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Title: Metabolism and Disposition of Pan-Genotypic Inhibitor of HCV NS5A
Ombitasvir in Humans
Authors: Jianwei Shen, Michael Serby, Bruce Surber, Anthony J. Lee, Junli Ma, Prajakta Badri,
Rajeev Menon, Olga Kavetskaia, Sonia M. de Morais, Jens Sydor, Volker Fischer
Drug Metabolism and Pharmacokinetics, Research & Development (J-W.S., M.S., A.J.L., J.M.,
P.B.), AbbVie, 1 N. Waukegan Road, North Chicago, IL 60064
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S.M., V.F.); Process Chemistry (B.S.); Drug Analysis (O.K., J.S.); CPPM-Clinical PK/PD (R.M.,
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Running Title
a) Metabolism and disposition of [14C]ombitasvir in humans.
b) Address Correspondence to: Dr. Jianwei Shen, Drug Metabolism, AbbVie, 1 N. Waukegan
Rd., North Chicago, IL 60064. Email: [email protected]
c) Number of text pages - 26
Number of tables - 6
Number of figures - 9
Number of words in the Abstract -263
Number of words in the Introduction -596
Number of words in the Discussion -768
d) ABBREVIATIONS:
HCV, hepatitis C virus; DAAs, direct-acting antiviral agents; SVR, sustained virologic response;
IFN, interferon; AUC, area under the curve; BID, twice a day; QD, once daily; CYP, cytochrome
P450; CID, collision- induced dissociation; LSC, liquid scintillation counting; HPLC, highperformance liquid chromatography; SPE, solid phase extraction; ABT-267, ombitasvir, ABT450, paritaprevir; paritaprevir/r, paritaprevir/ritonavir; ABT-333, dasabuvir.
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Number of reference - 23
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Abstract
Ombitasvir (also known as ABT-267) is a potent inhibitor of hepatitis C virus (HCV)
nonstructural protein 5A (NS5A), which has been developed in combination with paritaprevir /
ritonavir and dasabuvir in a three direct-acting antiviral oral regimen (DAAs) for the treatment of
patients infected with HCV genotype 1. This article describes the mass balance, metabolism and
disposition of ombitasvir in humans without co-administration of paritaprevir/ritonavir and
healthy male volunteers, the mean total percentage of the administered radioactive dose
recovered was 92.1% over the 192-hour sample collection in the study. The recovery from the
individual subjects ranged from 91.4 to 93.1%. Ombitasvir and corresponding metabolites were
primarily eliminated in feces (90.2% of dose), mainly as unchanged parent drug (87.8% of dose),
but minimally through renal excretion (1.9% of dose). Biotransformation of ombitasvir in
human involves enzymatic amide hydrolysis to form M23 (dianiline) which is further
metabolized through CYP-mediated oxidative metabolism (primarily by CYP2C8) at the tertbutyl group to generate oxidative and/or C-desmethyl metabolites. [14C]Ombitasvir, M23, M29,
M36 and M37 are the main components in plasma, representing about 93% of total plasma
radioactivity. The steady-state concentration measurement of ombitasvir metabolites by LC-MS
analysis in human plasma following multiple dose of ombitasvir, in combination with
paritaprevir/ritonavir and dasabuvir confirmed that ombitasvir is the main component (51.9% of
all measured drug related components), while M29 (19.9%) and M36 (13.1%) are the major
circulating metabolites. In summary, the study characterized ombitasvir metabolites in
circulation, the metabolic pathways, and the elimination routes of the drug.
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dasabuvir. Following the administration of a single 25-mg oral dose of [14C]ombitasvir to four
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Introduction
Hepatitis C virus (HCV) infection affects approximately 170 million individuals worldwide
(WHO, 2011). Untreated chronic HCV infection can result in cirrhosis or hepatocellular
carcinoma, both of which are leading causes of liver transplantation (Pawlotsky, 2004; Lavanchy,
2011; Mohd Hanafiah et al., 2013). Recently, several interferon (IFN)-free combinations of
direct-acting antivirals (DAAs) have been developed to cure chronic hepatitis C virus (HCV)
developed for the HCV genotype-1 infection in combination with an NS3 protease inhibitor
paritaprevir with ritonavir (r) and/or an NS5B non-nucleoside polymerase inhibitor (dasabuvir)
with or without ribavirin (RBV) (Feld et al., 2014; Ferenci et al., 2014; Kowdley et al., 2014;
Poordad et al., 2014). Ombitasvir is an inhibitor of HCV nonstructural protein 5A (NS5A)
(DeGoey et al., 2014; Krishnan et al., 2015). Ombitasvir exhibited picomolar activities against
HCV genotype 1a and 1b subgenomic replicons in vitro, with EC50 values of 14 and 5 pM,
respectively. Ombitasvir also demonstrated robust in vivo responses with mean maximum
decreases in HCV RNA up to 3.10 log10 IU/mL following 3-day monotherapy in treatmentnaïve HCV genotype-1 infected subjects (Lawitz et al., 2012).
Clinically, ombitasvir has favorable safety, tolerability and pharmacokinetic profiles when given
as a monotherapy or in combination therapy at doses administered to date (Dumas et al., 2011;
Menon et al., 2012). Ombitasvir shows linear pharmacokinetics with dose-proportional increases
in exposure over the range of 5 mg to 100 mg after once daily multiple-dose administration.
Ombitasvir has a t1/2 of approximately 24 hours when administered once daily. The mean Cmax
and AUC0-24h values of ombitasvir were 27% and 62% higher, respectively, on Day 10 compared
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infection with high success rates (Shah et al., 2013; Zeuzem, 2014). Ombitasvir has been
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to Day 1, following 5 mg to 200 mg QD multiple doses, suggesting minimal accumulation
(Dumas et al., 2011).
This report describes the metabolism, and disposition of a single 25-mg oral dose of
[14C]ombitasvir in four healthy human subjects. The purpose of this study was to assess the
mass balance, elucidate the routes and rates of excretion, identify and quantify the exposure of
circulating metabolites in human plasma, elucidate the metabolite structures, determine the
the circulating metabolites of ombitasvir at steady-state in the 3DAA regimen
(paritaprevir/ritonavir (r), dasabuvir and ombitasvir) were assessed and mechanisms of
metabolite formation are also described.
