Drug Metab Dispo0

0090-9556/10/3805-789–800$20.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
DMD 38:789–800, 2010

Vol. 38, No. 5
31252/3577279
Printed in U.S.A.

Excretion and Metabolism of Lersivirine (5-{[3,5-Diethyl-1-(2hydroxyethyl)(3,5-14C2)-1H-pyrazol-4-yl]oxy}benzene-1,3dicarbonitrile), a Next-Generation Non-Nucleoside Reverse
Transcriptase Inhibitor, after Administration of [14C]Lersivirine to
Healthy Volunteers
Manoli Vourvahis, Michelle Gleave, Angus N. R. Nedderman, Ruth Hyland, Iain Gardner,
Martin Howard,1 Sarah Kempshall, Claire Collins, and Robert LaBadie
Pfizer Global Research and Development, New London, Connecticut (M.V., R.L.); and Pharmacokinetics, Dynamics and
Metabolism, Pfizer Global Research and Development, Sandwich, United Kingdom (M.G., A.N.R.N., R.H., I.G., M.H., S.K., C.C.)
Received November 19, 2009; accepted February 2, 2010

Lersivirine [UK-453,061, 5-((3,5-diethyl-1-(2-hydroxyethyl)(3,514
C2)-1H-pyrazol-4-yl)oxy)benzene-1,3-dicarbonitrile] is a nextgeneration non-nucleoside reverse transcriptase inhibitor, with a
unique binding interaction within the reverse transcriptase binding
pocket. Lersivirine has shown antiviral activity and is well tolerated
in HIV-infected and healthy subjects. This open-label, Phase I
study investigated the absorption, metabolism, and excretion of a
single oral 500-mg dose of [14C]lersivirine (parent drug) and characterized the plasma, fecal, and urinary radioactivity of lersivirine
and its metabolites in four healthy male volunteers. Plasma Cmax
for total radioactivity and unchanged lersivirine typically occurred
between 0.5 and 3 h postdose. The majority of radioactivity was
excreted in urine (⬃80%) with the remainder excreted in the feces

(⬃20%). The blood/plasma ratio of total drug-derived radioactivity
[area under the plasma concentration-time profile from time zero
extrapolated to infinite time (AUCinf)] was 0.48, indicating that radioactive material was distributed predominantly into plasma. Lersivirine was extensively metabolized, primarily by UDP glucuronosyltransferase- and cytochrome P450-dependent pathways, with
22 metabolites being identified in this study. Analysis of precipitated plasma revealed that the lersivirine-glucuronide conjugate
was the major circulating component (45% of total radioactivity),
whereas unchanged lersivirine represented 13% of total plasma
radioactivity. In vitro studies showed that UGT2B7 and CYP3A4 are
responsible for the majority of lersivirine metabolism in humans.

The AIDS epidemic has reached pandemic proportions, culminating in the death of more than 2 million people in 2007 alone and
resulting in 33 million people currently estimated as living with
HIV/AIDS worldwide (UNAIDS/WHO, http://data.unaids.org/pub/
epislides/2007/2007_epiupdate_en.pdf). The widespread usage of
highly active antiretroviral therapy since 1996 resulted in a substantial

reduction in the mortality and morbidity of people infected with HIV
(Palella et al., 1998). However, the emergence of drug-resistant viruses in patients treated with highly active antiretroviral therapy,
together with the increasing transmission of these viruses in newly
infected patients, has increased the demand for further therapeutic
improvements (Cane et al., 2005; Daar and Richman, 2005). It is
unfortunate that the three currently approved first-generation nonnucleoside reverse transcriptase inhibitor (NNRTI) agents (efavirenz,
nevirapine, and delavirdine) and the next-generation NNRTI etravirine all have side effects and/or drug interactions that limit their use
for the treatment of HIV. In addition, all first-generation NNRTIs are
susceptible to rapid-resistance generation through single-point mutations (Turpin, 2003).

This research was sponsored by Pfizer Inc. and was conducted at Charles
River Clinical Services Ltd., Edinburgh, United Kingdom.
1
Current affiliation: Drug Metabolism and Pharmacokinetics, Pharmaceutical
Division, F. Hoffman-La Roche Ltd., Basel, Switzerland.
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
doi:10.1124/dmd.109.031252.

ABBREVIATIONS: NNRTI, non-nucleoside reverse transcriptase inhibitor; lersivirine/UK-453,061, 5-((3,5-diethyl-1-(2-hydroxyethyl)-1H-pyrazol4-yl)oxy)benzene-1,3-dicarbonitrile; P450, cytochrome P450; ADME, absorption, distribution, metabolism, and excretion; [14C]lersivirine, 5-{[3,5diethyl-1-(2-hydroxyethyl)(3,5-14C2)-1H-pyrazol-4-yl]oxy}benzene-1,3-dicarbonitrile; HPLC, high-performance liquid chromatography; AUCinf,
area under the plasma concentration-time profile from time zero extrapolated to infinite time; MS/MS, tandem mass spectrometry; AE, adverse
event; rUGT, recombinant human UDP-glucuronosyltransferase; PF-04580552, 2-[4-(3,5-dicyanophenoxy)-3,5-diethyl-1H-pyrazol-1-yl]ethyl
b-D-glucopyranosiduronic acid; PF-03139905, 5-{[3-ethyl-5-(1-hydroxyethyl)-1-(2-hydroxyethyl)-1H-pyrazol-4-yl]oxy}benzene-1,3-dicarbonitrile;
UK-533,713, [4-(3,5-dicyanophenoxy)-3,5-diethyl-1H-pyrazol-1-yl]acetic acid; UK-508,550, 5-{[5-ethyl-1,3-bis(2-hydroxyethyl)-1H-pyrazol-4yl]oxy}benzene-1,3-dicarbonitrile; PF-03230716, 5-{[5-ethyl-3-(1-hydroxyethyl)-1-(2-hydroxyethyl)-1H-pyrazol-4-yl]oxy}benzene-1,3-dicarbonitrile; JNJ-10198409, 3-Fluoro-N-(6,7-dimethyoxy-2,4-dihydroindeno[1,2-c]pyrazol-3-yl)phenylamine.
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ABSTRACT:

790

VOURVAHIS ET AL.

