Altering metabolic profiles of drugs by precision deuteration 2: Discovery of a deuterated analog of ivacaftor with differentiated pharmacokinetics for clinical development

Article abstract of DOI:10.1124/jpet.117.241497

Ivacaftor is currently used for the treatment of cystic fibrosis as both monotherapy (Kalydeco; Vertex Pharmaceuticals, Boston, MA) and combination therapy with lumacaftor (Orkambi; Vertex Pharmaceuticals). Each therapy targets specific patient population

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Altering Metabolic Profiles of Drugs by Precision Deuteration 2:
Discovery of a Deuterated Analog of Ivacaftor with
Differentiated Pharmacokinetics for Clinical Development
Scott L. Harbeson, Adam J. Morgan, Julie F. Liu, Ara M. Aslanian, Sophia Nguyen, Gary W.
Bridson, Christopher L. Brummel, Lijun Wu, Roger D. Tung, Lana Pilja, Virginia Braman, Vinita
Uttamsingh
Concert Pharmaceuticals, Inc., Lexington, Massachusetts
L.P. and L.W. are former employees of Concert Pharmaceuticals, Inc.
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Running Title
CTP-656: Deuterated Ivacaftor Analog with Differentiated PK
Corresponding Author
Dr. Scott L. Harbeson
Concert Pharmaceuticals, Inc
99 Hayden Ave., Suite 500
Lexington, MA 02421
Phone: 781-674-5208, fax: 781-674-5308, e-mail: sharbeson@concertpharma.com
Enumeration
Number of text pages: 37
Number of tables: 6
Number of figures: 5
Number of references: 33
Number of words in abstract: 246 (allowed: 250)
Number of words in Introduction: 750 (allowed: 750)
Number of words in Discussion: 1461 (allowed: 1500)
List of Non-Standard Abbreviations
APCI, atmospheric pressure chemical ionization; CF, cystic fibrosis; CFTR, cystic fibrosis
transmembrane conductance regulator protein; CTP-656, N-(2-(tert-Butyl)-4-(tert-butyl-d9)-5-
hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide; CYP, cytochrome P450; DIE,
deuterium isotope effect; FEV
, forced expiratory volume in 1 s; HBE, human bronchial
1
epithelial, HLM, human liver microsomes; IS, internal standard; Isc, short circuit current; LC,
liquid chromatography; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MBI,
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mechanism-based inhibition; MRM, multiple reaction monitoring; MS, mass spectrometry; PK,
pharmacokinetics; ppFEV , percent predicted FEV ; RODI, reverse osmosis deionized
1
1
Recommended Section Assignment:
Drug Discovery and Translational Medicine
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Abstract
Ivacaftor is currently used for the treatment of cystic fibrosis as both monotherapy (Kalydeco®)
and combination therapy with lumacaftor (Orkambi®). Each therapy targets specific patient
populations: Kalydeco treats patients carrying one of nine gating mutations in the cystic fibrosis
transmembrane conductance regulator protein (CFTR), while Orkambi treats patients
homozygous for the F508del CFTR mutation. In this study, we explored the pharmacological
and metabolic effects of precision deuteration chemistry on ivacaftor by synthesizing two novel
deuterated ivacaftor analogs, CTP-656 (d -ivacaftor) and d18-ivacaftor. Ivacaftor is administered
9
twice daily and is extensively converted in humans to major metabolites M1 and M6; therefore,
the corresponding deuterated metabolites were also prepared. Both CTP-656 and d18-ivacaftor
showed similar in vitro pharmacologic potency to ivacaftor, and the deuterated M1 and M6
metabolites showed equivalent pharmacology to the corresponding metabolites of ivacaftor,
consistent with previous studies of deuterated compounds. However, CTP-656 exhibited
markedly enhanced stability when tested in vitro. The deuterium isotope effects for CTP-656
D
D
metabolism ( V = 3.8, V/K = 2.2) were notably large for a CYP-mediated oxidation. The
pharmacokinetic (PK) profile of CTP-656 and d18-ivacaftor were assessed in six healthy
volunteers in a single-dose crossover study, which provided the basis for advancing CTP-656 in
development. The overall PK profile, including the 15.9 hour t1/2 for CTP-656 suggests that
CTP-656 may be dosed once daily thereby enhancing patient adherence. Together, these data
continue to validate deuterium substitution as a viable approach for creating novel therapeutic
agents with potentially differentiated properties to existing drugs.
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Introduction
Deuterium labeling of compounds has a long history of in vitro and in vivo use, primarily as a
method to probe enzymatic mechanisms and metabolic pathways or to allow non-radioactive
isotopic tracing (Rittenberg and Schoenheimer, 1937; Baillie, 1981). Deuterated agents as
pharmaceuticals are an area of growing interest as shown by the number of deuterated
compounds entering clinical development (Halford, 2016). Additionally, the first deuterated
compound, deutrabenazine (Austedo®), has recently been approved (Austedo, 2017). A key
feature of this technology is that deuterium substitution generally has minimal impact on the
structure, physical properties or pharmacology compared to the all-hydrogen version of a drug.
However, deuterium substitution can have a marked effect upon the pharmacokinetic profile of a
compound due to the deuterium isotope effect (DIE), which can lower the rate of carbon-
deuterium versus carbon-hydrogen bond cleavage (Kushner et al., 1999; Nelson and Trager,
2003; Harbeson and Tung, 2011). Although the theoretical limit for the DIE is 6–9 fold, effects
this large are rarely observed for enzyme-catalyzed reactions (Northrop, 1975; Jenks, 1987).
The magnitude of the DIE for a cytochrome P450 (CYP)-mediated oxidation is unpredictable
(
Fisher et al., 2006; Harbeson and Tung, 2014). Due to this unpredictability, deuterium-
substituted analogs must be synthesized and tested in preclinical studies to assess the
magnitude and direction of the DIE. Even then, preclinical observations may not quantitatively,
or potentially even qualitatively, predict human clinical results.
