1. Title Page Modeling and Simulation to Probe the Pharmacokinetic

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1. Title Page

Modeling and Simulation to Probe the Pharmacokinetic Disposition of Pomalidomide R-

and S-Enantiomers

Yan Li, Simon Zhou, Matthew Hoffmann, Gondi Kumar, and Maria Palmisano

Translational Development and Clinical Pharmacology (Y.L., S.Z., M.P.); Drug Metabolism and

Pharmacokinetics (M.H., G.K.); Celgene Corporation, 86 Morris Avenue, Summit, New Jersey

07920, USA

JPET Fast Forward. Published on May 15, 2014 as DOI:10.1124/jpet.114.215251

Copyright 2014 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on May 15, 2014 as DOI: 10.1124/jpet.114.215251

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2. Running Title Page

a) Running title [56 characters, max. 60 characters]

Pharmacokinetics of R- and S-enantiomers of pomalidomide

b) Corresponding author:

Simon Zhou, Translational Development and Clinical Pharmacology, Celgene Corporation, 86

Morris Avenue, Summit, NJ 07920, USA.

E-mail: [email protected]

Phone number: 908-673-9284

Fax: 908-673-2842

c) Manuscript details:

Number of text pages: 15

Number of tables: 3

Number of figures: 8

Number of references: 25 [max 60]

Total word count: 3831

Word count in Abstract: 247 [max. 250]


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Word count in Introduction: 573 [max. 750]

Word count in Discussion: 1274 [max. 1500]

d) Abbreviations:

AUC, area under the curve; CC-4047, pomalidomide; CC-5083, S-isomer of pomalidomide; CC-

6016, R-isomer of pomalidomide; CLR, apparent clearance of CC-6016; CLS, apparent

clearance of CC-5083; Cmax, peak concentration of pomalidomide in plasma; FDA, US Food and

Drug Administration; i.v., intravenous; kaR, first-order absorption rate constant for CC-6016; kaS,

first-order absorption rate constant for CC-5083; k_elim, elimination rate constant; k_inter, in vivo

interconversion rate constant between the R- and S- enantiomers of pomalidomide; LC/MS,

liquid chromatography tandem–mass spectrometry; PEG-400, polyethylene glycol 400; PK,

pharmacokinetic; p.o., per os; S.E., standard error; Tmax, time to reach Cmax; V, volume of

distribution of the central compartment in monkeys; V/F, volume of distribution of the central

compartment in humans; w/v, weight per volume.

e) Recommended section: Metabolism, Transport, and Pharmacogenomics


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3. Abstract

Background: Pomalidomide, a potent novel immunomodulatory agent, has been developed as

a racemic mixture of its R- and S-isomers. Pharmacokinetic (PK) analyses were conducted to

determine the PK disposition of the isomers from their PK profiles in humans and monkeys.

Modeling and simulation were performed to describe the observed PK profiles and explore

potential differences in isomer disposition and exposure. Methods: PK profiles of S- and R-

isomers were measured in a human absorption, distribution, metabolism, and excretion study

following oral administration of racemate. PK profiles of S- and R-isomers were measured in

monkeys following intravenous and oral administration of S- or R-isomers and pomalidomide

racemate. Modeling and simulation were performed using NONMEM 7.2 to describe the

observed PK profiles of S- and R-isomers in humans and monkeys. Results: In humans, the in

vivo elimination rate of pomalidomide isomers was lower than the R-/S- interconversion rate,

resulting in no clinically relevant difference in overall exposure to the two isomers. However, in

monkeys, the in vivo elimination rate was higher than the R-/S- interconversion rate, resulting in

1.72-fold and 1.55-fold difference in R- versus S-isomer exposure. Monte Carlo simulation

indicated that exposure to R- and S- enantiomers in humans should be comparable even if

single isomers were administered. Conclusion: In humans, rapid isomeric interconversion of

pomalidomide isomers results in comparable exposure to R- and S-enantiomers regardless of

whether pomalidomide is administered as a single enantiomer or as a racemate, therefore

justifying the clinical development of pomalidomide as a racemate.


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4. Introduction

Pomalidomide (CC-4047) (Fig. 1) is a novel immunomodulatory agent with pleiotropic cytotoxic

effects against multiple myeloma cells (Mitsiades et al., 2002; Zhu et al., 2008), as well as anti-

proliferative (Hideshima et al., 2000; Verhelle et al., 2007), anti-angiogenic (Gupta et al., 2001;

Reddy et al., 2008; Lu et al., 2009), and immunomodulatory actions (Corral et al., 1999; Hayashi

et al., 2005; Reddy et al., 2008). It has potent effects on key cytokines including tumor necrosis

factor-α (TNF-α), interleukin-10 (IL-10), and interferon- γ (IFN-γ) (Teo et al., 2003). Potential

therapeutic benefits have been shown in the treatment of various hematologic and non-

neoplastic hematologic disorders. Pomalidomide is currently approved in the US (Pomalyst®)

(Celgene Corporation, 2005-2013) and Europe (Imnovid®) for the treatment of

relapsed/refractory multiple myeloma in patients who have received at least two prior therapies

including lenalidomide and bortezomib, and have demonstrated disease progression on or

within 60 days of completion of the last therapy. It is also undergoing clinical evaluation for the

treatment of myelofibrosis and systemic sclerosis.

