Should We Expect to Able to

Predict PK in Individual
Patients?
Leslie Z. Benet
Department of Biopharmaceutical Sciences, UCSF
Lewis B. Sheiner Memorial
Symposium
Washington, DC December 4, 2006
Take Home Message

NO!!
 WHY?
To appreciate my answer I must
give you some background on our
latest work in enzyme-transporter
interplay and the
Biopharmaceutics Drug
Disposition Classification System
 Biopharmaceutical Classification
High Solubility Low Solubility

Permeability 1 Acetaminophen 2 Carbamazepine

Propranolol Cyclosporine
High

Metoprolol Digoxin
Valproic acid Ketoconazole
Tacrolimus

3 4
Permeability

Cimetidine Chlorothiazide
Ranitidine Furosemide
Low

Methotrexate

Amidon et al., Pharm Res 12: 413-420, 1995

 Biopharmaceutical Classification
High Solubility Low Solubility

Permeability Class 1 Class 2

High Solubility Low Solubility
High

High Permeability High Permeability

Rapid Dissolution

Class 3 Class 4
Permeability

High Solubility Low Solubility

Low

Low Permeability Low Permeability

Amidon et al., Pharm Res 12: 413-420, 1995

BCS High Solubility Criteria
A drug substance is considered
highly soluble when the highest
dose strength is soluble in 250 ml or
less of aqueous media over a pH
range of 1-6.8 at 37C.
BCS High Permeability Criteria
A drug substance is considered to
be highly permeable when the
extent of absorption in humans is
determined to be 90% of an
administered dose based on a mass
balance determination or in
comparison to an i.v. reference dose
Distribution of Drugs on the Market
vs. Small Molecule NMEs
High Solubility Low Solubility

Permeability Class 1 Class 2

Marketed Drugs Marketed Drugs
High

~35% ~30%
NMEs: 5% NMEs: 70%

Class 3 Class 4
Permeability

Marketed Drugs Marketed Drugs

Low

~25% ~10%
NMEs: 5% NMEs: 20%
 High Solubility Low Solubility

Class 1 Imipramine I Class 2

Ketorolac Itraconazole S,I
Abacavir Ketoprofen Amiodarone I
Acetaminophen AtorvastatinS, I Ketoconazole I
Labetolol LansoprazoleI
Acyclovirb LevodopaS Azithromycin S ,I
AmilorideS,I Carbamazepine S,I Lovastatin S,I
Levofloxacin S Mebendazole
Amitryptyline S,I LidocaineI Carvedilol
Antipyrine Chlorpromazine I Naproxen
Lomefloxacin Nelfinavir S,I
Atropine Meperidine CisaprideS
Buspironec Ciprofloxacin S Ofloxacin
Metoprolol Oxaprozin
Cyclosporine S, I
High Permeability

Caffeine Metronidazole
Captopril Danazol Phenazopyridine
MidazolamS,I Phenytoin S
ChloroquineS,I Minocycline Dapsone
Chlorpheniramine Diclofenac Piroxicam
Misoprostol Raloxifene S
Cyclophosphamide Nifedipine S Diflunisal
Desipramine Digoxin S Ritonavir S,I
Phenobarbital Saquinavir S,I
Diazepam Phenylalanine Erythromycin S,I
Diltiazem S,I Flurbiprofen Sirolimus S
Prednisolone Spironolactone I
Diphenhydramine PrimaquineS Glipizide
Disopyramide GlyburideS,I Tacrolimus S,I
Promazine TalinololS
Doxepin Propranolol I Griseofulvin
Doxycycline Ibuprofen Tamoxifen I
QuinidineS,I Terfenadine I
Enalapril Rosiglitazone Indinavir S
Ephedrine Indomethacin Warfarin
Salicylic acid
Ergonovine Theophylline
Ethambutol Valproic acid
Ethinyl Estradiol Verapamil I
Fluoxetine I Zidovudine
Glucose
 High Solubility Low Solubility

Class 3 Class 4
Acyclovir Fexofenadine S Amphotericin B
Amiloride S,I Folinic acid Chlorthalidone
Amoxicillin S,I Furosemide Chlorothiazide
Low Permeability

Atenolol Ganciclovir Colistin

Atropine Hydrochlorothiazide Ciprofloxacin S
Bisphosphonates Lisinopril Furosemide
Bidisomide Metformin Hydrochlorothiazide
Captopril Methotrexate Mebendazole
Cefazolin Nadolol Methotrexate
Cetirizine Pravastatin S Neomycin
Cimetidine S Penicillins
Ciprofloxacin S Ranitidine S
Cloxacillin Tetracycline
Dicloxacillin S Trimethoprim S
Erythromycin S,I Valsartan
Famotidine Zalcitabine
 Major Routes of Drug Elimination
High Solubility Low Solubility

