Laboratory Manual

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LABORATORY MANUAL OF

BIOPHARMACEUTICS AND PHARMACOKINETICS

Dr. S. B. Bhise

M. Pharm PhD Principal, Govt College of Pharmacy, Karad

Dr. R. J. Dias

M. Pharm PhD MBA Professor, Satara College of Pharmacy, Satara

Dr. S. C. Dhawale

M. Pharm PhD Professor, Govt College of Pharmacy, Karad

Shri. K. K. Mali

M. Pharm (Biopharm) Associate Professor, Satara College of Pharmacy, Satara

INNOVATE

P

TRINITY PUBLISHING HOUSE

PUBLISH

Serving Pharmacy Profession

Laboratory Manual of Bipharmaceutics & Pharmacokinetics Published by Mrs. Anita R. Dias For Trinity Publishing House, 475/8, F-3, Suryanandan Apartments, Near Hotel Suruban, Sadar Bazaar, Satara - 415 001. India. Tel: (02162) 236229. E. mail: [email protected]

© 2010 Trinity Publishing House All rights reserved. No part and style of this book be reproduced or transmitted, in any form, or by any means- electronic, mechanical, photocopying, recording or otherwise, without prior permission of the publishers and authors.

Disclaimer: As new information becomes available, changes become necessary. The editors/authors/contributors and the publishers have, as far as it is possible, taken care to ensure that the information given in this book is accurate and up-to-date. In view of the possibility of human error or advances in medical science neither the editor nor the publisher nor any other party who has been involved in the preparation or publication of this work warrants that the information contained herein is in every respect accurate or complete. Readers are strongly advised to confirm. This book is for sale in India only and cannot be exported without the permission of the publisher in writing. Any disputes and legal matters to be settled under Mumbai jurisdiction only.

Rs. 225/Printed at Print Om Offset, Satara. 269, B/2, Daulatnagar, Karanje, Satara. Phone- (02162)234049. Designed by Srushti Computers, Satara. G-2, ‘Venna’, Adarshnagar, Khed, Satara. [email protected].

PREFACE Right kind of laboratory manuals suitable to Indian conditions is a dire necessity for promoting professional sciences. Teaching in Pharmacy lacks it. The present laboratory manual is expected to fill up the yawning gap. Good quality articles in Journal of Chemical Education and American Journal of Pharmaceutical Education is a rich source of ready experiments to be implemented in laboratories; however all these publications have American perspectives. In recent years few research papers on laboratory experiments are being published in Indian Journal of Pharmaceutical Education and Research. We have included the simple experiments which are feasible in laboratory of undergraduate courses instead of experiments requiring sophisticated instruments and costly setup. This book is divided into seven sections comprising 32 experiments as per their feasibility in laboratories and we are in process of designing more such experiments for the benefits of students. We acknowledge the help and co-operation extended by various persons in bringing out this book. We are highly indebted to the authors of the various books and articles mentioned in bibliography which became a major source of information for writing this book. We also thank the publishers and designers who graciously worked hard to publish this book in time. Our request to all users of this book is to provide constructive criticism in improving further editions of the book. We sincerely hope that readers will certainly welcome the book.

Satara

Authors August 31, 2010.

CONTENTS Experiment No. Section 1 1 2 3 4 5 6 7 8 Section 2 9 10 11 12 13 Section 3 14 15 16 Section 4 17 18 Section 5 19 20 Section 6 21 22 23 24 25 26 27 28 29 30 31 Section 7 32

Title of the experiment

Page No.

Physicochemical properties of drugs and dosage forms Determination of partition coefficient and dissociation constant......................... 01 Verification of Noyes Whitney law of dissolution............................................... 07 Kinetic study of dissolution of drug......................................................................13 In vitro dissolution of compressed tablet..............................................................18 In vitro dissolution of fast dissolving tablet......................................................... 22 In vitro dissolution of sustained release tablet......................................................25 Effect of pH on dissolution of Benzoic acid sticks...............................................31 Effect of pH on dissolution behavior of drug....................................................... 36 Absorption of drugs Intestinal permeability using chicken intestine.................................................... 41 Effect of permeation enhancers on intestinal permeability of drug..................... 46 Percutaneous absorption of drug from various ointment bases............................ 50 Percutaneous absorption of drug through different membranes...........................56 In vitro permeation study using Franz diffusion cell............................................ 62 Protein binding of drugs Protein binding study using equilibrium dialysis method................................... 66 Protein binding study using dynamic dialysis method......................................... 71 Determination of binding sites using bovine serum albumin............................... 75 Metabolism of drugs Metabolism of drug using in vitro method............................................................79 Effect of food on metabolism of drug...................................................................84 Excretion of drugs Urinary excretion of drug..................................................................................... 87 Influence of urinary pH on excretion of drug.......................................................91 Pharmacokinetics Simulation of plasma elimination and urine excretion after an IV bolus dose.....95 Calculation of various pharmacokinetic parameters after an IV bolus injection.105 Calculation of urinary excretion rate constant and elimination rate constant......109 Simulation of plasma elimination after an IV Infusion........................................114 Calculation of various pharmacokinetic parameters after an IV infusion............119 Calculation of area under curve by Trapezoidal rule...........................................122 Calculation of absorption rate constant by method of residuals..........................125 Calculation of absorption rate constant by of Wagner-Nelson method................129 Calculation of various pharmacokinetic parameters after extravascular administration.......................................................................................................133 Pharmacokinetic study of drug using plasma and urinary data............................140 Pharmacokinetic study of drug using salivary drug concentration......................146 Bioavailability and Bioequivalence Bioequivalence testing of drug using salivary samples....................................... 151 Bibliography....................................................................................................... 159

ABBREVIATIONS A A0 API ARA Au AUC BCS BSA C Cmax C0 Css Cv CADD CDR Cl CYP ER F Fr FDTs HSA IAEC IEC Jss K K Ka Ka Kp Ku Papp PC PC pKa Ro t1/2 tmax V

Amount of drug in body at time t Amount of drug at zero time Active pharmaceutical ingredient Amount of drug remaining to be absorbed Amount of drug excreted in urine Area under the plasma drug concentration-time curve Biopharmaceutical classification system Bovine serum albumin Plasma concentration at time t Maximum plasma concentration Initial plasma concentration Steady state plasma concentration Initial concentration of drug in donar compartment Cumulative amount of drug diffused Cumulative drug release Clearance Cytochrome Excretion rate Absolute bioavailability Relative bioavailability Fast dissolving tablets Human serum albumin Institutional Animal Ethics Committee Institutional Ethics Committee Steady state flux Elimination rate constant (Pharmacokinetic studies experiment ) Dissolution rate constant (Dissolution studies experiment) Absorption rate constant Acid constant Permeability coefficient Excretion rate constant Apparent permeability Partition coefficient Apparent partition coefficient Dissociation rate constant Constant infusion rate Biological half life Time required to achieve maximum plasma concentration Volume of distribution

SECTION 1

Physicochemical properties of drugs and dosage forms Experiment 1 Determination of partition coefficient and dissociation constant Aim To determine Ka, pKa, and partition coefficient (PC) of Salicylic acid and study their relationship.

Learning objectives 1. To study partition coefficient (PC) and dissociation constant (pKa) of Salicylic acid. 2. To measure the extraction of Salicylic acid from aqueous buffer into organic solvent. 3. To understand the relationship between pKa and pH. Theory pH-partition hypothesis was put forth by Brodie et al., which states that drugs are absorbed from the gastrointestinal tract by passive diffusion depending on the fraction of undissociated drug at the pH of the intestine. Thus, the process of absorption is governed by: 1. the dissociation constant (pKa) of the drug 2. the lipid solubility of the unionized drug ( PC) and 3. the pH at the absorption site. Drug pKa and gastrointestinal pH pKa is a measure of the strength of an acid or a base. pKa is defined as the negative logarithm of the equilibrium coefficient of the neutral and charged forms of a compound. Calculation of pKa allows the proportion of neutral and charged species at any pH to be estimated, as well as the basic or acidic properties of the compound to be defined. Lower the pKa of an acidic drug, stronger is the acid i.e. greater the proportion of ionized form at a particular pH. Higher the pKa of a basic drug, the stronger is the base. Thus, from the knowledge of pKa of drug and pH at the absorption site, the relative amount of ionized and unionized drug in solution at a particular pH and the percent of drug ionized at this pH can be determined by Henderson-Hasselbalch equation: For weak acids, pH = pK a + log

ionized drug concentration unionized drug concentration

…1

10 pH - pKa ´100 1 + 10 pH - pKa

…2

% drug ionized =

For weak bases, pH = pK a + log

unionized drug concentration ionized drug concentration

...3 1

Laboratory Manual of Biopharmaceutics and Pharmacokinetics

2

% drug ionized =

10 pKa - pH ´100 1 + 10 pKa - pH

…4

Lipophilicity and drug absorption PC of a drug is a measure of how well a substance partitions between a lipid (oil) and water. It is defined as the ratio of concentration of compound in aqueous phase to the concentration in an immiscible solvent, as the neutral molecule. Partition Coefficient, PC = [Conc in organic phase] / [Conc in aqueous phase] If the drug exists predominantly in the unionized form, it will be poorly absorbed if it has poor lipid solubility. Ideally, for optimum absorption, a drug should have sufficient aqueous solubility to dissolve in the fluids at the absorption site and lipid solubility high enough to facilitate partitioning of the drug in the lipoidal biomembrane and into systemic circulation. The lipid solubility of a drug is determined from its oil/water PC value. PC is a measure of the degree of distribution of drug between one of the several organic, water immiscible, lipophilic solvent such as n-octanol, chloroform, n-heptane, etc. and an aqueous phase (water or a suitable buffer). In general, the octanol/pH 7.4 buffer partition coefficient value in the range of 1 to 2 of a drug is sufficient to predict passive absorption across lipoidal membranes. Principle Salicylic acid is a relatively polar, poorly aqueous soluble material. The salt form however is quite water soluble. By changing pH of the aqueous buffer, you are able to alter the ratio between the ionized and unionized form of the acid. Since the unionized form is extracted into the organic phase, the fraction extracted will vary with pH of the aqueous solution. The definition of ionization constant (Ka) can be useful. In aqueous buffer H+ [H + ] ×[ A- ] K a ×[H A] Ka = or [ A- ] = [H A] [H + ]

...5

The partition between organic and aqueous buffer can be described by a 'true' partition coefficient, PC, as

PC =

[ HA - organic ] [ HA - aqueous] or

[HA- organic] = PC [HA- aqueous]

…6

In laboratory an apparent partition coefficient, PC' is measured, which will vary with pH or [H+]. This apparent partition coefficient is given by: PC ¢ =

[ HA - organic ] [ HA] + [ A- ]

…7

Substituting for [HA-organic] from equation 6 and [A-] from equation 5 gives

Section 1

Physicochemical properties of drugs and dosage forms

PC ¢ =

PC ¢ =

3

[PC ]´ [H A] Ka ù é êë 1 + H + úû ´ [ H A ]

…8

PC K a ù é êë 1 + H + úû

…9

This gives us an equation with PC' as function of [H+]with two unknown parameters, PC and Ka. This can be converted into a straight line equation by taking the reciprocal of both sides of the equation. Thus,

1 [H + ] Ka = + PC ¢ PC ´ [H + ] PC ´ [H + ]

… 10

1 1 Ka = + PC ¢ PC PC ´ [H + ]

… 11

Therefore plotting 1/PC' versus 1/[H+] gives a straight line with a slope of Ka/PC and an intercept of 1/PC. 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 0

2000

4000

6000

8000

+

1/[H ] PC' can be estimated by determining concentration of Salicylic acid partitioned in organic phase. Salicylic acid present in organic phase is estimated by adding ferric nitrate solution. The reaction of Salicylic acid with ferric nitrate produces an intensely colored complex, whose maximum absorbance can be detected at 540 nm spectrophotometrically. Prerequisite 1. Concept of pH and pKa. 2. pH partition hypothesis. 3. Concept of drug absorption.

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4

Requirements 1. Glasswares: Volumetric flasks, pipettes, etc. 2. Chemicals: Salicylic acid, sodium hydroxide, hydrochloric acid, etc. 3. Instruments: Balance, spectrophotometer. Procedure 1. Prepare 100 ml of buffer at pH 2.5. 2. Weigh accurately 20 mg of Salicylic acid and transfer to 100 ml volumetric flask and adjust volume by buffer of pH 2.5. Similarly prepare 0.02 % solution of Salicylic acid with buffers of pH 2.8, 3.0, 3.5, 3.8, and 4.0. 3. Take 4 ml of the buffer pH of 2.5 containing Salicylic acid stock solution and add 1 ml of the ferric nitrate solution. Allow the color to form and measure the absorbance at 540 nm using spectrophotometer. This is absorbance ONE (ABS I). 4. Next, take 5 ml of the buffer pH 2.5 containing Salicylic acid stock solution and add 5 ml of the organic solvent hexane/ethyl acetate. Stopper and shake the test tube for 5 minutes to complete the extraction. Allow the two phases to settle, remove 4 ml of the aqueous phase, add 1 ml of ferric nitrate solution (0.55% ferric nitrate in 0.4 M nitric acid), allow the color to form, and measure the absorbance at 540 nm. This is absorbance two (ABS II). Using the two absorbance readings, calculate the apparent partition coefficient, PC', as

PC ¢ =

[total in organic] [total in aqueous]

…12

Where, [total in organic] = First absorbance - Second absorbance and [total in aqueous] = Second absorbance. Thus, PC ¢ =

(ABS I - ABS II) ABS I

…13

5. Repeat the experiment with the buffers of pH 2.8, 3.0, 3.5, 3.8, and 4.0. Plot 1/PC' versus 1/[H+] and calculate PC, Ka, and pKa. 6. Calculate H+ ion concentration with the help of equation, pH = -log10 [H+]. Observations Table 1.Apparent partition coefficient (PC') of Salicylic acid Sr. No.

pH

1

2.5

2

2.8

3

3

4

3.5

5

3.8

6

4

Absorbance I (A)

Absorbance II (B)

PC'-Apparent partition coefficient

Extraction of SA (A - B)

PC' = (A -B) / B

Section 1

Physicochemical properties of drugs and dosage forms

5

+

Table 2. Values of [H ] and PC' of Salicylic acid Sr. No.

pH

1

2.5

2

2.8

3

3

4

3.5

5

3.8

6

4

[H+]

1/[H+]

PC'

1/PC'

Calculations +

1. Concentration of H at given pH +

pH = - log10 [H ] [H+] = -Antilog (pH) 2.Apparent partition coefficient (PC') PC' = (ABS I -ABS II)/ABS II 3. Partition Coefficient (PC) +

Plot the graph of 1/PC' versus 1/H You will get straight line with a slope, m = Ka/PC and an intercept, c = 1/PC. Calculate intercept c, and determine PC = 1/c. 4.Acid Constant, Ka Substitute the values of slope (m) and PC in the formula Ka= m x PC 5. Dissociation Constant, pKa pKa = -log10 (Ka) Results 1. Extraction of Salicylic acid in organic layer at pH 2.5 was_______. 2. The partition coefficient (PC) of Salicylic acid was found to be ______. 3. pKa of Salicylic acid was found to be _____. Conclusion Dissociation constant, pKa value gives idea regarding the ionization of the drug at given pH while partition coefficient, PC gives idea regarding its lipid solubility at given pH. Thus, the drug existing in unionized form with higher lipophilicity at given pH ensures its absorption. As the pH goes on increasing, extraction of Salicylic acid in organic layer goes on increasing. Applications 1. Determination of pKa: We can calculate the relative amount of unionized (absorbable) and ionized (unabsorbable) forms of the drug and predict the extent of absorption at a given pH of gastro intestinal tract, if pKa of drug is known. 2. Determination of partition coefficient: We can predict the extent of absorption by knowing the lipid solubility

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6

because high lipid solubility facilitates the partitioning of the drug in the lipoidal biomembrane and into the systemic circulation. 3. The knowledge of value of pKa and PC of particular drug will be useful for designing appropriate dosage form for optimum bioavailability. Questions 1. Define PC, pKa, and pH. 2. How will you utilize the values of pKa and PC of drug while designing of drug deliveries? 3.An acid has a pKa of 5.2. What percentage of the acid will be ionized at pH 6.0? 4. Consider following compounds: Compound Toluene-4-sulphonic acid

Type Acid

pKa -1.3

Benzoic acid

Acid

4.2

Thiopental

Acid

7.6

Codeine

Base

8.2

Atropine

Base

10

Which will be best absorbed from the stomach ( pH = 2)? Which will be best absorbed from the small intestine (pH = 4.2)? (Assume partition coefficients of compounds are same.) Exercise Determine Ka, pKa and PC of Caffeine or any other weak base.

Section 1

Physicochemical properties of drugs and dosage forms

7

Experiment 2 Verification of Noyes Whitney law of dissolution Aim To verify Noyes Whitney law of dissolution using Benzoic acid sticks. Learning objectives 1. To study and understand the concept of Noyes Whitney law of dissolution. 2. To verify Noyes Whitney law of dissolution using Benzoic acid sticks. Theory When a drug is given orally in the form of a tablet, capsule or suspension the rate of absorption is controlled by how fast the drug dissolves in the fluids at the absorption site. Therefore, dissolution rate is often rate limiting step in the following sequence.

Disintegration

Permeability Dissolution

Fraction absorbed Drug in systemic circulation

Drug in solution Degradation

Complexation

Gut wall metabolism

Liver metabolism

Absorption

Bioavailability

Figure 1. Dissolution and absorption process of tablet When dissolution is the rate controlling step in the over all process, absorption is said to be dissolution rate limited.Absorption from the solution proceeds more rapidly than from a solid dosage form. In case of freely soluble drugs absorption is independent of dissolution while sparingly soluble drugs tend to result in dissolution limited absorption. Dissolution of a solid in a liquid involves transfer of mass from a solid to a liquid phase. The overall transfer process may be regarded as being composed of two consecutive steps. The first involving an interfacial reaction that results in the liberation of solute molecules from the solid phase, followed by the transport of solute molecules away from the interfacial boundary under the influence of diffusion or convection. Like any complex reaction that involves consecutive stages, the overall rate of mass transfer in dissolution will be determined by the slowest stage. If rates of two consecutive stages are comparable in magnitude, then both stages will influence the overall rate of transfer. In 1897, Noyes and Whitney described the quantitative analysis that correlated the amount of time it took to dissolve a drug from solid particles. Noyes and Whitney law states that the rate at which a solid

8

Laboratory Manual of Biopharmaceutics and Pharmacokinetics

substance dissolves in its own solution is proportional to the difference between the concentration of that solution and the concentration of the saturated solution. Mathematical expression of Noyes and Whitney law is as follows dc = K (C s - C) dt ...1 Where, dc/dt= rate of dissolution, Cs= saturation solubility of the substance, C= concentration at the expiration of the time t, and K= dissolution constant. Drug particle

Diffusion layer

Bulk solution

Cs

Ch

h

Figure 2. Dissolution of a drug according to diffusion layer model The current version of the equation is slightly modified from the original but remains based on a diffusion layer model of dissolution (figure 2) of drug from a particle into a large excess bulk medium. The rate of dissolution depends upon the surface area of the solid, which in turns depends upon how finely the drug is subdivided. It also depends upon energy and energy states within the crystals of drug. A general relationship descending the dissolution process was first observed by Mayer and Whitney. The modified Noyes Whitney equation states that

dc = K S (C s - C ) dt

…2 Where, S = surface area of dissolving solid, Cs = saturation solubility of drug (concentration of diffusion layer), C = concentration of drug in the dissolution medium at time t. K, the dissolution rate constant, is equal to the diffusion layer (D/h), like the unstirred water layer in the intestine and is a thin, stationary film of solution adjacent to the surface of solid. The layer is saturated with the drug. Thus the drug concentration in the layer is equal to Cs. The term Cs-C represents the concentration gradient between the diffusion layer and the bulk solution. In dissolution rate limited absorption, C is negligible. Then equation 2 is reduced to

dc D S C s = …3 h dt Noyes and Whitney equation assumes that the rate of mass transfer depends on the rate at which the solute diffuses from the thin boundary layer into the bulk of solution. Therefore K, will depend on the diffusion coefficient of the solute and the thickness of the diffusion pathway and it will be influenced by factors that influences the diffusion coefficient and film thickness. If surface area is kept constant, then KS = K' …4 Therefore, equation 2 can be reduced to

Section 1

Physicochemical properties of drugs and dosage forms

dc = K ¢(C s - C) dt

9

…5

On integrating above equation, log

K¢t Cs = ( C s - C) 2.303

…6

Dissolution rate for a particular drug in a particular solvent can be calculated since Cs K ¢ 2.303 K= = ´ log S St (C s - C ) …7 Principle The principle of this experiment is based on the fact that the dissolution rate constant remains same despite variation in surface area of Benzoic acid sticks. The dissolution rate constant of Benzoic acid is calculated by using equation 7. The saturation solubility (Cs) of Benzoic acid can be calculated by dissolving its excess amount in water while the concentration of Benzoic acid (C) in the solution at given time (t) can also be estimated. The dissolution rate constant for Benzoic acid stick A and stick B remains same even though the surface areas of both these sticks are different. Thus, Noyes and Whitney law can be verified using Benzoic acid sticks. Prerequisite 1. Concept of dissolution and factors affecting dissolution process. 2. Saturation solubility. 3. Logarithmic calculations. Requirements 1. Glasswares: Beakers, Nesseler's cylinder, test tubes, glass rods, burette and conical flask. 2. Chemicals: Benzoic acid, sodium hydroxide, oxalic acid and phenolphthalein. 3. Equipments: Vernier caliper. Procedure 1. Standardization of 0.05 N sodium hydroxide solution Take 10 ml of 0.05 N oxalic acid (dissolve 315 mg of oxalic acid in 100 ml of distilled water) solution into a conical flask and add 2 drops of phenolphthalein indicator. Titrate contents of the flask against sodium hydroxide solution until permanent pink color is obtained. Repeat the titration to get concordant values. 2. Preparation of saturated solution of Benzoic acid (BA) Prepare saturated solution of Benzoic acid in water by putting excess of Benzoic acid in 100 ml of water. Stir resulting solution using magnetic stirrer for 2 hours and filter. Withdraw 10 ml of filtered saturated solution and determine the quantity of Benzoic acid by titrating with 0.05 N NaOH (this will give value of Cs) 3. Preparation of Benzoic acid sticks 1. Place required quantity of pure crystals of Benzoic acid in a beaker and heat till it melts (heating should not be so vigorous that Benzoic acid gets discolored).

Laboratory Manual of Biopharmaceutics and Pharmacokinetics

10

2. Take a glass rod and test tube. Flatten one end of glass rod to fit into the test tube and place this test tube on a test tube stand vertically. 3. Hold glass rod in the center of the test tube and pour molten Benzoic acid carefully. Fill it to 9-10 cm height. Hold the rod in the center till acid begins to solidify. 4. After cooling at room temperature, place test tube on ice-bath for 10 to 15 minutes. Due to further cooling Benzoic acid will shrink and dislodge from surface of the test tube. 5. After thorough cooling, pull out the glass rod along with the Benzoic acid cylinder. Cut with a blade to a length of 6 to 8 cm. Measure the exact length with scale and also the diameter with the help of a vernier caliper. 6. Smear some soft paraffin on two opposite surfaces of stick (cylinder). The idea is to prevent the surfaces for dissolution. Excess of soft paraffin should be avoided as it diffuses to the Benzoic acid stick and spoils the circular surface. 7. Prepare Benzoic acid sticks of varying length and mark as StickAand Stick B. 4. Dissolution study of Benzoic acid sticks 1. Fill a pair of Nesseler's cylinder/measuring cylinder to 100 ml with distilled water. Then dip Benzoic acid sticks into cylinder and note initial time. Move sticks up and down for 10 minutes. Then remove 10 ml of solution and titrate with 0.05N NaOH using phenolphthalein as an indicator for analysis of Benzoic acid salt. Maintain sink conditions with water. 2. Similarly determine the concentration of Benzoic acid after 20, 30 and 40 minutes. 3. Perform the blank titration omitting the sample and substract the readings from the above said titrations and report the results. 5. Plotting of graph 1. Plot the graph of log (Cs/Cs-C) versus time (t) on graph paper. Observations Table 1. Standardization of 0.05 N NaOH Sr. No.

Volume of 0.05N Oxalic acid solution (ml)

1

10

2

10

3

10

Burette reading initial (ml)

Burette reading final (ml)

Vol. of NaOH used (ml)

Normality of NaOH

Table 2. Parameters of Benzoic acid stick Sr. No. 1

Stick A

2

Stick B

Stick

Diameter (D) (cm)

Length (L) (cm)

Surface area (S) (cm2)

Saturation solubility of Benzoic acid in water (Cs)= ___________g/l.

Normality

Section 1

Physicochemical properties of drugs and dosage forms

11

Volume of NaOH used (a-b)* (ml)

Normality of NaOH

Concentration of BA (A)

log Cs/(Cs-C)

Time (min)

Cs/(Cs-C)

Table 3. Observation table for stickA Slope

K' = K=K'/S slope x 2.303

10 20 30 40 Average K * a is volume of NaOH used for Benzoic acid titration, b is blank titration i.e. without Benzoic acid.

Volume of NaOH used (a-b)* (ml)

Normality of NaOH

Concentration of BA (B)

log Cs/(Cs-C)

Time (min)

Cs/(Cs-C)

Table 3. Observation table for stick B Slope

K' = K=K'/S slope x 2.303

10 20 30 40 Average K * a is volume of NaOH used for Benzoic acid titration, b is blank titration i.e. without Benzoic acid.