Materials and Methods
Drugs and Reagents
Ombitasvir, dimethyl ((2S,2'S)-((2S,2'S)-2,2'-(((((2S,5S)-1-(4-(tert-butyl)phenyl)pyrrolidine-2,5diyl)bis(4,1-phenylene))bis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(3-methyl-1oxobutane-2,1-diyl))dicarbamate; M23, 4,4'-((2S,5S)-1-(4-tert-butylphenyl)pyrrolidine-2,5diyl)dianiline; M29, 1-(4-((2S,5S)-2,5-bis(4-aminophenyl)pyrrolidin-1-yl)phenyl)ethanone; M36,
1-(4-((2S,5S)-2,5-bis(4-aminophenyl)pyrrolidin-1-yl)phenyl)-2-hydroxyethanone, these
reference standards were supplied by Process Chemistry, AbbVie, Inc (North Chicago, IL) and
were used as HPLC and mass spectrometric standards. [14C]Ombitasvir was supplied by Process
Chemistry, AbbVie, Inc (North Chicago, IL). The chemical structure of [14C]ombitasvir is
shown in Fig 1, (*) denotes the [14C]label position. The radiochemical synthesis of
[14C]ombitasvir started from para-nitroacetophenone[carbonyl-14C] ([14C]4'-NAP) and was
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metabolite profiles in excreta and the metabolic pathway of ombitasvir in humans. In addition,
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completed in six steps. Purification of the compound by crystallization provided >99%
radiochemical purity by HPLC.
Clinical Study
The clinical study was conducted at Covance Laboratories Inc., in conjunction with the Covance
Clinical Research Unit (Madison, WI). In this open-label study, a total of four adult male
subjects (N = 4) in general good health were selected to participate in the study according to the
[14C]ombitasvir under non-fasting conditions. The study drug, ombitasvir (25 mg active, 100
microcuries (μCi)), was administered as a single liquid filled capsule. The total amount of liquid
taken was approximately 240 mL, 30 minutes after starting a standardized breakfast. Subjects
were confined to the study site for a minimum of 192 hours, post-dose, or up to a maximum of
360 hours, post-dose. Subjects were released from the study site at any time after 192 hours post
dose if the preset release criteria were met.
Blood samples were collected by venipuncture into vacutainer collection tubes containing
potassium (K2) EDTA at the following times: 0 hour (predose), 1, 2, 4, 6, 8, 10, 12, 24, 48, 72,
96, 120, 144, 168, 192 hours after dosing of [14C]ombitasvir on day 1. Plasma was separated via
centrifugation and stored at –70°C.
Urine samples were collected over the following intervals: 0 to 12, 12 to 24, 24 to 48, 48 to 72,
72 to 96, 96 to 120, 120 to 144, 144 to 168, 168 to 192 hours after dosing of [14C]ombitasvir on
Study Day 1. Urine samples were collected into a collection jar containing approximately 1.2
grams of dodecylbenzenesulfonic acid (DBSA) sodium salt to minimize non-specific binding
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selection criteria. On the morning of Study Day 1, subjects received a single oral dose of
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with the container. Aliquots of the urine were frozen and maintained at –20°C prior to
metabolite profiling.
Fecal samples were collected pre-dose (upon check-in before dosing) and over the following
intervals after dosing: 0 to 24, 24 to 48, 48 to 72, 72 to 96, 96 to 120, 120 to 144, 144 to 168, 168
to 192 hours. All feces collected during a collection interval were kept frozen at -20 0C.
Total Radioactivity Measurement by Liquid Scintillation Counting
Company) and the resulting 14CO2 was trapped in a mixture of Perma Fluor and Carbo Sorb.
The oxidation efficiency was evaluated each day of sample combustion by analyzing a
commercial radiolabeled standard both directly in scintillation cocktail and by oxidation.
Acceptance criteria were defined as combustion recoveries of 95 to 105%. Ultima Gold XR
scintillation cocktail was used for samples analyzed directly. All samples were analyzed for
radioactivity in Model 2900TR liquid scintillation counters (Packard Instrument Company) for at
least 5 minutes or 100,000 counts. Each sample was homogenized and an aliquot was mixed
with scintillation cocktail before radioanalysis. All samples were analyzed in duplicate if the
sample size allowed unless the entire sample was used for analysis. If the results from sample
replicates (calculated as 14C dpm/g sample) differed by more than 10% from the mean value and
sample aliquots had radioactivity greater than 200 dpm, the sample was rehomogenized and
reanalyzed.
After mixing, duplicate blood samples were weighed (approximately 0.2 g), combusted, and
analyzed by LSC. The representative lower limit of quantitation for blood was 13.1 ng
equivalents/g. Plasma samples were mixed and duplicate weighed aliquots (approximately 0.2 g)
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All sample combustion was performed using a Model 307 Sample Oxidizer (Packard Instrument
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were analyzed directly by LSC. The representative lower limit of quantitation for plasma was
11.9 ng equivalents/g. The urine samples were mixed and duplicate weighed aliquots
(approximately 0.2 g) were analyzed directly by LSC. The representative lower limit of
quantitation for urine was 11.3 ng equivalents/g. Fecal samples were combined by subject at 24hour intervals and the weight of each combined sample was recorded. A weighed amount of
water was added and the sample was mixed. The sample was removed from the freezer and
homogenized, or immediately homogenized using a probe-type homogenizer. Duplicate
Sample Preparation for Metabolite Profiling
Plasma samples were thawed at room temperature and pooled across subjects at selected time
points in addition to AUC plasma pooling utilizing the Hamilton method (Hamilton et al., 1981).
Plasma samples were processed using a solvent extraction method. In brief, pooled plasma was
extracted with a four-fold volume of acetonitrile/methanol mixture (3:1, v/v), followed by
vortexing and sonication. The sample was then centrifuged at 3000 rpm (2465 x g) for 15 min at
4oC. The supernatant was transferred to a glass tube. The protein pellets were extracted
sequentially four times, each using two-fold the original sample volume of acetonitrile/methanol
mixture (3:1, v/v), with vortexing and sonication (15 min). After combining the supernatants
100 µL of formamide was added and the solution was concentrated to ~ 100 µL under a stream
of nitrogen. The residues were diluted with 75 µl of acetonitrile/methanol mixture (3:1, v/v) and
150 µl of water before HPLC-MS-radiochemical detection analysis. An aliquot of the
reconstituted sample was subjected to LSC analysis to determine total radioactivity recovery.
Another aliquot of the reconstituted sample was transferred to an HPLC autosampler vials and
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weighed aliquots (approximately 0.2 g) were combusted and analyzed by LSC.
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was injected for HPLC-MS-radiochemical detection analysis. The mean radioactivity recovery
in the processed plasma samples was about 93.1 ± 12.7% (S.D.).
