An outlook on bioavailability and bioequivalence studies and their significance in supporting NDA & ANDA
1.0 Introduction
1.1 Bioavailability
It is the rate and extent of active ingredient which is absorbed from drug and is available at the site of action
It provides an estimate of fractional amount of drug absorbed
1.2 Bioequivalence
Absence of difference in rate and extent of the active ingredient in pharmaceutical equivalent which is available at site of action on administration of same molar dose under same conditions in an appropriately designed study
These studies are important when manufacturing or formulation changes occur at drug development & post approval stage.
Here the profile of the test drug is compared to reference drug product.
1.3 Bioavailability
It provides the estimate as to the relative fraction of orally administered dose which is absorbed into systemic circulation
The comparison data produced should be documented
By measuring the concentration of active ingredient and active metabolites in the systemic circulation over time profile can be generated for the drug
These profiles generated reflect the release of drug substance from the product along with presystemtic and systemic actions on the released drug substance
The regulations for BA studies are given by FDA as in 21 CFR 320.25
As per the regulations the reference product should be either a solution or suspension or intravenous dosage form
BA study with an oral solution as reference is used to assess formulation impact on bioavailability
BA study with IV dosage forms as reference is used to assess the impact of route of administration on bioavailability and also provides the information of absolute BA of drug released from the drug product
1.4 Bioequivalence
1.4.1 Preapproval changes:
BE documentation during IND period is done to compare
Decision of documentation is taken by sponsor and it should be prepared according to Section II C 2, Post approval changes & III D, in vitro studies
Test product is the new formulation or the formulation produced by a new method or new strength of the candidate
Reference product is the prior formulation or product of prior method of manufacture or prior strength.
1.4.2 Post approval Changes
Before distributing a drug it should be submitted to FDA along with list of post approval changes under section 506A (c) (2) of federal food drug and cosmetic act (21 U.S.C 356 a(c) (2)) . Certain post approval changes may require studies to be submitted to FDA
Guidance given by the FDA provides information on immediate and modified release drug products
1.5 BE Considerations
They are conducted by crossover design and in these studies intrasubject variability is used when study sample size is determined.
In parallel design studies total variability and intersubject variability is considered rather than intrasubject variability
A test product may measure rate and extent of absorption to reference product outside acceptable higher or lower limits
When bioequivalence can’t be demonstrated then the sponsor should show that the difference in rate & extent of absorption doesn’t cause significant differences in safety and efficacy of dose or concentration response data. If evidence is not there then BE may indicate that test product should be reformulated or method of manufacture should be changed or efficacy data may be needed.
1.6 Approaches to support BA/BE
In vitro tests predictive of human in vivo BA
Pharmacodynamic studies
Clinical studies
In vitro studies
2.0 Design and conduction of studies for orally administered pharma products
A BE study is a comparative BA study which establishes if there is bioequivalence between reference and test product.
2.1 Design:
Design of the study should be in a way to differentiate between formulation and other effects. If number of formulations is two then two period, two sequences cross over design is preferred.
Parallel designs can be considered when scientifically based statistical and study design is available.
Single dose studies are considered generally
Proper washouts should be done to prevent carry over effects
To estimate the extent of absorption a planned sampling schedule should be given which provides estimation of Cmax and plasma drug concentration time curve. It can be achieved if the derived AUC from measurements is 80% of AUC which is extrapolated to infinity
By collecting the 3-4 samples above LOQ terminal half-life can be obtained in terminal log linear phase
For long life drugs with more than 24 hours the study must cover at least 72 hours until 80% is recovered before 72 hours
2.2 Number of subjects
Number of subjects should be at least 80% power of meeting the acceptance criteria
The subject number should not be less than 12 and if 12 subjects don’t meet the 80% power of acceptance criteria then more subjects should be included
The sample size can be calculated by power equations which should be indicated in the protocol
Any add-ons in the protocol should be made by including maximum number of subjects
2.3 Subjects selection
Subjects should be selected to reduce differences and variability between the pharma products
The inclusion and exclusion criteria should be included in the protocol
Characteristics
Gender – Subjects should be selected from both the genders
Age – The age should be between 18 and 55 years
Mass – According to the Body Mass Index the body mass in the normal range should be accepted
Informed consent – All the participant subjects should have the capability to give informed consent
Screening – subjects should be screened on the basis of clinical lab tests, review of medical history and thorough medical examination
During or after completion of study drugs class and safety profile and medical investigations may be carried out.
