The document discusses bioavailability (BA) and bioequivalence (BE) studies, outlining their definitions, significance, and methodologies according to CDSCO guidelines. It details requirements for conducting PK studies, different study designs, analysis criteria, and exceptions based on therapeutic window and drug characteristics. The conclusion emphasizes the importance of BE for generics in improving patient access to affordable medications while maintaining quality.

1. Bioavailability and Bioequivalence studies
Dr Pranav Sopory
Junior Resident (Academic)
Department of Pharmacology
AIIMS- New Delhi
Mob: +91-9999491690
Email: pranav.sopory@gmail.com
BA/BE
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2. Contents
1 Introduction on BA/BE
2 Discussion of a “single dose” PK study
3 CDSCO guidelines
4 Cases requiring a “steady state” PK study
5 Special considerations for modified-release drug products
6 Methods apart from PK studies
7 Approval without BE study
8 Biowaivers
9 Biosimilars
10 Conclusion
BA: Bioavailability
BE: Bioequivalence
CDSCO: Central Drugs Standard Control Organization
PK: Pharmacokinetic 2

3. BA and BE
• BA of a drug is defined as the extent and rate to which the active
ingredient or active moiety from the drug product is absorbed and
becomes available at the site of drug action.
• BE is achieved when the extent and rate of absorption are not
statistically significantly different from those of the reference product
when administered at the same molar dose (under similar conditions in
an appropriately designed study).
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4. CDSCO (2005) approved methods to establish BE:
1. In vivo study
A. PK study
• Highest order of preference among BA/BE study: USFDA
B. PD study
• Active drug/ its metabolite cannot be detected in biological matrix
• Can be detected but not with sufficient accuracy or sensitivity
• Can be detected but has no correlation to its action (topical)
C. Comparative clinical study
• Absence of PK parameters along with lack of meaningful PD parameters
• PK and PD studies are not feasible
2. In vitro study
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5. BE: in brief
BA is measured via AUC and Cmax
1. Measure AUC and Cmax for the test drug
2. Measure AUC and Cmax for the reference drug
3. If the ratio (test/reference) falls between 80-125% range: BE established
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6. PK study: For a single dose
Time after drug
administration
(hours)
Drug
concentration in
plasma (ng/ml)
0.017 (1 min) 0.49
0.08 (5 min) 0.99
0.17 (10 min) 10.12
0.5 (30 min) 50.01
1 90.10
2 87.10
4 65.71
8 41.11
12 20.22
24 6.00
36 1.31
PK parameters
1. Cmax
2. AUC(0-t)
3. AUC(0-∞)
4. Kel
5. t1/2
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7. Calculating the AUC (exposure to the drug)
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8. Calculating the AUC (exposure to the drug)
BE study requires
TOTAL EXPOSURE: AUC(0-∞)
This is AUC(0-t)
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9. Calculating: Elimination rate constant (Kel)
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10. Calculating AUC (0-∞) from AUC(0-t) and Kel
• Involves extrapolation of 2%
• Upto 20% allowed
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11. Why not just measure the exposure to the drug (AUC)?
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12. Statistical analysis & criteria to establish BE
• To assume normality of data, logarithmic transformation of PK
parameters: Cmax and AUC
• The 90% confidence interval for the ratio of the population means (test/reference)
OR
• Two one sided t-tests with the H0 for non-BE at the 5% significance level
• To establish BE:
1. 90% C.I. for Cmax and AUC should fall within the BE range (80-125%)
2. This is equivalent to the REJECTION of two one sided-t tests with the H0 for non-
BE at the 5% significance level
3. Assess Tmax if clinically relevant
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13. PK parameters are lognormally distributed
• A normal distribution reaches from (– ∞) to (+ ∞)
• A lognormal distribution reaches from 0 to (+ ∞)
There are physiologic limits to PK parameters. They cannot be
negative.
Also the data is highly skewed.
• To perform statistical tests: LOGARITHMIC TRANSFORMATION
• The values are normally distributed
• The values are less skewed
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14. Why the 80-125% range to establish BE?
• USFDA: differences in systemic drug exposure up to 20% are not clinically significant.
• Shouldn’t the appropriate range be 80-120% (100% ± 20%)?
• PK parameters are log-normally distributed.
• Thus, the symmetrical ± 20% has to be in the log-transformed space so that the statistical
test of bioequivalence will be valid.
Maximum BE
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15. Exceptions to the criteria
• USFDA: Distinct limits for permissible differences in BA required for drugs that
have:
1. A narrow therapeutic window (90% to 111.11%, ln ratio: -0.105 to + 0.105)
2. A wide therapeutic window (75% to 133%, ln ratio: -0.286 to +0.286)
3. A non-linear PK within the therapeutic dose range (tighter limits due to fluctuations in conc.)
• Suprabioavailability
a. Reformulation followed by a fresh BE study
b. Otherwise perform a clinical trial 15