The first paper in this series presented the data for CTP-347, a deuterated analog of paroxetine
(
Paxil®), which is a selective serotonin reuptake inhibitor for the treatment of a number of
psychological disorders (Paxil, 2012; Uttamsingh et al, 2015). Herein, we report the effects of
selective deuterium substitution on another therapeutic agent, ivacaftor (Figure 1). Ivacaftor
(
Kalydeco) was approved in 2012 as the first drug to target specific mutational defects in the
cystic fibrosis transmembrane conductance regulator (CFTR) protein (Kalydeco, 2015). It is
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currently labeled for the treatment of cystic fibrosis (CF) patients 2 years and older who have at
least one mutant allele from a group of 9 gating mutations in the CFTR gene, with G551D being
the most common (4.4% of US CF patients) (Aditya, 2012; Kalydeco, 2015). In 2016, ivacaftor
was approved as part of the fixed-dose combination drug Orkambi, which combines ivacaftor
and lumacaftor for the treatment of CF patients 12 years and older homozygous for the F508del
CFTR mutation (approximately 45% of the US CF patients) (Wainwright et al., 2015; Orkambi,
2016). Lumacaftor is a CFTR “corrector” that improves the formation of active CFTR at the
epithelial surface of cells (Van Goor et al., 2011). The CFTR protein is an anion channel that
regulates epithelial chloride and bicarbonate concentrations to maintain proper hydration and
pH of secretory epithelia (Riordan, 2008). CFTR mutations reduce anion secretion and the
resulting dysregulation of anion concentrations promotes the accumulation of thick, viscous
mucus. Ivacaftor is a CFTR “potentiator” and increases the channel open probability, thereby
increasing anion conductance, improving mucus hydration and decreasing mucus viscosity (Van
Goor et al., 2009). Viscous mucus is particularly problematic for lung function since it results in
an inability to efficiently clear airways, which leads to chronic inflammation and infections that
contribute to a decline in lung function over time (Ong and Ramsey, 2015).
In this study, deuterated analogs of ivacaftor (Figure 1) were prepared and characterized with
respect to pharmacology, in vitro metabolism and in vivo pharmacokinetics (PK). Since
ivacaftor is extensively metabolized in humans to two major circulating metabolites, M1 and M6,
these metabolites and the corresponding deuterated metabolites d
8 6
-M1 and d -M6 were also
9
prepared and studied (Figure 2) (Zha et al., 2011). Two compounds -- d -ivacaftor (CTP-656)
and d18-ivacaftor -- showed increased metabolic stability versus ivacaftor in vitro. Deuterium
modification did not impact pharmacology, as the two deuterated compounds demonstrated
equivalent potency to ivacaftor in multiple in vitro studies. In vitro metabolic stabilization did
translate to increases in in vivo exposure parameters when the compounds were orally dosed in
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rats and dogs, although species differences were observed. Subsequently, both compounds
were dosed in healthy volunteers in a single-dose crossover study. The new chemical entity
CTP-656 was identified as the agent for further clinical advancement due to its general trend of
increased exposure versus d18-ivacaftor. The observed increase in exposure and half-life
compared to that reported for ivacaftor supported the potential for CTP-656 to be dosed once
daily. These data continue to validate deuterium substitution as a viable approach to creating
novel therapeutic agents with potentially differentiated properties to existing drugs.
Materials and Methods
Reagents
Indiplon, CTP-656, d18-ivacaftor, ivacaftor, M1, M6, d
Pharmaceuticals, Inc. (Lexington, MA). Human liver microsomes (mixed gender, pool of 200,
0 mg/mL) were from Xenotech, LLC (Lenexa, KS). Cryopreserved human hepatocytes were
8
-M1 and d -M6 were provided by Concert
6
2
purchased from In Vitro ADMET Laboratories (IVAL, LLC), (Malden, MA). Human cDNA
expressed CYP3A4 SupersomesTM were obtained from BD Biosciences (Woburn, MA).
Dimethylsulfoxide (DMSO), acetonitrile, MgCl and NADPH were from Sigma-Aldrich (St. Louis,
2
MO) and PEG400 was from J.T. Baker (Center Valley, PA). CF-HBE cells were obtained from
Cystic Fibrosis Foundation Therapeutics organization. Forskolin was obtained from LC
Laboratories (Woburn, MA). Benzamil and bumetanide were obtained from Sigma-Aldrich. HBE
studies were conducted in the laboratory of Dr. Robert Bridges at Rosalind Franklin University of
Medicine and Science, Chicago Medical School, 3333 Green Bay Road, North Chicago, IL
60064.
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Synthetic Procedures for the Synthesis of CTP-656, d18-Ivacaftor, d
8
-M1, d -M6, M1 and
6
M6
The synthetic methods for the preparation of CTP-656 and d18-ivacaftor have been previously
described (Morgan, 2015). Analytical characterization of CTP-656 can be found in the
Supplemental Materials. These include liquid chromatography (LC) purity assessment
1
(
(
Supplemental Figure 1); mass spectrum (Supplemental Figure 2) and the H-NMR spectrum
Supplemental Figure 3). Analytical data for d18-ivacaftor in the Supplemental Materials include
LC purity assessment (Supplemental Figure 4); mass spectrum (Supplemental Figure 5) and
1
the H-NMR spectrum (Supplemental Fig. 6). Synthetic routes and methods for the preparation
8 6
of CTP-656 metabolites d -M1 and d -M6 are also in the Supplemental Materials (Supplemental
Figures 7 and 8, respectively). Analytical data for d
8
-M1 and d -M6 in the Supplemental Material
6
include LC purity assessments (Supplemental Figure 9), mass spectra (Supplemental Figure
1
1
0), and H-NMR spectra (Supplemental Figure 11). The M1 metabolite of ivacaftor was
prepared by a modification of a synthetic method previously described (Yang et al., 2010). The
modified synthetic route (Supplemental Figure 12) and methods for the preparation of M1 can
be found in the Supplemental Materials. Ivacaftor metabolite M6 was prepared as previously
described (Yang et al., 2010). Analytical data for M1 and M6 in the Supplemental Material
include LC purity assessments (Supplemental Figure 13), mass spectra (Supplemental Figure
1
1
4), and H-NMR spectra (Supplemental Figure 15).
Bioanalytical Methods
Preclinical Non-GLP Methods. All plasma samples, in vitro samples, quality control, and
standards were treated with acetonitrile during protein precipitation. Acetonitrile containing
internal standard (indiplon) was then added and samples were vortexed at 1450 rpm for
3
minutes and then centrifuged at 3500 rpm for 10 minutes. Ten microliters of prepared sample
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was injected onto an HPLC-tandem quadrupole mass spectrometer (LC-MS/MS) consisting of
an Agilent 1200 HPLC with a Leap auto-sampler (Leap HTS-PAL) equipped with a C6 Gemini
phenyl 2.1x50 mm column maintained at 30˚C. Analytes were eluted from the column using a
gradient, where mobile phase B was linearly increased from 0 to 100% over 1 minute and then
returned to 0% over another minute. The composition of mobile phase B was 0.1% formic acid
in acetonitrile while the composition of mobile phase A was 0.1% formic acid in reverse osmosis
deionized (RODI) water.
