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11/18/2011 1

BIOANALYTICAL METHODFOR

BIOAVAILABILITY/

BIOEQUIVALENCY ASSAY

Jutti Levita

BIOAVAILABILITY/BIOEQUIVALENCY ASSAYJUTTI LEVITA

2011


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Definition

Bioavailability:

Amount of a drug that is absorbed into

human systemic circulation in its activeform. It is determined by its concentrationin blood versus time, or its excretion inurine

Bioequivalency:

Therapeutic equivalency of two drugs whenboth of them are administered to the sameperson in the same dosage and those willshow similar efficacy and safety


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Why is BA/BE Assay Necessary to BeCarried Out?

Health cost

Substitute drug (with me-too product

or generic) will be preferred

Substitution drug should be equivalenttherapeutically (bioequivalent) with

the innovator


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BA/BE Laboratories

RequirementsCompetent Clinical

Laboratory ResourcesCompetent Analytical

Laboratory Resources

Subjects/

Healthy volunteersBioanalytical

proceduresBiological

sample

Good Clinical Practice

ICH-GCP E6

Good Laboratory Practice

ISO/IEC 17025


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ICH - GCP E6INTERNATIONAL 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 protectedICH Guidelines 1.24

GCP regulations are applied to all drug and device studies


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ISO/IEC 17025:1999- General Requirementsfor the Competence of Testing & CalibrationLaboratories ( 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 work4.10 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 Requirementsfor the Competence of Testing & CalibrationLaboratories ( 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 Sampling5.8 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 assayshould fulfilled the requirements in ICH

GCP

• The protocols should be approved byIEC


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Design of AssaySTANDARD TWO SEQUENCES IN

TWO PERIODS (CROSS-OVER DESIGN)

SUBJECTS

R

A

N

DO

M

I

Z

A

TI

O

N

I

II

PERIODE I : REF

WASH OUT

PERIODE II : TEST

PERIODE I : TEST

WASH OUT

PERIODE II : REF


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SUBJECT

• Healthy volunteers

• Male and female (2:1)

• Age between 18 – 55 years

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

• 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 shouldbe used to prepare solutions of known

concentrations. If possible, the reference

standard should be identical to the

analyte.


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The fundamental parameters for a bioanalytical

method validation are accuracy, precision,

selectivity, sensitivity, reproducibility, and stability

of analyte in spiked samples.


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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

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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 isrecommended.

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Principal Biometrics

Test Parameters

• Single dose studies:

- Cmax, tmax, AUCt, AUC∞

• Steady state studies- Cmax, Cmin, Cav, AUCτ,

• Fluctuation: (Cmax-Cmin/Cav)

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A U d th C t ti


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Area Under the Concentration –

Time Curve (AUC) A quantitative measure for

exposure from dosing time totime „t‟

An important parameter in PK

AUC(t) and AUC(inf)

Determined by trapezoidalmethod

AUC(inf) = AUC(t) + Ct/k

Proportional to Dose (linear PK)

Accuracy of the estimatedepends on frequency of

sampling

0.01

0.1

1

10

0 5 10 15 20

Time (hr)

C o n c e n t r a t i o n

( U n i t s / m l )

Area Under the Curve (AUC)

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

the drug products and substance.


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A

B

C

D

E

Sample Mixture

Chromatogram

0 5 10 15 20

Time (minutes)

A b u n d a n c e

A

B

C

D

E

Chromatograph

The Chromatogram of HPLC

The time that a peakappears (it’s retentiontime) is diagnostic for a

given compound

The relative size of a

peak (area or height)is proportional to therelative abundance ofthe compound in themixture

B i


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Basic

Separation

Principles

Example:

• Column elutionchromatography

• Introduce two solutes(A & B) onto a packedcolumn through whicha mobil phase (i.e.

solvent) or eluent iscontinuously pumped


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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.


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Increasing Mobil

phase Polarity,

Decreases

Elution Time

Reversed order

of elution


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HPLC ASSAY OF ORAL ANTICANCER EXEMESTANE

Exemestane is used to treat advancedbreast 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 μg/ml.


