dmd # 67496 - drug metabolism and...

DMD # 67496

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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.,

S.M., V.F.); Process Chemistry (B.S.); Drug Analysis (O.K., J.S.); CPPM-Clinical PK/PD (R.M.,

P.B.), AbbVie, 1 N. Waukegan Road, North Chicago, IL 60064

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on May 13, 2016 as DOI: 10.1124/dmd.115.067496

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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 reference - 23

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, high-

performance liquid chromatography; SPE, solid phase extraction; ABT-267, ombitasvir, ABT-

450, paritaprevir; paritaprevir/r, paritaprevir/ritonavir; ABT-333, dasabuvir.


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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

dasabuvir. Following the administration of a single 25-mg oral dose of [14C]ombitasvir to four

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 tert-

butyl 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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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)

infection with high success rates (Shah et al., 2013; Zeuzem, 2014). Ombitasvir has been

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 treatment-

naïve 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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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

metabolite profiles in excreta and the metabolic pathway of ombitasvir in humans. In addition,

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-2,5-

diyl)bis(4,1-phenylene))bis(azanediyl))bis(carbonyl))bis(pyrrolidine-2,1-diyl))bis(3-methyl-1-

oxobutane-2,1-diyl))dicarbamate; M23, 4,4'-((2S,5S)-1-(4-tert-butylphenyl)pyrrolidine-2,5-

diyl)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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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

selection criteria. On the morning of Study Day 1, subjects received a single oral dose of

[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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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

All sample combustion was performed using a Model 307 Sample Oxidizer (Packard Instrument

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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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 24-

hour 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

weighed aliquots (approximately 0.2 g) were combusted and analyzed by LSC.

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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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-MS-

radiochemical detection analysis. Aliquots were also cleaned up using Strata SAX solid phase

extraction (SPE) cartridge (Phenomenex) in order to remove the detergent in the urine sample.

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-MS-

radiochemical 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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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):

acetonitrile/methanol mixture (1:1, v/v); the flow rate was maintained at 1.0 mL/min. The

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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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.

Plasma concentration-time radioactivity data were analyzed with SAS software (version 9.2;

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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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

were pooled using an equal 100 µL volume across subjects at each time point for HPLC-MS/MS

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; 0.2-

0.22 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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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

Following a single oral dose of [14C]ombitasvir (25 mg, 100 µCi) to four healthy, male

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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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

summarized in Table 2. Metabolites M29, M36, M37 and M23 accounted for 32.9%, 25.7%,

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 (0-

192hr 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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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),

including M9 (0.7% of dose), M3 (0.6%), M2 (0.2%), M5 (0.2%), and M6 (0.2%). The

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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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

of one oxygen atom to M23. The predicted molecular formula was C24H26N3O+ (calculated mass

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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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-2,5-

diyl-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

418.2489. The collision-induced dissociation of M25 produced major fragment ions at 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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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

237.1382 and 144.0800. M35 was tentatively assigned as tert-butyl demethylation and di-

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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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,

547.3271, 491.2643 and 334.1904. M9 was tentatively assigned as a hydration metabolite of

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 ng�hr/ml. Two

downstream metabolites, M29 and M36, provided Cmax values more than 4-fold lower (31.4 and


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23.0 ng/ml, respectively), with correspondingly lower AUC values (669 and 442 ng�hr/ml,

respectively). Plasma concentrations of M37 (Cmax 16.1 ng/ml; AUC 312 ng�hr/ml) and M23

(Cmax 8.6 ng/ml; AUC 194 ng�hr/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

ombitasvir with paritaprevir/ritonavir and dasabuvir, and no additional new metabolites were

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 di-

hydroxyl) 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 carbon-


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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.

Discussion

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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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).

In humans, M23 further undergoes CYP2C8-mediated oxidative metabolism at the tert-butyl

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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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

replicons in vitro and are not expected to have clinically relevant on-target or off-target

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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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.

Performed data analysis: Shen, Serby, Ma, Badri , Menon.

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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Unnumbered Footnote to the Title

Disclosure Statement: The design, study conduct, and financial support for this study were

provided by AbbVie. AbbVie participated in the interpretation of data, writing, review and

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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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

25-mg (n=4).

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.


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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)

T1/2 (h)

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 30.3 ± 9.80


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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

Ombitasvir 8.5

M37 16.3

M36 25.7

M25 3.1

M29 32.9

M26 3.4

M23 10.0


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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)

Time 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


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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 Ombitasvir 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

Subtotal 87.83 0.2 0.7 0.6 0.2 0.2 0.6 0.57

* 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.

a Uf = unknown metabolites in feces. Uf1-3 is the combined radioactivity dose recovery for Uf1, Uf2 and Uf3. b Mu = unknown metabolites in urine. Mu1-5 is the combined radioactivity dose recovery for Mu1, Mu2, Mu3, Mu4 and

Mu5.