A
293.22

100
90
Relative abundance (%)

80
70
60
267.19

50
40
30
20
124.12137.12
150.16
95.15 109.09
167.24

10
0
80

100

120

140

160

196.29 212.20

180

200
m/z

220

239.21

240

265.13 278.14

260

280

306.65

300

320

FIG. 1. MS/MS spectrum of lersivirine (A) and proposed fragmentation of lersivirine (B).

B
N

N

O

137

124

N

N

267
293

HO

metabolism enzymes responsible for the metabolism of lersivirine
are described.
Materials and Methods
Chemicals and Reagents. [14C]Lersivirine was synthesized by GE Healthcare (Little Chalfont, Buckinghamshire, UK) at a specific activity of 3.7

5000

Blood radioactivity ng eq/g
Plasma radioactivity ng eq/g
Plasma lersivirine ng/mL

4500
4000
Concentration

Lersivirine (formerly UK-453,061), an NNRTI with a unique
binding interaction within the reverse transcriptase binding pocket
(Phillips et al., 2007), is currently in clinical development for the
treatment of HIV-1 infection. Lersivirine has shown potent in vitro
activity against both wild-type and clinically relevant drug-resistant viruses (Mori et al., 2008). Furthermore, lersivirine has shown
synergy in vitro with other classes of compounds, particularly
those in the nucleoside reverse transcriptase inhibitor class. In vitro
studies suggest that lersivirine has good membrane permeability
(Allan et al., 2008) and is predominantly cleared by metabolism,
via glucuronidation and cytochrome P450 (P450)-mediated oxidation (Vourvahis et al., 2009). Furthermore, the clinical safety,
tolerability, and pharmacokinetics of lersivirine have been studied
in both HIV-infected and healthy subjects (Davis et al., 2007;
Fatkenheuer et al., 2009). In a 7-day monotherapy study, lersivirine was found to be well tolerated at all the doses used, achieving significant mean viral load reductions of ⱖ1.62 log10 for
dosing regimens of 500 mg once daily, 750 mg once daily, and 500
mg b.i.d. in HIV-infected patients (Fatkenheuer et al., 2009).
This open-label Phase I study investigated the absorption, distribution, metabolism, and excretion (ADME) of a single oral 500-mg dose
of [14C]lersivirine (parent drug) to characterize the plasma, fecal, and
urinary radioactivity of lersivirine and its metabolites in healthy
male subjects. In addition, in vitro data identifying the major drug

3500
3000
2500
2000
1500
1000
500
0
0

12

24

60
36
48
Time post dose (hours)

72

168

FIG. 2. The mean blood and plasma concentration-time profiles for drug-derived
total radioactivity and lersivirine (parent drug) after a single oral administration of
500 mg of [14C]lersivirine.

791

EXCRETION AND METABOLISM OF LERSIVIRINE
TABLE 1
Lersivirine pharmacokinetic parameter values (geometric means and intersubject variability)
PK Parameter (Units)

n
AUCinf (ng 䡠 h/ml)a,b
Cmax (ng/ml)a,b
Tmax (h)c
t1/2 (h)d
CL/F (l/h)b
Vz/F (liter)b

Lersivirine (Plasma)

4
4100 (19)
1010 (40)
1.50 (0.50–3.00)
5.83 (26)
122 (18)
994 (24)

Total Drug-Related Radioactivity (Plasma)

Total Drug-Related Radioactivity (Whole Blood)

4
39,130 (4)
6042 (21)
1.50 (1.00–3.00)
9.41 (30)
12.78 (4)
169.4 (26)

4
18,650 (7)
2767 (27)
1.50 (1.00–3.00)
9.91 (19)
26.81 (7)
37,804 (15)

AUCinf, area under the plasma concentration-time profile from time zero extrapolated to infinite time; CL/F, apparent clearance; Cmax, maximum plasma concentration; t1/2, terminal half-life;
Tmax, time to reach maximum concentration; VZ/F, apparent volume of distribution.
a
Units for radioactive compound are ng-Eq 䡠 h/g for AUCinf and ng-Eq/g for Cmax.
b
Geometric mean (percentage coefficient of variation).
c
Median (range).
d
Arithmetic mean (percentage coefficient of variation).

80
% of lersivirine dose excreted in urine

A

Subject 1
Subject 2
Subject 3
Subject 4
Mean

70
60
50
40
30
20
10
0
0 to 12

12 to 24

24 to 36

36 to 48

48 to 72

72 to 96 144 to 168

Time post dose (hours)

B

20
% of lersivirine dose excreted in feces

kBq/mg and radiochemical purity of 97.6% as assessed by high-performance
liquid chromatography (HPLC) (Alliance 2695 Separations Module with 2487
Dual Absorbance Detector; Waters, Milford, MA). Authentic, nonradiolabeled
lersivirine was supplied by Pfizer Global Research and Development; purity
was 99.3% as assessed by HPLC. Authentic standards of known metabolites of
lersivirine [PF-04580552 (M15), PF-03139905 (M17), and UK-533,713
(M19)] were synthesized at Pfizer Global Research and Development (Sandwich, UK). Aquasafe 500 Plus liquid scintillation fluid was obtained from
Zinsser Analytic (Maidenhead, UK). Carbo-Sorb CO2 absorbing solution and
Permafluor E⫹ scintillation fluid, used in conjunction with the Packard TriCarb 307 Automatic Sample Oxidizer, were supplied by PerkinElmer Life and
Analytical Sciences (Waltham, MA). Spec-Check-14C, used to estimate combustion efficiency, was also from PerkinElmer Life and Analytical Sciences.
Luna C18 (for radiopurity; Phenomenex, Torrance, CA) and HIRPB (for urine,
feces, and plasma profiling; Hichrom Ltd., Theale, UK) columns were used for
HPLC analyses. All the other commercially available chemicals and solvents
were of analytical grade where available.
Clinical ADME Study Design. Study design. This study, conducted at
Charles River Clinical Services Ltd. (Edinburgh, UK), was an 8-day, openlabel, Phase I study of lersivirine in four healthy male subjects. All the subjects
gave written informed consent; the study was conducted in accordance with the
Declaration of Helsinki and was approved by an accredited institutional ethics
committee. After an overnight fast, all the subjects received a single oral
500-mg dose of [14C]lersivirine as a 5-ml suspension in aqueous Avicel RC
591, containing a maximum of 62 ␮Ci of [14C]; this amount of radioactivity
was chosen to comply with the International Commission on Radiological
Protection category IIa guidelines (radioactive exposure not exceeding 1 mSv),
followed by 240 ml of water. Participants remained in an upright position and
refrained from eating or drinking for 4 h postdose.
Sample collection. Blood samples (12 ml) were collected into nonbeaded
lithium-heparinized tubes immediately predose and at 0.5, 1, 2, 3, 4, 6, 8, 10,
12, 18, 24, 36, 48, 72, 96, 120, 144, and 168 h postdose. One milliliter of whole
blood per sample was transferred into a nonheparinized tube and stored at 4°C
for subsequent liquid scintillation counting. The remaining samples were
processed for plasma via centrifugation at 1500g for 10 min at 4°C; samples
were then divided into three aliquots for HPLC tandem mass spectrometry
(HPLC/MS/MS) analysis of lersivirine (1 ml), liquid scintillation counting (1
ml), and metabolite profiling (approximately 3 ml).
Urine samples were collected at 12-h intervals postdose until day 4 and then
at 24-h intervals until 168 h postdose. Urine was stored at 4°C until the end of
the collection period. A 10-ml aliquot of each sample was withdrawn for
scintillation counting, and 2 ⫻ 50-ml aliquots were frozen at ⫺20°C for
subsequent metabolite profiling.
Fecal samples were collected in polypropylene containers at 24-h intervals
until 168 h postdose and stored at ⫺20°C. After each collection, samples were
homogenized in water. Duplicate portions of the homogenate (0.4 g each) were
taken for combustion in oxygen, and a separate 100-g homogenate aliquot was
used for metabolite profiling. Sample collection was stopped when ⱖ90% of
administered radioactivity had been recovered in the combined urine and fecal
samples or when ⬍1% of radioactivity was recovered in 24 h.
Determination of total radioactivity. Whole blood, plasma, urine, and fecal
samples were assayed for drug-derived total radioactivity. Liquid scintillation