Equal volumes of urine were pooled across subjects at each time point before processing. The
pooled urine was centrifuged at 3220 x g for 15 min at 4°C. Aliquots were dried under a stream
of nitrogen at room temperature and reconstituted for metabolite profiling using HPLC-MSradiochemical detection analysis. Aliquots were also cleaned up using Strata SAX solid phase
In brief, an SPE cartridge (1 g/12 mL) was conditioned with 15 mL methanol and 15 mL
deionized water. Aliquots of pooled urine were loaded to the pre-conditioned column, followed
by washing with 10 mL of water. The elution was achieved by using 4 x 5 mL
acetonitrile/methanol mixture(3:1, v/v). The eluate was dried under the nitrogen stream at room
temperature. The residue was reconstituted in the initial mobile phase for HPLC-MSradiochemical detection analysis. The overall extraction recovery was about 59.3% ±
13.1%(S.D.).
The feces samples were processed using multiple solvent extractions with acetonitrile/methanol
mixture (3:1, v/v) using 1:3 sample:solvent ratio, followed by centrifugation at 3220 x g for 20
min at 4°C. The repeated extraction was stopped when either 80% of the radioactivity had been
recovered or until less than 2% of the radioactivity was extracted. Aliquots of extracted samples
were subjected to LSC counting for total radioactivity. The extract was dried under a nitrogen
stream at room temperature. The final residues were reconstituted in acetonitrile/methanol (3:1;
v/v) and further diluted with 30% water for HPLC-MS-radiochemical detection analysis. An
aliquot of the reconstituted solution was subjected to LSC analysis for extraction recovery
calculation. The overall extraction recovery for the fecal sample was 98.6% ± 24.1% (S.D.).
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extraction (SPE) cartridge (Phenomenex) in order to remove the detergent in the urine sample.
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Method for Metabolite Profiles and Identification
HPLC separation of ombitasvir and the corresponding metabolites was achieved using a Thermo
Accela HPLC system (Thermo Fisher, San Jose, CA), which consisted of Accela autosampler,
1250 Series binary pump and Accela PDA detector. The elution of metabolites in plasma and
urine was achieved at room temperature on an Agilent Eclipse XDB C18, 5 µm, 4.6 x 250 mm
HPLC column. Mobile phases were: A) 50 mM ammonium acetate aqueous solution, and B):
gradient was as follows: 0-3 min: 5% B; 3-10 min: 5%-40% B; 10-53 min: 40%-75% B; 53-72
min: 75%-95% B; 72-75min: 95% B; 75-76 min: 95% -5% B; 76-80 min: 5% B. The HPLC
system was interfaced with a mass spectrometer. The high resolution MS and MSn acquisitions
were performed with a Thermo Fisher Orbitrap DiscoveryTM mass spectrometer, fitted with an
ESI source (typical source parameter: sheath gas 25.0; auxiliary gas 10; ESI Source +4500 Volts;
capillary temperature 300°C; capillary voltage 43 V; tube lens 80 V). The instrument was
calibrated daily using external calibration reference compounds, with mass resolution set at
30000 for full scan and 7500 for MSn scan. Typical mass errors of analytes relative to theoretical
masses are less than ± 5 parts per million in daily operations. MS data were processed using
Thermo Xcalibur 2.10 and MetWorks 1.2 utilizing a multiple mass defect filtering (MMDF)
algorithm.
For metabolite analysis in fecal samples, separation was accomplished on an Agilent Eclipse
XDB C18, 5 µm, 4.6 x 250 mm HPLC column; mobile phases were: A) 0.1% formic acid in
water, and B): acetonitrile; the flow rate was maintained at 1.0 mL/min. The gradient was as
follows: 0-2 min: 15%-20% B; 2-5 min: 20%-30% B; 5-25 min: 30%-45% B; 25-50 min: 45%65% B; 50-53min: 65% -75% B; 53-60 min: 75% - 95% B; 60-61 min: 95% B.
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acetonitrile/methanol mixture (1:1, v/v); the flow rate was maintained at 1.0 mL/min. The
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Radioactive components in plasma, urine or feces samples were collected in TopCount 96 Deep
Well Luma Plate (Perkin Elmer, Waltham, MA) and counted by using a Perkin Elmer TopCount
NXT system. The HPLC eluent was split postcolumn between the mass spectrometer and
Agilent 1100 fraction collector at a ratio of 20:80. The Agilent 1100 fraction collector was set to
collect fractions at intervals of 0.3 min/well.
Pharmacokinetic Calculations.
SAS Institute Inc., Cary, NC). Maximum plasma concentration (Cmax), time at which Cmax was
achieved (Tmax), area under the concentration time curve from time zero to the last measurable
time point (AUC0-t) for total radioactivity, [14C]ombitasvir, and its metabolites in plasma were
estimated. Area under the concentration time curve from time zero to infinity (AUC0-∞) and
half-life (t1/2) for total radioactivity, [14C]ombitasvir in plasma were also calculated.
Metabolism of M23 by Recombinant CYP2C8.
Synthetic reference material of M23 was incubated with recombinant CYP2C8 enzyme (BD
Gentest) in the presence of NADPH. The incubation mixture (225 uL) included the substrate (10
µM final concentration), 0.1 mM phoshphate buffer pH 7.4, and recombinant CYP2C8 protein
(final concentration 100 pmol/mL). After a 5 minute pre-warm in 37°C water bath, 25 µL of 10
mM NADPH was added to initiate the reaction (NADPH final concentration 1 mM). The
samples were incubated in a 37oC water bath for 60 minutes. The reaction was stopped by
adding one volume of quenching solution (acetonitrile/methanol; 1:1, by volume) to the
incubation mixture. The mixture was centrifuged at 3220 x g for 20 min at 4°C. Aliquots of the
supernatant were subjected to HPLC-MS analysis.
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Plasma concentration-time radioactivity data were analyzed with SAS software (version 9.2;
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Human Plasma for Quantitative Analysis by LC-MS
Human plasma samples were obtained from 12 subjects in a Phase 1 open-label,
pharmacokinetics, safety and tolerability study. Healthy subjects were orally administered with
ombitasvir (25 mg tablet, QD), paritaprevir/ritonavir (150/100 mg tablet, QD) and dasabuvir
(400 mg tablet, BID) for 14 days. Blood samples for plasma concentration analysis were
collected on Day 14 at 0, 1, 2, 3, 4, 6, 9, 12, 16 and 24 hours after dosing. The plasma samples
quantitation.
