Bioavailability

and
Bioequivalenc
e Studies
Bioavailability
Measurement of the relative amount & rate at which,
the drug from administered dosage form,
reaches the systemic circulation &
becomes available at the site of action

Bioavailable fraction (F), refers to the fraction of administered dose that

enters the systemic circulation

F = Bioavailable dose
Administered dose
Therapeutic Relevance
 Absolute Bioavailability
Compares the bioavailability of the active drug in systemic
circulation following non-intravenous administration with
the same drug following intravenous administration

 For drugs administered intravenously, bioavailability is

100%

 Determination of the best administration route

Fab = (AUC)drug
(AUC)IV
Absolute Bioavailability of Nimodipine for different routes:
Oral : 1.17 %
Nasal : 67.4 %
Intravenous: 100%
 Relative Bioavailability/Comparative
Bioavailability
Compares the bioavailability of a formulation (A) of a certain drug
when compared with another formulation (B) of the same
drug, usually an established standard

( AUC) drug
F rel =
(AUC) standard
 Formulation B

Formulation A

Plasma concentration versus time curve

 AUC: Trapezoidal Rule

AUC2-3 = Cp2 + Cp3 x (t3 - t2)

2
 FACTORS AFFECTING DRUG ABSORPTION/ BIOAVILABILTY

 [Link] FACTORS

1. Physicochemical factors:
1) Drug solubility & dissolution rate
2) Particle size & effective surface area
3) Polymorphism & amorphism
4) Pseudoploymorphism (hydrates/solvates)
5) Salt form of the drug
6) Lipophilicity of the drug pH- Partition
7) pKa of drug & gastrointestinal pH hypothesis
8) Drug stability

9
[Link] form characteristics & Pharmaceutical
Ingredients/Pharmaco technical factors:
1) Disintegration time (tablets/capsules)
2) Dissolution time
3) Manufacturing variables
4) Pharmaceutical ingredients (excipients/adjuvants)
5) Nature & type of dosage form
6) Product age & storage condition

[Link] related factors:

7) Route of administration
8) Membrane physiology
a) Nature of cell membrane
b) Transport processes
9) Age
10)Gastric emptying time
11)Intestinal
10
transit time
 6) Gastrointestinal pH
7) Disease states
8) Blood flow through the GIT
9) Gastrointestinal contents:
a) Food- drug interactions
b) Fluids
c) Other normal GI contents
10) Presystemic metabolism by:
a) Luminal enzymes
b) Gut wall enzymes
c) Bacterial enzymes
d) Hepatic enzymes

11
Reference Product
 Identified by the Regulatory Authorities
as “Designated Reference Product”

 Usually the Global Innovator’s Product

Protected by a patent

Marketed under manufacturers brand

name

 Clinical efficacy & safety profile is well documented

in extensive trials

 All generics must be Bioequivalent to it

 In India, CDSCO may approve another product as

 Generic Drug
 Drug product which is identical or bioequivalent to Brand/ Reference
drug in:
• Active ingredient (s)
• Route of administration
• Dosage form
• Strength
• Indications
• Safety

May have different:

• Inactive ingredients
• Colour
• Shape

 Almost half of drugs in market have Generics

Price difference between
Reference & Generic Drugs

Reference Drug Generic Drug

• Expensive • 30-80% cheaper
• 5/5000 new drug candidates • Since already tested & approved,
tested in humans & 1 approved cost of simply manufacturing

• Takes 12-15 yrs • Fraction of the cost of testing &

development
• Costs around 1 billion $

• Drug Patents of 20yrs, applied • Approved for sale after drug

before clinical trials begin patent protection expires

• Effectively 7-12 yrs

Fundamental Bioequivalence Assumption

When a generic drug is claimed bioequivalent to a

Reference drug, it is assumed that they are
therapeutically equivalent
Indian Legislation

 In India, CDSCO provides “Guidelines for Bioavailability &

Bioequivalence Studies” mentioned in Schedule Y

 As per the Drugs & Cosmetic Rules (IInd Amendment) 2005,

all bioavailability and bioequivalence studies should be conducted
in accordance to these Guidelines
Objectives of BA & BE Studies
 Development of suitable dosage form for a New Drug Entity

