BIOANALYTICAL METHOD

FOR

BIOAVAILABILITY/
BIOEQUIVALENCY ASSAY
Jutti Levita

BIOAVAILABILITY/BIOEQUIVALENCY ASSAY
11/18/2011

JUTTI LEVITA
2011

Definition
Bioavailability:

Amount of a drug that is absorbed into

human systemic circulation in its active
form. It is determined by its concentration
in blood versus time, or its excretion in
urine

Bioequivalency:

Therapeutic equivalency of two drugs when

both of them are administered to the same
person in the same dosage and those will
show similar efficacy and safety

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Why is BA/BE Assay Necessary to Be

Carried Out?

Health cost
Substitute drug (with me-too product
or generic) will be preferred
Substitution drug should be equivalent
therapeutically (bioequivalent) with
the innovator

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BA/BE Laboratories
Requirements
Competent Clinical
Laboratory Resources

Subjects/
Healthy volunteers

Good Clinical Practice

ICH-GCP E6
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Competent Analytical
Laboratory Resources

Biological
sample

Bioanalytical
procedures

Good Laboratory Practice

ISO/IEC 17025
4

ICH - GCP E6
INTERNATIONAL CONFERENCE ON HARMONIZATION OF TECHNICAL
REQUIREMENTS FOR THE REGISTRATION OF PHARMACEUTICALS
FOR HUMAN USE

A standard for the design, conduct,

performance, monitoring, auditing, recording,
analyses, and reporting of clinical trials that
provides assurance that the data and
reported results are credible and accurate,
and that the rights, integrity, and
confidentiality of trial subjects are protected
ICH Guidelines 1.24

GCP regulations are applied to all drug and device studies

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ISO/IEC 17025:1999- General Requirements

for the Competence of Testing & Calibration
Laboratories ( Based on ISO9001:1994 )

Technical Criteria ISO/IEC17025:1999

4. Management Requirements

4.1 Organizaton
4.2 Quality system
4.3 Document control
4.4 Review of request, tenders or contract
4.5 Subcontracting of tests and calibrations
4.6 Purchasing services and supplies
4.7 Service to the client
4.8 Complaints
4.9 Control of NC Testing and/or calibration work
4.10 Corrective action
4.11 Preventive action
4.12 Control of records
4.13 Internal audits
4.14 Management Reviews
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ISO/IEC 17025:1999- General Requirements

for the Competence of Testing & Calibration
Laboratories ( Based on ISO9001:1994 )

5. Technical Requirements
ISO/IEC17025:1999
5.1 General
5.2 Personnel
5.3 Accommodation & environment conditions
5.4 Tests & calibration methods and methods validation
5.5 Equipment
5.6 Measurement traceability
5.7 Sampling
5.8 Handling of test and calibration items
5.9 Assuring the quality of test
5.10 Reporting the results
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Design of Assay

Ethics
Design
Subject
Sampling
Methods of Analysis and Validation
Requirements of Drug and Its Comparator
BA parameters and BE criterias
Statistical Analysis

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ETHICS
Bioequivalency is conducted to human
subjects
The design and protocols of the assay
should fulfilled the requirements in ICH
GCP
The protocols should be approved by
IEC
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Design of Assay
STANDARD TWO SEQUENCES IN
TWO PERIODS (CROSS-OVER DESIGN)

SUBJECTS

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R
A
N
D
O
M
I
Z
A
T
I
O
N

PERIODE I : REF
I

WASH OUT
PERIODE II : TEST
PERIODE I : TEST

II
WASH OUT
PERIODE II : REF

10

SUBJECT
Healthy volunteers
Male and female (2:1)
Age between 18 55 years

Normal body weight (BMI 18-25 or 15% of normal body

weight)
Not in pregnant condition or lactation
No more than 10 cigarrettes/day
No alcohol or drugs been taken within a month period
Number of subjects between 18-24 or minimum 12
(depends on the intra subject variability of the drug)

