Bioavailability and bioequivalence studies BIOAVIALABILITY STUDY BIOAVAILABILITY STUDIES

Bioavailability and bioequivalence studies

BIOAVIALABILITY STUDY ü BIOAVAILABILITY STUDIES FOR NEW DRUGS In vivo Bioavailability studies are performed for new drugs to establish essential pharmacokinetic parameters including the rate of absorption, extent of absorption, rates of excretion and metabolism, and elimination half life after a single and multiple doses administration. q Bioavailability studies are also conducted to determine the influence of excipients, manufacturing procedures, packaging materials and patient related factors on the biological performance of a new drug formulation. q

BIOAVAILABILITY STUDIES FOR APPROVED DRUGS q. Bioavailability studies for approved drugs are performed to develop a new dosage form to improve on existing dosage form. q. In approving a drug product for marketing, the FDA must ensure that the drug product is safe and effective for its labeled indications of use. q. In addition, the drug product must meet all applicable standards of identity, strength, quality and purity…hence a drug product is fisrt subjected to all applicable official laboratory tests.

q. Finally, the FDA requires bioavailability / pharmacokinetic studies and where necessary bioequivalence studies to ensure the safety and efficacy of the drug product. q. In bioequivalence studies, test drug product is compared with a reference standard ( FDA approved drug product). q. Test product is called bioequivalent if it produces a bioavailability equivalent to that of the reference standard.

BIOAVAILABILITY STUDY PROTOCOL n The assessment of bioavailability of several drug products most often requires the measurement of drug and/or metabolite levels in either the blood or the urine. n The bioavailability of a drug from a dosage form depends on the dose and route of administration, time of administration, subjects and dosage form. n The aim of bioavailability study is to find out the dosage form influence on the biological performance of the drug…. hence the bioavailability study protocol used should be of sufficient sensitivity to detect differences in the rate and extent of absorption that are attributable only to dosage form variability and should avoid variabilities due to other factors

n Sometimes, due to inherent properties of the drug, analytical difficulties are encountered that will preclude the measurement of drug or metabolite levels in body fluids, so other techniques are used to assess bioavailability. n The drug may be labeled with a radioactive tag, a pharmacological or clinical response may be measured, or studies may be performed on animals.

BIOAVAILABILITY STUDY PROTOCOL üSTUDY OBJECTIVE üSTUDY DESIGN • Experimental Design • Wash out period • Drug Products 1. Test Products 2. Recognized Standards • Route of administration • Dosage Regimen • Frequency and duration of sampling • Randomization of drug administration • Single- versus multiple dose study • Subjects 1. Healthy subjects versus patients 2. Subject selection –Medical History –Physical examination –Laboratory tests v. Study conditions • Analysis of biological fluids üMETHODS OF ASSESSMENT OF BIOAVAILABILITY • Plasma data • Urine data • Acute pharmacological effect • Clinical response üANALYSIS AND PRESENTATION OF DATA • Statistical treatment of data analysis of variance [ANOVA] • Format of data.

STUDY OBJECTIVE §The objective of bioavailability study decides the study protocol §A study meant for estimating essential pharmacokinetic parameters differs significantly from a bioequivalence study meant for comparing the test formulation with reference of standard.

STUDY DESIGN §Various factors to be considered in conducting a bioavailability study since the rate and extent of absorption of a drug into the systemic circulation, its distribution and elimination are influenced by a variety of factors. §Subject factors such as age, sex, disease states, food habits, general health condition, body weight, experimental design, time of administration, time of sampling, analytical method used and compartment model used in estimating pharmacokinetic parameters contribute to the observed blood concentration time profile. §It is necessary to consider all these important factors in a study design.

PARALLEL DESIGN §The aim of experimental designs is to minimize the experimental variables and to avoid a bias. §In a parallel design, two formulations are administered randomly to the volunteers. §Disadvantage – Intersubject variation CROSS- OVER DESIGN §Generally, a substantial inter subject variability exists in the drug levels achieved from any given dose of medication in a panel of subjects. §The cross- over design mininmizes the effect of inter- subject variability in the study by using each subject as his or her own control § §Types of Cross- over designs • Latin square Cross- over design • Balanced Incomplete Bock Design [ BIBD]

LATIN SQUARE CROSS- OVER DESIGN §A standard approach for conducting a comparative bioavailability study is to use a randomized, balanced, cross-over design called a Latin Square or Complete Cross-over design. §In this design… §Each subject receives just once each formulation §Each formulation is administered just once each study period