LC-MS/MS analysis was performed using atmospheric pressure chemical ionization (APCI),
followed by multiple reaction monitoring (MRM) of each analyte including the internal standard.
Transitions monitored were 393.2/172.2 m/z for ivacaftor, 402.2/172.2 m/z for CTP-656,
4
11.2/172.2 m/z for d18-ivacaftor, 417.2/172.2 m/z for d
-M1 and 409.2/172.2 m/z for M1, while
8
the transition for indiplon (the IS) was 377.2/293.2 m/z. For all analytes, a dwell time of 100 ms
was used.
Clinical GLP Method. The determination of CTP-656 and d18-ivacaftor in plasma was performed
using a validated method under GLP by CPR Pharma Services (28 Dalgleish Street, Thebarton
SA 5031, Australia) conducted in compliance with CPR Pharma Standard Operation
Procedures. Plasma analysis was performed following protein precipitation with 99% acetonitrile
and 1% formic acid using ivacaftor as the internal standard. The analytes were separated by
HPLC using a C18 column (Phenomenex; Luna C18, 50 x2 mm, 3µm), where mobile phase A
consisted of 10% acetonitrile and 90% water with 0.1 % formic acid and mobile phase B
consisted of 50% acetonitrile, 50% methanol with 0.1% formic acid. Elution of the analytes from
the column was performed at a flowrate of 0.3 mL/min using a gradient where the percent of
mobile phase B was increased linearly from 55% to 100% over 2.5 minutes and held at 100%
for 1 minute, after which the mobile phase composition was set to 10% B for 0.4 minutes and
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then returned to initial conditions (55% B) for re-equilibration. Total run time for the method was
minutes.
6
The analysis of the eluates was performed using an API4000 (PE Sciex) mass spectrometer in
positive multiple reaction monitoring (MRM) mode. The MRM transitions monitored were
402.2/172.2 m/z for CTP-656 and 411.2/172.2 m/z for d18-ivacaftor. Ivacaftor was the internal
standard for both CTP-656 and d18-ivacaftor. For all analytes, the method was linear and in all
cases the precision and accuracy met FDA & ICH criteria.
In Vitro Pharmacology Assessments
Chloride conductance was evaluated with an equivalent current (Ieq) assay system using fully
differentiated primary cultures of CF human bronchial epithelial (HBE) cells grown at an air
liquid interface for four to six weeks. Transepithelial voltage (V
measured under current clamp conditions using a custom-designed 24-channel current clamp
TECC-24) and a 24-well electrode manifold. For all experiments, cells were tested in symmetric
chloride solution (HEPES buffered Coon's F12 media without serum or bicarbonate) at
6 ± 0.5 °C, and all compounds and test articles were dissolved in DMSO. For chloride
T
) and conductance (G
T
) were
(
3
conductance measurements, epithelial sodium channels were blocked with 3 µM benzamil;
potentiator responses were measured following 10 µM forskolin addition. CFTR specificity was
determined by blocking chloride secretion with 20 µM bumetanide. Potentiator responses were
quantified by calculating the total peak area (AUC) of Ieq following addition of test article. For
studies using corrector, lumacaftor was added at 3 µM 24 hr prior to chloride conductance
measurements. All compounds were assayed in triplicate at a single concentration per well, and
AUC values were fit to a Hill equation to yield EC50 values.
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Metabolic Stability in CYP3A4 Supersomes
Stock solutions (10 mM) of CTP-656, d18-ivacaftor and ivacaftor, were prepared in DMSO. The
stock solutions were diluted to 50 µM in acetonitrile. Human CYP3A4 Supersomes
(
1000 pmol/mL) were diluted to 62.5 pmol/mL in 0.1 M potassium phosphate buffer, pH 7.4,
containing 3 mM MgCl . Metabolic stability in the diluted Supersomes was assessed as
2
previously described (Morgan, 2015).
Semi-Quantitative Metabolite Profile in Human Hepatocytes Stock solutions (5 mM) of CTP-
656, d18-ivacaftor and ivacaftor were prepared in DMSO. Cryopreserved human hepatocytes
were thawed, diluted in cold thawing media, and centrifuged. The supernatants were removed
and the cells resuspended in Krebs-Henseleit Buffer (KHB). The test articles were added to the
hepatocytes in KHB buffer to achieve a final concentration of 25 µM in 0.5 mL, containing 4 x
6
o
1
0 cells/mL hepatocytes. The reaction mixtures were incubated at 37 C in a cell culture
incubator (5% CO ). Aliquots of the mixture were removed at 0 and 4 hours and acetonitrile was
2
added. Samples were transferred micro-centrifuge tubes and centrifuged at 10,000 g for 8
minutes to pellet precipitated proteins. Supernatants were analyzed for amounts of parent
remaining and metabolites formed by LC-MS/MS. UV/Vis @ 254nm was used for semi-
quantitative analysis, wherein metabolites were measured as percentage of parent peak area.
Analysis was by UV/Vis since metabolite standards had not been synthesized at the time of this
experiment.
Determination of Deuterium Isotope Effect for CTP-656 in Human Liver Microsomes
Stock solutions (50 mM) of test compounds were prepared in DMSO. The 20 mg/mL human
liver microsomes (HLM) were diluted to 0.625 mg/mL in 0.1 M potassium phosphate buffer, pH
7
.4, containing 3 mM MgCl
. Aliquots (375 µL) of the HLM and test compound mixtures were
2
transferred to wells of a deep 96-well plate in triplicate and pre-warmed for 7 minutes. Reactions
were initiated by addition of 125 µL of pre-warmed NADPH solution. The final reaction volume
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was 0.5 mL and contained 0.5 mg/mL HLM, and 1, 2, 5, 10, 20, 40, 60, 80 and 100 µM of
ivacaftor or CTP-656, 2 mM NADPH and 3 mM MgCl . The reaction mixtures were incubated at
2
37ºC for 10 minutes after which 50 µL aliquots were removed and added to shallow 96-well
plates. The reactions were stopped by addition of 50 µL of ice-cold acetonitrile with internal
º
standard. The plates were stored at 4 C for 20 minutes, after which 100 µL of water was added
to the wells before centrifugation to pellet precipitated proteins. Supernatants were transferred
to another 96-well plate and analyzed for parent and metabolite by LC-MS/MS using an Applied
Bio-systems API 4000 mass spectrometer.
