Transcript Blue with White Corner

DRUG BIOAVAILABILITY
Prof. Dr. Basavaraj K. Nanjwade M. Pharm., Ph. D
Department of Pharmaceutics
KLE University’s College of Pharmacy
BELGAUM – 590010, Karnataka, India
Cell No; 0091 9742431000
E-mail: [email protected]
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Contents
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•
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•
•
Introduction
Objectives of bioavailability studies
Considerations in bioavailability studies
Bioavailability concept
Importance of bioavailability
Equivalent concept… Chemical, Pharmaceutical, Bioequivalence &
Therapeutic equivalence.
• Measurement of bioavailability
1. Pharmacokinetic method
2. Pharmacodynamic method
• Measurement of Rate of absorption
1. The method of ‘Residuals’
2. Wagner Nelson Method
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Contents
• Bioequivalence studies & Its importance
 Introduction
 FDA Determinations of Bioequivalence
Main Terms





FDA Methods to Determine Bioequivalence
Bases for Determining Bioequivalence
Design And Evaluation of Bioequivalence Studies
Evaluation of the Data
Clinical importance of bioequivalence studies
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Introduction
• The therapeutic effectiveness of a drug depends
upon the ability of the dosage form to deliver the
medicament to its site of action at a rate &
amount sufficient to elicit the desired P’cological
response.
• This attribute of the dosage form is referred to as
physiologic availability or bioavailability.
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Introduction
• The term bioavailability is defined as the rate
and extent of absorption of unchanged drug
from its dosage form.
• A rapid onset of action is desired as in the
treatment of acute conditions like asthma, pain,
etc.
• A slower absorption is desired when aim is to
prolong the duration of action.
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Introduction
• The influence of route of administration on
drug’s bioavailability is generally in the foll. order
parenteral > oral > rectal > topical
• Most drugs are administered orally, for reason of
stability and convenience.
• The dose available to patient – Bioavailable
dose.
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Introduction
• The amount of drug that reaches the systemic
circulation – systemic availability.
• Bioavailable fraction (F), refers to the fraction of
administered dose that enters the systemic
circulation.
• F =
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Bioavailable dose
Administered dose
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Objectives of bioavailability studies
•
It is important in
1. Primary stages of development of a suitable
dosage form for new drug entity.
2. Determination of influence of excipients,
patient related factors and interaction with
other drugs on the efficiency of absorption.
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Objectives of bioavailability studies
3. Development of new formulations of the
existing drugs.
4. Control of quality of a drug product during early
stages of marketing (to determine the influence
of processing factors, storage & stability of
absorption).
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Considerations in bioavailability studies
• Bioavailability – absolute vs. relative
• Single dose vs. multiple dose
• Human volunteer – healthy subject vs. patients
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Bioavailability – absolute vs. relative
• Absolute bioavailability : when the systemic
availability of the drug administered orally is
determined in comparison to its i.v.
administration.
• Relative bioavailability : when the systemic
availability of the drug administered orally is
compared with that of an oral standard of the
same drug.
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Plasma concentration
Plasma concentration vs. time profile of a drug ingested
orally & intravenously.
70
60
i.v. route
50
40
oral route
30
20
10
0
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4
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10
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Single dose vs. multiple dose
Single dose
• Very common & easy
Multiple dose
• Difficult to control
• Less exposure to
drug & less tedious
• More exposure to
drug & tedious
• Difficult to predict
steady state
• Time consuming
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Time to reach steady state
Concentration due to
repeated doses
Concentration due to a single dose
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Single dose vs. multiple dose
•
Multiple dose study has several advantages
like
1. More accurately reflects the manner in which
the drug should be used.
2. Drugs levels are higher due to cumulative
effect which makes its determination possible
even by less sensitive analytical methods.
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Single dose vs. multiple dose
3. Better evaluation of the performance of
controlled release formulation is possible.
4. Small intersubject variability is observed which
allows use of fewer subjects.
5. Nonlinearity in pharmacokinetics, if present,
can be easily detected.
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Human volunteer – healthy subject vs. patients
•
Ideally, the bioavailability study should be
carried out in patients for whom the drug is
intended to be used.