Permeability Class 1 Class 2

Metabolism Metabolism
High

Class 3 Class 4
Permeability

Renal & Biliary Renal & Biliary

Low

Elimination of Elimination of
Unchanged Drug Unchanged Drug
 What are the Implications?
If you know the intestinal absorption (or more
likely the Caco-2 permeability) of an NME, you
can predict whether the major route of
elimination of the NME will be metabolism.
Note that the permeability parameter does not
predict the ability for the NME to enter the liver/
hepatocytes (since a number of non-metabolized
Classes 3 & 4 compounds will be excreted in the
bile), but rather the access to the metabolic
enzymes within the hepatocytes.
Biopharmaceutics Drug Disposition Classification System

BDDCS
High Solubility Low Solubility
Metabolism
Extensive

Class 1 Class 2
High Solubility Low Solubility
Extensive Metabolism Extensive Metabolism
(Rapid Dissolution and
70% Metabolism for Biowaiver )
Metabolism

Class 3 Class 4
Poor

High Solubility Low Solubility

Poor Metabolism Poor Metabolism
This slide was not presented but it was added
here to give the reference for BDDCS.
Cellular and animal studies from our laboratory
over the past six years examining transporter-
enzyme interplay led us to make 22 predictions
concerning drug absorption and disposition
Some of these predictions were discussed
in my presentation but all may be found in
our January 2005 Pharmaceutical Research
paper
C-Y. Wu and L.Z. Benet. Pharm. Res. 22:11-23 (2005)
 Oral Dosing: Transporter Effects
High Solubility Low Solubility

Permeability Class 1 Class 2

Transporter Efflux transporter
High

effects minimal effects

predominate

Class 3 Class 4
Permeability

Absorptive Absorptive and

Low

transporter effects efflux transporter

predominate (but can
be modulated by efflux effects could be
transporters) important
Transporter effects will be minimal
for Class 1 compounds. The high
permeability-solubility of such compounds
allows large concentrations in the gut to saturate
any transporter, both efflux and absorptive, or
alternatively the intestine is sufficiently leaky
that small molecular weight, soluble, nonpolar
compounds readily pass the membrane. That is,
Class 1 compounds may be substrates for both
uptake and efflux in cellular systems under the
right conditions, but transporter effects will not
be important clinically.
 Efflux transporter effects will
predominate for Class 2
compounds.
The high permeability of these
compounds will allow ready
access into the gut membranes,
but the low solubility will limit the
concentrations coming into the
enterocytes, thereby preventing
saturation of the efflux
transporters.
 Transporter-enzyme interplay will
be primarily important for Class 2
compounds that are substrates for
CYP 3A and Phase 2 gut enzymes
(e.g. glucuronosyltransferases)
where efflux transporter effects
can control the access of the drug
to the gut enzymes. Absorption of
Class 2 compounds is primarily
passive and a function of
lipophilicity.
A series of cellular studies in our
laboratory with CYP3A4-
transfected Caco-2 cells
demonstrated that transporter
enzyme-interplay was an
important component of
metabolic extraction of Class 2
substrates in that transporters
were shown to control access to
the enzymes
 Summary of Our Cellular Studies

Inhibition of the efflux pump,

P-glycoprotein, with no effect on CYP
3A enzyme, will cause decreased
extraction ratio of Class 2 drugs in the
intestine resulting in increased AUC,
but increased extraction ratio in the
liver resulting in decreased AUC.
Current Drug Metabolism
Cover October 2003 Issue
Absorptive transporter effects
will predominate for Class 3
compounds. Sufficient drug will
be available in the gut lumen due
to good solubility, but an
absorptive transporter will be
necessary to overcome the poor
permeability characteristics of
these compounds.
Reflect on the drugs that Professors Yuichi
Sugiyama and Richard Kim study in their
double transfected systems.
Reflect on the drugs for which companies
such as Xenoport attempt to improve
absorption through a transporter prodrug
approach.
All of the substrates upon which they
work are nonmetabolized Class 3 and
Class 4 drugs.
However, since influx of Class 3
(and Class 4) compounds will be
rate limited by an absorptive
transporter, the counter effects
of efflux transporters will not be
saturated and can also be
important
 Post Absorption Effect and
Intravenous Dosing
Post intestinal absorption and
following intravenous dosing both
uptake and efflux transporters can
be important determinants of
Classes 2, 3 and 4 compounds.
Thus, for 95% of NMEs transporters can
have significant effects on drug
absorption,disposition & drug interactions
Hepatic Uptake and Efflux
Transporters in Rat Liver
Hepatocyte
Oatp1a1
ATP
Oatp1a4 Bse
ADP p
Oatp1b2 ATP
P-gp
Oat2 ADP
Bile duct
ATP Mrp2
Oat3
ADP
Oct1 ATP
rp
ADP Bc
ATP
Mrp3
ADP
ATP ATP
Ntcp
Na+ ADP ADP
Mrp6 Mrp4
 There Are No Useful CYP3A
Probes that Quantitatively Predict
the In Vivo Kinetics of Other
CYP3A Substrates and No
Expectation that One Will Be
Found.
L. Z. Benet
Molecular Interventions 5, 79-83 (2005)
 Consider first the EBT
(Erythromycin Breath Test)