Calculations 1. Calculation for normality of NaOH N1V1 = N2V2 N1= Normality of NaOH, V1= Volume of NaOH used, N2= Normality of Benzoic acid, V2= Volume of Benzoic acid used 2. Calculation for concentration of saturated solution of Benzoic acid (Cs) N1V1 = N2V2 NaOH Benzoic acid Concentration of BAsolution = N2 x Molecular weight of BA = N2 x 122.1 Saturation solubility = N2 x 122.1 3. Calculation for surface area of BAstick (S) S = p DL Where, D- diameter, L - length 4. Calculation for concentration of Benzoic acid sticks at time t (C) N1V1 = N2V2 NaOH Benzoic acid Concentration of BA solution = N2 x Molecular weight of BA = N2 x 122.1

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12

5. Calculation of dissolution rate constant (K) 0.4 0.35 0.3 log (Cs/Cs-C)

Plot the graph of log (CS/CS-C) versus time (t) on graph paper. Graphically calculate K' K' = Slope x 2.303 K=D/h= K'/S

0.25 0.2 0.15 0.1 0.05 0 0

10

20 Time (min)

30

40

Result Dissolution rate constant for Benzoic acid stick was found to be ________. Conclusion It can be concluded from this experiment that the dissolution rate constant 'K' remains constant for given drug at given conditions and hence Noyes and Whitney law of dissolution is obeyed. Applications 1. Dissolution tests are used in the pharmaceutical industry for quality control and to assist with the determination of bioequivalence. 2. Dissolution test provides useful information at several stages of drug development. 3. Dissolution test can be used as a prognostic tool of oral drug absorption. 4. Dissolution can also be an essential tool for the development and evaluation of sustained release formulations. 5. Dissolution test can be a tool for in vitro- in vivo correlation of drug. Questions 1. Define solubility. How will you determine saturation solubility? 2. Describe factors affecting dissolution rate of drug. 3. Describe the sequential events during the transfer of a drug from a solid dosage form in the gastrointestinal tract to the systemic circulation. 4. Define dissolution and describe film theory for dissolution. Exercise Verify Noyes Whitney law of dissolution using any weakly basic drug.

Section 1

Physicochemical properties of drugs and dosage forms

13

Experiment 3 Kinetic study of dissolution of drug Aim To study the kinetics of dissolution ofAspirin and report dissolution rate constant. Learning objectives 1. To study the kinetics of dissolution of solid substances by Guggenheim's method. 2. To correlate the obtained data for determination of dissolution rate constant. Theory Dissolution of solid substances is one of the heterogeneous processes occurring at the boundary between two phases, which is called as phase interface. Obviously one of the phase is solid, so it is reaction on the solid surface and it can be divided into following phases. 1. Diffusion of interacting substances to the surface, 2.Adsorption on the surface, 3. Reaction on the surface, 4. Desorption from the surface, 5. Diffusion of product from the surface, The total reaction rate of heterogeneous process is controlled by the rate of the slowest step and in the case of solid/liquid systems, the rate determining stage is sub processes involving diffusion. In biological systems, water represents the most frequent liquid environment-solvent. In the process of dissolution of crystalline solid compounds into aqueous solution, the above steps are supplemented with hydration of the surface, and the products of dissolution. Dissolution of solid substance is controlled by the slowest reaction stage, which is the diffusion of dissolved and hydrated compound from the solid surface. The diffusion transports the dissolved substance across a thin diffusion layer h, where the concentration of dissolved substance continuously decreases from the concentration of saturated solution (Cs) at the solid surface to the concentration level (C) in the bulk solution. The driving force of diffusion is the spatial concentration gradient in according to First Fick's Law:

dn dc = - DS dt dx

…1

Where, dn = amount of the dissolved substance within time interval dt, D = diffusion coefficient, S = total surface (phase interface) of the dissolved solid and finally dc/dx = concentration gradient. When the mixing is efficient, the diffusion layer is very thin (0.02-0.05 nm) and the concentration gradient may be replaced by a single linear approximation

dc (C - C S ) = dx h

…2

For calculating the amount of dissolved substance, ‘dn’ we can then write

dn = Vdc

…3

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Laboratory Manual of Biopharmaceutics and Pharmacokinetics

Where, V = total volume of solution and dc = concentration increment. The final shape of the Nernst equation is dc DS dc = (C S - C ) = K (C S - C ) or dt Vh dt

…4

where, K = rate constant of dissolution. After separation of variables and integration we obtain following equation: C = C S (1 - e - kt ) …5 which is formally equivalent to the equation for the first order reaction kinetics. Chemical kinetics can be defined as a quantitative study on concentration (or pressure) changes with time brought about by a chemical reaction. In other words, the chemical kinetics investigates velocities of various chemical reactions. Reaction rate is decrease of the concentration per unit time of one of the reactants. The rate constant is a measure of the rate of a given chemical reaction under specified conditions (pressure, temperature). It may be defined as the rate of changes in concentration of reactant or product with time for a reaction in which all the reactants are at unit concentration. The order of reaction is usually a small whole number, but in special case it may have a fractional value or be zero. It is formally defined as sum of the powers of the concentration terms that occur in the differential form of the rate law. If the chemical reaction proceeds in a series of sequential stages, then the rate of reaction is limited by the slowest stage. This stage is referred to as the rate determining stage. Principle Kinetic measurements are usually performed for determination of reaction rate (or rate constant) or reaction order at given conditions. Dissolution process of ionic substances can be observed by measuring the conductivity changes with time:

G ( t ) = G s (1 - e - k t )

. . .6

where G(t)= conductivity at time t, Gs = saturated conductivity Assume the surface changes of the dissolved substance are negligible. In case of less stable organic compounds, precise determination of the conductivity of saturated solution is impossible because of side reactions (e.g. acetylsalicylic acid hydrolyses to acetic and salicylic acid). Thus the saturated conductivity Gs is handled as unknown quantity and must be determined, too. The Guggenheim's method of evaluation of the rate constant will be used, because the final concentration is unknown. The essence of the method may be characterized as follows: there are some measurements series constructed within the experiment, where the time shift between the series remains constant. The concentration data are recorded at time t and t+dt (dt is the appropriate time shift). Using the Guggenheim's method the mentioned kinetic equation for the conductivity dependence may be written as:

In [G(t + dt ) - G(t )] = A - e - kt

. . .7

Where

A = In [GS (1 - e - kd )]

. . .8 The last equation is used for determination of the unknown saturated conductivity.

Section 1

Physicochemical properties of drugs and dosage forms

15

The series are not to be taken from the start and the end of measurement process, because the experimental errors are bigger at these points. Prerequisite 1. Theory of dissolution. 2. Chemical kinetics. Requirements Glasswares: Electrode holder, 400 ml beaker, perforated test tube. Chemicals: 4 tablets ofAcetylsalicylic acid (maximum solubility 2.5 g/l at 150C). Instruments: Conductometer, electromagnetic stirrers, stop watch. Procedure 1. Place the beaker with 200 ml of double distilled water on the electromagnetic stirrer. 2. Using the laboratory stand, fix the perforated test tube and conductivity cell into beaker. 3. Set the mixing rate at 800 rpm, which is constant for the experiment (note that no heating is needed). 4. Determine conductivity of pure redistilled water (it must be less than 2mS). Now carefully put the tablets into the test tube and start the stop-watch. Write down the conductivity data every minute and from the 5th minute every 5 minutes. Repeat the described conductivity determinations for 85 minutes. 5. After finishing the measurements switch off the stirrer and conductometer and rinse the conductivity cell with distilled water. 6. Plot the graph of function G=f(t) to characterize the overall development of the dissolution in time. Select two series from the measured conductivity data, which differ in reaction times by a constant time shift (t=40 min). Then calculate the appropriate quantity: log [G(t+dt) G(t)]. 7. The data set is given by conductivities at reaction time: t=5, 10, 15, 20, 25, 30, 35 min. The second data set is defined sequentially: (t+dt) =45, 50, 55, 60, 70, 75. 8. Using of least squares method determine parameters for the following linear function:

log [G(t+dt) - G(t)] = logA- Kt/2.303 Slope = -K/2.303

5.00 ln[G(t+dt)-G(t)]

In[G(t+dt) - G(t)] =A-kt where, slope = -K Where, the slope is negative value of the dissolution rate constant at a given temperature. or alternately

4.00 3.00 2.00 1.00 0.00 5

10

15 20 25 Time (min)

30

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Laboratory Manual of Biopharmaceutics and Pharmacokinetics

16

B A

Figure 1. Design of apparatus A- Electrode, B- Perforated tube, C- Beaker, D- Magnetic stirrer C

D

Observations Table1. Table of conductivity dependence on time

Time (t/min) 1 2 3 4 5 10

Conductivity G(t)/mS

Table 2. Guggenheim's method of determination of the rate constant t (min) 5 10 15 20 25

G (t) (mS)

t+dt /min 45 50 55 60 65

G(t+dt) (mS)

G(t+dt)-G(t)

In [G(t+dt)-G(t)]

Result Dissolution rate constant forAspirin tablet by Guggenheim's method was found to be _______ . Conclusion It can be concluded that dissolution rate constant can be determined by Guggenheim's method using conductometer. Applications The Guggenheim method for the evaluation of rate constants is shown to be applicable to a wide range of problems that are of pharmaceutical interest. These include: 1. Reaction kinetics in which more than one product is produced from a common reactant. 2. Consecutive first-order reactions.

Section 1

Physicochemical properties of drugs and dosage forms

3. Dissolution followed by partitioning into a lipid phase. 4. The use of dissolution kinetics to obtain drug solubility and 5. The analysis of drugs through kinetics. Question Enlist the applications of conductance property of drug in the field of pharmacy. Exercise Study dissolution kinetics of any weakly basic drug.

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18

Experiment 4 In vitro dissolution of compressed tablet Aim To study in vitro drug release of the given compressed tablet of Paracetamol. Learning objectives 1. To understand the working of dissolution test apparatus. 2. To understand the dissolution process of an uncoated tablet. Theory Dissolution is the process by which a solid solute enters a solution. In the pharmaceutical industry, it may be defined as the amount of drug substance that goes into solution per unit time under standardized conditions of liquid/solid interface, temperature and solvent composition. Dissolution is considered as one of the most important quality control tests performed on pharmaceutical dosage forms and is now developing into a tool for predicting bioavailability, and in some cases, replacing clinical studies to determine bioequivalence. Dissolution behaviour of drugs has a significant effect on their pharmacological activity. In fact, a direct relationship between in vitro dissolution rate of many drugs and their bioavailability has been demonstrated and is generally referred to as in vitro-in vivo correlation, IVIVC. Solid dosage forms may or may not disintegrate when they interact with gastrointestinal fluid following oral administration depending on their design (Figure 1). Tablet Tablet

Non disintegrating

D

isi

nt

eg

ra

Blood

tio

n

Dissolution

Granules

Figure 1: Schematic diagram of the dissolution process Dissolution kinetics is important in determining the bioavailability of a drug. The dissolution rate controls rate of build up of certain drugs in the blood stream. It was thus recognized that in-vitro dissolution kinetics provides useful information on the availability of drugs and their subsequent therapeutic effects invivo. This led to the inclusion of dissolution tests in the United States NF XIII (1970) and USP XVIII (1970) monographs for one capsule and twelve tablet preparations. In 1975, dissolution tests were included in the British Pharmacopoeia (amendment to BP 1973) for Digoxin tablets. Various pharmacopoeias contain specifications on the dissolution requirements of various drugs. A variety of designs of apparatus for dissolution testing have been proposed and tested, varying from simple beaker with stirrer to complex systems with lipid

Section 1

Physicochemical properties of drugs and dosage forms

19

phases and lipid barrier where an attempt is made to mimic the biological milieu. The choice of the apparatus to be used depends largely on the physicochemical properties of the dosage form. Compressed tablets are the standard uncoated tablets made by either direct compression or wet granulation or dry granulation or double compaction. They may be used for local action in gastro-intestinal tract or systemic action. When tablet exert local action, they are formulated as more water insoluble by means of selecting slow dissolving excipients and thus provides local action for long time period. e.g., antacids and adsorbents. The drugs that produce systemic action have some aqueous solubility and are designed to disintegrate and dissolve quickly so that the drug can be quickly absorbed and produce systemic action. Generally, an active pharmaceutical ingredient (API) exhibits bioavailability depending upon biopharmaceutical class, which is based on water solubility and gastro-intestinal membrane permeability criteria. But, it can be altered by appropriate selection of excipients and processing technology. Requirements a) Glasswares: Beaker, volumetric flasks, pipettes, test tubes, funnel. b) Equipments: Dissolution test apparatus, UV spectrophotometer. c) Chemicals: Pure Paracetamol, Paracetamol tablets, sodium hydroxide, potassium dihydrogen phosphate. Procedure A. Preparation of solutions 1. Preparation of 0.2 M potassium dihydrogen phosphate: Dissolve 27.22 gm of potassium dihydrogen phosphate in sufficient quantity of water to produce 1000 ml. 2. Preparation of 0.2 M sodium hydroxide: Dissolve 8 gm of sodium hydroxide in sufficient quantity of water to produce 1000 ml. 3. Preparation of phosphate buffer of pH 5.8: Place 50 ml of 0.2 M potassium dihydrogen phosphate in a 200 ml volumetric flask, add 3.6 ml of 0.2 M sodium hydroxide and then add water to make up the volume. B. Calibration curve of Paracetamol 1. Preparation of standard stock solution: Weigh accurately 100 mg of pure Paracetamol and transfer it into 100 ml volumetric flask and adjust volume (Stock I, 1 mg/ml). Transfer 10 ml stock I to another 100 ml volumetric flask and adjust volume (Stock II, 100 mg/ml). 2. Preparation of working solution: From stock solution II, pipette out 0.2, 0.4, 0.6, 0.8 and 1 ml into 10 ml volumetric flask and adjust volume to get concentration in the range of 2-10 mg/ml. 3. Measurement of absorbance: Measure absorbance of the respective dilutions at l max 243 nm using UVVisible spectrophotometer. Plot the graph of absorbance of Paracetamol against concentration in MS Excel and determine slope and intercept. C. Procedure for dissolution 1. Remove one of the dissolution vessels and fill acrylic tank with distilled water up to the level mark. Place dissolution vessel back. 2. Fix six stirring shafts with blades to the spindles projecting out of stirrer platform with the help of screw. 3. Fill the dissolution vessel with dissolution media up to mark, 900 ml.

Laboratory Manual of Biopharmaceutics and Pharmacokinetics

20

4. Connect the mains cord to the mains supply (230V). Put on mains switch. 5. Press the platform down key to lower down the platform. Place the acrylic covers on each vessel. 6. Set the parameters such as RPM, test temperature, test time and alarm time. Monitor the temperature of vessel by inserting the thermometer. 0

7. Once the temperature of vessels reaches to 37 C, insert three tablets of Paracetamol in first row of 3 vessels and press enter key to start test. 8. Withdraw samples at predetermined time interval. Filter and dilute sample if required and analyze spectrophotometrically at 243 nm. 9. From the absorbance values determine concentration of drug (mg/ml) and percent drug release. 10. With help of MS Excel, plot the graph of percent drug dissolved versus time. A. Parameters set for plotting of calibration curve 1) Beer's & Lambert range: 2) Solvent:

2-10 mg/ml Phosphate buffer pH 5.8

3) l max for Paracetamol:

243 nm

B. Parameters set for dissolution studies 1) Tablet: 2) Strength of tablet: 3) Apparatus: 4) Rotation Speed: 5) Test time: 6) Dissolution medium: 7) Volume of dissolution medium:

Paracetamol 500 mg IPType I dissolution apparatus (Paddle). 50 rpm 30 min Phosphate buffer (pH 5.8) 900 ml

Observations Table 1. Calibration curve of Paracetamol

Concentration (mg/ml) 2 4 6 8 10 Slope Intercept

Absorbance

Section 1

Physicochemical properties of drugs and dosage forms

21

Table 2. In-vitro dissolution of Paracetamol Time (min) 10 20 30

Percent cumulative drug release I

II

III

Mean ± SD

Calculations 1. Determination of concentration of dissolved drug (mg/ml) Y= m X + c Where, Y= absorbance, m= slope, X= concentration (mg/ml), c= intercept. 2.Amount of drug released (mg) Amount of drug released = [Concentration (mg/ml) x (volume of dissolution medium) x (dilution factor)]/1000 3. Dilution factor Dilution factor = volume of diluted sample (ml)/ volume of sample removed (ml) 4. Percent cumulative drug release Percent cumulative drug release = (amount of drug released) x 100 /strength of tablet Result From the release data it was found that _______ % of drug was released at 30 min. Conclusion Paracetamol tablet was investigated for in vitro drug release study and it passes/fails IP standards. Application As given in previous experiment (Expt No. 2). Questions 1. State difference between Type I and Type II dissolution apparatus. 2. Enlist the major parts and functions of Type I dissolution apparatus. Exercise Perform in vitro dissolution studies of an uncoated tablet of any weakly basic drug.

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Laboratory Manual of Biopharmaceutics and Pharmacokinetics

Experiment 5 In vitro dissolution of fast dissolving tablet Aim To study in vitro drug release of the fast release tablets of Domperidone. Learning objective To understand the dissolution process of fast release tablet of Domperidone. Theory Domperidone shows significant first pass metabolism and poor bioavailability. Thus, a strategy to improve bioavailability should aim at improving its aqueous solubility and overcoming first pass metabolism. Innovative drug delivery systems known as melt in mouth or mouth dissolving tablets are novel types of tablets that disintegrate/disperse/dissolve in saliva. Advantage of mouth dissolving tablets allowing administration without water anywhere anytime, leads to their suitability for geriatric and pediatric patients. They are also most suitable for drugs that undergo extensive first pass metabolism. The benefits, in terms of patient compliance, rapid onset of action as the drug goes directly into systemic circulation and good stability, make these tablets popular as a dosage form of choice in the current market. Fast dissolving tablets (FDTs) of Domperidone contains superdisintegrants, which accelerate the disintegration of tablets by virtue of their ability to absorb large amount of water when exposed to aqueous environment. This rapid disintegration of FDTs is due to penetration of saliva into the pores, which leads to swelling of superdisintegrants to create enough hydrodynamic pressure for quick and complete disintegration of the tablets. This increases bioavailability / rapid absorption through pre-gastric absorption of drugs from mouth, pharynx and esophagus as saliva passes down. Requirements a) Glasswares: Beaker, volumetric flasks, pipettes, test tubes, funnel. b) Equipments: Dissolution test apparatus, UV spectrophotometer. c) Chemicals: Domperidone tablets, pure Domperidone , methanol, hydrochloric acid, potassium dihydrogen phosphate. Procedure A. Calibration curve of Domperidone 1. Preparation of standard stock solution: Transfer 100 mg of Domperidone to 100 ml volumetric flask. Add 30 ml of methanolic HCl (0.1 N) so as to dissolve the drug. Make up the volume to 100 ml (Stock I). Withdraw 10 ml of solution and transfer to another 100 ml volumetric flask and make up volume (Stock II). 2. Preparation of working solutions: Transfer from Stock II, 0.2, 0.4, 0.8, 1.2, 1.6, and 2 ml solutions to 10 ml volumetric flasks (ten flasks) and make volume up to 10 ml with 0.1 N HCl in each flask so as to get concentration in the range of 2 to 20 µg/ml. 3. Measurement of absorbance: Measure absorbance of the respective dilutions at l max 284 nm using UVVisible spectrophotometer. Plot the graph of absorbance of Domperidone against concentration in MS Excel and determine slope and intercept.

Section 1

Physicochemical properties of drugs and dosage forms

23

B. Procedure for dissolution 1. Remove one of the dissolution vessels and fill acrylic tank with distilled water up to the level mark. Place dissolution vessel back. 2. Fix six stirring shafts with blades to the spindles projecting out of stirrer platform with the help of a screw. 3. Fill the dissolution vessel with dissolution media up to mark, 900 ml. 4. Connect the mains cord to the mains supply (230V). Put on main switch, heater switch & motor switch. 5. Press the platform down key to lower down the platform. Place the acrylic covers on each vessel. 6. Set the parameters such as RPM, test temperature, test time and alarm time. Monitor the temperature of vessel by inserting the thermometer. 0

7. Once the temperature of vessels reaches to 37 C, insert three tablets of Domperidone in first row of 3 vessels and press key to start test. 8. Withdraw samples at predetermined time interval (5 min). Filter and dilute sample if required and analyze spectrophotometrically at 284 nm. 9. From the absorbance values determine concentration of the drug (mg/ml) and percent drug release. 10. With the help of MS Excel, plot the graph of percent drug dissolved versus time. A. Parameters set for plotting of calibration curve 1) Beer's and Lambert range : 2-20 µg/ml 2) Solvent: Phosphate buffer pH 6.8 3) l max for Domperidone: B. Parameters set for dissolution studies 1) Tablet: 2) Strength of tablet: 3)Apparatus: 4) Rotation Speed: 5) Test time: 6) Dissolution medium: 7) Volume of dissolution medium:

284 nm Domperidone tablet 10 mg IPType I dissolution apparatus (Paddle). 50 rpm 30 min 0.1N HCl 900 ml

Observations Table 1. Calibration curve of Domperidone Concentration (mg /ml) 2 4 8 12 16 20 Slope Intercept

Absorbance

Laboratory Manual of Biopharmaceutics and Pharmacokinetics

24

Table 2. In vitro dissolution of Domperidone Time (min) 10 20 30

Percent cumulative drug release I

II

III

Mean ± SD

Calculations 1. Determination of concentration of Domperidone (mg/ml) Y= m X + c Where, Y= absorbance, m= slope, X= concentration (mg /ml), c = intercept. 2.Amount of drug released (mg) Amount of drug released = [Concentration (mg /ml) x (volume of dissolution medium) x (dilution factor)]/ 1000 3. Dilution factor Dilution factor = volume of diluted sample (ml)/ volume of sample removed (ml) 4. Percent cumulative drug release Percent cumulative drug release = Amount of drug released x 100 /strength of tablet Result It was found that Domperidone fast dissolving tablet shows 100 % drug release at _____ min. Conclusion Domperidone fast release tablet was investigated for in vitro drug release study. Applications 1. Fast dissolving tablets can improve the bioavailability of drug by virtue of overcoming first pass metabolism. 2. The comparison of various superdisintegrants can be done. 3. The drugs inactivated by gastric juice can be formulated as FDTs. Questions 1. Give the fast dissolving tablets available in market. 2. Enlist various superdisintegrants with their mechanisms. Exercise Study in vitro release of marketed fast dissolving tablet of Diclofenac sodium.

Section 1

Physicochemical properties of drugs and dosage forms

25

Experiment 6 In vitro dissolution of sustained release tablet Aim To study in vitro dissolution of the given sustained release Diclofenac sodium tablet. Learning objective To understand dissolution of a sustained release tablet of Diclofenac sodium. Theory Modified release tablet The main aim behind formulation of this dosage form is to release the medicament slowly for a long duration after administration of a single tablet. A widespread use of this type of tablet is mainly because of improvement in patient's compliance. As the dosage frequency is reduced, patient can take an undisturbed sleep at night. It's also beneficial for psychiatric patients who forget to take their tablets regularly and the dose related side effects and toxicities are reduced. Any adjuvant that can alter water uptake rate, swelling and gelling characteristics of matrixing agents can alter the release rate of API e.g., electrolytes in hydroxy propyl methyl cellulose (HPMC) matrix tablet. It's also possible to achieve pulsed drug release. Weakly basic drugs exhibit good solubility at low pH while less soluble drugs show at high pH conditions, which can result in incomplete drug release for sustained release formulations. The drug release can be modified by providing suitable micro environmental pH in the tablet e.g., acidic polymer, succinic acid, etc. Similarly, inclusion of alkaline polymers results in desirable drug release of acidic drugs. Classic approaches are usually based on adaptation of either film coated or multiparticulate technologies or those involving slow release matrices, which are discussed below: 1. Coating technology It combines semi permeable coatings and osmotic tablet cores to produce “zero order release” technology. Attention is also focused to trigger drug release at critical time point e.g., to achieve drug release 1 2 hours before the patient awakens. Alza's prolific research activities have yielded a technology called “Ringcap” which is based on a tablet, preferentially film coated, partially coated with a series of rings whose respective thickness provides the means of moderating the rate at which the drug is released from final dosage form. 2. Matrix technology Classically matrix products exhibit first order (or perhaps square-root-of-time) drug release characteristics. In order to achieve zero order release characteristics, it's necessary to employ specially designed materials or strategies that seek to manipulate tablet structure or geometry. Combination of conventional HPMC matrix technology with upper and lower layer helps to moderate drug release by increase in surface area with concomitant reduction in drug concentration within the device. Release of drug can follow various mechanisms 1. Diffusion is rate limiting Diffusion is driving force where the movement of drug molecules occurs from high concentration in the tablet to lower concentration in gastro intestinal fluids. This movement depends on surface area exposed to

26

Laboratory Manual of Biopharmaceutics and Pharmacokinetics

gastric fluid, diffusion pathway, drug concentration gradient and diffusion coefficient of the system. In practice, we can follow either of the two methods, 1. The drug is formulated in an insoluble matrix; the gastric fluid penetrates the dosage form and dissolves the drug and release it through diffusion. 2. The drug particles are coated with polymer of defined thickness so as the portion of drug slowly diffuse through the polymer to maintain constant drug level in blood. 2. Dissolution is rate limiting The drugs with poor water solubility (BCS class 2 and 4) are inherently sustained release forms. While for water soluble drugs, it's possible to incorporate a water insoluble carrier to reduce dissolution of the drug particles. They are coated with this type of materials e.g. Polyethylene glycol. One may skip the use of disintegrating agent to promote delayed release. 3. Osmotic pressure is rate limiting Osmosis is a phenomenon in which the flow of liquid occurs from lower concentration to higher concentration through a semi permeable membrane which allows transfer of liquid only. The whole drug is coated with a semi permeable membrane with a hole on one end of tablet made by a laser beam. The gastric fluid penetrates through the membrane, solubilizes the drug and increases the internal pressure which pumps the drug solution out of the aperture and releases the drug in gastric environment. The delivery rate is constant provided that the excess of drug is present inside the tablet. But, it declines to zero once the concentration drops below saturation. 4. Release is controlled by ion exchange Ion exchangers are water insoluble resinous materials containing salt forming anionic or cationic groups. While manufacturing, the drug solution is mixed with resin and dried to form beads which are tableted. The drug release depends upon high concentration of charged ions in gastro intestinal tract where, the drug molecules are exchanged and diffused out of the resin into the surrounding fluid. This mechanism relies upon the ionic environment of resin and not pH or an enzyme on absorption site. 3. Delayed action tablet Enteric coated tablet is such an example of delayed action tablet. This formulation is preferred when, 1. TheAPI irritates gastric mucosa e.g.,Aspirin or strong electrolytes. 2. Drugs that produce nausea and vomiting. 3.API is sensitive to low pH e.g., Erythromycin. 4. When it's necessary to release the drug undiluted e.g., intestinal antibacterial, antiseptic agents, etc. The commonly used coating agents are: Cellulose acetate phthalate, Hydroxy methyl propyl phthalate, polyvinyl acetate phthalate, Eudragit®, etc. This dosage form is intended to hydrate and begin to dissolve in duodenum (pH 4 to 6) or in small intestine where pH increases to 7 to 8. The presence of esterases or bile salts like surface active agents plays a role in drug release. Requirements a) Glasswares: Beaker, volumetric flasks, pipettes, test tubes, funnel. b) Chemicals: NaCl, NaOH, HCl, KH2PO4, Diclofenac sodium, methanol.

