Bioavailability and Bioequivalence

Studies
AZIB. S.
M.PHARM(PHARMACEUTI
 Bioavailability

Bioavailability describes the fraction of the

the dose of a drug that enters into
the systemic circulation.

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.
 Absolute Bioavailability

 Requires I.V. Administration

 Ratio of the oral :intravenous AUC

values, normalized for dose
oral/i.v. absolute bioavailability

Estimates the efficiency of

absorption of the drug from the
formulation versus an intravenous
bolus injection

F = AUC oral / AUC iv*

Dose iv /Dose oral
 Relative Bioavailability
 no I.V. reference
 comparison AUC values (ratio) of different
dosage forms / formulations

 Frel =(AUC a / AUC b) *(Dose b /Dose

a)
 5 5

4 4

Solution
Capsule
3 3
Conc. [mg/L]

Conc. [mg/L]
2 2

1
1

0
0

0 4 8 12 16 20 24 0 4 8 12 16 20 24
Time [hours] Time [hours]

20 mg administered as an i.v. 80 mg given as a solution and a

bolus capsule
 relative
bioavailability

Bioequivalence
absolute

Different
formulations

Interactions

food-
effect
 Bioequivalence (BE): Def inition
“The absence of a significant difference in the
rate and extent to which the active ingredient
or active moiety in pharmaceutical equivalents
or pharmaceutical alternatives becomes
available at the site of drug action when
administered at the same molar dose under
similar conditions in
an appropriately designed study.”
 Bioequivalence
Two medicinal products are bioequivalent if
they are pharmaceutical equivalents or
alternatives and if their bioavailabilities (rate
and extent) after administration in the same
molar dose are similar
to such degree that their effects, with
respect to both efficacy and safety, will
be
essential the same.
 Bioequivalence

Pharmaceutical equivalents:
Medicinal products are pharmaceutical equivalents if they contain the
same amount of the same active substance in the same dosage form
that meet the same or comparable standard.

Pharmaceutical alternatives:
Medicinal products are pharmaceutical alternatives if they contain the
same active moiety but differ in chemical form of that moiety or in the
dosage form or strength.
When do we do BE studies ?

 Clinical Service Form to Final Market

Form

 Change of formulations (capsules to

tablet)

