Bioavailability and bioeqivalance testing
- 2. ❖ DEFINITION ● Bioavailability is defined as the rate and the absorption of drug that reaches the biological system in an active form, capable of exerting the desired pharmacological effect, including its onset, intensity and duration of its action.
- 3. Objectives of bioavailability studies: • Development of new formulation. • Determination of influence of excipients, patient related factors and possible interaction with other drugs on the efficiency of absorption. • Control of quality of a drug product during the early stages of marketing in order to determine the influence of processing factors, storage, stability on drug absorption. • Primary stages of the development of a suitable dosage form for a new drug entity.
- 4. • CONSIDERATIONS IN IN-VIVO BIOAVAILABILITY STUDY DESIGN: ➢Bioavailability—Absolute versus Relative • ABSOLUTE BIOAVAILABILITY: • The systemic availability of a drug administered orally is determined in comparison to its iv administration. • Characterization of a drug's absorption properties from the e.v. site. • F = AUCev AUCiv • RELATIVE BIOAVAILABILITY: • The availability of a drug product as compared to another dosage form or product of the same drug given in the same dose. • Characterization of absorption of a drug from its formulation. • Fr=AUCA AUCB
- 5. Route Bioavailability (%) Characteristics Intravenous 100 (by definition) Most rapid onset (IV) Intramuscular 75 to ≤ 100 Large volumes often possible; may be (IM) painful Subcutaneous 75 to ≤ 100 Smaller volumes than IM; may be painful (SC) Oral (PO) 5 to < 100 Most convenient; first pass effects may be significant Rectal (PR) 30 to < 100 Less first-pass effects than oral Inhalation 5 to < 100 Often very rapid onset
- 6. • Single dose bioavailability studies : are very common. They are easy, offer less exposure to drugs and are less tedious. Limitation: Difficult to predict the steady-state characteristics of a drug and inter- subject variability. • Multiple dose study : • the bioavailability is determined at steady-state. • Better evaluation of a controlled- release formulation is possible. • Small inter-subject variability is observed, • Requires collection of fewer blood samples. • Limitations: – Poor compliance by subjects. – Greater exposure of subjects to the test drug, increasing the potential for adverse reactions. In multiple dose study the drug should be administered for 5 to 6 elimination half-lives until the steady-state has been reached, before collecting the blood samples ➢Single Dose versus Multiple Dose Studies: dose to be administered for bioavailability study is determined form of preliminary clinical experiment:
- 7. • ASSESSMENT OF BIOAVAILABILITY ➢Pharmacokinetics method – This method is more practical and discriminative. Pharmacokinetic methods are of two types. a) Determination of whole blood, plasma or serum concentration b) Urinary excretion method ➢ Pharmacodynamic methods: • Acute Pharmacologic Response Method : When bioavailability measurement by pharmacokinetic method is difficult, an acute pharmacologic effect such as effect on pupil diameter, heart rate or BP can be useful as an index of drug bioavailability. • Disadvantage: It tends to be complex, expensive, time-consuming and require a sensitive and quantitative measure of the desired response. • Therapeutic Response Method: Clinical response of the drug for which it is intended to be used is measured. • E.g.: heart rate, body temperature, blood sugar levels, and for anti- inflammatory drugs, reduction in inflammation is determined. • Drawbacks: quantification of observed response is too improper to allow for reasonable assessment of relative bioavailability between two dosage forms of the same drug.
- 8. • A) The blood (or serum or plasma) concentration-time curve - • These are based on the assumption that there is a direct relationship between the concentration of drug in blood or plasma and the concentration of drug at the site of action. • If the drugs are given to the volunteers through iv dose, the blood samples should be withdraw after 5min. And the frequency of sampling should be 15min. • In case of one compartment open model – at elimination phase 3 points are required to describe he kE value. • In case of two compartmental model – 5 to 6 points are required to describe kE value. • If Oral dose is given – 3 points are required to describe ka value.
- 9. • The key parameters for determining bioavailability
- 10. AUC: The AUC is proportional to the total amount of drug reaching the systemic circulation, and thus characterizes the extent of absorption. Cmax: Gives indication whether drug is sufficiently absorbed systemically to provide a therapeutic response. Tmax: The Tmax reflects the rate of drug absorption, and decreases as the absorption rate increases. MEC: The minimum plasma concentration of the drug required to achieve a given pharmacological or therapeutic response MSC: plasma concentration of the drug beyond which adverse effects are likely to happen.
- 11. • THERAPEUTIC RANGE-The range of plasma drug concentration in which the desired response is achieved yet avoiding adverse effect. The aim is clinical practice is to maintain plasma drug concentration within the therapeutic range. • ONSET OF ACTION-On set of action is the time required to achieve the minimum effective plasma concentration following administration of drug formulation. • DURATION OF ACTION-Duration of action of the therapeutic effect of the drug is defined as the time period during which the plasma concentration of the drug exceeds the minimum effective level. • INTENSITY OF ACTION-In general, the difference between the peak plasma concentration and the minimum effective plasma concentration provides a relative measure of the intensity of the therapeutic response of the drug.
