Bioavailability

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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

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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

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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

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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?

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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

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Chemical analysis

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

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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

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Pre-study phase

• The method used must be well characterised
• Stability
• Specificity
• Accuracy
• Precision
• Limit of quantitation
• Response function

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Validation objective

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

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Analytical method validation
Analytical Procedure
Stability
Selectivity
Robustness
Validation
Accuracy
Calibration curve
Precision - within-run- between-run
Limit of Quantitation LOQ
Recovery
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Analytical procedure
Separation
Detection
Sample preparation
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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?

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Accuracy

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

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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
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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

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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

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Precision and Accuracy
Conc.
Accuracy
Precision
n
nmol/l
()
Within-run
Between-run


0.76
0.6
5.1
6.1
18
23
3.6
1.7
1.8
18
122
3.9
1.3
1.3
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Recovery

• The extraction efficiency of an analytical method
• Recovery of an analyte need not be 100

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Lower limit of quantitation

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

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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

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LLOQ (1.50 nmol/l) for morphine
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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

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Calibration curve
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Sample dilution

• Any required sample dilutions should use like
  matrix
• Dilution QC sample should be used

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Robustness

• How many samples can be analysed in one run?

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Robustness
115
110
105
100
Found concentration
0
10
20
30
40
50
60
70
80
90
100
110
95
90
85
Sample No.
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Stability of your substance
In Freeze/Thaw tests
In room temperature (4 h)
In the automatic injector
In stock solutions
In plasma during storage
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Analytical method validation
Analytical Procedure
Stability
Selectivity
Robustness
Validation
Accuracy
Calibration curve
Precision - within-run- between-run
Limit of Quantitation LOQ
Recovery
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References

• Guidance for IndustryBioanalytical Method
  Validation FDA, May 2001
• Workshop Report Shah, V.P. Et al.,
  Pharmaceutical Research 1992 9588-592.
• Workshop Report Shah, V.P. et al.,
  Pharmaceutical Research 2000 171551-1557

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Costs

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

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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.

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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

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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

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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.

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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

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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

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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