Pomalidomide is a chiral compound with an asymmetric carbon center and can therefore exist

as the optically active forms of S (-) and R (+). The S-isomer is termed CC-5083 and the R-

isomer is termed CC-6016. The R- and S-isomers exist in a 50:50 ratio and interconvert in

plasma via enzymatic and non-enzymatic pathways (Hoffmann et al., 2013). Pomalidomide has

been developed as a racemic mixture of its R- and S-isomers even though the S-isomer of

pomalidomide has been reported to be the more potent enantiomer of the racemate (Teo et al.,

2003), and in vitro studies have shown that the anti-TNF-α and immunomodulatory activities are

primarily due to the S-isomer (Corral et al., 1996; Muller et al., 1999; Davies et al., 2001).


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Although the R- and S-isomers of pomalidomide are identical in terms of their molecular

formula, atomic bonds, and bond distances, they have different three-dimensional structures

and are non-superimposable (Teo et al., 2003). Biological systems are themselves chiral

environments (e.g. right-handed B-DNA and left-handed Z-DNA, D-carbohydrate forms, and L-

amino acid structures), and within the biochemical environment of living systems, specific

tertiary structural forms are often required for biological activity to occur (e.g. enzyme catalysis,

receptor bonding, molecular transport, etc). Therefore, in biochemical systems, racemic

mixtures (or individual enantiomers) can exhibit distinctive absorption, transport, protein binding,

metabolism, and elimination, leading to different pharmacokinetic (PK), pharmacodynamic, and

toxicity profiles depending on the composition of the enantiomers, and the rate of

interconversion between the stereoisomeric forms. Stereospecific variations have been reported

for many classes of pharmaceuticals, including antibiotics, cardiovascular drugs, chemotherapy

agents, and antirheumatics (Smith, 2009). Thus, it is important that clinical pharmacologic

evaluations during the development of chiral compounds take into account the potential

differences in enantiomer disposition, exposure, and dose-response relationships.

The aim of this analysis was to determine the potential differences in PK disposition of the R-

and S- pomalidomide isomers from the PK profile of the R- and S- pomalidomide isomers

following oral (per os, p.o.) administration of pomalidomide racemate in humans, and from R-

and S-isomer PK profiles in a monkey study following intravenous (i.v.) and p.o. administration

of S- or R-isomers and pomalidomide racemate. In addition, modeling and simulations were

performed to probe the interplay of isomer interconversion versus elimination on plasma

exposure of isomers. Simulations were also conducted to evaluate potential differences in

plasma exposure of R- and S-isomers when R- or S-isomers or racemate are administered.


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5. Materials and Methods

Pharmacokinetic Study of Pomalidomide and its Two Enantiomers in Monkeys

The plasma concentration–time profiles of CC-4047 (pomalidomide racemate) and its two

enantiomers (CC-5083 and CC-6016) were measured after i.v. and p.o. administration to

monkeys. These experiments were performed according to the Care and Use of

LaboratoryAnimals guidelines, as adopted and promulgated by the U.S. national Institute of

Health. CC-4047, CC-5083, and CC-6016 were formulated in aqueous 1.0% (weight per

volume, w/v) carboxymethyl cellulose for p.o. dosing, and 5% dimethylacetamide, 45%

polyethylene glycol 400 (PEG-400), and 50% saline for i.v. dosing. CC-4047, CC-5083, and CC-

6016 were administered to three groups of male cynomolgus monkeys (total of 9 monkeys). The

doses were 1 mg/kg (i.v.) and 2 mg/kg (p.o.) for CC-4047 and 0.5 mg/kg (i.v.) and 1.0 mg/kg

(p.o.) for the individual enantiomers. Serial blood samples (approximately 1 ml) were collected

from each animal at the following time points: predose, and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12,

24, and 48 hours post-dose for the i.v. cohort, and predose, and 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12,

24, and 48 hours post-dose for the p.o. cohort. Plasma samples were prepared by

centrifugation, then diluted into an equal volume of Sorenson’s citrate buffer, pH 2.5 to stabilize

the analytes.