TWO WAY CROSSOVER GROUP NUMBER SUBJECTS IN GROUP 1 2 TREATMENT FOR PERIOD I II 1, 2, 3, 4, 5, 6 A B 7, 8, 9, 10, 11, 12 B A THREE WAY CROSSOVER GROUP NUMBER SUBJECTS IN GROUP 1 TREATMENT FOR PERIOD I II III 1, 2, 3, 4, 5, 6 A C B 2 7, 8, 9, 10, 11, 12 B A C 3 12, 13, 14, 15, 16, 17, 18 C B A FOUR WAY CROSSOVER TREATMENT FOR PERIOD GROUP NUMBER SUBJECTS IN GROUP I II IV 1 1, 2, 3, 4, 5, 6 A B C D 2 7, 8, 9, 10, 11, 12 B D A C 3 12, 13, 14, 15, 16, 17, 18 C A D B 4 19, 20, 21, 22, 23, 24 D C B A

LATIN SQUARE CROSS- OVER DESIGN FOR BIOEQUIVALENCE STUDY OF THREE DRUG PRODUCTS IN SIX HUMAN VOLUNTEERS SUBJECT S TREATMENT FOR PERIOD I II III 1 A B C 2 B C A 3 A A B 4 A C B 5 C B A 6 B A C

LATIN SQUARE CROSS- OVER DESIGN FOR BIOEQUIVALENCE STUDY OF FOUR DRUG PRODUCTS IN SIXTEEN HUMAN VOLUNTEERS SUBJECTS TREATMENT FOR PERIOD I II IV 1 A B C D 2 B C D A 3 C D A B 4 D A B C 5 A B D C 6 B D C A 7 D C A B 8 C A B D 9 A C B D 10 C B D A 11 B D A C 12 D A C B 13 A C D B 14 C D B A 15 D B A C 16 B A C D

ADVATAGES OF LATIN SQUARE CROSS- OVER DESIGN • It minimizes the effect of inter subject variability in the study by using each subject as his or her own control. • It minimizes the carry over effects • It minimizes the time effect on bioavailability since each dosage form is administered in each study period. • It requires less number of subjects to get meaningful results. DISADVATAGES OF LATIN SQUARE CROSS- OVER DESIGN • It requires longer time to complete the study since a washout period exists between two study periods…. t 1/2 • The time to complete the trails depends on the number of formulations to be evaluated in the study…more no…. . more time • Increased number of study periods leads to high subject dropouts and the study becomes difficult • Medical ethics does not allow too many trails on a subject continuously for a longer time

BALANCED INCOMPLETE BLOCK DESIGN [BIBD] §BIBD eliminates many of the difficulties encountered with the Latin square design. §Salient aspects…. §Each subject receives not more than two formulations §Each formulation is administered the same number of times §Each pair of formulations occurs together in the same number of subjects. §In this design, each subject receives two formulations, each formulation is administered six times and each pair of formulations occurs together in two subjects (AB, AC, AD, BC, BD & CD)

BALANCED INCOMPLETE BLOCK DESIGN [BIBD] SUBJEC TS TREATMENT FOR PERIOD I II 1 A B 2 B A 3 A C 4 C A 5 A D 6 D A 7 B C 8 C B 9 B D 10 D B 11 C D 12 D C

WASHOUT PERIOD §In a Latin Square cross- over study design each subject receives each formulation and even in BIBD each subject receives two formulations at different occasions. §The time interval between the two treatments is called WASHOUT PERIOD §Washout period is required for the elimination of the administered dose of a drug so as to avoid carryover. §Official guidelines recommend 10 half lives should be allowed between successive treatments.

§Washout period is a function of the half life and the dose of the drug administered. §The no. of wash out periods depends upon the type of crossover design used and the no. of formulations to be evaluated. §Metabolites of the drug should also be eliminated from the body before the next treatment…. . more water soluble…rapid elimination.

DRUG PRODUCTS TEST PRODUCTS §Test products may be new drug formulations developed by a pharmaceutical technologist or new dosage forms of an existing drug. §A test product may be compared to a reference standard recognized by the FDA for getting approval for marketing the drug product. §Test products are evaluated for the following reasons…. §To select best dosage form of a new drug or existing drug among different dosage forms §To select best dosage form of a new drug or existing drug among different formulations that have shown equal performance in in-vitro tests. §To compare biological performance of a test product to that of a recognized standards ( Bioequivalence studies)

REFERENCE STANDARD §A chemical or generic product has to be compared with some standard dosage form to verify its in-vivo performance. §FDA accepts any innovator drug product as a reference standard.

