Bioavailability Metrological aspects Assessment of parameters

1
BioavailabilityMetrological aspectsAssessment
of parameters
Update july /211/2006
2
Bioavailability

• Bioavailability quantifies the proportion of a
  drug that is absorbed and available to produce
  its systemic effect
• extent
• rate

3
Bioavailability

• Definition
• Absolute
• amount of administered drug which enters the
  systemic (arterial) circulation and the rate at
  which the drug appears in the blood stream
• Relative
• to compare formulations (bioequivalence)
• to compare routes of administration

4
Bioavailability vs. absorption
5
Bioavailability vs. absorption

• Absorption movement of drug from the site of
  administration into the blood which drains the
  site of administration
• Bioavailability refers to the amount of drug
  which actually gains the access to the systemic
  (arterial) circulation

6
Bioavailability vs. Bioequivalence
7
Assessment of drug absorption and bioavailability
8
Assessment of drug absorption and bioavailability

• in silico predictive model
• Physicochemical methods
• in vitro methods
• in situ methods
• in vivo methods

9
Screening for absorption
screen order
increased degree of complexity
in vivo/ human data
intestinal loop
bioavailability absorption profile throughout
the gastro-intestinal tract "Loc-i-Gut"
perfusion in situ
awake animal absorption in specified region
perfusion in vitro
liver
Ussing chamber
blood flow
cell cultures
well defined flow Differentiates between
apsorption and absorption
intestinal sacs or rings
cell suspensions membrane vesicles
well defined flow
hetero-genous cells cell layers
W/O partition
Ungell, Drug Dev. Indus.Pharm. 1997 23 879-892
10
Assessment of drug absorption and bioavailability

• In silico
• models based on molecular structure
• many physicochemical parameters (H. bounding, MW,
  LogP, pKa, polar surface area) and solubility can
  be generated automatically from chemical
  structures
• many software to achieve these measurements

11
Assessment of drug absorption and bioavailability

• Physicochemical methods
• experimental determination of physicochemical
  parameters to predict permeability

12
Assessment of drug absorption and bioavailability

• In vitro methods cell based methods
• Caco2 (human colonic cell lines)
• drawback 21 days culture / overexpression of P-gp
  do not model paracellular passages (water soluble
  molecules of low MW)
• 3 days culture Caco-2
• Madin-Darby canine kidney (MDCK)
• 3 days correlation with Caco-2 culture
• Caco-2 cells engineering to express CYP3A4

13
Assessment of drug absorption and bioavailability

• In vitro methods tissue based methods
• Ussing chamber technique
• Everted gut sac
• perfused isolated intestinal segment
• unlike Caco-2 cells these models possess an
  apical mucus layer
• possibility to study drug transport in
  combination with intestinal metabolism

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Assessment of drug absorption and bioavailability

• In situ model
• perfusion of segment of small intestine
• sampling from mesenteric and portal vein

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Assessment of drug absorption and bioavailability

• IN VIVO METHODS

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Bioavailability in man prediction from rodents,
primates dogs
From Grass ADDR 2002 pp433
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Main steps for bioavailability (oral route)
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Main steps for bioavailability (oral route)
Administered dose
Disintegration
in vitro dissolution test
Fab
Dissolution
F
caco-2, everted sac ex vivo
Absorption
Fh
Hepatic first pass effect
hepatocytes culture (intrinsic clearance)
Pulmonary first pass effect
Fp
in vivo difference A.V.
bioavailable fraction of the dose (reaching the
systemic circulation)
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Bioavailability oral route and first pass effect
30 60
30
fp 0.50
Gut
Portal vein
PO
60
80
Lung
Vena cava
100
30
60
20
60 80
CD
GG
fh 0.75
80 100
20
Heart
fabs 0.8
30
arterial circulation 30
30
F fabs x fh x fp 0.8 x 0.75 x 0.5 0.3
20
First pass effect (oral route)
Gut Lumen
Gut Wall
Portal vein
CYP P-Gp
Liver
To site of measurement
Gut Metabolism
Hepatic Metabolism
To feces
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Bioavailability by oral route

• F fabs x ffirst pass
• ffirst pass fraction escaping the different
  first pass effects

fabs absorbed fraction
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Sites of first-pass elimination

• Intestinal mucosa
• - Cyp enzymes
• Liver
• - Cyp enzymes

- P-glycoprotein
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Non-nitrogenous Substances that Effect Drug
Metabolism by Forming Complexes with CYPs

• Grapefruit juice - CYP 3A4 inhibitor highly
  variable effects unknown constituents
• D.G. Bailey, et al. Br J Clin Pharmacol 1998,
  46101-110
• Isosafrole, safrole - CYP1A1, CYP1A2 inhibitor
  found in root beer, perfume
• Piperonyl butoxide alcohol -CYP1A1, CYP1A2
  inducer insecticide constituent