Pharmacokinetic Study of Pomalidomide in Healthy Human Subjects

This Phase 1 study was an open-label, single-center, single-dose study. Eight (8) healthy male

subjects were enrolled, and all completed the study. The study consisted of a 21-day screening

period, a baseline period (Day -1), a single p.o. dose treatment on Day 1, sample collection up

to Day 15 (336 hours post-dose), a study completion evaluation on Day 15 (or day of

discharge), and a follow-up visit 7 to 10 days from day of discharge.


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Prior to study initiation, the study protocol and Investigator's Brochure (IB), and Informed

Consent Form (ICF) were reviewed and approved by the Institutional Review Board (IRB;

Independent Investigational Review Board Inc., Plantation, Florida). All 8 subjects consented to

the study prior to dosing.

On Day 1, each subject received a single 2-mg p.o. dose of pomalidomide that contained

approximately 100 µCi of [14C]-CC-4047, after at least an 8-hour fast. Blood samples were

collected at predose and 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 24, 48, 72, 120, and 168 hours

post-dose. Plasma samples were prepared by centrifugation, then transferred to a tube

containing citric acid to stabilize the analytes. Samples were used for radioactivity counting;

metabolite profiling; and determination of concentration of the parent drug, its enantiomers, and

its metabolites, as applicable.

Bioassay

Plasma concentrations of the pomalidomide and the enantiomers CC-5083 and CC-6016 were

measured by using a chiral liquid chromatography tandem–mass spectrometry method

(LC/MS). The analytical range of the assay was 1 to 200 ng/ml.

Pharmacokinetic Analysis

Based on visual inspection of the log concentration–time profile, a one-compartment model with

first-order absorption and elimination was selected to characterize the PK disposition of CC-

4047 and the isomers CC-5083 and CC-6016 in monkeys and humans. A pictorial presentation

of the PK model is shown in Fig. 2. The PK model was parameterized in micro-constants of the

first-order absorption rate constant (kaS for CC-5083 and kaR for CC-6016), the volume of

distribution of the central compartment (V/F for humans and V for monkeys), the apparent

clearance (CLS for CC-5083 and CLR for CC-6016), and the in vivo interconversion rate constant

between R- and S- enantiomers (k_inter). Assuming a log-normal distribution for inter-individual


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variability in PK parameters, the inter-individual variability was modeled as:

Pi = P • eηi (Eq. 1)

where P was the typical value of the parameter in the population, Pi was the value of the

parameter for the ith individual, and ηi was a random inter-individual effect in the parameter for

the ith subject with a mean of zero and variance ω2 (i.e., η ~N[0, ω2]).

Intra-individual or residual variability (RV) was modeled as follows:

Ln(Cij) = Ln(Cmij) + εij (Eq. 2)

where Cmij was the model-predicted jth concentration in the ith subject, Cij was the observed jth

concentration in the ith subject, and εij was the random residual effect for the jth concentration in

the ith subject with a mean of zero and variance of σ2.

PK analysis of the concentration–time data of pomalidomide (CC-4047, CC-5083, and CC-

6016) in monkeys and humans was conducted using the nonlinear mixed-effect modeling

program (NONMEM) (version 7.2 or higher, Globomax, Ellicott City, MD, US). All fitting

procedures were performed under Windows XP with the Intel® Visual Fortran Compiler (version

9.1). Selection criteria during the model development process were based on the goodness-of-

fit plots, changes in the objective function value, residual distributions, and parameter estimates

and their relative standard error (relative S.E.) values.

Simulations to explore key attributes of kinetic properties of interconversion and elimination

driving potential exposure difference to individual isomers were performed using Berkeley

Madonna (version 8.3.18, University of California at Berkeley, CA, USA). Since the V/F of the R-

and S-enantiomer was similar, a simpler model was employed. While multiple values of full

permutation of interconversion rate constants and elimination rate constants were tested, the

exercise aimed to compare the exposure difference to the two enantiomers and settled on a

dimensionless factor of the relative ratio of the elimination rate versus interconversion rate


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constant. Different relative ratios of elimination rate constant (k_elim) versus interconversion rate

constant (k_inter) were simulated to check the difference in isomer exposures under different

scenarios.

Monte Carlo Simulation

A Monte Carlo simulation was performed using NONMEM to probe whether the anticipated

exposure of two enantiomers were comparable after dosing of each individual R- or S-isomer in

humans. The dose of each individual isomer of 2 mg was simulated at steady state. The PK

profiles and exposure for two individuals under each scenario were compared.


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6. Results

Pharmacokinetics in Monkeys

The plasma concentration–time profiles of CC-5083 and CC-6016 following i.v. and p.o.

administration of pomalidomide racemate (CC-4047), the S-isomer (CC-5083), or R-isomer

(CC-6016) in monkeys are shown in Figs. 3 and 4.

