, In: Novel drug delivery system, Marcel

Dekker, Inc. New York,(1992) 269-300.

6. K.C. Swan, The use of methylcellulose in ophthalmology, Arch. Ophthalmol. 33 (1945) 378–

380.

7. O.Dudinski, B.C. Finnin, B.L. Reed, Acceptability of thickened eye drops to human subjects,

Curr.Ther.Res.33 (1983) 322-337.

8. H.Ibrahim, C. Bindschaedler, E.Doelker, P. Buri, R.Gurny, Concept and development of

ophthalmic pseudo-lattices triggered by pH, Int.J.Pharm. (1991) 211-219.

9. S.C. Miller, M.D.Donovan, effects of poloxamer 407 gel on the miotic activity of pilocarpine

nitrate in rabbits, Int.JPharm. 12(1982) 147-152.

10. A.Rozier, C. Mazuel, J. Grove, B. Plazonnet, Gelrite®: a novel, ion activated in situ gelling

polymer for ophthalmic vehicles: Effect on bioavailability of timolol, Int.J.Pharm. 57 (1989)

163-168.

11. J. Burrows, J. Tslbouklls, J. Smart, Drug delivery to the eye, Biomaterials and drug delivery

group Pharmaventure Ltd. The drug delivery companies report, spring 2002.

12. A. Ludwig, The use of mucoadhesive polymers in ocular drug delivery, Adv. Drug.Del. Rev.

57(2005) 1595-1639.



13. J. W. Lee, J. H. Park, J. R. Robinson, Bioadhesive based dosage forms – the next generation,

J.Pharm.Sci.89 (2000) 850-866.

14. R. Krishnamoorthy, A. K. Mitra, Mucoadhesive polymers in ocular drug delivery, A. K. Mitra

(Ed.), Ophthalmic drug delivery system, Marcel Dekker, New York,(1993) 199-221.

15. S.W. Kim, Y.H. Bae, T. Okano, Hydrogel : swelling, drug loading and release, Pharm.Res.9

(1992) 283-290.

16. M. Zingnani, C.Tabataba, R.Gurny, Topical semisolid drug delivery: kinetics and tolerance of

ophthalmic hydrogels, Adv. Drug Deliv. Rev.16 (1995) 51–60.

17. Q.Hongyi ,W. Chena, C.Huanga, Li Lib,C. Chena, L.Wenmin ,W.Chunjie ,Development of

a poloxamer analogs/carbopol-based in situ gelling and mucoadhesive ophthalmic delivery

system for puerarin ,Int. J. Pharm. 337 (2007) 178–187

18. A.H.El-Kamel, In vitro and in vivo evaluation of pluronic F 127 based ocular delivery system

for timolol maleate, Int.J.Pharm. 241 (2002) 47-55.

19. Hong-R. Lina , K.C. Sung Carbopol / pluronic phase change solutions for ophthalmic drug

delivery, J. of Controlled Release, 69 (2000) 379–388

20. S. Mayazaki, S. Suzuki, N. Kawasaki, K. Endo, A. Takahashi, D. Attwood, In situ gelling

xyloglucan formulation for sustained release ocular delivery of pilocarpine hydrochloride, Int.

J. Pharm. 229 ( 2001) 29-36.

21. K.Y.Cho, T.W.Chuno, B.C. Kim, M.K.Kim, J.H.Lee, W.R.Wee, C.S.Cho, Release of

Ciprofloxacin from poloxamer-graft hyaluronic acid hydrogels in vitro, Int.J.Pharm. 260

(2003) 83–91.

22. Y.Cao, C. Zhang, W. Shen, Z. Cheng, L. Yu,Q.Ping, Poly (N-isopropyl acrylamide) chitosan

as thermosensitive in situ gel forming system for ocular drug delivery, J.Control.Release 120(

2007) 186–194.