24
Grapefruit Juice Facts

• GJ or G (not OJ) elevates plasma peak drug
  concentration, not elimination t1/2
• GJ reduced metabolite/parent drug AUC ratio
• GJ caused 62 reduction in small bowel enterocyte
  3A4 and 3A5 protein liver not as markedly
  effected (i.v. pharmacokinetics unchanged)
• GJ effects last 24 h, require new enzyme
  synthesis
• Effect cumulative (up to 5x Cmax) and highly
  variable among individuals depending upon 3A4
  small bowel basal levels

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Drugs with first-pass metabolism or
P-glycoprotein transport
Aldosterone morphine Cyclosporine nortriptyline
Isoproterenol organic nitrates Lidocaine proprano
lol
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Reasons for knowing the absolute bioavailability

• To assess a possible major source of therapeutic
  variability
• if mean F close to 100 no inter-individual
  variability of AUC
• if mean F is low (eg 10) large
  interindividual variability due to formulation or
  physiological condition

27
Absolute bioavailability and interindividual
variability
125
100
75
CV ()
50
25
0
0
25
50
75
100
125
150
F
Hellriegel et al, 1996 Clin. Pharmacol. Ther
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Drawback of a low absolute availability
overexposure of some animals (side effects)
AUC
undesired concentration
therapeutic concentration
Bioavailability
underexposure of some animals (therapeutic
failure, resistance)
1
3
2
Dose
29
Why is an intravenous PK study required ?

• To know absolute bioavailability because
• absolute low bioavailability is generally
  correlated with a poor intersubject
  reproducibility
• and
• poor intersubject reproducibility generally leads
  to a more than proportional increase in dose rate
  to ensure drug efficacy in animals with the
  lowest bioavailabilty

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Drawback of a low absolute availability

• Possible interaction with a spurious increase of
  bioavailabilty
• The case of felodipine

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Effect of Grapefruit Juice on Felodipine Plasma
Concentration
Review- D.G. Bailey, et al. Br J Clin Pharmacol
1998, 46101-110
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BioavailabilityRelevance of the rate of
absorption

• The 3 AUC are equal
• The rates of absorption are different

CE1
CE2
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The main steps of bioavailabilityImportance of
the rate of absorption
Administered dose
mean disintegration time
Disintegration
mean dissolution time
Dissolution
MRToral
Absorption
mean absorption time
First pass effect (Lung, Liver)
mean disposition time
Arrival in the systemic circulation
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The absolute bioavailability
35
Bioavailability

• By IV route 100 by definition
• (except if it is a prodrug e.g. Ramipril)

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Corticosteroid preparations

• Methylprednisolone (medrol)

CH3
37
Methylprednisolone (MP) and methylprednisolone
succinate (MPS) disposition after an IV
administration of MPS or MP (4 mg/kg)
plasma concentration (ng / ml)
105
104
MP, IV
103
MP after MPS
102
MPS
10
0
60
120
240
360
480 minutes
Toutain, J. Pharm. Sci.
38
How to measure an absolute bioavailability?

• Principle
• Dose IV AUC IV x Cl IV
• Dose EV AUC EV x Cl EV
• Assumption Cl IV Cl EV
• F x 100

Dose EV Dose IV
AUC EV AUC IV
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How to measure an absolute bioavailability?

• If the doses are equal
• F x 100
• If IV and EV doses are different
• F x x 100
• Other possible methods (metabolite, urinary data,
  in steady state conditions, without IV, )

AUCEV AUCIV
AUCEV DoseIV AUCIV DoseEV
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How to measure an absolute bioavailability?

• Assumption Cliv Clev
• crossover design risk of carryover effect
• induction / inhibition
• appropriate washout (PK and PD)

41
Bioavailability estimation by semisimultaneous
drug administration
Karlsson Breberg J Pharmacokinet Biopharm 1990
18 pp102
42
Influence of permanent cannulation of the jugular
vein on PK parameters of antipyrine(low
extraction drug, not bound to plasma protein)
2h 48h
T1/2 (Hr) 1.5 1.9
Vd (L/kg) 0.87 0.95
Cl (ml/kg/min) 6.84 5.66
Chindaviak et al JPET 1988 246 1075-1079
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Bioavailability of antipyrine in ratsinfluence
of permanent cannulation
Sequence Washout 4days IV then Oral Oral then IV
AUCiv 1219 (OK) 1735 (overestimated)
AUCoral 2038 (overestimated) 1294
F 173 74 (OK)
Torres-Molina et al Pharmac Res 1992 9 1587-1591
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How to measure an absolute bioavailability?