Following a single i.v. or p.o. dose of either R-isomer or S-isomer in monkeys, both isomers

were eliminated rapidly with a relatively short half-life, and the extent of in vivo conversion to S-

or R-isomer was small by visual inspection, suggesting that the in vivo elimination of the

individual R- or S-isomer is relatively faster than their in vivo interconversion. Following a single

i.v. or p.o. administration of racemate (CC-4047), the plasma concentration profiles showed

comparable plasma exposure to both the R- and S-isomers, with slightly higher concentrations

of R-isomer. Plasma exposure (area under the curve, AUC) to CC-6016 (R-isomer) was

approximately 1.5× as compared to that of CC-5083 (S-isomer) following i.v. or p.o.

administration of pomalidomide (1.72× and 1.55× respectively). After i.v. or p.o. administration

of CC-5083, plasma exposure to CC-6016 was smaller than that of CC-5083 (47% and 32%

respectively), and after i.v. or p.o. administration of CC-6016, plasma exposure to CC-5083 was

smaller than that of CC-6016 (22% and 21% respectively), suggesting only limited to mild

interconversion between R- and S-isomers.

The PK parameter estimates following i.v. or p.o. administration in monkeys are presented in

Table 1. The structure model parameters were estimated precisely as indicated by small S.E.

values (~5%) as compared to the population estimates. The diagnostic plots showed good

agreement between population-predicted and individual observed values, and no systematic

bias could be identified in the weighted residual plot (data not shown), suggesting that the


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proposed model characterized well the PK central trend and the associated inter-subject

variabilities for both the R- and S- isomers in monkeys.

Based on the PK modeling analysis, the in vivo interconversion rate between R- and S- isomers

in monkeys was characterized by a value of 0.223 (1/h), which is comparable to the value

characterized in vitro (0.4 1/h). In addition, the apparent in vivo elimination rates (Table 2) were

comparable or higher than the interconversion rate (0.223 1/h).

Pharmacokinetics in Humans

The plasma concentration–time profiles of CC-5083 and CC-6016 after p.o. administration of

pomalidomide are shown in Fig. 5. Both CC-6016 (R-isomer) and CC-5083 (S-isomer) were

rapidly absorbed, with peak concentrations (Cmax) observed at a Tmax of approximately 3 hours

for both isomers. Visual inspection of the PK profiles showed that the Cmax and AUC values for

CC-5083 and CC-6016 were comparable, indicating that the two enantiomers were present in

approximately equal amounts.

In humans, after a single 2-mg p.o. dose of CC-4047, the R- and S enantiomers were present in

approximately equal amounts. For CC-6016, the mean Cmax and AUC0-t values were

approximately 49% and 50% of those observed for pomalidomide, and for CC-5083, the mean

Cmax and AUC0-t values were approximately 52% and 49% of those observed for pomalidomide.

Different PK models and approaches were tested to identify the most stable and robust PK

model to describe the observed PK profiles from R- and S-isomers. Initially, the observed PK

differences between R- and S-enantiomers were forced to a single PK parameter (ka, or V/F or

clearance [CL/F]) in the tested model. It was shown that the relaxed model (different ka, V/F,

and CL/F PK parameters for R- and S-enantiomers) best described the PK profiles for both R-

and S-enantiomers. The PK parameter estimates in humans are presented in Table 3. The

structure parameters were estimated precisely as indicated by relatively small S.E. values


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(~20%) as compared to the population estimates. The diagnostic plots showed good agreement

between population-predicted and individual observed values, and no systematic bias could be

identified in the visual predict check plot (Fig. 6), suggesting the proposed model characterized

well the PK central trend and the associated inter-subject variabilities for both R- and S-isomers

in humans.

The model-identified in vivo interconversion rate between R- and S-isomers in humans was

0.353 (1/h), which is comparable to the value of 0.223 (1/h) in monkeys. In addition, the in vivo

elimination rates (0.056 and 0.091 1/h for S- and R- isomer, respectively) were slower than the

interconversion rate (0.353 1/h).

Simulation Exploring Interconversion and Elimination Rates

Simulation results for the impact of different ratios of the elimination rate constant (k_elim) versus

the interconversion rate constant (k_inter) on the drug exposures of R- and S-isomers after dosing

the S-isomer are presented in Fig. 7. As expected, the higher the ratio of the elimination rate

constant (k_elim) versus the interconversion rate constant (k_inter), the larger the differences of

drug exposures between R- and S-isomers. In monkeys, the elimination rate constant is

higher/comparable to the interconversion rate constant in vivo (k_elim/k_inter is ~2.5), and

significantly higher drug exposure for the isomer dosed is expected. The model-predicted ratio

of R- to S- enantiomers is approximately 30%, comparable to the observed ratio of a range from

20% to 40% following i.v. and p.o. administration. In humans, the elimination rate constant is

slower than the interconversion rate constant in vivo (k_elim/k_inter is ~0.25), and therefore,

comparable drug exposure for R- and S- isomers is expected. The model-predicted ratio of R- to

S- enantiomers is approximately 95%, comparable to the observed ratio of 94% following p.o.

administration

Monte Carlo Simulation


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Based on the final model derived from human data, Monte Carlo simulations were performed to

assess the difference in plasma exposures of the two enantiomers following p.o. dosing of each

individual R- or S-isomer in humans. The results are shown in Fig. 8.

