Bioavailability Bioavailability means the rate and extent to which the active substance is adsorbed from a pharmaceutical product and become available.

CPMP/EWP/QWP/1401/98Note for guidance on the

investigation of bioavailability and bioequivalence

Dr. Monika Johansson

Quintiles AB, Analytical Services

Bioavailability

Bioavailability means the rate and extent to which the active substance is adsorbed from a pharmaceutical product and become available at the site of action

Bioequivalence

Two medical products are bioequivalent if they are pharmaceutical equivalent or pharmaceutical alternatives and if their bioavailabilities after administration in the same molar dose are similar to such degree that their effects, with respect to both efficicy and safety, will be essentially the same

Design and conduct of studies

The study should be designed in such a way that the formulation effect can be distinguished from other effects.

Most common is a two-period, two-sequence crossover design, if the formulations to be compared is two

Single dose studies

Steady-state studies

Design and conduct of studies

Adequate wash out periods between treatments Sampling schedule

– to provide an adequate estimation of Cmax

– to cover the plasma concentration time curve long enough, 80% of AUC

– 24 hours cycle at steady state?

– drugs with long half-life?

Subjects

Healthy volunteers The inclusion/exclusion criteria should be clearly

stated in the protocol Both sex 18-55 years old Normal weight Screened for

– laboratory test– medical history– medical examination– preferable non-smokers and without a history of

alcohol or drug abuse

Chemical analysis

The bioanalytical part of bioequivalence trials should be conducted according to the applicable principle of Good Laboratory Practice (GLP)

Good Laboratory Practice (GLP)

Test plan (Analytical protocol) Sample traceability Documentation, possible to reconstruct the study Analytical method validation report Analytical report signed by responsible investigator

Pre-study phase

The method used must be well characterised

– Stability

– Specificity

– Accuracy

– Precision

– Limit of quantitation

– Response function

Validation objective

To demonstrate that the analytical procedure is suitable for its intended purpose

Analytical method validationAnalytical Procedure

Selectivity

Accuracy

Precision- within-run- between-run

Recovery

Limit of Quantitation LOQ

Calibration curve

Robustness

Validation

Stability

Sample Sample preparationpreparation

Sample Sample preparationpreparation Separation Separation Separation Separation DetectionDetectionDetectionDetection

Analytical procedure

Specificity (selectivity)

Ability of an analytical method to measure only what it is intended to measure

Blank samples from six different subjects Will other drugs, metabolites or endogenous

components interfere in the measurements?

Accuracy

The closeness of mean test results obtained by the analytical method to the true value (concentration) of the analyte.

Accuracy

Accuracy should be measured at minimum 3 levels At least 5 determinations per concentration The mean value should be within 15% of the actual

value At the lower limit of quantitation level within 20%

is accepted

xx xx xxxx

xxxx

Precision

The closeness of individual measurements of an analyte when the procedure is applied repeatedly to multiple aliquotes of a single homogenous volume of biological matrix

Precision

Precision should be measured at minimum 3 levels At least 5 determinations per concentration The calculated CV should not exceed 15% At the lower limit of quantitation level, CV should

not exceed 20% Subdivided into within-run and between-run

Precision and Accuracy

Conc.nmol/l

Accuracy(%)

Precision

% %

n

0.76 0.6 5.1 6.1 18

23 3.6 1.7 1.8 18

122 3.9 1.3 1.3 18

Within-run Between-run

Recovery

The extraction efficiency of an analytical method

Recovery of an analyte need not be 100%

Lower limit of quantitation

The lowest standard on the calibration curve should be accepted as the lower limit of quantitation (LLOQ)

if

Lower limit of quantification

The analyte responce at LLOQ is at least 5 times the blank response

The peak should be identifiable and discrete Precision within 20% CV Accuracy of 80-120%

LLOQ (1.50 nmol/l) for morphine

Sample No. nmol/l Accuracy LLOQ 1 1.58 5.3% LLOQ 2 1.61 7.3% LLOQ 3 1.46 -2.6% LLOQ 4 1.44 -4.0% LLOQ 5 1.50 0.0% LLOQ 6 1.49 -0.7% Mean: 1.51 0.9%

SD: 0.067 CV%: 4.5

Calibration/Standard curve

A calibration curve is the relation between instrument response and known concentrations of the analyte

Should be prepared in the same biological matrix as the samples

Should consist of 6-8 samples covering the expected range Should include LLOQ and a blank sample Should include a zero sample (with internal standard) Same curve fitting, weighting in prestudy and study Any changes should be documented

Calibration curve

Sample dilution

Any required sample dilutions should use like matrix

Dilution QC sample should be used

Robustness

How many samples can be analysed in one run?

85

90

95

100

105

110

115

0 10 20 30 40 50 60 70 80 90 100 110

Sample No.

Fo

un

d c

on

ce

ntr

ati

on

%

Robustness

Stability of your substance

In the automatic injectorIn the automatic injector

In plasma during storageIn plasma during storage

In room temperature (4 h)In room temperature (4 h)

In Freeze/Thaw testsIn Freeze/Thaw tests

In stock solutions

Analytical method validationAnalytical Procedure

Selectivity

Accuracy

Precision- within-run- between-run

Recovery

Limit of Quantitation LOQ

Calibration curve

Robustness

Validation

Stability

References

1. Guidance for IndustryBioanalytical Method Validation FDA, May 2001

2. Workshop Report: Shah, V.P. Et al., Pharmaceutical Research: 1992; 9:588-592.

3. Workshop Report: Shah, V.P. et al., Pharmaceutical Research: 2000; 17:1551-1557

Costs

Validation = 130-180.000 SEK Stability = 15-20.000 SEK for each time point QA = 11.000 SEK/study

The study phase (1)

...in which the validated bioanalytical method is applied to the actual analysis of samples from the biostudy mainly in order to confirm the stability, accuracy and precision.

The study phase (2)

Calibration curve in each run Six Quality Control samples in each run Pre-stablished SOPs for procedures (method) Acceptance criteria for a run

- accuracy and precision of the calibration curve- accuracy and precision of the QC samples- repeat analysis

It is preferable to analyse all study samples from a subject in a single run

The study phase (3)

The QC samples should be used to accept or reject the run (2 samples at 3 levels)

Four QC samples out of six should be within 15% of their nominal value

Two QC samples can be outside ±15% but not both at the same concentration

System suitability test

Signal to noise ratio is above 5 for the substance.

The peak shape is acceptable after visual inspection of the chromatogram

The retention times are within 10% of the previous run.

The lowest calibration sample is injected before each run.

The system is accepted if:

The analytical report should include

Results for all calibration curves Results for all quality control samples Representative number of chromatograms Should include data from subjects who eventually

dropped-out Reanalysed samples and the reason for reanalyses The analytical validation report The responsible investigator(s) should sign for their

respective section of the report

Chiral active substances

The bio-analytical method should be enantiomeric Unless

– Both products contain the same stable singel enantiomer

– Both products contain the racemate and both enantiomers show linear pharmacokinetics

Also guidance for

Reference and test product Data analysis In vitro dissolution comlementary to a

bioequivalence study Reporting of results Application for products containing new active

substances Application for products containing approved

active substances

is given