Drug abuse – subjects included in the study shouldn’t smoke or drink alcohol or any other drugs
2.4 Inclusion of patients
If an investigational API is known to cause adverse effects or risks pharmacologically then it should not be given to healthy volunteers instead patients may be included with proper justification for their inclusion
2.5 Phenotyping/ Genotyping
Phenotyping or genotyping can be considered for exploratory BA studies. This criteria can be considered for cross over studies like food interaction, dose proportionality and BE studies for pharmacokinetic reason or for safety profile. If a drug causes genetic polymorphism then studies can be done in study cohorts for genotype or phenotype polymorphism
2.6 Standardization of study conditions
Following criteria should be considered to minimize the variability and to standardize the test conditions
Dosing
Fluid and food intake
Fluid intake at dosing
Concomitant medication
Physical activity and posture
2.7 Collection of sample and sample times
To measure the concentrations of drug the sample is collected from blood or serum or plasma
2.7.1 When blood is used
Blood sampling duration in the study should be sufficient to justify 80% of the known AUC to infinity (AUC∞). This duration is three terminal half-lives of the drug
Most of the drugs require 12 to 18 samples with a pre dose sample collected subject per dose.
Sample collection should be done so that maximum concentration of drug (Cmax) in blood can be estimated along with terminal elimination rate constant (Kel)
To estimate Kel by linear regression analysis at least 3-4 samples should be above LOQ during the terminal log-linear phase
When samples are collected the actual clock time and elapsed time relative to drug administration should be recorded.
If low drug concentrations are obtained in blood and more than 40% or a notable amount of drug is eliminated by urine then urine analysis can be done
2.7.2 When urine is collected
Immediately after collection the sample volume should be measured and recorded in report
The collection of urine is over an extended period and it should not be less than seven times of terminal elimination half-life so estimation of the amount excreted to infinity (Ae∞) can be done.
Sufficient number of samples should be collected to estimate the rate & extent of renal excretion
For a 24 hour study appropriate sampling should be done on 0 – 2, 2-4, 4-8, 8-12, 12-24hours post dose
When samples are collected the actual clock time and elapsed time relative to drug administration should be recorded.
2.8 Characteristics to investigate
2.8.1 Blood or plasma or serum concentration Vs Time profiles
The following parameters should be estimated
AUCt, AUC∞, Cmax, tmax for plasma concentration Vs Time profiles
AUC∞ , Cmax , Cmin , fluctuation (%PTF) and Swing (% Swing) studies should be conducted at steady state
Any justifiable characteristics
Estimation method of AUC- Values should be specified
2.8.2 Urinary Excretion Profiles
If the investigational API is excreted in major amount renally then urine excreted data can be advantageous
The following parameters should be estimated
Aet, Ae∞, as per urinary excretion studies
Any justifiable characteristics
Estimation method of AUC- Values should be specified
2.8.3 Pharmacodynamic Studies
The following parameters should be estimated
Dose response relation should be demonstrated
Pharmacodynamic response profile should be demonstrated by appropriate measurements
The dose effect curve should be below maximum physiological response
2.8.4 Chirality
When the following parameters are met then individual enantiomers are measured in BE studies
They are:
Enantiomers depict dissimilar Pharmacodynamic & pharmacokinetic parameters
Minor enantiomer attributes to safety or primary efficacy
At least one enantiomer has nonlinear absorption
If the above parameters are not met then achiral assay method is followed
2.8.5 Bio analysis
In the BE studies, bio analysis should be done in accordance to GLP and cGMP
These methods help in determining active moiety or metabolic products in blood, serum, plasma and urine. They are validated and documented
Validation should address the following parameters
Stability of stock solutions and analyte in biological matrix
Accuracy
Specificity
Precision
Response function
Detection and quantification limits
Robustness & Ruggedness
Calibration curve should be generated for every analyte and it should calculate analyte concentration in unknown samples
All the performed procedures should be according to standard operating procedures
3.0 Study Products
3.1 Test & Reference Products
Test products are compared with a comparable dosage form in case of a generic product
If the reference product is outside of WHO reference list then it should be justified by the applicant
Oral solid forms for systemic action test products should originate from batch of one tenth of the production scale otherwise it should be justified
3.2 Retention samples
Appropriate number of retention samples for test & reference products in BE studies should be stored for 1 year in excess of accepted shelf life or 2 years after approval or completion of trial. Out of both whichever is longer it is considered as it allows in retesting if required
3.3 Sample handling
Audit trial of procurement, use, storage and transport of reference and test product is to be recorded.