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BIOAVAILABILITY AND
BIOEQUIVALENCY ASSAY

FOR HIGHLY VARIABLE DRUGS

Drugs with low and variable absorption
e.g biphosphonates [etidronate, aledronate,
clodronate]
Drugs with variable clearance
e.g spironolactone, verapamil

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 Analysis of drugs and their metabolites in a

biological matrix is carried out using samples
spiked with calibration (reference)
standards and using quality control (QC)
samples. QC samples are matrix spiked with
analyte.
An authenticated analytical reference
standard of known identity and purity should
be used to prepare solutions of known
concentrations. If possible, the reference
standard should be identical to the
analyte.

The fundamental parameters for a bioanalytical

method validation are accuracy, precision,
selectivity, sensitivity, reproducibility, and stability
of analyte in spiked samples.

ASSESMENT OF METABOLITES
BA studies: for parent drug and major
metabolite
BE studies: only for parent drug
If parent drug is not measurable by the
analytical method used, measurement of a
metabolite [active or inactive] is recommended
If metabolite contributes meaningfully to safety
or efficacy, both parent drug and metabolite
should be measured
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CHIRAL DRUGS
BA studies: measurements of
individual enantiomers must be
important
BE studies: measurements of the
racemate using an achiral assay is
recommended.
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Principal Biometrics
Test Parameters
Single dose studies:
- Cmax, tmax, AUCt, AUC
Steady state studies
- Cmax, Cmin, Cav, AUC,
Fluctuation: (Cmax-Cmin/Cav)

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Concentration (Units/ml)

Area Under the Concentration

Time Curve (AUC)
A quantitative measure for
exposure from dosing time to
time t
An important parameter in PK
AUC(t) and AUC(inf)
Determined by trapezoidal
method
AUC(inf) = AUC(t) + Ct/k

10

0.1

0.01
0

10

Time (hr)
Area Under the Curve (AUC)

15

20

Proportional to Dose (linear PK)

Accuracy of the estimate
depends on frequency of
sampling

HPLC method is one of the most important analytical methods in characterizing

the drug products and substance.

The Chromatogram of HPLC

A

The time that a peak

appears (its retention
time) is diagnostic for a
given compound

Chromatograph

D
B

Sample Mixture

Chromatogram
B

Abundance

D
0

10
15
Time (minutes)

20

The relative size of a

peak (area or height)
is proportional to the
relative abundance of
the compound in the
mixture

Basic
Separation
Principles
Example:
Column elution
chromatography
Introduce two solutes
(A & B) onto a packed
column through which
a mobil phase (i.e.
solvent) or eluent is
continuously pumped

Separation Principles in HPLC

General Rule of Thumb:
Polarity of analytes polarity of stationary
phase polarity of mobile phase
To achieve good separation, the analytes
should interact with the stationary phase, but
not too strongly or the retention time will
become very long.

Reversed order
of elution

Increasing Mobil
phase Polarity,
Decreases
Elution Time

HPLC ASSAY OF ORAL ANTICANCER EXEMESTANE

Exemestane is used to treat advanced
breast cancer. It is practically insoluble
in water and its bioavailability is
approximately 5%.
An HPLC method with UV detection at
249 nm was used to assay this drug in
methanol medium, using Hichrom
Nucleosil C18 column.
Exemestane peak area was linear over
the concentration range 2.5-50 g/ml.

The size of the peak

area is proportional to
the concentration of
exemestane

BA/BE Biometrics
Cmax
tmax
AUC: calculated using trapezoidal method

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 Pharmacokinetics
what the body does to the
drug
Absorption
Distribution
Metabolism
Elimination

Pharmacodynamics
what the drug does to
the body
wanted effects efficacy
unwanted effects toxicity

Pharmacokinetics
Dose regimen

Exposure

Pharmacodynamics

Site of action

Response

Basic Concepts
Easy to understand using intravenous route

Drug
Product

Drug in
Blood

Excretion

No absorption phase
Simple to follow
Concepts clear with less assumptions
Need some math background

Distribution to
Tissue and Receptor sites

Metabolism

IV administration

Following dose administration, we

need to follow its drugs disposition
to understand its PK characteristics.