Subject 1
Subject 2
Subject 3
Subject 4
Mean

15

10

5

0
0 to 24

24 to 48

48 to 72

72 to 96

144 to 168

Time post dose (hours)

C
Total recovery

103.7

Feces

23.2

Urine

80.4

0

20
40
60
80
100
% recovery post lersivirine dose

120

FIG. 3. Excretion of total radioactivity after a single oral administration of 500 mg
of [14C]lersivirine in urine (A), feces (B), and in total (C).

792

VOURVAHIS ET AL.

counting was used for both urine (2300 TR; Canberra Industries, Meriden, CT)
and plasma (Guardian; PerkinElmer Life and Analytical Sciences) samples
after mixing with scintillation mixture. Whole-blood samples were first mixed
with SOLVABLE (PerkinElmer Life and Analytical Sciences), EDTA, and
H2O2 before mixing with scintillation mixture and subjected to liquid scintillation counting (Guardian; PerkinElmer Life and Analytical Sciences). Fecal
samples were homogenized in water using a Waring (Stamford, CT) industrial
blender, combusted in a biological oxidizer (model 307 MK2 Tri-Carb;
PerkinElmer Life and Analytical Sciences), and the evolved 14CO2 was
trapped and measured by liquid scintillation counting in Permafluor E⫹
scintillation mixture (PerkinElmer Life and Analytical Sciences). A 1-ml
aliquot of each plasma sample was analyzed by scintillation counting. The
detection limit was 2.5 dpm above background. Samples of plasma (⬃0.2 g)
were mixed with scintillation mixture (16 ml, Starscint; PerkinElmer Life
and Analytical Sciences) before liquid scintillation counting (Guardian;
PerkinElmer Life and Analytical Sciences). Total radioactivity (percentage of
dose) was determined for urine, feces, and their combination.
Plasma assay. Plasma samples were analyzed for lersivirine concentrations
using a validated HPLC/MS/MS method (Allan et al., 2008). The method was

linear over the range of 1 to 2000 ng/ml. The lower limit of quantification was 1
ng/ml.
Metabolite profiling. Urine samples (0 –36 h) were prepared for metabolite
profiling by centrifugation (3500 rpm, 15 min, 4°C); resultant pools (2 ml/
subject) were profiled by HPLC using PU-2080 Plus pumps coupled to an
HIRPB column (250 ⫻ 7.75 mm; Hichrom Ltd.). Separation was achieved
with a binary solvent gradient at 2 ml/min, comprising methanol and 0.1 M
ammonium acetate, pH 5. The gradient consisted of 30% methanol held for 10
min, increased to 50% over 1 min, held at 50% for 14 min, increased to 80%
methanol over 20 min and held for 5 min, before returning to 30% methanol
over 1 min, which was maintained for a further 4 min. Fractions (6.88 s) were
collected into 96-well Scintiplates (PerkinElmer Life and Analytical Sciences),
which were then dried and counted on a Microbeta scintillation counter
(PerkinElmer Life and Analytical Sciences). Composite plasma samples (0 –24
h time-normalized pools) (Hamilton et al., 1981; Hop et al., 1998) were treated
1:4 with methanol, centrifuged, and resultant supernatants were collected,
dried, and profiled by HPLC in a manner similar to that of the urine samples.
Drug-related material was extracted from composite fecal homogenate samples
(0 –96 h) by mixing with methanol, followed by sonication. The mixture

Counts per minute

A Plasma (0 to 24 hours)
M15

120.0
110.0
100.0
90.0
80.0
70.0
60.0
50.0
40.0
30.0
20.0
10.0
0.0

P

M8

M9

M3, M4
M6

M1

0.0
B Urine (0 to 36 hours)

M2

10.0

M10

M11

M17
M19
M20

M7

20.0

30.0

M21

40.0

50.0

mins

800.0
M15

700.0
Counts per minute

600.0
500.0

FIG. 4. Representative radiochromatograms showing lersivirine
metabolites in the plasma (A), urine (B), and feces (C) of one
healthy male subject after a single oral (500 mg) administration of
[14C]lersivirine.

