Altering metabolic profiles of drugs by precision deuteration: Reducing mechanism-based inhibition of CYP2D6 by Paroxetine

Article abstract of DOI:10.1124/jpet.115.223768

Selective deuterium substitution as a means of ameliorating clinically relevant pharmacokinetic drug interactions is demonstrated in this study. Carbon-deuterium bonds are more stable than corresponding carbon-hydrogen bonds. Using a precision deuteration platform, the two hydrogen atoms at the methylenedioxy carbon of paroxetine were substituted with deuterium. The new chemical entity, CTP-347 [(3S,4R)-3-((2,2-dideuterobenzo[d][1,3]dioxol-5-yloxy)me thyl)-4-(4-fluorophenyl)piperidine], demonstrated similar selectivity for the serotonin receptor, as well as similar neurotransmitter uptake inhibition in an in vitro rat synaptosome model, as unmodified paroxetine. However, human liver microsomes cleared CTP-347 faster than paroxetine as a result of decreased inactivation of CYP2D6. In phase 1 studies, CTP-347 was metabolized more rapidly in humans and exhibited a lower pharmacokinetic accumulation index than paroxetine. These alterations in the metabolism profile resulted in significantly reduced drug-drug interactions between CTP-347 and two other CYP2D6-metabolized drugs: tamoxifen (in vitro) and dextromethorphan (in humans). Our results show that precision deuteration can improve the metabolism profiles of existing pharmacotherapies without affecting their intrinsic pharmacologies.

Full text of DOI:10.1124/jpet.115.223768

Supplemental material to this article can be found at:
http://jpet.aspetjournals.org/content/suppl/2015/05/05/jpet.115.223768.DC1.html
1521-0103/354/1/43–54$25.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
http://dx.doi.org/10.1124/jpet.115.223768
J Pharmacol Exp Ther 354:43–54, July 2015
Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics
Altering Metabolic Profiles of Drugs by Precision Deuteration:
Reducing Mechanism-Based Inhibition of CYP2D6
by Paroxetines
Vinita Uttamsingh, Richard Gallegos, Julie F. Liu, Scott L. Harbeson, Gary W. Bridson,
Changfu Cheng, David S. Wells, Philip B. Graham, Robert Zelle, and Roger Tung
Concert Pharmaceuticals, Inc., Lexington, Massachusetts
Received February 24, 2015; accepted April 15, 2015
ABSTRACT
Selective deuterium substitution as a means of ameliorating clinically paroxetine. However, human liver microsomes cleared CTP-347
relevant pharmacokinetic drug interactions is demonstrated in this faster than paroxetine as a result of decreased inactivation of
study. Carbon-deuterium bonds are more stable than corresponding CYP2D6. In phase 1 studies, CTP-347 was metabolized more rapidly
carbon-hydrogen bonds. Using a precision deuteration platform, the in humans and exhibited a lower pharmacokinetic accumulation
two hydrogen atoms at the methylenedioxy carbon of paroxetine index than paroxetine. These alterations in the metabolism profile
were substituted with deuterium. The new chemical entity, CTP-347 resulted in significantly reduced drug-drug interactions between
[(3S,4R)-3-((2,2-dideuterobenzo[d][1,3]dioxol-5-yloxy)methyl)-4-(4- CTP-347 and two other CYP2D6-metabolized drugs: tamoxifen (in
fluorophenyl)piperidine], demonstrated similar selectivity for the vitro) and dextromethorphan (in humans). Our results show that
serotonin receptor, as well as similar neurotransmitter uptake precision deuteration can improve the metabolism profiles of existing
inhibition in an in vitro rat synaptosome model, as unmodified pharmacotherapies without affecting their intrinsic pharmacologies.
bonds. Consequently, as commented on by others (Foster, 1984;
Introduction
Kushner et al., 1999), deuterium modification might be a useful
Compounds labeled with deuterium, a nonradioactive iso-
tool to alter beneficially the metabolism of existing pharmaco-
tope of hydrogen bearing a neutron, have long been used as
therapies without affecting their intrinsic pharmacology.
metabolic probes (Nelson and Trager, 2003), but few studies
The purpose of this study was to examine whether the me-
have assessed the potential of deuterated agents as pharma-
tabolism of a widely used drug, paroxetine, could in fact be
cotherapies (Harbeson and Tung, 2011; Braman et al., 2013;
changed without altering its principal pharmacologic activities
Gant, 2014). The molecular structures and pharmacologic ac-
via deuterium substitution. It had the additional goal of directly
tivities of deuterated compounds (Harbeson and Tung, 2011),
correlating the effects of deuteration at the molecular level with
and even the structure and function of fully substituted enzymes
clinically meaningful changes in human pharmacokinetics.
(Di Costanzo et al., 2007), are extremely similar to their all-
Paroxetine (Paxil; GlaxoSmithKline, Research Triangle Park,
hydrogen analogs. Nonetheless, the metabolism of deuterated
NC) is a selective serotonin reuptake inhibitor currently in-
agents can differ substantially from nondeuterated compounds
dicated for major depressive, obsessive compulsive, panic, social
as a result of a phenomenon known as the deuterium isotope
anxiety, general anxiety, and posttraumatic stress disorders
effect (Wiberg, 1955; Shao and Hewitt, 2010), which occurs as
(Paxil, 2012). Paroxetine and other serotonin reuptake inhibitors
a result of the greater atomic mass of deuterium relative to
also have proven effective in the treatment of vasomotor symp-
hydrogen, causing deuterium-containing bonds with carbon,
toms (e.g., hot flashes, night sweats) in women undergoing
oxygen, and nitrogen to have lower vibrational frequencies than
menopausal transition (Stearns et al., 2005; Deecher and
corresponding hydrogen-containing bonds. As a result of their
Dorries, 2007) and in patients receiving antiestrogenic cancer
lower ground-state energies, deuterium-containing bonds re-
therapy (Fisher et al., 1998; Bordeleau et al., 2007). Paroxetine
quire higher activation energy to reach the transition state for
undergoes significant first-pass metabolism (Kaye et al., 1989),
scission and, thus, are more stable than hydrogen-containing
catalyzed largely but not exclusively by CYP2D6 (Bloomer et al.,
1992; Brosen et al., 1993). Repeat administrations of paroxetine,
however, have been shown to inactivate CYP2D6, resulting in
nonlinear pharmacokinetics and increased exposure over time
C.C., D.S.W., and R.Z. are former employees of Concert Pharmaceuticals,
Inc.
dx.doi.org/10.1124/jpet.115.223768.
s
This article has supplemental material available at jpet.aspetjournals.org. (Bourin et al., 2001). The inactivation of CYP2D6 occurs by
ABBREVIATIONS: AUC, area under the concentration-time curve; CTP-347, (3S,4R)-3-((2,2-dideuterobenzo[d][1,3]dioxol-5-yloxy)methyl)-4-(4-
fluorophenyl)piperidine; DM, dextromethorphan; DX, dextrorphan; EM, extensive metabolizer; LC, liquid chromatography; LC-MS/MS, liquid
chromatography–tandem mass spectrometry; MBI, mechanism-based inhibition; MIC, metabolic-intermediate complex; MRM, multiple reaction
monitoring; MS, mass spectrometry; PM, poor metabolizer.
43