 Determination of influence of excipients, patient related factors &

possible interactions with other drugs

 Development of new drug formulations of existing drugs

 Control of quality of drug products, influence of

→ processing factors, storage & stability

 Comparison of availability of a drug substance

from different form
or same dosage form produced by different
manufacturers
NDA vs ANDA Review Process
NDA Requirements ANDA Requirements

1. Chemistry 1. Chemistry
2. Manufacturing 2. Manufacturing
3. Controls 3. Controls
4. Labeling 4. Labeling
5. Testing 5. Testing
6. Animal Studies
7. Clinical Studies 6. Bioequivalence
8. Bioavailability
Orange Book

All FDA approved drugs listed

(NDA’s, ANDA’s & OTC’s)

 Expiration of patent dates

 Drug, Price and Competition

Act (1984)
FDA required to publish Approved Drug Products with
Therapeutic Equivalence & Evaluations
Elements of Bioequivalence
Study Protocol
[Link]
[Link] investigator
[Link] number and date
[Link] objective
[Link] design
[Link]
[Link] Products
i. Test products(s)
[Link] product
[Link] regimen
[Link] collection schedule
[Link]
[Link] /Meals schedule
[Link] methods
[Link] population
[Link]
[Link] selection
[Link] history
[Link] examination
[Link] tests
[Link]/exclusion criteria
[Link] criteria
[Link] criteria
[Link]/prohibitions
[Link] procedures
[Link] and Drug Administration
[Link] sampling schedule
[Link] of subjects
[Link] considerations
[Link] principles
[Link] review board
[Link] consent
[Link] for subject withdrawl
[Link] reaction and emergency procedures
[Link]
[Link] Analysis
[Link] validation procedure
[Link] treatment of data
[Link] Accountability
[Link]
Methods used to assess Bio Equivalence

I. Pharmacokinetic Studies

II. Pharmacodynamic Studies

III. Comparative Clinical Studies

IV. Dissolution Studies

Pharmacokinetic
Studies
Parameters to be measured
Pharmacokinetic Parameters measured
are:
• Cmax
• Tmax
• AUC0-t
• AUC0-∞

Techniques used for Determination of BE Studies

[Link] Period Cross Over Design
[Link] Square Design
[Link] Group Design
[Link] Cross over Study Design
 I. Two-Period Crossover Design
2 formulations, even number of subjects,
randomly divided into 2 equal groups

First period , each member of one group receive a single dose of the
test formulation; each member of the other group receive the standard
formulation

Afte rPeriod
Subjects a wash1 pe riod 2(5 half lives), in second period , each member
Period of
the respective groups will receive an alternative formulation &
iment wil l be repeated.
1-8
exper T S

9-16 S T
II. Latin Square Design
 More than two formulations

 A group of volunteers will receive formulations in the sequence

shown
III. Parallel-Group Design
Even number of subjects in two groups

Each receive a different formulation

No washout necessary

For drugs with long half life

Treatment A Treatment B
1 2
3 4
5 6
7 8
9 10
11 12
IV. Replicate Crossover-study design
For highly variable drugs

 Allows comparisons of within-subject variances

 Reduce the number of subjects needed

 Four-period, two-sequence, two-formulation design (recommended)

OR
 Three-sequence, three-period, single-dose, partially
replicated
Period 1 2 3 4
Group 1 T R T R
Group 2 R T R T
Subject selection

Healthy adult volunteers

Age: 18-45 yrs

Age/Sex representation corresponding to therapeutic & safety profile

Weight within normal limits→ BMI

Women: Pregnancy test prior to 1st & last dose of study; OC pills C/I

Drug use intended in Elders (Age >60yrs)

Teratogenic Drugs→ Male volunteers

Highly toxic drugs: Patients with concerned disease (stable) eg. Cancer
Exclusion Criteria
H/o allergy to test drug
H/o liver or kidney dysfunction
H/o jaundice in past 6 months
Chronic diseases eg. Asthma, arthritis
Psychiatric illness
Chronic smoker, alcohol addiction, drug abuse
Intake of enzyme modifying drug in past 3 months
Intake of OTC/Prescription drugs past 2 weeks
HIV positive
BA & BE studies in past 3 months
H/o bleeding disorder
To establish BE:

