Compositions and Methods of Use of Ritonavir for Treating HCV
[001] Related Applications
[002] This application claims priority to U.S. Patent /Application Serial No. 60/779,50l, filed March 6, 2006, and entitled, "COMPOSITIONS AND METMODS OI' USE OI RITONAY1R FOR TREATING HCY."
[003] Background of the Invention
[004| I Iepatitis C is a blood-borne disease that infects approximately 150-200 million individuals worldwide. I Iepatitis C is a viral disease that is caused bv a hepatropic virus, I ICY (I Iepatitis C Yirus). Infection with HCV results in liver inflammation which can ultimately result in cirrhosis and liver cancer. Although manv individuals do not exhibit symptoms related to hepatitis C infection, it is the leading cause of liver transplants in the United States.
[005| Although science was aware ot the hepatitis A and B viruses for decades, it was not until the late 1980s that discovery of hepatitis C virus was published tor the medical and scientific communities. The discovery confirmed that most post-transfusion hepatitis cases were not due to hepatitis A and B viruses, but instead were due to the ncwlv discovered liepatitis C \irus. With the discovery ot the hepatitis C virus, the need arose for methods to treat the virus and to understand the urstructural and replication process performed b\ the virus.
[006] MCY is a small, enveloped, single stranded, positive sense RNA \ irus in the family Flaviv iridae. HCY mainly replicates within hepatoevtcs. 1 ICY particles bind to receptors on the surfaces of hepatoevtes and subsequently enter the cells. The RNA genome encodes a single stranded polypeptide comprising of approximately 3000 amino acids.
[007] Tberapies for Hepati/i/isC Vins (HCV')
[008] Several different types of I ICY therapies exist. One ot the most common therapies involves using the combination of alpha interferon and ribavirin. Ex en with this t\ pe of therapy main patients do not exhibit a reduction in viral activity. Accordingly, there is a ckar long-felt and unresolved need to develop new etlcetivc therapeutics in the treatment of HCY infection.
[009] The inventors have herein developed compositions and methods of improving the pharmacokinetics ot I ICY pharmaceutical agents (or pharmaceutical!y acceptable salts, esters, and prodnigs thereof) which are
metaboilized by cytochrome P450 monoxygenase comprising coadministering ritonavir or a pharmaceutically acceptable salt, ester, and prodrug thereof with such HCV compounds.
[0010] Summary of the Invention
[0011] In accordance with the present invention, there is disclosed compositions and a method of improving the pharmacokinetics of pharmaceutical agents (or pharmaceutically acceptable salts, esters, and prodrugs thereof) which are metaboilized by cytochrome P450 monoxygenase comprising coadministering ritonavir or a pharmaceutically acceptable salt, ester, and prodrug thereof.
[0012] Brief Description of the Figures
[0013] Figure 1 shows the structures and chemical names of YX-950 and SCH 503034.
[0014] Figure 2 shows enhancement of the plasma levels of YX-950 by coadministering with ritonavir in rats.
[0015] Figure 3 shows . enhancement of the plasma levels of SCI I 503034 by coadministering with ritonavir in rats.
[0016] Detailed Description of the Invention
[0017] In accordance with the present invention, there is disclosed compositions and a method of improving the pharmacokinetics oi pharmaceutical agents (or pharmaceutically acceptable salts, esters, and prodrugs thereoi) which are metaboilized by cytochrome P45(> monoxygenase comprising coadministering ritonavir or a pharmaceutically acceptable salt, ester, and prodrug thereof.
[0018] "Coadministered" or "coadministering" means that the therapeutic agents can be formulated as separate compositions which are administered at the same time or different times, or alternatively that the therapeutic agents can be co-formulated and administered as a single composition.
[00 19] Drugs which are metabolized by cytochrome P450 monoxygenase and which benefit from
coadministration with ritonavir include 2-(2- {2-cyclohexyl-2|(pyrazine-2-carbonyl)-amino]-acetylamino}-3,3-
dimethyl-butynl)-octahydro-cyclopenta[c]pyrrole-l -carboxylic acid(l-cyclopropylaminooxaryl-buty!)-amide
(YX-950), and 3-[2-(3-tert-butyl-ureido)-3,3-diemthyl-hunyry,l|-6,6-dimethyl-3-aza-bicyclo[3.1.0]hexane-2-
carboxylic acid (2-carbamoyl-l-cyclobutylmethyl-2-oxo-ethyl)-amide (SCH 503034).
[0020] In a preferred embodiment of the present invention, there is disclosed a composition wherein 2-(2-
{2-cyclohexyl-2[(pyrazine-2-carbonyl)-amino]-acetylamino)}-3,3-dimnethyl-butyryl)-octahydro-
cyclopenta|c]pyrro]e-l-carboxylic acid(l-cyclopropylaminoi)xalvl-hut\l)~ainidc (VX-950) is coadministered with ritonavir.