44
Uttamsingh et al.
(3S,4R)-3-((Benzo[d][1,3]Dioxol-(2,2-d2)-5-Yloxy)Methyl)-4-(4-
Fluorophenyl)-1-Methylpiperidine (IV). To flask containing
metabolism or mechanism-based inhibition (MBI) whereby an
intermediate metabolite of paroxetine forms a covalent complex
known as a metabolic-intermediate complex (MIC) with the
cytochrome P450-Fe(II) form of the enzyme and inhibits its
activity in a quasi-irreversible fashion (Murray, 2000; Bertelsen
et al., 2003; Orr et al., 2012). Since the antidepressant effect of
paroxetine exhibits a relatively flat dose-response curve (Bourin
et al., 2001), the observed MBI appears to have limited effect on
the agent’s overall efficacy, but drug-drug interactions of sig-
nificant clinical relevance have been observed between parox-
etine and other coadministered drugs that are also metabolized
by CYP2D6 (Paxil, 2012).
CTP-347 [(3S,4R)-3-((2,2-dideuterobenzo[d][1,3]dioxol-5-
yloxy)methyl)-4-(4-fluorophenyl)piperidine] is a new chemical
entity that is structurally identical to paroxetine except for
two deuterium atoms, rather than hydrogen atoms, at the
methylenedioxy carbon (Fig. 1). Here we demonstrate using in
vitro model systems that CTP-347 abrogated the MBI observed
with paroxetine and that this effect reduced drug-drug in-
teractions with other CYP2D6-metabolized drugs. In addition,
in phase 1 studies, we showed that CTP-347 was metabolized
more rapidly than paroxetine, most likely as a result of the
substantial decrease in the inactivation of CYP2D6. These
results validate deuterium substitution as a potentially impor-
tant approach to creating improved therapeutic agents.
a
crude II (approximately 8.96 mmol) were added toluene (45 ml, benzo
[d][1,3]dioxol-(2,2-d2)-5-ol [III], 99.7% isotopic purity, 1.26 g, 8.96 mmol),
tetra-n-octylammonium bromide (245 mg, 0.448 mmol), and 3M aqueous
NaOH (22.4 ml, 67.2 mmol) with stirring. The resulting pale yellow
turbid bilayer was stirred and heated in a 90°C oil bath under a vented
air condenser for 5 hours. The reaction mixture was cooled to room
temperature and diluted with water (100 ml) and toluene (50 ml). The
mixture was poured into a separatory funnel and shaken, and the layers
were separated. The organic layer was washed with saturated aqueous
NaHCO3 and with brine, then dried over magnesium sulfate, filtered,
and concentrated on a rotary evaporator to afford approximately 4 g of
IV, which contained some residual toluene. This material was suitable
for use in crude form. MS m/z: 346.3 (M 1 H).
(3S,4R)-4-Nitrophenyl 3-((Benzo[d][1,3]Dioxol-(2,2-d2)-5-Yloxy)
Methyl)-4-(4-Fluorophenyl)Piperidine-1-Carboxylate (V). To
a flask containing crude IV (approximately 8.96 mmol) were added
toluene (60 ml), diisopropylethylamine (0.312 ml, 1.79 mmol), and
4-nitrophenylchloroformate (1.81 g, 8.96 mmol). The mixture was
stirred and heated in an 80°C oil bath under a vented air condenser for
5 hours. The reaction mixture was cooled to room temperature and
diluted with toluene (50 ml). The mixture was poured into a separatory
funnel, and the flask was rinsed with an additional 50 ml of toluene.
A 100-ml portion of water was added to the separatory funnel, and the
layers were shaken and separated. The aqueous layer was extracted
with an additional 25 ml of toluene. The combined organic layers were
washed with brine, dried over magnesium sulfate, filtered, and con-
centrated on a rotary evaporator to afford an amber oil. The material
was purified via column chromatography (5% –30% EtOAc/hexanes) to
provide 2.16 g of V. MS m/z: compound does not ionize well.
Materials and Methods
CTP-347 (hydrochloride salt), paroxetine, N-desmethyl tamoxifen,
and endoxifen were provided by Concert Pharmaceuticals, Inc.
(Lexington, MA). Human liver microsomes were from Xenotech LLC
(Lenexa, KS). Human cDNA-expressed CYPD6 supersomes were
obtained from Corning Life Sciences (Tewksbury, MA). Dextromethor-
phan (DM) and NADPH were from Sigma-Aldrich (St. Louis, MO).
(3S,4R)-3-((Benzo[d][1,3]Dioxol-(2,2-d2)-5-Yloxy)Methyl)-4-(4-
Fluorophenyl)Piperidine, HCl Salt (VI). To a solution of V (2.16 g,
4.35 mmol) in dioxane (29 ml) was added 2M aqueous NaOH (43.5 ml,
87.0 mmol), and the mixture was stirred and heated in a 70°C oil bath
under a vented air condenser for 3 hours. The reaction mixture was
cooled to room temperature and concentrated on a rotary evaporator to
remove most of the dioxane. The aqueous residue was poured into
a separatory funnel and extracted three times with dimethyl ether
(Et2O). The combined organic layers were washed with 1 N aqueous
NaOH, then dried over magnesium sulfate, filtered, and concentrated
on a rotary evaporator to afford the free base of VI as a pale yellow oil
(1.2 g). This material was purified via preparative high-performance
LC-MS to provide 710 mg of the free base of VI, which was then taken
up in a minimal volume of acetone and added slowly to a stirred
solution of 1 M HCl/Et2O (5 ml), Et2O (15 ml), and hexanes (60 ml). The
resulting cloudy white mixture was held at 0°C for 1 hour and then
concentrated to a reduced volume on a rotary evaporator. The resulting
white solids were filtered, washed with hexanes/Et2O, and dried in
a vacuum oven at 35–40°C overnight to provide 651 mg of the HCl salt
of VI: MS m/z: 332.0 (M 1 H), NMR (300 MHz, dimethylsulfoxide-d6):
d 9.04 (br s, 2H), 7.25–7.14 (m, 4H), 6.74 (d, 1H, J 5 8.3), 6.48 (d, 1H,
J 5 2.9), 6.18 (dd, 1H, J 5 2.4, 8.3), 3.58 (dd, 1H, J 5 3.4, 10.2), 3.52–3.47
(m, 2H), 3.39–3.35 (m, 1H), 3.01–2.91 (m, 2H), 2.86 (dt, 1H, J 5 3.4,
12.2), 2.47–2.39 (m, 1H), 2.05–1.94 (m, 1H), and 1.88–1.85 (m, 1H).
Dideuterocatechol (VIII). 3,4-Dihydroxybenzaldehyde VII (40 g)
was dissolved in tetrahydrofuran (160 ml), and then D2O (160 ml)
was added and the resulting mixture stirred overnight at room
temperature under nitrogen. The solvent was removed in vacuo and,
the residue was obtained dried in vacuo at 40°C overnight to provide
the exchanged aldehyde VIII as a solid (40 g). Analysis by 1H NMR
(dimethylsulfoxide-d6) showed an H/D exchange level of about 85%.
d2-Piperonal (IX). K2CO3 (29.6 g, 0.22 mol) was suspended in
N-methylpyrollidinone (270 ml) and heated to 110°C under N2. A
solution of 3,4-dideuteroxybenzaldehyde VIII (15 g, 0.11 mol) and
CD2Cl2 (68 ml, 1.1 mol) in N-methylpyrollidinone (30 ml) was added
dropwise over 45 minutes. The reaction was stirred at 110°C for an
additional 90 minutes, after which time analysis (TLC, LC) indicated
Synthetic Procedures for the Synthesis of CTP-347
The synthetic procedures for the synthesis of CTP-347 are shown in
Fig. 2 and described in the following sections. The liquid chromatog-
raphy (LC) purity assessment (Supplemental Fig. 1), mass spectrom-
etry (MS) (Supplemental Fig. 2), and 1H NMR spectra (Supplemental
Fig. 3) are shown in the Supplemental Material.
((3S,4R)-4-(4-Fluorophenyl)-1-Methylpiperidin-3-yl)Methyl
Methanesulfonate, HCl Salt (II). ((3S,4R)-4-(4-Fluorophenyl)-1-
methylpiperidin-3-yl)methanol I (2.00 g, 8.96 mmol) was dissolved in
dichloroethane (20 ml), and methanesulfonyl chloride (0.73 ml) was
added. The reaction was stirred for 6 hours at room temperature. The
reaction mixture was concentrated on a rotary evaporator to afford II
as a white solid residue, which was suitable for use in crude form. MS
m/z: 302.1 (M 1 H).
Fig. 1. Structure of CTP-347. D, deuterium atoms.