The kinetic parameters, K and Vmax, were calculated from the Michaelis-Menten equation. The
m
enzyme velocity (enzyme velocity=concentration of hydroxyl methyl/time/protein concentration)
from three separate experiments was input into GraphPad Prism 6.04 and reported values for
m
K and Vmax were determined from the fit using the average value.
Preclinical Pharmacokinetic Studies
All animal studies were carried out in accordance with the Guide for the Care and Use of
Laboratory Animals, as adopted and promulgated by the U.S. National Institutes of Health, and
were approved by the Institution’s Animal Care and use Committee or local equivalent.
Pharmacokinetics of CTP-656, d18-Ivacaftor and Ivacaftor in Rats. Male, Sprague-Dawley rats
(
234-276 g) were obtained from Harlan Labs (Indianapolis, IN) and housed at VivoPath, LLC
(Worcester, MA) where the studies were conducted. The PK of CTP-656, d18-ivacaftor and
ivacaftor in rats was assessed as previously described (Morgan, 2015). The plasma PK
parameters for the dosed compounds were calculated by non-compartmental analysis using
Phoenix 64 WinNonlin software.
Pharmacokinetics of CTP-656, d18-Ivacaftor and Ivacaftor in Dogs. Four male beagle dogs were
housed at Agilux Labs (Worcester, MA) where the studies were conducted. The dogs were
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administered 3 mg/kg of each test compound orally, in a discrete-dose crossover study with one
week wash out period between the dosing phases. All dogs were fasted overnight before
administration of each test article. Each compound was formulated as a solution in 100%
PEG400 at a concentration of 1.5 mg/mL. Blood samples were collected from each dog at 15
and 30 minutes, and 1, 2, 4, 6, 8, 16, 24, 48, 72, 84 and 96 hours post-dose. Blood samples
were centrifuged to obtain plasma samples that were analyzed for concentrations of the dosed
compound at each time point using LC-MS/MS. The limit of quantitation of each compound was
1
ng/mL. The plasma PK parameters for the dosed compounds were calculated by
non-compartmental analysis using Phoenix 64 WinNonlin software.
Evaluation of Pharmacokinetic Profiles of CTP-656 and d18-Ivacaftor in Healthy Human
Volunteers
The first-in-human study was a two-part open-label, randomized, sequential, crossover, single-
dose study to evaluate the safety, tolerability and pharmacokinetics of CTP-656 and d18
-
ivacaftor amorphous dispersion aqueous suspension in healthy volunteers, with a
pharmacokinetic comparison to ivacaftor. This manuscript will report on the first part of this
clinical study, which was a single-dose, randomized, 2-period, 2-treatment, crossover study
conducted in healthy male and female volunteers to evaluate the safety, tolerability and
pharmacokinetics of CTP-656 and d18-ivacaftor under fasted conditions. The study was
performed at a single center (CMAX [a division of IDT Australia Limited], Adelaide, Australia)
under the Clinical Trial Notification (CTN) scheme through Therapeutic Goods Administration.
The study was conducted in accordance with the Declaration of Helsinki, e.g., the clinical study
protocol, amendments, informed consent document(s), and any other study-related documents
were reviewed and approved by the Bellberry Human Research Ethics Committee, and
informed consent was obtained prior to any study-related procedures.
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Six healthy volunteers were enrolled in this part of the study. On Day 1, 3 subjects received a
single oral dose of 25 mg CTP-656 and 3 subjects received a single oral dose of 25 mg d18
-
ivacaftor as an aqueous suspension. On Day 8 following the first dose, subjects crossed over
and the dosing was repeated. A total of 21 PK blood samples (4 mL each) were collected for
assessment of plasma concentrations of CTP-656 and d18-ivacaftor. Time points for collections
were immediately prior to dosing (0) and 0.25, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 16, 20, 24, 36, 48,
60, 72, 84 and 96 hours post-dose. Plasma levels of CTP-656 and d18-ivacaftor were measured
by LC-MS/MS. The plasma PK parameters for the dosed compounds were calculated by
non-compartmental analysis using Phoenix 64 WinNonlin software. Each subject was monitored
for safety throughout the study, including vital signs, physical examinations, clinical laboratory
measurements and 12-lead electrocardiograms.
Results
Deuterium Substitution Does Not Alter the Pharmacology of Ivacaftor and its Major
Metabolites
The pharmacological properties of CTP-656 and its major metabolites, d
8
-M1 and d -M6, were
6
compared to ivacaftor, M1, and M6 using a chloride conductance assay with fully differentiated
HBE cells expressing mutant CFTR. It has been reported for the G551D CFTR mutation that
th
th
M1 has approximately 1/6 the potency and M6 less than 1/50 the potency of ivacaftor
Kalydeco, 2015). In G551D/F508del HBE cells, ivacaftor and CTP-656 had similar potencies
for potentiating forskolin responses, with EC50 values of 336 nM and 255 nM, respectively
Table 1). The potencies of the M1 and d -M1 metabolites were also similar (0.96 µM and 1.35
µM, respectively), and both metabolites were less potent than the parent: 3-fold for M1 and 5-
fold for d -M1. The EC50 values for M6 and d -M6 were 11 µM and 15 µM, respectively, in
(
(
8
8
6
agreement with prior reports that the M6 metabolite does not contribute to the therapeutic
efficacy of ivacaftor in CF patients (Kalydeco, 2015).
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To compare the activities of CTP-656, ivacaftor, and metabolites in the context of
pharmacologically-corrected F508del-CFTR, the potencies of all compounds for potentiating
forskolin responses were tested in F508del/F508del HBE cells following a 24-hour incubation
with lumacaftor. Similar to results obtained with G551D/F508del HBE, the potencies of
non-deuterated and deuterated parent and metabolite compounds were very similar (Table 2).
d18-Ivacaftor was also tested in G551D-CFTR-expressing FRT cells and lumacaftor-corrected
F508del/F508del HBE cells; no significant differences in potentiation activity or potency were
observed between d18-ivacaftor, CTP-656, and ivacaftor (data not shown).