 The calculated 90% CI for Cmax & AUC, should fall within range:
80-125% (Range of Bioequivalence)

 Non-parametric data 90% CI for Tmax should lie within

clinical acceptable range
BE Results

Demonstrate BE

Fail to Demonstrate BE

Demonstrate BE

Fail to Demonstrate BE Fail to Demonstrate BE

80% T/R (%) 125%

Conduct of Study
 Pre-study Requirements
 IEC approved protocol

 Written procedure (SOPs) for all the study related activities

 In accordance with ICH-GCP Guidelines

 Adequate infrastructure- Clinical facility

 Trained Study personnel

Healthy Volunteers
 Screening of Healthy volunteers

 Recruitment through advertisements

 Written consent for Screening & Consent for HIV testing
 Height & weight
 Medical History
 Physical examination, ECG & vital signs examination
 Blood & Urine sample
(Lab testing,; tests for HIV, Hepatitis A, B & C; UPT→ females)
 Volunteer Selection & Recruitment
 Volunteers called 1 day before study & admitted
 Written ICF taken

 During the Study

 Standardized study environment
 Vital signs examination at scheduled times
 Standardised amount of water [~240ml]
 No concomitant medications [including herbal remedies]
 Administration of the study medication is supervised by the
investigator
 Same time of dosing (multiple dosing)
 Sampling time with deviation of 2 mins allowed
 Uniform & identical meals at identical times in all periods
 Restriction of xanthines, grapefruit, citrus fruits, smoking,
alcohol
 Physical activity & posture standardized→
limit effects on GI flow & motility
All activities recorded in CRFs with time & date
Documentation
• Signed detailed protocol
• Approval by Ethics Committee
• Volunteer Information sheet
• Informed Consent Form (ICF)
• Case Record Form (CRF)
• Undertaking by investigator
• CV of investigator
• Randomization chart
• Laboratory certification
• Analytical method validation details
• Chromatograms of all volunteers including any aberrant ones
• Tabulated Raw Data of volunteers
Maintenance of Records & Retention of
Study Samples

 All Records of in vivo tests on any marketed batch of a

drug product should be maintained by the Sponsor
for atleast 2 years after expiry date of the batch

 All Drug samples to be retained for a period of

atleast 3 years after conduct of the study

OR

1year after expiry of the batch

[Stored in conditions consistent with the product
labeling]
METHODS FOR ENHANCEMENT OF DISSOLUTION
RATE/ BIOAVAILABILITY

Presented By
[Link]
Assistant Professor
Dept of Pharmaceutics
Shri Vishnu College of Pharmacy,
Bhimavaram
• Bioavailability means the rate and extent to which the
active substance or therapeutic moiety is absorbed from a
pharmaceutical form and becomes available at the site of
action.

• In the oral bioavailability of poorly water soluble

compounds, the insufficient dissolution rate is the limiting
factor. Some new technologies have been recently
developed to improve wet ability and aqueous solubility of
APIs.
The biopharmaceutical classification system
(BCS)

CLASS SOLUBILITY PERMEABILITY ABSORPTION RATE

PATTERN LIMITING
STEP IN
ABSORPTION

I High High Well absorb Gastric

emptying

II Low High variable Dissolution

III High Low Variable Permeabili

t y

IV Low Low Poorly Case

absorb by
case
• Class I: High Permeability and High Solubility:
Formulation independent:- The bioavailability of class I
compounds is determined only by delivery of the drug
solution to the intestine.
Examples: Benzapril, Loxoprofen, Sumatriptan etc.

• Class II: High Permeability but Low solubility:

Formulation dependent:- The bioavailability of
class II compounds is limited by drug
solubility/dissolution.
Examples: Valsartan, Nimesulide, Loratadine, Aceclofenac
etc
⚫Class III: Low Permeability but High Solubility:
Dependent on barrier properties:- The bioavailability
of class III compounds is limited by
intestinal permeability.
Examples: Gabapentine, Topiramate, Atropine etc.

