Transcript Bioavailability

CPMP/EWP/QWP/1401/98
Note 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)
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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 validation
Analytical
Procedure
Stability
Selectivity
Robustness
Validation
Accuracy
Calibration curve
Precision
Limit of Quantitation
LOQ
- within-run
- between-run
Recovery
Analytical procedure
Sample
preparation
Separation
Detection
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
x
x
x
x
x
x
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
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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
Within-run Between-run
0.76
0.6
%
5.1
23
122
3.6
3.9
1.7
1.3
%
6.1
18
1.8
1.3
18
18
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.
LLOQ 1
LLOQ 2
LLOQ 3
LLOQ 4
LLOQ 5
LLOQ 6
Mean:
SD:
CV%:
nmol/l
1.58
1.61
1.46
1.44
1.50
1.49
1.51
0.067
4.5
Accuracy
5.3%
7.3%
-2.6%
-4.0%
0.0%
-0.7%
0.9%
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?
Robustness
115
Found concentration %
110
105
100
0
10
20
30
40
50
60
95
90
85
Sample No.
70
80
90
100
110
Stability of your substance
In Freeze/Thaw tests
In room temperature (4 h)
In the automatic injector
In stock solutions
In plasma during storage
Analytical method validation
Analytical
Procedure
Stability
Selectivity
Robustness
Validation
Accuracy
Calibration curve
Precision
Limit of Quantitation
LOQ
- within-run
- between-run
Recovery
References
1. Guidance for Industry
Bioanalytical 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)
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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
The lowest calibration sample is injected before
each run.
The system is accepted if:
 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 analytical report should include
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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