This document discusses concepts related to bioavailability and bioequivalence studies. It defines key terms like bioavailability and provides an overview of different methods to measure bioavailability including pharmacokinetic methods like plasma level time studies and urinary excretion studies. It also discusses pharmacodynamic methods, in vitro dissolution studies and their relationship to bioavailability, in vitro-in vivo correlations, and different study designs used in bioequivalence studies.

1. Bioavailability
&
Bioequivalence
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2. CONTENTS Definitions
 Objectives of Bioavailability studies
 Methods of Bioavailability measurement
--Pharmacokinetic methods:
1. Plasma level time studies
2. Urinary excretion studies
--Pharmacodynamic methods:
1. Acute pharmacological response
2. Therapeutic response
 In vitro dissolution studies and bioavailability
 IVIVC
 Bioequivalence experimental study designs
1. Completely randomized designs
2. Randomized block designs
3. Repeated measures, cross over, carry-over designs
4. Latin square designs
 Statistical interpretation of bioequivalence data
1.Analysis of variance (ANOVA)
2.Confidence interval approach
Methods for enhancement of Bioavailability
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3. Bioavailability:
It is rate and extent of absorption of unchanged drug from its dosage
form.
Rate (time or rapidity) - acute conditions- asthma, pain etc
Extent (amount) – chronic conditions- hypertension.
If the size of the dose to be administered is same, then bioavailability of
a drug from it’s dosage form depends upon 3 major factors –
1} Pharmaceutical factors
2} Patent related factors
3} Route of administration
Influence of route of administration
PARENTRAL> ORAL> RECTAL>TOPICAL ------- with few exceptions
Definitions
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Systemic availability:- The amount of drug that reaches the
systemic circulation is C/A Systemic availability or simply
Availability. Denoted by F.
F = Bioavailable dose / Administered dose
Objective of Bioavailable studies
1. Development of a dosage form for a new drug entity.
2. Determination of influence of excipients & patient related
factors on the efficacy of absorption.
3. Development of new formulations of the exciting drugs.
4. Control of quality of a drug product during the early stages of
marketing, in order to determine the influence of processing
factors, storage and stability on drug absorption.

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CONSIDERATIONS OF BIOAVAIABILITY STUDY DESIGN
Absolute bioavailability:
When systemic availability of a drug administered orally is determined in
comparison to its I.V. administration, denoted by F.
Relative bioavailability:
When systemic availability of a drug after oral administration is
compared with that of oral standard of the same drug
(Solution or suspension) and denoted by Fr.
1. Bioavailability – Absolute Vs Relative

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2. Single dose Vs Multiple dose studies
Single dose versus multiple dose studies:
Single dose bioavailability studies are very common, easy, less
exposure to drugs,
less tedious. However it is difficult to predict the steady state
characteristics
and inter subject variability with these studies.
Advantages of multiple dose study:
-Accurately reflects manner in which drug will be used
clinically.
-Requires collection of few blood samples.
-Drug levels are higher due to cumulative effect and
useful for less sensitive analytical methods
-Better evaluation of controlled released formulation.
-Nonlinearity if present , can be easily determined.
-No need of long wash out periods.

7. Healthy subjects versus Patients:
Generally bioavailability study should be carried out in patients, as
patient get benefited from the study, reflects better therapeutic efficacy,
drug absorption pattern in disease state can be studied, avoids ethical
quandary of administering drug to healthy subjects. but there are also
various drawbacks like diseases, other drugs, physiological changes,
fasting state is difficult etc. Hence usually these studies performed on
young (20-40 yrs) healthy male adult volunteers (body weight ± 10%)
under restricted dietary and fixed activity conditions.
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3. Human volunteers – Healthy subjects Vs Patients

8. Methods of Bioavailability measurement
Pharmacokinetic methods
1. Plasma level time studies:
most reliable method of choice comparison to urine data method
Single dose: serial blood samples collection – 2-3 half lifes
Plot concentration vs time
For I.V. Sampling started within 5 min and subsequent samples at 15
min intervals
For oral dose at least 3 points taken on absorption curve (ascending
part)
Parameters considered important in plasma level time studies
1. Cmax : It is peak plasma concentration. It increases with dose as well
as increase in rate of absorption.
2. Tmax: The peak time at which Cmax attended.
3. AUC: Area under curve explains about amount of drug.
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For Single dose study
For Multiple dose study
For study state conc.