23. K.Edsman ,M. Paulsson, H. Hangerstrom, , Rheological studies of the gelation of deacetylated

gellan gum (Gelrite ®) in physiological condition, Eur.J.Pharm.Sci.9 (1999) 99-105.



24. J.K.Pandit, D.Bharathi, A. Srinatha, D.N. Ridhurkar, S.Singh, Long acting ophthalmic

formulation of Indomethacin, evaluation ogf alginate gel systems, Ind.J. Pharm.Sci. (2007) 37-

40.

25. D.M.Mourice, S.P. Srinivas, Use of flurometry in accessing the efficacy of a cationic –

sensitive gel as an ophthalmic vehicle: Comparison with scintigraphy, J.Pharm.Sci. 81 (1992)

615-619.

26. Z.Liu, J.Li, S.Nie, Hui-Liu, P.Ding, W.Pan, Study of alginate / HPMC based in situ gelling

ophthalmic delivery system for gatifloxacin, Int.J.Pharm. 315 (2006) 12-17.

27. B.Srividya, R.M.Cardoza, P.D.Amin, Sustained ophthalmic delivery of ofloxacin from a pH

triggered in situ gelling system, J.Control.Release 73 (2001) 205–211.

28. J.R. Robinson, G.M. Mlynek, Bioadhesive and phase change polymer for ocular drug delivery,

Adv.Drug Deliv.Rev. 16 (1995) 45–50.

29. K.Lindell, S.Engstrom, In vitro release of timolol maleate from an in situ gelling polymer

system, Int. J.Pharm.95 (1993) 219–228.

30. Wen-Di Ma, Hui Xu, Chao Wang, Shu-Fang-Nie, Wei san Pan, Pluronic F 127 –g- poly

(Acrylic acid) copolymers as in situ gelling system, Int.J.Pharm.350 (2008) 247-256

31. N.A.Charoo, K.Kohli, A.Ali, and Preparation of in situ forming ophthalmic gels of

ciprofloxacin for the treatment of bacterial conjunctivitis: In vitro and in vivo studies,

J.Pharm.Sci. 92 (2003) 407-413.

32. Z. Liu, , J. Li, S. Nie, H. Liu, P. Ding, W. Pan. Study of an alginate/HPMC-based in situ

gelling ophthalmic delivery system for gatifloxacin. Int.J. Pharm. 315 (2006)12–17.

33. C. A.Le Bourlais,A. L.Treupel , H. Zia, P. A. Sado, T. Needham, R. Leverge, Ophthalmic drug

delivery system, Prog. Retin- Eye Res. 17 (1998) 33-58.

34. H.Z.Klein, M.Lugo, B.Shields, J.Leon, E.Duzman, A dose response study of piloplex for

duration of action, Am.J.Ophthalmol. 99(1985) 23-26.



35. Ama L Gomes das Santos, A. Bochot, F. Poyle, N. Tsapis, J. Siepmann, F. Siepmann, J.

Schmaler, M. Besnard, F. Behar-Lohen, E. Fattal, Sustained release of nanosized complexes of

polyethyle diamine and anti-TGF-β 2 oligonucleotide improve the outcome of glaucoma

surgery, J. Control. Release, 112 (2006) 369-381.

36. E. Gavini, P. Chetoni, M. Cassu, M. G. Alvarez, M. F.Saettone, P.Giunched, PLGA

microsphere for the ocular delivery of a peptide drug vancomycin using emulcification / spray

drying as the preparation method: in vitro/ in vivo studies, Eur. J. Pharm. Biopharm. 57 (2004)

207-212.

37. J. L. Cheland, E. T. Duenas, A.Park, A. Dougherty, J. Kahn, J. Kowalski, A. Cuthertson,

Development of poly-(D,L-Lactide- co- glycolide ) microspheres formulations containuing

recombinant human vascular endothelial growth factor to promote local angiogenesis,

J.Controll.Release 72 (2001) 13-24.