Section 1

Physicochemical properties of drugs and dosage forms

27

c) Equipments: USP dissolution apparatus type II, Balance and UV spectrophotometer. Procedure A. Preparation of solutions 1.Acidic buffer of pH 1.2: Dissolve 2 gm of sodium chloride and 7 ml of concentrated HCl in sufficient quantity of water to produce 1000 ml. 2. Preparation of phosphate buffer (pH 6.8): Place 50 ml of 0.2 M potassium dihydrogen phosphate in 200 ml volumetric flask, add 22.4 ml of 0.2 M sodium hydroxide and make up the volume. B. Plotting of calibration curve 1. Preparation of standard stock solution: Transfer 100 mg of Diclofenac to 100 ml volumetric flask. Add 30 ml of methanol to it so as to dissolve the drug. Make up the volume to 100 ml (Stock I). Withdraw 10 ml of solution and transfer to another 100 ml volumetric flask and make up the volume to 100 ml with methanol (Stock II). 2. Preparation of working solutions: From Stock II, pipette out 0.2, 0.4, 0.6, 0.8, 1, 1.2 and 1.4 ml into seven 10 ml volumetric flasks and adjust the volume to 10 ml with respective buffer (acidic buffer (pH 1.2) or phosphate buffer (pH 6.8)) to get concentration in the range of 2 to 14 µg/ml. 3. Measurement of absorbance: Measure the absorbance of respective dilutions at 276 nm using UV-Visible spectrophotometer. Plot the graph of absorbance of Diclofenac versus concentration in MS Excel and determine slope and intercept. C. Procedure for dissolution 1. Remove one of the dissolution vessels and fill acrylic tank with distilled water up to the level mark. Replace back the dissolution vessel. 2. Fit six stirring shafts with blades in the spindles projecting out of stirrer platform with the help of screw. 3. Fill the dissolution vessel with dissolution medium (acidic buffer solution) up to mark, 900 ml. 4. Connect the mains cord to the mains supply (230V). Put on mains switch, heater switch and motor switch. 5. Lower down the platform and fit covers on each vessel. 6. Set parameters such as temperature, RPM, test time and alarm time according to experimental conditions. 0

7. Once the temperature of vessels reaches 37 C insert three Diclofenac SR tablets (75 mg) in first row of 3 vessels and start the dissolution. 8. Withdraw 2 ml sample at regular time interval, filter, dilute and analyze by UV spectrophotometer at 276 nm. Replace same quantity of fresh dissolution medium. 9.After 2 h, replace dissolution medium with phosphate buffer and continue dissolution for further 10 h. 10. From the absorbance values, determine concentration of drug (mg/ml) and percent drug release along with standard deviation. A. Parameters set for plotting of calibration curve 1) Beer's & Lambert range: 2) Solvent: 3) l max for Diclofenac: B. Parameters set for dissolution studies 1) Tablet:

2-14 mg/ml Acidic buffer/phosphate buffer pH 6.8 276 nm Diclofenac sodium SR

Laboratory Manual of Biopharmaceutics and Pharmacokinetics

28

2) Strength of tablet: 3)Apparatus: 4) Rotation Speed: 5) Test time: 6) Dissolution medium: 7) Volume of dissolution medium:

75 mg IPType I dissolution apparatus (Paddle) 50 rpm 12 h Acidic buffer/ phosphate buffer pH 6.8 900 ml

Observations Table 1. Calibration curve of Diclofenac sodium (acidic buffer) Concentration (mg/ml) 2 4 6 8 10 12 14 Slope Intercept Coefficient of correlation

Absorbance

Table 2. Calibration curve of Diclofenac sodium (phosphate buffer) Concentration (mg/ml) 2 4 6 8 10 12 14 Slope Intercept Coefficient of correlation

Absorbance

Section 1

Physicochemical properties of drugs and dosage forms

29

Table 3. Drug release profile of Diclofenac sodium tablet Time (h)

1

Percent drug release 2 3 Acidic buffer

Mean

% CDR

0.5 1 1.5 2 Phosphate buffer 3 4 5 6 7 8 9 10 11 12 % CDR- Percent cumulative drug release

Calculations 1. Determination of concentration of Diclofenac sodium (mg/ml) Y= m X + c Where, Y= absorbance, m= slope, X= concentration (mg /ml), c = intercept. 2.Amount of drug released (mg) Amount of drug released = [Concentration (mg /ml) x (volume of dissolution medium) x (dilution factor)]/ 1000 3. Dilution factor Dilution factor = volume of diluted sample (ml)/ volume of sample removed (ml) 4. Percent cumulative drug release Percent cumulative drug release =Amount of drug released x 100 /strength of tablet Result In vitro drug release from Diclofenac sodium sustained release tablet was found to be______% at 12 h. Conclusion Diclofenac SR tablet was investigated for in vitro drug release study.

Applications 1. Dissolution of sustained release tablet can be studied. 2. Sustained release dosage form reduces frequent dosing thereby improving patient compliance. 3. The behaviour of tablets in fluids of varying pH can be studied.

30

Laboratory Manual of Biopharmaceutics and Pharmacokinetics

Questions 1. Why enteric coating of tablets is done? 2. Give various approaches of modified release tablets. Exercise Study the dissolution of Theophylline SR tablets.

Section 1

Physicochemical properties of drugs and dosage forms

31

Experiment 7 Effect of pH on dissolution of Benzoic acid sticks Aim To study the effect of pH on dissolution of Benzoic acid sticks. Learning objectives 1. To study and understand the concept of Noyes and Whitney law of dissolution at different pH. 2. To study its applications. 3. To study the effect of pH on dissolution of Benzoic acid sticks. Theory The solubility of a weak electrolyte usually varies considerably as a function of pH. Hence, differences are expected in the dissolution rate of a weak acid or weak base in different regions of the GIT, which has large differences in surface areas as well as a notable difference in H+ ion concentration. The pH range of the fluids in various portions of the GIT varies between 1-8. The degree of acidity or alkalinity of biological fluids at the absorption site is one of the most critical factors in the absorption of many drugs. Majority of drugs used in therapeutics are weak acids or bases. The organic electrolytes tend to exists in solution as unionized or ionized species. The relative fraction of each species present in solution depends on the pH of the fluid in which the drug is dissolved. Since, the GIT barrier is selectively permeable to unchanged, lipid soluble solutes, a drug may be well absorbed from one portion of the tract where a favorable pH exists and poorly absorbed from another portion where a much less favorable pH is found. The absorption of the weakly basic drug is favored in intestine, where they exist in essentially unionized form. Conversely, the acidic gastric fluids tend to retard absorption of weakly basic drugs, but promote absorption of weakly acidic drugs. The transition from stomach to duodenum usually involves an abrupt and marked change in acidity. Beyond the pylorus, the pH of intestinal fluids gradually increases to the maximum of pH 8 in the colon. The total solubility (CS) of a weak acid is given by Cs = [HA] + [A-] …1 Where [HA] is the intrinsic solubility of the nonionized acid (denoted as C0) and [A-] is the concentration of its anion, which is infinitely soluble. The concentration of the anion can be expressed in terms of the dissociation constant, Ka and C0; that is Cs = C0 +

KaC 0 [H + ]

…2

In a similar manner, the solubility of a weak base is given by:

Cs = C0 +

C 0 [H + ] Ka

…3

Substituting equations 2 and 3 into modified Noyes Whitney law, the following dissolution rate equations are obtained,

Laboratory Manual of Biopharmaceutics and Pharmacokinetics

32

For weak acids é C + K aC 0 dc = K' ê 0 dt [H + ] êë

ù ú úû

é Ka ù ú ê1 + [ H + ] úû êë For weak bases

dc = K'C0 dt

dc = K'C dt

0

é [H + ] ù ú ê1 + K a úû êë

…4

…5

…6

Where, K'=DS/h Equations 5 and 6 indicate that the dissolution rate of weak acids increases with increasing pH, whereas the dissolution rate of weak bases decreases with increasing pH. The dissolution rate of weak bases is at a maximum in gastric fluids but that of weak acids is at a minimum. The dissolution rate of weak acid increases as the undissolved drug particles are transported to the more alkaline regions of the GIT. According to equation 5, a linear relationship should exist between dissolution rate (dc/dt) of a weak acid and the reciprocal of [H+] concentration (i.e. 1/H+). In practice, however a plot of dc/dt versus 1/H+ shows linearity only at low pH. As, [H+] decreases, negative deviation from linearity is observed. The reason for this deviation is that [H+] concentration of the bulk solution is not equal to [H+] concentration of the diffusion layer except at low pH values. So, in case of weak acid [H+]d >[H+] Where, [H+]d is the H+ concentration of the diffusion layer Since the diffusion layer is saturated with respect to drug, it is reasonable to accept that in solutions with a pH greater than pKa of the drug, the relatively large acid drug concentration in the layer may overcome the buffer capacity of the solution. In this case, the pH of the diffusion layer would be lower than the pH of the bulk solution. Principle Dissolution rate of weak acids increases with increasing pH, whereas dissolution rate of weak bases decreases with increasing pH. The dissolution rate of weak bases is at maximum in gastric fluids but that of weak acid is at a minimum. The dissolution rate of weak acid increases as the undissolved drug particles are transported to the more alkaline regions of the gastrointestinal tract. As Benzoic acid is a weak acid, its ionization is less in low pH and hence the dissolution rate is low. However, as the pH increases the ionization of Benzoic acid increases thereby increasing dissolution rate. Prerequisite 1. Understanding of concept of dissolution. 2. Understanding of Noyes Whitney law of dissolution. 3. Understanding of the concept of pH and Henderson Hasselbalch equation.

Section 1

Requirements 1. Glasswares: 2. Chemicals: 3. Equipments:

Physicochemical properties of drugs and dosage forms

33

Beakers, test tubes, glass rods, burette and conical flask. Benzoic acid, sodium hydroxide, phenolphthalein, potassium dihydrogen orthophosphate. Vernier caliper, pH meter.

Procedure A. Preparations of solutions 1. Preparation of 0.05 N sodium hydroxide Dissolve 2 gm of sodium hydroxide in 150 ml of carbon dioxide free water, cool the solution to room temperature, and filter through filter paper. Dilute clear filtrate to 1000 ml. 2. Preparation of phosphate buffer of pH 4.0 Dissolve 5.04 g of disodium hydrogen phosphate and 3.01 g of potassium dihydrogen phosphate in sufficient water to produce 1000 ml.Adjust the pH with glacial acetic acid. 3. Preparation of phosphate buffer of pH 5.5 Solution I- Dissolve 13.61g of potassium dihydrogen phosphate in sufficient water to produce 1000 ml. Solution II- Dissolve 35.81 g of disodium hydrogen phosphate in sufficient water to produce 1000 ml. Mix 96.4 ml of solution I with 3.6 ml of solution II. 4. Preparation of phosphate buffer of pH 6.8 Dissolve 28.8 g of disodium hydrogen phosphate and 11.45 g of potassium dihydrogen phosphate in sufficient water to produce 1000 ml. B. Dissolution of Benzoic acid sticks at different pH 1. Prepare three Benzoic acid sticks as per previous experiment (Expt No. 2). The prepared stick should have same length and diameter. 2. Prepare three buffers of pH 4.0, 5.5 and 6.8 and check their pH with pH meter. If required adjust their pH. 3. Prepare the saturated solution of Benzoic acid at various pH (4.0, 5.5 and 6.8) as per previous experiment (Expt No. 2) and calculate saturation solubility of Benzoic acid at various pH. 4. Perform the dissolution of Benzoic acid sticks as per previous experiment (Expt No. 2) at different pH values e.g. stickAat pH 4.0, stick B at pH 5.5 and stick C at pH 6.8. 5. Calculate the dissolution rate constant, for buffers of pH 4.0, 5.5 and 6.8. 6. Plot a graph between pH and dissolution rate constant. Observations Table 1. Parameters of Benzoic acid stick

Sr. No.

Stick

1 2 3

Stick A Stick B Stick C

Diameter (D) (cm)

Length (L) (cm) 8 8 8

Surface area (S) (cm2)

1. Saturation solubility of Benzoic acid at pH 4.0 = ____________g/l.

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Volume of NaOH used (a-b)* (ml)

Normality of NaOH

Concentration of BA (A)

log Cs/(Cs-C)

Time (min)

Cs/(Cs-C)

2. Saturation solubility of Benzoic acid at pH 5.5 = ____________g/l. 3. Saturation solubility of Benzoic acid at pH 6.8 = ____________g/l. B. Observation table for Benzoic acid stickAat pH 4.0 Slope

K' = K=K'/S slope x 2.303

10 20 30 40 Average K * a is volume of NaOH used for Benzoic acid titration, b is blank titration i.e. without Benzoic acid.

Volume of NaOH used (a-b)* (ml)

Normality of NaOH

Concentration of BA (B)

log Cs/(Cs-C)

Time (min)

Cs/(Cs-C)

C. Observation table for Benzoic acid stick B at pH 5.5 Slope

K' = K=K'/S slope x 2.303

10 20 30 40 Average K * a is volume of NaOH used for Benzoic acid titration, b is blank titration i.e. without Benzoic acid.

Volume of NaOH used (a-b)* (ml)

Normality of NaOH

Concentration of BA (C)

log Cs/(Cs-C)

Time (min)

Cs/(Cs-C)

D. Observation table for Benzoic acid stick C at pH 6.8

Slope

K' = K=K'/S slope x 2.303

10 20 30 40 Average K * a is volume of NaOH used for Benzoic acid titration, b is blank titration i.e. without Benzoic acid.

Calculations 1. Calculation for concentration of saturated solution of Benzoic acid (Cs) N 1V 1 = N2V2 NaOH Benzoic acid N1= Normality of NaOH, V1= Volume of NaOH used, N2= Normality of Benzoic acid, V2= Volume of Benzoic acid used Concentration of BAsolution (g/l) = N2 x Molecular weight of BA = N2 x 122.1

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Physicochemical properties of drugs and dosage forms

35

2. Calculation for surface area of BAstick (S) S = p DL where, D- diameter, L - length. 3. Calculation for concentration of Benzoic acid sticks at time t (C) N1V1 = N2V2 NaOH Benzoic acid Concentration of BA solution = N2 x Molecular weight of BA = N2 x 122.1 4. Calculation of dissolution rate constant (K) Graphically calculate K' K = Slope x 2.303 K'=D/h= K/S Result The dissolution rate constant (K) of Benzoic acid at pH 4.0, pH 5.5 and pH 6.8 are __________, __________ and ____________ respectively. Conclusion It can be concluded from this experiment that, the dissolution rate of Benzoic acid (weak acid) increases, as the pH of the dissolution medium increases. Application Thorough understanding of effect of pH on dissolution rate of weakly acidic or basic drug will help in designing various dosage forms. Questions 1. Describe effect of pH on weak acid and weak base. 2. Correlate your results with theoretical results of Benzoic acid. 3. Why Benzoic acid is a weak acid? Describe effect of pH on Benzoic acid solubility. Exercise Study the effect of pH on dissolution rate of a weakly basic drug.

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Experiment 8 Effect of pH on dissolution behavior of drug Aim To study effect of pH on dissolution behavior of commercially available enteric coated Diclofenac sodium tablets. Learning objectives 1. To understand the effect of pH on dissolution of a drug. 2. To utilize Hixon and Crowell cube root law for determining dissolution rate constant. 3. To compare dissolution of Diclofenac sodium at different pH. 4. To compare dissolution of different brands of Diclofenac sodium at same pH. Theory Enteric-coated dosage forms are those that remain intact in stomach but dissolve and release their contents on arriving in small intestine. Factors responsible for this include the difference in pH of gastric and intestinal fluids; where the coatings that are acid functionally or acid ester functionally remain unionized and remain intact in the low pH (pH 1-4) gastric environment, they ionize and thus disintegrate in the intestinal fluids, pH may vary from 5 in the duodenum to around 7.4 further down the intestinal tract. Other factors responsible for loss of film integrity include hydration and the presence of esterase in the intestinal fluid that are responsible for cleavage of ester linkage present in some kinds of enteric films. Dissolution kinetics may be influenced by the physicochemical characteristics of the drug and the formulation factors and in this case the most probable variants may be the kind of coating materials used, the coating material thickness, the amount of plasticizer used and the age of the coating film. To make the comparisons of test formulations easier, dissolution rate constants for every brand at different pH can be calculated by using Hixon and Crowell cube root law. Hixon and Crowell equation takes into account the changing surface area of the dissolving molecule. Mathematical form of Hixon and Crowell cube root law is given as follows: 1/3

1/3

[W0] - [W]

=kt

…1

where, W0 = initial amount of drug in grams, W = amount of drug remaining to dissolve, k = cube root dissolution rate constant. For the calculation of “k”, percent dissolution of Diclofenac sodium is subtracted from 100% to get the percentage of the drug that remains undissolved. The percentage of undissolved Diclofenac sodium is then converted into grams (W) and used to compute “k” by using equation (2) 1 - [W]1/3 = k t …2 Based on the dissolution rate constants of various brands, it can be predicted that the brand with lowest dissolution rate constant is least bioavailable. Principle As the pH in GIT goes on increasing the enteric coat gets dissolved and the dissolution rate of Diclofenac goes on increasing. The release of drugs from enteric-coated tablets is highly dependent on pH of the buffer solution.

Section 1

Physicochemical properties of drugs and dosage forms

37

At any particular pH, the difference in dissolution rates among different brands may be due to the use of different coating materials by different manufacturers. Commonly used enteric coating materials are: cellulose acetate derivatives such as cellulose acetate phthalate (CAP), hydroxyl propyl methyl cellulose (HPMC) and the two grades of hydroxy propyl methylcellulose phthalate (HP-50 and HP-55), polymethacrylate polymers (Eudragit L and Eudragit S), polyvinyl acetate phthalate (PVAP). All the enteric coatings in current use possess ionizable acid groups, usually a free carboxylic acid. The equilibrium between unionized insoluble polymer and ionized soluble polymer will be determined by pH of the medium and pKa of the polymer. As pH of the dissolution medium increases, the coating materials ionize and start breaking and dissolving which cause release of the drug. At lower pH, however, the polymer remains unionized and the integrity of the film is maintained. pKa of the film former and nature of the polymer backbone are the two most important factors that cause variation in the dissolution behaviour of film formers. The enteric coating materials with respect to their resistance to the gastric dissolution media are as follows: CAP > PVAP > HP55 > HP50. Numerous other factors can play their part in causing the observed variation in drug release behaviour. These factors include film thickness, plasticizers added to the coating material and the amount of diluent used. Requirements 1. Glasswares: Funnel, beakers, pipettes, test tubes, etc. 2. Chemicals: Two compressed oral formulations of enteric-coated Diclofenac sodium tablets (same strength 50 mg), pure Diclofenac sodium used as reference standard, analytical grade sodium hydroxide, potassium dihydrogen phosphate, 37 % HCl, distilled water. 3. Instruments: Balance, dissolution test apparatus, UV spectrophotometer. Procedure 1. Plot a standard curve of Diclofenac sodium by making standard dilutions of pure Diclofenac sodium in 0.1N HCl in the range of 1-10 mg/ml and note their absorbance at 276 nm. Similarly prepare the dilutions of Diclofenac sodium at phosphate buffer pH 4.0, 4.5, 5.5, 6.0, 6.5 and 7.0 and measure the absorbance at 276 nm and plot the calibration curves at different pH. 2. Conduct the dissolution test for all brands of Diclofenac sodium tablets as specified in USP XXII for Diclofenac sodium. 3. Use USP dissolution apparatus type II for dissolution study. 4. Place 900 ml of simulated gastric (0.1N HCl adjusted to pH 1.2) dissolution medium in hemispherical bottom flask. 5. Maintain the temperature of dissolution medium at 370C and rotation speed at 50 rpm. Place three tablets of brandAand three tablets of brand B in dissolution vessels. 6. Withdraw 10 ml samples from the dissolution vessel at predetermined time intervals (0, 15, 30, 60, 90, and 120 min) and add same volume of fresh dissolution medium to maintain sink conditions. 7. Analyze samples spectrophotometrically at 276 nm and determine percent cumulative drug release of Diclofenac sodium using calibration curve.

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8. Repeat the experiment in simulated intestinal media (mixed phosphate buffer) at pH 4.0, 4.5, 5.5, 6.0, 6.5 and 7.0 for 240 minutes. 9. Determine dissolution rate constants for every brand at different pH by using Hixon and Crowell cube root 1/3 1/3 law. Plot the graph of (W0 -W ) versus time, t. Slope of line is the dissolution rate constant. 10. Plot the graph of dissolution rate constant versus pH of dissolution medium. 11. Report the effect of pH of dissolution medium on dissolution rate constant. A. Parameters set for plotting of calibration curve 1) Beer's & Lambert range: 2) Solvent: 3) l max for Diclofenac sodium: B. Parameters set for dissolution studies 1) Tablet: 2) Strength of tablet: 3) Apparatus: 4) Rotation Speed: 5) Test time: 6) Dissolution medium: 7) Volume of dissolution medium:

1-10 mg/ml 0.1 N HCl or phosphate buffer (pH 4.0, 4.5, 5.5, 6, 6.5, 7.0) 276 nm Diclofenac sodium 50/75/100 mg IPType I dissolution test apparatus 50 rpm 420 min 0.1N HCl, phosphate buffer (pH 4.0, 4.5, 5.5, 6, 6.5, 7.0) 900 ml

Observations Table 1. Calibration curve of Diclofenac sodium pH 0.1 N HCl PB pH 4.0

Slope

Intercept

PB pH 4.5 PB pH 5.5 PB pH 6.0 PB pH 6.5 PB pH 7.0 PB- phosphate buffer

Table 2. Percent cumulative drug release of brandA Time (min) 0 15 30 60 90 120 150 180 210 240

pH 1.2

pH 4

pH 4.5

pH 5.5

pH 6

pH 6.5

pH 7.0

Physicochemical properties of drugs and dosage forms

Section 1

39

Table 3. Cumulative percent drug release of Brand B Time (min) 0 15 30 60 90 120 150 180 210 240

pH 1.2

pH 4

pH 4.5

pH 5.5

pH 6

pH 6.5

pH 7.0

Calculations 1. Concentration of dissolved drug (mg/ml) Plot the graph of absorbance versus concentration and determine slope and intercept. Y= m X + c Where, Y= absorbance, m= slope, X= concentration (mg/ml), c = intercept. 2.Amount of drug released (mg) Amount of drug released = [Concentration (mg/ml) x (volume of dissolution medium) x (dilution factor)]/1000 3. Dilution factor Dilution factor = volume of diluted sample (ml)/ volume of sample removed (ml) 4. Percent cumulative drug release Percent cumulative drug release = (amount of drug released) x 100 /strength of tablet 5. Determination of dissolution rate constant 1/3

1/3

[W0] - [Wt] = k t Where, W0- Initial amount of drug in grams, Wt - Amount of drug released at time t in grams. 1/3

Plot the graph of [W0] - [Wt]

1/3

versus time

Calculate the slope of the line as a dissolution rate constant. Result 1. The dissolution of the brandAvaries from ____ % to ____ % as the pH rises from 1.2 to 7.5. 2. The dissolution of the brand B varies from ____ % to ____ % as the pH rises from 1.2 to 7.5. 3. Dissolution rate constant of Brand A is low/high as compared to brand B at 7.0. Hence Brand A is more/less dissolved with given time. Conclusion It can be concluded that the enteric coating is greatly affected by changes in pH of dissolution medium. As the pH increases the dissolution becomes rapid and as the pH of the dissolution medium decreases, the dissolution becomes slow.

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Laboratory Manual of Biopharmaceutics and Pharmacokinetics

It can also be concluded that variation in dissolution behavior of the brands tested may be due to any of the factors like coating material used, film thickness, diluent used etc. Applications 1. Comparison of various brands of Diclofenac sodium in terms of bioavailability. 2. Studying the effect of pH of dissolution medium on various parameters like coating material, film thickness, plasticizers or diluents used in coating of tablet. 3. Studying drug release of enteric coated tablet throughout GIT. 4. Estimation of dissolution rate constant. Questions 1. Why enteric coating is required for Diclofenac sodium? 3. Enlist drugs requiring enteric coating. Exercise Similarly students can study effect of pH on dissolution behavior of commercially available Domperidone DT tablet. Dissolution medium for the study are 0.1N HCl and phosphate buffer pH 6.8. Correlate pH of medium and dissolution of Domperidone.