16. Study design: CROSSOVER DESIGN
• Standard: 2 sequence 2 period (2X2) crossover design
• Test drug: T
• Reference drug: R
• Study design (TR, RT)
Randomisation
Washout
period
Test drug
Reference drugTest drug
Reference drug
Advantages:
• Each subject serves as his/her
control (decreased confounding
variation)
• Statistically efficient; require lesser
subjects (≥16 subjects: CDSCO)
Disadvantages:
• Order effect
• Carry-out effect
• Intra-subject variability 16

17. PK study: Study design: Other designs
1. REPLICATE CROSSOVER STUDY DESIGN
• For highly variable drugs
• Study design (TRTR, RTRT)
• Minimum no. of subjects: 24 (USFDA)
• Allows comparisons within subject variances
2. PARALLEL STUDY DESIGN
• For drugs with long half-lives
• Administer each treatment to a separate group of subjects with similar demographics
• USFDA: Measure truncated AUC [eg. AUC(0-24)]: PK simulations show it to be an
equally effective parameter.
Period 1 2 3 4
Group 1 T R T R
Group 2 R T R T
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18. Requirement of BA/BE studies
Establish BE in case of:
1. NDA: Early and late clinical trial formulations
2. Post-marketing changes: Clinical trial formulations and to be
marketed formulations
3. ANDA: for generics
NDA: New Drug Application
ANDA: Abbreviated New Drug Application
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19. Regulations for generic drugs
• 1984: USFDA authorized “Drug Price Competition and Patent Term
Restoration Act”.
• USFDA approved generic drugs under this act based on BA/BE studies.
• MCI: Indian Medical Council (Professional Conduct, Etiquette and Ethics)
(Amendment) Regulations, 2016 – Part – I.
• “Every physician should prescribe drugs with generic names legibly and preferably
in capital letters and he/she shall ensure that there is a rational prescription and use of
drugs.”
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20. CDSCO guidelines: Introduction
1. Reasonable assurance that the test product when compared to the
reference product is therapeutically and clinically equivalent and
interchangeable.
2. Assessing this via clinical trial is cumbersome.
3. Systemic exposure profile during clinical trials in early drug
development serves as a benchmark for subsequent BE studies.
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21. Definitions in the guideline
Reference product:
• Identified by licensing authority as “Designated Reference Product”/ “Global
Innovator Product”.
• Contains the same active ingredient(s) as the new drug.
• Applicants seeking approval to market a generic equivalent must refer to the DRP to
which all generic versions must be shown to be bioequivalent.
• For subsequent new drug application, the Licensing Authority, may however, approve
another Indian product as DRP.
Active Moiety Salt/ester Dosage form Excipient Equivalence
Pharmaceutical
equivalents
+ + + - BA/BE outcome
Pharmaceutical
alternatives
+ +/- +/- N/A BA/BE outcome
Therapeutic
equivalents
+ +/- +/- N/A +
(Clinical efficacy)
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+ : Similar - : Not similar