• Advantages :
1. Patient is benefited from the study.
2. Reflects better therapeutic efficacy of drug.
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Human volunteer – healthy subject vs. patients
3. Drug absorption pattern in disease state can
be evaluated.
4. Avoids the ethical quandary of administering
drug to healthy subjects.
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Human volunteer – healthy subject vs. patients
• There are some drawbacks of using patients as
volunteers.
• Stringent conditions such as fasting state
required is difficult to be followed by the patients.
• Studies are therefore performed in young (20-40
yrs.), healthy males adult volunteers.
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Human volunteer – healthy subject vs. patients
• Female volunteers are used only when drugs
such as oral contraceptives are to be tested.
• No. of subject- extent of intersubject variability –
minimum required to obtain reliable data.
• They must be informed about the importance of
 Study
 conditions to be followed

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Human volunteer – healthy subject vs. patients
• Medical examination should be performed.
• Drug washout period for min. of ten biological
half lives must be allowed for between two
studies in same subject.
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Bioavailability Concept
• Rate and extent at which therapeutically active drug
reaches systemic circulation.
• The fraction of administered dose that reaches the
systemic circulation in contrast to that stated on label.
• Rate & extent of absorption of unchanged drug from its
dosage form.
• A measure relative to some standard of rate & amount
of drug ,which reaches the systemic circulation
unchanged following the administration of dosage form.
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Why bioavailability studies…?
• FDA requires that the drug product is safe
and effective.
• Measure of bioavailability: AUC/dose
• Absolute availability - for drugs with
approved NDA, bioavailability studies are required
for new drug formulations -bioequivalence to the
reference formulation
• Relative availability - for drugs without full NDA,
bioequivalence to the reference drug in the standard
formulation
• For determining safety & efficacy of drug product.
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Concept of Equivalents
• A comparative term.
• One drug product is similar with respect to a specific
characters or function to another .
• Defined a set of standard.
• Four types …
1. Chemical
2. Pharmaceutical
3. Bioequivalence
4 .Therapeutic
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Concept of Equivalents
1. Chemical Equivalence
Two or more drug products which contain same amount of
same active ingredient.
2. Pharmaceutical Equivalence
Identical in strength,quality,purity, content uniformity &
disintegration – dissolution characters … they may differ
in containing different excipient.
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Concept of Equivalents
3. Bioequivalence
A relative term which denotes ..the drug substance in
two or more identical dosage form reaches the systemic
circulation at the same relative rate & to same relative
extent.
4. Therapeutic Equivalence
Two or more bioequivalent having same therapeutically
active ingredient elicit pharmacological effects & can
control the disease to same extent.
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Measurement of
bioavailability
• Pharmacokinetic methods ( indirect )
1. Blood analysis
2. Urinary excretion data
• Pharmacodynamic methods ( direct )
1. Acute pharmacological response
2. Therapeutic response
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Blood analysis
• Plasma level time studies or The plasma concentration – time
curve or blood level curve.
• A direct relationship exists concentration of drug at the site of
action & concentration of drug in the plasma.
• Serial blood samples are taken after drug administration &
analyzed for drug concentration.
• A typical blood level curve obtained after oral administration of
drug.
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Parameters determined
Pharmacokinetic parameters
• Peak Plasma Concentration (Cmax)
• Time of Peak concentration (tmax).
• Area Under Curve (AUC)
•
•
•
•
•
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Pharmacodynamics parameters
Minimum Effective Concentration (MEC) / Minimum
Inhibitory Concentration (MIC).
Maximum Safe Concentration (MSC) / Maximum Safe
Dose (MSD).
Duration of action
Onset of action.
Intensity of action.
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Parameters determined
•
AUC or Extent of absorption can be measured by 3
methods…
1.Planimeter
Instrument for mechanically measuring the area
2. Cut & weigh method
AUC is cut & weighed on analytical balance. The weight
obtained is converted to proper unit by dividing it by the wt
of a unit area of same paper.
3. Trapezoidal method
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Parameters determined
3. Trapezoidal method
AUC = ½ ( C1 + C2) (t2 – t1) + ½ (C2 + C3) (t3 – t2) +…….