 Generic Formulations

 Change of Process or manufacturing site

(some times)
 Approaches to Determining BE

• In vivo measurement of active moiety in

biologic f luid
• In vivo pharmacodynamic comparison
(Topical Corticosteroid)
• In vivo clinical comparison (Nasal
suspensions)
• In vitro comparison (Nasal Solution,
Topical solution, Oral solution)
 Methods of Assessment
• Pharmacokinetic Methods
– Based on assumption that Pharmacokinetic
prof ile ref lects the therapeutic effectiveness of
a drug.
• Plasma Level- Time Studies
• Urinary Excretion Studies
• Acute pharmacological Response Method
• Therapeutic Response Method
• Clinical Trials
 Plasma Level- Time Studies
• Most common type of human bioavailability studies.
• Based on the assumption that there is a direct
relationship between the concentration of drug in blood
or plasma and the concentration of drug at the site of
action.
• Following the administration of a single dose of a
medication, blood samples are drawn at specific time
intervals and analyzed for drug content.
• A profile is constructed showing the concentration of
drug in blood at the specific times the samples were
taken.
• Bioavailability is generally assessed by the
determination of following three parameters-
– Cmax (Peak plasma concentration)
– tmax (time of peak)
– Area under curve (AUC)
 Plasma Level- Time Studies
• Cmax (Peak plasma concentration):
– Maximum concentration of the drug obtained after the
administration of single dose of the drug.
– Expressed in terms of μg/ml or mg/ml.
• tmax (time of peak):
– Time required to achieve peak concentration of the drug
after administration.
– Gives indication of the rate of absorption
– Expressed in terms of hours or minutes.
• Area under curve (AUC)
– Is the measurement of the extent of the drug
bioavailability
– It is the area under the drug plasma level-time curve
from
t =0 & t = ∞, and is equal to the amount of unchanged
drug reaching the general circulation divided by the
 Measurement of AUC
• Trapezoidal method:
– Most common method of estimating AUC.
– Divide the plasma conc-time curve into several
trapezoids.
– Count the trapezoids & find the area.
– Total area of the trapezoids will approximate the
area under the curve.
– More number of trapezoids formed, more
accurate will be the result.
– The area of one trapezoid between time t1 and t2
is (C1 +C2) /2.(t2 – t1 )
 Measurement of AUC
• CUT & WEIGH
– Preparing calibrated plot.
– Squares of graph are cut &weights are recorded
– Plot of weight Vs area.
– Sample curve is cut & weight is recorded.
– By interpolation method area of sample graph is found.
• PLANIMETER:
– Instrument for mechanically measuring the area under
the curve.
– Measures area by tracing outline of curve.
– Degree of error is high due to instrumental & human error.
•
• COUNTING THE SQUARE
– Total no. of squares enclosed in the curve is counted.
– Area of each square determined using relationship:
AREA=(height) (width)
 Urinary Excretion Studies
• Urinary excretion of unchanged drug is
directly proportional to plasma concentration
of drug.
• Thus, even if a drug is excreted to some
extent (at least 10 to 20%) in the urine,
bioavailability can be determined.
– Thiazide diuretics, Sulphonamides.
• Method is useful when there is lack of
suf ficiently sensitive analytical technique to
measure drug concentration.
• Non-invasive method, so better patient
compliance.
 Urinary Excretion Studies
• Bioavailability is generally assessed by the
determination of following three
parameters-
– (dxu/dt)max : (Maximum urinary excretion rate)
Its value increases as rate and/or extent of
• absorption increases.
• Obtained from peak of plot between rate of
excretion versus midpoint time of urine collection
period.
• Related to Cmax of plasma level data.
– (tu)max :Time for maximum excretion rate
• Its value decreases as absorption rate increases.
• Analogues of tmax of plasma level data.
– Xu∞ : Cumulative amount of drug excreted in
urine
• Related to AUC of plasma level data.
• It increases as the extent of absorption increases..
Bioequivalence – Extent of
Absorption

Plasma data-
AUC

Urine data-
Cumulative amount
excreted unchanged in
urine (Xu)

21
Bioequivalence – Extent of
Absorption
Plasma
data

Urine
data

22
Acute Pharmacologic Response
Method
• When bioavailability measurement by
pharmacokinetic method is difficult, an acute
pharmacologic effect related to time course of
drug, such as
– Effect on pupil diameter,
– EEG & ECG readings.
• Bioavailability can then be determined by
construction of pharmacological effect- time curve
as well as dose response graphs.
• Disadvantage:
– It tends to be more variable.
– Observed response may be due to an active
metabolite whose concentration is not
proportional to concentration of parent drug.
 Therapeutic Response Method
• This method based on observing the
clinical response to a drug formulation
given to patient suffering from disease.
• Drawbacks:
– Quantitation of observed response is
difficult to correlate with relative
bioavailability between two dosage forms
of the same drug.
• Anti-inflammatory drugs.
– Many patients receive more than one drug
simultaneously.
 Clinical Trials
• Clinical trials in humans establish the
safety and effectiveness of the drug
products and also used to determine
bioavailability.
• The FDA consider this approach only
when analytical methods and
pharmacodynamic methods are not
available.
• Comparative clinical studies have been
used to establish bioequivalence for
topical antifungal drug product. E.g.:
 Drug dissolution studies
• Under certain conditions, dissolution
prof ile may give an indication of drug
bioavailability.
• Dissolution studies are often performed in
several test formulations of the same drug.
• The test formulation that demonstrates
the most rapid rate of drug dissolution in-
vitro will generally have the most rapid rate
of drug bioavailability in-vivo.
• The FDA may also use the other in-vitro
approaches for establishing
bioequivalence.
 Study designs
• Cross over study Design
• Parallel Design
• Replicate Design

