Elements of Bioequivalence Study Protocol

The bioavailability studies are done by measuring the concentration of the administered drug in the plasma or blood. This is done by following the systemic protocol of studies and is documented over time.

The protocol is helpful for clinical trials in the early drug development, and the data obtained is used in subsequent bioequivalence studies.

They are carried out to distinguish between two pharmaceutical products containing the same active substance.


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The elements of bioequivalence study protocol are as follows:  

    Study Objective

    If a new product is intended as a substitute for an approved medicinal product as a pharmaceutical equivalent or alternative, the equivalence with this product should be shown or justified.

    To ensure the clinical performance of such drug products, the bioequivalence studies should be performed.

    Bioequivalence studies are conducted if there is:

    A risk of bio-inequivalence and/or

    A risk of pharmacotherapeutic failure or diminished clinical safety.


    Study Design

    For many drugs the FDA, division of bio equivalence, office of generic drugs provides guild lines for the performance of in-vivo and in-vitro bioequivalence studies.


    a) Experimental Design

    The experimental designs are of two types: 

    Parallel design, and

    Cross over design.


    i. Parallel Design

    In this design, two formulations are administered to two groups of volunteers. To avoid bias, they are administered randomly.


    Disadvantages

    Inter-subject variability is not corrected.

    But parallel design is considered in the following conditions:

    Inter-subject variability is relatively small compared with intra-subject variability.

    If the drug is potentially toxic and has a long elimination half-life.

    The population of interest consists of ill patients.

    The cost of the increasing number of subjects is less than adding additional treatment periods.


    ii. Cross Over Design

    The cross over design is of following types:


    1. Latin Cross Over Design

    The main features are:

    Each subject receives just once each formulation.

    Each formulation is administered only once in each study period.


    Advantages

    It minimizes the effect of inter-subject variability.


    Disadvantages

    It requires a long time to complete the study since the washout period exists between two study periods and to complete the study as the number of formulations increase.

    An increase in the number of study periods leads to dropouts.

    Medical ethics do not allow too many trials continuously on a subject for a longer time.


    Two-way Crossover

     


    Three-way Crossover




    2. Balanced Incomplete Block Design (BIBD)

    It eliminates the difficulties encountered with Latin square crossover design.


    Salient Features

    Each subject receives not more than two formulations.
    And each formulation is administered equally.
    Each pair of formulations occur together in the same number of subjects.

    Drugs: A B C D.

    Subjects: 12.

    Combinations: 6 (AB, AC, AD, BC, BD, CD).




    3. Replicated Crossover Design

    It is useful to estimate the intra-subject variants in an individual by bioequivalence approach for both test and standard processes.

    Following is the four study periods, two formulations, two sequences recommended to replicate the bioequivalence study:



    b) Washout Period

    It is the time interval between two periods. The number of washout periods mainly depends upon the type of crossover design and number of formulations to be evaluated.

    For Digitoxin (elimination half-life=6-9days), if four formulations are to be administered by Latin square crossover design, the study period may exceed upto one year.


    c) Drug Products


    i. Test Product

    It may be a new drug formulation developed by a pharmaceutical technologist or a new dosage form of an existing drug. 

    It may be compared with the reference product recognized by USFDA. 

    It is evaluated for the following reasons:

    To select the best dosage form of a new drug.
    To compare the biological performance of the test product with that of a recognized standard.

    ii. Reference Product (Recognized Standard Product)

    It is the standard dosage form to which a new drug is compared to verify its in-vivo performance. 

    The USFDA accepts any innovator drug product as a reference standard.

    The innovator is the one who originally received approval from the FDA to market the product in the country.


    d) Route of Administration

    Oral administered drugs or dosage forms are subjected to bioavailability studies.

    Dosage forms administered by other routes such as transdermal, buccal are also evaluated for their biological performance.


    e) Dosage Regimen

    It is the frequency at which a drug is administered.


    f) Frequency and Duration of Sample

    Before the commencement of bioavailability studies, one has to decide which biological fluid has to be selected for the determination of drug concentration.

    If it is the blood sample, one has to estimate the plasma concentration-time profile (Cmax, Tmax, AUC).