Deuterated Ivacaftor Analogs Show Increased Metabolic Stability in In Vitro Assays
Metabolic stability was initially determined by incubating each compound in the presence of
CYP3A4 Supersomes for 30 minutes and monitoring the decrease in compound versus time.
Additionally, compounds were incubated with human hepatocytes and the amounts of parent
remaining and metabolites formed were measured. In CYP3A4 supersomes, the in vitro t1/2
value for ivacaftor was 5.5 minutes, whereas the t1/2 values for CTP-656 and d18-ivacaftor were
both 8.1 min (average of two experiments for the 3 compounds tested), which is a 47% increase
in t1/2 .for CTP-656 and d18-ivacaftor versus ivacaftor (Figure 3A). The data from human
hepatocytes showed an approximate 2-fold reduction of the M1 metabolites as percent of parent
for both CTP-656 and d18-ivacaftor with respect to ivacaftor (Table 3). Additionally, no new
metabolites were identified in the human hepatocyte incubations of the two deuterium modified
compounds.
Deuterium-enhanced metabolic stability was also assessed by incubating increasing
concentrations of ivacaftor and CTP-656 in the presence of HLM for 10 minutes and measuring
the rate of formation for M1 and d
form of the Michaelis-Menten equation to derive the kinetic parameters K
substrate at ½ Vmax), Vmax (enzyme velocity at saturating substrate: [S] >> K
8
-M1, respectively. The data were then fit to the hyperbolic
m
(concentration of
m
) and Vmax/K
m
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(
m
enzyme velocity at low substrate concentrations: [S] << K ). The ratios of the kinetic
parameters Vmax and Vmax/K
m
for ivacaftor versus CTP-656 measure the deuterium kinetic
D
D
isotope effects V and V/K for the CYP3A4-catalyzed formation of M1 in HLM (Northrop, 1975).
D
Significant deuterium isotope effects were measured for both kinetic parameters: V = 3.8 and
DV/K = 2.2 (Figure 3B). The observed increase in t1/2 for CTP-656 and d18-ivacaftor versus
ivacaftor in CYP3A4 Supersomes, the decreased metabolism in human hepatocytes and the
isotope effects observed in HLM supported progression of the compounds to in vivo
pharmacokinetic studies in rats and dogs.
Deuterated Ivacaftor Analogs Show Differentiated Non-clinical Pharmacokinetics Upon
Oral Dosing
The PK profiles of the 3 compounds were initially assessed in male Sprague-Dawley rats. Each
compound was dosed at 10 mg/kg to each of 3 rats, with a total of 9 rats in the study. The
plasma levels of compound were measured over 72 hours. The plasma concentration versus
time curves for drug exposure of the compounds are shown in Figure 4A (only the 0–24 hour
data are shown for clarity). Although d18-ivacaftor showed greater exposure than either CTP-656
or ivacaftor, the error bars for the time points are overlapping, indicating modest differentiation
for d18-ivacaftor versus ivacaftor. This modest differentiation is supported by the PK parameters
for the 3 compounds (Table 4). Across the parameters Cmax, AUC0-24 hr, C24hr and t1/2, d18
-
ivacaftor showed a trend to larger PK parameters in rats compared to both CTP-656 and
ivacaftor.
The PK profiles of the 3 compounds were also studied in male beagle dogs. Each compound
was dosed by oral gavage at 3 mg/kg to each of 4 dogs in a cross-over fashion with a 1 week
washout between doses, and the plasma levels of compound were measured over 96 hours. As
shown by the plasma concentration versus time profiles for each compound (Figure 4B), CTP-
656 had much greater exposure than ivacaftor and appeared to exhibit greater exposure than
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d
18-ivacaftor (only the 0–24 hour data are shown for clarity). The PK parameters for CTP-656
versus ivacaftor also supported the differentiated profile versus ivacaftor in dogs (Table 4). The
max, AUC0-24 hr and C24hr show 62%, 69% and 83% increases vs. ivacaftor, respectively.
C
Although the PK data for CTP-656 and d18-ivacaftor in rats and dogs demonstrated deuterium-
mediated slowing of ivacaftor metabolism, the data did not clearly identify which compound
should be advanced to human studies. The Pharmacology Review section of the Summary
Basis of Approval for ivacaftor noted that exposure to ivacaftor was significantly greater than M1
and M6 in non-clinical test species (mouse, rat and dog)
(
Center for Drug Evaluation and Research, 2012a). However, in humans, the exposure to M1
and M6 is significantly greater than ivacaftor. In view of the extensive metabolism of ivacaftor
that occurs in humans but is not observed in either rats or dogs, both CTP-656 and d18-ivacaftor
were advanced to a first-in-human study to identify the preferred compound for continued
clinical development.
CTP-656 Identified as the Deuterated Ivacaftor Analog for Clinical Advancement
This first-in-human study of CTP-656 and d18-ivacaftor was designed as a 2-part cross-over
study of a low (25 mg) single oral dose of the two compounds as an aqueous suspension of
their respective solid, amorphous dispersions. The baseline demographics of the study
participants are summarized in Table 5. In the first part, six healthy volunteers (4M and 2F) were
administered study drug on Day 1: three received CTP-656 and three received d18-ivacaftor. On
Day 8, the subjects were crossed-over and were administered the other compound. Blood
samples were collected over a 96-hour period on Days 1 and 8, and the PK parameters were
determined (Table 6). The plasma concentration versus time curves (0–24 hours shown for
clarity) for each compound are also shown (Figure 5).
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The PK parameters reflect the general trend shown in the exposure curves in that Cmax and
AUC0–inf are greater for CTP-656 than for d18-ivacaftor. The approximate 16-hour half-life of
CTP-656 is also compatible with identifying a dose that would enable once-daily dosing. PK
data for a 25mg dose of ivacaftor have been previously reported as part of a single-ascending
dose study (Center for Drug Evaluation and Research, 2012b). For comparative purposes, the
half-life for ivacaftor was 11.1 hr and the exposure (AUC0-inf) was 1627 ng/mL-hr. These data
for ivacaftor suggest that at the same dose, CTP-656 may have greater exposure and a longer
half-life versus ivacaftor in humans. In subsequent clinical studies, CTP-656 as a solid oral 150
mg dose was compared directly to a single dose of ivacaftor (150 mg tablet) and was shown to
have an approximate 15-hour half-life (Uttamsingh et al., 2016).