This is achieved by analyzing the

changes of the drug and/or its
metabolite(s) in blood/plasma and
urine.

A simple approach is to generate

Drug Concentration-Time profile

Dosing

Sampling at
Pre-determined
Time intervals

Blood withdrawal

Bio-analytics

Concentration vs time
profiles

Concentration versus Time Profiles

Broadly the concentration time profiles can be viewed as two different ways

OneCompartment
Model

Dose
1

Assumes body as one

compartment

Two-Compartment
Model
Central compartment (drug entry and
elimination)

Tissue compartment (drug distributes)

Dose
2

The one compartment

model linear assumes
that the drug in question
is evenly distributed
throughout the body
into a single
compartment.
This model is only
appropriate for drugs
which rapidly and
readily distribute
between the plasma
and other body tissues.

The distribution phase for aminoglycosides is

only 15-30 minutes, therefore, we can use a
one-compartment model with a high degree
of accuracy

Drugs which exhibit a slow equilibration with

peripheral tissues, are best described with a
two compartment model

Vancomycin is the classic example, it's distribution

phase is 1 to 2 hours. Therefore, the serum level time
curve of vancomycin may be more accurately
represented by a 2-compartment model.

Conc. [mg/L]

Conc. [mg/L]

Solution
Capsule
3

0
0

12

16

20

Time [hours]

20 mg administered as an i.v.
bolus (Diovan)

24

12

16

20

Time [hours]

80 mg given as a solution and a

capsule (Diovan)

24

How Absorption
affects
Bioavailability?

Absorption

Absorption is defined as the

process by which a drug
proceeds from the site of
administration to the site of
measurement.
Drugs are frequently
administered extravascularly
oral, sublingual
intramuscular,
topical, patches,
inhalation

Absorption is a prerequisite for

a drug to exert its
pharmacologic effect (other
than local effect)

Several possible sites

contribute to the loss

Absorption

Drug
Product

Drug in
Blood

Excretion

Distribution to
Tissue and
Receptor sites

Metabolism

Plasma Concentration-Time Profile for

a Drug Following a Single Oral Dose

Interactions in Oral Drug Absorption

Pharmacokinetic Assessment of Absorption

Interactions
Clinically significant interactions are
typically assessed in terms of:
Rate of Absorption:
peak plasma drug concentrations
(Cmax)
time to Cmax (tmax)
Extent of Absorption:
area under the concentration-time
curve (AUC)

Effect of Absorption Interactions on Drug Plasma

Concentration Profiles

Effect of Food
A required study helps for dosage
administration in Clinical Trials
Measure PK parameters (rate and
extent) under Fasted and Fed
conditions.
Single dose cross over study is
recommended.
FDA Guidance gives type of food

High Fat Meal (breakfast)

total of 800 1000 calories
of which 150 cal from
Proteins, 250 cal from
carbohydrates and 500
600 cal from fat.

Test Meal
2 eggs fried in butter
2 strips of bacon
2 slices of toast with
butter
4 oz of hash-brown
potatoes
8 oz of whole milk

Effect of Food on Rivastigmine

Absorption
MEAN RIVASTIGMINE PLASMA LEVELS (ng/mL)

7
6
5
4

3 mg (fasted) N=20
3 mg (fed) N=19

3
2
1
0
-1
0

TIME (hrs)

10

12

14

Concentration (ng/mL)

Effect of Food on Lescol XL

150
Fasted
Fed

120
90
60
30
0
0

12
Tim e (h)

18

24

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)

References
Clinical Pharmacokinetics: Concepts and
Application - 3rd Edition
By Malcolm Rowland & Thomas N. Tozer

http://www.fda.gov/cder/guidance/index.ht
m
http://www.access.gpo.gov/nara/cfr/waisidx
_03/21cfr320_03.html