½ (C n-1 + C n ) (tn – tn-1 )
C = Concentration
t = time
subscript= sample number
AUC = Area Under Curve
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Parameters determined
Relative bioavailability
F rel =
( AUC) drug . (Dose) standard
(AUC) standard .(Dose) drug
Absolute bioavailability
Fab =
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(AUC)drug . (Dose) IV
(AUC)IV . (Dose) drug
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Parameters determined
•From the following blood data obtained after the oral administration of 50mg of
drug A. calculate the AUC?
Time in hr
Plasma drug con in mcg/ml
1
2
5.5
9.2
3
14.9
4
5
10.3
7.3
6
2.2
AUC = ½ (5.5 +9.2) (2-1) + ½ (9.2+14.9) (3-2) + ½ (14.9+10.3) (4-3)
½ (10.3+ 7.1)(5-4) + ½ (7.1 +2.2) (6-5)
AUC = 45.35 mcg/ml hr
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Parameters determined
• The AUC of a new sustained release diclofenac sodium
developed in the lab after giving in a dose of 100mg was
found to be 250.30 mcg/ml hr.The AUC of the standard
marketed sustained release tablets of the same at the
same dose was found to be 261.35 mcg/ml hr. what is
the the relative bioavailability of he same drug.
F rel = 250.30 X 100
261.35 X 100
= 0.9577 or 95.77%
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Parameters determined
• The AUC of salbutamol sulphate from a 10 mg
IV dose was found to be 94.6mcg/ml hr.when
the same dose was given orally, the AUC was
found to be 60.5 mcg/ml hr. What is the
absolute bioavailability of the drug?
Fabs = 60.5 X 10
94.6 X 10
Fabs = 0.6395 or 63.95
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Urinary excretion data
• The method of determination bioavailability
provided that the active ingredient is excreted
unchanged in the significant quantity of urine.
• The cumulative amount of active drug excreted in
urine is directly proportional to extent of systemic
drug absorption.
• The rate of drug excretion is directly proportional
to rate of systemic drug absorption.
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Advantages
• Useful when there is lack of sufficiently sensitive analytical
techniques to measure concentration of drug in plasma.
• Noninvasive method therefore better subject compliance.
• Convenience of collecting urine samples in comparison to
drawing of blood periodically.
• If any case the urine drug concentration is low, assaying of
larger sample volume is relatively more.
• Direct measurement of bioavailability, both absolute &
relative is possible without the necessity of fitting the data
to the mathematical model.
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Advantages
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Advantages
• Bioavailability is determined by….
F= (U
(U
) oral . D IV
) IV . D oral
U
= Cumulative amt of unchanged drug excreted in
urine
D IV = IV dose
D oral = oral dose
F = absolute bioavailability
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Advantages
• When equal doses are administered Intravenously & Orally…..
F = (U
(U
) oral
) IV
F = (U
(U
) test
) standard
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Advantages
• When drug A was administered IV to a group of volunteers, 80%
of the 500mg dose was recovered unchanged in the urine. When
the same drug was administered to the same volunteers
orally.280 mg was recovered unchanged in urine. What is the
absolute bioavailability of Drug A following oral administration.
Absolute bioavailability = (cumulative amt.of drug excreted)sample
(cumulative amt.of drug excreted)IV
=
280
400
= 0.7 or 70%
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Acute pharmacological response
• Bioavailability can be determined from the acute
pharmacologic effect – time curve as well as from dose
response graph.
• DISADVANTAGE is that pharmacological response tends to
more variable & accurate correlation between the measured
response & drug available from the formulation is difficult.
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Therapeutic response
• This method is based on the observing the clinical response to a
drug formulation given to a patients suffering from disease for
which it is intended to be used.
• Ex …for anti inflammatory drugs, the reduction in the
inflammation is determined.
• The major DRAWBACK is …quantification of observed response
is too improper to allow for reasonable assessment of relative
bioavailability between two dosage forms of a same drug.
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Rate of Absorption
• AUC/dose gives an average extent of bioavailability.
• The rate of absorption is usually also important
for the onset of drug action.
• The time of peak plasma concentration is used
often as a measure of the rate of drug absorption.
• The peak plasma concentration is also an
important parameter - for keeping the drug
concentration within the therapeutic window.
• Absorption can be characterized by evaluating the absorption rate
constant Ka from the plasma concentration –time data.
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The method of ‘Residuals’
• Also called as Feathering or peeling or stripping.