⚫Class IV: Low Permeability and Low Solubility:

Formulation and barrier properties dependent:- The
bioavailability of class IV compounds is limited both by
solubility/dissolution and intestinal permeability.
Examples: Hydrochlorthiazide, Furosemide, Meloxicam
etc.
CAUSES OF LOW BIOAVAILABILITY

•First pass metabolism

•Poorly water soluble, slowly absorbing
oral drugs
•Low Permeability through GI Membrane
•Poor dissolution
•Age, stress, disorders, surgery etc
•Chemical reactions
•Stability problems
METHODS FOR ENHANCEMENT OF
SOLUBILITY/DISSOLUTION RATE/ BIOAVAILABILITY
I. Physical Modifications
A. Particle size
reduction 3. Sonocrystalisation
1. Micronization 4. Supercritical fluid
2. Nanosuspension
B. Modification of the crystal process
habit
1. Polymorphs 2.
Pseudopolymorphs
C. Drug dispersion in carriers
1. Eutectic mixtures 2. Solid dispersions
3. Solid solutions
D. Complexation
Use of complexing agents
E. Solubilization by
surfactants
Microemulsions
II. Chemical Modifications
1. Change in the pH
2. Use of buffer

III. Other methods

[Link]-crystallisation
[Link]-solvency
[Link]
4. Solubilizing
agents
5. Selective
adsorption on
insoluble carrier
6. Solvent deposition
[Link] soluble prodrug
[Link] polymer technology
[Link] Porous method
[Link] technology
METHODS TO ENHANCE THE DISSOLUTION RATE AND
BIOAVAILABILITY OF POORLY SOLUBLE DRUGS

1) Bioavailability enhancement through enhancement of

drug solubility or dissolution rate:
a. Micronization
b. Nanonization
c. Sonocrystalisation
d. Supercritical fluid process
e. Use of surfactants
f. Molecular encapsulation with cyclodextrins
g. Use of Salt forms
h. Solid Dispersions
2. Bioavailability enhancement through
Enhancement of drug permeability
a. Lipid technologies
b. Ion Pairing
c. Penetration enhancers
3. Bioavailability enhancement through Enhancement
of drug stability
d. Enteric coating
e. Complexation
f. Use of metabolism inhibitors
4. Bioavailability enhancement through Enhancement
of gastrointestinal retention
a. Gastro Retentive Drug Delivery System
[Link] size
reduction:
Particle size reduction can be achieved by
a. Micronization
[Link]
[Link]
[Link] fluid
process
1. Micronization: Colloid mill
• Micronization increases the dissolution rate of drugs through
increased surface area.
• Micronization of drugs is done by milling techniques using jet mill,
Fluid energy mills etc.
• The process involves reducing the size of the solid drug particles
to 1 to 10 microns commonly by spray drying or by use of attrition
methods. The process is also called micro-milling.
 2. Nanosuspension :

Nanosuspensions are sub-micron colloidal

dispersion of pure particles of the drug, which are stabilized by
Nanosuspension
surfactants. technology is used for efficient delivery of
hydrophobic drugs . The particle size distribution of the
solid particles in nanosuspensions is usually less than one micron
with an average particle size ranging between 200 and 600 nm.

Advantage :
Increased dissolution rate due to larger surface area exposed.