LC-MS Quantitation Method for Ombitasvir and Metabolites in Plasma
A bioanalytical method was developed for the simultaneous quantitation of parent drug and the
metabolites M23, M29, M36 and M37 in human plasma. In brief, the method separated the
components of interest and internal standards from a plasma aliquot using protein precipitation
with a mixture of acetonitrile and methanol (9:1, v/v). Spiked plasma standards were analyzed
simultaneously with the samples. Parent drug, the selected metabolites and the internal standards
(D13- ombitasvir and [6-(4-fluoro-benzoyl)-1H-benzoimidazol-2-yl]-carbamic acid methyl ester)
were separated at room temperature on an Ascentis Express C18, 2.7 µm, 30 x 3 mm HPLC
column. Mobile phases were: A) acetonitrile, and B) 20 mM ammonium acetate pH 7.6. The
flow rate was maintained at 0.8 mL/min. The gradient was as follows: 0-0.2 min: 20% A; 0.20.22 min: 20%-30% A; 0.22-0.35 min: 30%-45% A; 0.35-0.9min: 45%; 0.9-0.92min: 45% 20%A; 0.92-1.1 min: 20% A. Analysis was performed on an ABSciex API5500™ Biomolecular
Mass Analyzer with a turbo-ionspray interface, with ionization of the analytes in the positive ion
mode; detection was in the multiple reaction monitoring (MRM) mode. The peak areas of all
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were pooled using an equal 100 µL volume across subjects at each time point for HPLC-MS/MS
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components of interest were determined using Sciex Analyst™ software. The concentration of
each sample was calculated by least squares linear regression analysis of the peak area ratio
(compound / internal standard) of the spiked human plasma standards versus concentration.
Results
Excretion of Radioactivity
volunteers, the excretion of radioactivity in urine and feces from all the subjects was measured
over a period of up to 192 hours post dose. Fig. 2 presents the mean cumulative recovery of total
radioactivity in excreta expressed as percentage of dose. The overall mean recovery of
radioactivity in urine and feces samples was 92.2% (± 0.82% S.D.) over the 192 hour collection
period, with recovery in individual subjects ranging from 91.4 to 93.1%. The radioactivity was
excreted primarily through fecal elimination (mean, 90.2% of dose). Renal excretion was
relatively minor (mean, 1.9% of dose).
Pharmacokinetic Data Analysis
The mean concentration - time profiles of ombitasvir and total radioactivity in human plasma
after oral administration of [14C]ombitasvir are graphically depicted in Fig. 3. The
pharmacokinetic parameters for ombitasvir and total radioactivity are summarized in Table 1.
The concentration of total radioactivity was measured by LSC, expressed as ng-equivalent/g.
The concentrations of ombitasvir were determined using a validated LC-MS/MS bioanalytical
method, expressed as ng/mL. Mean peak plasma concentrations (Cmax) for the parent drug and
total radioactivity were 27.8 ng/mL and 142.5 ng-eq/g, respectively. The AUC0-192h for the
parent drug, and total radioactivity were 359 ng•h/mL and 17411 ng-eq•h/g, respectively.
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Following a single oral dose of [14C]ombitasvir (25 mg, 100 µCi) to four healthy, male
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Metabolite Profiles of [14C]Ombitasvir in Circulation and Excreta
Plasma. A representative HPLC radiochromatogram of [14C]ombitasvir and its metabolites in
pooled human plasma using the Hamilton method (t = 0-192 h postdose) is shown in Fig. 4.
[14C]Ombitasvir and metabolites M29, M36, M37 and M23 were the main components in plasma.
In addition, M25 and M26 were also detected at low levels. The relative amounts of ombitasvir
and metabolites in human plasma, expressed as percent of radioactivity in plasma, are
16.3% and 10.0% of drug-related material in plasma, respectively. The unchanged drug
represented approximately 8.5% of radioactivity. Concentration-time profile of ombitasvir and
its metabolites is summarized in Table 3. Concentrations of metabolites are generally low,
approximately in nanomolar range.
Urine and feces. The recovered radioactivity of administered [14C]ombitasvir in urine was
relatively low. The mean cumulative recovery of the dose in the entire sample collection (0192hr post dose) is only 1.91% (±0.36). Chromatographic evaluation of selected pooled urine
samples showed several small poorly separable radiochemical peaks (Mu1 – Mu5) in the HPLC
retention time between 8-20 min. The representative HPLC radiochromatogram of pooled
human urine (48-72 h post dose) is shown in Fig. 5A. Due to the detergent interference in the
urine and low radioactive concentration of analytes, LC-MS analysis of SPE enriched urine
samples failed to provide molecular identities of these peaks. Only trace levels of
[14C]ombitasvir was observed in 12-24h pooled urine (0.03% of dose, Table 4) but not observed
in late time points.
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summarized in Table 2. Metabolites M29, M36, M37 and M23 accounted for 32.9%, 25.7%,
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The representative HPLC radiochromatogram of pooled human feces is shown in Fig. 5B.
Unchanged parent drug was the most abundant radiochemical component in feces throughout the
sample collection periods from 0-192 h post dose. The amount of ombitasvir and corresponding
metabolites in urine and feces, expressed as the mean percentage of the administered radioactive
dose, is tabulated in Table 4. The unchanged parent drug represents about 87.8% of dose,
indicating both absorbed and unabsorbed [14C]ombitasvir was mainly eliminated as unchanged
parent drug in feces. Metabolites detected in feces are very minor ( ≤1 % of total dose),
proposed metabolic scheme for ombitasvir in humans is shown in Fig. 6.
LC-MS/MS Characterization of Ombitasvir and Metabolites
As described under Method for Metabolite Profiles and Identification, metabolites of ombitasvir
were characterized using a combination of positive ionization high resolution full scan MS and
product ion scan (MS/MS) analyses. The structures of metabolites M5, M23, M29, M36, and
M37 were confirmed against the synthesized materials, while the structures of other metabolites
were proposed based on the high resolution MS/MS fragmentation pattern analysis. The
measured accurate masses and characteristic fragment ions are listed in Table 5.
Ombitasvir yielded a protonated molecular ion [M+H]+ at m/z 894.5113 (calculated mass m/z
894.5124, chemical formula C50H68N7O8+) in positive ion mode. The key MS/MS fragment ions
were m/z 737.4377, 640.3844, 588.3170, 547.3270 , 431.2433 and 334.1914 (Table 5). The CID
spectrum and detailed assignment of the fragments was provided in supplemental materials.
Metabolite M23. M23 was detected in human plasma with a protonated molecular ion at m/z
386.2587 (calculated mass m/z 386.2591, C26H32N3+). The characteristic MS/MS fragments of
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including M9 (0.7% of dose), M3 (0.6%), M2 (0.2%), M5 (0.2%), and M6 (0.2%). The
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M23 include m/z 369.1820 (-NH3), 293.2005 (loss of aniline), 237.1379 (loss of tert-butyl
aniline), 144.0802 (loss of aniline and tert-butyl aniline). Metabolite M23 was further confirmed
by comparing MS/MS fragmentation pattern and HPLC co-injection analysis using the synthetic
reference standard.
Metabolite M29. M29 was detected in human plasma by LC-MS with a protonated molecular
ion at m/z 372.2068, indicating a loss of two methyl groups and two hydrogens, plus the addition
m/z 372.2070). The characteristic fragment ions of M29 are m/z 355.2841 (loss of NH3),
279.1485 (loss of aniline), and common fragment ions at m/z 237.1379 and 144.0802 as in M23.
