BIOAVAILABILITY STUDIES By A. S. Adebayo, Ph.D. 11/21/2018

To define various terms relating to bioavailability studies Objectives: To define various terms relating to bioavailability studies To understand some of the past problems with bioavailability To evaluate the components and results of a bioavailability study 11/21/2018

Purposes of Bioavailability Studies Bioavailability or drug product evaluation studies, may be designed to compare: One type of dosage form with another, e.g. regular tablet with sustained release tablet. For this type of tablet ka values should be slower; but F values should be similar Two (or more) dosage forms made by two different manufacturers. 11/21/2018

Definition of Terms Bioavailability - indicates a measure of the rate and the extent (amount)) of therapeutically active drug reaching the general circulation. Bioequivalent Drug Products - means pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose of the therapeutic moiety under similar experimental conditions, either single dose or multiple dose. Some pharmaceutical equivalents or pharmaceutical alternatives may be equivalent in the extent of their absorption but not in their rate of absorption. 11/21/2018

Definition of Terms (Cont.) Bioequivalence Requirement means a requirement imposed by the Food and Drug Administration for the in vitro and/or in vivo testing of specified drug products which must be satisfied as a condition for marketing. Brand Name is the trade name of the drug. Chemical Name is the name used by the organic chemist to indicate the chemical structure of the drug. Drug Product means a finished dosage form, e.g., tablet, capsule, or solution, which contains the active drug ingredient, generally, but not necessarily, in association with inactive ingredients. 11/21/2018

Definition of Terms (Cont.) Generic Name - the established, non-proprietary or common name of the active drug in a drug product. (as listed in official books/Pharmacopoeias) Pharmaceutical Alternatives - drug products that contain the identical therapeutic moiety, or its precursor, but not necessarily in the same amount or dosage form or as the same salt or ester. 11/21/2018

Definition of Terms (Cont.) Pharmaceutical Equivalent - drug products that contain identical amounts of the identical active drug ingredient, i.e., the salt or ester of the same therapeutic moiety, in identical dosage forms, but not necessarily containing the same inactive ingredients, and that meet the identical compendia or other applicable standard of identity, strength, quality, and purity, including potency and where applicable, content uniformity, disintegration times and/or dissolution rate. 11/21/2018

Reasons for Bioequivalence Testing Chlorpropamide - Peak plasma concentration after one brand was less than ½ the peak after the other two products. 11/21/2018

Reasons for Bioequivalence Testing (Cont.) Digoxin – Reported toxicity due to change of formulation resulting in a two-fold increase in bioavailability. Phenytoin. Incidence of phenytoin toxicity in Australia in 1968 and 1969 following change of diluent from Calcium sulphate to lactose. 11/21/2018

Reasons for Bioequivalence Requirements by Regulatory Authorities The FDA may require bioavailability studies for a variety of reasons including: Results from clinical studies indicate that different drug products produce different therapeutic results. Results from bioavailability studies indicate that different products are not bioequivalent. Drug has a narrow therapeutic range. Low solubility and/or large dose. Absorption is considerably less than 100% 11/21/2018

Bioavailability study characteristics Drug The drug substance in each product must be the same. Compare “like with like“ In the case of pro-drug administration, we may compare the delivery of a dosage form containing the drug with another dosage form containing a pro-drug. This testing is generally conducted to evaluate the usefulness of the pro-drug, rather than a strict comparison of the drug products. Once the usefulness of the pro-drug is demonstrated comparisons between dosage forms all containing the pro-drug should be undertaken to evaluate the drug product performance. 11/21/2018

Drug product 11/21/2018

Cp versus Time for A and B with B having Slower Absorption 11/21/2018

A number of factors are of concern: Health Age Weight Enzyme status Subjects A number of factors are of concern: Health Age Weight Enzyme status Number of subjects. 11/21/2018

Health of subjects A cross- over design is usually employed to reduce/eliminate the effect of individual subject Even though each subject will act as their own control it is usually best to have subjects of similar kinetic characteristic so that major variations are not introduced. Thus healthy volunteers are often preferred by drug product evaluation studies. Informed consent should be obtained from each volunteer and some biochemical and medical examination will be used to confirm their medical state. For some drugs there may be special disease states which may cause the exclusion of some volunteers. 11/21/2018

Age *** As you will see later, age can have a significant effect on drug pharmacokinetics. Elderly patients and young children can have quite different kinetics compared with young adults. For better matched group, subjects between the ages of 18 to 35 years are preferred. Kinetic changes usually are not important until age greater than 60. 11/21/2018

Weight The apparent volume of distribution is usually proportional to weight in subjects of normal weight for height. However, in overweight (or underweight) subjects the Vd in L/kg maybe somewhat different. 11/21/2018

Enzyme status Smokers or subjects taking certain other drugs may have altered kinetics for the drug of interest. This can be caused by alteration of enzyme activity or by drug-drug interactions. These effects add complications to a study and an attempt is usually made to minimize these factors. 11/21/2018