Altering Metabolic Profiles of Drugs with Precision Deuteration
45
Fig. 2. Synthesis of CTP-347. See Materials and
Methods for details.
complete reaction. The mixture was allowed to cool, filtered, the
extracted with CH2Cl2 (2 liters, then 2 Â 1.5 liters). The combined
filtrate poured into water (900 ml) and extracted with EtOAc (3 Â organics were dried (magnesium sulfate), filtered, and concentrated in
600 ml). The combined organics were washed with water (2 Â 600 ml), vacuo; the residue was combined with material from another 165 g batch
dried (magnesium sulfate), filtered, and concentrated in vacuo. The and purified by column chromatography (hexane/EtOAc 4/1) to give III
residue was purified by silica-gel chromatography (4/1 hexane/EtOAc)
to give IX (14.2 g, 87%) of a pale brown oil that solidified on standing.
d2-Sesamol (III). To a stirred solution of d2-piperonal IX (165 g,
1.08 mol, 1.0 eq.) in CH2Cl2 (5.5 liters) were added 30% hydrogen
peroxide (343 ml, 3.01 mol, 2.75 eq.) and 96% formic acid (182 ml, 4.63 mol,
4.3 eq.), and the resulting mixture was stirred overnight at reflux.
Analysis (i.e., by LC) indicated the presence of residual starting material
(13%). The reaction mixture was cooled to 0–5°C, 1.5 M NaOH (5.9 liters,
(185 g, 61% combined yield) as a white solid with a purity of .95% by 1H
NMR and 99% by LC.
Bioanalytical Methods
The samples were analyzed by LC–tandem mass spectrometry (LC-
MS/MS) using an API 4000 mass spectrometer (Applied Biosystems,
Carlsbad, CA) equipped with TurboIonSpray source, Agilent 1200 RR
8.85 mol, 8.2 eq.) was added portionwise over 30 minutes (exotherm to pumps (Agilent Technologies, Santa Clara, CA), and a Leap auto-
25–30°C), and the mixture stirred for 30 minutes. The layers were sampler (Leap Technologies, Carrboro, NC). Analyst Software was
separated, the organics were concentrated in vacuo, and the residue was used to acquire the data.
obtained dissolved in methanol (3.6 liters), added to the aqueous, and the
mixture stirred at room temperature for 30 minutes. The methanol was
CTP-347. Aliquots (10–20 ml) of the in vitro or human plasma
samples were injected onto a C8-reverse phase column (SB-C8 Zorbax,
removed in vacuo, and the aqueous was washed with CH2Cl2 (2 liters 3.5 mm, 2.1 Â 30 mm) equilibrated with 95% solvent A (0.1% formic acid
and then 1.5 liters), acidified to pH 3 with concentrated aq. HCl, and in water) and 5% solvent B (0.1% formic acid in acetonitrile), followed