SECTION 2

Absorption of drugs Experiment 9 Intestinal permeability using chicken intestine Aim To perform in vitro absorption-permeation study of marketedAcyclovir tablet. Learning objectives 1. To study dissolution and in vitro absorption ofAcyclovir tablet. 2. To study and understand continuous dissolution absorption system using isolated everted intestine. Theory Good oral bioavailability occurs when the drug has maximum permeability and maximum solubility at the site of absorption. The extent of absorption of drug in vivo, thus, could be predicted based on permeability and solubility measurements. Hence, the intestinal permeability represents one essential part in the prediction of oral bioavailability. The intestinal permeability data can be used in preformulation studies to evaluate the effects of various pharmaceutical excipients on drug absorption. A number of in vitro methods for assessing the intestinal permeability of a given drug have been developed. In vitro absorption (permeability) studies based on isolated intestinal sacs are routinely performed. The advantages of this model are that it contains all the types of cells and mucus layer; it is relatively fast and inexpensive; and it can be used for preformulation studies. This kind of model is suitable for measuring kinetic parameters with high reliability and reproducibility. Several animal species including rat, rabbit, pig, dog, and monkey have been used in permeability studies based on isolated intestinal sacs. The chicken small intestine could be a useful model for intestinal absorption based on the assumption that membrane permeability of drugs is not species-dependant, since the composition of plasma membrane of intestinal epithelial cells is similar across the species. Thus the permeability across the chicken intestinal segment could be expected to be the same. Furthermore, the model would have advantages as less labor intensive, less time consuming, lower cost per assay and, since slaughtered chicken is used, no special permission from animal ethical committees would be required. Design of continuous dissolution-absorption system using everted intestine segment The in vitro continuous dissolution absorption system design is illustrated in Figure 1. The system consists of USP dissolution apparatus 1 and a side-by-side perfusion apparatus holding isolated everted intestine segment. In this system, drug dissolution from the slow release tablet and permeation across everted intestine occurs simultaneously. Use 1000 ml of distilled water as dissolution medium maintained at 37 ± 0.5 °C. The perfusion apparatus is consisted of two glass tubes, A and B, connected together (Figure 1). Tube B is a bent cannula at its lower end, and tube A, a straight cannula at its lower end. The distance between two cannula is kept constant. The isolated everted intestinal segment is fixed between the ends of tubes A and B as shown in the figure 1. The ends of the intestine are tied in position with a thread. The apparatus is immersed completely into the dissolution vessel. 41

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Laboratory Manual of Biopharmaceutics and Pharmacokinetics

Principle Acyclovir [9-(2-hydroxyethoxymethyl) guanine], a synthetic purine nucleoside analogue derived form guanine is the most widely used antiviral agent. According to BCS classification, Acyclovir is categorized as class III drug i.e. having high solubility and less permeability. Its absorption in GIT is slow, variable and incomplete. The bioavailability of Acyclovir after oral administration ranges from 10-30%. Approximately 80 % of an oral dose is never absorbed and excreted through feces. The continuous-absorption system using everted intestine segment can be used to study the dissolution of the Acyclovir tablet and at the same time the absorption of Acyclovir through intestinal membrane can be studied. High solubility and low permeability of Acyclovir can be substantiated by this experiment as both solubility and permeability can be simultaneously studied. The absorption ofAcyclovir is permeation rate limited.

Figure 1. Design of in vitro continuous dissolution-absorption system. Labels:1. Dissolution flask, 2. Rotating shaft, 3. Dissolution medium, 4. Basket, 5. Tablet, 6. Oxygen tube, 7. Tube B, 8. TubeA, 9. Everted intestine, I. Dissolution absorption system, II.Absorption (perfusion) apparatus. Prerequisite 1. Intestinal absorption of drugs. 2. Ex vivo absorption models. Requirements Glasswares: Beaker, test tube, funnel, volumetric flask, etc. Chemicals: Sodium lauryl sulphate, phosphate buffer pH 5, distilled water, Krebs ringer solution pH 7.4, Acyclovir tablet 200 mg. Instruments: Weighing balance, dissolution apparatus, UV spectrophotometer. Procedure 1. Preparation of solutions Krebs ringer solution: Prepare the Krebs ringer solution by combining 6.3 g NaCl, 0.35 g KCl, 0.14 g CaCl2, 0.16 g KH2PO4, 0.15 g MgSO4·7 H2O, 2.1 g NaHCO3, and 5 g glucose in one liter of distilled water. Phosphate buffer pH 5.0: Dissolve 6.8 g of potassium dihydrogen phosphate in 1000 ml water and adjust the pH

Section 2

Absorption of drugs

43

to 5.0 with 10 M potassium hydroxide. 2. Plotting of calibration curve Transfer accurately weighed 100 mg of Acyclovir in to 100 ml volumetric flask containing sufficient quantity of phosphate buffer pH 5.0. Finally adjust volume to get stock solution containing 100 µg/ml Acyclovir. Withdraw adequate quantities of aliquots from standard stock solution in 10 ml volumetric flask and dilute with phosphate buffer pH 5.0 to get the concentration of 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 µg/ml of Acyclovir. Measure the absorbance of these dilute solutions at a l max of 254 nm by using double beam UV spectrophotometer against a blank of phosphate buffer pH 5.0. Plot the graph of absorbance versus concentration and determine slope, intercept and coefficient of correlation. 3. Isolation of everted intestine Bring male white Leghorn chicks weighing between 500 and 600 g from the local market. For isolation of everted intestine, slaughter the chicks, perform a median incision of the abdomen and free the small intestine. Clean the lumen carefully from mucus by rinsing with a pH 7.4 buffer solutions (Krebs ringer solution). Remove an intestinal segment of the first 6 cm length and transfer to oxygenated Krebs ringer solution. Wash it thoroughly with warm Krebs ringer solution. Turn back the proximal extremity of the intestine and ligate on a glass rod to form an everted bag. Alternatively, chick intestine can be obtained from slaughter house by taking proper care of providing oxygenated Krebs ringer solution to intestinal segment removed. This will eliminate the requirement of taking permission of Ethics Committee for performing this experiment. 4. Dissolution absorption studies 1. Maintain the dissolution medium consisting of 900 ml of phosphate buffer pH 5.0 at 37 ± 0.5 °C. Clamp a fresh intestinal segment to perfusion apparatus, as shown in the figure 1. 2. Fill Krebs ringer solution in perfusion apparatus and record total volume of absorption compartment (tube A and tube B of perfusion apparatus). 3. Place the perfusion apparatus into dissolution medium and aerate it. 4. Place marketedAcyclovir tablet into basket and rotate it at 50 rpm. 5. Withdraw 2 ml sample at interval of 10 min up to 120 min and determine released Acyclovir spectrophotometrically at 254 nm. 6. Withdraw the transported drug from absorption compartment with replacement of Krebs ringer solution at 13 min to 123 min at the interval of 10 min and analyze it spectrophotometrically for transported Acyclovir at 254 nm. To allow time for drug to circulate from the dissolution vessel to the everted intestine surface, absorption samples should be collected 3 min later than their corresponding dissolution samples. 7. Repeat whole experiment in triplicate (n=3) using fresh dissolution medium as well as fresh intestinal segment each time. 8. Plot the graph of percent drug released versus time. 9. Plot the graph of cumulative amount of drug diffused versus time and determine slope of linear portion as steady state appearance rate (mg/min) namely the amount of a compound traversing the tissue in time t (min).

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Observations Table 1. Percent cumulative drug release (% CDR) Time (min)

Absorbance

Concentration (mg/ml)

Amount of drug released (mg)

% CDR

0 10 20 30 40 50 60 70 80 90 100 110 120

Table 2.Amount ofAcyclovir diffused Time (min)

Absorbance

Concentration (mg/ml)

Amount diffused (mg)

CADD

0 13 23 33 43 53 63 73 83 93 103 113 123 CADD= Cumulative amount of drug diffused in mg

Calculations 1. Concentration of diffused drug (mg/ml) Plot the graph of absorbance versus concentration and determine slope and intercept. Y= mX + c Where, Y= absorbance, m= slope, X= concentration (mg/ml), c = intercept. 2. Cumulative amount of drug diffused (mg) Cumulative amount of drug diffused (CADD) = [Concentration (mg/ml) x (volume of diffusion medium) x (dilution factor)]/1000

Section 2

Absorption of drugs

45

3. Surface area (A) of chicken intestine (cm2) A= 2prl Where, r = internal radius of everted intestine, l = length of everted intestine. 4. Dilution factor Dilution factor = volume of diluted sample (ml)/ volume of sample removed (ml) 5.Amount of drug released in dissolution medium (mg) Amount of drug released = [Concentration (mg/ml) x (volume of dissolution medium) x (dilution factor)]/1000 6. Percent cumulative drug release Percent cumulative drug release = (Amount of drug released) x (100 /strength of tablet) 7.Apparent permeability (cm/s)

Papp =

dQ 1 ´ dt C0 ´ A ´ 60

Where, dQ/dt = steady state appearance rate, namely the amount of a compound traversing the tissue in 2 time t (min), A = exposed area of the tissue (cm ), C0 = initial concentration of the drug in the donor compartment. Results 1. Percent drug release of marketedAcyclovir tablet in phosphate buffer is _________ % in 2 h. 2.Amount ofAcyclovir diffused through intestinal membrane is _________ in 2 h. Conclusion It can be concluded from this experiment that the in vitro continuous dissolution-absorption system design can be successfully used for simultaneous determination of dissolution and apparent permeability of Acyclovir. Applications 1. Simultaneous determination of dissolution and apparent permeability is possible using this model. 2. In vitro absorption studies can be done by using this model. Question Give various models used for in vitro absorption studies. Exercise Perform ex vivo absorption-permeation study of marketed Metformin HCl tablet.

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Experiment 10 Effect of permeation enhancers on intestinal permeability of drug Aim To study the effect of permeation enhancer on absorption of Acyclovir by using in vitro dissolutionabsorption system.

Learning objectives 1. To study dissolution and in vitro absorption ofAcyclovir tablet. 2. To study the effect of permeation enhancer on absorption ofAcyclovir. Theory As per previous experiment (Expt No. 9). Principle One of the approaches to improve the permeability of poorly absorbed drug from GIT is co administration of absorbance enhancers including surfactants, bile salts, fatty acids and some mucoadhesive polymers. Surfactants are often included in the oral solid dosage forms in order to improve their wetting or dissolution properties of drug. It is important to know whether these surfactants at concentrations that are achieved in the intestinal lumen enhances the permeability of the active ingredients. Sodium lauryl sulphate (SLS) is a commonly used surfactant to study the permeability at various concentrations. Increase in permeability is due to the ability of permeation enhancer (SLS) to promote permeability in the absorptive direction by opening the tight junctions and/or by inhibiting the active efflux system. Thus, by use of SLS the problem of less permeability can be solved which may lead to increased absorption of Acyclovir which in turn may increase its bioavailability. Increasing concentrations of SLS in the dissolution medium increases the permeability coefficient and enhancement ratio. Prerequisite 1. Knowledge about permeation enhancers. 2. In vitro absorption methods. 3. Concept of dissolution. Requirements Glasswares: Beaker, test tube, funnel, volumetric flask, etc. Chemicals: Sodium lauryl sulphate, phosphate buffer pH 5, distilled water, Krebs ringer solution pH 7.4, Acyclovir tablet 200 mg. Instruments: Weighing balance, dissolution apparatus, UV spectrophotometer. Procedure 1. Preparation of solutions Krebs ringer solution: Prepare the Krebs ringer solution by combining 6.3 g NaCl, 0.35 g KCl, 0.14 g CaCl2, 0.16 g KH2PO4, 0.15 g MgSO4·7 H2O, 2.1 g NaHCO3, and 5 g glucose in one liter of distilled water.

Section 2

Absorption of drugs

47

Phosphate buffer of pH 5.0: Dissolve 6.8 g of potassium dihydrogen phosphate in 1000 ml water and adjust the pH to 5.0 with 10 M potassium hydroxide. 2. Plotting of calibration curve Transfer accurately weighed 100 mg of Acyclovir in a 100 ml volumetric flask containing sufficient quantity of phosphate buffer pH 5.0. Finally adjust volume to get stock solution containing 100 µg/ml Acyclovir. Withdraw adequate quantities of aliquots from standard stock solution in ten, 10 ml, volumetric flasks and dilute with phosphate buffer pH 5.0 to get the concentration of 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 µg/ml of Acyclovir. Measure absorbance of these dilute solutions at a lmax of 254 nm by using double beam UV spectrophotometer against a blank of phosphate buffer pH 5.0. Plot the graph of absorbance versus concentration and determine slope, intercept and coefficient of correlation. 3. Isolation of everted intestine Bring male white Leghorn chicks weighing between 500 and 600 g from the local market. For isolation of everted intestine, slaughter the chicks, perform a median incision of the abdomen and free the small intestine. Clean the lumen carefully from mucus by rinsing with a pH 7.4 buffer solutions (Krebs ringer solution). Remove an intestinal segment of the first 6 cm length and transfer to oxygenated Krebs ringer solution. Wash it thoroughly with warm Krebs ringer solution. Turn back the proximal extremity of the intestine and ligate on a glass rod to form an everted bag. Alternatively, chick intestine can be obtained from slaughter house by taking proper care of providing oxygenated Krebs ringer solution to intestinal segment removed. This will eliminate the requirement of taking permission of Ethics Committee for performing this experiment. 4. Dissolution absorption studies 1. Maintain the dissolution medium consisting of 900 ml of phosphate buffer pH 5.0 at 37 ± 0.5 °C. Clamp a fresh intestinal segment to perfusion apparatus. 2. Fill Krebs ringer solution in perfusion apparatus and record total volume of absorption compartment (tube A and tube B of perfusion apparatus). 3. Place the perfusion apparatus into dissolution medium and aerate it. 4. Place marketedAcyclovir tablet into basket and rotate it at 50 rpm. 5. Withdraw 2 ml sample at interval of 10 min up to 120 min and determine released Acyclovir spectrophotometrically at 254 nm. 6. Withdraw the transported drug from absorption compartment with replacement of Krebs ringer solution at 13 min to 123 min at the interval of 10 min and analyze it spectrophotometrically for transported Acyclovir at 254 nm. 7. Repeat whole experiment in triplicate (n=3) using fresh dissolution medium as well as fresh intestinal segment each time. 8. Repeat the same procedure for other tablets except that, add SLS at three varying concentrations of 1%, 2% and 3% in the dissolution medium. 9. Plot the graph of percent drug released versus time. 10. Plot the graph of cumulative amount of drug diffused versus time and determine slope of linear portion as steady state appearance rate (mg/min) namely the amount of a compound traversing the tissue in time t (min).

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Observations Table 1. Percent cumulative drug release Time (min) 10 20 30 40 50 60 70 80 90 100 110 120

0% SLS

Percent cumulative drug release 1% SLS 2% SLS

3% SLS

Table 2. Amount of drug diffused Time (min) 13 23 33 43 53 63 73 83 93 103 113 123

Percent cumulative amount of drug diffused 0% SLS 1% SLS 2% SLS 3% SLS

Calculations 1. Concentration of diffused drug (mg/ml) Plot the graph of absorbance versus concentration and determine slope and intercept. Y= mX + c Where, Y= absorbance, m= slope, X= concentration (mg/ml), c= intercept. 2. Cumulative amount of drug diffused (mg) Cumulative amount of drug diffused (CADD) = [Concentration (mg/ml) x (volume of diffusion medium) x (dilution factor)]/1000 2

3. Surface area (A) of chicken intestine (cm ) A= 2prl Where, r = internal radius of everted intestine, l = length of everted intestine.

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49

4. Dilution factor Dilution factor = volume of diluted sample (ml)/ volume of sample removed (ml) 5.Amount of drug released in dissolution medium (mg) Amount of drug released = [concentration (mg/ml) x (volume of dissolution medium) x (dilution factor)]/1000 6. Percent cumulative drug release Percent cumulative drug release = (amount of drug released) x (100 /strength of tablet) 7.Apparent permeability (cm/s) dQ 1 ´ dt C0 ´ A ´ 60 Where, dQ/dt = steady state appearance rate, namely the amount of a compound traversing the tissue 2 in time t, A = exposed area of the tissue (cm ), C0 is the initial concentration of the drug in the donor compartment. 6. Enhancement ratio (ER) Papp of drug with enhancer ER = Papp of drug without enhancer Papp =

Results 1. Percent drug release of marketedAcyclovir tablet in phosphate buffer is ________ % in 2 h. 2.Amount ofAcyclovir diffused through intestinal membrane is ________ in 2 h. 3. The apparent permeability and enhancement ratio of Acyclovir at different concentrations of SLS is given below: 0% SLS 1% SLS 2% SLS 3% SLS Apparent permeability Enhancement Ratio

Conclusion It can be concluded from this experiment that as the concentration of SLS is increased permeability of Acyclovir is also increased. Applications 1. Permeability of Class III drugs with low permeability can be improved by using permeation enhancers. 2. The effect of various absorption enhancers on absorption of drugs can be studied. Questions 1. Give list of various permeation enhancers used in oral dosage forms. 2. Give the mechanisms by which permeation enhancers increase the permeability of drugs. Exercise Study the effect of bile salts on permeability ofAcyclovir.

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Experiment 11 Percutaneous absorption of drug from various ointment bases Aim To study the ex vivo percutaneous absorption of Metronidazole from water soluble and oleaginous vehicles. Learning objectives 1. To study the ex vivo percutaneous absorption method. 2. To understand the concept of permeability coefficient and its estimation. 3. To study the effect of ointment bases on percutaneous absorption of Metronidazole. Theory Stratum corneum is the principle barrier for cutaneous penetration allowing slow absorption for majority of drugs. In any case, the permeability of the stratum corneum is increased by using appropriate vehicle. It is generally assumed that nature of the selected vehicle strongly influences the rate and extent of drug release. Release may be improved by selecting the appropriate vehicle. The best vehicle for topical use has been described as the one which contributes to reversible decrease in the stratum corneum resistance and allows diffusion of molecules into the vehicle itself. Most in vitro experimental designs aim to mimic as closely as in vivo situation. The most common in vitro design is one where a membrane (usually the epidermis) separates two compartments. One compartment contains the drug in a vehicle, possibly a simple aqueous or buffer solution (termed the donor solution), and the other compartment contains a receptor (or receiver) solution that provides sink conditions (i.e. essentially zero concentration). After sufficient time, steady-state permeation across the membrane is achieved when concentration gradient of the drug across the membrane is constant. Under these conditions following equation can be used.

dM DC0 = dt h

…1 Where M = cumulative mass of the drug that passes through per unit area of the membrane in time t, C0 = concentration of the drug in the first layer of the membrane (at the skin surface, in contact with the donor solution) and h = membrane thickness. In practical terms, it is very difficult to measure C0 , concentration of the drug in first layer of the membrane; removal of the outer layer for assay is problematic and contamination from the applied donor solution is almost inevitable. However, concentration of the drug in the vehicle (donor solution) bathing the skin membrane (Cv) is usually known or can be determined relatively easily. Since C0 and CV are simply related by:

P=

C0 CV

so

C0 = PCV

…2

Where P is the partition coefficient of the drug between the membrane and the vehicle. Substitution of equation 2 into equation 1 gives:

Section 2

Absorption of drugs

51

dM DPCV = dt h

…3 This is most widely applied equation in examining transdermal drug delivery data. A plot of M, 2 cumulative amount of the drug passing through a unit area of membrane (mg/cm ) against time gives the typical permeation profile. The permeability coefficient (KP), rate of drug transport per unit concentration of a drug through a membrane can be defined by equation,

DP h Which can be substituted into equation 3 to give:

KP =

…4

dM = J SS = K P CV dt

…5 The steady state flux (JSS) is simply obtained as the gradient of the linear portion of the permeation profile (by plotting the graph of cumulative amount of drug diffused per unit area versus time) and if concentration of the drug in the applied vehicle is known, then the permeability coefficient can be determined. Higher the value of KP and JSS obtained for the formulation, higher will be permeation. Principle Release of the drug from the dosage forms depend directly on the physicochemical properties of the vehicle and the drug employed. Solubility of the drug is one of the most important physical properties that affect release in both base and its surrounding medium. Metronidazole (MTD) is soluble in phosphate buffer. Hence the solubility does not constitute a limiting factor in the absorption process. Lipophilicity is a very useful physicochemical parameter reflecting transfer properties of a compound. The partition coefficient (aqueous phase:n-octanol) is commonly used in the pharmaceutical industry to reflect lipophilicity of a drug. The partition coefficient of MTD is 0.56 and 0.69 in water and phosphate buffer (pH 7.4) respectively. So, MTD has more affinity to phosphate buffer than water. Release of MTD is dependent on the solubilizing effect of the vehicle. MTD will be released fast and high from water-soluble bases than oleaginous bases due to favorable partitioning of MTD toward the aqueous phase. Free concentration of MTD will be greater in water soluble vehicles. In contrast, for the oleaginous vehicles, partitioning towards the internal aqueous phase would render the drug almost unavailable in the external oil phase. It is possible to assume based on solubility of the drug in the vehicle, that formulation containing water soluble base will be good candidates for the topical delivery of MTD. Prerequisite 1. Permeation through skin. 2. Ointment Bases.

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Requirements 1. Glasswares: Beaker, open diffusion cell, beakers, test tubes. 2. Chemicals: Metronidazole, potassium dihydrogen orthophosphate, etc. 3. Equipments: UV spectrophotometer. 4. Membrane: Rat membrane. Procedure 1. Preparation of phosphate buffer 0.05M, pH 7.4: Dissolve required quantity of potassium dihydrogen phosphate in sufficient quantity of water to produce 1000 ml and adjust the pH to 7.4. 2. Calibration curve: 1. Preparation of standard stock solution: Weigh accurately 100 mg of pure MTD and transfer it into 100 ml volumetric flask and adjust volume (Stock I, 1mg/ml). Transfer 10 ml stock I to another 100 ml volumetric flask and adjust volume (Stock II, 100 mg/ml). 2. Preparation of working solution: From stock solution II, pipette out 0.5, 1, 1.5, 2, 2.5 and 3 ml into 10 ml volumetric flasks and adjust volume to get concentration in the range of 0-30 mg/ml. 3. Measurement of absorbance: Measure absorbance of the respective dilutions at lmax 320 nm using UVVisible spectrophotometer. Plot the graph of absorbance of MTD against concentration in MS Excel and determine slope and intercept. 4. Preparation of formulations 0

1. Heat separately aqueous and oil phases at 70 C, and mix together with continuous stirring. 0

2. After cooling to (40-50 C), add MTD (1%) and mix to obtain a homogeneous and uniform mixture as per formula given below Ingredients Beeswax White Vaseline Stearic acid Cetyl alcohol Spermaceti wax Glycerin Potassium hydroxide Distilled water

F1 5g 95 g -

F2 20 g 1g 1g 5g 1.05 g 71.95 g

3. In vitro release and permeation 1. Use dialysis cell method to determine the amount of the drug diffused from different ointment formulations. 2. Shave abdominal hair of the rat and cut the skin samples in full thickness. Remove rat skin and wash with water. Carefully remove fat and connective tissues and keep the skin in contact with receptor liquid (0.05 M phosphate buffer; pH 7.4) for 1 h.

Absorption of drugs

Section 2

53

Hollow tube

Beaker Membrane

Magnetic Stirrer

Figure 1. Design of diffusion assembly 3. Cover the tubes with a rat skin. Immerse the tubes into a 100 ml beaker containing 50 ml of the receptor phase (0.05 M phosphate buffer; pH 7.4). 4. Stir the receptor phase continuously with a small magnetic bar at a speed of 100 rpm during the experiments 0 to ensure homogeneity and maintain at 37 C. 5. Apply known quantity of the formulation F1 (oleagineous base) on membrane. At predetermined time interval remove samples and measure absorbance spectrophotometrically at 320 nm. 6. Repeat the experiment for formulation F2 (water soluble base). 7. Use the calibration curve for determination of the amount of MTD diffused. 8. Plot graph of amount of the drug diffused per unit area versus time and determine slope of linear portion of graph. 9. Calculate permeability coefficients by using following formula Kp = JSS/CV 2

Where, KP = permeability coefficient (cm/h), JSS = flux (mg/cm /hr), A = area of the diffusion 2

membrane (cm ); CV = initial concentration of the drug in the formulations (mg). A. Parameters set for plotting of calibration curve 1. Beer's & Lambert range: 2. Solvent: 3. l max for Metronidazole: B. Parameters set for diffusion studies 1. Formulation: 2. Strength: 3.Apparatus: 4. Rotation Speed: 5. Test time: 6. Diffusion medium: 7. Volume of diffusion medium:

5-30 mg/ml 0.05 M phosphate buffer, pH 7.4 320 nm Ointment 1% Open tube dialysis diffusion cell 100 rpm 8h 0.05 M phosphate buffer, pH 7.4. 50 ml

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Observation Table 1. Calibration curve of Metronidazole Concentration (mg/ml) 5 10 15 20 25 30 Slope Intercept

Absorbance

Table 2. Ex vivo absorption of formulation F1 through rat skin

Time (h) 0.5 1 2 3 4 6 8

Absorbance

Concentration (mg/ml)

Total amount diffused (mg)

CADD/unit area

Table 3. Ex vivo absorption of formulation F2 through rat skin Time (h) 0.5 1 2 3 4 6 8

Absorbance

Concentration (mg/ml)

Total amount diffused (mg)

CADD/unit area

CADD- cumulative amount of drug diffused

Calculations 1. Concentration of diffused drug (mg/ml) Plot the graph of absorbance versus concentration and determine slope and intercept. Y= m X + c Where, Y = absorbance, m = slope, X = concentration (mg/ml), c = intercept.

Absorption of drugs

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55

2. Cumulative amount of drug diffused (mg) Cumulative amount of drug diffused (CADD) = [Concentration (mg/ml) x (volume of diffusion medium) x (dilution factor)]/1000 3. Surface area (A) of rat skin A= pr Where, r = radius of rat skin. 2

2

4. Cumulative amount of drug diffused per unit area (CADD/cm ) 2

CADD/cm = CADD/Area of rat skin used 5. Dilution factor Dilution factor = volume of diluted sample (ml)/ volume of sample removed (ml) 6. Flux (JSS) Slope (JSS) of linear portion of plot of amount of drug diffused per unit area versus time 7. Permeability coefficient (KP)

KP =

J ss Cv

Results 1. Permeability coefficient and flux for formulation F1 were found to be ___cm/h and ___mg/cm /h respectively. 2. Permeability coefficient and flux for formulation F2 were found to be ___cm/h and ___mg/cm /h respectively. 2

2

Conclusion It can be concluded from this study that the MTD shows higher permeability in water soluble base as vehicle than oleaginous base. Applications 1. Determination of suitable vehicle for the topical delivery of drug. 2. Estimation of flux and permeation coefficient of the drug. 3. Comparison of various brands available in the market by estimating flux and permeation coefficient. Questions 1. What is permeation coefficient? 2. Define flux. 3. Discuss different types of vehicle bases. Exercise Study the effect of various bases on percutaneous absorption of Diclofenac gel.