22. In vivo BE studies are necessary
A. Oral immediate-release with systemic
action when one or more of the
following criteria applies:
1. Indicated for serious conditions requiring assured
therapeutic response;
2. Narrow therapeutic window
3. Complicated PK (Non-linear)
B. Non-oral and non-parenteral
formulations designed to act by
systemic absorption
(e.g. transdermal patches, suppositories)
C. Modified-release drug formulations
D. FDC products with systemic action
E. Non-solution pharmaceutical products
for non-systemic use (nasal, ocular,
dermal, rectal, etc.): Act without
systemic absorption.
In these cases, the PK parameter concept is not
suitable. Perform comparative clinical study or
pharmacodynamic study
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23. Study conditions: Characteristics to be investigated
Measurement of active drug substance(s) in the biological matrix
Measurement of drug metabolites
• Limitations in the analytical method
• Drug concentration is too low to be recorded
• Prodrugs
• Drugs with a very short half-life
Racemates (90% of drugs in the market) should be measured using an achiral assay
method
Measure individual enantiomer in case of
• Different PD parameters (eg: S-enantiomer of citalopram more potent)
• Different PK parameters (eg: l-enantiomer of MTX: 40 times higher AUC than d-enantiomer)
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24. CDSCO - Bioanalytical methodology: Pre-study phase
Sensitivity:
• Reliable lowest limit
quantification of
plasma concentration
• Limit of detection
should be lower than
limit of quantification
Precision and Accuracy
• Document on 3 concentrations (lowest measurable,
medium and Cmax)
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Intra-assay COV ≤ 20%
Range of accuracy: ± 15%
Accuracy: (Accepted value – Observed value*) X 100
Accepted value

25. Cases requiring an additional “Steady state study”
1. Toxic drugs requiring multiple dose therapy e.g. many cytotoxics.
2. Drugs with long terminal half life and blood concentrations after a
single dose cannot be followed for a sufficient time.
3. For modified-release products where it is necessary to assess the
fluctuation in plasma conc. over a dosage interval at steady state.
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26. PK parameters assessed in steady state study
• Statistical analysis of
1. AUC(0-𝛕)SS: AUC over one dosing interval.
2. Cmax
3. Cmin
4. Degree of fluctuation
• Criteria to establish BE
Same as single dose study
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27. AUC(0-tau), C(max), C(min) and Degree of fluctuation
Dose No Cmax
(ug/mL)
Cmin
(ug/mL)
Cavg
(ug/mL)
1 3.2 1.6 2.4
2 4.8 2.4 3.6
3 5.6 2.8 4.2
4 6 3 4.5
5 6.2 3.1 4.65
6 6.3 3.15 4.725
Calc. Cmax SS Cmin SS CavgSS
CavgSS (ug/mL): Cmax SS + Cmin SS
2
Degree of Fluctuation: Cmax SS - Cmin SS
CavgSS
Why measure AUC(0-tau)
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28. Special considerations for Modified-release drug products
Consideration 1:
RP: FOOD AFFECTS ABSORTION?
• ?/YES: Two 2X2 crossover
• NO: 3X3 crossover
Consideration 2:
ACCUMULATION?
 AUC(0-tau) /AUC(0-∞) ?
• ≥0.8: Unlikely: SINGLE DOSE Study
• ≤0.8: Likely: Additional SS Study
3. If test product first market entry: RP: IR
4. Additional parameter: Cpd: pre-dose
conc. just before steady state is achieved
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29. Requirements of a PD study
1. Response measured should be a pharmacological effect.
(substitute for plasma conc.)
2. Response should be measured in a double blinded fashion.
3. Pilot study can be conducted to check non-responders
4. If placebo effect can occur, include a third period/phase in
the study design
5. Crossover or parallel study design can be used
6. Conventional acceptance rate applicable for PK study is
not appropriate (too large). The range should be defined in
the protocol on a case-to-case basis
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30. • PK evaluation of acarbose is difficult because <2% is absorbed
systemically.
• Glucobay® 100 mg tablet (Bayer Healthcare) Vs. Generic acarbose
100‐mg tablet
• PD parameter: Gmax (Serial blood samples over 24 hours)
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31. Cases requiring Comparative clinical trials
1. Absence of PK parameters along lack of meaningful PD parameters
2. PK and PD studies are not feasible
3. Parameter: Clinical endpoint
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32. PK: Analytical studies for dihydroartemesnin: Not feasible
45 patients followed up for 28 days
• Mean time to fever subsidence (clinical parameter)
• Cure rate by Day 28 (clinical parameter)
• Time to parasite clearance 32