400.0
M9 M10

300.0

M8

M17

M3, M4

200.0

M11

M18

M7

100.0

M2

M6

M19

M14

P

0.0

Counts per second

0.0
C Feces (24 to 96 hours)

10.0

26.0
24.0
22.0
20.0
18.0
16.0
14.0
12.0
10.0
8.0
6.0
4.0
2.0
0.0

20.0

30.0

40.0

50.0

mins

50.0

mins

M9
M19

M12

M13
M22
M5

M18
M17
M21

M2
P

0.0

10.0

20.0

30.0

40.0

793

EXCRETION AND METABOLISM OF LERSIVIRINE
was centrifuged at 3500 rpm for 15 min, and the supernatant was collected.
This extraction procedure was repeated with a mixture of methanol (1 ml) and
Tris buffer (0.1 M, pH 6, 24 ml) and a mixture of methanol (23 ml) and Tris
buffer (0.1 M, pH 9, 2 ml). The extracts were combined and reduced to dryness
under nitrogen at 37°C in a Turbovap (Zymark, Hopkinton, MA), and the
residue was suspended in mobile phase for HPLC analyses. The fecal extracts
were profiled using the same HPLC system as urine and plasma; radiochromatographic peaks were isolated manually via on-line radiochemical detection
(␤-RAM; IN/US Systems, Tampa, FL) and reduced to dryness under nitrogen
at 37°C in a Turbovap (Zymark).
Metabolite identification. Initial identification of drug-related metabolites isolated from human plasma and excreta was determined by direct-infusion MS on a
Sciex API 4000 Q Trap mass spectrometer (Applied Biosystems, Foster City, CA)
using appropriate precursor and neutral loss experiments based on the MS/MS
fragmentation of lersivirine (Fig. 1, A and B). The mass spectrometer was operated
with a Turbo Spray source and controlled by Analyst 1.4.1 software (Applied
Biosystems). For MS/MS experiments, the collision energy was typically 40 V;
declustering potential was set to 50 V; and the ion spray voltage was set to 5000
eV. Additional confidence in structural assignments was obtained by HPLC/
MS/MS comparison with authentic standards and by the acquisition of additional
MS3 and accurate mass data using a Thermo LTQ Orbitrap mass spectrometer
(Thermo Fisher Scientific, Waltham, MA). The mass spectrometer was operated in
positive ion mode with data-dependent acquisition, typically consisting of full-scan
accurate mass data (100 – 800 mass units) triggering MS/MS experiments from a
parent ion list and subsequently MS3 experiments on the two largest ions in the
MS/MS spectrum. The Fourier transform MS resolution was set at 15,000. Both
MS/MS and MS3 experiments were performed with a normalized collision energy

A

of 40 V and an activation time of 30 ms. The HPLC/MS system consisted of
Agilent Technologies (Santa Clara, CA) 1100 binary pumps coupled to a Sunfire
C18 column (3.5 ␮m, 100 ⫻ 2.1 mm; Waters), with a binary solvent gradient (200
␮l/min) of formic acid (0.1% aqueous) and acetonitrile (plus 0.1% formic acid)
held at 5% acetonitrile for 1 min, increased to 98% over 7 min, held for 1 min, then
returned to 5% over 0.1 min and held for 3.9 min.
Glucuronide conjugates isolated from human urine were subjected to deconjugation using Helix pomatia extract (Sigma-Aldrich, St. Louis, MO) to provide
additional structural information on these metabolites. Dried isolates were reconstituted in water and incubated with an equal volume of H. pomatia extract for up
to 24 h. Incubations were terminated by addition of acetonitrile, diluted with 0.1 M
ammonium acetate, pH 5, and centrifuged (3500 rpm, 15 min, 4°C) to enable
analysis of each sample on the same HPLC system used for urine profiling.
Regions of interest were then analyzed by HPLC/MS/MS using a Thermo LTQ
Orbitrap MS (Thermo Fisher Scientific) as described previously.
Safety and tolerability. Blood pressure, pulse rate, ECG, and physical exams,
urine drug screening, and monitoring of adverse events (AEs) were reviewed on an
ongoing basis throughout the study. The investigator obtained and recorded all the
observed or volunteered AEs, the severity (mild, moderate, or severe) of the
events, and the investigator’s opinion of the relationship to lersivirine.
Statistics. Pharmacokinetic and radioactivity parameters were calculated for
each subject by using noncompartmental analysis of lersivirine and total
radioactivity plasma and blood concentration versus time profiles. Values were
summarized using descriptive statistics.
In Vitro Preclinical Metabolism Studies. Pooled human liver microsomes and microsomes prepared from baculovirus-infected insect cells
engineered to individually express recombinant human UDP-glucuronosyl-

B
N

N

N

N

N

N
N

N

N

N

N

N

N
O

O
O

NH

N
N

N

Gluc
O

M10
2%

N

O
O
Gluc

O

NH
N

M2 & M7
2% & 1%

N

N

SO3H
O

N

O

M18
2%

N
N

N

N

M16
1%

N

O

1%

N

Gluc

N

N

N

O

N

NH

NH
N

O

M10 & M14
2% & 1%

O

O
O

O

OH

M1
<1%

N

<1%

OH
O

O

O

N
N

N

N

OH

O Gluc

N

NH2
O

O
N

Gluc

OH
N

N

O

OH

M12
1%

N

O

OH

O

Gluc

N

N

O

N
N

HO

<2%
N

N

<1%

O

HO

N

O

O

N

OH

M4
<2%

N

N

N

OH
Gluc

M6;
3%

8%

O

O
N

M8 & M11;
4% & 1%

O

Gluc

N
N
N

OH
Gluc
OH

M4
<1%

N

N

2xO

M5
1%

N

OH
N

N

O
OH

54%

O

N

N

NH2

OH
N

HO

N

N

OH
N

O

O Gluc

N

HO

N

N

N

8%

N

N

N

OH
Gluc

N

OH

N

N

NH2

OH

N

O

N

OH

HO

N NH

N

OH

O

N

N

N

OH

M1
1%

N

M13
3%

N

45%
N

OH

Lersivirine
<1%

N

N

N

O

N

N

Lersivirine
13%

O

OH

N

N

N

N

N

N

N

M22
1%

N

OH

NH

N

OH
NH
N

N

O

N

N

O
Gluc

M2 & M7
2% & 1%
NH2

O
O
N

M20
1%

N

O

3%

OH

Gluc

O

N

O

NH

M9
10%

N

OH

N
N

O

NH

Gluc

M9
6%

O

O

N

N

2%

O

OH

OH
O

N

HO

N

OH
Gluc

N

M6;

M8 & M11;

2%

3% & 2%

N

Gluc
OH

FIG. 5. Proposed metabolic pathways of lersivirine in human excreta (A), with percentage of total excreted dose, and in human plasma (B), with percentage of AUC data
for composite (0 –24 h) samples after oral (500-mg single dose) administration of lersivirine.