ROUTE OF ADMINSTRATION §Generally orally administered dosage forms are subjected for bioavailability studies. §Others such as buccal, transdermal, intramuscular should also be evaluated for their biological performance. §Orally administered dosage forms show a much variation in their performance because of inter- subject and intra- subject variations. §All the dosage forms administered by an extra vascular route do require a bioavailability assessment.

SINGLE- VERSUS MULTIPLE- DOSE STUDY DESIGN §Single dose studies? …. . Multiple dose studies? . . . Better? ? ? §If the dosage forms are to be evaluated only for bioequivalence purposes, single dose studies are usually sufficient…. since relative BA of most tablets and capsules can be determined on a single dose basis…which is a predictive of multiple doses levels. §Certain dosage forms designed to achieve special release profiles of drugs may require multiple studies. Ex: Time release products, enteric coated tablets, intramuscular injections.

§Drugs that undergo first pass metabolism do require multiple dose study. §Use of an improper study design leads to the collection of insufficient or inappropriate data §If special dosage regimens such a s loading dose or twice- aday dosings are to be used, a multiple dose study design may also be necessary.

ADMINISTRATION OF DRUG PRODUCTS • Adminstration of drug products to the subjects should be based on randomization. • After the administration of the drug products, blood samples are withdrawn from the subjects at fixed time points. • It takes some time to take a sample from each subject, and the total time difference between first and last subject may range from 10 to 20 minutes depending upon the no. of subjects and technicians.

§This 10 -20 minutes difference would represent a substantial change in the drug concentrations observed in the blood. §If so, error between the time of administration and sampling will gradually increase from treatment group to treatment group. §To avoid this type of effect, randomized administration of drug products is used. §The order of dosing is not sequential but a part of each treatment is given first, a part in the middle and a part in the last.

SAPMLING §The biological sample to be used in the study has to be decided before the commencement of a BA study. §To have as estimate of the parameters, frequency and duration of sampling are very important since they can markedly influence the apparent results obtained in the study. §The sampling scheme should be frequent enough to define the absorption phase, the peak, and the elimination phase during a drugs time course in the body. §Estimate of Ka…. more data points in absorption phase

§Estimate of relative amount of absorption…. AUC curve…more points §To estimate the AUC from the data, sampling has to be carried out till the concentration of the drug reaches the linear elimination phase §In case of Urinary excretion studies…. bit difficult during absorption phase §Inorder to obtain useful data, it is necessary to plan the frequency and duration of sampling carefully §If possible, urine samples should be collected for 10 half lives of the drug to ensure 99. 9% of elimination

SELECTION OF SUBJECTS HEALTHY SUBJECTS VERSUS PATIENTS §BA studies are designed to find out the dosage form biological performance, hence it is necessary to minimize all possible variations if it is not possible to eliminate them §Use of healthy volunteers avoids much of variations that are possible with patients. §The variables associated with most diseases states make it impossible to design a meaningful BE test.

Some problems associated with testing in patients are… üIt is difficult to obtain many patients in a given place üThe severity of a disease varies from one patient to another üEthical considerations do not allow withdrawl of many blood samples from the patients for a longer time. üIt is not ethical to administer a dosage form to a patient whose therapeutic efficacy is unknown. ü Treatment of a disease involves use of several drugs simultaneously , the effect of these drugs on the bioavailability of the drug to be tested should be known before the interpretation of the bioavailability test results. Normal subjects are preferred in the bioavailability studies over patients.

SELECTION OF SUBJECTS §It is accepted that healthy volunteers should be used in bioavailability studies. §Healthy volunteers ? §Healthy means a person having an overall good state of physical health, which is asceratained by vital signs such as temperature, pulse, respiration, B. P and laboratory tests on blood ( RBC count, WBC count, haemoglobin, blood sugar), urine(p. H, albumin, sugar) and also liver function tests ( serum alkaline phosphatase and serum glutamic oxaloacetictransaminase) §Age, sex and body weight also influence the blood level profile of a drug product.

§In general, 21 year old, male subjects weighing 150 ld are ideal to act as volunteers in the study. §Difficult to obtain sufficient number…hence acceptable normal ranges are 20 -50 years of age and 102 to 220 lb body weight. §Males are preferred over females because menstrual cycle, pregnancy, lactation and menopause stages that occur in females may affect the blood level profiles of the drug. §Medical history of the subjects has to be reviewed critically by a panel of experts. §The selected subjects should be distributed randomly to different groups in order to achieve a uniform distribution of the available volunteers with respect of age, sex, and body weight and to avoid bias.