• Completely Randomized
Design
• Randomized Block Design
• Latin Square method
 REPEATED MEASURES/CROSS OVER/CARRY
OVER DESIGNS
• Administration of two or more treatments one after the
other in a specified or random order to the same group
of patients.eg: clinical trials to monitor safety and side
effects.
– Complete randomization is used to randomize the order of
treatments for each subject.
– Randomizations for different subjects are independent of
each other.
• ADVANTAGES :
– Good precision for comparing treatments because all
sources of subject variability are excluded.
– It is economic on subjects.
• DISADVANTAGES :
– There may be an order effect connected with the position in
treatment order.
– There maybe a carryover effect connected with the
preceding treatment or treatments.
 Crossover Designs
• Advantages
– Globally applied standard protocol for
bioequivalence, drug-drug interaction, food effect
studies.
– Straigthforward statistical analysis.
• Disadvantages
– Not suitable for studies in patients with unstable
diseases
– Not optimal for drugs with long half life
– Not optimal for highly variable drugs / drug
products
 Parallel design
• Advantages
– Sometimes faster than crossover.
– Straigthforward statistical analysis.
– Drugs with long half life.
– Potentially toxic drugs or effect and/or adverse effects
unacceptable in healthy subjects.
– Studies in patients, where the condition of the disease
irreversibly changes.
• Disadvantages
– Lower statistical power than crossover
– High sample sizes.
– Tight inclusion-/exclusion criteria to reduce between-
subject variability.
 Replicate Designs
• Each subject is randomly assigned to
sequences, where at least one of the
treatments (generally the reference) is
administered at least twice
– Not only the within-subject variability, but also
the within-subject variability per treatment may
be estimated.
– Smaller subject numbers compared to a
standard 2×2×2 design
– Increased number of periods.
– study costs directly related to number of
biosamples & hence more.
 Completely Randomized
Designs
• Treatments are randomly allocated among all experimental subjects.
 All subjects are labeled with same no of digits eg.for
20 subjects, numbers 1 to 20 are allotted.
 Non repeating numbers selected at random for f irst
treatment and then repeat for all other treatments.
ADVANTAGES :
 Easy to construct.
 It can accommodate any no of treatments and
subjects.
 Easy and simple to analyze even though sample size
may be different for each treatment.
DISADVANTAGES :
 It is suitable mostly for situations in which there are
few treatments.
 All subjects need to be as homogenous as possible.
 Randomized Block
• The subjects Design
are sorted into homogenous groups called blocks
and treatments are assigned at random within the blocks.
– subjects having similar characteristics are formed into blocks.
The t r e a t m e n t s a r e r a n d o m i z e d w i t h i n e a c h b l o c k .
Randomization for different blocks are done independent
of each other.
ADVANTAGES ;
– With systematic grouping ,it can provide more accurate results
than randomized designs of comparable size.
– It can accommodate any no of treatment or replications.
– Treatments need not have equal sample size.
– Design is easy and simple to construct.
– In case of spoiled results, an entire treatment or block can be
dropped thus avoiding complications.
– Scope for variability without sacrificing precision.
• DISADVANTAGES :
– Missing observations require more complex analysis.
– Degrees of freedom for error not as large as in random designs.
 LATIN SQUARE DESIGNS
• It is a two factor design where the subject
and treatment are two factors with one
observation in each cell. Such design is
useful when three or more treatments are
to be compared and carry over effects are
balanced.
• In a Latin square, rows represent subject
and columns represent treatments.
• EgTable below shows latin square design
for 12 subjects with formulation A,B,C
Subject Study Washout Study Washout Study
number period 1 period period 2 period period 3

1,7 A B C

2,8 B C A

3,9 C A B

4,10 A C B

5,11 C B A

6,12 B A C
 LATIN SQUARE DESIGNS
• ADVANTAGES :
– Minimum inter-subject variability in plasma drug levels.
– Minimizes variation due to time effect.
– Helps to focus more on formulation variables.
– Treatment effects can be studied from small scale
experiment.