11. 2. Urinary excretion studies:
This method is based on the principle that the urinary excretion of unchanged drug is
directly proportional to the plasma concentration of drug.
It can be performed if
-At least 20% of administered dose is excreted unchanged in urine.
The study is useful for
- Drugs that extensively excreted unchanged in urine eg. Thiazide diuetics
- Drugs that have urine as site of action eg. Urinary antiseptics like nitrofurontoin.
Steps involved:
-collection of urine at regular intervals for 7 half lifes.
- Analysis of unchanged drug in collected sample.
- Determination of amount of drug at each interval and cumulative as well.
Criteria's must be followed
- At each sample collection total emptying of bladder is necessary.
- Frequent sampling is essential in the beginning to compute correct rate of
absorption.
- The fraction excreted unchanged in urine must remain constant.
Parameters considered important in Urinary excretion studies
1. (dXu/dt)max: Maximun urinary excretion rate
2. (tu)max: Time for maximum excretion rate
3. Xu: Cumulative amount of drug excreted in the urine.
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For Single dose study
For Multiple dose study to study state conc.

13. 1. Acute pharmacological response:
When bioavailability measurement by pharmacokinetic methods is difficult,
inaccurate or non reproducible this method is used. Such as ECG, EEG, Pupil
diameter etc.
It can be determined by dose response graphs. Responses measure for at least 3
half lifes.
Disadvantages:
- Pharmacological response is variable and accurate correlation drug and
formulation is difficult.
-Observed response may be due to active metabolite.
2. Therapeutic response:
This method is based on observing clinical response in patients.
Drawbacks:
- Quantitation of observed response is too improper.
-The physiological status of subject assumed that does not change significantly over
duration of study.
-If multiple dose protocols are not involved. Patient receive only single dose for few
days or a week
-The patient s receiving more than one drug treatment may be compromised due to
drug-drug interaction.
Pharmacodynamic methods
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14. Dissolution Rate – Greatest influence on drug absorption
In Vivo Bioavailability – best way to assess therapeutic efficacy –
CPCSEA guidelines & cost.
Thus to assure batch to batch variations, inexpensive in vitro
methods are used.
In vitro dissolution testing models:
The physicochemical property of most drugs that has greatest
influence on absorption from GIT is dissolution rate. However in vitro
dissolution is good substitute for in vivo study in terms of saving cost
and time. The best available tool today which can at least
quantitatively assure about the bioavailability of drug from its
formulation is in vitro dissolution test.
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Drug Dissolution Rate & Bioavailability

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Factors must be considered in the design of dissolution test are
2. Factors related to Dissolution Media/Fluid
3. Process Parameters for Dissolution Study
1. Factors related to Dissolution Apparatus
Types of Dissolution Apparatus
I. Closed Compartment – as per USP, 7 types
II. Open Compartment
III. Dialysis System

16. CLASS SOLUBILITY PERMEABILITY
Class – I High High
Class – II Low High
Class – III High Low
Class – IV Low Low
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Biopharmaceutical Classification System for Drugs

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It is defined as the predictive mathematical model that describes the
relationship between in vitro property (rate & extent of dissolution) and
in vivo response (plasma drug concentration).
The main objective of developing and evaluating IVIVC is to use
dissolution test to serve as alternate for in vivo study in human beings.
In vitro- in vivo correlations (IVIVC):
Objective of IVIVC:
1. To ensure batch to batch consistency
2. To serve as a tool for the development of new dosage form
Approaches of IVIVC:
1. By establishing a relationship, usually linear, between the in vitro
dissolution & the in vivo bioavailability parameters.
2. By using the data from previous bioavailability studies to modify
the dissolution methodology in order to arrive meaningful IVIVC.

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Correlations of IVIVC:
1. Correlations based on the plasma level data
2. Correlations based on the urinary excretion data
3. Correlations based on the pharmacologic response
Levels IVIVC:
Level A: The highest category of correlation. It represents point to point
correlation between in vitro dissolution and in vivo rate of absorption.
Advantages: serves as alternate for in vivo study, change in manf.
Procedure or formula can be justified without human studies.
Level B: The mean in vitro dissolution time is compare with mean in vivo
residence time. It is not point to point correlation. Data can be used for
quality control standards.
Level C: It is single point correlation. e.g. t50%, Tmax, Cmax. This level is only
useful as guide for formulation development or quality control.