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Experiment 12 Percutaneous absorption of drug through different membranes Aim To study ex vivo percutaneous absorption of marketed Metronidazole ointment/gel through different membranes. Learning objectives 1. To understand the concept of permeability coefficient and its estimation. 2. To study the percutaneous absorption of Metronidazole through different membranes. Theory An ideal way to determine percutaneous absorption of a compound in human is to do the actual study in humans. However, many compounds are potentially toxic to be tested in vivo in humans. Besides this, evaluation of these systems in vivo using human beings is difficult from the viewpoint of cost, time consumption, and ethical restrictions. Therefore the studies must be conducted in vitro using excised skin (human cadaver, animals). Membranes from rats, mice, pigs, guinea pigs, snakes, rabbits and humans as well as synthetic membranes have been used for these drug diffusion studies. The animal skins differ significantly from human skin (HS) due to differences in thickness, nature of stratum cornea, density of hair follicles and sweat glands. In this respect, in vitro studies are generally conducted using a diffusion cell system with either static or a flow-through cell. The difficulty in obtaining excised skin and the variation in their permeability due to race, age, sex, anatomical site and concern for restricted use of animals has led the workers to use simulated skin or an artificial membrane. Permeation of drugs through various artificial membranes, such as collagen membrane and oil saturated membrane filter have been investigated. In addition, many studies have been reported on drug release from ointments using silicon rubber membrane, cellulose membrane and membrane filter. However, results obtained with these artificial membranes do not always reflect percutaneous absorption. Natural membranes such as peach and tomato skin, inter-lamellar layer of the onion, and inner layer of the egg could be applied instead of HS, synthetic, simulated or artificial skin in in vitro drug permeation studies. Natural membranes can be good alternatives for permeability studies because of their easy availability, very low cost and no ethical issues involved in it. Various artificial and natural membranes are compared with animal skin for permeation studies, in this experiment. Principle Metronidazole (MTD), being intermediate lipophilic with low molecular weight (171.2), shows highest permeability through cellophane membrane than all other membranes. Therefore for permeation study of low molecular weight drugs, cellophane membranes cannot be the membranes of choice for in vitro studies, instead of human skin. Some of the natural membranes are better models for such drugs. Hydrophilic drugs will permeate well through onion membrane while lipophilic drugs permeate well through egg membrane. Natural membranes have pores and channels with hydrophilic properties which permeate small to middle size hydrophilic drugs to diffuse in a manner similar to human skin, and because of their availability thus can be used for in vitro diffusion studies.

Section 2

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57

Prerequisite 1. Permeation through various membranes. 2. Permeability coefficient and flux. Requirements 1. Glasswares: Beaker, open diffusion cell, beakers, test tubes etc. 2. Chemicals: Metronidazole, marketed Metronidazole gel or ointment, potassium dihydrogen orthophosphate, etc. 3. Membranes: Rat skin, cellophane membrane, egg membrane and onion membrane. Procedure 1. Preparation of phosphate buffer 0.05 M, pH 7.4 Dissolve required quantity of potassium dihydrogen phosphate in sufficient quantity of water to produce 1000 ml and adjust the pH to 7.4. 2. Plotting of calibration curve 1. Preparation of standard stock solution: Weigh accurately 100 mg of pure Metronidazole and transfer it into 100 ml volumetric flask and adjust volume (Stock I, 1mg/ml). Transfer 10 ml stock I to another 100 ml volumetric flasks and adjust volume (Stock II, 100 mg/ml). 2. Preparation of working solution: From stock solution II, pipette out 0.5, 1, 1.5, 2, 2.5 and 3 ml into 10 ml volumetric flask and adjust volume to get concentration in the range of 0-30 mg/ml. 3. Measurement of absorbance: Measure absorbance of the respective dilutions at lmax 320 nm using UVVisible spectrophotometer. Plot the graph of absorbance of MTD against concentration in MS Excel and determine slope and intercept. 3. Preparation of membranes 1. Shave abdominal hair of the rat and cut the skin samples in full thickness. Remove rat skin and wash with water. Carefully remove fat and connective tissues and leave the skin in contact with receptor liquid (0.05 M phosphate buffer; pH 7.4) for 1 hour. 2. Peal the middle membrane of the Allium cepa L. (onion) with caution to prepare at least 10 cm2 uniform membrane without any crack or orifice. 3. The outer shell membrane of the egg of Gallus domesticus that is just located inside the shell exactly under the hard calcified layer is taken. This membrane is prepared by immersing the egg in 0.01N HCl solution for 6 h to dissolve the calcified layer without any further process. Cut the membrane cautiously to expel the content of the egg and wash it with normal saline solution. 4. Inspect the membrane by a microscope to assure about its integrity and uniformity. 4. In vitro release and permeation 1. Use the dialysis cell method to determine the amount of the drug diffused from MTD ointment using different membranes prepared as given above. 2. Cover the tube with a rat skin. Immerse the tube into a 100 ml beaker containing 50 ml of the receptor phase (0.05 M phosphate buffer; pH 7.4). 3. Stir the receptor phase continuously with a small magnetic bar at a speed of 100 rpm during the experiments to

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58 0

ensure homogeneity and maintain at 37 C. 4. Apply known quantity of the formulation on membrane. At predetermined time intervals, remove sample and measure absorbance spectrophotometrically at 320 nm. 5. Repeat the same experiment by using cellophane, onion and egg membrane. 6. Use the calibration curve for the determination of the amount of MTD diffused. 7. Plot the graph of amount of drug diffused per unit area versus time and determine slope of linear portion of graph. 8.Calculate permeability coefficients by using following formula KP = JSS/CV 2

Where, KP = permeability coefficient (cm/h), JSS = flux (mg/cm /hr), A = area of the diffusion membrane (cm2); CV = initial concentration of the drug in the formulation (mg). A. Parameters set for plotting of calibration curve 1. Beer's & Lambert range for MTD: 2. Solvent: 3.l max for MTD: 4. Instrument: B. Parameters set for diffusion studies 1. Formulation: 2. Strength: 3. Apparatus: 4. Rotation Speed: 5. Test time: 6. Diffusion medium: 7. Volume of diffusion medium:

5-30 mg/ml 0.05 M phosphate buffer, pH 7.4 320 nm UV spectrophotometer Ointment 1% Open tube dialysis diffusion cell 100 rpm 8h 0.05 M phosphate buffer, pH 7.4 50 ml

Observations Table 1. Calibration curve of Metronidazole Concentration mg/ml 0 5 10 15 20 25 30 Slope Intercept

Absorbance

Section 2

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59

Table 2. Ex vivo absorption of drug through rat skin Time (h) 0.5 1 2 3 4 6 8

Absorbance

Concentration (mg/ml)

Total amount diffused (mg)

CADD/unit area

CADD = cumulative amount of drug diffused

Table 3. Ex vivo absorption of drug through cellophane membrane Time (h) 0.5 1 2 3 4 6 8

Absorbance

Concentration (mg/ml)

Total amount diffused (mg)

Table 4. Ex vivo absorption of drug through onion membrane Time Absorbance Concentration Total amount (h) diffused (mg) (mg/ml) 0.5 1 2 3 4 6 8

CADD/unit area

CADD/unit area

Table 5. Ex vivo absorption of drug through egg membrane Time (h) 0.5 1 2 3 4 6 8

Absorbance

Concentration (mg/ml)

Total amount diffused (mg)

CADD/unit area

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Calculations 1. Determination of concentration of diffused drug (mg/ml) Plot the graph of absorbance versus concentration and determine slope and intercept. Calculate unknown concentration using formula Y= m X + c Where, Y = absorbance, m = slope, X= concentration (mg /ml), c = intercept. 2. Cumulative amount of drug diffused (mg) Cumulative amount of drug diffused (CADD) = [Concentration (mg /ml) x (volume of diffusion medium) x (dilution factor)]/1000 3. Surface area (A) of membrane used A=p r Where, r = radius of rat skin / cellophane membrane/ onion membrane / egg membrane. 2

2

4. Cumulative amount of drug diffused per unit area (CADD/cm ) 2

CADD/cm = CADD/Area of membrane 5. Dilution factor Dilution factor = volume of diluted sample (ml)/ volume of sample removed (ml) 6. Flux (JSS) Plot the graph of amount of drug diffused per unit area versus time. The slope of linear portion of plot will give flux, JSS. 7. Permeability coefficient (KP) Permeability coefficient calculated by using formula J K P = ss Cv Where CV is the initial concentration of the drug in donor compartment (mg). Results 1. Permeability coefficient and flux of MTD through rat membrane is ____cm/h and ____mg/cm /h respectively. 2. Permeability coefficient and flux of MTD through cellophane membrane is ____cm/h and ____mg/cm /h respectively. 3. Permeability coefficient and flux of MTD through onion membrane is ___cm/h and ___mg/cm /h respectively. 4. Permeability coefficient and flux of MTD through egg membrane is ____cm/h and ____mg/cm /h respectively. 5. The permeability of MTD is in the order _____> _____> _______ > _______. 2

2

2

2

Conclusion It can be concluded from this study that the formulation of MTD shows highest permeability through cellophane membrane while lowest permeability through rat membrane as the drug is hydrophilic in nature.

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61

Applications 1. Determination of suitable membrane for the permeability studies. 2. Estimation of flux and permeation coefficient of the drug. 3. Comparison of various brands of drugs available in the market can be done by estimating flux and permeation coefficient. Questions 1. Discuss various in vitro and ex vivo models of percutaneous absorption studies. 2. Which membrane is ideal membrane for percutaneous absorption studies and why? 3. How will you calculate the flux of absorption through membrane? 4. Describe permeability coefficient of drug. Exercise Perform this experiment using tomato, peach and capsicum membrane.

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Experiment 13 In vitro permeation study using Franz diffusion cell Aim To study the in vitro permeation of Diclofenac gel using Franz diffusion cell. Learning objectives 1. To understand construction of Franz diffusion cell. 2. To study the percutaneous absorption of Diclofenac using Franz diffusion cell. Theory As per previous experiment (Expt No. 12). Principle The in vitro permeation of drug can be studied by Franz diffusion cells. The Franz diffusion cells are made of glass with a contact area of 4.7 cm2 and pretreated with a silanizing agent. The Franz diffusion cell consists of a donor compartment (A) and a receptor compartment (B). The membrane is mounted between two cell compartments. The two cell compartments are held together with a clamp. The receptor compartment has a volume of 37 ml and is filled with diffusion medium. It is kept at 37°C by circulating water through an external water jacket. After 30 min of equilibration of the membrane with the receptor solution, specific quantity of the drug/ formulation is applied in the donor compartment. The donor compartment is then covered with parafilm to prevent evaporation of the solvent. The receptor solution is continuously stirred by means of a spinning bar magnet, at 400 rpm. Receptor solution samples, 2.0 ml aliquots, are withdrawn through the sampling port of the receptor compartment at various time intervals. The cells are refilled with receptor solution to keep the volume of receptor solution constant during the experiment. Sample holder

A

Sample port

Membrane

Figure 1. Diffusion study cell assembly B

Stir bar Magnetic stirrer

Prerequisite As per previous experiment (Expt No. 12). Requirements 1. Glasswares: Franz diffusion cell, test tubes, pipettes, etc. 2. Chemicals: Pure Diclofenac sodium, marketed Diclofenac gel, potassium dihydrogen orthophosphate, etc. 3. Membrane: Cellophane membrane.

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63

4. Equipments: UV Spectrophotometer, magnetic stirrer, thermostatic water bath, pump. Procedure 1. Preparation of Sorensen's phosphate buffer, pH 7.4 Solution A: Take 3.5 g of disodium hydrogen phosphate and dissolve it in 100 ml distilled water. Solution B: Take 2.76 g of sodium dihydrogen phosphate and dissolve it in 100 ml distilled water. Mix 40.5 ml of solution A with 9.5 ml of solution B and make volume to 100 ml. 2. Plotting of calibration curve of Diclofenac sodium 1. Preparation of standard stock solution: Weigh accurately 100 mg of pure Diclofenac sodium and transfer it into 100 ml volumetric flask and adjust volume with water (Stock I, 1mg/ml). Transfer 10 ml stock I to another 100 ml volumetric flask and adjust volume (Stock II, 100 mg/ml). 2. Preparation of working solution: From stock solution II, pipette out 0.4, 0.8, 1.2, 1.6, 2, and 2.4 ml into 10 ml volumetric flasks and adjust volume to get concentration in the range of 4-24 mg/ml. 3. Measurement of absorbance: Measure absorbance of the respective dilutions at lmax 270 nm using UVVisible spectrophotometer. Plot the graph of absorbance of Diclofenac sodium versus concentration in MS Excel and determine slope and intercept. 3. In vitro release and permeation 1. For the permeation study use Franz diffusion cells with a diffusion area of 4.7 cm2 and 37 ml capacity. 2. Place cellophane membrane between the donor and receptor compartments of the cells. 3. Fill receptor compartment with approximately 37 ml of Sorensen's phosphate buffer (pH 7.4). Maintain temperature of cell at 370C by means of circulating contents of a thermostatic water bath by a pump through the surrounding layer of the cell. Stir content of receptor compartment at 600 rpm with Teflon-coated magnetic bar placed inside cell throughout experiment. 4. Ensure that membrane should be in contact with the receptor medium. 5. Place 1 gm Diclofenac gel in a donor compartment and cover donor cell with aluminum foil to avoid evaporation. 6. Remove 2 ml sample from receptor compartment at predetermined time intervals and immediately replace with 2 ml of the receptor solution, at the same temperature. 7. Dilute withdrawn samples if required and analyze spectrophotometrically at 270 nm. 8. Use the calibration curve for the determination of the amount of Diclofenac sodium diffused. 9. Plot the graph of amount of drug diffused per unit area versus time and determine slope of linear portion of graph. 10. Calculate permeability coefficients by using following formula: KP = JSS/CV Where, KP = permeability coefficient (cm/h), JSS = flux (mg/cm2/hr), A = area of the diffusion membrane (cm2); CV = initial concentration of the drug in donor compartment (mg).

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Obervations Table 1. Calibration curve of Diclofenac sodium Concentration (mg/ml) 0 4 8 12 16 20 24 Slope Intercept

Absorbance

Table 2. In vitro permeation of Diclofenac sodium through cellophane membrane

Time (h)

Absorbance

Concentration (mg/ml)

Cumulative amount of drug diffused (mg)

CADD/unit area

0.5 1 1.5 2 Calculations 1. Determination of concentration of diffused drug (mg/ml) Plot the graph of absorbance versus concentration and determine slope and intercept. Y= m X + c Where, Y = absorbance, m = slope, X = concentration (mg/ml), c = intercept. 2. Cumulative amount of drug diffused (mg) Cumulative amount of drug diffused (CADD) = [Concentration (mg/ml) x (volume of diffusion medium) x (dilution factor)]/1000 3. Surface area (A) of cellophane membrane A= pr Where, r = radius of cellophane membrane exposed to diffusion medium. 2

2

4. Cumulative amount of drug diffused per unit area (CADD/cm ) 2

CADD/cm = CADD/Area of cellophane membrane 5. Dilution factor Dilution factor = volume of diluted sample (ml)/ volume of sample removed (ml)

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6. Flux (JSS) Slope (JSS) of linear portion of plot of amount of drug diffused per unit area versus time. 7. Permeability coefficient (KP)

KP =

J ss Cv

Where, CV = initial concentration of the drug in donor compartment (mg). Result Permeability coefficient and flux of Diclofenac gel through cellophane membrane is __________cm/h and ________mg/cm /h respectively. 2

Conclusion It can be concluded from this study that in vitro permeation of Diclofenac gel through cellophane can be studied using Franz diffusion cell. Applications 1. This experiment allows us to study in vitro permeation of various transdermal formulations. 2. This model can be used to study effect of formulation variables on permeation of drugs. 3. Estimation of flux and permeation coefficient is possible. 4. This model can be utilised for studing permeation of drug through nasal and buccal route. Question Give various in vitro permeation models Exercise Perform in vitro permeation study of Ketoconazole gel using Franz diffusion cell.

SECTION 3

Protein binding of drugs Experiment 14 Protein binding study using equilibrium dialysis method Aim To study the protein binding of Salicylic acid by method of equilibrium dialysis. Learning objectives 1. To understand the protein binding process of a drug. 2. To confirm protein binding of Salicylic acid by equilibrium dialysis method. Theory The desired therapeutic response of a drug is elicited as a result of its interaction with the specific receptor site. On its way to that receptor, the drug may bind to other constituents of biological system, such as plasma proteins like albumin, high density lipoproteins and globulins. It is the free drug that can diffuse to and from the extra vascular fluid across the endothelium and other biological barriers. The unbound fraction of drug in plasma/serum is available for binding to a specific site. The nature and magnitude of drug protein interaction significantly influence biologic activity of a drug by affecting its pharmacokinetic fate and pharmacodynamic performance. A drug in the body can interact with several tissue components of which two major categories are blood and extra vascular tissues. The interacting molecules are generally macromolecules such as proteins, DNA, etc. Proteins are particularly responsible for such an interaction. The phenomenon of complex formation with proteins is called as protein binding of a drug. Importance of such a binding derives from the fact that the bound drug is both pharmacokinetically as well as pharmacodynamically inert. Binding of drugs generally involves weak chemical bonds such as hydrogen bonds, ionic bonds or van der Waal's forces and, therefore, is a reversible process. Binding of a drug to protein contained in the body influence their action in a number of ways. 1. Protein may facilitate the distribution of a drug throughout the body. 2. It may inactivate the drug by not enabling a sufficient concentration of free drug to develop at the receptor site. 3. It may retard excretion of a drug. 4. The interaction of a drug with protein may cause a) The displacement of body hormones or a co administered agent. b) Aconfigurational change in the protein. c) Formation of a drug protein complex that itself is biological active. Methods to study protein binding of drug Number of methods are available for studying protein binding of drugs. Apart from satisfying the general criteria for any analytical technique, they must not upset the equilibrium between free and bound drug. 66

Section 3

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67

Equilibrium dialysis, dynamic dialysis, ultra filtration and electrophoresis are the classic techniques widely used to study protein binding. In recent years other methods such as gel filtration and nuclear magnetic resonance have been used with satisfactory results, all of them having their own inherent advantages and disadvantages. Spectral changes Most drugs have distinct UV spectra because of the conjugated chromophores in the molecule. When a drug interacts with a protein the UV or visible spectrum may be changed because of alterations in the electronic configuration. These alterations can be quantitated and used to determine the extent of binding. Changes in fluorescence spectra can be used in the same way. Gel filtration This involves the use of porous gels that are molecular sieves. They separate components on the basis of size. Low molecular weight drugs are held on the gel whereas bound drugs and proteins are washed through. Equilibrium dialysis Membrane Protein Drug

Free drug

Buffer Drug

Buffer

Figure 1. Equilibrium across a semi-permeable membrane The protein solution (e.g. plasma) containing a drug and a buffer solution are placed on opposite sides of a dialysis membrane. After a sufficient time (may be 12- 24 h), free drug concentration will be same on either side of the membrane. Protein binding can be determined by measuring the concentration of a drug on either side of the membrane. On left the concentration will involve free and bound drug, whereas on the right there is no binding and the concentration will be equal to the free drug concentration. Ultrafiltration Drug and protein solution Membrane

Free drug filtrate

Figure 2. Ultrafiltration as a method of measuring protein binding A quicker method of separating free and bound drug is the ultrafiltration method. Drug and protein solutions are placed in a filter membrane and liquid containing free drug is forced through the membrane by centrifugation. Dynamic dialysis One of the methods widely used to study in vitro protein binding of drug is dynamic dialysis. Dynamic

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dialysis uses flow dynamics to increase both the rate and efficiency of dialysis. Circulating the sample and/or the dialysis creates the highest possible concentration gradient to significantly decrease dialysis time. Other benefit of the sweeping action is that it prevents membrane fouling and in some situation generates a pressure differential. This supplemental driving force increases the hypo-osmotic mass transfer rate across the semipermeable membrane and allow for sample concentration during the dialysis process. Principle TubeAcontains only Salicylic acid with no protein in it. The equilibrium of Salicylic acid in tubeAand beaker is achieved through semipermeable membrane and the concentration of Salicylic acid in beaker can be measured. However, Tube B contains Salicylic acid in presence of protein (0.5 ml egg albumin) and hence the protein binding of Salicylic acid takes place. This allows only free Salicylic acid left after protein binding to equilibrate through semipermeable membrane, which reduces the concentration of Salicylic acid in beaker. Further, reduction in concentration of Salicylic acid takes place in tube C because of increased presence of protein (1 ml egg albumin). This increases protein binding of salicylic acid, thereby reducing free Salicylic acid in the tube and in beaker too. Salicylic acid present in beaker (non protein compartment) is estimated by adding ferric nitrate solution. The reaction of Salicylic acid with ferric nitrate produces an intensely colored complex (deep wine), whose maximum absorbance can be detected at 540 nm spectrophotometrically. Prerequisite 1. Concept of protein binding. 2. Significance of protein binding. Requirements Chemicals: Salicylic acid, egg albumin, ferric nitrate, hydrochloric acid, mercuric chloride, cellophane membrane, and distilled water. Instruments: UV spectrophotometer, magnetic stirrer and equilibrium dialysis unit. Procedure 1. Calibration curve of Salicylic acid 1. Preparation of coloring agent: Dissolve mercuric chloride (4 g) in about one-fifth volumes of water. Separately dissolve ferric nitrate (4 g) in 0.12 N HCl (12 ml). Mix these two solutions and adjust volume to 100 ml, filter and use the filtrate as a coloring agent. 2. Preparation of standard stock solution: Weigh accurately 100 mg of Salicylic acid. Dissolve in 100 ml of distilled water using 5 ml alcohol. Take 10 ml of this solution and dilute to 100 ml with water. 3. Preparation of working solution: From stock solution, pipette out 0.2, 0.4, 0.6, 0.8 and 1 ml into 10ml volumetric flask and add 4 ml coloring agent. Dilute resulting solution to 10 ml with water to get concentration in range of 2-10 µg/ml. 4. Measurement of absorbance: Record absorbance of the working solution at lmax of 540 nm using UVVisible spectrophotometer against water as a blank. Plot a graph of absorbance versus concentration and determine slope and intercept.

Section 3

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2. Protein binding study of drug 1. Take three hollow tubes and mark them asA, B and C. 2. To one end, tie a semipermeable membrane in such a way that a sack is formed. 3. Place 1ml of 1% Salicylic acid and 1 ml of distilled water in tubeA. 4. Place 1ml of 1% Salicylic acid and 0.5 ml of egg albumin and 0.5 ml distilled water in tube B. 5. Place 1ml of 1% Salicylic acid and 1 ml of egg albumin in tube C. 6. Place the tubes in a 50 ml of distilled water and pipette out 5 ml of sample from beaker at the intervals of 15, 30, 45, 60, 75 and 90 min. 7. Add 4 ml of coloring agent and 1 ml of distilled water and determine absorbance at 540 nm. Replace the fluid with 5 ml of distilled water in the beaker. 8. Plot comparative curves of amount of Salicylic acid present in non protein compartment versus time.

Figure 3. Equilibrium dialysis unit Observations Table 1. Calibration curve of Salicylic acid

Concentration of SA Absorbance mg/ml 2 4 6 8 10 Slope Intercept

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Table 2. Percent cumulative drug release with and without protein through cellophane membrane Time (min) 15 30 45 60 75 90

Percent cumulative drug release Tube A Tube B Tube C

Calculations A. Determination of unknown concentration of Salicylic acid Determine unknown concentration of Salicylic acid from receptor compartment by using following equation Y = mX +c Where, Y = absorbance, m = slope, X = concentration, c = the intercept. B.Amount of drug diffused (mg) Amount of drug diffused = [Concentration (mg/ml) x (volume of diffusion medium) x (dilution factor)]/1000 C. Dilution factor Dilution factor = volume of diluted sample (ml) / volume of sample removed (ml) D. Percent cumulative drug release Percent cumulative drug release = (amount of drug diffused x 100) /total amount of dose Result The percent release of Salicylic acid without any proteins was found to be _______, with 0.5 ml of egg albumin was______, and with 1 ml of egg albumin was_______. Conclusion The protein binding of Salicylic acid is confirmed and the increase in protein binding is found to be proportional to the amount of protein available. Applications 1. It can be used for studying protein binding of various other drugs. 2. Equilibrium dialysis method provides the non invasive in vitro method for studying protein binding. Questions 1. Discuss about protein binding of Salicylic acid. 2. Give clinical significance of protein binding. Exercise Study the protein binding of Ranitidine hydrochloride using equilibrium dialysis method.

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Experiment 15 Protein binding study using dynamic dialysis method Aim To study the effect of protein binding on drug diffusion by dynamic dialysis method. Learning objective To compare protein binding of human serum and plasma with a model drug Tetracycline. Theory Theory as per previous experiment (Expt No. 14). Principle Percentage of Tetracycline release is estimated by knowing its concentration in non protein compartment at various time intervals. The release of a drug is very less in presence of serum and plasma than that of the release of a dug without any proteins. This represents well that plasma proteins, serum proteins have great affinity towards the drug and bind with it. While comparing the drug release in presence of serum or plasma, the percentage of drug release in presence of plasma is less than that in presence of serum, since the serum lacks the clotting factors that are normally present in plasma but have been consumed during the process of coagulation. It indicates that the extent of protein binding is directly proportional to the amount of protein. Due to the affinity of plasma proteins towards drugs they bind with the drug and release the free drug slowly to maintain the equilibrium between free drug and bound drug. This may help to prevent the quick elimination of drug and since the therapeutic action of a drug depends upon the concentration of free drug alone, protein binding may also help to prolong action of the drug. Prerequisite As per previous experiment (Expt No. 14). Requirements Chemicals: Tetracycline, human serum, human plasma, hydrochloric acid, egg membrane and distilled water. Instruments: UV spectrophotometer, magnetic stirrers, dynamic dialysis setup. Procedure 1. Calibration curve of Tetracycline a) Preparation of standard stock solution: Weigh accurately 100 mg of Tetracycline and dissolve it in 100 ml of distilled water. Take 10 ml of this solution and dilute to 100 ml with water. b) Preparation of working solution: From stock solution, pipette out 0.2, 0.4, 0.6, 0.8, 1 and1.5 ml into 10 ml volumetric flask and adjust the volume to get concentration in range of 2-15 µg/ml. c) Measurement of absorbance: Record absorbance of working solution at lmax of 360 nm using UV-Visible spectrophotometer against water as a blank. Plot a graph of absorbance versus concentration and determine slope and intercept.