Number of subjects The number of subjects included in the study should be sufficient to see any real (maybe 20% variation) differences in bioavailability. Usually 10 - 20 subjects are used in these studies. In clinical studies where the end-point is some clinical response, much larger numbers are required because of the greater variability in clinical response. 11/21/2018

The same assay method should be used for all phases of the study. The assay method should be sensitive and specific. 11/21/2018

Usually a complete cross-over design is used. With this design each subject receives all products with a wash-out period between each dose administration A factorial design may be used to study multiple source of variation. 11/21/2018

Cross over design (2 products) Week 1 Week 2 Group 1 A B Group 2 for two products 11/21/2018

Cross over design (3 products) Week 1 Week 2 Week 3 Group 1 A B C Group 2 Group 3 Group 4 Group 5 Group 6 for three products 11/21/2018

Sample collection Biological fluids are usually collected. The commonest biological fluids collected are blood, urine and, at times, the saliva. Blood samples are collected using sterile needles and syringes into heparinized tubes. Two to 5 ml samples are collected at pre-determined time intervals. The frequency and duration of collection is usually related to the t1/2 of the drug: blood samples should not be less than 3 times the t1/2 of the drug. 11/21/2018

Sample collection/Treatment Blood samples are more frequently collected within the first 12 hr, such that the tmax of the drug can be ascertained. Thereafter, collection could be done every day or every other day depending on the t1/2 of the drug. Whole blood, plasma or serum can be used. When using whole blood, the white blood cells or red blood cells should be lysed by keeping the sample in the freezer so as to free the drug. The whole blood should then be centrifuged at 2000 revolutions/min. to obtain the plasma. 11/21/2018

Sample collection/Treatment To use the serum, the blood should be collected into an ordinary tube (with no heparin). The blood clots and the serum fraction is separated. The analyses of blood in most cases are normally done in plasma because analysis done on whole blood gives wide intra- and inter-individual variations as a result of difference in white blood cell count, a problem not encountered with plasma analysis. 11/21/2018

Urine/salivary analysis When performing urine analysis, samples must be collected for at least 10 times the t1/2 of the drug. The total urine voided should be collected. Saliva samples may also be collected at predetermined time intervals. Before using saliva, studies must have shown that correlation exists between plasma and saliva levels of the drug. 11/21/2018

Analysis of the Samples Requirements of analytical method: Specificity Sensitivity Reproducibility Precision Rapidity of rate of assay Accuracy 11/21/2018

The statistical determination of precision Where C.V = coefficient of variation s= sample standard deviation; = average value or mean. Precision requires a C.V value below 12%. 11/21/2018

Reproducibility, Accuracy & Specificity Reproducibility – The possibility of obtaining the same result if another individual performs the same experiment in another place using similar set up. Accuracy – Is a measure of deviation between the known and the obtained result. The lower the value, the more accurate the method Specificity – The ability of the test to differentiate between the drug and the metabolite. 11/21/2018

UV Assay Methods Fluorimetric Method The sensitivity of UV spectrophotometers are low (cannot measure up to nanogram level). The specificity is also poor, may not differentiate between the parent drug and the metabolite. Fluorimetric Method Useful for compounds that can fluoresce e.g. quinine. Its specificity is still poor but better than in UV spec. (about 10 X). 11/21/2018

TLC-UV Method The Thin layer chromatograph separates the metabolite from the parent drug, while the UV quantifies the amount of unchanged drug. The sensitivity is poor although the specificity is improved. 11/21/2018

TLC-Fluorimetry This gives a very good result. TLC separates and Fluorimeter analyzes. However, assay time is very long. 11/21/2018

HPLC This is most popular method for analysis of drugs in biological fluids. It can detect as low as 1ng/ml or 0.1ng/ml. It combines both the separation and the quantification 11/21/2018

GLC/HPLC Gas-liquid chromatography is also highly sensitive, selective and reproducible. HPLC has some advantages over GLC because: HPLC leads to higher selectivity (better separation of drug from metabolite) because both the column and the solvent can be changed GLC is more expensive GLC is not suitable for thermolabile (heat sensitive) substances 11/21/2018

Data analysis & Statistics The rate of absorption can be characterized by the ka value and the time of peak concentration. The extent of drug absorption is characterized by the F value or the peak concentration or total AUC values. Any differences in the average values of these parameters can then be analyzed statistically to determine the significance of the differences. The 5 % confidence level is usually used as the criteria for acceptance. The analysis of variance is a technique for separating the effect of product, subject, and sequence of treatment/administration. 11/21/2018

Analysis of Variance Table for Three-Way Cross-Over Study Source of Variation d.f. SS MS F Significance Level Total 35 44.6 - Subject 11 28.3 2.58 10.1 p < 0.001 Week 2 0.14 0.068 0.27 n.s. Treatment 11.0 5.55 21.8 P < 0.001 Residual 20 5.09 0.255 11/21/2018

THE END THANK YOU 11/21/2018