After administration of 2 mg of S-isomer (CC-5083) or R-isomer (CC-6016), comparable PK

profiles between R- and S-isomers were observed. Monte Carlo simulation showed that after

administration of 2 mg of S-isomer (CC-5083), the drug exposures (AUCinf) were 105.7 and 92.7

ng/ml*h for S-isomer (CC-5083) and R-isomer (CC-6016), respectively, and after administration

of 2 mg of R-isomer (CC-6016), the drug exposures (AUCinf) were 90.0 and 104.9 ng/ml*h for S-

isomer (CC-5083) and R-isomer (CC-6016), respectively, indicating comparable drug exposure

between R- and S-isomers after dosing of each individual R- or S-isomer in humans.


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7. Discussion

Pomalidomide exists as a racemic mixture of R- and S-isomer (50:50). The S-isomer of

pomalidomide has been reported to be the more potent enantiomer of the racemate (Teo et al.,

2003), and in vitro studies have shown that the anti-TNF-α and immunomodulatory activities are

primarily due to the S-isomer (Corral et al., 1996; Muller et al., 1999; Davies et al., 2001). In the

present study, we analyzed in vivo PK data and identified interesting disposition differences

between the two enantiomers CC-5083 and CC-6016 in monkeys and humans. A PK model

was developed to describe the difference in kinetics of S- and R-isomer (CC-5083 and CC-

6016) disposition in monkeys and humans, and it derived a dimensionless factor of the relative

ratio of the elimination rate constant versus the interconversion rate constant that drives the PK

exposure difference between the two enantiomers in vivo.

Evaluation of stability and interconversion of CC-6016 (R-isomer) and CC-5083 (S-isomer) in

humans and monkeys in vitro and ex vivo showed gradual degradation (approximate half-life of

24 h) and interconversion of the enantiomers (Teo et al., 2003). Both degradation (approximate

half-lives of 3 to 4 h) and interconversion (1:1 ratio achieved by approximately 4 h) occurred

rapidly, suggesting that both processes may occur via enzymatic and non-enzymatic pathways

(Teo et al., 2003). The model identified in vivo interconversion rate constants between R- and S-

isomers of 0.223 in monkeys and 0.353 1/h humans, which are comparable to that identified in

vitro (0.4 1/h); however, the model identified different elimination rate constants in monkeys and

humans in vivo.

For pomalidomide, in monkeys, the in vivo elimination rate constants of R- and S-isomers are

comparable or higher than the interconversion rate constant (Table 2), resulting in differences in

isomer exposure after dosing individual isomers or racemate (Figs. 3 and 4). However, in


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humans, the in vivo elimination rate constants are lower than the interconversion rate constant

between R- and S-isomers, which allows the racemization between R- and S-isomers in a

clinically meaningful time frame, resulting in small difference in isomer exposures after dosing

the racemate (Fig. 5). In addition, the Monte Carlo simulation of human data in this study

showed the comparable PK profiles and comparable drug exposures between R-and S-isomers

after administration of a single dose of either S- or R-isomer provided the scientific basis for the

development of racemate pomalidomide (Fig. 8). Therefore, the development of a single

pomalidomide enantiomer will not confer a clinical/therapeutic advantage over the racemate.

A chiral center confers different spatial orientation of the enantiomers, and thus often results in

differing pharmacologic effects. It has been long accepted that most of the biological activity

observed for a racemate often likely resides within a single enantiomer. It was estimated that

before the 1990s, nearly 50% of all drugs used were racemate (Walther and Netscher, 1996).

However, regulatory agencies’ interest in evaluating each enantiomer in a racemate spurred

development of single enantiomers, which now account for 55% of the new molecular entities

approved by the US Food and Drug Administration (FDA) (Agranat et al., 2012).