19. Types of bioequivalence studies
In vivo bioequivalence studies: when needed,
1. Oral immediate release product with systemic action
-Indicated for serious conditions requiring assured response.
-Narrow therapeutic window.
- complicated pharmacokinetic, absorption <70%, presystemic
elimination>70%, nonlinear kinetics.
2. Non-oral immediate release products
3. Modified release products with systemic action.
In vitro bioequivalence studies: If none of the above criteria is applicable
comparative in vitro dissolution studies can be done.
Biowaivers: In vivo studies can be exempted under certain conditions.
1. Drug product only differ in strength of drug provided,
- Their pharmacokinetics are linear, Drug & excipient ratio is same,
- both products manufactured by same manuf. at same site.
- BA/BE study done for original product, disso. rate same under same conditions.
2. The method of production slightly modified in a way that not affect bioavailability
3. The drug product meet following requirements: The product is in solubilised form,
no excipients affecting absorption, Topical use, Oral but not absorbed, inhalation as
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Equivalence :- It is a relative term that compares drug product with respect to
a specific characteristic or function or to a defined set of standards.
1} Chemical equivalence:- When two or more drug products contain the same
chemical substance as an active ingredient in the same amount it is called
chemical equivalence.
2} Pharmaceutical equivalence:- When two or more drug products are
identical in strength, quality, purity, content uniformity, disintegration,
dissolution characteristics, they may however differ in containing different
excipients, called as pharmaceutical equivalence.
3} Bioequivalence:- It is relative term that denotes drug substance in two or
more identical dosage forms, reaches the systemic circulation at the same
relative rate and to the same relative extent i.e. their plasma concentration-
time profiles will be identical without significant statistical differences.
When statistical differences are observed in the bioavailability of two or more
drug products, Bioinequivalence is indicated.
4} Therapeutic Equivalence :- When two or more drug products that contain
the same therapeutically active ingredient, elicit identical pharmacologic
effects and can control the disease to the same extent.
Bioequivalence Studies

21. • Approaches for enhancement of bioavailability –
o The pharmaceutical approach
o The pharmacokinetic approach
o The biological approach

22. • METHODS
1. Micronization
2. Use of surfactant
3. Use of salt forms
4. Alteration of pH of the drug microenvironment
5. Use of metastable polymorphs
6. Solute – solvent complexation

23. 7. Solvent deposition
8. Selective absorption on insoluble carrier
9. Solid solution
Use of solid solution
Use of eutectic mixture
Use of solid dispersion
10. Molecular encapsulation with
cyclodextrins

24.  Micronization .
Methods:
- spray drying
- air attrition methods.
E.g. : Aspirin
Griseofulvin
Steroidal compounds
Sulfa drugs

25. Use of surfactants :
1. ‘Surfactants promote wetting & penetration of
fluids into solid drug particles.’
2. Better membrane contact.
3. Enhanced membrane permeability.
- Surfactants are used below CMC(critical micelle
concentration)
- E.g. Spironolactone
Use of salt forms:
E.g. Alkali metal salts of acidic drugs like penicillins
Strong Acid salt of basic drugs like atropine.

26. Alteration of pH of drug microenvironment:
1)Insitu salt formation
2)Buffered formulation e.g. Aspirin
Use of Metastable Polymorphs :
- more stable than stable polymorph
e.g. Chloramphenicol palmitate B.

27. Solute-solvent complexation:
- Solvates of drugs with organic solvents (
pseudo polymorphs) have higher aqueous
solubility than their respective hydrates or
original drug .
E.g. 1:2 Griseofulvin – Benzene solvate

28.  Selective adsorption on insoluble carriers :
- A highly active adsorbent like inorganic clay e.g. Bentonite,
enhance dissolution rate by maintaining concentration gradient at its
maximum.
E.g. Griseofulvin
Indomethacin
Prednisone.

29. Solid solution (Molecular dispersion/mixed crystals ) :
- It is a binary system comprising of solid solute
molecularly dispersed in a solid solvent.
- Systems prepared by Fusion method : Melts
- e.g. Griseofulvin-succinic acid
Solid dispersions (Co evaporators/co precipitates) :
- Both the solute and solid carrier solvent dissolved in
common volatile liquid e.g. Alcohol
- The drug is precipitated out in an amorphous form
as compared to crystalline forms in solid
solutions/eutectics.
E.g. Amorphous sulfathiazole in crystalline urea.

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31.  Eutectic mixture :
-It is intimately blended physical mixture of two crystalline components.
- Paracetamol -urea
- Griseofulvin – urea
- Griseofulvin-succinic acid
 Disadvantage :
Not useful in :
a) Drugs which fail to crystallize from mixed melt.
b) Thermo labile drugs
c) Carrier like succinic acid decompose at their
melting point.

32. Molecular encapsulation with Cyclodextrins :
-β and γ Cyclodextrins have ability to form inclusion complexes with
hydrophobic drug having poor aqueous solubility.
- These molecules have inside hydrophobic cavity to accommodate lipophilic
drug , outside is hydrophilic.
E.g. Thiazide diuretics
Barbiturates
Benzodiazepines
NSAIDS.

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34. References
 Biopharmaceutics and pharmacokinetics – A Treatise ,
D. M. Brahmankar, Sunil B.Jaiswal. Vallabh prakashan IInd
edition, pp- 315-366.
 Basics of Pharmaokinetics, Leon Shargel, fifth edition, willey
publications, pp- 453-490.
 Internet sources.
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