- 12. B) Urinary Excretion Data: • These studies are based on the principle that urinary excretion of the unchanged drug is directly proportional to the plasma concentration of total drug. • This technique of studying bioavailability is most useful for those drugs that are not extensively metabolized prior to urinary elimination.
- 13. • The three major parameters examined in urinary excretion data are as follow: 1.(dXu/dt)max : maximum urinary excretion rate, gives the rate of appearance of drug in the urine is proportional to its concentration in systemic circulation. Its value increases as the rate of and/or extent of absorption increases 2. (tu)max : time for maximum excretion rate, is analogous to the of plasma level data, its value decreases as the absorption rate increases. 3. Xu : cumulative amount of drug excreted in the urine is related to the AUC of plasma level data and increases as the extent of absorption increases. • These studies used for certain thiazide diuretics and sulfonamides and for drugs that have urine as the site of action-for example, urinary antiseptics.
- 15. ● DEFINITIONS ● Equivalence – Equivalence is more relative term that compares one drug product with another or with a set of established standards. Equivalence may be defined in several ways: ● Chemical equivalence indicates that two or more dosage forms contain the labeled quantities of drug. ● Clinical equivalence occurs when the same drug from two or more dosage forms gives identical in vivo effects as measured by a pharmacological response or by control of a symptom or a disease. ● Therapeutic equivalence implies that one structurally different chemical can yield the same clinical result as another chemical.
- 16. The design and conduct of the study should follow ICH regulations on Good Clinical Practice, including reference to an Ethics Committee. A bioequivalence study is basically a comparative bioavailability study designed to establish equivalence between test and reference products. 1. Design: The various types of test designs that are usually employed in bioequivalence stdies; i. Completely randomised designs ii. Randomised block designs iii. Repeated measures, cross-over and carry-over designs iv. Latin square designs
- 17. i. Completely randomised designs ⬜ Method of randomisation ⬜ Label all subjects with the same number of digits, for e.g., if there are 20 subjects, number them from 1 to 20. ⬜ Randomly select non-repeating random numbers (like simple randomisation) with among these labels for the first treatment, and then repeat for all other treatments. ⬜ Advantages ⬜ The design is extremely easy to construct. ⬜ It can accommodate any number of treatments and subjects. ⬜ Disadvantages ⬜ Although the design can be used for any number of treatments, it is best suited for situations in which there are relatively few treatments. ⬜ Any unrelated sources of variability will tend to increase the random error term, making it difficult to detect differences among the treatment (or factor level) mean responses.
- 18. ii. Randomised block designs ⬜ First, subjects are sorted into homogeneous groups, called blocks and the treatments are then assigned at random within the blocks. ⬜ Method of Randomisation ⬜ Subjects having similar background characteristics are formed as blocks. ⬜ Then treatments are randomised within each block, just like the simple randomisation. ⬜ Randomisations for different blocks are done independent of each other. ⬜ Advantages ⬜ It can accommodate any number of treatments or replications. ⬜ Different treatments need not have equal sample size. ⬜ The statistical analysis is relatively simple. The design is easy to construct. ⬜ Disadvantages ⬜ Missing observations within a block require more complex analysis. ⬜ The degrees of freedom of experimental error are not as large as with a completely randomised design.
- 19. iii. Repeated measures, cross-over and carry-over designs ⬜ This is essentially a randomised block design in which the same subject serves as a block. ⬜ The administration of two or more treatments one after the other in a specified or random order to the same group of patients is called a crossover design or change-over design ⬜ Method of Randomisation ⬜ Complete randomisation is used to randomise the order of treatments for each subject. Randomisations for different subjects are independent of each other. ⬜ Advantages ⬜ They provide good precision for comparing treatments because all sources of variability between subjects are excluded from the experimental error. ⬜ It is economic on subjects. This is particularly important when only a few subjects can be utilized for the experiments. ⬜ Disadvantages ⬜ There may be an order effect, which is connected with the position in the treatment order. ⬜ There may be a carry-over effect, which is connected with the preceding treatment or treatments.
- 20. iv. Latin square designs ⬜ A Latin square design is a two-factor design (subjects and treatments are the two factors) with one observation in each cell. ⬜ Such a design is useful to compare the earlier ones when three or more treatments are to be compared and carry-over effects are balanced. ⬜ In a Latin square design, rows represent subjects, and columns represent treatments. ⬜ Advantages ⬜ It minimizes the inter-subject variability and carry-over effect in plasma drug levels. ⬜ Minimizes the variations due to time effect. ⬜ Treatment effects can be studied from a small-scale experiment. This is particularly helpful in preliminary or pilot studies. ⬜ Disadvantages: ⬜ The randomisation required is somewhat more complex than that for earlier designs considered. ⬜ The study takes a long time since an appropriate washout period between two administrations is essential which may be very long if the drug has a long t½.