ASSUMPTIONS
• Absorption & elimination process follows 1st order kinetics.
• Absorption from the dosage form is complete.
• Ka is at least five times larger than Ke
• Kinetic model is
AG
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Ka
AB
Kc
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The method of ‘Residuals’
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The method of ‘Residuals’
• This technique is used to resolve a multiexponential
curve into its individual components.
• For a drug that follows one compartment kinetics &
administered e.v, the concentration of drug in plasma is
expressed by…
C=
Ka F X0
Vd ( Ka –KE )
[ e –kEt – e –Kat ]
1
If Ka F X0 / Vd ( Ka – kE ) = A, a hybrid constant then,
C=Ae
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–kEt
-A e –Kat
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The method of ‘Residuals’
• During the elimination phase, when the absorption is
almost over, Ka > > KE & the value of second exponential e
–Kat approaches zero whereas the 1st exponential e –kEt
retains some finite value….at this time the equation is
C = A e –kEt
in log form
log C = log A – KEt/ 2.303
3
4
Where C is the back extrapolated plasma concentration
value.
A plot of log C versus t yields a biexponential curve with a
terminal linear phase having slope KE/ 2.303.
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The method of ‘Residuals’
• Back extrapolation of this straight line to zero yields
y-intercept equal to log A.
• Subtraction of true plasma concentration values that
is equation 2 from the extrapolated plasma
concentration values that is equation 3 yields a series
of residual concentration values Cr
( C - C ) = Cr = A e –Kat ,
in log form the equation is :
log Cr = log A - Kat/ 2.303
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The method of ‘Residuals’
• A plot of logCr versus t yields a straight line with
slope –Ka / 2.303 & y intercept log A.
• Absorption half life can be computed from Ka using
the relation 0.693/Ka …thus the method of residual
enables resolution of the biexponential plasma level
time curve into its exponential components.
• The technique works best when the difference
between Ka & KE is large ( Ka >= 3)
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Wagner Nelson Method
ASSUMPTIONS
• The body behaves as a single homogenous compartment.
• Drug elimination obeys 1st order kinetics.
DISADVANTAGES
• The absorption & elimination processes can be quite similar &
still accurate determination of Ka can not be made.
• The absorption process doesn’t have to be 1st order.
• The kinetics of absorption may be zero order, mixed order,
mixed zero order & 1st order or even more complex.
• This method involves determination of Ka from percent
absorbed –time plot & does not require the assumption of zero .
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Wagner Nelson Method
• The amount of drug in the body X & the amt of drug eliminated
in the body XE thus :
XA = X + X E
If the amt of drug in the body is X = V.dC…the amt of drug
eliminated at any time t can be calculated as ..
XE = KE Vd ( AUC)to
Substitution of values of X & XE in above equation…
XA = Vd C + KE Vd ( AUC)to
from this equation we can get the value for drug absorbed in to
the systemic circulation from time zero to
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Wagner Nelson Method
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BIOEQUIVALENCE STUDIES &
ITS IMPORTANCE
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BIOEQUIVALENCE STUDIES
• Bioavailability & bioequivalence data is
required to be furnished with application of new
drugs, as required under schedule Y, depending
upon the type of application being submitted.
• Both bioavailability & bioequivalence focus on
release of drug substance from its dosage form
& subsequent absorption into systemic
circulation.
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•
1.
2.
3.
4.
Several test methods are available to
asses equivalence, including;
Comparative bioequivalence studies, in
which the active drug substance or one
or more metabolites is measured in
accessible biological fluid such as
plasma, blood or urine.