794

VOURVAHIS ET AL.

transferase (rUGT) isoforms UGT1A1, UGT1A3, UGT1A4, UGT1A6,
UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15,
and UGT2B17 were obtained from BD Gentest (Woburn, MA). Human
recombinant P450s (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4,
and CYP3A5) were obtained from PanVera Corp. (Madison, WI), whereas
isozymes CYP2C8 and CYP2B6 were obtained from BD Gentest.
For glucuronidation studies, lersivirine (50 ␮M) was incubated with
human liver microsomes (0.5 mg/ml) or each of the rUGTs (0.5 mg/ml) for
up to 120 min at 37°C. Incubations comprised 50 mM Tris-HCl (pH 7.4 at
25°C), 5 mM saccharolactone, alamethicin at 50 ␮g/mg protein, 10 mM
MgCl2, and 5 mM UDP-glucuronic acid. Microsomes and rUGTs were
activated and incubated as described previously (Hyland et al., 2009). The
reaction was terminated by adding 3⫻ volume ice-cold acetonitrile at
specific times throughout the duration of the incubation, centrifuged at
3000 rpm for 45 min at 4°C, and analyzed for lersivirine and lersivirine
glucuronide on a Sciex API 3000 mass spectrometer (Applied Biosystems).
For UGT, enzyme kinetic study incubations were initially conducted to
optimize incubation time and protein concentration before the kinetic
study, which was conducted over a lersivirine range of 10 to 1500 ␮M.
Rates of glucuronide formation were quantified against an authentic standard and used to obtain values for Km and Vmax.
For P450 studies, lersivirine (1 ␮M) was incubated with each of the
recombinant P450 isozymes (150 pmol/ml) for 60 min. Incubations comprised
50 mM phosphate buffer, pH 7.4, 5 mM MgCl2, and 1 mM NADPH, regenerated in situ from an isocitric acid/isocitric acid dehydrogenase regenerating
system (Youdim et al., 2008). Reactions were terminated by the addition of
acetonitrile containing midazolam (as internal standard), followed by centrifugation at 3000 rpm for 45 min at 4°C. Samples were then analyzed for
lersivirine and two of the major oxidized products identified from the human
ADME study (M19 and M17) on a Sciex API 4000 mass spectrometer
(Applied Biosystems).

Results
Clinical ADME Study. Study subjects. Four healthy white male
subjects completed the study; mean age (⫾S.D.) was 37.3 years
(⫾7.4; range, 31– 48 years), and mean body weight was 81.3 kg
(⫾12.8).

Pharmacokinetics. The mean plasma concentration-time profiles
for total drug-derived radioactivity and lersivirine are shown in Fig. 2,
and the geometric means and intersubject variability for pharmacokinetic parameters for lersivirine and total drug-derived radioactivity are
shown in Table 1. There were appreciable differences in the Cmax and
AUCinf of total drug-derived radioactivity and lersivirine, indicating
that extensive metabolism of lersivirine had occurred. Tmax for total
drug-derived radioactivity in plasma and blood and lersivirine in
plasma occurred between 0.5 and 3 h postdose, and the mean blood/
plasma ratio of total drug-derived radioactive material AUCinf was
0.48.
Mass balance. The majority of radioactivity was excreted in urine,
accounting for 80% of the dose, whereas 23% was recovered in feces
(Fig. 3C). Mean total recovery of radioactivity (urine plus feces) was
103.7%. By 120 h postdose, all the radioactivity had been excreted,
with the largest recovery occurring within the first 12 h for urine and
first 48 h for fecal samples (Fig. 3, A and B).
Metabolite profiling. HPLC profiling of precipitated plasma (0 –24
h) after time-normalized pooling, pooled urine (0 –36 h), and pooled
fecal homogenate (0 –96 h) using radiochemical detection showed
extensive metabolism for all the matrices in all the subjects (Fig. 4).
Based on cochromatography, unchanged lersivirine was a major component of human plasma but only a minor component in excreta.
Metabolite identification. MS analysis of components isolated from
human plasma and excreta identified 22 metabolites of lersivirine
(Fig. 5, A and B). The MS/MS spectrum and proposed fragmentation
of lersivirine are shown in Fig. 1, A and B, with major ions at m/z 293
and 267 representing loss of water and loss of C2H4O, respectively.
Analysis of precipitated plasma (0 –24 h; Table 2) revealed that the
glucuronide conjugate (M15) was the major circulating component
(45% of total radioactivity). The MS/MS spectrum and proposed
fragmentation of M15 (m/z 487) is shown in Fig. 6, A and B, showing
the major fragment ion at m/z 311, representing the characteristic loss
of the glucuronic acid moiety (176 atomic mass units). As expected,

TABLE 2
14

Relative quantitation of [ C]lersivirine metabolites in human plasma and excreta
%Dose
Metabolite

M1
M2
M3
M4
M5
M6
M7
M8
M9
M10
M11
M12
M13
M14
M15
M16
M17
M18
M19
M20
M21
M22
Lersivirine
Total

m/z

345
459
503
521
343
503
459
503
443
501
503
341
330
501
487
286
327
391
325
325
267
283
311

%Circulating Radioactivity
Urine

Feces

Total

mean (range)

mean (range)

mean (range)

mean (range)

1 (N.D.–3)
2 (2–3)
⬍2 (⬍1–⬍2)
⬍2 (⬍1–⬍2)
N.D. (N.A.)
3 (2–3)
1 (N.A.)
4 (3–5)
6 (4–7)
2 (N.A.)
1 (1–2)
N.D. (N.A.)
N.D. (N.A.)
N.D. (N.A.)
45 (41–49)
N.D. (N.A.)
8 (6–10)
N.D. (N.A.)
3 (2–3)
1 (1–2)
⬍1 (N.D.–1)
N.D. (N.A.)
13 (10–16)
92 (90–92)

⬍1 (N.D.–⬍1)
2 (1–2)
⬍1 (N.A.)
⬍1 (N.A.)
N.D. (N.A.)
2 (N.A.)
1 (N.A.)
3 (3–4)
5 (3–6)
2 (N.A.)
2 (1–2)
N.D. (N.A.)
N.D. (N.A.)
1 (1–2)
54 (49–66)
N.D. (N.A.)
1 (N.A.)
1 (N.A.)
3 (2–3)
N.D. (N.A.)
N.D. (N.A.)
N.D. (N.A.)
⬍1 (N.A.)
77 (69–89)