Although the decision to develop a single enantiomer product should include both scientific and

economic rationales (Blake and Raissy, 2013), poor understanding of the kinetics of each

individual enantiomer, especially the interplay between enantiomer racemization and difference

in enantiomer elimination, can still lead to redundant investment in developing an enantiomer

over the racemate. Levalbuterol, the R-enantiomer of albuterol, was introduced in the US

market in 1999 based on the fact that the R-enantiomer is stereo-selective at the target receptor

with a 68-fold greater potency than the S-enantiomer (Brittain et al., 1973). However, a

randomized crossover study in which the racemate and the R- and S-enantiomers were

administered demonstrated that the therapeutic ratio of the R-enantiomer was comparable with

that of the racemate in asthma patients (Lotvall et al., 2001), which might be due to the limited


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understanding of the interplay between enantiomer racemization and enantiomer elimination in

vivo. Similarly, arformoterol, the (R,R)-enantiomer of formoterol, having a 1000-fold greater

potency than the (S,S)-enantiomer, was approved by the FDA in 2006 (Blake and Raissy,

2013). Available data indicate that there is no evidence to suggest an advantage of the (R,R)-

enantiomer, which might be due to the different kinetic profiles. Another example is thalidomide.

The R-enantiomer of thalidomide is effective against morning sickness, while the S-enantiomer

is teratogenic, causing birth defects (Trapnell, 1998). However, there is no rationale for

developing a single enantiomer drug of thalidomide, since the single enantiomer is converted

into a racemate within a clinically meaningful time frame in vivo (Eriksson et al., 2001). All these

cases indicate that kinetic properties including racemization and elimination of enantiomers play

a critical role in the decision whether to develop an enantiomer or a racemate in addition to

using only data on in vitro stereo-selectivity at the target receptor. However, the development of

single enantiomer drugs from established or previously marketed racemates may still have the

potential benefits of less complex, more selective pharmacodynamic profiles, potential for an

improved therapeutical index, less toxicity, and reduced potential for complex drug interactions

(Hutt and Valentova, 2003; Mansfield et al., 2004). This was successfully demonstrated by the

non-steroidal anti-inflammatory drugs dexketoprofen and dexibuprofen, the proton pump

inhibitor esomeprazole, the antimicrobial levofloxacin, the selective serotonin reuptake inhibitor

escitalopram, the anesthetic ketamine, the histamine H1-receptor antagonist levocetirizine, etc.

(Hutt and Valentova, 2003). The decision of whether to develop the racemate or its enantiomers

requires scientific justification based on quality, safety, and efficacy, together with the risk–

benefit ratio.

The scientific decision tree to inform the development of an active enantiomer from a racemate

begins with confirmation of stereo-selectivity in receptor recognition and includes in vivo efficacy

(Blake and Raissy, 2013). Importantly, the active enantiomer must not racemize (interconvert


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between enantiomers) in any clinically meaningful time frame and extent, have no

pharmacodynamic or PK interaction with the inactive enantiomer, and have no adverse effects

not already known for the racemate. Lastly, the enantiomer must represent a therapeutic

advantage for the patient by having a more selective pharmacodynamic profile, improved

therapeutic index, a less complex PK profile, such as a more direct relationship between

concentration and effect, and reduced potential for drug interactions (Blake and Raissy, 2013).

In order to explore the sensitivity of the drug exposure of the R- and S-isomers in relation to the

elimination rate constant and the interconversion rate constant, a dimensionless factor for the

relative ratio of elimination/interconversion rate was proposed. The PK exposure ratios between

the R- and S-isomers were simulated following different scenarios of dimensionless factors.

The simulation showed that the higher the ratio of the elimination rate constant over the

interconversion rate constant, the larger the differences in PK exposures between the R- and S-

isomers. Therefore, the potential difference to isomer exposure is primarily determined by the

ratio of the rate of elimination over the rate of the interconversion, and secondarily to potential

differences in isomer PK disposition. This quantitative measure can aid in determining if a new

single enantiomer product might or might not confer any clinical advantages over the racemate.

In conclusion, a population PK model was successfully developed to describe the PK disposition

of R- and S-isomers in monkeys and in humans in vivo. The model was able to predict the

comparable PK profiles and comparable drug exposures of R- and S-isomers after dosing of a

single isomer. The relatively slower elimination rate constant as compared to the

interconversion rate constant in humans may result in fast racemization between R- and S-

isomers, indicating no clinical/therapeutic advantage of developing single-enantiomer

pomalidomide versus developing racemate pomalidomide.