• Assumption Cliv Clev
• correction by the terminal half-life

AUCEV AUCIV
t1/2IV t1/2EV
F x x 100
Warning! illicit correction if flip-flop
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How to measure an absolute bioavailability?
With a metabolite
AUC EV,metab AUC IV,métab
Dose IV Dose EV
x 100
F x
N.B.1 the metabolite should not be formed at
the administration site or by a first-pass
effect N.B.2 note 1 does not hold for a
relative bioavailability
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How to measure an absolute bioavailability?

• Problem of the analytical techniques
• The case of Enrofloxacin
• Using microbiological assay
• ? overestimation of F

Ciproflox
Liver Enroflox
Enroflox
Enrofloxacin
47
Absolute bioavailability

• Using urinary drug concentrations
• Drug itself

?
Xu,EV Xu,IV
Dose IV Dose EV
x 100
F x
?

• A metabolite
• Not formed by a first pass effect

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How to measure an absolute bioavailability when
an IV administration is not possible
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How to measure an absolute bioavailability
without IV administration?
Dose AUCIV

• IV Cltot ClR ClnR
• PO Cltot/F

ClR F
Dose AUCPO
ClnR F
50
How to measure an absolute bioavailability
without IV administration

• Measurement possible from oral data only
• Condition the renal clearance is known and
  displays a large interindividual variance

constant
variable
1 F
1 F
Dose AUCpo
x ClR ClNR
Y a X b
51
How to measure an absolute bioavailability
without IV administration
Measure possible with oral data only
Dose AUCpo
1 F
1 F
ClNR
Clrenal
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METHODS FOR ASSESSMENT OF ABSOLUTE BIOAVAILABILITY

• Conventional method iv and oral doses
• Usually given on two separate occasions
• requires two study sessions
• Requires two sets of blood samples
• Assumes no change in disposition
• Parameters between studies.

• Stable isotope method
• One study and set of blood samples
• Special synthesis requirements
• Mass spectrometer assay required

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NAPA-13C2
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SIMULTANEOUS ADMINISTRATION OF ORAL NAPA AND IV
NAPA-C13
From Atkinson AJ Jr, et al. Clin Pharmacol Ther
198946182-9.
55
Thoughts about absolute bioavailability studies

• Absolute bioavailability is usually
• Studied in healthy subjects, not in
• The patient population for whom its
• use is intended.
• The stable isotope method is ideally
• Suited for studies in special
• populations (e.G. Pediatrics, pregnant
• Women) and other patient groups.

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Relative bioavailability
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Relative bioavailability

• Single dose

AUCA AUCB
x 100
F
AUCA
AUCB
58
Relative bioavailability

• Under steady state conditions

Formulation A Formulation B (after
equilibrium) (after new equilibrium)
Plasma
AUCA
AUCB
Time
Condition linearity and stationarity
F (AUCA / AUCB) ? 100
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Bioavailability

• Evaluation of AUC

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Bioavailability
Measurement of AUC sampling strategy (1)

• If the samples are numerous and appropriately
  spaced, the AUC is accurately determined

61
Bioavailability
Measurement of AUC sampling strategy (2)

• Not enough samples in the ascending phase. The
  AUC is under-estimated

62
Bioavailability
Measurement of AUC sampling strategy (3)

• Not enough samples in the descending phase. The
  AUC is over-estimated

63
BioavailabilityAssessment of the rate of
absorption
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BioavailabilityRate of absorption

• Cmax et Tmax

Cmax
Tmax
65
Are Cmax and Tmax suggestive of the absorption
rate of the drug ?
this can be very misleading
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BioavailabilityRate of absorption

• Cmax and Tmax are hybrid parameters
• Cmax
• F, Ka, K10
• Tmax
• Ka, K10

67
Tmax

• Monocompartmental model

Ka
Ln Ka - Ln K10
Tmax
Ka - K10
K10
!
Ka varies with bioavailability
!
flip-flop situation
68
Terminal half-life
Ka1 absorption
Ka2 Irreversible loss of drug from the injection
site
K10 Elimination from the central compartment
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Tmax

• Monocompartmental model

1
1
0
0.2
0.2
1
Ln1 - Ln0.2
Ln2 - Ln0.2
Tmax 2.01 h
Tmax 1.27 h
1 - 0.2
2 - 0.2
F 50
F 100
70
Baignoire avec fuite
(K10)
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Tmax bioavailabilty

• Tmax is observed more early in case of low
  bioavailability .