N.D. (N.A.)
1 (N.D.–1)
N.D. (N.A.)
N.D. (N.A.)
1 (N.D.–2)
N.D. (N.A.)
N.D. (N.A.)
N.D. (N.A.)
5 (3–7)
N.D. (N.A.)
N.D. (N.A.)
1 (⬍1–1)
4 (3–5)
N.D. (N.A.)
N.D. (N.A.)
1 (N.D.–2)
1 (N.D.–1)
1 (1–2)
6 (4–7)
N.D. (N.A.)
1 (1–2)
1 (N.D.–2)
⬍1 (N.D.–1)
22 (14–27)

⬍1 (N.D.–⬍1)
2 (2–3)
⬍1 (N.A.)
⬍1 (N.A.)
1 (N.D.–2)
2 (N.A.)
1 (N.A.)
3 (3–4)
10 (9–11)
2 (N.A.)
2 (1–2)
1 (⬍1–1)
4 (3–5)
1 (1–2)
54 (49–66)
1 (N.D.–2)
2 (1–2)
2 (2–3)
8 (6–10)
N.D. (N.A.)
1 (1–2)
1 (N.D.–2)
⬍1 (N.D.–1)
99 (96–103)

N.A., not available; N.D., not detected. Data from all the subjects were included in the calculation of the mean, N.D. being equal to zero for the calculation. Where there is no range stated it
is because there was no variability between subjects.

EXCRETION AND METABOLISM OF LERSIVIRINE
A

795

311.19

100
Relative abundance (%)

90
80
70
60
50
40
30
20
10
0

141.03 159.05

210.24 235.13

150

200

267.29 293.32

250

312.21

300
m/z

353.33

350

399.17 411.49 451.15 469.23

400

450

500

293.21

100
Relative abundance (%)

90
80
70

267.26

60
50
40

FIG. 6. MS/MS and MS3 spectrum of M15 (A) and proposed
fragmentation of M15 (B).

30
20
10

124.22
80.26 95.22109.18

0

80

100

137.19
150.23
153.23 167.24 183.16

120

140

160

180

210.23

200
m/z

220

239.28

265.32

278.30

240

260

280

294.16 311.20

300

320

B

N

N
O
N N
OH
O

267

293

O

O
311

HO

OH
HO

the MS3 spectrum of m/z 311 is essentially identical to the MS/MS
spectrum of lersivirine. A product of mono-oxidation of one of the
ethyl side chains (M17) accounted for 8% of the total radioactivity,
whereas unchanged lersivirine accounted for 13%. The MS/MS spectrum and proposed fragmentation of M17 (Fig. 7, A and B) showed a
major ion at m/z 309, consistent with loss of water. The MS3 spectrum
indicated a further loss of water to yield m/z 291 and a loss of C2H4O
to give m/z 265. Nine additional products of glucuronidation were
detected; metabolites M3, M6, M8, and M11 were derived by glucuronidation in conjunction with monohydroxylation, and each accounted for between 1 and 4% of the total radioactivity; metabolites
M2 and M7 were produced by glucuronidation in conjunction with
N-dealkylation and mono-oxidation, both accounting for 3% of circulating radioactivity; M9 was a product of N-dealkylation and glucuronidation (6%); M4 was a product of oxidation, glucuronidation,
and hydrolysis of one of the nitrile moieties to form an amide (⬍2%);
whereas M10 formation was postulated to involve oxidation to form
a ketone on one of the ethyl side chains and glucuronidation (2%).

Additional characterization of four of the glucuronic acid conjugates was achieved by enzymatic hydrolysis with ␤-glucuronidase
followed by HPLC/MS/MS analysis of the deconjugated analog.
Figure 8, A and B, shows the MS/MS spectrum and proposed fragmentation of M3, whereas Fig. 8C shows the radiochromatogram for
M3 before and after hydrolysis together with chromatographic comparison with the mono-oxidized authentic standard UK-508,550. The
approach resulted in identification of M3 as a product of oxidation on
the terminal carbon of one of the ethyl side chains followed by
glucuronidation. The same approach was used to provide further
structural information for M6, M8, and M11, which shows that M6
was a glucuronic acid conjugate of the hydroxylated metabolite PF03230716, whereas M8 and M11 were both glucuronides of the
hydroxy metabolite M17, involving conjugation on both alcohol moieties. The MS/MS data for the glucuronic acid metabolite M9 showed
the characteristic loss of 176 mass units to yield a major fragment ion
at m/z 267 (Fig. 9, A and B). The specific site of glucuronidation for
M9 was not determined but is postulated as a product of glucuronida-

796
A

VOURVAHIS ET AL.
309.18

100
Relative abundance (%)

90
80
70
60
50
40
30
20
10
123.28 141.29

0
80

100

120

166.29 181.17

140

160

180

226.40

200 220
m/z

251.23 265.10 283.24 291.25

240

260

280

310.27

300

320

340

291.19

100
Relative abundance (%)

90
80
70

265.20

60
50

FIG. 7. MS/MS and MS3 spectrum of M17 (A) and proposed fragmentation of M17 (B).

40
30
20
10

95.12 107.12

0
80

B

100

122.17

120

148.18

140

166.16 181.15

160

180

208.15

200
m/z

220

236.17

240

263.19

260

276.17

280

294.15 309.07

300

320

N

N
O

N
N
HO

265
-C2H4O
309

291

HO

tion of the pyrazole ring. Further low-level drug-related products of
N-dealkylation, hydroxyethyl side chain oxidation, glucuronidation,
nitrile group hydrolysis, and ethyl side chain oxidation were also
detected in the plasma (Fig. 4A).
Profiling of human urine and extracted fecal homogenates also
revealed similar and extensive metabolism in all four subjects (Fig. 4,
A and B). The major components were the glucuronide of lersivirine,
M15, accounting for 54% of the dosed radioactivity; a metabolite
involving N-dealkylation and glucuronidation (M9, 10%); and M19, a
product of oxidation of the hydroxyethyl moiety to a carboxylic acid
(8%). The MS/MS data for the carboxylic acid metabolite (M19) are
shown in Fig. 10, A and B, with the major ion at m/z 279 representing
loss of CH2O2. Sulfation of the parent compound and nitrile group
hydrolysis were also detected in excreta, whereas unchanged lersivirine accounted for ⬍1% of the dose in both urine and feces.
Safety. There were no serious AEs and no discontinuations as a
result of AEs after administration of radioactive lersivirine (ADME
study). The only AE reported during the ADME study was mild
headache, which was not considered treatment-related and resolved
during the course of the study.