Eg., Nanosuspension approach has been employed drugs like

paclitaxel, tarazepide, amphotericin B which are still on research
stage.
[Link]
n: Particle size reduction on the basis of crystallisation by
using ultrasound is Sonocrystallisation . Sonocrystallisation utilizes
ultrasound power for inducing crystallisation . It not only enhances
the nucleation rate but also an effective means of size reduction
and controlling size distribution of the active pharmaceutical
ingredients. Most applications use ultrasound in the range 20 kHz-
5 MHz.
B. Modification of the crystal
habit:
Polymorphs
Enantiotropic Monotropic
One polymorphs form can No reversible
change reversibly into another transition is possible.
at a definite transition
temperature
below the melting point.
• Metastable forms are associated with higher energy and thus
higher solubility. Similarly the amorphous form of drug is always
more suited than crystalline form due to higher energy associated
and increased surface area.
• The anhydrous form of a drug has greater solubility than the
hydrates. This is because the hydrates are already in interaction
 with water and therefore have less energy for
crystal breakup in comparison to the
anhydrates.
• They have greater aqueous solubility than the
crystalline forms because they require less
energy to transfer a molecule into solvent. Thus,
the order for
Amorphous dissolution
> metastable of different
polymorph > stablesolid forms
polymorph
of drug is
• Melting followed by a rapid cooling or
recrystallization from different solvents can
produce metastable forms of a drug.
C. Drug dispersion in
carriers:
The term “solid dispersions” refers to the dispersion of one or
more active ingredients in an inert carrier in a solid state, frequently
prepared by the

• Hot melt mehod

1.
• Solvent evaporation
2.
method
• Hot melt extrusion method
3.
 1. Hot melt method :

Drug + vehicle (m.p low, organic solvent – insoluble)

(heating) Important
A molecular requisites :
dispersion can 1. Miscibility of the
Melting
be achieved or drug & carrier
.
not, depends on in the molten
the degree of form,
Freezing
supersaturation 2. Thermostability
quickly
and rate of cooling of the drug &
used in the carrier.
Dosage
process.
forms
Suitable to drugs and vehicles with promisingheat
stability.
2. Solvent evaporation method:
Drug + vehicle ( both soluble in
solvent)
organic solvent
solution The solvent
evaporation can
be done by spray
drying or freeze
evaporate the
drying.
solvent
Temperatures
coprecipitates used for solvent
evaporation
generally lie in the
dosage range 23-65 C.
forms
[Link]-melt Extrusion:
Hot melt extrusion of miscible components results in
amorphous solid solution formation, whereas extrusion of an
immiscible component leads to amorphous drug dispersed in
crystalline excipient. The process has been useful in the
preparation of solid dispersions in a single step.
D. Complexation

: Complexation is the reversible association between

two or more molecules to form a nonbondedentity with a
well defined stoichiometry . Complexation relies on relatively
weak
forces such as van-derwaal forces, hydrogen bonding and
hydrophobic interactions. Hydrophillic
Molecular Encapsulation with Cyclodextrin
These are
formed by the insertion of the nonpolar
molecule or the nonpolar region of one
molecule into the cavity of another molecule
or group of molecules. The most commonly
used host molecules are
cyclodextrins . Cyclodextrins are non-
reducing, crystalline , water soluble,
Hydrphobic
cyclic, oligosaccharides.
glucose monomers arranged in a donut
Cyclodextrins
shape ring. consist of CYCLODEXTRIN
The surface of the cyclodextrin molecules makes them water
soluble, but the hydrophobic cavity provides a microenvironment
for appropriately sized non-polar molecules. Based on the structure
and properties of drug molecule it can form 1:1 or 1:2 drug
cyclodextrin complex. Three naturally occurring CDs are α
Cyclodextrin, β Cyclodextrin, and γ Cyclodextrin.
E. Solubilization by surfactants:
Surfactants are molecules with distinct polar and
nonpolar regions.
Most surfactants consist of a
hydrocarbon segment connected to
a polar group. The polar group can
be anionic, cationic, zwitter ionic or
nonionic. The presence of surfactants
may lower the surface tension and increase the solubility of
the drug within an organic solvent .
Microemulsion : A microemulsion is a four-component system
composed of external phase, internal phase, surfactant and co
surfactant . The addition of surfactant, which is predominately
soluble in the internal phase unlike the co surfactant , results in the
formation of an optically clear, isotropic, thermodynamically stable
emulsion. It is termed as microemulsion because of the internal
phase is <0.1 micron droplet diameter.
26
 The surfactant and the co surfactant alternate each other and
form a mixed film at the interface, which contributes to the
stability of the microemulsion .
Non-ionic surfactants, such as Tweens ( polysorbates ) and
Labrafil ( polyoxyethylated oleic glycerides ), with high hyrophile-
lipophile balances are often used to ensure immediate formation
of oil-in- water droplets during production.
Advantages :
 Ease of preparation due to spontaneous formation.
 Thermodynamic stability,
transparent and elegant appearance,
enhanced penetration through the biological membranes,
increased bioavailability and
less inter- and intra-individual variability in drug
pharmacokinetics.
 II. CHEMICAL MODIFICATIONS
By change of pH:
For organic solutes that are ionizable, changing
the pH of the system is the simplest and most effective means of
increasing aqueous solubility .