Comparatives pharmacodynamic studies
in humans
Comparative clinical trials
In-vitro dissolution tests
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FDA Determinations of Bioequivalence
Main Terms
•
•
•
•
Pharmaceutical equivalents
Pharmaceutical alternatives
Therapeutic equivalents
Bioequivalence
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Pharmaceutical Equivalents
• Drug products are considered pharmaceutical
equivalents if they contain the same active
ingredient (s), have the same dosage form and
route of administration, and are identical in
strength or concentration
• Equivalent products contain the same amount of
ingredient in the same dosage form but may
differ in characteristics, such as shape, release
mechanisms, and packaging
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Pharmaceutical Alternatives
• Drug products are considered pharmaceutical
alternatives if they contain the same therapeutic
moiety, are different salts, esters, or complexes
of the same moiety, are different dosage forms,
or are different strengths
• Other pharmaceutical alternatives
– Different dosage forms and strengths within a single
product line by a single manufacturer
– Extended-release formulations when compared with
immediate- or standard-release formulations
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Therapeutic Equivalents
• Drug products are considered therapeutic
equivalents if they are all of the following
–
–
–
–
–
Pharmaceutical equivalents
Bioequivalent
Approved as safe and effective
Adequately labeled
Manufactured in compliance with current Good
Manufacturing Practice regulations
• Therapeutic equivalents are expected to have
the same clinical effect and safety profile
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Bioequivalence
• Bioequivalence of a drug product is
achieved if its extent & rate of absorption
are not statistically significantly different
from those of the reference product when
administered at the same molar dose.
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FDA Methods to Determine
Bioequivalence
• Generic drug manufacturers must
demonstrate that a drug is bioequivalent to
a reference drug product
• In order of FDA preference, methods used
to define bioequivalence
– Pharmacokinetic studies
– Pharmacodynamic studies
– Comparative clinical trials
– In vitro studies
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Important PK parameters
Cmax:
the observed maximum concentration of a drug
 measure of the rate of absorption
AUC:
tmax:
time at which Cmax is observed
area under the concentration-time curve
 measure of the extent of absorption  measure of the rate of absorption
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Studies
Different approach for
establishing equivalence
PD studies
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clinical
studies
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in vitro
methods
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Methods for Assessing
Bioavailability & Bioequivalence
• Plasma Drug Concentration:1. The time for peak plasma (blood)
concentration (tmax)
2. The peak plasma drug concentration
(cmax)
3. The area under the plasma drug
concentration versus time curve (AUC)
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Contd…
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Plasma concentration time profile
Cmax
AUC
Tmax
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•
Urinary Drug Excretion:-
1. Cumulative amount of drug excreted in
the urine (Du)
2. The rate of drug excretion in the urine
(dDu/dt)
3. The time for maximum urinary excretion (t)
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• Acute pharmacodynamic effect:1. Max. pharmacodynamic effect (Emax)
2. Time for max. pharmacodynamic effect
3. Area under pharmacodynamic effect
versus time curve
4. Onset time for pharmacodynamic effect
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• Clinical observations
Well controlled clinical trials
• In-Vitro studies
Drug dissolution.
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• 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 clearance
[ AUC]0∞ = ∫0∞Cpdt
[ AUC]0∞ = FD0
= FDO/ kVD
Clearance
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Where F = fraction of dose absorbed
Do = dose, k = elimination rate constant,
VD = volume of distribution
AUC is independent of route of administration
and process of drug elimination as long as the
elimination process don’t change.
The AUC can be determined by numerical
integration procedure such as trapezoidal rule
method. The units for AUC are concentration
time ( μg hr/ml)
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• For many drugs, AUC is directly proportional to the
dose, for eg. If a single dose of drug is increased from
250- 1000mg the AUC will also show a four fold
increase fig
Plasma level time curve
following administration
of single doses of
(A)250mg, (B)500mg &
(C)1000mg of drug.
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Linear relationship between AUC & dose
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• In some cases the AUC is not directly proportional to the
administered dose for all dosage levels, for eg. As the
dosage of the drug is increased, one of the pathway for drug
elimination becomes saturated, fig
Relationship between AUC
& dose when metabolism
is saturable
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• Drug elimination includes metabolism and
excretion.
• for drugs such as salicylates and
phenytoin continuous increase of dose
causes saturation of one of the enzyme
pathway for drug metabolism and
consequent prolongation of elimination of
half life.
• The AUC thus increases disproportionally
to the increase in dose because smaller
amount of drug is being eliminated.
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• When AUC is not directly proportional to
the dose, bioavailability of the drug is
difficult to evaluate because drug kinetics
may be dose dependent.
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Urinary Drug Excretion Data
• It is an indirect method for estimating
bioavailability.
• The drug must be excreted in significant
quantities as unchanged drug in the urine,
timely urine samples must be collected
and total amount of urinary excretion must
be obtained.