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8. Acknowledgments: [To be inserted]


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9. Authorship contributions:

Participated in research design: Hoffmann

Conducted experiments:

Contributed new reagents or analytic tools: Performed data analysis: Li, Zhou, Hoffmann

Wrote or contributed to the writing of the manuscript: Li, Zhou, Hoffmann, Kumar, Palmisano


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10. References

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SP, Richardson P, and Anderson KC (2005) Molecular mechanisms whereby

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RL, Richardson P, Muller G, Stirling DI, and Anderson KC (2000) Thalidomide and its

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Surapaneni S, and Kumar G (2013) Absorption, metabolism and excretion of

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11. Footnotes

a) Financial support

The authors acknowledge the financial support for this study from Celgene Corporation and

received editorial assistance from Kathy Boon, PhD (Excerpta Medica) sponsored by Celgene.

b) Thesis information, citation of meeting abstracts where the work was previously

presented, etc.: not applicable.

c) Name, full address, and e-mail address of person to receive reprint requests: Simon

Zhou, Translational Development and Clinical Pharmacology, Celgene Corporation, 86 Morris

Avenue, Summit, NJ 07920, USA. E-mail: [email protected]

d) Numbered footnotes, using superscript numbers, to the manuscript text: not applicable


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12. Figure Legends

Fig. 1. Structure of pomalidomide

Fig. 2. Pharmacokinetic model of pomalidomide for monkeys and humans. CC-5083, S-isomer

of pomalidomide; CC-6016, R-isomer of pomalidomide; CLR, apparent clearance of CC-6016;

CLS, apparent clearance of CC-5083; kaR, first-order absorption rate constant for CC-6016; kaS,

first-order absorption rate constant for CC-5083; k_inter, in vivo interconversion rate constant

between the R- and S- enantiomers; V/F, volume of distribution of the central compartment in

humans.

Fig. 3. Plasma concentration–time profiles of CC-5083 and CC-6016 after intravenous (i.v.)

administration of CC-4047, CC-5083, and CC-6016 in monkeys. (A) Concentration profiles of

CC-5083 after i.v. administration of CC-4047 (1 mg/kg); (B) concentration profiles of CC-6016

after i.v. administration of CC-4047 (1 mg/kg); (C) concentration profiles of CC-5083 after i.v.

administration of CC-5083 (0.5 mg/kg); (D) concentration profiles of CC-6016 after i.v.

administration of CC-5083 (0.5 mg/kg); (E) concentration profiles of CC-5083 after i.v.

administration of CC-6016 (0.5 mg/kg); and (F) concentration profiles of CC-6016 after i.v.

administration of CC-6016 (0.5 mg/kg).

Fig. 4. Plasma concentration–time profiles of CC-5083 and CC-6016 after oral (p.o.)

administration of CC-4047, CC-5083, and CC-6016 in monkeys. (A) Concentration profiles of

CC-5083 after p.o. administration of CC-4047 (2 mg/kg); (B) concentration profiles of CC-6016

after p.o. administration of CC-4047 (2 mg/kg); (C) concentration profiles of CC-5083 after p.o.


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administration of CC-5083 (1 mg/kg); (D) concentration profiles of CC-6016 after p.o.

administration of CC-5083 (1 mg/kg); (E) concentration profiles of CC-5083 after p.o.

administration of CC-6016 (1 mg/kg); and (F) concentration profiles of CC-6016 after p.o.

administration of CC-6016 (1 mg/kg).

Fig. 5. Plasma concentration–time profiles of CC-5083 (blue solid lines) and CC-6016 (red solid

lines) after oral administration of CC-4047 in humans. Different panels are data from different

subjects.

Fig. 6. Visual prediction check (VPC) plots of the model fittings for the time profiles of CC-5083

and CC-6016. (A) VPC plot for the time profiles of CC-5083 after p.o. administration of CC-4047

in humans (2 mg); (B) VPC plot for the time profiles of CC-6016 after p.o. administration of CC-

4047 in humans (2 mg); (C) VPC plot for the time profiles of CC-5083 after i.v. administration of

CC-4047 in monkeys (1 mg/kg); (D) VPC plot for the time profiles of CC-6016 after i.v.

administration of CC-4047 in monkeys (1 mg/kg); (E) VPC plot for the time profiles of CC-5083

after i.v. administration of CC-5083 in monkeys (0.5 mg/kg); (F) VPC plot for the time profiles of

CC-6016 after i.v. administration of CC-5083 in monkeys (0.5 mg/kg); (G) VPC plot for the time

profiles of CC-5083 after i.v. administration of CC-6016 in monkeys (0.5 mg/kg); (H) VPC plot

for the time profiles of CC-6016 after i.v. administration of CC-6016 in monkeys (0.5 mg/kg).

Blue circles represent observed data. Red solid lines represent the 50th percentiles of the

observed data. Pink shaded areas represent nonparametric 95% confident intervals about the

50th percentiles for the corresponding model predicted percentiles.


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Fig. 7. Simulation results for exposure (area under the curve, AUC) ratios of R- and S-isomer

under different ratios of elimination rate (k_elim) versus inter-conversion rate (k_inter) after dosing

the S-isomer in humans.

Fig. 8. Pharmacokinetic profiles of R- and S-isomers when 2 mg of S-isomer (CC-5083) or R-

isomer (CC-6016) was administered in Monte Carlo simulations. (A) Concentration profiles of

CC-5083 and CC-6016 after p.o. administration of CC-5083 (2 mg/kg); and (B) concentration

profiles of CC-5083 and CC-6016 after p.o. administration of CC-6016 (2 mg/kg).