38. S.E.Leucut, The kinetics of in vitro release and the pharmacokinetics of miotic response in

rabbits of gelatin and albumin microspheres with pilocarpine, Int.J.Pharm. 54 (1989) 71-78.

39. A.M. Durrani, S.J. Farr, I.W. Kellaway, Precorneal clearance of mucoadhesive microspheres

from the rabbit eye, J. Pharm.Pharmacol. 47 (1985) 23-26.

40. R. Diepold, J. Kreuter, P. Guggenbuhl, J.R. Robinson, Distribution of poly-hexyl 2 cyano [314

C] acrylate nanoparticles in healthy and chronically inflamed rabbit eyes, Int.J. Pharm.

54(1989) 149-153.

41. A.Zimmer, J. Kreuter, J.R. Robinson, Studies on the transport pathway of PBCA nanoparticles

in ocular tissues, J.Microcapsul. 8 (1991) 497-504.

42. R. Pignatello, C.Bucolo, P. Ferrara, A. Maltese, A. Puleo, G. Puglisi, Eudragit RS 100®

nanosuspension for ophthalmic controlled delivery of Ibuprofen, Eur.J. Pharm, 16 (2002) 53-

61.



43. R.Pignatello, C.Bucolo, G. Puglisi, Ocular tolerability of Eudragit RS 100 ® and RL 100®

nanosuspension as carrier for ophthalmic controlled drug delivery, J.Pharm.Sci.91( 2002)

2636–2641.

44. R.Pignatello, C.Bucolo, G.Spedalieri, A. Maltese, G. Puglisi, Flurbiprofen loaded acrylate

polymer nanosuspensions for ophthalmic application, J.Biomaterials 23 (2002) 3247-3255.

45. A.K. Gupta, S.Madan, D.K. Mujumdar, A. Maitra, Ketorolac entrapped in polymeric micelles

preparation, characterization and ocular anti-inflammatory studies, Int.J. Pharm. 209 (2000) 1-

14.

46. G.H. Hsiue, S.H. Hsu, C.C.Yang, S.H. Lee, I.K.Yang, Preperation of controlled release

ophthalmic drops for glaucoma therapy using thermosensitive poly-N- isopropyl acrylamide,

Biomaterials 23(2002) 467-462.

47. H.Kimura, Y.Ogura, Biodegradable polymers for ocular drug delivery, Ophthalmologica

215(2001) 143-155.

48. C. Giannavola, C.Bucolo, A.Maltese, D.Paolino, M.A. Vandeli, G.Puglisi, V.H.L. Lee,

M.Fresta, Influence of preparation of acyclovir-loaded poly-D, L-lactic acid nanospheres and

effect of PEG coating on ocular drug bioavailability, Pharm.Res. 20(2003) 584-590.

49. M.Fresta, G.Fontana, C. Bucolo, G.Cavallaro, G. Glammona, G. Puglisi, Ocular tolerability

and in vivo bioavailability of poly(ethylene glycol )(PEG) –coated polyethyl-2- cyanoacrylate

nanospheres-encapsulated acyclovir, J.Pharm.Sci. 90(2001) 288-297.

50. J.Vandervoort, A.Ludwig, Preperation and evaluation of drug loaded gelatin nanoparticles for

topical ophthalmic use, Eur.J.Pharm.Biopharm. 57(2004) 251-261.

51. K.Kyyronen, L.Hume, L.Benedetti, A.Urtti, E.Topp, V.Stella, Methylprednisolone, esters of

hyaluronic acid in ophthalmic drug delivery, in vitro and in vivo release, Int.J.Pharm.80 (1992)

161-169.



52. S.Rossi, F.Ferrari, M.C. Bonferoni, C.Caramella, and Characterization of chitosan

hydrochloride – mucin rheological interaction: influence of polymer concentration and

polymer: mucin weight ratio, Eur.J.Pharm.Sci. 12(2001) 479-485.