Discussion
This study reports the results of precision deuteration of ivacaftor (Kalydeco), which is the first
approved CFTR modulator for the treatment of cystic fibrosis. Two deuterated analogs, d
9
-
ivacaftor (CTP-656) and d18-ivacaftor were tested in vitro and in vivo to assess the
pharmacology, pharmacokinetics and metabolism of the compounds with respect to ivacaftor.
The studies of the deuterated ivacaftor analogs were designed to assess whether a deuterium
isotope effect could reduce metabolism in humans and provide an agent with a differentiated
clinical profile. The data for the deuterated analogs of ivacaftor align with the previous paper in
this series that assessed the effects of precision deuteration on paroxetine pharmacology and
pharmacokinetics (Uttamsingh et al., 2015). Deuterated paroxetine (CTP-347) retained the
intrinsic in vitro pharmacology profile of paroxetine with greatly reduced mechanism-based
inactivation of CYP2D6 in in vitro studies. This preclinical profile of CTP-347 translated to a
reduced drug-drug interaction liability with dextromethorphan in a clinical study. However,
further clinical development of CTP-347 as a non-hormonal therapy for hot flashes was stopped
for commercial reasons.
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To confirm that deuterium substitution did not affect the intrinsic pharmacology of ivacaftor, in
vitro studies were performed in HBE derived from CF patients. The potency as a potentiator of
CFTR with a gating mutation was measured in G551D/F508del HBE. For homozygous F508del
HBE, the cells were first corrected with lumacaftor prior to measuring the potentiation of
conductance. Ivacaftor is extensively metabolized in humans to two major circulating
metabolites, M1 and M6 (Zha et al., 2011). At steady state, the metabolites are the major
circulating species in humans, with mean exposure ratios (metabolite/parent) of approximately
5
-fold and 2-fold for M1 and M6, respectively (Center for Drug Evaluation and Research,
012b). These metabolites of ivacaftor and CTP-656 were, therefore, also evaluated in the
2
HBE assays.
Overall, the in vitro pharmacology data confirmed that deuteration did not change the CFTR
activity of ivacaftor and the major metabolites. The equivalent activity of CTP-656 and ivacaftor
measured in G551D/F508del HBE supports the use of CTP-656 in the same patient population
as ivacaftor. Likewise, the potentiation of CFTR function in lumacaftor-corrected F508del
homozygous HBE supports the clinical use of CTP-656 in combination with lumacaftor and
potentially other correctors currently in development.
Although precision deuteration of ivacaftor did not change the intrinsic pharmacology of the
compound, decreases in metabolism for the deuterated analogs were shown both in vitro and in
vivo. High protein binding, non-specific binding and low solubility prevented the metabolic
stability assessment of ivacaftor in our standard HLM assay due to the slow apparent rate of
metabolism (unpublished data). Since ivacaftor is metabolized primarily by CYP3A4 (Kalydeco,
2015), CYP3A4 Supersomes were used to assess metabolic stability. The two deuterated
analogs exhibited t1/2 values approximately 50% greater than ivacaftor in this study, which
signaled that precision deuteration could positively impact the PK of ivacaftor.
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Once the respective M1 and d -M1 metabolites of ivacaftor and CTP-656 were synthesized, a
8
more sensitive assay to measure metabolism in HLM was possible. By measuring the formation
of metabolites instead of loss of parent, a direct measurement of metabolite formation in HLM
was possible and enabled calculation of the DIE using classical Michaelis-Menten kinetics. The
D
D
measured V/K and V values for CYP3A4 catalyzed metabolism in HLM were large.
D
Although a V/K isotope effect of 2.2 was measured, the literature reports that an isotope effect
on V/K should not be observed unless it has been unmasked, which can occur under certain
conditions (Korzekwa et al., 1989; Nelson and Trager, 2003). Unmasking could arise due to
oxidation at a different site on CTP-656 to produce an alternate metabolite (“metabolic
switching”) (Horning et al., 1975). Preclinical and clinical data, however, do not suggest
metabolic switching. Incubations of CTP-656 and d18-ivacaftor in human hepatocytes showed
only reductions in the formation of the M1 metabolites vs. ivacaftor; no other metabolites were
detected. Clinical data also showed the same plasma metabolites for CTP-656 and ivacaftor in
humans (manuscript in preparation). Another possible mechanism for unmasking the isotope
effect is direct reduction of the activated enzyme-substrate complex to produce free enzyme,
substrate and water (Atkins and Sligar, 1987). Studies were not conducted to assess this
D
mechanism but it appears to be a reasonable explanation for the observed V/K isotope effect
for CTP-656 in the absence of metabolic switching.
A recent review on the potential issues for deuterium in drug development notes than an isotope
effect in vitro needs to translate to a PK effect in vivo to have a practical application to drug
development (Guengerich, 2013). Although slower metabolism was observed in vitro, the
translation to in vivo is unpredictable since non-CYP clearance mechanisms may predominate
in vivo and clearance mechanisms may vary between species. PK studies in rats and dogs
were particularly challenging since neither rat nor dog metabolize ivacaftor as extensively as
humans; therefore, these preclinical species may not be adequate models for human
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metabolism (Center for Drug Evaluation and Research, 2012a). The PK results in rats and dogs
for CTP-656 were encouraging with a longer half-life and greater exposure vs. ivacaftor in both
species. d18-Ivacaftor, however, showed increased exposure versus ivacaftor in rats, but not
dogs. These results emphasize the unpredictable effects of deuterium substitution. Although
positive preclinical PK effects were very encouraging, these models were not sufficient to
choose a preferred compound for clinical development. The more rigorous approach involved
testing both compounds in humans and selecting a compound for advancement based upon
human PK data.
The clinical crossover study of orally dosed CTP-656 and d18-ivacaftor identified the preferred
compound for further clinical development. CTP-656 exposure was only modestly increased
versus d18-ivacaftor; however, the increases were consistent across the PK parameters Cmax
1/2, AUC0-inf, C24hr and CL/F. These data, coupled with the reduced amount of deuterium in CTP-
56, were sufficient to identify CTP-656 for continued clinical development.