46
Uttamsingh et al.
by a linear gradient to 50% solvent B over 5.0 minute and then
preincubation, aliquots of the reaction mixtures were diluted 1:10 in
returned to the original conditions in 1.0 minute. The flow rate was buffer (0.1 M potassium phosphate, pH 7.4 with MgCl2 and NADPH).
0.5 ml/min. The multiple reaction monitoring (MRM) transitions for DM (25 mM) was added to the diluted preincubations, and these
CTP-347 were m/z 332.2 to 192.2 and m/z 377.0 to m/z 293.0 for the mixtures were incubated for another 5 minutes, after which the samples
internal standard, indiplon. The internal standard for the human were analyzed by LC-MS/MS. The formation of DX, a CYP2D6-specific
plasma bioanalysis of CTP-347 was paroxetine-d6, which had MRM metabolite of DM, was monitored as a measure of CYP2D6 activity.
transitions of m/z 336.3 to m/z 198.2.
Similar reactions were performed with paroxetine in parallel. The
DM and Dextrorphan. Aliquots (10–20 ml) of the in vitro or experiment was repeated twice, and the results of a representative
human urine samples were injected onto a C18-reverse phase column experiment are shown and discussed.
(Xbridge phenyl, 3.5 mm, 2.1 Â 100 mm) equilibrated with 100%
solvent A (0.1% formic acid in water), followed by a linear gradient to
Reversible Inhibition of CYP2D6
100% solvent B (0.1% formic acid in acetonitrile) over 10 minutes and
Human liver microsomes were incubated with the CYP2D6-selective
then returned to the original conditions in 1.0 minute. The flow rate
marker substrate DM, and 1:3 serial dilutions of CTP-347 (highest
was 0.45 ml/min. The MRM transitions for DM were m/z 272 to m/z
concentration used was 10 mM) in 0.1 M potassium phosphate buffer,
215.3 and for dextrorphan (DX) were m/z 261.2 to m/z 157.1.
pH 7.4 with 3 mM MgCl2. The reactions were initiated by the addition
Indiplon was used as the internal standard.
of NADPH and were incubated for 15 minutes at 37°C in triplicate
N-Desmethyl Tamoxifen and Endoxifen. Aliquots (10–20 ml)
wells of a 96-well plate and then analyzed by LC-MS/MS for the
of the in vitro samples were injected onto a C18-reverse phase column
amounts of DX formed. Similar incubations were performed with
(Xbridge phenyl, 3.5 mm, 2.1 Â 100 mm) equilibrated with 90% solvent
paroxetine and quinidine (positive control inhibitor) in parallel. The
A (0.1% formic acid in water), followed by a linear gradient to 90%
percent inhibition relative to blank (no CTP-347 or paroxetine) samples
solvent B (0.1% formic acid in acetonitrile) over 2.0 minutes and then
was calculated, and the IC50 value was determined by nonlinear
returned to the original conditions in 1.0 minute. The flow rate was
regression analysis using the log (inhibitor) versus response sigmoidal
0. 5 ml/min. The multiple MRM transitions for N-desmethyl tamoxifen
fit using GraphPad Prism. The experiment was repeated, and the
were m/z 358 to m/z 58 and for endoxifen were m/z 374 to m/z 152.
reported IC50 is the mean of two experiments.
Indiplon was used as the internal standard.
Spectral Analysis of Metabolite Intermediate Complex
Formation
In Vitro Pharmacology Assessments
The inhibition of the uptake of serotonin, norepinephrine, and
The formation of a metabolite intermediate complex after preincu-
dopamine by CTP-347 and paroxetine was tested in rat brain,
bation of CYP2D6 supersomes (0.5 nmol/ml) with 10 mM CTP-347 or
hypothalamus, and striatum synaptosomes, respectively. Serial dilu-
paroxetine and NADPH (2 mM) was monitored as described by
tions of CTP-347 and paroxetine were incubated with 3H-labeled
Bertelsen et al. (2003). Difference spectra were obtained by scanning
serotonin, norepinephrine, or dopamine for 15–20 minutes at 37°C. The
between 500 and 400 nm every 2 minutes for 26 (paroxetine) or 36
concentration range of CTP-347 and paroxetine tested were 0.1–40,
(CTP-347) minutes using an Olis DW-2000 spectrophotometer (Olis,
1–400, and 10–4000 nM for inhibition of serotonin, norepinephrine,
Inc., Bogart, GA). The increase in absorption (Abs) at 456 nm over
and dopamine uptake, respectively. The percent of specific activity in
time was observed to determine intermediate complex formation with
the presence of CTP-347 or paroxetine relative to control specific
CTP-347 and paroxetine.
activity was determined. The IC50 values for inhibition of the incor-
poration of 3H-labeled neurotransmitters were determined by non-
linear regression analysis of the percent of control specific activity
Determination of Binding Affinity Ks for CYP2D6
versus log (concentration) curves using Hill equation curve fitting.
An additional battery of tests against 166 molecular targets was
also conducted to characterize other pharmacologic effects of CTP-
347. Radioligand binding or enzymatic assays were used, and CTP-
347 was tested at concentrations of 10 and 1 mM and compared with
that of paroxetine at a concentration of 1 mM.
Type 1 binding difference spectra were determined by spectral
scanning (350–500 nM) of a mixture of CYP2D6 supersomes
(250 pmol/ml) and several concentrations (0 to 145 mM) of CTP-347 and
paroxetine using an Olis DW-2000 spectrophotometer. The difference in
absorption (DAbs) at 390 nm and 424 nm was calculated, and Ks values
were calculated by nonlinear regression analysis.
Assessment of the Drug-Drug Interaction Potential with
Tamoxifen
Metabolic Stability in Human Liver Microsomes
The metabolic stability of CTP-347 in human liver microsomes was
evaluated by the in vitro half-life (t1/2) method described by Obach
et al. (1997). The metabolic stability of paroxetine was also determined
similarly in parallel. CTP-347 or paroxetine was incubated with human
liver microsomes for 30 minutes at 37°C in triplicate wells of a 96-well
plate. Aliquots of the reaction mixtures were removed at 0, 3, 7, 12, 20,
and 30 minutes and analyzed for amounts of parent remaining by LC-
MS/MS. The experiment was repeated on 4 separate days, and mean
values at each time point were reported. The in vitro t1/2 values for
CTP-347 and paroxetine were calculated from the slopes of the linear
regression of % parent remaining (LN) versus the incubation time
relationship.
CTP-347 was preincubated in triplicate wells of a 96-well plate with
human CYP2D6 supersomes (500 pmol/ml) at concentrations of 0, 0.5,
0.66, 1, 2, 5, 10, and 25 mM for 20 minutes in the presence of NADPH,
after which aliquots of the reaction mixture were diluted 1:10 in buffer
containing NADPH. N-Desmethyl tamoxifen (50 mM) was then added
to the diluted preincubation mixtures and further incubated, following
which samples were analyzed by LC-MS/MS for amounts of endoxifen
formed. Similar incubations with paroxetine were performed in
parallel. The percent activity remaining after a 20-minute preincuba-
tion with CTP-347 or paroxetine, measured as percent endoxifen
formed, was calculated.
Evaluation of Effect of CTP-347 on CYP2D6 Activity in
Healthy Subjects
Time-Dependent Inactivation of CYP2D6
This study was conducted in accordance with the procedure es-
tablished by Bertelsen et al. (2003) for paroxetine. Several concen-
A randomized, double-blind, placebo-controlled, single-center study
trations of CTP-347 (0, 0.5, 0.66, 1, 2, 5, 10, 25 mM) were preincubated was conducted in healthy female subjects. The study protocol was
with human liver microsomes (5 mg/ml) for 0, 10, 25, and 30 minutes in approved by the PRACS Institute (Fargo, ND) institutional review
the presence of NADPH in triplicate wells of a 96-well plate. After the board. The study was conducted in accordance with the guidelines set