53. I.Genta, B.Conti, P. Prugini, P.Pavanetto, A. Spadaro, P. Puglisi, Bioadhesive microspheres for

ophthalmic administration of acyclovir, J.Pharm.Pharmacol. 49(1997) 737-742.

54. M. J. Alonso, A. Sanchez, The potential of chitosan in ocular drug delivery, J. Pharm.

Pharmacol. 55(2003) 1451-1463.

55. P.Giunchedi, U. Conte, P.Chetoni, M.F. Saettone, Pectin microspheres as ophthalmic carrier

for piroxicam –evaluation in vitro and in vivo in albino rabbits, Eur.J.Phar.Sci. 9 (1999) 1-7.

56. S.Leucutta, The kinetics of in vitro release and the pharmacokinetics of miotic response in

rabbits of gelatin and albumin microspheres with pilocarpine, Int. J. Pharm. 54 (1989) 71-78.

57. P.Giunchedi, P.Chetoni, O.Conte, M.F. Saettone, Albumin microspheres for ocular delivery of

piroxicam, Pharm.Pharmacol.Commun.6 (2000) 149-153.

58. A.K.Zimmer, P. Chetoni, M.F.Saettone, H.Zerbe, J.Kreuter, Evaluation of pilocarpine –loaded

albumin particles as controlled drug delivery system for the eye: part II, Co-administration

with bioadhesive and viscous polymers, J. Control.Release 33 (1995) 31-46.

59. M.Marodio, J.M.Irache, F.Valamaresh, M.Mirshahi, Ocular disposition and tolerance of

ganciclovir –loaded albumin nanoparticles after intravitreal injection in rats, J.Biomaterials

13(2002) 1587-1594.

60. R.Cavalli, M.R.Gasco, P.Chetoni, S.Burgalssi, M.F.Saettone, Solid lipid nanoparticles (SLN)

as ocular delivery system for Tobramycin, Int.J.Pharm. 238(2002) 241-245.

61. E. Barbu, L. Verestiuc, T. G. Nevell, J. Tsibouklis, Polymeric materials for ophthalmic drug

delivery: trends and perpectives, J.Material Chem. 16(2006) 3439-3443.

62. A.Kupferman, W.J.J.Ryan, H.M.Leibowitz, Prolongation of anti-inflammatory effect of

prednisolone acetate, influence of formulation in higher viscosity gel, Arch.Ophthalmol.99

(1981) 2028-2029.



63. I.P.Kaur, A.Garg, A.K.Singla, D.Aggrawal, Vesicular systems in ocular drug delivery- an

overview, J.Pharm.269 (2004) 1-14.

64. A.D.Bangham, M.M.Standish, J.C.Watkins, Diffusion of univalent ions across the lamellae of

swollen phospholipid, J.Mol.Biol. 13(1965) 238-252.

65. D.Kurz, T.A. Ciulla, Novel approaches for retinal drug delivery, Ophthalmol. Clin.North Am.

15(2002) 405-410.

66. J.M. Megaw, Y.Tukei, S.Lerman, Lectin mediated binding of liposomes to ocular lens,

Exp.Eye Res. 32(1981) 395-405.

67. P.N.Shek, R.A.Barber, Liposomes are effective carrier for the ocular delivery of propylactics,

Biochem, Biophysic Acta 902(1987) 229-236.

68. S.L.Law, K.J. Huang, C.H. Chiang, Acyclovir-containing liposomes for potential ocular

delivery –corneal penetration and absorption, J.Control.Release 63(2000) 135-140.

69. D.A. Dean, J.N.Byrd Jr, B.S. Dean, Nuclear targeting of plasmid DNA in human corneal cells,

Curr.Eye 19(1999) 66-75.

70. A.Bochot, E.Fattal, V.Boutet, P. Couvreur, Intravitreal administration of antisens

oligonucleotides: A potential of liposomal delivery, Prog.Retin. Eye Res. 19(2000) 131-147.