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2. Preparation of egg shell membrane Soak a whole chicken egg in 0.5 N HCl solution. The outer calcareous shell gets dissolved, then cutoff a part of the egg shell membrane and remove the inner contents. Wash obtained membrane thoroughly in distilled water and store it in a refrigerator and use within a week. 3. Protein binding study of Tetracycline 1. Tie egg membrane to one end of an open-ended glass cylinder and use it as a protein compartment (donor). 2. Use a glass beaker capacity of 25 ml as a non-protein compartment (receptor) and fill it with 20 ml of distilled water. 3. Place drug solution (1 mg/ml) of 2 ml to the inner tube and immerse into the beaker. Take care to maintain the level of the drug solution coincide with the water level in outside compartment and fix it with a stand. 4. Keep this whole set-up on a magnetic stirrer and stirr the outer compartment continuously with an optimal 0 speed (see figure 1). Maintain the temperature at 35 ± 2 C for the whole experiment. 5. At predetermined time intervals (5, 10, 15, 30, 60, 90 min), remove 1 ml of the sample from beaker and replace it with same volume of fresh distilled water. 6. Determine the concentration of Tetracycline spectrophotometrically. 7. Repeat the experiment by using 1 ml of human blood serum and 1 ml of drug solution (2 mg/ml) in the protein compartment and determine the percentage of drug released from the protein compartment into the non-protein compartment in the same time period as above. 8. Also repeat this experiment using 1 ml of human blood plasma and 1 ml of drug solution (2 mg/ml) in the protein compartment and determine the percentage of the drug released. 9. Plot the graph of percent cumulative drug release versus time.

Figure 1. Dynamic dialysis unit

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Observations Table 1. Calibration curve of Tetracycline

Concentration (mg/ml) 2 4 6 8 10 15 Slope Intercept

Absorbance

Table 2. Percentage cumulative drug release with and without protein through egg membrane

Time (min) 5 10 15 30 60 90

Percent cumulative drug release Without any protein In presence of serum In presence of plasma

Calculations 1. Determination of unknown concentration of Tetracycline For the determination of unknown concentration of Tetracycline from receptor compartment following equation is used. Y = mX + c Where, Y = absorbance, m = slope, X = concentration, c = intercept. 2.Amount of drug diffused (mg) Amount of drug diffused = [Concentration (mg/ml) x (volume of diffusion medium) x (dilution factor)]/1000 3. Dilution factor Dilution factor = volume of diluted sample (ml)/ volume of sample removed (ml) 4. Percent cumulative drug release Percent cumulative drug release = amount of drug diffused x 100 /drug dose

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Result The percent release of Tetracycline without any proteins was found to be _______, with plasma was _______, and with serum was ______. Conclusion The protein binding of Tetracycline is confirmed and is dependant on the amount of proteins available. Therefore, the extent of protein binding in plasma is more giving lesser drug release. Applications 1. Protein binding of drug can be studied. 2. The displacement of one drug from protein binding site in presence of another drug can be studied. 3.Affinity of various drugs towards protein can be studied. 4. Protein binding study is very important for calculation of dose of the drug. Questions 1. Give significance of protein binding of a drug. 2. Give five examples of displacement of drugs from protein binding site and their pharmacological effects. Exercise Study the protein binding of Phenylbutazone using dynamic dialysis method.

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Experiment 16 Determination of binding sites using bovine serum albumin Aim To determine binding site of Propranolol using Warfarin sodium/Diazepam as a site-I/II specific probe. Learning objectives 1. To understand and study binding of drugs to albumin. 2. To determine binding site of Propranolol HCl. Theory Bovine serum albumin (BSA) is a large multi-domain protein folded into three domains, each of which is built of three loops. On the basis of probe displacement method, there lie at least three relatively high specific binding sites on the BSA molecule. These sites are generally called the warfarin-binding site, the benzodiazepine-binding site and digoxin-binding site and are denoted as site-I, II and III, respectively. Site-II is more specific than site-I whereas site-III is an independent binding site. Serum albumin, the most abundant protein in the blood, plays a very important role in the binding phenomenon and serves as a depot protein and transport protein for numerous endogenous compounds. Displacement of a drug is defined as reduction in the extent of binding of a drug to other agent, the displacer. This type of interaction may occur when two drugs or agents, capable of binding to proteins, are administered concurrently. Competitive displacement is more significant, when two drugs or agents are capable of binding to the same sites on the protein. From different investigations, it has been suggested that human serum albumin (HSA) has limited number of binding sites. Since the number of protein binding sites is limited, competition will exist between drugs or drugs with metals or other agents and the agent with higher affinity will displace the other causing increased free drug concentration leading to higher toxicity or short duration of action. Ability of one drug to inhibit the other is a function of their relative concentration, binding affinities and specificity of binding. Principle Interactions of Propranolol HCl and its binding characteristics on BSA can be studied by equilibrium dialysis method. It provides the possible way of in vitro estimates of protein binding to Propranolol HCl. The relative strength and specificity of binding to BSA is determined by its ability to displace the probes (warfarin as site-I specific probe and diazepam as site-II specific probe) specific for particular sites (site-I or site-II) on the BSA molecule. By measuring the free concentration of the site specific probe it is inferred with regard to the binding of Propranolol HCl to BSA. BSA and HSA have structural similarity. In this study BSA, in lieu of human serum albumin (HSA), can be used because of its low cost and easy availability. Propranolol HCl is known to increase the free concentration of Warfarin to a greater extent than that of Diazepam, showing its high affinity binding to site-I and low affinity to site-II in the BSA. So, in patients suffering from hypertension, if they take drugs having high affinity for site-I, it may result in rapid action or rapid excretion from the body or even may cause toxicity at the normal doses. Prerequisite As per previous experiment (Expt No. 14).

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Requirements 1. Drugs: Warfarin sodium, Diazepam, Propranolol HCl. 2. Reagents: Disodium hydrogen phosphate (Na2HPO4), potassium dihydrogen phosphate (KH2PO4), borax (NaB4O7.10H2O), cellulose membrane (M.W. 1200 Daltons), bovine serum albumin (fatty acid free, fraction V, 96-98%, M.W. 66500). 3. Instruments: UV/VIS spectrophotometer, pH Meter, metabolic shaking incubator, micro syringes. 4. Method: Equilibrium dialysis method. Procedure Calibration curve of Warfarin 1. Preparation of standard stock solution: Weigh accurately 100 mg of Warfarin. Dissolve in 100 ml of phosphate buffer pH 7.4. Take 10 ml of this solution and dilute to 100 ml with buffer (100 mg/ml). 2. Preparation of working solution: From stock solution pipette out 0.2, 0.4, 0.6, 0.8 and 1 ml into 10 ml volumetric flasks and make volume to 10 ml with buffer to get concentration in range of 2-10 µg/ml. 3. Measurement of absorbance: Record absorbance of working solution at l max of 308 nm using UV-Visible spectrophotometer against water as a blank. Plot a graph of absorbance versus concentration and determine slope and intercept. Calibration curve of Diazepam 1. Preparation of standard stock solution: Weigh accurately 100 mg of Diazepam. Dissolve in 100 ml of phosphate buffer pH 7.4. Take 10 ml of this solution and dilute to 100 ml with buffer (100 mg/ml). 2. Preparation of working solution: From stock solution, pipette out 0.2, 0.4, 0.6, 0.8 and 1 ml into 10 ml volumetric flask and make volume to 10 ml with buffer to get concentration in range of 2-10 µg/ml. 3. Measurement of absorbance: Record absorbance of working solution at max of 235 nm using UV-visible spectrophotometer against water as a blank. Plot a graph of absorbance versus concentration and determine slope and intercept. Protein binding study Perform the experiment in the following successive steps: -5

1. Prepare 2×10 M BSAsolution and transfer 3 ml in each of fifteen clean and dry test tubes. -3

2.Add 1×10 M Warfarin solution to seven test tubes so that the final ratio of protein and Warfarin is maintained -5 -5 -3 1:1 (2×10 M: 2×10 M) in each of first seven test tubes. Now, add 1×10 M Diazepam solution to next seven -5 -5 test tubes so that the final ratio of protein and Diazepam is maintained 1:1 (2×10 M: 2×10 M) in each of next seven (8-14) test tubes. Mark the fifteenth test tube containing only BSAsolution as "Blank" or "Control". 3. Allow these mixtures to stand for 10 minutes in order to allow binding of the Warfarin to its particular binding site (to site-I) and that of Diazepam to binding site II. -5

4. Add Propranolol HCl solution with increasing concentrations (0-12 x 10 ) into six out of seven test tubes containing 1:1 mixture of protein and Warfarin and also into six out of seven test tubes containing 1:1 mixture, protein and Diazepam. The final ratios of protein: Warfarin: Propranolol HCl or, protein: Diazepam: Propranolol HCl are 1:1:0, 1:1:1, 1:1:2, 1:1:3, 1:1:4, 1:1:5 and 1:1:6. The test tube No. 7 will contain only protein-Warfarin mixture and test tube No. 14 will contain only, protein-Diazepam mixture in the ratio 1:1.

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5. Mix the solution properly and allow to stand for 10 minutes to ensure maximum binding of Propranolol HCl to site-I and site-II and thereby displacing the probes from site-I and site-II on BSA. 6. Take 2 ml of solution from each test tube into fourteen different semi-permeable membrane tubes. Clip the two ends of the membrane tube and ensure that there is no leakage. 7. Immerse the membrane tubes in fourteen separate 50 ml conical flasks containing 30 ml of phosphate buffer solution pH 7.4. 0

8. Place the conical flasks in a metabolic shaker for dialysis at 25 C and shake at 20 rpm. Continue the shaking for 10 hours. 9. At the end of dialysis, collect samples from each flask. Measure the free concentrations of Warfarin and Diazepam by a UV spectrophotometer at lmax 308 and 235 nm respectively. 10. Plot the graph of free concentration of Warfarin sodium and free concentration of Diazepam as percent of initial concentration versus ratio of Propranolol to BSAconcentration. Observations Table I. Calibration curve of Warfarin Concentration mg/ml

Absorbance

Slope Intercept

Table 2. Calibration curve of Diazepam Concentration mg/ml

Slope Intercept

Absorbance

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Table 3. Displacement of Warfarin/ Diazepam by Propranolol HCl Propranolol to BSA ratio

Warfarin concentration

Free concentration of Warfarin as percent of initial

Diazepam Concentration

Free concentration of Diazepam as percent of initial

0 1 2 3 4 5 6

Results 1. The free fraction of Warfarin (site-I specific probe) increased from 100% (as % of initial) to _______ with an increasing concentration from 0-12×10-5 M of Propranolol HCl. 2. The free fraction of Diazepam (site-II specific probe) increased from 100% (as % of initial) to _______ with the same increment range of Propranolol HCl. Conclusion As % increment of the free fraction Warfarin is more than that of Diazepam, it can be concluded that Propranolol shows high affinity binding to site-I (Warfarin binding site) than to site-II (Diazepam binding site). Applications As per previous experiment (Expt No. 14). Questions 1. Give various drug binding sites on human serum albumin. 2. Give various factors affecting protein-drug binding. Exercise Study displacement of Warfarin and Diazepam by Tetracycline.

SECTION 4

Metabolism of drugs Experiment 17

Metabolism of drug using in vitro method Aim To study metabolism of Hexobarbitone sodium by enzymes of the liver microsomal fraction. Learning objectives 1.To study the metabolism of Hexobarbitone sodium. 2.To understand the activity of enzymes most concerned in metabolism of exogenous compounds. Theory Metabolism is the process of transformation of water insoluble, lipophilic, non polar drugs into polar and water soluble products that can be easily excreted by the kidneys. Hence metabolism is a detoxification process. The enzymes that biotransform drugs are broadly divided into two categories: microsomal and non microsomal. The microsomes are isolated from endoplasmic reticulum of liver homogenate. The large varieties of microsomal enzymes catalyse a number of oxidative, reductive, hydrolytic and glucuronidation reactions. The non microsomal enzymes are those that are present in soluble form in the cytoplasm, that catalyse few oxidative reactions, a number of reductive and hydrolytic reactions and conjugation reactions other than glucuronidation.

Principle The preparation of enzymes first involves separation of the supernatant fraction of the liver homogenate by centrifugation at 10,000 g. The supernatant fraction contains soluble enzymes and the microsomes. Metabolism reactions are first performed with the supernatant fraction. Conversion of hexobarbitone takes place due to oxidation by several routes given below: CH3

H O

CH3

N

H 3C

OH N

O

N

H 3C

O

OH

N

H3C

OH N

N O

O

O

OH

Desmethylhexobarbitone Hexobarbitone 3 -Hydroxyhexobarbitone Microsomes are then prepared and same reactions are performed. The enzymes concerned require NADPH and oxygen. NADPH can be added directly but it is more satisfactory to use a generating system. The soluble fraction can be used to generate NADPH Mg++ Glucose-6-phosphate + NADP 6-phosphogluconolactone + NADPH glucose-6phosphate dehydrogenase 79

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When the 10,000 g supernatant is used it is necessary to reinforce endogenous glucose-6 phosphate and NADP. When microsomes are used, it is necessary to add glucose-6 phosphate dehydrogenase as well. In addition, nicotinamide is added to prevent destruction of NAD by tissue nucleosides. It is necessary also to add Mg++ ions when hydroxylation is desired, but demethylation apparently proceeds satisfactorily with no addition of magnesium. The enzymes involved are most commonly named cytochrome P-450 and b5, and these enzymes are characterized by their spectral properties. Their concentrations are related to pre-treatment of the animals and microsomal protein. Metabolism of the test compound can be related to amounts of the microsomal enzymes. Requirements Apparatus: Refrigerated centrifuge, tissue homogenizer, spectrophotometer, water bath, surgical instruments. Chemicals: KCl (1.15%) in phosphate buffer (0.01M, pH 7.6), phosphate buffer (0.5M, pH 7.6), phosphate buffer (0.05M, pH 7.6), hexobarbitone sodium, NADP, glucose-6-phosphate, nicotinamide, magnesium chloride, glucose-6-phosphate dehydrogenase, citrate buffer (0.5M, pH 5.5) saturated with NaCl, n-heptane, amyl alcohol. Animal: Rats (150-250g, fasted overnight before the experiment). Procedure 1. Preparation of the 10,000 g supernatant fraction 1.Take the approval of the study from institutional animal ethics committee. 2. Kill the rat with a blow on the head. 3. Remove the liver(s) immediately (25 g of liver is required) into the beaker containing ice and KCl/0.01M buffer. Weigh the liver and add more KCl/0.01 M buffer to form a 25% suspension of liver in water (volume approximately 100 ml). 4. Chop or mince the liver to form small pieces. This can be done on a cold tile or under the buffer in the tared beaker. 5. Homogenize in the tissue homogenizer taking small quantity at a time subjecting each fraction to six or eight vertical excursions.Also keep everything cold by using ice bucket. 6. Centrifuge the suspension at 10,000 g for 20 min at 40C. The tubes should be as full as possible and balanced, and generally 2 x 50 ml will be sufficient. 7. Remove the supernatant (designated 10,000 g supernatant) and store on ice to be used on the day of preparation. One milliliter is equivalent to 250 mg of liver. 2. Preparation of microsomes 1. Centrifuge 4 x 14 ml of the 10,000 g supernatant at 100,000 g for 60 min. 2. Decant the supernatants and discard. 3. Re-suspend the pellets in 10 ml of KCl/0.01M buffer in the centrifuge tubes and fill the tube after resuspension is complete. 4. Re-centrifuge as before for 30 min and decant the supernatant. 5. Overlay pellets with 1 ml (or more if needed) of 0.05 M phosphate buffer pH 7.6 for storage. Store at -180 for further study.

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3. Preparation of a cofactor solution for metabolic reactions using the 10,000 g supernatant and microsomes Table 1. Formula for preparation of a cofactor solution Code a

Quantity for each incubation flask 0.65 mmol

Constituent NADP

Formula for experiment 5 mg

b

Glucose-6-phosphate

10 mmol

28.2 mg

c

Nicotinamide

50 mmol

61.05 mg

d

Magnesium chloride

25 mmol

50.8 mg

e

Glucose-6-phosphate dehydrogenase

2 units

20 units

f

Phosphate buffer pH 7.6 (0.5M)

to 3 ml

to 30 ml

4. Metabolism of Hexobarbitone sodium in liver homogenate 1. Prepare an aqueous solution of Hexobarbitone sodium at 1mg/ml. Salt is freely soluble. 2. Using 25 ml Erlenmeyer flasks (wide mouthed conicals) prepare a series of solutions as given in table 2. Prepare these solutions from ice cold reagents and keep the mixtures ice cold until starting the experiment, as preliminary incubation can inactivate microsomal enzymes. The cofactor solution must contain constituents of table 1 except (e). Table 2. Solutions required to demonstrate Hexobarbitone metabolism Flask No 1 2 3 4 5 6 7 8

Hexobarbitone solution (1mg/ml) (ml) 1 1 1 1 1 1

Distilled water (ml) 1 1 -

Cofactor solution (ml) 3 3 3 3 3 3 3 3

KCl/buffer (ml) 2 2 -

10,000g supernatant 2 2 2 2 2 2

0

3. Incubate flasks 1 to 6 with gentle shaking for 30 min at 37 C; retain flasks 7 and 8 on ice. 4.At the end of 30 minutes cool flasks 1 to 6 upto 40C. Store all eight flasks if necessary at -180C. 5.Assay of Hexobarbitone 1. Prepare distilled water blanks and hexobarbitone sodium standards in water at 10, 20, 50, 100 and 200 mg/ml from a 1mg/ml solution prepared. 2. Add 0.5 ml aliquots of these preparations to screw capped tubes containing 1 ml of 0.5 M citrate buffer pH 5.5 saturated with NaCl and 10 ml of n-heptane containing 1.5 % amyl alcohol. 3. Shake for 10 min and centrifuge. 4. Remove the aqueous layer completely with pipettes. 5.Add 1.5 ml of the citrate buffer to the same tubes and once again shake for 10 minutes and centrifuge. 6. Transfer 8 ml of the heptane layer to a fresh tube containing 4 ml of 0.8 M phosphate buffer pH 11. 7. Shake and centrifuge. 8. Record the spectrum of the aqueous layer over the range 200-400 nm with water in the reference position.

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9. Determine values of (OD245-OD280) and compare experimental unknowns with standard curves. Hexobarbitone sodium is relatively unstable at pH 11 and delays in reading the OD values should be avoided. 6. Metabolism of Hexobarbitone by microsomes Follow the procedures in the previous sections except 0

1. Resuspend one microsomal pellet in 14 ml of 0.05 M phosphate buffer pH 7.6 at 4 C (use the Teflon pestle after decanting and discarding the storage overlay buffer). 2. Substitute cofactor solutions containing constituents (e) in table 1 in addition to other materials. 3. Use 2 ml microsomal suspension prepared in (1) above in place of the 10,000 g supernatant. Observations A. Hexobarbitone sodium metabolism in liver homogenates 1. Hexobarbitone sodium concentration at start _______ mg/ml 2.Absorbance differences for standards Concentration (mg/ml) 10 20 50 100 200

Absorbance difference

3.Absorbance differences for flasks 1 to 8 Flask No 1 2 3 4 5 6 7 8

Absorbance difference

B. Hexobarbitone sodium metabolism in microsomes 1. Hexobarbitone sodium concentration at start ______ mg/ml 2.Absorbance differences for standards Concentration (mg/ml) 10 20 50 100 200

Absorbance difference

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3.Absorbance differences for flasks 1 to 8 Flask No 1 2 3 4 5 6 7 8

Absorbance difference

Calculations 1. Metabolism of Hexobarbitone sodium after incubation = Initial concentration -Average concentration obtained for flask 3 & 4. 2. Metabolism of Hexobarbitone sodium without incubation = Initial concentration -Average concentration obtained for flask 7 & 8. Results 1. Metabolism of Hexobarbitone sodium with and without incubation was found to be ______mg/ml and ______ mg/ml respectively. 2. Metabolism of Hexobarbitone sodium in liver microsomes with and without incubation was found to be ______mg/ml and ______ mg/ml respectively. Conclusion It can be concluded that metabolism of Hexobarbitone sodium takes place by oxidation in both liver homogenate and microsomes. Applications 1. This experiment allow us to study in vitro metabolism of drugs. 2. The activity of enzymes most concerned in metabolism of particular drug can be studied. 3. Metabolism of drug by liver homogenate and microsomes can be separately studied. 4. Various metabolic processes viz. oxidation, reduction etc. can be studied. 5. The enzyme induction and inhibition due to drugs can be studied in vitro. Questions 1. Give the various enzymes involved in metabolism of drugs. 2. Give the significance of metabolism. 3. Explain the process of enzyme induction and inhibition. Exercise Study the metabolism of thiopental sodium by this method.

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Experiment 18 Effect of food on metabolism of drug Aim To study the effect of food products on metabolism of drugs. Learning objectives 1. To understand the metabolism of drug. 2. To understand the effect of food on metabolism of Thiopental. Theory Majority of drugs undergo metabolic change. Metabolism usually results in increased polarity, and this reduces the degree of tissue penetration and increase the rate of urinary excretion. Metabolism commonly results in reduction of pharmacological potency, but this is not inevitable, and there are cases of metabolism leading to enhancement or induction of activity. Metabolism often occurs in two phases. Phase I is usually an oxidation, a reduction, or a hydrolysis. Phase II is always a synthetic reaction, such as a conjugation. Phase I often leads to introduction of a substituent, which can then combine with conjugation group. Phase II is the major mechanism for increasing polarity, as Phase I is often an intermediate step making increase in polarity possible. The commonest combination is aromatic hydroxylation followed by conjugation with glucuronic acid. Major site of metabolism of exogenous drugs is liver. A wide variety of compounds is oxidized by a non specific mixed function oxidase system found in the microsomal fraction of the liver homogenate. Endogenous drugs tend to depend on enzymes of higher specificity for their metabolism, located more specifically at places relevant to their sites of action. Principle Biotransformation of drugs is primarily carried out by mixed-function oxidase system localized in the endoplasmic reticulum of the liver. The relationship between the nutritional status of an animal and the ability to metabolize drugs is well established. The protein rich diet induces cytochrome-p-450 (CYP), a metabolizing enzyme leading to increased metabolism of drugs. Deficiency of various nutrients such as calcium, zinc, iron, vitamin C, thiamin, or lipid have been shown to alter the rate of drug metabolism. The rate of metabolism in liver from rats fed with gluten diet (normal diet) is significantly lower than that rats fed with casein diet. There is a direct relation between metabolism of thiopental and sleeping time. As drug metabolism is reduced by feeding gluten diet, more thiopental is available for action and hence sleeping time is increased while rats fed with casein diet show increased metabolism of thiopental sodium thus decreasing seeping time. Drug concentration is relatively lower in the animals fed with gluten than in the corresponding groups pair-fed with casein. The effective dietary variable is probably the imbalance and/or deficiency of amino acids in gluten. Either amino acid imbalance or deficiency or both might be hindering the de novo synthesis of microsomal enzymes or of some cofactors or components of the mixed function oxidase system for drug metabolism.

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Thiopental is an ultra short acting barbiturate which produces sleep in mice within 15-20 seconds and it regenerates within 10-20 mins. Elimination half life is 7-12 h and is metabolized by hepatic metabolism. Requirements Apparatus: Righting reflex chamber, tuberculin syringe. Chemicals: Food products (gluten diet, casein diet), thiopental injection, water for injection I.P. Animal: Mice. Procedure 1. Select twelve mice weighing around 30-50 gm for the study after approval fromAnimal Ethics Committee. 2. Keep the animals in controlled laboratory condition for 2 days. 3. Divide animals into two groups, six each in a group. 4. Group I is fed with gluten diet and group II with casein diet for 5 days. 5.After 5 days, weigh the animals and note their weights. 6. Thiopental in saline is injected intraperitoneally at a dose of 3-5 mg/kg body weight to the mice of both groups. 7. Record the time between the loss and the recovery of righting reflex, after Thiopental administration. Observations Table 1. Weight gain in group I and II Animal No

Group I Initial Wt

th

Wt on 5 day

Group II Gain in Wt

Initial Wt

Wt on 5th day

1 2 3 4 5 6 Mean SD

Table 2. Sleeping time observed after Thiopental injection Animal No 1 2 3 4 5 6 Mean SD

Group I

Sleeping time Group II

Gain in Wt

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Results 1. Mean weight of mice with casein diet is __________g while mean weight of mice fed with glutein diet is _____ g. 2. Mean Thiopental sleeping time found for mice fed with casein diet is ______min while mean sleeping time for mice fed with gluten diet is ______min. The mean Thiopental sleeping time for mice fed with gluten is significantly longer than for mice with casein diet. Conclusion It can be concluded that mice fed with casein diet (protein diet) show significantly more body weight and increased metabolism of Thiopental than the mice fed with gluten diet (normal diet). Applications 1. The effect of food products on metabolism of various drugs can be studied. 2. Metabolism of drug in vivo can be studied. Questions 1. Give various steps involved in process of metabolism. 2. Why protein rich diet increases the metabolism of drug? Exercise Study the effect of deficiency of calcium, zinc, iron and lipid on metabolism of Thiopental sodium

SECTION 5

Excretion of drugs Experiment 19 Urinary excretion of drug Aim To study the urinary excretion of Acetylsalicylic acid (Aspirin) in healthy, male volunteers after its oral administration. Learning objective To determine urinary excretion of aspirin in free and conjugated forms. Theory Acetylsalicylic acid, most commonly known asAspirin is a member of salicylate group of compounds. It is a non steroidal anti-inflammatory drug that possesses analgesic, anti- inflammatory and antipyretic properties.Aspirin is hydrolyzed in stomach and in blood to salicylic acid and acetic acid. Salicylates are excreted mainly by kidney as salicyluric acid (75%), free salicylic acid (10%), salicylic phenol (10%) and acyl (5%) glucuronides, and gentisic acid (> K and the value of second -Kat

exponential e retains some finite value.At this time, the equation 2 reduces to: s -K t C = Xe

…3

Converting equation 3 into logarithmic form s Kt log C = log X 2.303 …4 s Where C represents the back extrapolated plasma concentration values. A plot of log C verses t yields a biexponential curve with a terminal linear phase having slope K/2.303. Back extrapolation of this straight line to time zero yields y intercept equal to log (X). Substraction of true plasma concentration values i.e. equation 2 from the extrapolated plasma concentration values i.e. equation 3 yields a series of residual concentration values Cr: s (C - C ) = Cr = Xe - K t …5 a

In log form, the equation is log Cr = log A -

K at 2.303

…6

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Aplot of log Cr versus t yields a straight line with slope Ka/2.303 and y intercept log X.Absorption halflife can then be computed from Ka using the relation 0.693/Ka. Thus, the method of residuals enables resolution of the biexponential plasma level-time curve into its two exponential components. Ideally, the extrapolated and the residual lines intersect each other on y axis i.e. at time t = zero and there is no lag in absorption. If an intersection occurs at a time greater than zero, it indicates time lag. The method is best suited for drugs which are rapidly and completely absorbed and follow one compartment kinetics.