Altering Metabolic Profiles of Drugs with Precision Deuteration
47
forth by the International Conference on Harmonization (ICH)
Guidelines for Good Clinical Practice (ICH Guideline E6), the Code
of Federal Regulations for Good Clinical Practice (21 CFR Parts 50
and 56), and the Declaration of Helsinki regarding the treatment of
human subjects in a study. Written informed consent was obtained
from all subjects at the screening visit.
other molecular targets, including the human serotonin, norepi-
nephrine, and dopamine transporters; human muscarinic recep-
tors; and rat L-type calcium channels (Table 1). These results are
consistent with previous studies indicating that deuterium
substitution had little impact on pharmacologic activity and
molecular structure relative to their all-hydrogen analogs (Di
Costanzo et al., 2007; Harbeson and Tung, 2011).
CTP-347 Exhibits Little or No Mechanism-Based
Inhibition of CYP2D6 In Vitro. Metabolic stability of the
two drugs was determined by incubating each in the presence
of human liver microsomes for 30 minutes. The in vitro t1/2
values for CTP-347 and paroxetine were 21 6 2 and 49 6
8 minutes (n 5 4 for both compounds), respectively; that is, the
t1/2 value for CTP-347 was 57% shorter than that for paroxetine
(Fig. 3B). Thus, CTP-347 was cleared faster than paroxetine by
human liver microsomal preparations. As measured by type 1
difference spectra (Bertelsen et al., 2003), however, the binding
affinity, Ks, of CTP-347 (11.2 mM) was similar to that of
paroxetine (8.5 mM) (Fig. 3C), indicating that the higher rate of
CTP-347 metabolism was unlikely attributable to differences
in binding of the drug to CYP2D6.
A plausible explanation for the rapid metabolism of CTP-
347 posits that substitution of deuterium atoms at the
methylenedioxy carbon might decrease MIC formation with
CYP2D6, thereby allowing for more rapid turnover. To examine
this possibility, we analyzed CTP-347 metabolism using a
method that was previously applied to paroxetine and takes
into account the effects of MBI on reaction kinetics (Kitz and
Wilson, 1962; Silverman, 1995; Bertelsen et al., 2003). This
method assumes that a dual reactant-inactivator like parox-
etine can have three separate fates on binding to CYP2D6:
The primary objectives of the first in-human study with CTP-347
were to investigate the safety, tolerability, and pharmacokinetics of
CTP-347 after single and multiple doses. The inactivation of CYP2D6
by CTP-347 after once-daily oral dosing for 14 days to healthy subjects
was investigated as a secondary objective, and only the methods and
results of this objective are discussed here. CTP-347 5-mg immediate-
release tablets were administered to healthy women age 25–65 years.
Three cohorts of nine subjects received once-daily doses of 10, 20, or
40 mg, and a fourth cohort of seven subjects received twice-daily doses
of 10 mg (20 mg/day) for 14 days. In addition, one cohort of 10 subjects
received once-daily paroxetine at a dose of 10 mg for the same time.
On the evening before the first CTP-347 dose, 30 mg of DM solution
was administered orally to each subject. Urine was collected for
8 hours, and the levels of DM and DX were determined by LC-MS/MS.
The urinary DM/DX ratio was used to determine the baseline
CYP2D6 phenotype. This procedure was repeated on day 14 of CTP-
347 administration to determine the extent of inhibition of CYP2D6
activity. CYPD26 genotyping was performed on blood samples by
a contract research organization according to standard validated PCR
methods. CTP-347 plasma levels at several time points postdose on
day 1 and day 14 were also measured by LC-MS/MS. Each subject was
monitored for safety throughout the study including vital signs,
physical examinations, clinical laboratory tests, 12-lead electrocardio-
gram, and cardiac telemetry.
Results
CTP-347 Is a Selective Serotonin Uptake Inhibitor In
Vitro. Neurotransmitter uptake inhibition was assessed release without further chemical modification (reversible
using an in vitro rat synaptosome model that measured uptake binding), conversion to product (productive catalytic cycle), or
3
formation of an MIC (Fig. 3A). Solution of the mathematical
of H-labeled neurotransmitter in the presence of CTP-347 or
paroxetine (see Materials and Methods). In this system, IC50 relationships describing these outcomes results in two key
levels for serotonin uptake were similar with CTP-347 (0.89 kinetic parameters of interest, KI and kinact. Making simplifying
assumptions, KI represents the equilibrium disassociation
nM) and paroxetine (0.72 nM) (Table 1). Furthermore, CTP-
347 was highly selective for the serotonin transporter, as the constant for paroxetine (reversible binding), whereas kinact
IC50 for the deuterated agent was 35-fold (31 nM) and 300-fold represents the rate constant to form inactive MIC from the
(270 nM) greater for norepinephrine and dopamine uptake, enzyme-drug complex (Mayhew et al., 2000).
KI and kinact for MBI are typically determined in a two-step
respectively, again similar to paroxetine. At a concentration of
1 mM, the deuterated and all-hydrogen molecules were reaction assay (Kitz and Wilson, 1962; Silverman, 1995). In
approximately equipotent in their inhibitory interactions with the first step, enzyme is incubated for varying times with
TABLE 1
CTP-347 Pharmacologic effects in vitro
Species
CTP-347
Paroxetine
Reuptake inhibition, IC50 (nM)
Serotonin
Rata
Ratb
Ratc
0.89
31
270
0.72
43
230
Norepinephrine
Dopamine
Percent inhibition at 1 mM concentration, %
Serotonin transporter
Norepinephrine transporter
Dopamine transporter
Muscarinic M1
Human
Human
Human
Human
Human
Human
Rat
88
87
48
50
56
51
55
19
90
89
67
57
63
50
45
47
Muscarinic M3
Muscariic M5
Calcium channel L-type, benzothiazepine
Calcium channel L-type, dihydropyridine
Rat
aTested in rat brain synaptosomes.
bTested in rat hypothalamus synaptosomes.
cTested in rat striatum synaptosomes.

48
Uttamsingh et al.
Fig. 3. Metabolism and binding of CTP-347. (A) Early steps of paroxetine metabolism. Paroxetine is metabolized by CYP2D6 to a hydroxylated
methylene intermediate that has two potential fates: 1) opening to the formate ester and conversion to the catechol (productive catalysis); or 2)
dehydration to form a carbene that can bind to the heme iron of CYP2D6 and form an MIC (enzyme inactivation). (B) Metabolism of CTP-347 and
paroxetine by human liver microsomes. (C) Binding affinities of paroxetine and CTP-347 as assessed by type 1 binding spectra (only spectra for
paroxetine binding are shown, left). Bmax, maximum specific binding; [L], ligand concentration; Ks, equilibrium binding constant.
different concentrations of test inactivator, in this case, either Limited inhibition of CYP2D6 was observed with CTP-347 at
CTP-347 or paroxetine, to allow for variable enzymatic in- higher concentration (25 mM), although it remains unclear
activation. This step is followed by a second incubation with whether this reflected MBI, reversible inhibition (IC50 values
a probe substrate, in this case DM, to measure the remaining were approximately 3 and 1 mM for CTP-347 and paroxetine,
activity. Analysis of the data on a Kitz-Wilson plot determines respectively, in the assay), or a combination of both (Fig. 4A).
the key kinetic parameters (Kitz and Wilson, 1962; Silverman,
Decreased MIC formation with CTP-347 was confirmed by
1995). Using this approach in human liver microsomes (Fig. spectral difference scanning. Previous studies showed that the
4A, right), we found that KI and kinact for paroxetine were 1.96 MIC formed between the methylenedioxyphenyl substituent of
mM and 0.08 minute–1, respectively, similar to previously paroxetine and CYP2D6 was characterized by peaks of
reported values (KI 5 4.85 mM and kinact 5 0.17 minute–1
)
absorbance at 430 and 456 nm, referred to as the type 3 binding
(Bertelsen et al., 2003). By comparison, little if any time- spectrum (Bertelsen et al., 2003). Consistent with earlier
dependent inactivation was observed after preincubation with studies, we observed that incubating CYP2D6 with paroxetine
CTP-347 in the 0.125- to 10-mM concentration range (Fig. 4A, over approximately 30 minutes resulted in increasing peaks of
left). Because of the absence of time-dependent inhibition, KI absorbance at 455 nm, characteristic of MIC formation (Fig. 4B,
and kinact could not be calculated for CTP-347, suggesting that right). By contrast, similar absorbance changes were absent
the bulk of the in vitro reaction comprised productive catalysis. when CYP2D6 was incubated with CTP-347 (Fig. 4B, left). In