LOWER UNIONISED INSOLUBLE

pH FORM PPT
HIGHER IONISED MORE SOLUBLE DRUG
pH FORM

Lower Ionized More soluble drug

pH form
Higher UNIONISED INSOLUBLE
pH FORM PPT
III. OTHER
METHODS.
[Link]-crystallization:
A co-crystal may be defined as a crystalline material
that consists of two or more molecular species held together by
non-covalent forces.
•Co-crystals are more stable, particularly as the co-
crystallizing
agents are solids at room temperature.
•Co-crystals can be prepared by evaporation of a
heteromeric
solution or by grinding the components together.
•Another technique for the preparation of co-crystals
includes
sublimation, growth from the melt, and slurry preparation.
•Only three of the co-crystallizing agents are classified as generally
recognised as safe (GRAS) it includes saccharin, nicotinamide and
2. Cosolvency : Co solvents are prepared by mixing miscible
or
partially miscible solvents. Weak electrolytes and nonpolar
molecules have poor water solubility and it can be improved by
altering polarity of the solvent. It is well-known that the addition
of an organic cosolvent to water can dramatically change the
solubility of drugs. Cosolvent system works by reducing the
interfacial tension between the aqueous solution and hydrophobic
solute.
Aquous solvent - Etahnol, sorbitol, glycerin,
propylene glycol.
Non aquous solvent - glycerol dimethyl ketal,
glycerol formal, glycofurol,
dimethyl acetamide.
SOME PERANTRALPRODUCT THAT
CONTAIN COSOLVENT
1. Diazepam - 10% ethanol + propylene
glycol
 6. Solvent deposition: In this method,the poorly aqueous solubledrug
such
as Nifedipine is dissolved in an organic solvent like alcohol and deposited on an
inert , hydrophilic, solid matrix such as starch or microcrystalline cellulose by
evaporation of solvent.
•7. Use of soluble prodrug : Prodrug stratergy involves the
incorporation of polar or ionizable moiety into the parent compound to
improve aqueous solubility. Example : prodrug of established drugs
has been successfully used to improve water solubility of
corticosteroids benzodiazepines.
[Link] Polymer Technology : Functional polymer enhances
the dissolution rate of poorly soluble drugs by avoiding the lattice
energy of the drug crystal, which is the main barrier to rapid
dissolution in aqueous media. The dissolution rate of poorly
soluble , ionizable drug like cationic, anionic and amphoteric
actives can be enhanced by this technology. Applied to heat
sensitive materials and oils also.
9. Precipitation: In this method, the poorly aqueous soluble drug
such as cyclosporine is dissolved in a suitable organic solvent
followed by mixing with a non-solvent to
its size effect particles.
rapid precipitation
prepared is also called of nano
as hydrosol. The product
drug in so
 Conclusion
Concept of BE has been adopted by the pharmaceutical industry & national regulatory
authorities throughout the world for over 20 years

There is a continuing attempt to understand & develop more efficient & scientifically valid
approaches to assess bioequivalence of various dosage forms including some of the tough
complex special dosage forms

Bioequivalence industry always existed in India→ become more matured now

 Changes in patent laws has added tremendous fuel to this growth

 Many BA/BE CROs in India

 Generics help patients by making drugs available at affordable price while retaining
their quality

 Balance public interests especially in diseases like Cancer & AIDs which have high
prevalence in developing countries & patented drugs are steeply priced

 Translated into increased opportunities for Indian Pharmaceutical

Industry → Export of generics to the regulated markets