In Vitro Preclinical Metabolism Studies. Of the UGTs investigated only rUGT2B7 was able to metabolize lersivirine. Further
investigations through enzyme kinetic experiments performed in human liver microsomes and rUGT2B7 showed that formation of glucuronide was linear with time up to 60 min and protein up to 1 mg/ml.
Kinetic studies were performed at 0.5 mg/ml for 20 min in both
rUGT2B7 and human liver microsomes over a substrate concentration
range of 10 to 1500 ␮M (Fig. 11).
Data analysis indicated that in human liver microsomes glucuronidation exhibited typical Michaelis-Menten kinetics, characterized
by a Km of 224.7 ⫾ 56.7 ␮M and a Vmax of 1583.3 ⫾ 468.0
pmol/min/mg protein. Enzyme kinetics in rUGT2B7 also exhibited
Michaelis-Menten kinetics with a Km of 120.2 ⫾ 31.8 ␮M and a Vmax
of 725.1 ⫾ 64.8 pmol/min/mg protein.
In incubations with recombinant P450 enzymes, CYP3A4 metabolized lersivirine most rapidly with an intrinsic clearance of 0.9
␮l/pmol P450/min. The only other enzyme shown to metabolize
lersivirine based on a substrate depletion approach was CYP3A5, with
a greater than 10-fold lower rate of metabolism (⬍0.08 ␮l/pmol
P450/min). The in vitro clearance in rCYP3A4 was scaled to activity

797

EXCRETION AND METABOLISM OF LERSIVIRINE
A

C
327.14

100

800.0
700.0

80

Counts per minute

Relative abundance (%)

90
70
60
50
40
30
20
10
161.16

0

243.47 262.20 291.09 309.17

198.98

150

200

250

328.14
369.22 381.36 408.95 444.39 467.21 485.35 514.02

300

350

400

450

600.0
500.0
400.0
M3
300.0
200.0
100.0

500

0.0

m/z

90
80

Counts per minute

Relative abundance (%)

0.0

10.0

20.0

0.0

10.0

20.0

309.17

100

70
60
50
40
30
20
310.28

10
100.23

0
80

100

153.20 165.94

122.13

120

140

160

209.87 219.26 236.25

180

200 220
m/z

240

291.23

265.10

260

280

311.26

300

320

340

30.0
mins

40.0

50.0

30.0

40.0

50.0

40:00

50:00

34.0
32.0
30.0
28.0
26.0
24.0
22.0
20.0
18.0
16.0
14.0
12.0
10.0
8.0
6.0

mins

B
N
N

mV

O
N
N

OH
327

HO

O

-H 2O

309

OH

HO

1100.0
1000.0
900.0
800.0
700.0
600.0
500.0
400.0
300.0
200.0
100.0
0.0
0:00

O

OH

HO

10:00

20:00

30:00
mm:ss

FIG. 8. MS/MS and MS3 spectrum of M3 (A), proposed fragmentation of M3 (B), and radiochromatogram for M3 before and after hydrolysis and comparison with
UK-508,550 (C).

Relative
abundance (%)

A
100
80
60
40
20
0

309.17

95.18 109.35 122.06 136.16 153.21 166.08 186.47 209.66 225.93 236.24

80

100

120

140

160

180

200 220
m/z

240

265.19

260

291.15

280

300

310.20
320.62

320

340

B

N
N

FIG. 9. MS/MS and MS3 spectrum of M9 (A) and proposed fragmentation of M9 (B).

O

N
N
HO

309

OH

798

VOURVAHIS ET AL.

A

267.11

100
Relative abundance (%)

90
80
70
60
50
40
30
20
10
141.05 159.02 176.66

0

150

226.82 239.20

200

250

309.12

340.42 353.30 382.27 407.25

300
m/z

350

425.13

400

434.15

450

124.10

100

123.09

90
Relative abundance (%)

268.12

239.11

80
70
60
50
109.02

40

FIG. 10. MS/MS spectrum of M19 (A) and proposed fragmentation
of M19 (B).

30
20
10

96.08

81.14

0
60

80

100

145.08 156.04 167.06

120

140

160

183.90 195.14

180
m/z

200

212.11

220

238.37

240

250.18

267.25

260

280

B

N

N

O
123

NH N
HO

267
H4

O

2

239

OH
O
HO

OH

in human liver microsomes using a relative activity factor of 0.28
(determined in-house) and a CYP3A4 abundance of 120 pmol
P450/mg microsomal protein. The scaled recombinant P450 clearance
of 8.55 ␮l/min/mg liver microsomal protein compared well with the
human liver microsomal clearance of 8.23 ␮l/min/mg. In addition,
CYP3A4 was shown to be capable of forming the oxidative metabolites seen in vivo (M19 and M17).
Discussion
In this human ADME study, recovery of the oral [14C]lersivirine
dose was complete (103.7%) and occurred within 5 days after ingestion in healthy volunteers. The high urinary excretion of radioactivity
(80.4%) is evidence that lersivirine is well absorbed after oral dosing
in humans. After absorption lersivirine was extensively metabolized,
with unchanged lersivirine constituting 13% of total plasma radioactivity (AUCinf) and ⱕ1% of excreted radioactivity.
The blood/plasma ratio of total drug-derived radioactivity AUCinf was
0.48, indicating limited distribution of radiolabeled material to red blood
cells. This result likely reflects limited penetration of the polar lersivirine

glucuronide (the major circulating metabolite in plasma) into red blood
cells because in vitro studies with [14C]lersivirine show a higher blood/
plasma ratio of 0.77 (Pfizer Inc., data on file).
Characterization of the radioactivity in plasma and excreta of
humans dosed with [14C]lersivirine showed that at least 22 different
metabolites were present and indicated that glucuronidation is the
primary metabolic pathway of lersivirine. In both plasma and excreta
the major metabolite was a glucuronide conjugate of lersivirine
(M15). In vitro studies showed that of the isozymes studied only
UGT2B7 was capable of forming lersivirine glucuronide. Furthermore, 9 of the 22 metabolites identified in both plasma and excreta
were products of glucuronidation.
The renal clearance of M15 was 2 ml/min/kg. Although the plasma
protein binding of M15 has not been determined, given the greater
polarity of M15 compared with lersivirine and that the plasma protein
binding of lersivirine is itself low to moderate (fraction unbound ⫽
0.424), M15 would be expected to have minimal plasma protein
binding. In this case, the unbound renal clearance of M15 would be
essentially equivalent to glomerular filtration rate.