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Table 5. Molecular ions and characteristic fragment ions of ombitasvir and metabolites in human plasma, urine or feces

Compound

[M+H]+

(Measured)

[M+H]+

(Theorectical) Chemical formula Δppm Key Fragment Ions (m/z)

Ombitasvir 894.5113 894.5124 C50H68N7O8

+ -1.2 737, 640, 588, 547, 431

M6 640.3856 640.3857 C38H50N5O4

+ -0.2 547, 491, 473, 334, 255

M9 912.5224 912.5230 C50H70N7O9

+ -0.7 894, 755, 640, 547, 491, 334

M3 910.5055 910.5073 C50H68N7O9

+ -1.9 892, 737, 588, 431

M5 910.5056 910.5073 C50H68N7O9

+ -1.9 Accurate mass only

M23 386.2587 386.2591 C26H32N3

+ -1.0 369, 293, 237, 144

M26 402.2538 402.2540 C26H32N3O

+ -0.5 370, 309, 237, 144

M34 388.2383 388.2383 C25H30N3O

+ 0.0 371, 237, 144

M29 372.2068 372.2070 C24H26N3O

+ -0.5 355, 279, 237, 144

M25 418.2490 418.2489 C26H32N3O2

+ 0.2 237, 144

M28 416.2338 416.2333 C26H30N3O2

+ 1.2 237, 144

M36 388.2015 388.2020 C24H26N3O2

+ -1.3 371, 295, 237, 144

M35 404.2330 404.2333 C25H30N3O2

+ -0.7 237, 144

M37 390.2171 390.2176 C24H28N3O2

+ -1.3 297, 237, 144

M7 273.1441 273.1445 C12H21N2O5

+ -1.5 255, 227, 116


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DMD # 67496

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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*

Compound % Total AUCt AUCt (ng●hr/mL)** 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

* 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.

**Pharmacokinetic samples were collected on Day 14 up to 24 hours and pooled across the subjects for quantitative HPLC-MS/MS analysis.


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Fig. 1.

Ombitasvir


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Time (h)

% o

f D

ose

0 24 48 72 96 120 144 168 192

0

20

40

60

80

100

Urine

Feces

Total

Fig. 2


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Time (h)

Concentr

ation (

ng/m

L o

r ng

-equiv

/g)

0 24 48 72 96 120 144 168 1920.01

0.1

1

10

100

1000

Plasma Total Radioactivity (ng-equiv/g)

Plasma ombitasvir (ng/mL)

Fig. 3


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R T (m in )

CP

M

0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0

2 0

4 0

6 0M29

M23Ombitasvir

M26

M36

M37 M25

Plasma AUC0-192h

A)

RT: 0.21 - 78.71

10 20 30 40 50 60 70

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance M23

Ombitasvir

M26M37

M36

M25

B)M29

Fig. 4


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Fig. 5

RT (min)

CP

M

0 10 20 30 40 50 60 70 800

10

20

30

40

RT (min)

CP

M

0 10 20 30 40 50 600

100

200

300

6000

8000

10000

Ombitasvir

M2

M9M3

UNKM6

M5

(B) Pooled feces48-72 h

OmbitasvirMu1

Mu2

Mu3Mu4

Mu5

(A) Pooled Urine12-24 h

Note – Mu stands for uncharacterized urinary metabolites


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Fig. 6

NN

N

N

ON

O ON

O

NO

O

O

O

NN

N

N

ON

O ON

O

NO

O

O

O

OH

NN

N

N

ON

O ON

O

NO

O

O

O

OH

NN

N

N

ON

O ON

O

NO

O

O

O

NN

N

NH2

OO

NO

O

N

OO

NO

O

OH

M5

M9

M7 M6

HO

ABT-267

H2N

N

NH2

M23

H2N

N

NH2

OH

M34

H2N

N

NH2

OH

H2N

N

NH2

OH

OH

M25

M26 H2N

N

NH2

COOH

M28

H2N

N

NH2

OH

M35

HO

H2N

N

NH2

O

M29

H2N

N

NH2

O

M36

HO

H2N

N

NH2

OHHO

M37

M3

amide hydrolysis

hydroxylation

oxidation

oxidation/C-demethylation

oxidation

ombitasvir


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RT: 0.00 - 79.99 SM: 3B

0 10 20 30 40 50 60 70

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bu

nd

an

ce

ombitasvir

M23 M29

M7 M37

M36

M25 M26 M6 M9

Fig. 7


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RT: 0.00 - 79.99

0 10 20 30 40 50 60 70

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bu

nd

an

ce

M23

M29

M34

M28

M38/M39

M35

M25

M37

M36

M26

M33

Fig. 8.


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NNH2H2N

NNH2H2N

OH

NNH2H2N

OHO

NNH2H2N

OH

OH

NNH2

O

H2NN

NH2

OH

H2NN

NH2

OH

H2N

HO

NNH2

O

H2NN

NH2

HO

H2N

HO

OH

M34M35

M26

M25

M28

M29

M37 M36

NNH2

OH

H2N

M38

NNH2

OH

H2N

M39

O

NH2H2N O

M33

M23

Oxidation/C-demethylation

Oxidation

Fig. 9


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dmd # 67496 - drug metabolism and...

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