Erythromycin is a Class 3 drug that is a

substrate of OATP hepatic uptake
transporters, the hepatic efflux
transporter P-glycoprotein and in
humans to a minor extent CYP3A4.
In man the drug is primarily eliminated
unchanged by biliary excretion
Why should the EBT be expected to predict
the clearance of any other CYP3A4 substrate?
 Effects of Uptake and Efflux
Transporter Inhibition on
Erythromycin Breath Test Results
S. Poon, L.A. Frassetto, C. Tsourounis, C. Valera
and L.Z. Benet
Clinical Pharmacology and Therapeutics, in press

Sixteen healthy volunteers (8 M, 8F) randomized to receive EBT

on 3 occasions: at baseline, after a 30 min iv infusion of rifampin
600mg (OATP inhibitor) and after a 30 min iv infusion of
lansoprazole 30 mg (MDR1 [P-gp] inhibitor)
EBT at Baseline, and after Rifampin and Lansoprazole (14C met/hr)

All Males Females M vs F P-value

Baseline 2.630.70
2.210.61 3.040.53 0.830.72 0.014

EBT + 2.180.65
Rifampin 1.730.45 2.640.52 0.920.38 0.0003
Rifampin -0.440.40 0.0005
from -0.490.45 0.077
Baseline 0.018
-0.400.36
EBT + 2.880.92
Lansopra 2.24 0.49 3.51 0.80 1.270.81 0.0003
Lansopra +0.250.51 0.071
from +0.03 0.51 0.89
Baseline 0.018
+0.47 0.44
My reaction to the many studies that
use midazolam, diazepam and
verapamil as model substrates?
The science is great and the correlations
are excellent, but so much of the work is
carried out with Class 1 compounds,
where we can ignore transporter effects.
Will the methodology be useful and
reliable when we investigate NMEs or in
predicting drug kinetics in patients?
 So ignoring transporters
(and 95% of NMEs), will one
Class 1 CYP3A4 substrate
predict the clearance of
another CYP3A4 substrate?
This question was addressed by
Masica et al. (Clin. Pharmacol. Ther. 76,
341-349, 2004) using midazolam,
triazolam and alprazolam
 Published Mean Pharmacokinetic and Metabolic
Pathway Parameters for Alprazolam, Midazolam
and Triazolam with the Mean Experimental Values
of Masica et al. (2004) Given in Parentheses
F %EU CL V t_ 1'-OH 4-OH
urine mlmin/kg L/kg Metab % Metab %
% hr
Alprazolam 88 20 0.74 0.72 12 20 60
(14)
Midazolam 44 <1 6.6 1.1 1.9 5 95
(30) (5.5) (1.3)
Triazolam 44 2 5.6 1.1 2.9 49 49
(55) (3.2) (2.8)
 Spearman Rank Correlations for Relationships
Between Clearance Values Studied by
Masica et al. (2004)
CL iv CL oral CL iv CL oral
Midazolam Midazolam Triazolam Triazolam
CL oral 0.36 0.60 0.70 0.66
Alprazolam
CL iv 0.70 0.66 0.48
Midazolam
CL oral 0.77 0.68
Midazolam
CL iv 0.71
Triazolam
Masica et al. (2004) found that the Spearman rank
correlation coefficients were statistically significant
for all of the relationships but CLoral alprazolam vs
CLiv midazolam. However, finding a significant
correlation coefficient between two parameters
does not guarantee that a clinically useful
relationship exists. Such a relationship depends on
the measure of the coefficient of determination (r2).
As Masica et al. (2004) note, only ~13 to 59% of
the variability in the clearance of one of these
structurally similar CYP3A drugs could be
accounted for by the measure of CYP3A activity as
determined by the clearance of a second probe.
Use of microdosing to predict pharmacokinetics at
the therapeutic dose: Experience with 5 drugs
Lappin et al. Clin. Pharmacol. Ther. 80:203-215(Sept. 2006)
Warfarin CL/F 65%; V/F 380%, t1/2 560%
oral 5mg/oral 100 g
ZK253 F = 0.0016; Fmicro <1
oral 50mg/iv 100 g (oral 100 g below limit of detection)
Diazepam CL 106%, V 137%, t1/2 79%
iv 10 mg/iv 100 g
Midazolam CL/F 99%, V 52%, t1/2 84%, F 97%
oral 7.5 mg/oral 100 g
Erythromycin t1/2 99%
oral suspension 250 mg/iv 100 g (oral 100 g below limit of detection)
Authors conclude that microdose data from 3 of the 5 drug
candidates tested would have predicted the therapeutic dose PK well
 In evaluating the validity of a new
predictive algorithm (e.g., predictive
ADME , QSAR, allometric scaling
proposals, microdosing, systems biology)
check the drugs used in the validation.
If the validation primarily uses Class 1
drugs, there is no assurance that the