71. N.M. Davies, S.J. Farr, J. Hadgraft, I.W. Kellaway, Evaluation of mucoadhesive polymers in

ocular drug delivery, II polymer coated vesicles, Pharm. Res. 9(1992) 1137-1144.

72. D.Meisher, M.Mezei, Liposomes ocular systems, Adv.Drug Deliv.Rev.16 (1995) 75-93.

73. A.M.Durrani, N.M.Davies, M.Thomas, I.W.Kellaway, Mucoadhesive liposomal drug delivery

system, Int.J.Pharm.88 (1992) 409-415.

74. A. Bochot, E. Fattal, J. L. Grossiord, F. Puisieux, P. Couvreur Characterization of a new ocular

delivery system based on a dispersion of liposomes in a thermosensitive gel, Int.J.Pharm.

162(1998) 119-127.



75. A.Bochot, E.Fattal, A.Gulik, G.Couarraze, P.Couvreur, Liposomes dispersed within a

thermosensitive gel: a new dosage form for ocular delivery of oligonucleotides, J.Pharm. Res.

615(1998) 1364-1369.

76. L.Budai, M.Hajdu, M.Budai, P.Grof, S.Beni, B.Noszal, I.Klebovich, I.Antal, Gels and

liposomes in optimized ocular drug delivery: Studies on ciprofloxacin formulations,

Int.J.Pharm. 343(2007) 34-40.

77. Th.F. Vandamme, L. Brobeck, Poly (amidoamine) dendrimers as ophthalmic vehicles for

ocular delivery of pilocarpine nitrate and tropicamide Jr.Control.release,102.(2005),23-38.

78. B. Devarakonda, N. Li , MM de Villiers, Effect of Polyamidoamine (PAMAM) Dendrimers on

the In Vitro Release of Water-Insoluble Nifedipine From Aqueous Gels. AAPS PharmSciTech.

6(2005) 504-512.

79. R.J. Marano, I. Toth, N. Wimmer, M. Brankov, P. E. Rakoczy, Dendrimer delivery of an anti-

VEGF oligonucleotide into the eye: a long-term study into inhibition of laser-induced CNV,

distribution, uptake and toxicity Gene Therapy 12 (2005) 1544–1550.

80. Kataoko, Kazunori, Tamaki, Yasuhiro, Harado, Atsushi,Ophthalmic drug delivery system

using polymer micelle, US Patent 20060110356, (2006).

81. G.L. Khromow, A.B. Davydov, A.B. Maychuk, I.F. Tishina, Base for ophthalmic medicinal

preparation and ophthalmological medicinal film, US Patent 3935303 (1976).

82. M.F.Saettone, L.Salminen, Ocular inserts for topical delivery, Adv, Drug Delv, Rev. 16(1995)

95-106.

83. V. Rao, S.Shyale, Formulation and Evaluation of Ocular Inserts Containing Norfloxacin, urk.

J, Med, Sci, 34 (2004) 239-246.

84. Y. Sultana,M. Aqil A. Ali, Ocular inserts for controlled delivery of pefloxacin mesylate:

Preparation and evaluation, Acta Pharm. 55 (2005) 305–314.



85. M.Hornof, W.Weyenberg, A. Ludwig, A.Bernkop-schneurch, Mucoadhesive ocular insert

based on thiolated poly (acrylic acid): development and in vivo evaluation in humans,

J.Control.Release 89(2003) 419-428.

86. H.Sasaki, T.Nagalso, K.Sakanaka, S.Kawakami, J.Iwashito, T.Nakamura, M.Nakashima, One

side coated insert as a unique ophthalmic drug delivery system, J.Control.Release, 92(2003)

241-247.

87. R. Bawa, Ocular inserts In: A.K.Mitra (Ed.) Ophthalmic drug delivery systems, Marcel

Dekker, Inc. New York (1993) 223-260.

88. J.G. Lowrenson, D.F.Edgar, A.C. Gudegeon, J.M. Burns, M.Geraint, B.A.Nas, Comparison of

the efficacy and duration of action of topically applied proxymethocaine using a novel

ophthalmic delivery system versus eye drop in healthy young volunteers, Br.J.Ophathalmol.