,
t
6
The potential of CTP-656 to be dosed once-daily was predicted based on available information
for ivacaftor including single-ascending dose (SAD) PK in healthy volunteers and PK/PD
relationships in patients (Center for Drug Evaluation and Research, 2012b). In the SAD study
of ivacaftor, which was conducted in the fasted condition, exposure increases were dose
proportional from 25mg to 375mg. Additionally, the 150mg twice-daily dose of ivacaftor was
selected based on simulations that showed a plasma Cmin at steady-state of ~250 ng/mL
achieved the predicted EC90 for FEV
1
and the EC84 for sweat chloride. The 25mg dose of CTP-
656 in the fasted state provided a C24hr (Cmin) of 53 ng/mL in plasma. Assuming dose
proportional increases in exposure for CTP-656, a once-daily dose of approximately 118mg in
the fasted condition would achieve the target C24hr of 250 ng/mL even in the absence of
accumulation on multiple-dosing. Ivacaftor shows an increase in exposure of 2-3 fold when
dosed with food; therefore, CTP-656 has the potential to exceed the 250 ng/mL Cmin at steady-
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state if a similar food effect is observed. Based on this analysis of the 25mg dose, CTP-656
was projected to achieve the desired once-daily profile in future studies, which was confirmed in
subsequent Phase 1 studies.
Once-daily dosing may translate to improved adherence in real-world use. A small clinical study
assessed ivacaftor adherence in patients and found the adherence to be highly variable
(
Siracusa et al., 2015). The ivacaftor label (Kalydeco, 2015) specifies dosing every 12 hours;
however, this study found the median duration between doses was 16.9 hours, with a range of
2.1 to 43.7 hours. The overall adherence rate—calculated as (total doses taken)/(total days
monitored x 2 doses/day)—was only 61%. It is also noteworthy that the 10.4% improvement in
absolute ppFEV
observed in the clinical study (12+ years old) is much greater than the 5.4%
improvement observed over 12 months in a non-clinical trial patient population (Fink et al.,
015). Although this may be due to a number of factors, the suboptimal adherence could be an
1
1
2
important contributing factor, which may be positively impacted by a once-daily potentiator.
CTP-656 has completed three Phase 1 studies in healthy volunteers, including single-ascending
dose (SAD), multiple-ascending dose (MAD) and food effect studies. The tablet formulation was
reported to have an average half-life of 15 hours after a single oral dose and an average half-life
of 18 hours in the MAD studies (Uttamsingh et al., 2016). Of particular note is the reported CTP-
6
56 to d
-M1 AUC ratio of 1.5 to 1 after a single dose and also at steady state. This is much
8
greater than ivacaftor, which has a parent to M1 AUC ratio of 1 to 5 at steady state. With a
longer half-life and greater exposure to the more active parent, the PK profile for CTP-656 may
translate into an improved clinical profile in CF patients with gating mutations. A Phase 2 safety
and efficacy study in CF patients is currently underway (NCT02971839).
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Acknowledgments
The authors would like to thank Doug Williams, Kinga Chojnacka, and Junfeng Huang (Adesis
Inc., New Castle, DE) for metabolite synthesis and David Norris (Ecosse Medical
Communications, Falmouth, MA) for editorial assistance.
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Authorship Contributions
Participated in research design: Aslanian, Braman, Brummel, Harbeson, Liu, Tung, Uttamsingh,
Wu.
Conducted experiments: Aslanian, Bridson, Morgan, Nguyen, Pilja, Uttamsingh.
Contributed new reagents or analytic tools: Bridson, Brummel, Liu, Morgan.
Performed data analysis: Aslanian, Brummel, Nguyen, Uttamsingh
Wrote or contributed to the writing of the manuscript: Aslanian, Braman, Brummel, Harbeson,
Liu, Tung, Uttamsingh
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References
Aditya S (2012) Ivacaftor in cystic fibrosis: the first disease modifying agent. Int J Basic Clin
Pharmacol 1:225-229.
Atkins WM and Sligar SG (1987) Metabolic switching in cyctochrome P-450cam: deuterium
isotope effects on regiospecificity and the monooxygenase/oxidase ratio. J Am Chem
Soc 109:3754-3760.
Austedo (2017) Prescribing Information. Teva Pharmaceuticals USA, Incorporated: North
Wales, PA.
Baillie TA (1981) The use of stable isotopes in pharmacological research. Pharmacol Rev
3
3:81-132.
Center for Drug Evaluation and Research (2012a). Pharmacology Review(s): Ivacaftor
Application Number: 203188Orig1s000).
http://www.accessdata.fda.gov/drugsatfda_docs/nda/2012/203188Orig1s000PharmR.pdf
Accessed November 2016.
(
.
Center for Drug Evaluation and Research (2012b). Clinical Pharmacology and
Biopharmaceutics Review(s): Ivacaftor (Application Number: 203188Orig1s000).
http://www.accessdata.fda.gov/drugsatfda_docs/nda/2012/203188Orig1s000ClinPharm
R.pdf. Accessed November 2016.
Fink A, Sawicki GS, Morgan WJ, Schechter MS, Rosenfeld M, and Marshall BC (2015)
Treatment response to ivacaftor in clinical practice: analysis of the US CF Foundation
patient registry. Ped Pulmonol 50: S41, p. 361 (Abstr. 450).
Fisher MB, Henne KR and Boer J (2006) The complexities inherent in attempts to decrease
drug clearance by blocking sites of CYP-mediated metabolism. Curr Opin Drug Discov
Devel 9:101-109.
25

JPET Fast Forward. Published on June 13, 2017 as DOI: 10.1124/jpet.117.241497
This article has not been copyedited and formatted. The final version may differ from this version.
JPET #241497
Guengerich FP (2013) Kinetic deuterium isotope effects in cytochrome P450 oxidation
reactions. J Labelled Comp Radiopharm 56:428-431.
Halford B (2016) Deuterium swithcheroo breathes life into old drugs. Chem Eng News 94:32-36.
Harbeson SL and Tung RD (2011) Deuterium in drug discovery and development. Annu Rep
Med Chem 46:403-417.
Harbeson SL and Tung RD (2014) Deuterium medicinal chemistry: a new approach to drug
discovery and development. Med Chem News (Japan) 4:8-22.
Horning MG, Haegele KD, Sommer KR, Norwin J and Stafford M (1975) Metabolic switching of
drug pathways as a consequence of deuterium substitution, in International Conference
on Stable Isotopes (Klein ER and Klein PD eds) pp 41-54, Argonne National Lab.