M29 was assigned as 2,5-bis(4-aminophenyl)pyrrolidin-1-yl)phenyl)ethanone, and was further
confirmed by comparing MS/MS fragmentation pattern and HPLC co-injection analysis using
the synthetic reference standard.
Metabolite M36. M36 was detected in human plasma by LC-MS with a protonated molecular
ion at m/z 388.2015. The measured accurate mass data suggested the molecular formula of
C24H26N3O2+ (calculated mass m/z 388.202). The major fragment ions of M36 included m/z
371.2258 (loss of NH3), 295.1435 (loss of aniline), and common fragment ions at m/z 237.1380
and 144.0802 as in M23. M36 was assigned as a hydroxylated metabolite of M29; hydroxylation
occurred at the 1-(4-aminophenyl)ethanone moiety. The structure of M36 was further confirmed
by comparing MS/MS fragmentation pattern and HPLC co-injection analysis using the synthetic
reference standard.
Metabolite M37. M37 was detected in human plasma by LC-MS with a protonated ion at m/z
390.2171, suggesting a molecular formula of C24H28N3O2+ (calculated mass: 390.2176). The
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of one oxygen atom to M23. The predicted molecular formula was C24H26N3O+ (calculated mass
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collision-induced dissociation of M37 produced major fragment ions at m/z 297.1611 (loss of
aniline), m/z 237.1380 and 144.0803. M37 is di-hydroxylated 4-ethylphenyl-pyrrolidine-2,5diyl-dianiline. The structure of M37 was further confirmed by comparing MS/MS fragmentation
pattern and HPLC co-injection analysis using the synthetic reference standard.
Metabolite M25. M25 was detected at low levels in plasma, yielded a protonated molecular ion
at m/z 418.2490. The predicted molecular formula was C26H32N3O2+ with calculated mass of m/z
237.1378 and 144.0804. M25 was tentatively assigned as tert-butyl di-hydroxyl metabolite of
M23.
Metabolite M26. M26 was detected at low levels in plasma; yielded a protonated molecular ion
at m/z 402.2538 in LC-MS, indicating addition of 16amu (+O) to M23. The predicted molecular
formula is C26H32N3O+ with calculated mass of m/z 402.2540. The collision-induced
dissociation of M26 produced major fragment ion at m/z 309.1947, 237.1377 and 144.0803. The
presence of m/z 237.1379 indicated the hydroxylation occurred at the tert-butylaniline moiety.
Therefore, M26 was assigned as a tert-butyl hydroxyl metabolite of M23.
Metabolite M28. M28 was observed as a trace metabolite in plasma; it gave a protonated
molecular ion at m/z 416.2338. The predicted molecular formula is C26H30N3O2+ (calculated
mass: 416.2333). The collision-induced dissociation of M28 produced major fragment ions at
m/z 237.1382 and 144.0805. Metabolite M28 was tentatively assigned as tert-butyl carboxylic
acid metabolite of M23.
Metabolite M34. M34 was detected as a trace metabolite in plasma; it gave a protonated
molecular ion at m/z 388.2383, suggesting a molecular formula of C25H30N3O+ (calculated mass:
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418.2489. The collision-induced dissociation of M25 produced major fragment ions at m/z
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388.2383). The collision-induced dissociation of M34 generated major fragment ions at m/z
371.2265 (loss of NH3), 237.1379 and 144.0801. M34 was tentatively assigned as tert-butyl
demethylation and hydroxylation metabolite of M23.
Metabolite M35. M35 was detected as a trace metabolite in plasma; it produced a protonated
molecular ion at m/z 404.2330, suggesting a molecular formula of C25H30N3O2+ (Calculated mass:
404.2333). The collision-induced dissociation of M35 generated major fragment ions at m/z
hydroxylation metabolite of M23.
Metabolite M5. M5 was present as low level in both plasma and feces; it gave a protonated
molecular ion at m/z 910.5056, suggesting an addition of oxygen to parent drug. Due to low ion
intensity, no MS/MS spectrum was obtained. M5 was tentatively assigned as a hydroxylation
metabolite of parent drug.
Metabolite M6. Metabolite M6 was present at a trace level in both plasma and feces, gave a
protonated molecular ion at m/z 640.3856, suggesting a molecular formula of C38H50N5O4+
(calculated mass: 640.3857). The collision-induced dissociation of M6 generated major
fragment ions at m/z 547.3279 (loss of aniline), 491.2649 (loss of tert-butyl aniline), 334.1912
(further cleavage of aniline amide from 491), and 255.1336 (aniline amide cleavage ). M6 was
assigned as a monohydrolysis product of the parent drug.
Metabolite M7. M7 was only detected by LC-MS and gave a protonated molecular ion at m/z
273.1441, suggesting a molecular formula of C12H21N2O5+ (calculated mass: 273.1445). The
collision-induced dissociation of M7 generated major fragment ions at m/z 255.1337, 227.1380
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237.1382 and 144.0800. M35 was tentatively assigned as tert-butyl demethylation and di-
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and 116.0703. M7 was confirmed by co-injection HPLC-MS analysis using reference standard
of (S)-1-((S)-2-(methoxycarbonylamino)-3-methylbutanoyl)pyrrolidine-2-carboxylic acid.
Metabolite M9. M9 was a trace metabolite present in both plasma and feces; it gave a
protonated molecular ion at m/z 912.5224, suggesting an addition of water to parent drug, with
predicted molecular formula of C50H70N7O9+ (calculated mass: 912.5230). The collision-induced
dissociation of M9 generated major fragment ions at m/z 755.4494, 738.4403, 640.3842,
parent drug.
Trace levels of M2 (measured m/z 910.5075) and M3 (measured m/z 910.5075) were also
detected in fecal samples. Due to their overall low abundance in feces (each <1% of dose, Table
4), no further characterization was performed. M2 and M3 were assigned as hydroxylated
metabolites of parent drug.