• DISADVANTAGES :
– Very small no of degrees of freedom for error when only
few experiments are studied.
– The randomization required is more complex.
– Longer duration of study as adequate washout period is
required
– When large no of formulations are studied the study
becomes more difficult and subject dropout rates are high.
 BIOWAIVER
• The term biowaiver is applied to a
regulatory approval process when the
application is approved on the basis of
evidence of equivalence other than an in
vivo BE test.
• For solid oral dosage forms, the evidence
of equivalence is determined on the basis
of an in vitro dissolution profile
comparison (IVIVC).
 BIOWAIVER
1.The drug product differs only in strength of
the a c t i v e s u b s t a n c e s i t c o n t a i n s ,
provided all the following conditions
hold –
• Pharmacokinetics are linear
• The qualitative composition is the same
• The ratio between active substance and
excipients is the same ( or in case of small
strength, the ratio between the excipients
is the same)
 BIOWAIVER
• Both products are produced by the same
manufacturer at the same production site
– A bioavailability or bioequivalence study has
been performed with the original product
– Under the same test conditions , the in vitro
dissolution rate is the same
2. The drug product has been slightly
reformulated or manufacturing method has
slightly modified by the original manufacturer
in ways that can convincingly be argued to be
irrelevant for the bioavailability .
 BIOWAIVER
3. The drug product meets all of the following
requirements –
• The product is in the form of solution or
solubilised form ( elixir, syrup, tincture etc)
• The product contains active ingredient in the same
concentration as the approved drug product
• The product contains no excipients known to
signif icantly affect absorption of the active
ingredient.
4. Acceptable IVIVC and the in vitro dissolution rate
of the new product is equivalent with that of the
already approved medicinal product.
 BIOWAIVER Based on
the Pharmaceutical
• TheDosage
product isForm
intended for topical
administration (cream , ointment , gel , etc.)
for local effect
• The product is for oral administration but
not intended to be absorbed ( antacid or
radio opaque medium)
• The product is administered by inhalation
as a gas or vapour .
 Biowaiver Based on the
Pharmaceutical Dosage
• Form
Aqueous solutions to be administered
parenterally
• Solutions for oral use that do not contain an
excipient that affects GI transit or absorption of
the API
• Gases
• Powders for reconstitution as a solution
• Otic or ophthalmic products prepared as aqueous
solutions
• Topical products prepared as aqueous solutions
• Inhalation products or nasal sprays to be
administered with essentially the same device.
 Biowaiver Based on the BCS
• In case of biowaiver consideration based on
BCS system, the dissolution study results (in
vitro test) of the reference & test dosage
forms are compared.
• On the basis of dissolution rate, the
pharmaceutical dosage forms are classified
as -
– very rapidly dissolving, if 85% or greater of the the
dosage form dissolves in 15 minutes or less;
– rapidly dissolving, if 85% or greater of the dosage
form dissolves in 30 minutes; or
– Slowly dissolving, if the dosage form takes more
than 30 minutes for 85% of drug dissolution.
 Biowaiver Based on the BCS
• For biowaiver, the dissolution tests should
be carried out for both generic and
reference product under the same test
conditions (using media of pH 1.2, 4.5
&6.8).
• For the generic product to be eligible for
biowaiver, the reference product should
belong to the same BCS class and should
meet dissolution profile comparison
criteria.
 Biowaiver Based on the
BCS Class BCS Criteria

I • 85% or more of the dosage form dissolves in 30 minutes

or less in all 3 media and
• The dissolution profile of the generic product is similar to
that of the reference product
II • 85% or more of the dosage form dissolves in 30 minutes
or
less in pH 6.8 buffer media and
• The dissolution profile of the generic product is similar to
that of the reference product
III • Both the reference and the generic dosage forms are
very rapidly dissolving (85% dissolution in 15 minutes or
less in all three media under the test conditions given
above), and
• They do not contain any excipients and/or inactive
substances that are known to alter gastrointestinal
motility and/or permeability or influence drug absorption
Elements of Bioequivalence Study
Protocol
1. Title
a) Principle investigator
b) Project number and date

2. Study objective

3. Study design
a) Design
b) Drug products
i. Test product(s)
ii. Reference product
c) Dosage regimen
d) Sample collection
schedule
e) Housing
f) Fasting/meals schedule
g) Analytical methods
Elements of Bioequivalence Study
Protocol
4. Study population
a) Subjects
b) Subject selection
i. Medical history
ii. Physical examination
iii. Laboratory tests
c) Inclusion/exclusion criteria
d) Restrictions/prohibitions

5. Clinical procedures
a) Dosage and drug administration
b) Biological sampling schedule
c) Activity of subjects
Elements of Bioequivalence Study
Protocol
6. Ethical considerations
a) Basic principles
b) Institutional review board
c) Informed consent
d) Indications for subject withdrawal
e) Adverse reactions and emergency
procedures

7. Facilities

8. Data analysis
a) Analytical validation procedure
b) Statistical treatment of data

9. Drug accountability

10. Appendix