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The size of the peak

area is proportional tothe concentration ofexemestane


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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


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Dose regimen ResponseExposure

Site of action

Pharmacokinetics Pharmacodynamics


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B i C


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Basic Concepts

Drug

Product

Drug in

Blood

Distribution to

Tissue and Receptor sites

MetabolismExcretion

Easy to understand using intravenous route

No absorption phase

Simple to follow

Concepts clear with less assumptions

Need some math background


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IV administration Following dose administration, we

need to follow its drug‟s disposition

to understand its PK characteristics.

This is achieved by analyzing the

changes of the drug and/or its

metabolite(s) in blood/plasma andurine.

A simple approach is to generate

Drug Concentration-Time profile

DosingSampling at

Pre-determined

Time intervals

Bio-analytics Concentration vs tim

profiles

Blood withdrawal


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Concentration versus Time Profiles

One-Compartment

Model Assumes body as onecompartment

1

Two-CompartmentModelCentral compartment (drug entry and

elimination)

Tissue compartment (drug distributes)

1 2

k

k

Dose

Dose

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


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The one compartmentmodel linear assumesthat the drug in questionis evenly distributedthroughout the bodyinto a single

compartment.

This model is onlyappropriate for drugs

which rapidly andreadily distributebetween the plasmaand other body tissues.

The distribution phase for aminoglycosides is


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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


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Drugs which exhibit a slow equilibration with

peripheral tissues, are best described with a

two compartment model

V i i th l i l it' di t ib ti


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Vancomycin is the classic example, it's distributionphase is 1 to 2 hours. Therefore, the serum level timecurve of vancomycin may be more accurately

represented by a 2-compartment model.


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Time [hours]

0 4 8 12 16 20 24

Conc.[mg/L]

0

1

2

3

4

5

Time [hours]

0 4 8 12 16 20 24

Conc.[mg/L]

0

1

2

3

4

5

SolutionCapsule

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

80 mg given as a solution and acapsule (Diovan)


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How Absorptionaffects

Bioavailability?

Absorption


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Absorption is defined as the

process by which a drugproceeds from the site ofadministration to the site ofmeasurement.

Drugs are frequently

administered extravascularly oral, sublingual intramuscular, topical, patches,

inhalation

Absorption is a prerequisite fora drug to exert it’spharmacologic effect (otherthan local effect)

Several possible sites

contribute to the loss

DrugProduct

Drug inBlood

Distribution toTissue and

Receptor sites

MetabolismExcretion

Absorption

Absorption

Plasma Concentration-Time Profile for


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Plasma Concentration Time Profile for

a Drug Following a Single Oral Dose


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Interactions in Oral Drug Absorption


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Pharmacokinetic Assessment of AbsorptionInteractions

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


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p gConcentration Profiles

Effect of Food


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Effect of Food A required study – helps for dosage

administration in Clinical Trials

Measure PK parameters (rate andextent) 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 fromcarbohydrates and 500 –

600 cal from fat.

Test Meal2 eggs fried in butter2 strips of bacon2 slices of toast with

butter4 oz of hash-brownpotatoes8 oz of whole milk

Effect of Food on Rivastigmine


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Effect of Food on RivastigmineAbsorption

TIME (hrs)

0 2 4 6 8 10 12 14

MEANRIVA

STIGMINEPLASMAL

EVELS(ng/mL)

-1

0

1

2

3

4

5

6

7

3 mg (fasted) N=20

3 mg (fed) N=19

Eff t f F d L l XL


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Effect of Food on Lescol XL

0

30

60

90

120

150

0 6 12 18 24

Time (h)

C

o n c e n t r a t i o n ( n g / m L

Fasted

Fed


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Clinical Service Form to FinalMarket Form

Change of formulations(capsules to tablet)

Generic Formulations

Change of Process ormanufacturing site (some times)

When do we do BE studies ?


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References

Clinical Pharmacokinetics: Concepts andApplication - 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

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