Jenks WP (1987) Catalysis in Chemistry and Enzymology. Dover Publications Inc., Mineola,
NY; p. 243.
Kalydeco (2015) Prescribing Information. Vertex Pharmaceuticals Incorporated: Boston, MA.
Korzekwa KR, Trager WF and Gillette JR (1989) Theory for the observed isotope effects from
enzymatic systems that form multiple products via branched reaction pathways:
cytochrome P-450. Biochemistry 28:9012-9018.
Kushner DJ, Baker A and Dunstall TG (1999) Pharmacological uses and perspectives of heavy
water and deuterated compounds. Can J Physiol Pharmacol 77:79-88.
Morgan AJ (2015) Deuterated CFTR potentiators. US Patent 9181192.
Nelson SD and Trager WF (2003) The use of deuterium isotope effects to probe the active site
properties, mechanism of cytochrome P450-catalyzed reactions, and mechanisms of
metabolically dependent toxicity. Drug Metab Dispos 31:1481-1498.
Northrop DB (1975) Steady-state analysis of kinetic isotope effects in enzymic reactions.
Biochemistry 14:2644-2651.
Ong T and Ramsey BW (2015) Update in Cystic Fibrosis 2014. Am J Respir Crit Care Med
192:669-675.
26

JPET Fast Forward. Published on June 13, 2017 as DOI: 10.1124/jpet.117.241497
This article has not been copyedited and formatted. The final version may differ from this version.
JPET #241497
Orkambi (2016) Prescribing Information. Vertex Pharmaceuticals Incorporated: Boston, MA.
Paxil (2012) Prescribing Information. GlaxoSmithKline, Research Triangle Park, NC.
Riordan JR (2008) CFTR function and prospects for therapy. Annu Rev Biochem 77: 701-726.
Rittenberg D and Schoenheimer R (1937) Deuterium as an indicator in the study of intermediate
metabolism: XI. further studies on the biological uptake of deuterium into organic
substances, with special reference to fat and cholesterol. J Biol Chem 121:235-253.
Siracusa CM, Ryan J, Burns L, Wang Y, Zhang N, Clancy JP and Drotar D (2015) Electronic
monitoring reveals highly variable adherence patterns in patients prescribed ivacaftor. J
Cyst Fibros 14:621-626.
Uttamsingh V, Gallegos R, Liu JF, Harbeson SL, Bridson GW, Cheng C, Wells DS, Graham PB,
Zelle R and Tung R (2015) Altering metabolic profiles of drugs by precision deuteration:
reducing mechanism-based inhibition of CYP2D6 by paroxetine. J Pharmacol Exp Ther
354:43-54.
Uttamsingh V, Pilja L, Grotbeck B, Brummel CL, Uddin N, Harbeson SL, Braman V, and
Cassella J (2016) CTP-656 tablet confirmed superiority of pharmacokinetic profile
relative to Kalydeco ® in Phase 1 clinical studies. J Cyst Fibros 15: S1, p. S22 (Abstr.
WS13.6)
Van Goor F, Hadida S, Grootenhuis PD, Burton B, Cao D, Neuberger T, Turnbull A, Singh A,
Joubran J, Hazlewood A, Zhou J, McCartney J, Arumugam V, Decker C, Yang J, Young
C, Olson ER, Wine JJ, Frizzell RA, Ashlock M and Negulescu P (2009) Rescue of CF
airway epithelial cell function in vitro by a CFTR potentiator, VX-770. Proc Natl Acad Sci
U S A 106:18825-18830.
Van Goor F, Hadida S, Grootenhuis PD, Burton B, Stack JH, Straley KS, Decker CJ, Miller M,
McCartney J, Olsen ER, Wine JJ, Frizzell RA, Ashlock M and Negulescu PA (2011)
Correction of F508del-CFTR protein processingg defect in vitro by the investigational
drug VX-809. Proc Natl Acad Sci U S A 108: 18843-18848.
27

JPET Fast Forward. Published on June 13, 2017 as DOI: 10.1124/jpet.117.241497
This article has not been copyedited and formatted. The final version may differ from this version.
JPET #241497
Wainwright CE, Elborn JS, Ramsey BW, Marigowda G, Huang X, Cipolli, Colombo C, Davies
JC, De Boeck, K, Flume PA, Konstan MW, McColley SA, McCoy K, McKone EF, Munck
A, Ratjen F, Steven M. Rowe SD, Waltz D, and Boyle, MP (2015) Lumacaftor–Ivacaftor
in Patients with Cystic Fibrosis Homozygous for Phe508del CFTR. N Engl J Med 373:
220-231.
Yang X, Hadida Ruah, S, Grootenjuis, P, Van Goor FF, Botfield, M, and Zlokarnik, G (2010)
Modulators of cystic fibrosis transmembrane conductance regulator. International
Publication Number WO 2010/108155.
Zha J, Zhang J and Ordonez C (2011) Pharmacokinetics (PK), metabolism and excretion of
[
14C] VX-770, a potentiator of cystic fibrosis transmembrane conductance regulator
CFTR), in healthy subjects. J Clin Pharmacol 51:1358-1359 (Abstr. 1124131).
(
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Footnotes
*
Research supported by Concert Pharmaceuticals, Inc., Lexington, Massachusetts.
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Figure Legends
9
Figure 1. Structures of CTP-656 (d -ivacaftor), d18-ivacaftor and ivacaftor. D = deuterium atom.
Figure 2. CTP-656 and ivacaftor are metabolized in humans by CYP3A to two major
metabolites.
Figure 3. In Vitro metabolism. (A) t1/2 values of ivacaftor and d-ivacaftor analogs in human
CYP3A4 supersomes. (B) Deuterium kinetic isotope effect as measured by rate of formation of
M1 and d -M1 metabolites in human liver microsomes.
8
Figure 4. In Vivo metabolism. (A) Exposure profiles in male Sprague-Dawley rats (n=3/group)
following oral dosing of CTP-656, d18-ivacaftor and ivacaftor. (B) Exposure profiles in male
Beagle dogs (n=4) following oral dosing of CTP-656, d18-ivacaftor and ivacaftor in a crossover
study.
Figure 5. Exposure profiles in healthy volunteers (n=6) following oral dosing of CTP-656 and
d18-ivacaftor.
30