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Tmax and Bioavailability of Cefadroxil in foal
Age (months) 0.5 1 2 3 5
F 99.6 67.6 35.1 19.5 14.4
Tmax (h) 2.1 1.60 1.60 0.96 0.90
Duffee JVPT 1997 20 427
73
Tmax and flip-flop situation
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Tmax and flip-flop situation
K10 of A or Ka of B
Ka of A or K10 of B
Tmax 2.55 h

• Drug A Ka 1.0 K10 0.1 t1/2 abs 0.693 h
• Drug B Ka 0.1 K10 1.0 t1/2 abs 6.93 h

75
Bioavailability and half-life

• In case of flip-flop, bioavailability may
  influence the terminal half-life

76
BioavailabilityHow to evaluate Ka?

• Curves fitting
• Interpretation of Ka when F lt 100

K12
Ka1
Ka
K21
Ka2
K10
Ka1 Ka1 Ka2
What is measured is not Ka1 but Ka Ka1Ka2 t1/2
Ka measures the rate of drug disappearance at the
administration site
F
77
What is the meaning of the terminal half-life
after an extravascular drug administration?
Half-absorption or half-elimination ?

a rate-limited absorption
(flip-flop) must be recognized
(C)
100
10
EV rate of absorption
IV
EV rate of elimination
1
time
0.1
0
5
10
15
20
25
30
78
Terminal half-life and the flip-flop case
K12
Slow process of absorption
Ka1
K21
Ka2 negligible
K10
(ng/ml)
100
KaKa1Ka2 Ka1 flip-flop
10
elimination
1
Ka1 Ka1 Ka2
F ? 100
0.1
Time
0
5
10
15
20
25
30
79
Terminal half-life and the flip-flop case
K12
Ka1
K21
K10
Ka2 ()
Ka1 Ka1 Ka2
F low
(ng/ml)
100
Lack of flip-flop due to low bioavailability not
to an increase of the rate of absorption (Ka1)
10
1
elimination
KaKa1Ka2 Ka2
Time
0.1
0
5
10
15
20
25
30
80
Tmax and flip-flop situation

• For the same drug
• Formulation Ka (h-1) K10 (h-1) Tmax (h) t1/2abs
  (h)
• A 1.0 0.1 2.56 0.693
• B 0.1 0.1 10.0 6.93
• C 0.01 0.1 25.6 69.3

!
The Tmax ratios do not reflect the t1/2 abs
ratios
81
The true meaning of Ka

• Remember
• Ka is the apparent first order absorption rate
  constant derived from plasma data
• This parameter may also contain processes
  parallel to the true absorption step such as
  degradation of drug in the administration site

82
The true meaning of Ka
Ka1 absorption
Ka1Ka2
Ka1Ka2
Ka2 Irreversible loss of drug from the injection
site
K10 Elimination from the central compartment
83
Lufenuron SQ vs oral administration
interpretation of the terminal phase
SC
Ka1 Ka1 Ka2
Ka1
F
Skin
Blood
Ka2
Ka Ka1 Ka2
If Ka2 ? ?F ? and t1/2 ?

• In flip-flop conditions, F and t1/2 are linked

84
How to evaluate the rate of absorption
85
How to evaluate Ka accurately

• 1- Directly from the ascending phase
• 2- By peeling method
• 3- Wagner-Nelson, Loo-Riegelman (deconvolution)
• 4- Statistical moments

86
BioavailabilityHow to evaluate Ka?

• Fitting plasma concentrations

Ka
Ka

• Computation of Ka, constant of absorption
• t1/2 Ka 0.693 / ka

87
BioavailabilityHow to evaluate Ka?

• Curves fitting
• How to know Ka

k a1
k 10
?
k a
k a2
?
IM route
?
?
k a1
k 12
k a
k 21
k a2
k 10
k 10
88
BioavailabilityHow to evaluate Ka?

• Curves fitting
• Interpretation of Ka when F lt 100

K12
Ka1
K21
Ka2
K10
Ka1 Ka1 Ka2
What is measured is not Ka1 but Ka Ka1Ka2 t1/2
Ka measures the rate of drug disappearance at the
administration site
F
89
BioavailabilityHow to evaluate Ka?

• Curves fitting
• Problem of undistinguishability

K12
Ka
K21
K10
If Ka K21, the plasma concentrations curve will
be identical to the classical Bateman curve and
Ka will not be properly estimated
90
BioavailabilityHow to evaluate ka?

• Remember
• Ka is the apparent first order absorption rate
  constant derived from plasma data
• This parameter may also contain processes
  parallel to the true absorption step such as
  degradation of drug in the administration site

91
BioavailabilityAssessment of the rate of
absorption

• The Wagner-Nelson method
• Principle calculate the fraction that remains
  to be absorbed
• Advantage allows identification of several Ka
  or zero order input processes
• Conditions monocompartmental model

92
BioavailabilityAssessment of the rate of
absorption

• The Loo-Riegelman method
• Principle to calculate the fraction that
  remains to be absorbed
• Advantage allows identification of several Ka
  or 0 order input processes
• Conditions know the parameters of the
  disposition model (IV required)

93
BioavailabilityAssessment of the rate of
absorption

• General deconvolution method
• Allows identification of an input signal

94
FIRST-PASS METABOLISM