[0021 ] In another preferred embodiment of the present invention, there is disclosed a method for improving the pharmacokinetics of HCV compounds by coadministering 2-(2-{2-cyclohexyl-2[(pyrazine-2-carbonyl)-aminr)]-acetylamino}-3,3-dimethyl-butyryl)-octahydro-cyclopenta[c|pyrrole-l-carboxylic acid(l-cyclopropylaminooxalyl-butyl)-amide (VX-950), with ritonavir.
[0022] In another preferred embodiment of the present administration, there is disclosed a composition wherein 3-[2-(3-tert-buty-l-ureido)-3,3-diemthyl-butyryl|-6,6-dinuthyl-3-aya-bicyclo[3.1.l)]hexane-2-carboxylic acid (2-carbamoyl-l-cydobutylmethyl-2-oxo-ethyl)-amide (SCH 503034) is coadministered with ritonavir.
[0023] In another preferred embodiment of the present administration, there is disclosed a mediod for improving the pharmacokinetics of HCV compound by coadmistering 3-[2-(3-tert-butyl-ureido)-3,3-diemthyl-butyryl]-6,6-dimethyl-3-aza-bic.yclo|3.1.0|hexane-2-carbox)lic acid (2-carbamoyl-1 -cyclobutylmethyl-2-oxo-ethyl)-amide (SCH 503034) and ritonavir.
[0024] In another preferred embodiment of the present invention, there is disclosed a mediod of inhibiting HCV in a mammal comprising coadministering 2-(2- 12-cyclohexyl-2[(pyrazine-2-carbonyl)-amino]-acetylamino}-3,3-dimethyl-butyry-l)-octahydro-cyclopenta|c|pyrrole-l-carboxylic acid(l~ cyclopropylaminooxalyl-butyl)-amide (VX-950), or a salt, ester, or prodrug thereof and ritonavir or a salt, ester, or prodrug thereof.
[0025] In another preferred embodiment of the present invention, there is disclosed a mediod of inhibiting HCV comprising coadminstering 3-[2-(3-tert-butyl-ureido)-3,3-diemth\l-butyryl]-6,6-diniethyl-3-aza-bicyclo|3.1.0|hexane-2-carboxylic acid (2-carbamoyhl-c\dobut\lmethyl-2-oxo-ethyl)-amide (SCH 503034) or a salt, ester, or prodrug diereof and ritonavir or a salt, ester, or prodrug thereof.
[0026] Ritonavir is (2S, 3S, 5S)-5-(N-(N-((N-methyhl-((2-isopropyl-4-thiaz(>yl)methyl)amino)carbonyl)-I/-valinyl)aniino)--2-(N-((5-thiazoyl)methoxycarbonyl)ainino)-l,6-diphenyl-3~hydn)xyhexane. Ritonavir can be synthesized by the procedures described in PCT Patent Application No. \Vo94/14436, published July 7, 1994, and US5541206 issued July 30, 1996.
[0027] 2-(2-{2-cyclohexyl-2[(pyrazine-2-carbonyl)-amino]-acetylaniinc)}-3,3'dimethyl-butyryl)-octahydro-cyclopenta[c|pyrrole-l-carboxylic acid(l-cyclopropylaminooxalyi-butyl) amide (VX-950), can be synthesized according to the procedures described in published PCT application WO02/18369, published March 7,2002
[0028] 3-[2-(3-tert-butyl-ureidr))-3,3-dienithyl-butyni]-6,6-diniethyl-3-aza-bicyclo[3.1.0]hexane-2-carboxytic acid (2-carbamoyl-1-cyclobutylmethyl-2-oxo-cthyl)-amide (SCH 503034), can be synthesized according to the
procedures described in published patent application US 2003/0216325, having a publication date of November 20, 2003.
[0029] The compositions of the present invention are useful for treating I ICY infections in mammals, particularly in humans. Accordingly, ritonavir can be coadministered with either VX-950 or SCH 503034 to treat HCV infection. Furthermore, the compositions of the present invention can also be coadministered with one or more anti-viral agents, including, but not limited to, entry inhibitors, protease inhibitors, polymerase inhibitors, and the like. In particular, the compositions of the present invention can be coadministered with anti-viral agents such as interferons and ribavirin. Examples of interferons suitable for use with ritonavir/YX 950 and ritonavir/SCH 503034 compositions of the present invention include, but arc not limited to, interferon alpha-2a, interferon alpha-2b, pegylated interferon, pegylated interferon alpha-2a, pegylated interferon alpha-2b, concensus interferon alpha, pegylated concensus-inteferon Alpha, interferon fused to a protein such as, but not limited to, interferon fused to serum human albumin (albuferon). The ritonavir/YX 950 and ritonavir/SCH 503034 compositions of the present invention can also be coadministered with other anti-viral agents. In a preferred embodiment, the ritonavir/YX 950 and ritonavir/SCH 50.3034 compositions of the present invention can be coadministered one or more pharmaceutical agents such as ribavirin and viramidine.
|o030] The following Examples are illustrative of the ability of ritonavir to improve the pharmacokinetics of an HCY compound.