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• Du∞:-
The cumulative amount of drug excreted in
the urine, Du∞ is directly related to total
amount of drug absorbed. Experimentally,
urine samples are collected periodically
after administration of the drug product.
Each urine specimen is analyzed for free
drug with a specific assay.
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Graph is plotted between cumulative drug excreted to the
collection time interval.
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• The relationship between cumulative
amount of drug excreted in urine and
plasma level time curve is shown in fig.,
( 1)
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• When the drug is almost completely
eliminated (point c), the plasma
concentration approaches zero and
maximum amount of the drug excreted in
the urine, Du∞ is obtained
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• dDu/dt
Because most drugs are eliminated by
first order rate process, the rate of drug
excretion is dependent on first order
elimination rate constant k and the
concentration of drug in the plasma Cp.
In fig, the maximum rate of drug excretion
would be at point B, the minimum rate of
drug excretion would be at point A and C.
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(2)
Graph comparing rate of drug excretion w.r. to time
should be similar in shape as the plasma level- time
curve for that drug.
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• t∞ :it is the total time for the drug to be
excreted. In fig 1 & 2, slope of curve
segment A-B is related to the rate of drug
absorption, whereas point C is related to
the total time required after drug
administration for the drug to be absorbed
and completely excreted ( t = ∞).
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Acute Pharmacodynamic effect
• An Acute Pharmacodynamic effect such
as an effect on pupil diameter, heart rate,
or B.P can be used as an index of drug
bioavailability.
• In this case, an acute pharmacodynamic
effect- time curve is constructed.
Measurement of pharmacodynamic effect
should be made with sufficient frequency
to permit a reasonable estimate of the total
AUC for a time period at least three times
a half life of the drug.
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• In this to determine bioavailability requires
demonstration of a dose related response.
• Bioavailability is determined by
characterization of dose-response curve.
• PD parameters that are obtained include
total area under the acute PD effect- time
curve, peak PD effect, & time for peak PD
effect.
• onset time & duration of PK effect may
also be included in the analysis of data.
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Clinical Observations
• Well-controlled clinical trials in humans
that establish the safety & effectiveness of
the drug product.
• This approach is least accurate, least
sensitive & least reproducible of the
general approaches for determining in-vivo
bioavailability.
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• The FDA only consider this approach
when analytical methods are not available
to permit use of one of the approaches
described above.
• An E.g. of this approach is for the
determination of bioequivalence of two
topical antifungal products by different
mfgs. Containing the same active
antifungal agent (ketoconazole)
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In-Vitro Studies
• Under certain conditions give an indication
of drug bioavailability. Ideally, in-vitro
dissolution rate should correlate with invivo drug bioavailability.
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Bases for Determining
Bioequivalence
• Bioequivalence is established if the in-vivo
bioavailability of a test drug product (Generic
product) does not differ significantly in the
products rate & extent of drug absorption, as
determined by comparison of measured
parameters (eg, concentration of active drug
ingredient in blood, urinary excretion rate, or PD
effects), from that of the reference material
(Brand name product).
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Design And Evaluation of
Bioequivalence Studies
• Bioequivalence studies are performed to
compare bioavailability of generic drug
product to the brand name product.
• Design and evaluation of bioequivalence
studies requires comparative inputs from;
PKs, statisticians, clinicians, bioanalytical
chemist, & others.
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• Basic design for BE is determined by ;
1. The scientific questions to be answered,
2. The nature of reference material &
dosage form to be tested,
3. The availability of analytical methods,
4. Benefit-risk considerations with regards
to testing in humans.
• For some generic drugs, FDA offers
general guidelines for conducting these
studies.
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•
Eg, “Statistical procedure for bioequivalence
studies using a standard two-treatment
crossover design”
• The publication addresses three specific
aspects, including;
1. Logarithmic transformation of PK data,
2. Sequence effect
3. Outlier consideration.
• With such guidelines, the principal
investigator should prepare a detailed
Protocol for the study.
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• For bioequivalence studies, both test &
reference drug formulations contain
pharmaceutical equivalent drug in the
same dose strength, in similar dosage
forms (immediate release or controlled
release), & both are given by the same
route of administration.
• Both single &/or a multiple dose (steady
state) study required.