    The sampling should be frequent to define the absorption phase, peak, elimination phase during drug time course in the body.

    Urinary excretion studies are suggested when it is not possible to measure the given drug concentration in blood and when ethical considerations do not allow the collection of samples for a longer time.


    g) Randomization of Drug Administration

    Generally, after the administration of the drug at a fixed time interval, the drug samples are withdrawn.

    If treatment is administered to a subject sequentially, during sampling, a time gap of 5 to 10mins occurs between the first and last subject, which leads to the increase in plasma drug concentration, which is due to the difference between the time of administration and sampling. 

    To avoid this, drug products are randomly administered.


    h) Single versus Multiple Dose Study Design

    For bioequivalence purposes, a single-dose study is sufficient for dosage forms (tablets or capsules). Dosage form meant for single-dose administration for a therapeutic benefit such as analgesic needs only a single-dose study.

    A multiple-dose study is done for time-release products, enteric-coated preparations, and IM injections.


    i) Subjects

    Ideally, the bioavailability study should be carried out in patients for whom the drug is intended to be used because of:

    The patient will be benefited from the study.
    Reflects better on the therapeutic efficacy of a drug.
    Drug absorption patterns in the disease states can be evaluated.
    Avoids the ethical quandary of administering drugs to healthy subjects.

    Patients are generally preferred in multiple-dose bioavailability studies.

    Drawbacks include disease, other drugs, physiological changes, etc., which may modify the drug absorption pattern.

    Healthy male volunteers under restricted dietary and fixed activity conditions are preferred. 

    Female volunteers are used only when drugs such as oral contraceptives are to be tested.

    The number of subjects to be selected depends upon the extent of inter-subject variability but should be kept to a minimum required to obtain reliable data. 

    Before starting the study, the consent of volunteers must be taken, and they must be informed about the importance of the study, conditions to be followed during the study, and possible hazards.

    Medical examination should be performed to exclude subjects with any kind of abnormality or disease.

    The drug washout period for a minimum of ten biological half-lives must be allowed between any two studies in the same subject.


    j) Analysis of Biological Fluids

    The volume and type of biological fluids must be specified and analyzed for the studies.


    Methods of Assessment of Bioavailability

    The methods useful in the quantitative evaluation of Bioavailability can be broadly divided into two categories:

    Pharmacokinetic methods, and 

    Pharmacodynamic methods.


    a) Pharmacokinetic Methods

    These are widely used and based on the assumption that the pharmacokinetic profile reflects the therapeutic effectiveness of a drug. Thus, these are indirect methods. 

    The two major pharmacokinetic methods are:

    Plasma level- time studies, and 

    Urinary excretion studies.


    i. Plasma Level - Time Studies

    It is the most reliable and method of choice in comparison to urine data. The method is based on the assumption that two dosage forms exhibit superimposable plasma level - time profiles in a group of subjects result in an identical therapeutic activity.

    In the case of a single-dose study, the samples must be collected for a period of 2-3 biological half-lives after drug administration to obtain a plasma concentration-time profile.


    For IV dose administration, the sample must be collected within 5mins after administration, and subsequent samples are withdrawn at an interval of 15mins.

    If drug administration follows a one-compartment open model, then at least 3 sample points must be considered to describe the disposition phase.

    If drug administration follows a two-compartment open model, then 5-6 sample points are to be considered to describe the disposition phase. 


    For oral administration, at least 3 sample points should be taken on the ascending part of the curve, and for accurate determination of Ka and remaining sample points in the disposition phase are similar to IV bolus dose.


    In the case of the multiple-dose study, the method involves drug administration for at least five biological half-lives, with a dosing interval equal to or greater than half-life to reach a steady state.

    The blood samples should be taken at the end of the previous dosing interval, and 8-10 samples are collected after administration of the next subsequent doses. 

    The extent of bioavailability is given as:


    `F_r = \frac{AUC_{(test)}}{Dose_{(test)}}\times\frac{Dose_{(std)}}{AUC_{(std)}}\times \frac{\tau_{(test)}}{\tau_{(std)}}`


    Fr = Relative Bioavailability.