- 21. ⬜ WASHOUT PERIOD ⬜ The time interval between the 2 treatments is called as WASHOUT PERIOD ⬜ It is required for the elimination of the administered dose to avoid the carry over effect ⬜ Large no. of drugs have been found to have half – life between 1-10 hrs, awashout period of 1 week was usually found suitable in most cases.
- 22. 2. Subjects: A. Selection of subjects: ⬜ Aim to minimize variability and permit detection of differences between pharmaceutical products. ⬜ The studies should normally be performed with healthy volunteers. ⬜ They should be screened for suitability by means of clinical laboratory tests, review of medical history, and medical examination. B. Standardization of the study: ⬜ The test conditions should be standardized in order to minimize the variability of all factors involved. ⬜ Standardization of the diet, fluid intake and exercise is recommended. ⬜ The subjects should not take other medicines during a suitable period before and during the study.
- 23. C. Inclusion of patients: ⦿ If the investigated active substance is known to have adverse effects and the pharmacological effects or risks are considered unacceptable for healthy volunteers it may be necessary to use patients instead, under suitable precautions and supervision. D. Genetic pheno-typing: ⦿ Pheno-typing of subjects should be considered as well in crossover studies. ⦿ If a drug is known to be subject to major genetic polymorphism, studies could be performed in panels of subjects of known phenotype or genotype. 3. Characteristics to be investigated: ⦿ In most cases evaluation of bioavailability and bioequivalence will be based upon the measured concentrations of the parent compound.
- 24. ● In some situations, measurements of an active or inactive metabolite is carried out. ● The plasma concentration versus time curves are mostly used to assess extent and rate of absorption. ● The use of urine excretion data may be advantageous in determining the extent of drug input in case of products predominately excreted renally. ● Specificity, accuracy and reproducibility of the methods should be sufficient. 4.Chemical analysis: ● It is conducted according to the applicable principles of Good Laboratory Practice (GLP). ● Determination of the active moiety and/or its biotransformation product(s) in plasma, urine or any other suitable matrix must be well characterized, fully validated and documented to yield reliable results that can be satisfactorily interpreted.
- 25. 5 Reference and test product: • The choice of reference product should be justified by the applicant and agreed upon by the regulatory authority. • The test products used in the biostudy must be prepared in accordance with GMP-regulations. 6.Data analysis: • To quantify the difference in bioavailability between the reference and test products and to demonstrate that any clinically important difference. A.Statistical analysis: • The statistical analysis (e.g. ANOVA) should take into account sources of variation that can be reasonably assumed to have an effect on the response variable. • Pharmacokinetic parameters derived from measures of concentration, e.g. AUC, Cmax should be analyzed using ANOVA.
- 26. B. Acceptance range for pharmacokinetic parameters: 1.AUC-ratio: ● It should lie within an acceptance interval of 0.80-1.25. 2.Cmax-ratio: ● It should lie within an acceptance interval of 0.80-1.25. ● The wider interval must be 0.75-1.33. ➢ For tmax if there is a clinically relevant claim for rapid release or action or signs related to adverse effects. The interval should lie within a clinically determined range. C. Handling deviations from the study plan: ● The protocol should also specify methods for handling drop- outs. D.A remark on individual and population bioequivalence: ● Bioequivalence studies are designed for population and individual is limited.
- 27. 7.In vitro dissolution for bioequivalence study: ✕ The term commonly used to describe is "in-vitro/in-vivo correlation". ✕ The specifications for the in vitro dissolution of the product should be derived from the dissolution profile of the batch. A. Official dissolution tests: ✕ Apparatus 1, (basket method). ✕ Apparatus 2 (paddle method). B.Parameters used: 1. Degree of agitation 2. Size and shape of container 3. Composition of dissolution medium • pH, ionic strength, viscosity 4. Temperature of dissolution medium 5. Volume of dissolution medium
- 28. 8.Reporting of results: ● The report of a bioequivalence study should give the complete documentation of its protocol, conduct and evaluation complying with GCP-rules and ICH guideline. ● Drop-out and withdrawal of subjects should be fully documented. ● The method used to derive the pharmacokinetic parameters should be specified. ● The analytical report should include the results for all standard and quality control samples.
- 29. APPLICATIONS FOR PRODUCTS CONTAINING APPROVED ACTIVE SUBSTANCES: A. Bioequivalence studies: ⦿ In vivo bioequivalence studies are needed when there is a risk that possible differences in bioavailability may result in therapeutic in equivalence. ⦿ oral immediate release drug formulations with systemic action ⦿ Non-oral and non-parenteral drug formulation designed to act by systemic absorption. ⦿ Sustained or otherwise modified release drug formulation. ⦿ Fixed-dose combination products.
- 30. ● REFERENCES: ● Brahmankar.D.M,Sunil.B.Jaiswal,Vallabh Prakashan Biopharmaceutics and Pharmacokinetics-A Treatise ,page no.282-305. ● LeonShargel & Andrew Yu Applied Biopharmaceutics & pharmacokinetics, page no 247-260. ● Madan.PL Biopharmaceutics & pharmacokinetics page no 118-127. ●