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• Prior to initiation of the study, the Institutional Review
Board (IRB) of the clinical facility in which the study is
to be performed must approve the study.
• Generally study is performed in normal, healthy male
volunteers.
• The number of subjects depend upon the expected
intersubject variability.
• The subject is generally fasted 10 to 12 hrs (overnight)
prior to drug administration & may continue to fast for
2 to 4 hr period after dosing.
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Reference standard
• Before beginning an in-vivo bioequivalence
study, the total content of active drug
substance in the Test product must be within
5% of reference product.
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Study Design
• The FDA, Division of Bioequivalence,
Office of Generic Drugs provides a
guidance for the performance of In-vitro
dissolution & in-vivo bioequivalence
studies.
• Currently, three different studies may be
required for solid oral dosage forms,
including;
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1. Fasting study
2. Food intervention study
3. Multiple dose (steady state)
 Fasting study:• Bioequivalence studies are evaluated by
single dose, two-period, two-treatment,
two-sequence, open label, randomized
crossover design comparing equal doses
of the test and reference products in
fasted, adult, healthy subjects.
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• This study required for all immediate release
and modified release oral dosage forms.
• Both male and female subjects used in the
study.
• Blood sampling is performed just prior (zero
time) to the dose and at appropriate intervals
after the dose to obtain an adequate plasmadrug concentration versus time profile.
• No other medication is normally given to subject
for atleast one week prior to the study.
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Food intervention study:• This study is single dose, randomized,
three-treatment, three-period, sixsegments, crossover, limited food effect
study comparing equal dosage of the test
product given under fasting conditions with
those of the test and reference products
given immediately after a standard high fat
content breakfast.
• For eg. Ibuprofen
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
Multiple dose ( steady state study ):-
• A multiple dose steady state, randomized, twotreatment, two-way, crossover study comparing
equal dosage of the test and reference products in
the adult, healthy subject is required for the oral
extended release (controlled release) drug
products in addition to a single dose fasting study
and food intervention study.
• Three consecutive trough concentrations (Cmin) on
three consecutive days should be determined to
ascertain that the subjects are at steady state.
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• The last morning dose is given to the
subject after an overnight fast with
continuous fasting for atleast two hours
following dose administration.
• Blood sampling is performed similarly to
the single dose study.
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Crossover Designs
• Subjects who meet the inclusion and exclusion
study criteria and have given informed consent
are selected at random.
• Each subject recieves the test drug product and
the reference product.
• E.g. of Latin square crossover design for a
bioequivalence study In human volunteers,
comparing three different drug formulations (A,
B, C) are described in tables
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Crossover Designs
Subject
Drug Product
1
Study
period 1
A
Study
period 2
B
Study
period 3
C
2
B
C
A
3
C
A
B
4
A
C
B
5
C
B
A
6
B
A
C
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• The Latin square design plans the clinical trial so
that each subjects receives each drug product only
once, with adequate time between medications for
the elimination of drug from the body.
• In this design, each subject is his own control, and
subject-to-subject variation is reduced.
• The variation due to sequence, period and
treatment (formulation) are reduced, so that all
patients don’t receive the same drug product on the
same day and in the same order
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• Possible carry-over effect from any particular
drug product are minimized by changing the
sequence in which the drug products are given
to the subjects as in the table.
• This Latin square design for bioequivalence
study of 4 drug products in 16 volunteers may
also be performed.
• After each subject recieves the drug product,
blood samples are collected at the appropriate
time intervals so that valid blood drug level-time
curve is obtained.
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• The time intervals should be spaced so
that peak blood concentration, total AUC,
and the absorption and the elimination
phases of the curve may be well
described.
• In some cases the measurement of drug in
urine may be necessary.
• Period refers to the time period in which
study is performed.
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• A two period study, referred to as two
legged study, means that the study is
performed on two different days (time
periods) seperated by a wash out period
during which most of drug has been
eliminated from the body- generally 10
elimination half lives.
• A sequence refers to the number of
different orders in the treatment groups in
a study.
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• E.g. two-sequence, two-period study
designed as follows:Period 1
Period 2
Sequence 1
T
R
Sequence 2
R
T
R = reference , T = treatment
As shown in the table
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Evaluation of the Data
• Analytical method:The method for measurement of the drug
must be validated for accuracy, precision,
sensitivity, and specificity.