STUDY CONDITIONS §The selected subjects should be maintained on a uniform diet and none of them should have taken any drug at least one weak prior to study. §Before the commencement of the study it is necessary to define the study conditions such as the fasting period before the administration, time period after drug product administration during which fasting is continued, standard diet to be given after fasting, fluid intake and volume to be allowed. §The quality and quantity of food intake drastically affects the bioavailability of some drugs §Generally bioavailability trails are conducted on subjects that have fasted overnight.

ANALYSIS OF BIOLOGICAL SAMPLES §The biological samples collected as per the sampling procedures have to be analyzed immediately after the study. §Most of the times, the samples are stored for several days before they are subjected to a chemical analysis. §The storage of biological samples is an important aspect in a bioavailability study, since during storage the sample may undergo a chemical degradation, adsorption on the walls of the container. §The analytical method used for the estimation of the active ingredient responcible for therapeutic efficacy must be selective and sensitive.

§If the analytical method used in nonspecific, then results of the study may not reveal the differences in therapeutic efficacy that exists between drug products. §Only one analytical method should be used for the analysis of all the samples of study. §Analytical method used must be specific to the active chemical moiety and should exhibit high sensitivity.

ESTIMATION OF DRUGS &/or METABOLITES IN BIOLOGICAL FLUIDS §Since BE testing is based on the premise that equivalent blood levels produce equivalent pharmacological responses, it is obvious that one must be certain that equivalent components are measured in the biological fluids after administration of chemical equivalent products. §The development of highly sensitive and specific instrumental techniques, coupled with advances in selective extraction and separation procedures has enabled a residue analysis of drugs and their metabolites in biological fluids with a high degree of precision and accuracy. §In the analysis of blood and urine samples, the most easily obtainable and useful biological fluids, the major problem is to extract quantitatively and then separate the intact drug from its major metabolites or even to separate a mixture of two or more drugs from their metabolites.

METHODS OF ASSESSMENT OF BIOAVAILABILITY The assumption in using pharmacokinetic methods for the assessment of bioavalilability of drug products is that there exists a linear relationship between the drug level in the biological fluid and therapeutic response…. Indirect methods. INDIRECT METHODS or PHARMACOKINETIC METHODS The parameters that are useful in determining the BA of a drug from drug product based on indirect methods are…. PLASMA DATA 1. Tmax 2. Cmax 3. AUC URINE DATA 1. d. Xu / dt 2. Xu α 3. tu α

DIRECT METHODS or PHARMACOKINETIC METHODS §The pharmacokinetic methods are based on the assumption that the drug concentration in blood or the drug excretion in the urine are related to the observed therapeutic effect. §Pharmacodynamic methods are used when the assessment of bioavailability by pharmacokinetic methods is not possible due to non availability of a sensitive analytical method for the measurement of the drug or the analytical methods lacks sufficient accuracy or precision. §Two Pharmacodynamic methods used for the estimation of bioavailability are based on the measurement of …. §Acute Pharmacological Effect §Clinical Response

Measurement of Acute Pharmacological Effect §In order to estimate the bioavilaibility of a drug product accurately by this method, the following criteria should be met…. §An established dose related response curve §An easily measurable pharmacological response such as heart rate, ECG, blood pressure, pupil diameter. §Experimentally, the pharmacological effect is measured at different time intervals following the admi 9 nistration of drug product. §A plot of observed pharmacological effect versus time is made inorder to get a smooth curve §is difficult to obtain.

§The AUC is the measure of the performance of the drug product and is used for the estimation of the bioavailability. §The study should be conducted for at least three times the half life of the drug and a measurement of the pharmacological response should be made with sufficient frequency to permit a reasonable estimate of the total area under the curve §Main drawback of this method is that an accurate linear relationship between the drug level and observed pharmacological response

CLINICAL RESPONSE §Theoretically best among all…. practically it is not. §The drug may be available to the systemic circulation from a drug product at a sufficient rate and extent, but may not elicit a clinical response in an individual because his receptors are less sensitive to the drug compared to others.

STATISTICAL ANALYSIS OF THE DATA §The purpose of the BA test is to find out whether the test formulation gives a blood level profile identical to that observed for a reference standard product or not. §Due to biological and experimental variations, some differences always exist and it is necessary to ascertain whether these differences are simply chance occurrences or due to actual differences in treatment administered to subjects. §Statistical methods are used to evaluate the data in order to identify the different sources of variation and, if possible, to measure the contribution of each identified variable and isolate the specific observation of primary interest. §The Analysis of Variance [ANOVA], a statistical procedure used for cross over design, is used widely in bioavailability testing and is the procedure that will be encountered most frequently by health scientists.