    Bioavailability can also be determined from the peak plasma concentration at steady-state as:


    `F_r = \frac{(C_{ss,max})_{test}}{Dose_{(test)}}\times\frac{Dose_{(std)}}{(C_{ss,max})_{std}}\times \frac{\tau_{(test)}}{\tau_{(std)}}`

     

    Css = Concentration at steady state.


    ii. Urine Excretion Studies

    This method of assessing Bioavailability is based on the principle that urinary excretion of unchanged drugs is proportional to the plasma concentration of the drugs.

    Urinary data collection Method is considered for the following drugs:

    A 20% unchanged drug should be excreted in the urine.

    Drugs were extensively excreted unchanged in the urine. 

    E.g., thiazides and sulphonamides


    Drugs having urine as the site of action.

    E.g., antiseptics, nitrofurantoin.


    The method involves:

    Collection of the urine samples at regular intervals for some time equal to seven biological half-lives.
    Analysis of unchanged drug in collected samples by using accurate methods.
    Determination of the amount of the drug excreted in each time interval and the cumulative amount excreted.
    Total emptying of the bladder must be done during sample collection to avoid errors.

    The major parameters examined in urinary excretion data are: 


    1. (dXu / dt)max:

    It is the maximum urinary excretion rate, which is obtained from a peak of a plot between the rate of excretion versus mis point time of urine collection period.

    It is analogous to Cmax since the rate of appearance of the drug in urine is proportional to its concentration in the systemic circulation. It increases as the rate and extent of absorption increases.


    2. (tu)max:

    It is the time for maximum excretion rate, which is analogous to tmax of plasma level data.


    3. Xu ∞:

    It is the cumulative amount of drug excreted in the urine, which is related to the AUC of plasma level data.

    The extent of Bioavailability is calculated from the following equation:


    `F_a = \frac{(X_u^\infty)_{Oral}}{D_{Oral}}\times \frac{D_{IV}}{(X_u^\infty)_{IV}}`



    `F_r = \frac{(X_u^\infty)_{test}}{D_{test}}\times \frac{D_{std}}{(X_u^\infty)_{std}}`


    With multiple-dose study at steady-state concentration, the equation for Bioavailability is: 

     

    `F_a = \frac{(X_{u,ss})_{test}}{D_{test}}\times \frac{D_{std}}{(X_{u,ss})_{std}}\times \frac{\tau_{(test)}}{\tau_{(std)}}`


    b) Pharmacodynamic Methods

    These methods are complementary to pharmacokinetic approaches and involve direct measurement of drug effect on a physiological process as a function of time.

    This is generally useful when there is a lack of a sensitive analytical method for the measurement of the drug in plasma.

    The two pharmacodynamic methods involved in the determination of Bioavailability are:

    Acute Pharmacological response, and 

    Therapeutic response.


    i. Acute Pharmacological Response

    In this method, acute Pharmacological effects such as changes in ECG, EEG, BP, pupil diameter are related to the time course of a drug. 

    It can be determined by the construction of the pharmacological effect versus time graph.

    This response is calculated for at least three biological half-lives to get a good estimate of AUC.


    Disadvantage

    It is difficult to obtain an accurate linear relationship between the drug level and pharmacological effect.


    ii. Therapeutic Response

    It is the most definitive method which is based on observing the clinical response of a patient to a drug formulation, but practically, it is not possible because of the difference in clinical response, observed among individuals, not only due to formulations but also due to difference in both pharmacokinetic and pharmacodynamic behavior.

    Various factors such as age, drug tolerance, drug interactions may also influence the pharmacodynamic behavior of the drug.


    Analysis of Variance (ANOVA)

    This indicates the deviation of the test product in comparison to the standard product. The type of deviation and also the cause of deviation can be estimated.

    It is important in bioavailability and bioequivalence studies.

    ANOVA compares the Bioavailability of the test and standard formulations. In some cases, the metabolites of drugs are also concentrated in the sample that interferes with the estimation of the unchanged drug in the sample. 

    Urinary samples should be collected for ten biological half-lives of the drug.


    Reference

    1. Bioavailability and Bioequivalence Studies | IntechOpen.com

    2. BIBD Study | authorSTREAM.

    3. Bioavailability - SlideShare.

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