Figure 1. Semilog plot of plasma concentration versus time profile after oral administration of a drug When using the method of residuals, a minimum of three points should be used to define the straight line. Data points occurring shortly after tmax may not be accurate, because drug absorption is still continuing at that time. Because this portion of the curve represents the post-absorption phase, only data points from the elimination phase should be used to define the rate of drug absorption as a first order process. Prerequisite 1. Concept of one compartment extravascular administration. Requirements Regular graph paper, semilog graph paper, calculator, pencil. Given data The following data were obtained when a 500 mg of an antibiotic was given orally. Assume 100% of the administered dose was absorbed. Table 1. Plasma data obtained after oral administration of 500 mg dose of drug Time h Conc. mg/ml

1

2

3

4

5

6

8

16

18

20

26.501

36.091

37.512

36.055

32.924

29.413

22.784

7.571

5.734

4.343

Calculate absorption rate constant.

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127

Solution 1. Plot the drug concentration (C) versus time on semilog paper with the concentration values on logarithmic axis. 2. Extrapolate elimination phase back upto Y axis. 3. Denote concentration from extrapolated line as C . 4. Obtain C concentrations for time (t1, t2, ….tn) versus concentration (C1, C2,….Cn) from back extrapolated line ( C1, C 2,......, Cn ). 5. Obtain residual concentrations (Cr) by subtracting C from C with respect to time. 6. Plot a residual concentration (Cr) versus time on same semilog paper. 7. Determine slope of residual line. Table 2. Estimation of residual concentrations Time (h) 1 2 3 4 5 6 8 16 18 20

PDC (mg/ml) C 26.501 36.091 37.512 36.055 32.924 29.413 22.784 7.571 5.734 4.343

Extrapolated PDC C

Residual PDC

Cr = C - C

PDC: Plasma drug concentration Calculations 1.Absorption rate constant (Ka) Calculate absorption rate constant by using slope of residual line. s s log C 2 - log C1 Slope = t 2 - t1 K a = -(Slope ´ 2.303)

2.Absorption half life Calculate absorption half life by using following formula

t

1/ 2

=

0.693 Ka

Alternatively, half life can be determined graphically.

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Results By using method of residuals absorption rate constant (Ka) and absorption half life (t1/2) for given data set were found to be ________ h-1 and __________ h respectively. Conclusion It can be concluded that absorption rate constant and absorption half life (t1/2) can be estimated using method of residuals. Applications 1. Determination of absorption rate constant from oral absorption data is possible. 2. Method of residuals is easy method of calculating absorption rate constant. Questions 1. What is absorption rate constant? 2. How absorption rate constant can be determined by method of residuals? Exercise 1. The following data were obtained when a 500 mg of an antibiotic was given orally. Assume 100% of the administered dose was absorbed. Time h Conc. mg/l

0.25

0.5

0.75

1

1.5

2

3

4

5

6

7

1.91

2.98

3.54

3.80

3.84

3.62

3.04

2.49

2.04

1.67

1.37

Calculate absorption rate constant and absorption half life. 2. Plasma samples from a patient were collected after an oral bolus dose of 10 mg of a new benzodiazepine solution and the following data was obtained. Time h Conc. ng/ml

0.25

0.50

0.75

1

2

4

6

10

14

20

2.85

5.43

7.75

9.84

16.20

22.15

23.01

19.09

13.90

7.97

Calculate absorption rate constant and absorption half life.

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129

Experiment 28 Calculation of absorption rate constant by Wagner-Nelson method Aim To calculate absorption rate constant (Ka) using Wagner-Nelson method. Learning objective To understand Wagner-Nelson method for estimating absorption rate constant (Ka). Theory Estimation of absorption rate constant: Wagner-Nelson method After a single oral dose of a drug, at any time, the amount of drug absorbed into the systemic circulation Aa, is the sum of amount of drug in the bodyAand the amount of drug eliminated from the bodyAe. Thus Aa = A + Ae …1 The amount of drug in the body is A= VC while the amount of drug eliminated at any time t can be calculated as follows Ae = KV [ AUC ]t0 …2 Substituting the values ofAandAe in equation 1 gives

Aa = VC + KV [ AUC ]t0

…3 ¥

The total amount of drug absorbed into the systemic circulation from time zero to infinity Aa can be given as

Aa¥ = VC ¥ + KV [ AUC ]¥0

…4

Since at t = ¥ , C= 0, the above equation can be reduced to

Aa¥ = KV [ AUC ]¥0

…5

The fraction of drug absorbed at any time t is given as

Aa VC + KV [ AUC ]t0 = Aa¥ KV [ AUC ]¥0

…6

Aa C + K [ AUC ]t0 = Aa¥ K [ AUC ]¥0

…7

Percent drug unabsorbed at any time is therefore é A ù é C + K [ AUC ]t0 ù % ARA = ê1 - ¥a ú100 = ê ú100 ¥ ë Aa û ë K [ AUC ]0 û

…8

The method requires collection of blood samples after a single oral dose at regular intervals of time till the entire amount of drug is eliminated from the body. K is obtained from semilog plot of C versus t. A semilog plot of percent unabsorbed (i.e percent ARA) versus t yields a straight line whose slope is Ka/2.303. If a regular

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plot of the same is a straight line, then absorption is zero order.

Ka can also be similarly estimated from urinary excretion data. The biggest disadvantage of WagnerNelson method is that is applies only to drugs with one compartment characteristics. Given data Bioavailability of phenylpropanolamine hydrochloride was studied in 24 adult male subjects. The following data represent the mean blood phenylpropanolamine hydrochloride concentrations (ng/ml) after the oral administration of a single 25 mg dose of phenylpropanolamine hydrochloride solution. Time (h) Conc (ng/ml)

0.25

0.5

0.75

1

1.5

2

3

4

6

8

12

18

24

51.33

74.05

82.91

85.11

81.76

75.51

62.98

52.32

36.08

24.88

11.83

3.88

1.27

Determine absorption rate constant and absorption half life by using Wagner-Nelson method. Solution Wagner-Nelson Method 1. Plot the semilog graph of plasma concentration versus time.

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131

2. Determine elimination rate constant from the slope of terminal part of line. 3. Plot the regular graph of plasma concentration versus time and determine [ AUC ]t0 by trapezoidal rule. t Determine cumulative [ AUC ]0. t t 4. Determine K [ AUC ]0 by multiplying each [ AUC ]0 by K. ¥ 5. Find [ AUC ]0 by adding up all theAUC pieces from zero to infinity. 6. Determine the fraction unabsorbed value corresponding to each time point t by using observation table. 7. Plot fraction unabsorbed versus time on semilog paper. 8. Determine slope of line. Observation table for determination of fraction of drug unabsorbed Time

C

0.25 0.5 0.75 1 1.5 2 3 4 6 8 12 18 24

51.33 74.05 82.91 85.11 81.76 75.51 62.98 52.32 36.08 24.88 11.83 3.88 1.27

[ AUC ]t0

Cumulative

K x Cum

[ AUC ]t0

[ AUC ]t0

C + K x Cum

[ AUC ]t0

(amount absorbed)

Ab/Ab¥ (Fraction absorbed)

¥

Consider (C + K x Cum [ AUC ]024 ) value as amount absorbed at infinity (Ab ) Calculations 1. Determination of absorption rate constant Determine totalAUC by the trapezoidal rule. ( A U C ) tt nn -1 =

C n -1 + C n ( t n - t n -1 ) 2

( A U C ) t¥ =

C la s t K

Determine absorption rate constant. 2.Absorption half life Determine slope of line and calculate absorption half life by using formula 0.693 t1/ 2 = K a Alternatively, half life can be determined graphically.

1 ( Ab/A b¥ ) (Fraction unabsorbed)

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Result By using Wagner-Nelson absorption rate constant (Ka) and absorption half life (t1/2) for given data set -1

was found to be ______h and ________h respectively. Conclusion It can be concluded that absorption rate constant and absorption half life (t1/2) can be estimated using Wagner-Nelson method. Application This method allows calculation of absorption rate constant (Ka). Questions 1. How absorption rate constant can be determined by Wagner Nelson method? 2. What are the disadvantages of this method? Exercise The following data were obtained when a 100 mg of an antibiotic was given orally. Assume 100% of the administered dose was absorbed. Time h Conc. mg/ml

0.25

0.5

1

2

3

4

6

8

10

12

1.6

2.7

3.7

3.5

2.7

2

1.02

0.49

0.26

0.12

Calculate absorption rate constant and absorption half life.

Pharmacokinetics

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133

Experiment 29 Calculation of various pharmacokinetic parameters after extravascular administration Aim To calculate various pharmacokinetic parameters after extravascular administration of drug (one compartment model). Theory Administration of drug dose by an extravascular route involves passage of the drug by absorption through a biological membrane. The plasma profile obtained following extravascular administration of a drug is different from plasma profile of same drug obtained after the drug administered as a rapid intravenous bolus injection because the entire dose of administered drug is not absorbed all at once.

Absorption phase

Elimination phase

Time (h)

For a drug that enters the body by a first order absorption process, gets distributed in the body according to one-compartment kinetics and is eliminated by a first order process, the model can be depicted as follows Dose

Ka (absorption) First order

Central Compartment

Blood and body tissue

K (Elimination) First order

After extravascular administration, the rate of change in the amount of drug in the body dA/dt is the difference between the rate of input (absorption), dAa/dt and rate of output (elimination), dAe/dt. Amount of drug in the body = Rate of absorption - Rate of elimination

dA dAa dAe = dt dt dt

…1

The differential equation that follows relates changes in drug concentration in the blood with time to the absorption and the elimination rates …2 dA

dt

= K a Aa - KA

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where dA/dt = rate of change of amount of drug in the blood; A = amount of drug in the blood at time t; Aa = amount of absorbable drug at the absorption site at time t; Ka = absorption rate constant; K elimination rate constant, respectively, KaAa = first order rate of absorption and KA= first-order rate of elimination. Integration of equation 2 yields

A=

FK a A0 (e - Kt - e - Kat ) (K a - K )

…3

Where A = amount of drug in the body at time t; A0 = amount of drug at the site of administration at t=0 (the administered dose), F = fraction of drug absorbed. Equation 3 shows that the amount of drug in the body or blood follows a biexponential profile, first rising and then declining. For orally or extravascularly administered drugs, generally Ka>>K; therefore, the rising portion of the graph denotes the absorption phase. If K>>Ka (perhaps indicating a dissolution rate-limited absorption) the exact opposite will hold true. Converting equation 3 into concentration form, as V=A/C

C=

FK a A0 (e - Kt - e - Kat ) V (K a - K ) Intercept=

…4 FAoKa V (Ka- K)

Slope=

t=0

-K 2.303

Time (h)

Determination of elimination rate constant (K) This parameter can be estimated from elimination phase of the plasma level time curve profile. For most drugs administered extravascularly, absorption rate is significantly greater than the elimination rate i.e. Kat>>Kt. Hence, one can say that e approaches to zero much faster than does e-Kt . At this stage, when absorption is complete, the change in plasma concentration is dependent only on elimination rate and equation 4 becomes -Kat

C=

FK a A0 e - Kt V (Ka - K )

...5

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135

Transforming into log form, the equation 5 becomes:

log C = log

FK a A0 Kt V ( K a - K ) 2.303

…6

Aplot of C versus t yields a straight line with slope K/2.303. Elimination half life One can estimate elimination half life form elimination rate constant.

t1/ 2 =

0.693 K

…7

Apparent volume of distribution For a drug administered by oral, or any other extravascular route of administration, the apparent volume of distribution cannot be calculated from plasma drug concentration data alone. The reason is that the value of F (the fraction of administered dose that reaches the general circulation) is not known. From equation 6 we get,

Intercept =

FK a A0 V (Ka - K )

…8 In the absence of data for the fraction of administered dose that reaches the general circulation, the best one can do is to obtain the ratio of V/F:

K a A0 V 1 = ( ) F ( K a - K ) intercept

…9

Time of maximum drug concentration, peak time (tmax) The peak time (tmax) is the time at which the body displays the maximum plasma concentration, (Cmax). It occurs when the rate of absorption is equal to the rate of elimination. At the peak time, therefore, KaAa = KA. The rate of change in plasma drug concentration dc/dt = zero. This rate can be obtained by differentiating following equation 4.

FK a A0 dC = ( - Ke - Kt + K a e - Kat ) dt V ( K a - K ) On simplifying, above equation becomes:

Ke - Kt = K a e - Kat Converting into logarithmic form,

log K -

Ka Kt = log K a 2.303 2.303

…10

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When t is tmax, rearrangement of above equation yields

t max =

2.303 log( K a / K ) Ka - K

...11 Equation 11 indicates that peak time depends on, or is influenced by, only the absorption and elimination rate constants; therefore, any factor that influences the absorption and the elimination rate constants will influence the peak time value; however, the peak time is always independent of the administered dose of a drug. Maximum (peak) plasma concentration (Cmax) There are three methods available for determining peak plasma concentration Cmax. Two are given here: Method 1. Peak plasma concentration obtained from the graph of plasma concentration versus time. Method 2. Peak plasma concentration can also be obtained by using an equation 4,

C=

FK a A0 (e - Kt - e - Kat ) V (K a - K )

If tmax is substituted for t then

Cmax =

FK a A0 (e -Kt max - e -Kat max ) V (K a - K )

…12

Where,

FA 0 K a = Intercept V (Ka - K ) Obtain intercept by plotting plasma concentration versus time profile on semilog paper. Given Data The bioavailability of phenylpropanolamine hydrochloride was studied in 24 adult male subjects. The following data represent the mean blood phenylpropanolamine hydrochloride concentrations (ng/ml) after the oral administration of a single 25 mg dose of phenylpropanolamine hydrochloride solution. Plasma concentration time profile of phenylpropanolamine hydrochloride is given below: Time h Conc ng/ml

0.25

0.5

0.75

1

1.5

2

3

4

6

8

12

18

24

51.33

74.05

82.91

85.11

81.76

75.51

62.98

52.32

36.08

24.88

11.83

3.88

1.27

1. Determine elimination rate constant and elimination half life. 2. Determine totalAUC. 3. Determine Cmax and Tmax.

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Solution Table 1. Determination ofAUC Time h 0.25 0.5 0.75 1 1.5 2 3 4 6 8 12 18 24

C mg/ml 51.33 74.05 82.91 85.11 81.76 75.51 62.98 52.32 36.08 24.88 11.83 3.88 1.27

Segment

Cn-1 + Cn 2

tn-tn-1

AUC

A B C D E F G H I J K L M AUC0-24 Clast/K Total AUC= AUC 0-24 + Clast/K

Table 2. Determination of residual concentrations Time h 0.25 0.5 0.75 1 1.5 2 3 4 6 8 12 18 24

Conentration (mg/ml) 51.33 74.05 82.91 85.11 81.76 75.51 62.98 52.32 36.08 24.88 11.83 3.88 1.27

Extrapolated C

Residual Conc. Cr = C - C

Calculations 1. Elimination rate constant and elimination half life Plot a graph of decline in plasma concentration versus time on semilog paper and determine slope of line.

K = - ( Slope ´ 2 . 303 )

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2.Area under curve Plot a graph of plasma concentration versus corresponding time on simple graph paper and prepare segmentsAto M. C1 + C 0 AUC0-1 = ´ (t1 - t 0 ) 2

AUC0-¥ = AUC0-0.25 + AUC0.25-0.5 + ..... + AUC18- 24 +

C24 K

3.Absorption rate constant (Ka) Calculate absorption rate constant by using slope of line. s s log C 2 - log C1 Slope = t 2 - t1 K a = -( Slope ´ 2.303)

4.Absorption half life Calculate absorption half life by using following formula

t

1/ 2

=

0.693 Ka

5. Determination of tmax and Cmax

I=

FAoKa V (Ka- K) Cmax Elimination phase

Slope=

Absorption phase

tmax Time (h)

-K 2.303

Section 6

t max =

Pharmacokinetics

139

2.303 log( K a / K ) Ka - K

Cmax =

FA 0 K a (e - Kt max - e - Kat max ) V (Ka - K )

Where (FA0Ka)/V(Ka-K) is the y intercept of semilog plot of plasma concentration versus time curve. Results The various pharmacokinetic parameters calculated from given plasma data of phenylpropanolamine hydrochloride are given as follows: Sr. No.

Parameter

Result

1

Elimination half life

2

Elimination rate constant

3

Cmax

4

t max

5

Total AUC

6

Absorption rate constant

7

Absorption half life

Conclusion It can be concluded that various pharmacokinetic parameters can be calculated after extravascular administration of drug. Applications 1.Various pharmacokinetic parameters can be estimated from given plasma data after extravascular administration of drug. 2.Bioequivalence testing between various brands can be done. 3.Bioavailability of drug can be studied. Exercise After an oral administration of 500 mg drug, the following data was obtained

Time (h) Plama Conc (mg/l)

0.5 2.4

1 3.8

1.5 4.2

2 4.6

4 8.1

6 5.8

10 5.1

16 4.1

24 3.0

32 2.3

48 1.3

Calculate elimination half, elimination rate constant, Cmax, tmax, AUC, absorption rate constant and absorption half life.

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Experiment 30 Pharmacokinetic study of drug using plasma and urinary data Aim To study the pharmacokinetics of amoxicillin capsules by using plasma and urinary data. Learning objectives 1. To study the pharmacokinetics of amoxicillin in humans. 2. To estimate various pharmacokinetic parameters using plasma and urinary data. Theory 1. Calculations of pharmacokinetics ofAmoxicillin using plasma data As per previous experiment ( Expt No. 29 ). 2. Calculations of pharmacokinetic parameters using urinary data When plasma drug level-time data is not available, one can estimate pharmacokinetics of drug by using urinary excretory data. It is possible to estimate first-order elimination, excretion and absorption rate constant, fraction excreted unchanged and renal clearance of drug. If volume of distribution is known then total clearance and non renal clearance can also be calculated. Urinary drug excretion data can be used for calculation of the first order elimination rate constant. The rate of drug excretion after a single oral dose of drug is given by

dAu FK a K u A0 = - (e - Kt - e - Kat ) dt Ka - K

…1

Where dAu/dt = rate of urinary drug excretion, Ku = excretion rate constant, K = elimination rate constant, Ka = absorption rate constant and F = fraction of drug absorbed. A graph of rate of urinary drug excretion (dAu/dt) versus time yield a curve identical in appearance to the plasma level-time curve for the drug. After complete drug absorption -e-kat approaches to zero and equation is reduced to

dAu FK a K u A0 - Kt = e dt Ka - K

…2

We can rewrite equation 2 in logarithmic form as follows

log

dAu FK a K u A0 Kt = log dt Ka - K 2.303

…3

When a graph of rate of urinary drug excretion versus time (t* is midpoint of the collection period) is plotted on semilog paper yields straight line with a slope of –K/2.303. The first order elimination rate constant can be calculated from urine data by using sigma-minus method. Following equation will be utilized to determine rate constant.

ARE = ( Au¥ - Au ) =

Au¥ ( K a e- Kt - Ke- Kat ) Ka - K

…4

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141

Where, ARE = amount remaining to be excreted, K = first-order elimination rate constant, A¥ is amount excreted at infinity, Au is amount excreted at time t, Ka is first order absorption rate constant and t is the time. A plot of ARE versus t results in a biexponential curve and if (Ka>K), the slope of the terminal linear portion of the curve will define K of the drug. Intercept of equation 4 is equal to Au /(Ka-K) so it is possible to determine absorption rate constant. The absorption rate constant Ka can be estimated by method of residuals using the same data. Urinary excretion data after oral administration can also be treated according to Wagner-Nelson method to calculate Ka by construction of percent amount remaining to be absorbed (ARA) plots. The method requires urine collection for sufficiently long time to ensure accurate estimation of K but need not be collected to time infinity. The equation derived to relate %ARAwith urinary excretion rate is: é dA / dt + KAu ù A % ARA = (1 - ¥a )100 = ê1 - u ú100 …5 Aa KAu¥ ë û Where ARA = amount remaining to be absorbed, Aa = amount of drug absorbed in to systemic circulation at time t, Aa ¥= total amount of drug absorbed into the systemic circulation from time zero to infinity, dAu/dt = rate of urinary drug excretion (proportional to the amount of drug in body) and K = elimination rate constant. A semilog plot of %ARAversus t yields a straight line with slope -Ka/2.303. u

Requirements Glassware: Petri plates, micropipettes, test tubes etc. Chemicals: Pure amoxicillin, nutrient agar etc. Instrument:Autoclave, UV Spectrophotometer, centrifuge etc. Procedure Assay of amoxicillin 1. Maintain Streptococcus pneumoniae cultures in nutrient broth. Maintain stock cultures of Streptococcus 0 pneumoniae from isolates on nutrient agar plates and store at 4 C. Before each assay, run subculture into nutrient broth. Give at least two transfers in nutrient broth before the test assay. 0

2.Keep the nutrient broth cultures at 37 C for 24 h. Measure absorbance of broth cultures at 630 nm in a spectrophotometer on the day of the assay. Select the culture showing an optical density ranging from 0.275 to 0.325. 3. Dissolve 11.5 gm of nutrient agar in 500 ml of distilled water and adjust pH to 7.0 using 0.4 N sodium hydroxide solution. Autoclave nutrient agar for 20 min at 15 lbs pressure. Then, bring temperature of agar 0 solution to 37 to 40 C and then seed the organism kept in nutrient broth (0.4 ml culture/500 ml solution). Then pour this into sterile petri plates and wait till it gets solidify. Finally with the help of sterile borer cut the wells of 2 mm. 4. Prepare 1 mg/ml solution ofAmoxicillin by dissolving it in minimal amount of suitable sterile solvent. 5. Collect blank plasma and urine samples from volunteers, centrifuge and remove supernatant. Dilute

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supernatant with distilled water and prepare standard samples of Amoxicillin in concentration range of 0-12 mg/ml in quadruplicate. 0

6. Load ten micro liters of the standard solutions of drug into the wells and keep the petri plates at 4-10 C for 1-2 h and incubate the plates at 370C for 24 h. 7. Measure the zone of inhibition and its diameter. Plot the graph of log concentration of Amoxicillin versus diameter of zone of inhibition and determine unknown concentration of drug from regression line. Study participants 1. Take approval of the study protocol from Institutional Ethics Committee and obtain written informed consent from all the volunteers. 2. Select six healthy male volunteers with age ranging between 20-30 years and a mean body weight ranging between 45-69 kg. 3. Assess the volunteers as healthy on the basis of medical history, hepatic and renal function tests. All subjects should be non-smokers and should not ingest any medicine two weeks before or during the study for any ailment. 4. Exclude volunteers with history of penicillin allergy or who takes alcohol. 5. Instruct subjects to avoid antibiotics and beverages for 14 days before the study. Study design 1. After an overnight fast, administer amoxicillin 500 mg on an empty stomach after emptying the bladder with 180 ml drinking water. 2.Allow food, 4 h after ingestion of amoxicillin. Sample collection and processing 1. Collect blood samples (5 ml) from a peripheral vein before drug administration and then at 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 8, and 12 hours after dosing into heparinised tubes. 2. Centrifuge collected samples within 10 minutes at 3000 rpm for 15 min and separate plasma. Store plasma samples at -400C prior to analysis. 3. Collect urine samples prior to drug administration considered as blank sample and then in block samples at 0-2, 2-4, 4-6, 6-8, 8-10 and 10-12 h post dosing. 4. Measure the volume of urine, collected during each period from each volunteer. Collect the urine samples in coded eppendorf and store at -400C until further analysis. Pharmacokinetic analysis: Plasma data Assume that Amoxicillin follows one compartment open model with first order absorption and calculate pharmacokinetic parameters as given in calculations. Pharmacokinetic analysis: Urinary excretion data 1. Plot the graph of percent cumulative amount ofAmoxicillin excreted versus time. 2. Plot the graph of excretion rate ofAmoxicillin versus mid point of urine collection period on semilog paper. 3. Plot the graph of amount ofAmoxicillin to be excreted ( Au¥ - Au ) versus mid point of urine collection period on semilog paper. 4. Calculate pharmacokinetic parameters as given in calculations.