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13. Tables

Table 1. Population pharmacokinetic parameter estimates for pomalidomide in monkeys

Structure

model

Population

estimate

Relative S.E.

(%)

Inter-individual

variability (%)

Relative S.E.

(%)

V (l) 2.9 4.0 0.00747 66

CLS (l/h) 1.61 11.0 0.0954 38

CLR (l/h) 0.228 8.7 0.0625 122

k_inter (1/h) 0.223 5.8 0.0388 62

Residual

error

σ2 0.123 16.8

S.E., standard error; V, volume of distribution; CLS, total body clearance of S-isomer; CLR, total

body clearance of R-isomer; k_inter, in vivo interconversion rate between S- and R-isomers.


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Table 3

Population pharmacokinetic parameter estimates for pomalidomide in humans

Structure

model

Population

estimate

Relative S.E.

(%)

Interindividual

variability (%)

Relative S.E.

(%)

V (l) 135 23 0.0263 50

CLS (l/h) 7.58 18 0.685 45

CLR (l/h) 12.4 21 0.399 43

k_inter (1/h) 0.353 15 0 FIXED N/A

Residual

error

σ2 0.0166 22

S.E., standard error; V, volume of distribution; CLS, total body clearance of S-isomer; CLR, total

body clearance of R-isomer; k_inter, in vivo interconversion rate between S- and R-isomers.


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Table 2

Interconversion constant versus elimination constant in monkeys and humans

Monkeys Humans

k_inter (1/h) 0.22 0.35

k_elim (1/h) (S-isomer) 0.56 0.056

k_elim (1/h) (R-isomer) 0.08 0.091

k_elima/k_inter 2.49 0.258

aFaster k_elimin constants from S- and R-isomer were used.


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OHN

O

O

N

H

ONH2

Figure 1


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k k

k inter

kas kaR

_

k_inter

CC-5083

V/F

CC-6016

V/F

CLs CLR

Figure 2


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Panel: A

0 10 20 30 40

Panel: B

400

600

800

0

200

Panel: C

800

Panel: D

ng/m

L)

200

400

600

once

ntra

tion

(n

0

600

800

Panel: E Panel: F

Co

0

200

400

0 10 20 30 40

Time (hr)

Figure 3


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Panel: A

0 10 20 30 40

Panel: B

400

600

800

1000

0

200

Panel: C

1000

Panel: D

ng/m

L)

200

400

600

800

once

ntra

tion

(n

0

600

800

1000

Panel: E Panel: F

Co

0

200

400

600

0 10 20 30 400 10 20 30 40

Time (hr)

Figure 4


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ID: 005

0 10 20 30 40

ID: 006 ID: 007

0 10 20 30 40

ID: 008

6

8

10

2

4

(ng/

mL)

10

ID: 001 ID: 002 ID: 003 ID: 0040

once

ntra

tion

(

4

6

8Co

0

2

0 10 20 30 40 0 10 20 30 400 10 20 30 40 0 10 20 30 40

Time (hr)

Figure 5


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2.5

Panel: A

2.5

Panel: B Panel: C Panel: Dg(

Con

cent

ratio

n).0

1.5

2.0

g (C

once

ntra

tion)

.01.

52.

0

g(C

once

ntra

tion)

46

g(C

once

ntra

tion)

46

Log

0.0

0.5

1

Log

0.0

0.5

1

Log

02

Log

02

Time (hr)

0 5 10 15 20

Time (hr)

0 5 10 15 20

Time (hr)

0 10 20 30 40

Time (hr)

0 5 10 15 20

Panel: E Panel: F Panel: G Panel: H

(Con

cent

ratio

n)4

6

( Con

cent

ratio

n)4

6

( Con

cent

ratio

n)4

6

(Con

cent

ratio

n)4

6

Log(

02

Log(

02

Log(

02

Log(

02

Time (hr)

0 5 10 15 20

Time (hr)

0 5 10 15 20

Time (hr)

0 5 10 15 20

Time (hr)

0 5 10 15 20

Figure 6


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0er

s .81.

0

Human

- and

S-is

ome

0.6

0.e

Rat

ios

of R

-0.

40

Exp

osur

e0.

20

Monkey

0 2 4 6 8 10

k_elim/k_inter

Figure 7


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Panel: ACC 5083

0 50 100 150

Panel: BCC 5083CC-5083

CC-6016CC-5083CC-6016

1

(ng/

mL)

Con

cent

ratio

n

10^-1

C

0 50 100 150

Time (hr)

Figure 8


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Documents

1. Title Page Modeling and Simulation to Probe the Pharmacokinetic