Data should be presented in tabulated and
graphical form for evaluation.
The plasma drug conc. Versus time curve
for each drug product and each subject
should be available.
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• Pharmacokinetic evaluation of the
data:For single dose studies, including fasting
study or food intervention study, the PK
analyse include calculation for each
subject of the AUC to the last quantifiable
concentration (AUC0-t) and to infinity
(AUC0-∞), Tmax and Cmax.
Elimination rate constant k, elimination
half life t ½ may be estimated.
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• Statistical evaluation of the data:To prove bio-equivalence, there must be no
statistical difference between the bioavailability
of test product and reference product.
Statistical approaches assume that the data are
distributed according normal distribution or bell
shaped curve.
The log transformation of the bioavailability data
(Cmax, AUC) is performed prior to statistical data
evaluation for bioequivalence determination.
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• Analysis of variance (ANOVA):It is a statistical procedure used to test the
data for difference within and between
treatment and controlled groups. The
ANOVA may evaluate variability in
subjects, treatment groups, study periods,
formulation and other variables depending
on study design.
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• One sided tests procedure:This is also referred as confidence interval approach.
This method is used to demonstrate if the
bioavailability of the drug from the test formulation is
too low or high in comparison to the reference drug.
For a single dose, fasting study, analysis of variance
(ANOVA) is usually performed on the log-transformed
AUC and Cmax. There should be no statistical
difference between mean AUC and Cmax parameters
for the test (generic) and refernce drug products.
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In addition, 90% confidence intervals about
the ratio of the mean for AUC and Cmax values
of the test drug should not be less than 0.80
(80%) nor greater than 1.25 (125%) of the
reference product based on log-transformed
data.
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Clinical importance of
bioequivalence studies
• Bioequivalence of different formulations of the same
drug substance involves equivalence with respect to
the rate and extent of systemic absorption.
• Generally two formulations whose rate and extent of
absorption differ by 20% or less are considered
bioequivalent
• When a therapeutic objectives of the drug are
considered, an equivalent clinical response should be
obtained from the comparison dosage form if the
plasma drug concentration remain above MEC for
appropriate interval and don’t reach the MTC.
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Clinical importance of
bioequivalence studies
• Bioequivalence studies should be conducted for
the comparison of two medicinal products
containing the same active substance.
• Two products marketed by different licensees,
containing same active ingredient(s), must be
shown to be therapeutically equivalent to one
another in order to be considered
interchangeable.
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Special concerns in bioavailability
& bioequivalence studies.
• The bioequivalence determination of the
drugs that are not absorbed systemically
form the site of application is difficult to
assess. For this non-systemic absorbable
drugs surrogate marker is needed for the
bioequivalence determination
• Possible surrogate markers for
bioequivalence studies are given in table.
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Drug product
Possible surrogate
marker for
bioequivalence
MDI
Albuterol
FEV1
Topical steroid
Hydrocortisone Skin blanching
Anion exchange cholestryamine Binding to bile
resin
acids
Antacids
Mg and Al
Neutraliztion of
hydroxide gel acid
Topical
ketoconazole
Drug uptake into
antifungal
stratum corneum
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Drug
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Reference standard
• Before beginning an in-vivo bioequivalence
study, the total content of active drug
substance in the Test product must be within
5% of reference product.
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References
• Dissolution, bioavailability & bioequivalence –
Hamed M. Abdou, Mack publishing company,
Pg.no. 297-299.
• Biopharmaceutics & pharmacokinetics –
D.M.Bramhankar, S.B.Jaiswal, Pg.no. 297-299.
• Biopharmaceutics & pharmacokinetics –
Robert E. Notari, 2nd edition, Marcel Dekker.
•18/11/2010
www.google.com
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References
• “Applied Biopharmaceutics & pharmacokinetics”, Leon
Shargel & Andrew B.C.
• Leon Shargel, Andrew Yu; “Applied biopharmaceutics
& Pharmacokinetics”, 4th edition
• Peter G. Welling, Francis L.S. Tse; “Pharmaceutical
Bioequivalence”, Vol 48, Dekker Series.
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THANK YOU
Cell No: 0091 9742431000
E-mail: [email protected]
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