799

EXCRETION AND METABOLISM OF LERSIVIRINE
A

1600
1400
1200
1000

Rate
(pmol/min/mg protein)

Lersivirine glucuronide formation
(pmol/min/mg protein)

1800

800
600
400

800
600
400
200
0

200

0

2

4
v/s

6

8

0
0

200

400

600

800

1000 1200 1400 1600

Lersivirine concentration (uM)
B

600

Rate
(pmol/min/mg protein)

Lersivirine glucuronide formation
(pmol/min/mg protein)

800

400

200

800
600
400
200
0
0

2

4
v/s

6

0
0

200

400

600

800

1000 1200 1400 1600

Lersivirine concentration (uM)

FIG. 11. Kinetics of in vitro glucuronidation of lersivirine in human liver microsomes (A) and rUGT2B7 (B) with Eadie-Hofstee plots as inset enzymes. Data points
represent the mean of three determinations.

Although M15 is the major circulating metabolite in humans, it
does not circulate in the plasma of rat, mouse, or dog, but it is formed
in the rat and excreted into bile. This glucuronide is significantly less
potent than the parent compound (see later in Discussion) and does
not contribute to the activity of lersivirine in vivo. As an unreactive,
inactive ether glucuronide M15 does not raise any particular safety
concerns, and in fact this type of glucuronide metabolite is specifically
exempted in the Food and Drug Administration Human Metabolites in
Safety Testing guidance (http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm079266.pdf).
The formation of a pyrazole N-glucuronide (M9) has previously
been identified for the investigative anticancer agent JNJ-10198409 in
human, rat, and monkey liver microsomes (Yan et al., 2006). M9 was
resistant to hydrolysis with ␤-glucuronidase, as was observed for the
major pyrazole N-glucuronide of JNJ-10198409 (Yan et al., 2006).
A number of oxidative metabolites of lersivirine were identified, of
which metabolites with oxidation of the alcohol to a carboxylic acid
group (M19) and a hydroxylated metabolite (M17) were the most
abundant. In vitro, CYP3A4 was found to be the predominant P450
isozyme tested that metabolized lersivirine. In addition, CYP3A4 was
shown to be capable of forming the major oxidative metabolites
formed in vivo (M19 and M17). Although these data are consistent
with previous observations that CYP3A4 is the primary contributor to
oxidative metabolism of lersivirine, in a previous study, using slightly
different buffers and reagents, other P450 enzymes were able to
metabolize lersivirine (Allan et al., 2008). This result most likely
reflects the different source of the P450 isozymes used in the two
studies and the inherent metabolic capabilities of the two systems.

CYP3A5*1, a polymorphic-expressed enzyme in which the allelic
frequency of wild-type is only 10 to 15% in the white population
(increasing to approximately 50% in blacks) (Daly, 2006), was also
assessed in this study. Although it is not currently possible to make a
quantitative extrapolation from recombinant CYP3A5 data because of
the lack of an appropriate CYP3A5 probe substrate for the generation
of relative activity factors, the low rate of metabolism of lersivirine by
CYP3A5 compared with CYP3A4 suggests that no dosage adjustment
will be needed in populations in which the expression of this polymorphic enzyme is high.
Compared with the parent compound (IC50, 2 nM), the metabolites
M17 (138 nM), M15 (5 ␮M), and M19 (15.8 ␮M) are significantly
less potent against the laboratory-adapted HIV-1 strain NL 4-3. In
fact, the potency of either glucuronide could be accounted for by 0.1%
parent in the glucuronide standard or by a small amount of degradation of M15 to parent in the culture. When the average for each of the
metabolites is compared with the IC50 of these four components, the
ratio is 325, 3, 0.5, 0.01 for lersivirine, M17, M15, and M19, respectively, suggesting that the antiviral activity is mainly associated with
the lersivirine parent molecule.
In addition to glucuronidation and oxidative metabolism, metabolic
pathways of lersivirine also involve sulfation and nitrile hydrolysis. It
is well recognized that the extent of a drug-drug interaction as a result
of enzyme inhibition will depend on the fraction of the substrate
metabolized by that enzyme (Brown et al., 2005; Ito et al., 2005). The
balanced clearance of lersivirine is expected to reduce the interindividual variability and drug-drug interaction liability of the compound
by reducing the reliance on a single enzyme to clear the compound.
Furthermore, the predominant clearance pathway in humans in vivo is
UGT2B7-mediated glucuronidation. In general, glucuronidation is a
high-capacity low-affinity metabolic pathway, and drug-drug interactions mediated through UGTs are known to be small in magnitude
(Williams et al., 2004; Kiang et al., 2005). Coadministration with
valproic acid, a potent inhibitor of UGT2B7, results in increased
exposure of a number of compounds that undergo glucuronidation via
UGT2B7, including zidovudine and lamotrigine; however, the magnitude of the changes in exposure is low [1.8-fold (Lertora et al.,
1994) and 2.6-fold (Morris et al., 2000), respectively]. It is not
expected that the plasma levels of lersivirine will increase dramatically on coadministration with medications that inhibit UGT metabolism because only a 1.3-fold increase in lersivirine AUC was observed after coadministration with valproic acid (Langdon et al.,
2008).
Conclusion
In conclusion, this open-label Phase I study has shown that lersivirine is almost completely absorbed, extensively metabolized by both
UGT- and P450-dependent pathways, and radiolabeled material is
eliminated entirely by renal and fecal routes. The major human
circulating metabolites examined thus far do not appear to contribute
to the antiviral activity of lersivirine.
Acknowledgments. We thank Drs. Kristin Larsen and Clemence
Hindley of Complete Medical Communications for editorial support
(funded by Pfizer Inc.).
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Address correspondence to: Manoli Vourvahis, Pfizer Global R&D, 50 Pequot
Avenue, New London, CT 06320. E-mail: [email protected]