technique will work for NMEs.
(Thats the scientific equivalent of only
looking at night for your lost wallet
under the street light.)
Can we make some general conclusions
as to when metabolic and transporter
genetic polymorphisms will be important
clinically in terms of drug disposition?
CYP 2D6 MDR1
CYP 2C19 OATPs
CYP 2C9 OCTs
CYP 3A5 OATs
CYP 1A2 MRP2
UGT1A1 Other ABC
NAT2 transporters
Can we make some general conclusions
as to when metabolic and transporter
genetic polymorphisms will be important
clinically in terms of drug disposition?
CYP 2D6 For sure MDR1 No
CYP 2C19 Yes OATPs Yes
CYP 2C9 Yes OCTs Probably
CYP 3A5 Maybe OATs Probably
CYP 1A2 Maybe MRP2 Maybe
UGT 1A1 Maybe Other ABC
NAT2 No transporters ??
 Cytochrome P450 2D6 Substrates
Antiarrhythmics: encainide, flecainide, mexilitene,
propafenone, propylajmaline, sparteine
-Adrenoceptor antagonists: metoprolol,
propranolol, timolol
Antihypertensive: debrisoquine guanoxan,
indoramin
Antidepressants: amitryptyline, clomipramine,
desipramine, fluoxetine, nortriptyline, paroxetine
Neuroleptics: clozapine, perphenazine,
thioridazine
Opiods: codeine, dextromethorphan
 Cytochrome P450 2D6 Substrates
Appear to be predominantly Class 1 substrates
Therefore there will be no transporter interplay
(I am unaware of a CYP2D6 substrate that has been
shown to be an efflux transporter substrate)
Therefore they will exhibit good absorption
The enzyme shows marked genetic differences in
enzyme activity between EMs and PMs
(i.e., marked activity vs. no activity)
There is no significant gut CYP2D6 activity
All factors that minimize nongenetic
variability
Yet, I suspect that even for this best
case genetic polymorphism scenario
No probe CYP2D6 substrate will provide
sufficient predictability to choose the final
dose for a narrow therapeutic agent
Neither will genotyping be sufficiently
accurate
That is TDM will always be needed
(But it is also unlikely that an NTI
CYP2D6 substrate will achieve a large market
or even be approved)
 Cytochrome P450 2D6 Substrates
Appear to be predominantly Class 1 substrates
Therefore there will be no transporter interplay
(I am unaware of a CYP2D6 substrate that has been
shown to be a transporter substrate)
Therefore they will exhibit good absorption
The enzyme shows marked genetic differences in
enzyme activity between EMs and PMs
(i.e., marked activity vs. no activity)
There is no significant gut CYP2D6 activity
Conversely the opposite of these
characteristics for CYP3A4 suggest that no
clinically significant polymorphism will be
found for CYP3A4.
Can we make some general conclusions
as to when metabolic and transporter
genetic polymorphisms will be important
clinically in terms of drug disposition?
CYP 2D6 For sure MDR1 No
CYP 2C19 Yes OATPs Yes
CYP 2C9 Yes OCTs Probably
CYP 3A5 Maybe OATs Probably
CYP 1A2 Maybe MRP2 Maybe
UGT 1A1 Maybe Other ABC
NAT2 No transporters ??
 There have been many contradictory studies
of the importance of MDR1 polymorphisms on
drug disposition, and it is highly unlikely that
any significant consistent effect will be found

P-gp effects are always imbeded with an enzyme for

Class 2 compounds and with an uptake transporter for
Classes 3 and 4 compounds.
The transporter does not show marked genetic
differences in transporter activity and this may be
further confounded by an inverse relationship between
transporter amounts and transporter activity.
For interplay with CYP3A, the effects in the gut are
different than in the liver.
 Conclusions
a. Predicting the PK of a drug in an individual patient is
best carried out by measuring the PK of the drug of
interest in the patient. It is highly unlikely that any
probe substrate will provide clinically useful predictions.

b. Predicting the PK of an NME may be facilitated by

characterizing the BDDCS class of the compound.

c. When evaluating new predictive algorhythms (e.g.,

predictive ADME , QSAR, allometric scaling,
pharmacogenomics, microdosing, systems biology) be
sure that the validation includes drugs that are not just
Class 1.