3.4 Data analysis
The primary role is to find the difference between bioavailability of test & reference products and any significant clinically important difference should not be there
3.5 Statistical analysis
The statistical method is based on 90% confidence interval for test/ reference ration of the parameters of population means
The concentration measures like AUCt, AUC∞, Cmax i.e. pharmacokinetic parameters should be analysed by ANOVA analysis. Using logarithmic transformation the data should be transformed to analysis.
To analyse tmax the technique involved should be non-parametric and it should be applied to untransformed data
Additionally geometric means, arithmetic means, SD, %RSD and pharmacokinetic ranges should be provided
4.0 Acceptance Range
Parameters to be tested and the testing procedure with acceptance ranges should be given in the protocol
4.1 Single Dose Studies
Following are the parameters which determine average BE
4.1.1 AUCt ratio
The 90% confidence interval for test to reference ratio should be within acceptance interval of 0, 80-1, 25 (80-125%)
In few cases alternative approaches can be accepted but they should be stated in the protocol before but should not be assed retrospectively
4.1.2 Cmax ratio
The 90% confidence interval for test to reference ratio should be within acceptance interval of 75 to 133%, for narrow therapeutic ranges, APIs with an acceptance interval of 80 to 125% can be applied
In a highly variable API a wider interval can be considered but it should be stated in the protocol before hand
4.1.3 Steady State Studies
Immediate release dosage forms
Acceptance criteria are same as single dose studies but AUC∞ instead of AUCt
Controlled or Modified release dosage forms
Acceptance criteria should be as following:
AUC∞ ratio
The 90% confidence interval for test to reference ration should be within acceptance interval of 0, 80 – 1, 25 (80-125%)
Cmax (ss) and Cmin (ss)
The 90% confidence interval for test to reference ration should be within acceptance interval of 0, 75 – 1, 33 (75-133%)
% Swing and % PTF
The 90% confidence interval for test to reference ration should be within acceptance interval of 0, 80 – 1, 25 (80-125%)
5.0 Study report
The bioequivalence or bioavailability study should include the complete document of the protocol in accordance with GMP, GLP, GCP
5.1 Clinical report
The clinical section of bioequivalence study should include
Statement indicating the ethics committee
Document proof of ethical approval of study
List of members of ethics committee their affiliations and qualifications
Investigator names & affiliations
Site of study and period of execution
Names & batch numbers of products tested
Reference & test products applicants name & address
Reference product expiry date and test products manufacture date
Dissolution and assay profiles of reference and test products
Certificate of Analysis of API used
Summary on adverse events and their influence and outcome of study
Document on subjects who dropped out or withdrew from the study
5.2 Analytical report
The analytical section of bioequivalence study should include:
Complete analytical validation report
Individual subject concentration data
Calibration data and calibration curve parameters
QC samples for complete study
Chromatograms from analytical runs and also for standard & QC samples
Summary on protocol deviations and their influence and outcome of the study
5.3 Pharmacokinetic & statistical report
This report should include the following
Data on plasma concentration Vs time profiles on linear and log scale and hard copy of the data should be submitted on processing by SAS software
Programmes and methods to derive pharmacokinetic parameters from raw data
Detailed ANOVA and non-parametric analysis
Summary of pharmacokinetic & statistical data
Statistical report should include detailed analysis so it can be repeated in future
5.4 Quality Assurance
QA statement should be signed confirming the release of document
Declaration should be given as study was performed subjected to pre study audit and in compliance with GCP, GLP, and GMP
The audit certificates should indicate audit date, name, address and qualification of auditor
An independent certificate on clinical portion of study should be submitted by the applicant
Bioavailability, bioequivalence, phenotyping, genotyping, subjects, bio analysis, statistical analysis, data analysis,single dose studies