Altering Metabolic Profiles of Drugs with Precision Deuteration
49
Fig. 4. Reduced mechanism-based inhibition with CTP-347. (A) Metabolism of DM by human liver microsomes after preincubation with paroxetine (left)
or CTP-347 (right). (B) Spectral difference scanning of CYP2D6 after incubation with paroxetine (left) or CTP-347 (right). Abs, absorbance.
summary, the previous kinetic and spectral analyses indicated (25 mM), 8-fold more endoxifen was produced in the presence
that substituting deuterium for hydrogen at the methylenedioxy of CTP-347 than in the presence of paroxetine, demonstrat-
carbon significantly decreased the rate of MIC formation and ing a significantly lower level of drug-drug interaction
stimulated productive catalysis, although it should be noted between CTP-347 and tamoxifen compared with paroxetine
that the time frames of these experiments were shorter than and tamoxifen.
what might occur during a typical clinical exposure to the drug
(see the following section).
CTP-347 Is Metabolized More Efficiently Than Parox-
etine in Humans. The reduced MBI and drug-drug interac-
CTP-347 Reduces Drug-Drug Interactions with Tamox- tion of CTP-347 relative to paroxetine might be beneficial in
ifen. Next, we assessed whether the apparent in vitro metabolic a variety of clinical settings; however, a range of complicating
effects of CTP-347 extended beyond the probe substrate DM to factors in whole organisms may mask or even reverse the
encompass a more frequently used drug, tamoxifen (Hertz et al., theoretical consequences of drug deuteration (Harbeson and
2012). Previous studies have shown that tamoxifen is metabo- Tung, 2011), and it was therefore of interest to determine
lized to N-desmethyl tamoxifen, which is further metabolized by whether any effects observed in vitro with the deuterated agent
CYP2D6 to the primary active metabolite endoxifen (4-OH-N- translated into the clinical setting.
desmethyl-tamoxifen) (Stearns et al., 2003). When N-desmethyl
To examine this issue, we first characterized the multidose
tamoxifen was incubated with human liver microsomes in the pharmacokinetics of paroxetine in humans to provide com-
presence of increasing concentrations of paroxetine, the metab- parative values for subsequent CTP-347 studies. For these
olism of N-desmethyl tamoxifen decreased substantially with initial analyses, we chose paroxetine 10 mg daily as our test dose.
higher paroxetine concentrations, presumably because of MIC Thus, 10 healthy adult women (Table 2) received paroxetine
formation with CYP2D6 (Fig. 5). Conversely, little or no change 10 mg daily for 14 days, blood samples were collected over
in endoxifen formation was observed over the range of tested a 24-hour period on days 1 and 14, and pharmacokinetic para-
CTP-347 concentrations (0–25 mM). At the highest concentration meters were determined (Table 3). The resulting concentration-

50
Uttamsingh et al.
TABLE 2
Subject demographics
CTP-347
Paroxetine
N
34
10
NA
Median age, yr (range)
Median weight, kg (range)
Median body mass index, kg/m2 (range)
Female, n (%)
Race, n (%)
48 (22–64)
68 (51–87)
26 (19–30)
34 (100)
NA
NA
10 (100)
African American
Asian
1 (3)
2 (6)
30 (88)
1 (3)
—
—
—
8 (80)
1 (10)
1 (10)
White
Native Indian
Unknown
CYP2D6 status, n (%)
EM phenotype
PM phenotype
Genotype
33 (97)
1 (3)
10 (100)
—
*1*1
18 (53)
13 (38)
1 (3)
1 (3)
1 (3)
NA
NA
NA
NA
NA
Fig. 5. Reduced drug-drug interaction with CTP-347. Metabolism of N-
desmethyl tamoxifen to endoxifen by human liver microsomes in the
presence of CTP-347 or paroxetine.
*1*4
*1*10
*10*10
*1*7(2XN)
—, none; NA, not available.
time curve on day 14 was significantly elevated compared with
the curve on day 1 (Fig. 6A), consistent with inactivation of
CYP2D6-mediated metabolism over the 2-week study period. collected for 8 hours, and the levels of unmetabolized DM versus
The overall exposure to paroxetine on day 14, as assessed by the DX metabolite were determined by LC-MS/MS. Consistent
median area under the plasma concentration-time curve during with the presence of some residual CYPD6 inactivation, urinary
the dosing interval at steady state (AUCss), was 248.1 hour*ng/ml, DM/DX ratios rose in all patients after 14 days of CTP-347
whereas the median area under the plasma concentration- exposure (i.e., more unmetabolized DM was present in urine on
time curve during the first 24 hours of exposure on day day 14 (Fig. 7; Table 4). Nonetheless, 25 of 32 (78%) subjects who
1 (AUC0–last) was 17.8 hour*ng/ml (Table 3). In other words, were extensive metabolizers (EMs) at baseline, defined as
the subjects were exposed to 13.9-fold more paroxetine on day having DM/DX ratios ,0.3 (Schmid et al., 1985; Alfaro et al.,
14 than day 1, despite the equivalent 10-mg oral doses on the 2000), retained their EM phenotype at day 14, and of those who
2 days.
converted from an EM to a poor metabolizer (PM) phenotype,
We next characterized the multidose pharmacokinetics of four of seven (57%) were in the 40-mg daily group, two of seven
CTP-347 in an escalating-dose study. Thirty-four healthy were in the 20-mg daily group, and one of seven was in the
female subjects received once-daily doses of 10 mg (n 5 9), 10-mg twice daily group. No EM→PM conversion was observed
20 mg (n 5 9), or 40 mg (n 5 9) CTP-347 for 14 days; a fourth in the CTP-347 10-mg daily group. Finally, the DM/DX ratios
cohort received 10-mg doses twice daily (n 5 7) for the same observed in this study were substantially lower than those
amount of time. Blood samples were again collected on day 1 reported in 12 subjects who had received paroxetine 20 mg daily
and day 14, and pharmacokinetic parameters were deter- for 8 days (the mean DM/DX ratios for paroxetine in this study
mined (Table 3). Subjects receiving CTP-347 10 mg daily had went from 0.017 6 0.018 to 0.601 6 0.417; 11 of 12 subjects
mean concentration-time curves that were very similar on day converted from EM→PM) (Fig. 4B) (Alfaro et al., 2000). The sum
1 and day 14, unlike those in the earlier paroxetine 10-mg of the clinical data is in accord with in vitro results suggesting
daily study (Fig. 6A). The median AUCss in the CTP-347 that deuterium substitution greatly reduced inactivation of
group was 18.0 hour*ng/ml on day 14, whereas the median CYPD6 in vivo.
AUC0–last was 8.3 hour*ng/ml on day 1, for an accumulation
index of 2.9 (Table 3). Thus, not only were the AUC values
lower in the CTP-347 10-mg group compared with the
Discussion
paroxetine 10-mg group, but the accumulation of drug over
The current study assessed the potential of precision
time was significantly reduced. Overall exposure levels to deuteration of paroxetine as an approach to positively impact
CTP-347 at the 10 mg twice daily and 20 mg daily dosages the metabolism profiles of drugs. The substitution of two
were also lower than for paroxetine 10 mg daily on day 14 deuterium atoms at the methylenedioxy carbon of paroxetine
(Table 3). Thus, CTP-347 was metabolized more efficiently resulted in a new chemical entity, CTP-347, that demon-
than paroxetine in humans and the faster clearance observed strated substantially reduced inactivation of CYP2D6 while
for CTP-347 in vitro translated into the clinical setting.
retaining selective serotonin uptake inhibition in rat synap-
The rise in both AUCs and accumulation indices with in- tosomes and pharmacologic binding to a battery of receptors.
creasing CTP-347 dosages nonetheless displayed a small level of By these criteria, deuterium substitution appeared to have
nonlinearity (Table 3), suggesting some residual inhibition of limited effects on the pharmacologic and physicochemical
CYP2D6. Further insight into this issue was gleaned by ex- properties of paroxetine. Despite the apparent functional
amining data on the metabolism of DM, a CYP2D6-metabolized similarities, however, the rate of CYP2D6-mediated metabo-
drug, in the same dose-escalation study (Table 4). On the lism was significantly greater for CTP-347 than for parox-
evenings before the first and last doses of CTP-347, 30 mg of DM etine, as manifested by shorter t1/2 values for metabolism in
solution was administered orally to each subject, urine was human liver microsomes. This observation did not appear to