33. Biowaiver: in vivo BA/BE studies are waived
Applicant has to provide data to substantiate in vitro studies
Different strengths of drug manufactured by the same manufacturer where all of the following
criteria are fulfilled:
1. The ratio of active ingredients and excipients between the strengths is essentially the same, or, in
the case of small strengths, the ratio between the excipients is the same;
2. The method of manufacture is essentially the same
3. An appropriate equivalence study has been performed on the highest strength, unless a lower
strength is chosen for reasons of safety
4. In case of systemic availability- pharmacokinetics have shown to be linear over the therapeutic
dose range
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34. CDSCO Guideline: BE study not necessary for approval
New drug Active substance(s) Excipient
A. Parenteral (IV, IM, SC, IT) adm. as aqueous
solution
Same concentration Comparable
concentrations
B. Solution for oral use Same concentration Not suspected to affect
GI transit or absorption
of active substance
C. Gas - -
D. Powder for reconstitution as a solution Meets criterion A. or
B.
Meets criterion A. or
B.
E. Otic or ophthalmic or topical product
prepared as an aqueous solution
Same concentration Comparable
concentration
F. Inhalational product or a nasal spray: adm.
with/without the same device as the
reference product (in vitro testing to
document device performance between
reference and test product)
Same concentration Same concentration
For E. and F. the applicant is expected to demonstrate that the excipients in the new drug are
comparable concentrations as those in the reference product
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35. What about Biopharmaceuticals
• Vaccines, , blood components, gene therapies, tissues, monoclonal antibodies
• TERM: Biosimilar
• Establishing biosimilarity
1. Animal study
2. A clinical study or studies [including the assessment of immunogenicity and
pharmacokinetics (PK) or pharmacodynamics (PD)] that are sufficient to demonstrate
safety, purity, and potency in 1 or more appropriate conditions of use for which the
reference product is licensed and for which licensure is sought for the biosimilar product.
CDSCO guidelines available:
http://www.cdsco.nic.in/writereaddata/Proposed%20Guidelines%20for%20Similar%20Biologic%202016.pdf 35

36. Retrospective analysis comparing BE parameters between innovator
drugs and USFDA approved generic drugs b/w 1996-2007 (12 y)
Result:
1. The average difference in Cmax: 4.35% and in AUC: 3.56%
between generic and innovator products
2. 98% of BE studies: the generic product AUC differed from that of
the innovator product by less than 10%.
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37. FDA Orange book:
Mobile appPK/PD software
www.certara.com
cdsco.nic.in
ONLINE
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38. Conclusion
• Concept of BE has been adopted by the pharmaceutical industry &
national regulatory authorities throughout the world.
• Generics help patients by making drugs available at affordable price
while retaining their quality.
• Translates into increased opportunities for Indian pharmaceutical
companies (Current value: US $ 55 billion)
Thank you38

39. PK terms
1. Cmax: maximum drug concentration achieved in systemic circulation following drug
administration
2. Cmin: minimum drug concentration achieved in systemic circulation following
multiple dosing at steady state
3. Cpd: the pre-dose concentrations determined immediately before a dose in given at a
steady state
4. AUC 0-t: from 0 h to the last quantifiable concentration, to be calculated using the
trapezoid rule
5. AUC 0-∞: from 0h to infinity to be calculated as the sum of AUC0-t plus the ratio of
last measureable concentration to the elimination rate constant
6. AUC 0-𝛕: AUC over one dosing interval following single dose or modified release
products
7. AUC 0-𝛕 (SS): AUC over one dosing interval in multiple dose study at steady state
8. Kel: Terminal elimination rate constant calculated from a semi-log plot of plasma
concentration versus time curve
9. t1/2: time necessary to reduce the drug concentration in the blood, plasma, serum to
one-half after equilibrium in reached
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