77(1993) 713-715.

89. T.P.O.Brien, M.R.Sawusch, J.D.Dick, T.R.Hamburg, J.D.Gattsch, Use of collagen corneal

shields versus soft contact lenses to enhance penetration of topical tobramycin,

J.Catar.Refra.Surg. 4 (1988) 505-507.

90. H.A.Quigley, I.P.Pollack, T.S.Harbin, Pilocarpine ocuserts, long term clinical trials and

selected pharmacodynamics, Arch. Ophthalmol. 93 (1975) 771-775.

91. G.Di Colo, Y.Zambito, A study of release mechanism of different ophthalmic drugs from

erodible ocular inserts based on poly (ethylene oxide), Eur.J.Pharm.Sci.54(2002), 193-199.

92. P.Chetoni, G.Di Colo, M.Grandi, M.Morells, M.F.Saettone, S.Darougar, Silicon rubber /

Hydrogel composite ophthalmic insert: preparation and preliminary in vitro / in vivo

evaluation, Eur.J.Pharm.Biopharm. 46(1998) 125-132.

93. F.G.Urtler, V.Kalsatos, B.Boisrame, R.Gurny, Long acting soluble bioadhesive ophthalmic

drug insert (BODI) containing gentamicin for veterinary use: optimization and clinical

investigation, J.Control.Release 33(1995)231-236.



94. M.Diestelhorst, S.Grunthal, R. Suverkrup, Dry drops: A new preservative free drug delivery

system, Graefes Arch.Clin.Exp.Ophthalmol. 237-(1999) 394-398.

95. P.Simamora, S.R.Nadkaril, Y.C.Lee, S.H.Yalkowsky, Controlled delivery of pilocarpiness II -

in vivo evaluation of Gelfoam device, Int.J.Pharm. 170(1998) 209-214.

96. G.J.Jaffe, D.Martin, D.Callonan, P.A.Pearson, B.Levy, T.Comstock, Fluocinolone, acetonide

uveitis study group, fluocinolon acetonide implant (Retisert) for noninfectious posterios

uveitis: thirty four week results of multicenter randomized clinical study, Ophthalmol.

113(2006) 1020-1027.

97. E.Sakurai, M.Nozaki, K.Okabe, N.Kunou, H.Kimura, Y.Ogura, Scleral plug of biodegradable

polymers containing tacrolimus (FK 506) for experimental uveitis, Invest.Ophthalmol.Visual

Sci. 44(2003) 4845-4852.

98. B.C.Gilger, J.H.Salmon, D.A.Wikkie, A novel bioerodible deep scleral lamellar cyclosporine

implant for uveitis, Invest.Ophthalmol.Visual Sci. 47 (2006) 2596-2605.

99. E. Eljarrat -Binstock, A. J. Domb, Iontophoresis: A non-invasive ocular drug deliver, Jr.

Control.Rel. 110(2006) 479-489

100.S.A.Molokhia, E.Jeong, W.I.Higuchi, S.KevinLi, Examination of penetration routes and

distribution of ionic permeants during and after transcleral iontophoresis with magnetic

resonance imaging, Int.J.Pharm. 335(2007), 46-53.

101.G.A.Fischer, T.M.Parkinson, M.A.Szlek, OcuPhorTM –The future of ocular drug delivery,

drugdeliverytechnology, 2(2002).

102. T.M. Parkinson, E. Ferguson., S. Febbraro, A. Bakhtyari, M. Mundasad, Tolerance of

ocular iontophoresis in healthy volunteers. Invest Ophthalmol Vis Sci.,43(2002) Abstract

No.1854.

103.W.L. Zielinski, T. R. Sullivan, Ophthalmic drug delivery- challenges and advances in front of

the eye delivery, Ocular delivery, TouchBriefings (2007) 44-45.

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