Altering Metabolic Profiles of Drugs with Precision Deuteration
51
TABLE 3
Pharmacokinetic parameters of paroxetine and CTP-347 on days 1 and 14
Cmax
Day 1 AUC0–last
Day 14 AUCss
Accumulation Indexa
Day 1
Day 14
ng/ml
h×ng/ml
Paroxetine: Multidose PK
Paroxetine 10 mg daily
Patient no.
1
0.5
0.5
3.2
3.2
1.3
0.3
1.6
0.4
1.6
4.0
1.6
1.4
1.4
4.3
6.9
17.0
15.6
14.0
1.1
25.3
1.0
14.1
35.9
13.5
11.0
14.0
4.3
7.1
40.2
39.1
15.2
2.6
22.9
3.0
20.4
60.0
21.5
19.4
17.8
57.5
122.3
296.5
262.2
261.8
14.8
494.1
12.2
234.5
649.3
240.5
207.1
248.1
13.4
17.2
7.4
2
3
4
6.7
5
17.2
5.7
21.6
4.1
11.5
10.8
11.2
10.7
13.9
6
7
8
9
10
Mean
S.D.
Median
CTP-347: Escalating dose PK
CTP-347 10 mg daily
Patient no.
49
2.8
10.7
1.0
0.2
0.5
0.4
1.6
0.3
0.8
2.0
7.1
34.2
2.3
31.6
195.7
8.7
91.0
650.0
29.0
8.0
7.0
5.0
18.0
4.0
65.0
97.4
209.4
18.0
2.9
3.3
3.3
6.2
1.9
1.5
1.2
1.9
7.8
3.3
3.3
2.2
52
54
55
0.6
1.3
56
0.9
3.6
57
0.5
3.3
58
1.5
15.1
2.1
59
0.5
60
4.7
8.3
Mean
5.8
30.0
62.9
8.3
S.D.
3.36
0.8
10.9
1.5
Median
CTP-347 10 mg twice daily
Patient no.
85
1.5
0.8
0.2
0.6
2.3
0.5
0.2
0.8
0.8
0.8
16.9
1.6
0.9
1.3
33.2
5.2
1.0
8.6
11.4
5.3
0.6
2.0
20.0
4.0
1.1
6.3
7.1
5.3
164.0
11.0
6.0
11.0
318.0
50.0
5.0
80.7
119.2
50.0
14.4
2.1
10.0
5.5
15.9
12.5
4.5
12.8
16.8
9.4
86
88
89
90
91
92
Mean
S.D.
12.3
5.2
Median
CTP-347 20 mg daily
Patient no.
61
2.0
1.5
12.1
0.6
0.8
3.4
2.6
3.8
1.2
3.1
3.6
2.0
6.2
29.8
15.6
1.7
21.7
20.4
145.1
4.7
11.8
32.6
33.4
37.5
14.0
35.7
42.5
21.7
72.0
523.0
233.0
24.0
112.0
136.0
597.0
585.0
134.0
268.4
232.6
136.0
3.3
25.6
1.6
62
63
64
5.1
66
7.9
9.5
67
11.6
33.4
37.2
9.0
4.2
69
17.9
15.6
9.6
71
72
Mean
16.9
13.1
11.6
7.5
S.D.
5.5
Median
6.3
CTP-347 40 mg daily
Patient no.
74
75
1.0
1.3
31.7
24.0
75.4
124.2
57.9
132.1
39.3
122.8
59.7
74.1
42.2
59.7
11.5
13.7
28.9
220.0
60.3
132.0
15.2
112.4
66.8
73.4
70.2
60.3
489.0
342.0
1231.0
2170.0
963.0
2090.0
562.0
42.5
25.0
42.6
9.9
16.0
15.8
37.0
17.1
15.0
16.3
10.1
16.7
77
2.4
78
14.7
4.7
79
81
10.5
1.1
82
83
9.5
1924.0
1004.0
1197.2
707.2
84
5.9
Mean
S.D.
Median
5.7
4.9
4.7
1004.0
ND, not determined; PK, pharmacokinetics.
aDay 14 AUCss/Day 1 AUC0–last
.