Quantification of Ombitasvir Metabolites following Multiple Oral Dosing in Human
As ombitasvir is not intended for use as a single agent, the metabolic profile of ombitasvir in
human plasma was further investigated at steady state, following administration of 3DAA
combination. Plasma samples obtained from 12 healthy subjects, following 14 days of dosing
with ombitasvir (25 mg QD), administered in combination with paritaprevir/ritonavir (150/100
mg QD) and dasabuvir (400 mg, BID), were pooled. Concentrations of M23, M29, M36 and
M37 were measured against synthetic reference standards. The measured concentrations and
AUCs of these metabolites are listed in Table 6. Unchanged parent drug was the major
component in plasma, with a Cmax of 125 ng/ml and an AUC0-24 of 1745 nghr/ml. Two
downstream metabolites, M29 and M36, provided Cmax values more than 4-fold lower (31.4 and
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547.3271, 491.2643 and 334.1904. M9 was tentatively assigned as a hydration metabolite of
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23.0 ng/ml, respectively), with correspondingly lower AUC values (669 and 442 nghr/ml,
respectively). Plasma concentrations of M37 (Cmax 16.1 ng/ml; AUC 312 nghr/ml) and M23
(Cmax 8.6 ng/ml; AUC 194 nghr/ml) were even lower. Parent drug accounted for 51.9% of the
drug related material, followed by M29 (19.9%), M36 (13.1%), M37 (9.3%) and M23
(5.8%).The same set of plasma samples was also pooled using the Hamilton method, extracted
and analyzed using a high resolution mass spectrometer for metabolite profiling (Fig. 7).
Qualitatively similar metabolites were detected in the plasma following multiple doses of
identified.
Metabolism of M23 by Recombinant CYP2C8
Since the enzymatic amide hydrolysis product M23 is a precursor to M29 and M36 , the
metabolic pathway of M23 was characterized using an in vitro hepatic system. In vitro
cytochrome P450 reaction-phenotyping indicated that CYP2C8 is the primary enzyme to
metabolize M23. Fig. 8 showed the extracted ion chromatogram of downstream metabolites of
M23 following in vitro incubation of M23 in recombinant CYP2C8 enzyme. At least eleven
downstream metabolites of M23 were identified. M28 (tert-butyl acid), M25 (tert-butyl dihydroxyl) and M29 are the most abundant components in HPLC-MS analysis. The proposed
metabolic pathway of M23 by CYP2C8 was illustrated in Fig. 9. M23 undergoes a tert-butyl
hydroxylation (Rodrigues et al., 1995; Weber et al., 1999; Polsky-Fisher et al., 2006; Prakash et
al., 2008) to form M26, followed by further oxidation to generate oxidative and/or desmethyl
metabolites such as M25, M29, M34, and M35. The uncommon metabolic pathway involving
oxidation at the tert-butyl group followed by C-demethylation has been reported previously
(Prakash et al., 2008; Yoo et al., 2008). Although the exact chemical mechanism for the carbon20
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ombitasvir with paritaprevir/ritonavir and dasabuvir, and no additional new metabolites were
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cleavage reaction from M23 to M29, M36 or other demthylated metabolites is yet to be further
characterized, it is likely that it involves a similar reaction mechanism postulated by Prakash and
co-workers showed (Prakash et al., 2008) that C-demethylation may involve the oxidation of the
tert-butyl alcohol to form an aldehyde metabolite, followed by P450-mediated deformylation to
lose formic acid, to produce an unstable carbon-centered radical which reacts with water to form
downstream demethylated metabolites.
The mass balance, disposition, and metabolism of ombitasvir were evaluated in four healthy
human subjects. Following the administration of a single 25-mg oral dose of [14C]ombitasvir,
the mean total recovery of the administered radioactive dose was 92.2% (± 0.82% S.D.), with
recovery in individual subjects ranging from 91.4 to 93.1%. Nearly all of the administered
radioactive dose (90.2% of dose) was recovered in feces, while a very limited amount of
radioactivity (1.9%) was recovered in urine through the last collection interval, indicating that
ombitasvir and metabolites were predominantly eliminated in humans through feces and
minimally through renal clearance.
Metabolites of ombitasvir in plasma, urine and feces were profiled using HPLC-radioactivity
detection and structures of metabolites were characterized using HPLC-high resolution mass
spectrometry. Of the total radioactivity excreted in human feces, unchanged parent drug
constituted 87.8% of total dose, and metabolites M2, M3, M5, M6 and M9, each accounting only
for < 1% of total dose. In urine, only trace levels of parent drug and several small polar
components (Mu1-Mu5) were present. In human plasma, [14C]ombitasvir, M23, M29, M36 and
M37 are the main components after a single 25-mg dose of [14C]ombitasvir alone, representing
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Discussion
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about 93% of total plasma radioactivity, with at least nine additional metabolites, M5, M6, M7,
M9, M25, M26, M28, M34 and M35, observed at either minor or trace levels. Similar to
preclinical toxicology species, biotransformation of ombitasvir in humans primarily involves
enzymatic amide hydrolysis at the aniline amide linker to generate metabolite M6 (mono-aniline),
M7 (pyrrolidine acid) and M23. It is not clear what enzymes are involved in the amide
hydrolysis and where the process occurs. Only trace levels of metabolite M6 and M7 were
produced in in vitro hepatocytes or liver microsomes across species (Abbvie unpublished data).
group to generate oxidative and/or C-desmethyl metabolites such as M26 (hydroxy tert-butyl
dianiline), M25 (dihydroxy tert-butyl dianiline), M34 (tert-butyl desmethyl hydroxy dianiline),
M35 (tert-butyl desmethyl dihydroxy dianiline), M36 (hydroxyacetophenone dianiline), M37
(tert-butyl desmethyl dihydroxy dianiline) and M29 (acetophenone dianiline).
Based on the assessment of metabolite exposures at steady state, M29 and M36 were defined as
major circulating metabolites, representing 19.9% and 13.1%, respectively, of the total drug
related material in plasma, while the parent drug accounted for 51.9% of the total drug-related
material. M29 and M36 are downstream metabolites of M23 which is present in preclinical
species at higher levels than in humans, providing safety coverage in all toxicology species.
M29 and M36 have not been observed in studies using animal and human-derived hepatic in
vitro systems, in plasma, or excreta of in vivo preclinical animals used in ADME or toxicology
studies. These two metabolites were further characterized independently(i.e., not combined), and
proved to be negative in in vitro Ames tests and chromosomal aberration assay. There were no
adverse findings in independent four-week repeat-dose and embryo-fetal developmental toxicity
studies in mice with M29 and M36.
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In humans, M23 further undergoes CYP2C8-mediated oxidative metabolism at the tert-butyl
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While the ICH M3 R2 and FDA Guidance on Safety Testing of Metabolites focus on the relative
abundance of metabolites, there has been considerable emphasis on the fact that absolute
exposures (as circulating concentrations or total body burden) of metabolites need to be taken
into consideration, especially for drugs at low doses (Smith et al. 2008). These disproportionate
metabolites M29 and M36 are present at low nanomolar plasma concentrations (average
concentration 17–31 ng/mL) in humans receiving a 25-mg dose of ombitasvir as a part of the
3DAA treatment regimen. They are highly bound to plasma proteins, not active against HCV
pharmacologic activity.