[0031] Example 1. Inhibition of the metabolism of VX-950 and SCH 503034 in human liver microsomes
[0032] Liquid handling was carried out on a Tecan EVO robotic system. Triplicate incubations were carried out at a final test compound concentration of 1µM with 0.5 mg/ml microsomal protein, and 1 mM NADPH. Pooled human liver microsomes (1 mg/ml protein) and NADPH cofactor (2 mM) were prepared in 50 mM phosphate buffer at pH 7.4. Stock solutions (10 mM) of VX-950 or SCH 503034 were prepared in DMSO and then diluted to 100 µM in 1:1 acetonitrile/water. The solutions of compounds were added into the NADPH cofactor solution containing 0, 0.8 or 8 µM of ritonavir in a 2 ml 96-well plate. The resulting solution was added to the microsomes (1:1) that had been pre-incubated for 10 minutes at 37°C. Samples (0.1 ml) were incubated in 96-well plates at 37°C for 0, 10, 20 and 30 min in a Tecan 4-slot incubator. At each time point, the robotic arm removed one of the replicate plates and the reactions were stopped by adding 1 volume (100 µ1) of acetonitrile with internal standard (0.05µM buspirone) to each well. All plates
were centrifuged at 3500 rpm for 30 min, and the supernatant was transferred to a 96-well injection plate. The plates were stored at 4°C until analyzed.
[0033] LC-MS/MS analysis: The samples were analyzed in positive mode using die turbospray ion source of PE/Sciex API 4000 Q-Trap mass spectrometer with Shimadzu HPLC system. Samples were injected (5 µL) onto a Lancer CI8 column (5 µm, 30 x 2.1 mm) from Analytical Sales and Services Inc. (Pompton Plains, NJ) and separation occurred via a gradient: The flow rate was 0.5 mL/min; starting conditions, of 7.5% B, 2.5%C increasing to 30% B and 10 % C at 0.4 min. The percentage of B and C were rapidly increased to 74 and 21%, respectively, over 0.5 min and held for 0.7 min, then decreased back to the initial conditions over 0.1 min, and held for 0.4 min, for a total run time of 2.5 min. Mobile phase A was 95/5 water/methanol (v/v) with 10 mM ammonium acetate and 60 µL/1. acetic acid. Mobile phase B was methanol containitig 10 mM ammonium acetate and 60 µL/L acetic acid. Mobile phase C was acetonitrile.
[0034] Using the above conditions, the presence of ritonavir inhibited the metabolism ofVX-950 and SCH : 503034 in the following manner as shown in Table 1
(Table Removed)

[0035] Example 2. Inhibition of the metabolism of VX-950 and SCH 503034 in rat liver microsomes
(Table Removed)
[0037] Example 3. Enhancement of the plasma levels of VX-950 by coadministering with ritonavir in rats
[0038J The pharmacokinetic behavior of VX-950 was characterized following a single 5 mg/kg intravenous or oral dose in Sprague-Dawley derived rats (n=3 per group); an additional group of three rats received a 5 mg/kg oral dose of VX-950, coadministered with a 5 mg/kg oral dose of ritonavir. VX-950 (± ritonavir) was prepared as 5 mg/ml, solution in a 10% DMSO: 90% PKG-400 vehicle for both oral (± ritonavir) and intravenous administration. The 1 mL/kg intravenous dose was administered as a slow bolus (~1 minutes) in a jugular vein of the rats under isoflurane anesthetic; the 1 ml/kg oral dose (± ritonavir) was administered by gavage. Serial blood samples were obtained from a tail vein of each animal 0.1 (IV only), 0.25, 0.5, 1, 1.5, 2, 3, 4, 6 and 8 hours after dosing. The heparinized samples were placed on ice immediately following collection. Plasma was separated by centrifugation and stored frozen tor subsequent analysis.