52
Uttamsingh et al.
TABLE 4
Urinary DM/DX ratios for CTP-347 on days 1 and 14
Urinary DM/DX
Baseline
Day 14
CTP-347 10 mg daily
Patient no.
49
0.0110
0.9720a
0.0150
0.0001
0.0020
0.0020
0.0040
0.0010
0.0180
0.1139
0.3219
0.0040
0.0260
1.4050a
0.0720
0.0050
0.0070
0.0050
0.0090
0.0040
0.1050
0.1820
0.4600
0.0090
52
54
55
56
57
58
59
60
Mean
S.D.
Median
CTP-347 10 mg twice daily
Patient no.
85
0.0140
0.0010
0.0002
0.0010
0.0160
0.0020
0.0010
0.0050
0.0069
0.0020
0.1640
0.0020
0.0010
0.0050
0.3530
0.0330
ND
86
88
89
a
90
91
92
Mean
0.0930
0.1419
0.0985
S.D.
Median
CTP-347 20 mg daily
Patient no.
61
0.0030
0.0050
0.1020
0.0020
0.0040
0.0380
0.0090
0.0070
0.0060
0.0196
0.0328
0.0060
0.0270
0.2580
0.2530
0.0220
0.0430
0.1230
0.4820a
0.3120a
0.0500
0.1744
0.1609
0.1230
62
63
64
66
67
69
71
Fig. 6. CTP-347 pharmacokinetics. Mean plasma concentration-time
curves after administration of 10 mg CTP-347 or paroxetine (A) and
median urinary DM/DX ratios (B) after 14 days of dosing to humans. The
paroxetine bar in (B) is an historic control (Alfaro et al., 2000), as assessed
8 days after initiation of 20 mg daily administration.
72
Mean
S.D.
Median
CTP-347 40 mg daily
Patient no.
74
75
0.0040
0.0030
0.0010
0.0380
0.0010
0.0020
0.0010
0.0010
0.0110
0.0069
0.0121
0.0020
0.1670
0.0730
0.1800
0.8500a
0.1370
0.7230a
0.1200
0.4960a
0.3880a
0.3482
0.2849
0.1800
be attributable to differences in binding affinities for CYP2D6
as Ks values for CTP-347 and paroxetine were similar.
Moreover, the effect did not appear to be due to changes in
metabolism pathways, as studies in human hepatocytes
demonstrated that the metabolic profiles of CTP-347 and
paroxetine were qualitatively very similar (data not shown).
Instead, kinetic analyses indicated that the higher rate of
CTP-347 metabolism was due to a decrease in MIC formation
with CYP2D6 and a consequent reduction in MBI. Consistent
with this, CTP-347 exhibited significantly less drug-drug
interaction with tamoxifen, another substrate for CYP2D6
metabolism. To our knowledge, this is the first demonstration
of a targeted deuterium substitution approach that greatly
diminished MBI liability of an approved drug.
77
78
79
81
82
83
84
Mean
S.D.
Median
ND, not determined.
aPoor metabolizer.
daily, because of its more rapid metabolism, had an exposure at
To examine whether the in vitro results translated into the steady state (AUCss 5 268.4 hour*ng/ml) that was roughly
clinical setting, we assessed the steady-state pharmacokinet- similar to paroxetine 10 mg daily (AUCss 5 240.5 hour*ng/ml).
ics of CTP-347 in healthy female volunteers. The results At 20 mg daily, CTP-347 exhibited lower DM/DX ratios and
demonstrated that the exposure (AUCss) of CTP-347 was lower EM→PM conversions than historical paroxetine 20 mg daily
than that of paroxetine after administration of equivalent data (Alfaro et al., 2000), demonstrating its potential for
doses (10 mg daily), establishing that the metabolism of CTP- eliminating drug-drug interactions in vivo and again mirroring
347 was more rapid in humans than paroxetine. Thus, the in the in vitro results.
vitro results were recapitulated in humans, a prerequisite for
A closer analysis of the pharmacokinetic results and the
making deuterium substitution a practical approach in drug urinary DM/DX ratios suggested that there was some residual
development. Further, subjects treated with CTP-347 20 mg inactivation of CYP2D6 in human, although it was greatly

Altering Metabolic Profiles of Drugs with Precision Deuteration
53
Fig. 7. Exposure (AUC) versus baseline urinary DM/
DX ratios for CTP-347 on day 1 and day 14. Extensive
metabolizers are to the left, and poor metabolizers
are to the right of the dashed lines.
diminished compared with paroxetine. The decrease in ground- development for diabetic neuropathy, although the effects of
state energy for the C-D bond raises the transition energy this latter agent on metabolic and pharmacokinetic profiles
necessary for its scission (Wiberg, 1955; Shao and Hewitt, were not detailed (Braman et al., 2013). Other new agents are
2010). Whether this effect is enough to completely eliminate or, being investigated (http://investor.auspexpharma.com/release-
instead, substantially lower, its overall rate is likely to be detail.cfm?releaseid5887958; Harbeson and Tung, 2011; Lu
dependent on the specific context of each reactant-enzyme pair. et al., 2015). It is important to note that the effects observed in
These effects may become more noticeable in the in vivo setting, this study (i.e., reduced MBI and a consequent increase in first-
where the characteristics of drug distribution, as well as the pass metabolism) may be quite different from the metabolism-
repeat administrations, may provide conditions whereby a small related effects imparted by deuteration of other drugs. For
level of MIC formation can occur. From a practical standpoint, instance, in other contexts, decreasing bond scission rates
however, it is likely that, even in this case, the overall reduction could reduce first-pass metabolism, which could be beneficial in
in MBI could provide substantial clinical benefit.
certain scenarios. Alternatively, deuteration could affect the
Heavy water (D2O) is used as a moderator in nuclear proportions of metabolic end products. It is hoped that
reactors, and multi-ton quantities of deuterium are commer- empirical characterization of this expanding class of drugs,
cially available at reasonable cost (Marter et al., 1982). using approaches similar to those described in this report, will
Moreover, deuterium exposure in the form of D2O appears to provide better tools to predict how deuterium substitution can
have low systemic toxicity; 15%–23% deuterium replacement be leveraged in the future and, as a result, to improve the
in whole body plasma has been reported to have no evident safety and efficacy of existing therapeutic agents.
adverse effects in humans (Blagojevic et al., 1994). The
abundant availability, reasonable cost, and low toxicity,
Acknowledgments
coupled with the potential to modify metabolism properties,
The authors thank Rheem Totah at the University of Washington
suggest that deuterium substitution may play a larger role in
School of Pharmacy, Department of Medicinal Chemistry, for
drug development in the future. One recent report, for
instance, described a phase 1 evaluation of another novel
technical assistance with the spectra analysis and review of the
manuscript, and David Norris (Ecosse Medical Communications,
deuterium-containing, controlled-release drug candidate under Falmouth, MA) for editorial support.

54
Uttamsingh et al.
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Authorship Contributions
Participated in research design: Uttamsingh, Harbeson, Wells,
Zelle, Tung, Graham.
Conducted experiments: Uttamsingh, Gallegos, Liu, Bridson, Cheng.
Contributed new reagents or analytic tools: Bridson, Cheng, Liu.
Performed data analysis: Uttamsingh, Liu, Graham, Wells, Zelle.
Wrote or contributed to the writing of the manuscript: Uttamsingh,
Liu, Harbeson.
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Address correspondence to: Dr. Vinita Uttamsingh, Concert Pharmaceut-
icals, Inc., 99 Hayden Ave., Suite 500, Lexington, MA 02421. E-mail:
vuttamsingh@concertpharma.com