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143

Observations Table 1. Calibration curve ofAmoxicillin Concentration (mg/ml) 1 2 4 6 8 10 12 Slope Intercept

Log C

Zone of Inhibition (mm)

Table 2. Plasma data obtained after oral administration of 500 mgAmoxicillin Time (h)

Plasma Concentration mg/ml S1 S2 S3 S4 S5

Average (SD) S6

0.25 0.5 0.75 1 1.5 2 4 8 12 S-Subject, SD-standard deviation

Table 3. Urinary excretion data obtained after oral administration of 500 mgAmoxicillin Time of urine collection (h) 0-2 2-4 4-6 6-8 8-10 10-12

Time interval of urine collection t (h) 2 2 2 2 2 2

Mid point of urine collection period t*

Urine volume (ml)

Conc (mg/ml)

Amount excreted Au (mg)

Au/t ER (mg/min)

Cumulative Au (mg)

1 3 5 7 9 11 ¥

ER- excretion rate;Au- amount of drug excreted in mg;Au CumulativeAu at time 12h.

ARE (A¥u - A u)

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Calculations Plasma data 1. Plasma concentration ofAmoxicillin (mg/ml) Plot the graph of absorbance versus log concentration and determine slope and intercept. Y= m X + c Where, Y = absorbance, m= slope, X = log concentration (mg/ml), c = intercept. 2. Peak amoxicillin concentration (Cmax, mg/ml) in plasma and time to peak concentration (tmax, h) Plot the graph of plasma concentration versus time and determine peak concentration (Cmax) and time to peak (tmax) from the graph. 3. Elimination half life (t½, h) and Elimination rate constant (K, /h) Plot the graph of plasma concentration of Amoxicillin versus time on semilog paper and determine slope of decline in plasma concentration. Elimination rate constant can be determined using following equation K = -2.303 (slope) Elimination half life can be determined using following formula t1/2 = 0.693/K 4. Area under curve (AUC, mg/h/ml) DetermineAUC from 0-t by using trapezoidal rule. C1 + C 0 AUC0-1 = + (t1 - t 0 ) 2

AUC0-t = AUC0- 25 + .......... + AUC8-12 Determine totalAUC by using following formula

AUC12-¥ =

C12 K

TotalAUC =AUC0-12 +AUC12- ¥ Urinary data 1. Concentration of excreted drug in urine (mg/ml) Plot the graph of absorbance versus log concentration and determine slope and intercept using formula Y= m X + c Where, Y= absorbance, m= slope, X= log concentration (mg/ml), c= intercept. 2. Amount of drug excreted in urine,Au (mg/ml): Au = Conc. of drug in urine (mg) x Total volume of urine voided (ml) 3. Excretion rate Excretion rate =Amount excreted (Au) / Time interval of urine collection

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145

4. Elimination rate constant (sigma-minus method) Plot the graph of amount remaining to be excreted versus mid point of urine collection period on semilog paper. Determine slope of terminal linear portion (last three to four points) of the curve and determine elimination rate constant. Slope=(-K)/2.303 5. Excretion rate Excretion rate =Amount excreted (Au) / Time interval of urine collection 6. Elimination half life Determine elimination half life using following formula t1/2 = 0.693/K Result 1. The concentration ofAmoxicillin at 1.5 h was found to be ________mg/ml. 2. The pharmacokinetic parameters ofAmoxicillin were found as follows: Parameter

Plasma data

Urinary data

Elimination rate constant Elimination half life Total AUC

-

Volume of distribution

-

Conclusion It can be concluded that the pharmacokinetic parameters can be calculated from both plasma and urinary excretion data. Applications 1. Estimation of pharmacokinetic parameters using urinary excretion data is possible. 2. Comparison of pharmacokinetic parameters obtained by plasma and urinary data is possible. 3. Bioequivalence testing of different brands can be done. Questions 1. Give two methods for calculating various pharmacokinetic parameters using urinary excretion data. 2. What is tmax and Cmax? Exercise Study pharmacokinetics of Ofloxacin by both plasma and urinary excretion data.

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Experiment 31 Pharmacokinetic study of drug using salivary drug concentration Aim To study the pharmacokinetics of ofloxacin tablets using salivary drug concentration. Learning objectives 1. To study the salivary pharmacokinetics of ofloxacin in humans. 2. To estimate various pharmacokinetic parameters using salivary drug concentration. Theory Determination of drug concentrations in the saliva has gained widespread acceptance in a variety of settings. Estimation of drugs in the saliva has been employed for therapeutic drug monitoring and for calculation of pharmacokinetic variables. The rational use of such determinations can provide knowledge of patient-specific pharmacokinetic parameters leading to improved therapy. Saliva can serve as an alternative body fluid for pharmacokinetic investigations. It can be collected with minimal patient discomfort and can be easily obtained on multiple occasions. It is particularly suitable for investigations in geriatrics and pediatrics. Pharmacokinetic studies have shown that Ofloxacin penetrates into saliva and its concentration correlates well with serum levels. Therefore, once the salivary concentration of Ofloxacin is determined, the pharmacokinetic data can be generated by the following equations 1. Elimination rate constant (K) can be determined from the slope of concentration time plot Slope=(-K)/2.303 2. Elimination half life (t1/2) = 0.693/K 3. TotalAUC C AUC0-¥ = AUC0-0.5 + AUC0.5-1 + ..... + AUC5-6 + AUC6-8 + last K Principle Ofloxacin is a synthetic fluorinated carboxy quinolone which is reported to have a broad antimicrobial spectrum. Pharmacological studies of ofloxacin require sensitive and specific methods for the estimation of the drug in biological fluids such as blood, saliva and urine. HPLC methods using fluorescence detector are available but they involve a series of steps for preparation of the sample before analysis. More over, all the laboratories cannot afford to have such sophisticated equipment. Since E.coli is sensitive to Ofloxacin, it can be used as test organism for the microbiological assay. The zone of inhibition exhibited by Ofloxacin against E.coli is proportional to the concentration of the drug present. Hence the estimation of Ofloxacin in saliva can be done by microbial method. Here, simple spectrophotometric method is also discussed for estimation of Ofloxacin in saliva. Prerequisite 1. Basic pharmacokinetics. 2. Plotting of graphs

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Requirements Glassware: Petri plates, pipettes, test tubes etc. Chemicals: Pure Ofloxacin, nutrient broth, nutrient agar etc. Instrument:Autoclave, UV Spectrophotometer. Procedure Assay of Ofloxacin Method I (Spectrophotometric estimation) 1. Collect blank saliva samples from volunteers, centrifuge and remove supernatant. Dilute supernatant with distilled water and prepare standard samples of Ofloxacin in concentration range of 0-16 mg/ml. 2. Determine the absorbance with UV spectrophotometer at 283 nm. Determine the unknown concentration of Ofloxacin in saliva from the calibration curve. Method II (Microbiological assay) 1. Maintain E. coli cultures in nutrient broth. Maintain stock cultures of E. coli from isolates on nutrient agar 0 plates and store at 4 C. Before each assay, run subculture onto nutrient broth. Give at least two transfers in nutrient broth before the test assay. 0

2. Keep the nutrient broth cultures at 37 C for 24 h. Measure the absorbance of broth cultures at 630 nm in a spectrophotometer on the day of the assay. Select the culture showing an optical density ranging from 0.275 to 0.325. 3. Dissolve 11.5 gm of nutrient agar in 500 ml of distilled water and adjust pH to 7.0 using 0.4 N sodium hydroxide solution. Autoclave nutrient agar for 20 min at 15 lbs pressure. Then, bring temperature of agar 0 solution to 37 - 40 C and then seed the organism kept in nutrient broth (0.4 ml culture/500 ml solution). Pour this solution into sterile petri plates and wait till it gets solidify. Finally with the help of sterile borer cut the wells of 2 mm. 4. Prepare 1 mg/ml solution of Ofloxacin by dissolving it in minimal amount of sterile 0.1 N hydrochloric acid solution. 5. Collect blank saliva samples from volunteers, centrifuge and remove supernatant. Dilute supernatant with distilled water and prepare standard samples of Ofloxacin in concentration range of 0-16 mg/ml in quadruplicate. 6. Load ten micro liters of the standard solutions of drug onto the wells and keep the petri plates at 4-100C for 1-2 h and incubate the plates at 370C for 24 h. 7. Measure the zone of inhibition and its diameter. Plot the graph of log concentration of Ofloxacin versus diameter of zone of inhibition and determine unknown concentration of drug from regression line. Study design 1. Take approval of the study protocol from Institutional Ethics Committee and obtain written informed consent from all the volunteers. 2. Select six healthy male volunteers with age ranging between 20-30 years and a mean body weight ranging between 45-69 kg. 3. Assess the volunteers as healthy on the basis of medical history, hepatic and renal function tests. All subjects

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should be non-smokers and should not ingest any medicine one week before or during the study for any ailment. 4. After an overnight fast, administer Ofloxacin 200 mg on an empty stomach after emptying the bladder with 180 ml drinking water. 5. On each occasion, collect 2-3 ml of saliva samples in clean test tubes at 0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 and 8.0 h period after oral administration of the drug. 6. Instruct the volunteers participated in the study to wash their oral cavity and use a piece of unsweetened, unflavored chewing gum to stimulate salivary secretion. 0

7. Collect saliva sample from volunteers, centrifuge and separate the supernatant and store the samples at -20 C till assayed. Pharmacokinetic analysis 1.Assume that Ofloxacin follows one compartment open model with first order absorption. 2. Plot the regular graph of salivary concentration of drug versus time. Fit data into bell shape curve and obtain peak Ofloxacin concentration (Cmax) in saliva and time to peak (tmax) concentration from the graph. 3. Calculate various pharmacokinetic parameters as per given in calculations section. Observations Table 1. Calibration curve of Ofloxacin Spectrophotometric method Concentration (C) Absorbance (mg/ml) 0.5 1 2 4 6 16 Slope Intercept

Microbial assay Log C Zone of Inhibition (mm)

Table 2. Salivary concentrations and pharmacokinetic parameters calculated for individuals Time (h)

Salivary Concentration mg/ml S1 S2 S3 S4 S5 S6

0 0.5 1.0 2.0 3.0 4.0 5.0 6.0 8.0 S-Subject, SD-standard deviation

Average (SD)

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Calculations 1. Salivary concentration of Ofloxacin (mg/ml) Plot the graph of absorbance versus concentration and determine slope and intercept. Y= m X + c Where, Y= absorbance, m = slope, X= concentration (mg/ml), c = intercept. or Plot the graph of log concentration versus diameter of zone of inhibition. Y= m X + c Where, Y = absorbance, m = slope, X = log concentration (mg/ml), c = intercept. 2. Peak Ofloxacin concentration (C , mg/ml) in saliva and time to peak concentration (t , h) Plot the graph of salivary concentration versus time and determine peak concentration (C ) and time to peak (tmax) from the graph. max

max

max

-1

3. Elimination half life (t½, h) and Elimination rate constant (K, h ) Plot the graph of salivary concentration of Ofloxacin versus time on semilog paper and determine slope of decline in salivary concentration. Elimination rate constant can be determined using following equation K = -2.303 (slope) Elimination half life can be determined using following formula t1/2 = 0.693/K 4.Area under curve (AUC, mg/h/ml) DetermineAUC from 0-t by using trapezoidal rule.

AUC0-1 =

C1 + C 0 + (t1 - t 0 ) 2

AUC0-8 = AUC0-0.5 + AUC0.5-1 + ........ + AUC6-8 Determine totalAUC by using following formula C AUC8-¥ = 8 K TotalAUC =AUC0-8+AUC8- ¥ Results 1. The mean salivary concentration of Ofloxacin at 8 h is found to be_______(mg/ml) . 2. The pharmacokinetic parameters of Ofloxacin found as follows: Cmax: tmax: Elimination rate constant: Absorption rate constant:

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Elimination half-life: TotalAUC: Conclusion It can be concluded that the pharmacokinetic parameters can be calculated from salivary concentration (noninvasive method) provided if the correlation between plasma to saliva concentration exists. Applications 1. Non invasive method determining drug concentration. 2. Various pharmacokinetic parameters can be calculated from salivary concentrations of drug at various time points. Questions 1. Why estimation of drug concentration has gained popularity? 2. Give the principle of microbiological assay of Ofloxacin. Exercise Estimate the salivary concentration of Theophylline.

SECTION 7

Bioavailability and Bioequivalence Experiment 32 Bioequivalence testing of drug using salivary samples Aim To study bioequivalence of Paracetamol tablets by means of salivary samples. Learning objectives 1.To understand the concepts of bioavailability and bioequivalence. 2.To study the use of saliva in estimating bioavailability of different brands of Paracetamol. 3.To estimate various pharmacokinetic parameters of Paracetamol through salivary samples. Theory The term “bioavailability” refers to the extent to which a drug/nutrient reaches its site of action or a biological fluid such as blood that has access to its site of action. “Relative bioavailability” is assessed using a reference product and “absolute bioavailability” is determined using an IV as 100%. The term “bioequivalence” refers to pharmaceutically equivalent drug products where the rates/extents of bioavailability of the actives are not significantly different under suitable test conditions. In other words, this is a comparison of two or more products with respect to their bioavailability. Bio-equivalent means that one brand or dosage form of a drug or supplement is equivalent to a reference brand or dosage form of the same drug or supplement in terms of various bioavailability parameters measured via in vivo testing in human subjects. Bio-equivalence cannot be claimed based on in vitro testing only or on the basis of animal studies only. Bio-equivalence of human drugs must be determined in humans via established measures of bioavailability. Likewise, animal drugs must be tested for bio-equivalence in the animal species for which the drug in intended. Once bio-equivalence has been established via bioavailability testing in a statistically significant manner subsequent batches of the same product are deemed bio-equivalent based on in vitro measures such as drug dissolution. There are two types of bioavailability; absolute and relative. Absolute bioavailability Absolute bioavailability is assessed by comparing the values of (AUC)¥0 and/or cumulative mass of drug excreted in the urine (Au), obtained following the administration of a drug in an extravascular dosage form and an equal dose of the same drug intravenously (intravenous bolus). From plasma data, it can be calculated by following equation

F=

( AUC0¥ ) oral DoseIV ´ ( AUC0¥ ) IV Doseoral

...1 151

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From urinary data, it can be calculated by following equation

F=

( Au¥ ) oral DoseIV ´ ( Au¥ ) IV Doseoral

...2

Relative bioavailability The ratio comparative (relative) bioavailability is assessed by comparing the bioavailability parameters derived from plasma drug concentration–time plot data and/or urinary excretion data following the administration of a drug in two different dosage forms (i.e. tablet and syrup, capsule and suspension, etc.) and/or two different extravascular routes of administration. It also compares a generic formulation with a standard formulation of the same dosage form of the same drug. From plasma data, it can be calculated by following equation

( AUC0¥ ) test Dosestd ´ ...3 ( AUC0¥ ) std Dosetest From urinary data, it can be calculated by following equation ( A¥ ) Dosestd Fr = u¥ test ´ ( Au ) std Dosetest ...4 Paracetamol (N-acetyl-p-aminophenol) has been in use as analgesic and antipyretic drug over 50 years. It is used for an effective medication for the relief of pain and fever in adults and children. It is rapidly absorbed and has a elimination half life of around 2 h. Serum concentrations between 10 to 20 mg/ml are generally considered to be therapeutically effective, while >150 mg/ml may produce hepatic necrosis. According to current Biopharmaceutical Classification System (BCS) criteria, Paracetamol is a BCS class III compound. Differences in the composition of Paracetamol formulation show differences in the rate of absorption, as in the case of tablets containing high amount of sodium bicarbonate which increases absorption of Paracetamol by an effect on gastric emptying. It is suggested that these differences are due to differences in disintegration and/or gastric emptying rates. Pharmacokinetics of Paracetamol in human is found to be affected by formulation. The present experiment examines the relative bioavailability of one generic formulation of Paracetamol tablets in comparison to the standard brand. Assessment of the bioequivalence of one locally manufactured tablet brand is undertaken with reference to standard tablet brand by generating in vivo data from saliva concentration of twelve volunteers. The use of saliva concentration data for bioavailability assessment of Paracetamol is considered feasible since the ease of collection and analysis of saliva samples besides the good correlation between saliva and plasma concentration of Paracetamol. The spectrophotometric method is applied for the determination and assessment of the bioavailability of Paracetamol using saliva samples. Fr =

Principle The spectrophotometric method is based on the reduction of iron (III) by p-aminophenol which is formed due to the hydrolysis of Paracetamol. A Prussian blue color is formed due to the reaction of iron (II) with potassium ferricyanide, whose intensity is proportional to the concentration of Paracetamol. P-aminophenol is a reducing agent due to the presence of phenolic hydroxyl and aromatic amine groups. Absorbance of colored complex can be estimated at lmax 700 nm and hence the estimation of

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Paracetamol in saliva can be done. Figure 1: Oxidation and hydrolysis of Paracetamol in spectrophotometric method NHCOCH3 +

O

NH2 3Fe(III)

OH

Paracetamol

heat

-3H+ -3e

Hcl OH

p-aminophenol

+

3Fe(II)

O

Benzoquinone

Requirements Glassware: volumetric flasks, test tubes, micropipettes, etc. Chemicals: Pure Paracetamol pure drug, different brands of Paracetamol (one standard & one generic), Na2SO4, diethyl ether, ferric sulfate, potassium ferricyanide, HCl and distilled water. Instruments: UV spectrophotometer. Procedure 1. Plotting of calibration curve 1. Preparation of standard stock solution: Prepare standard stock solution of Paracetamol (100 m g/ml, Stock I). From stock solution I, pipette out 2.5 ml into 100 ml volumetric flask and adjust volume with water to get concentration of 2.5 mg/ml. 2.Preparation of working solution: Transfer 0.25 ml of drug free saliva into 10 test tubes using micropipette. From stock solution II, pipette out 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.6 and 2 ml into test tubes. Adjust volume with water to 2.5 ml so as to get concentration in the range of 0-2 µg/ml. The final concentrations of Paracetamol in each saliva sample are 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.6 and 2 µg/ml. 3. Extraction of samples: Transfer 0.25 ml of working solution into test tube. Add 1.0 g of Na2SO4 and 10 ml of diethyl ether into it and mix thoroughly. Separate diethyl ether layer and evaporate to dryness. Reconstitute residue in 2.5 ml distilled water. 4. Color formation: Take 2.5 ml of working solution in test tube and add 0.5 ml of 1.0 M HCl solution followed by 1ml of ferric sulfate. Heat the mixture on boiling water bath for 10 min and cool it. Add 1 ml of potassium ferricyanide and if required dilute with water to make volume 5 ml. Keep solutions for 24 min and measure absorbance. 5. Measurement of absorbance: Measure absorbance at lmax 700 nm using UV Visible spectrophotometer. Plot the graph of absorbance of Paracetamol against concentration in MS Excel and determine slope and intercept. 2. Study design 1.Prepare study protocol and take approval from IEC. 2. Take informed consent from all participants involved in study. 3. Select 12 healthy volunteers and make two groups each comprising six subjects. 4. After an overnight fast, give 2 tablets (500 mg x 2) of Paracetamol brand A (standard) orally along with 150

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ml of water to group I. Similarly give brand B (generic) of Paracetamol to group II. 5. Rinse the mouth promptly with another 100 ml of water and swallow it. 6. Withheld food for a further period of 4 h to ensure complete absorption of the drug. 7. Collect 3 ml saliva samples at 0, 0.25, 0.5, 0.75, 1, 1, 1.25, 1.5 1.75, 2, 4, 6 and 8 h into a centrifuge tube. Instruct volunteers to drink water regularly during study to keep saliva flow. 3. Salivary analysis of drug 1. Centrifuge the samples at about 5000 rpm for 5 min to remove mucous and particulate matter from saliva. 0 Separate the salivary supernatant and keep in freezer at 20 C till analysis. 2. Transfer 0.25 ml of working solution into test tube. Add 1.0 g of Na2SO4 and 10 ml of diethyl ether into it and mix thoroughly. Separate diethyl ether layer and evaporate to dryness. Reconstitute residue in 2.5 ml distilled water. 3. Take 2.5 ml of working solution in test tube and add 0.5 ml of 1.0 M HCl solution followed by 1ml of ferric sulfate. Heat the mixture on boiling water bath for 10 min and cool it. Add 1 ml of potassium ferricyanide and if required dilute with water to make volume 5 ml. Keep solutions for 24 min and measure absorbance at lmax 700 nm using UV-Visible spectrophotometer. 4. Pharmacokinetic analysis: The estimation of pharmacokinetic parameters is done as per given in calculations. Observations Table 1. Plotting of calibration curve Concentration mg/ml 0.1 0.2 0.4 0.6 0.8 1 1.2 1.6 2 Slope Intercept

Absorbance

Table 2. Salivary data for Group I, who have taken standard brand (A) of Paracetamol Time (h) 0.25 0.5 0.75 1 1.25 1.5 1.75 2 4 6 8

Salivary concentration (mg/ml) A1 A2 A3 A4 A5 A6

Mean Conc (mg/ml)

SD

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Table 3. Salivary data for Group II, who have taken generic brand (B) of Paracetamol Time (h) 0.25 0.5 0.75 1 1.25 1.5 1.75 2 4 6 8

Mean Conc (mg/ml)

Salivary concentration (mg/ml) A1 A2 A3 A4 A5 A6

SD

Table 4. TotalAUC using the Trapezoidal rule for standard brand of Paracetamol (A) Time (h) 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 4 6 8

Mean Conc. (mg /ml) -

Segment

Cn -1 + Cn 2

A B C D E F G H I J K

-

tn-tn-1

AUC

0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 2 2 2

-

Table 5. TotalAUC using the Trapezoidal rule for generic brand of Paracetamol (B) Time (h) 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 4 6 8

Mean Conc. (mg /ml) -

Segment A B C D E F G H I J K

Cn -1 + Cn 2

-

tn-tn-1

AUC

0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 2 2 2

-

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Calculations 1. Concentration of drug in saliva (mg/ml) Plot the graph of absorbance versus concentration and determine slope and intercept. Y= m X + c Where, Y= absorbance, m= slope, X= concentration (mg/ml), c= intercept. 2. Elimination rate constant · Plot graph of salivary concentrations of Paracetamol versus time on a semi-logarithmic graph paper. Calculate elimination rate constant by using slope of terminal portion of line. log C 2 - log C1 Slope = t 2 - t1

K = -( Slope ´ 2.303)

3. Elimination half life Calculate elimination half life by using following formula

t

1/ 2

=

0.693 K

Alternatively, half life can be determined graphically. 4. Peak concentration and time to peak Determine mean peak salivary concentration (Cmax) and mean time to peak salivary concentration (tmax) by plotting salivary concentrations versus time on regular graph paper.

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5.Area under curve (AUC) DetermineAUC from 0 to 8 h by using trapezoidal rule. DetermineAUC from 8 to infinity by using following equation:

AUC8-¥ =

Clast K

TotalAUC is sum ofAUC from 0 to 8 h andAUC from 8 to infinity. 6. Relative bioavailability (Fr) Determine relative bioavailability by using following formula: AUCTest % Fr = ´100 AUCStd Results 1. The pharmacokinetic parameters estimated for both brands are summarized below: Pharmacokinetic parameter

Standard Brand (A) Generic Brand (B)

Elimination rate constant (h-1) Elimination half life (h) Mean maximum salivary concentration (mg/ml) Mean time to peak saliva concentration (h) AUC0-8 mg h/ml Total AUC

2. Relative bioavailability for generic Paracetamol tablet is found to be ________. Conclusion It can be concluded from this experiment that the generic brand (B) is bioequivalent / not bioequivalent to standard brand (A) of Paracetamol. Applications Bioavailability and bioequivalence studies can provide useful information regarding the drug such as: 1.Bioavailability studies provide an estimate of the fraction of the orally administered dose that is absorbed into the systemic circulation when compared to the bioavailability for a solution, suspension, or intravenous dosage form that is completely available. 2.Bioavailability studies provide other useful information that is important to establish dosage regimens and to support drug labeling, such as distribution and elimination characteristics of the drug. 3.Bioavailability studies provides indirect information regarding the presystemic and systemic metabolism of the drug and the role of transporters such as p-glycoproteins. 4.Bioavailability studies are designed to study the effect of food and other nutrients on the absorption of the drug substance. 5.Bioavailability studies provide information regarding the performance of the formulation and subsequently

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are a means to document product quality. 6. Bioequivalence studies provide a link between the pivotal and early clinical trial formulation, a link between formulation used in the pivotal clinical trial, and the stability studies, the pivotal clinical trial and the to-bemarketed drug product, and other comparisons as appropriate. 7. Bioequivalence studies are the basis for determination of the therapeutic equivalence between a pharmaceutically equivalent generic drug product and a corresponding reference listed drug. 8. Bioequivalence studies provide information on product quality and performance when there are changes in components, compositions, and method of manufacture after approval of the drug product. Questions 1. Give the significance of bioequivalence studies. 2. What is the difference between relative and absolute bioavailability. 3.Comparison of plasma concentrations (mg/ml) of antibiotic taken from 10 humans (average weight 70 kg), as related to dosage form and time are given below: Time (h) 0.5 1.0 1.5 2.0 3.0 4.0 6.0 8.0 10.0 12.0 AUC

IV solution (2 mg/kg) 5.94 5.30 4.72 4.21 3.34 2.66 1.68 1.06 0.67 0.42 29

Oral solution (10 mg/kg) 23.4 26.6 25.2 22.8 18.2 14.5 9.14 5.77 3.64 2.30 145

Oral tablet (10 mg/kg) 13.2 18.0 19.0 18.3 15.4 12.5 7.92 5.00 3.16 1.99 116

Oral capsule (10 mg/kg) 18.7 21.3 20.1 18.2 14.6 11.6 7.31 4.61 2.91 1.83 116

a. Which of the four drug products would be preferred as a reference standard for the determination of relative bioavailability? Why? b. From which oral drug product is absorbed more rapidly? c. What is the absolute bioavailability of the drug from the oral solution? d. What is the relative bioavailability of the drug from the oral tablet compared to the reference standard? e. From the data determine volume of distribution, elimination half life, elimination rate constant and total clearance. Exercise Perform the bioequivalence testing of various brands of Ofloxacin by salivary means.

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