Clinical drug-drug interactions of the complete 3DAA regimen including ombitasvir have been
extensively characterized and summarized elsewhere (Menon et al., 2015). The disproportional
metabolites M29 and M36 were de facto tested as part of ombitasvir administration. No safety
findings were attributed to ombitasvir and its metabolites. Detailed in vitro studies to profile
CYP450 enzymes and drug transporters for ombitasvir and other DAA components have been
conducted (Shebley et al., 2016), and physiologically based pharmacokinetic models were
established to provide mechanistic understanding of potential DDIs. In summary, the overall
disposition and metabolism of 25-mg [14C]ombitasvir in healthy volunteers was investigated.
The overall study objectives were met, with good recovery of radioactivity dose from all subjects.
The mass balance results confirm that orally administered ombitasvir is primarily eliminated by
the biliary-fecal route. The metabolite structures of the main circulating metabolites were
elucidated, with a proposed metabolic pathway of enzymatic amide hydrolysis of ombitasvir
followed by tert-butyl hydroxylation of M23 to generate secondary oxidative or C-demethylation
metabolites.
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replicons in vitro and are not expected to have clinically relevant on-target or off-target
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Acknowledgements: Special thanks to Anthony R. Haight, Benoit Cardinal-David, Shashank
Shekhar and Brian Kotecki for preparation of M29 and M36 reference materials.
Authorship Contributions
Participated in research design: Shen, Menon, Kavetskaia, Fischer.
Conducted experiments: Serby, Ma.
Contributed new reagents or analytic tools: Serby, Surber.
Wrote or contributed to the writing of the manuscript: Shen, Kavetskaia, Lee, de Morais, Sydor,
Fischer, Serby, Ma, Badri , Menon, Surber.
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Performed data analysis: Shen, Serby, Ma, Badri , Menon.
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Unnumbered Footnote to the Title
Disclosure Statement: The design, study conduct, and financial support for this study were
approving the publication. All authors are current employees of AbbVie, except Olga
Kavetskaia who was an AbbVie employee at the time the manuscript was developed (her current
affiliate is: Global Clinical Pharmacology, Pfizer, Groton, CT).
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provided by AbbVie. AbbVie participated in the interpretation of data, writing, review and
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Figures
Fig. 1. Structure of [14C]ombitasvir. Asterisk denotes position of [14C] radiolabel.
Fig. 2. Mean cumulative percent of radioactive dose recovered in urine and feces at specified
intervals after a single 25-mg (100-µCi) oral dose of [14C]ombitasvir to healthy male subjects.
Fig. 3. Mean (standard deviation) plasma concentration-time curves for ombitasvir (ng/mL) and
total radioactivity (ng-eq/g) in male subjects administered a single oral dose of [14C]ombitasvir
Fig. 4. Representative HPLC radiochromatogram (A) and HPLC extracted ion chromatogram (B)
of ombitasvir and its metabolites in AUC(0-192h) pooled human plasma after a single 25-mg oral
dose of [14C]ombitasvir.
Fig. 5. Representative HPLC radiochromatograms of ombitasvir and its metabolites in human
excreta, (A) urine and (B) feces, after a single 25-mg oral dose of [14C]ombitasvir.
Fig. 6. Proposed metabolic pathways of ombitasvir in humans.
Fig. 7. HPLC MS extracted ion chromatogram of metabolites generated from pooled human
plasma following oral administration of ombitasvir (25mg once daily), paritaprevir/r (150/100
mg once daily) and dasabuvir (400mg twice daily) for 14 days.
Fig. 8. HPLC MS extracted ion chromatogram of metabolites generated from in vitro incubation
of M23 in human recombinant CYP2C8 enzymes.
Fig. 9. Proposed metabolic pathways of M23 mediated by human CYP2C8.
30
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25-mg (n=4).
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Tables
Table 1. Mean ± SD pharmacokinetic parameters of total radioactivity and ombitasvir
Analyte
Cmax
(ng-eq/g or
ng/mL)
Tmax (h)
AUC0-last
(ng-eq●h/g or
ng●h/mL)
AUC0-∞
(ng-eq●hr/g or
ng●h/mL)
Total
Radioactivity
142.5 ± 14.3
24 ± 0
17411 ± 1704
30470 ± 6568
Ombitasvir
27.8 ± 6.08
4.0 ± 0
359 ± 63.9
366 ± 63.1
T1/2 (h)
30.3 ± 9.80
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31
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Table 2. Percentages of radioactivity for ombitasvir and its metabolites in pooled human
plasma (n=4, AUC0-192h) following administration of a single 25-mg oral dose of
[14C]ombitasvir (n=4)
Percentage of Radioactivity in Plasma
8.5
M37
16.3
M36
25.7
M25
3.1
M29
32.9
M26
3.4
M23
10.0
32
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Ombitasvir
DMD Fast Forward. Published on May 13, 2016 as DOI: 10.1124/dmd.115.067496
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Table 3. Concentration (ng-eq/g) - time profile of [14C]ombitasvir metabolites in pooled
plasma at selected time point across subjects (n=4)
Radioactive Concentration (ng-eq/g)
A-1233617
M37
M36
M25
M29
M34
M26
M23
M5
2-4hr
38.2
5.7
8.5
0.0
11.4
0.0
0.0
5.7
5.1
4-6hr
51.4
8.6
11.8
0.0
20.4
0.0
0.0
7.5
5.4
6-8h
29.2
7.5
13.2
5.5
29.4
0.0
6.0
21.0
0.0
8-10hr
36.3
9.4
14.3
6.3
27.9
3.1
3.9
16.7
3.1
12-24hr
25.5
15.9
22.1
5.8
32.7
3.9
7.7
28.4
0.0
24-48h
2.5
12.1
23.0
3.4
37.9
0.0
5.3
29.8
0.0
48-72h
3.4
17.6
22.7
7.8
37.3
0.0
4.4
8.8
0.0
72-96hr
5.5
13.5
22.6
0.0
27.7
4.4
3.3
10.9
0.0
96-120h
2.1
10.8
20.0
3.1
30.1
0.0
2.6
10.4
0.0
120-144h
2.6
11.2
18.1
3.0
25.0
0.0
3.3
6.6
0.0
144-168h
2.2
11.3
15.9
2.7
19.3
0.0
3.2
7.8
0.0
168-192 h
3.8
13.6
15.3
2.7
16.4
0.0
0.0
6.5
0.0
33
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Time
DMD Fast Forward. Published on May 13, 2016 as DOI: 10.1124/dmd.115.067496
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Table 4. Percentages of excretory metabolites of ombitasvir in humans following
administration of a single 25-mg oral dose of [14C]ombitasvir (n=4)
Compound
M2
M9
M3
M6
M5
Uf1-3a
Mu1-5b
Feces
0-192h*
87.8
0.2
0.7
0.6
0.2
0.2
0.6
-
Urine
**
0.03
-
-
-
-
-
-
0.57
87.83
0.2
0.7
0.6
0.2
0.2
0.6
0.57
Subtotal
*
Ombitasvir
Sum of radioactivity dose recovery from 0-192 hr pooled feces.
** Sum of radioactivity dose recovery from 12-24 hr, 48-72 hr and 168-192 hr pooled urines.
Uf = unknown metabolites in feces. Uf1-3 is the combined radioactivity dose recovery for Uf1, Uf2 and Uf3.
Mu = unknown metabolites in urine. Mu1-5 is the combined radioactivity dose recovery for Mu1, Mu2, Mu3, Mu4 and
Mu5.
34
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a
b
DMD Fast Forward. Published on May 13, 2016 as DOI: 10.1124/dmd.115.067496
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Table 5. Molecular ions and characteristic fragment ions of ombitasvir and metabolites in
human plasma, urine or feces
Compound
Ombitasvir
M6
[M+H]+
(Measured)
[M+H]+
(Theorectical)
894.5113
894.5124
640.3856
640.3857
Chemical
formula
Δppm
C50H68N7O8+
-1.2
+
-0.2
C38H50N5O4
+
M9
912.5224
912.5230
C50H70N7O9
+
M3
910.5055
910.5073
C50H68N7O9
+
M23
910.5056
386.2587
910.5073
386.2591
C50H68N7O9
-1.9
+
-1.0
C26H32N3
+
M26
402.2538
402.2540
C26H32N3O
+
M34
388.2383
388.2383
C25H30N3O
+
M29
372.2068
372.2070
C24H26N3O
+
M25
418.2490
418.2489
C26H32N3O2
+
M28
416.2338
416.2333
C26H30N3O2
+
M36
388.2015
388.2020
C24H26N3O2
+
M35
404.2330
404.2333
C25H30N3O2
+
M37
390.2171
390.2176
M7
273.1441
273.1445
-1.9
-0.5
0.0
-0.5
0.2
1.2
-1.3
-0.7
C24H28N3O2
-1.3
C12H21N2O5+
-1.5
35
737, 640, 588, 547, 431
547, 491, 473, 334, 255
894, 755, 640, 547, 491, 334
892, 737, 588, 431
Accurate mass only
369, 293, 237, 144
370, 309, 237, 144
371, 237, 144
355, 279, 237, 144
237, 144
237, 144
371, 295, 237, 144
237, 144
297, 237, 144
255, 227, 116
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M5
-0.7
Key Fragment Ions (m/z)
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Table 6. Estimated Relative Amounts (%AUCt) of Ombitasvir and its Metabolites in
Human Plasma following Multiple Doses of ombitasvir
Multiple Dose (Steady-State)
3DAA combo regimen*
AUCt (ng●hr/mL)**
Compound
% Total AUCt
Cmax (ng/mL)
Ombitasvir
51.9
1745
125
M23
5.8
194
9
M29
19.9
669
31
M36
13.1
442
23
M37
9.3
312
16
**Pharmacokinetic samples were collected on Day 14 up to 24 hours and pooled across the subjects for quantitative HPLCMS/MS analysis.
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* 3 DAAs regimen (paritaprevir/ritonavir (150/100 mg), ombitasvir (25 mg) and dasabuvir (400 mg twice daily)) was
administered to healthy human subjects for 14 days.
Fig. 1.
Ombitasvir
Fig. 2
100
% of Dose
80
Urine
60
Feces
Total
40
20
0
0
24
48
72
96 120 144 168 192
Time (h)
Fig. 3
Concentration (ng/mL or ng-equiv/g)
Plasma Total Radioactivity (ng-equiv/g)
1000
Plasma ombitasvir (ng/mL)
100
10
1
0.1
0.01
0
24
48
72
96 120 144 168 192
Time (h)
Fig. 4
A)
CPM
60
Plasma
AUC0-192h
M29
M36
M37
40
M25
M26
M23
20
0
10
20
30
RT: 0.21 - 78.71
60
70
80
M29
100
90
40
50
R T ( m in )
Ombitasvir
B)
80
70
50
40
30
M23
M25
60
M36
Ombitasvir
M26
M37
20
10
0
10
20
30
40
Time (min)
50
60
70
Fig. 5
(A) Pooled Urine
12-24 h
40
Mu3
CPM
30
Mu4
Mu2
Mu5
20
Ombitasvir
Mu1
10
0
0
10
20
30
40
50
60
70
80
RT (min)
10000
CPM
8000
(B) Pooled feces
48-72 h
Ombitasvir
6000
300
M5
200
UNK
100
0
0
10
20
M3M9
M6
M2
30
40
50
60
RT (min)
Note – Mu stands for uncharacterized urinary metabolites
Fig. 6
OH
OH
N
N
N
tion
O
O
oxyla
hydr
O N
N
N
O
O
N
O
N
O
O
O
O
N O
M5
O
N
O
N
N
N
N
O
N
N
O
O
N O
N
M3
O
O
N
O
O
N O
N
ombitasvir
ABT-267
O
O
N
N
O
O
amide hydrolysis
N
O
N
N
O
HO
N
OH
O
N O
M9
O
OH
O
H2 N
OH
OH
N
O
O
oxidation
O
N
N
N
O
O
O
M7
NH2
N
NH2
O
N
H2N
H2N
NH2
N
N
M6
O
NH2
M25
oxidation
COOH
N
M23
M26
H2N
NH2
N
oxidation/C-demethylation
M28
OH
H2N
NH2 H2N
N
M34
O
OH
HO
NH2 H2N
O
HO
NH2
H2 N
OH
HO
NH2
H2N
NH2
N
N
N
N
M35
M29
M36
M37
Fig. 7
RT: 0.00 - 79.99 SM: 3B
100
ombitasvir
90
80
70
60
M29
50
M36
40
M7 M37
M25
30
M23
M9M6
M26
20
10
0
0
10
20
30
40
Time (min)
50
60
70
Fig. 8.
M28
RT : 0.00 - 79.99
100
90
80
M25
70
M36
60
M29
50
M38/M39
40
30
M35
M33
M34
M26
M23
20
M37
10
0
0
10
20
30
40
T ime (min)
50
60
70
Fig. 9
OH
OH
H2N
OH
M25
Oxidation
H2N
NH2
N
H2N
H2 N
O
NH2
O
H 2N
M26
M28
OH
M35
H2 N
O
OH
HO
M34
NH2 H2N
N
NH2
N
Oxidation/C-demethylation
M33
OH
NH2
N
M23
NH2
N
M29
NH2
N
H 2N
NH2
N
HO
HO
O
OH
M36
M37
H2 N
N
NH2
H 2N
OH
O
M39
N
NH2
H 2N
OH
M38
N
NH2 H2N
N
NH2