[0039] Concentrations of parent drug (and ritonavir) were determined by HP1.C-MS/MS following liquid-liquid extraction of the plasma samples. Analysis was performed on a Seiex API 2000™ Biomolecular Mass Analyzer using Turbo Ion Spray. Peak areas of the title compounds and internal standards were detennined using the Sciex MacQuan™ software. Calibration curves were derived from peak area ratio (parent drug/internal standard) of the spiked plasma standards using least squares linear regression of the ratio versus the theoretical concentration. The maximum plasma concentration (C,iMS) and the time to reach the maximum plasma concentration (Tmax) were read directlv from the observed plasma concentration-time data. The plasma concentration data were submitted to multi-exponential curve fitting using WihNonlin. The area under the plasma concentration-time curve from 0 to t hours (last measurable plasma concentration time point) after dosing (AUCut) was calculated using the linear trapezoidal rule for the plasma-time profiles. The
residual area extrapolated to infinity, determined as the final measured plasma concentration (Ct) divided by the terminal elimination rate constant (ß), was added to AUG., to produce the total area under the curve (AUCo 50). 'Hie apparent total plasma clearance (CL,,) was calculated by dividing the administered dose by the AUGi.*. The volume of distribution, \\, was estimated h\ dividing the dose by the extrapolated plasma concentration at time zero (Co). The volume of distribution at steady state, Vss, was estimated as a product of the plasma clearance (CLp,) and the mean residence time (MRT); the terminal-phase volume of distribution, Vß, was derived from the plasma clearance value (Cß,) divided b\ the plasma elimination rate constant (£5). The bioavailability was calculated as the dose-normalized AUG. / from the oral dose divided by the corresponding value derived from an intravenous dose.
[0040] As shown in Figure 2 and below, the following mean (± standard error) plasma levels were obtained, indicating that coadministering with ritonavir substantially elevated the plasma levels of VX-950:
[0041] The following mean (± SEM, n=3) pharmacokinetic parameters were obtained:

(Table Removed)

Mean (±SEM, n=3); t,/2 (hr); AUC (ug*hr/ml); C„MS (ug/ml); Timx (hr); l; (%);
0-8 hr AUG. PO+ = oral solution dose of \'X-950 + 5 mg/kg dose of ritonavir; nf — unable to estimate plasma elimination half-life.
[0042] Example 4. Enhancement of the plasma levels of SCH 503034 by coadministering with ritonavir in rats
[0043] Using the procedure of Example 3, but substituting SCH 503034 for VX-950, as shown in Figure 3 and below, the following mean (± standard error) plasma levels were obtained, indicating that coadministering with ritonavir substantially elevated the plasma levels of SCH 503034:
[0044] The following mean (± SEMVl, n=3) pharmacokinetic parameters were obtained:
(Table Removed)
Mean (+SEM, n=3); t}/2 (hr); AUG (p.g»hr/ml); G„M>: (ug/ml); l; (%); * 0-8 hr AUG. PO+ = oral solution dose ofSGH 503034 + 5 nig/kg dose of ritonavir; nf- unable to estimate plasma elimination half-life.

What is claimed:
1. A pharmaceutical coadministered composition comprising 2-(2- i 2-cyclohexyl-2[(pyrazine-2-carbonyl)-amino]-acetylainino} -3,3-dimethyl-butyryl)-octahydro-cyclopenta[c]pyrrole-l -carboxylic acid(l-cyclopropyrlaminooxalyl-butyl)-amide (\'X-950) or a salt, ester, or prodrug thereof and ritonavir or a salt, ester, or prodrug thereof.
2. A method for improving the pharmacokinetics of 2-(2- j 2-cyclohexyl-2|(pyrazine-2-carbonyl)-amino]-acety-laminoS-3,3-dimethyl-butyryl)-octahydro-cyclopenta|c|pyrrole-l -carboxylic acid(l-cyclopropylaminooxalyl-butyl)-amide (VX-950), or a salt, ester, or prodrug thereof comprising coadministering 2-(2-i2-cyclohexyl-2|(pyrazine-2-carbonly)-anmino)]-acetylamino}-3,3-dimethyl-buryryl)-octahydro-cyclopenta[c]pyrrole-l-carbox\lic acid(l-cyclopropylaniinooxalyl-butyl)-amide (VX-950) or a salt, ester, or prodrug thereof with ritonavir or a salt, ester, or prodrug thereof.
2. A pharmaceutical coadministered composition comprising 3-[2-(3-tert-butyd-ureido)-3,3-diemthyl-butyryl|-6,6-dimethyl-3-aza bicyclo|3.1.0]hexane-2-carboxylic acid (2-carbamoyl-l-cyclobutylmethyl-2-oxo-ethyl)-amide (SCH 503034) or a salt, ester, or prodrug thereoi and ritonavir or a stilt, ester, or prodrug thereof.
4. A method for improving the pharmacokinetics of 3-[2-(3-tert-butyl-ureido)-3,3-diemthyl-butyryl-
6,6-dimethyl-3-aza-bicyclo(3.1.0]hexane-2-carboxylic acid (2-carbamoyl-1 -cyclobutylmethyl-2-oxo-
ethyl)-amide (SCH 503034) or a salt, ester, or prodrug thereof comprising coadministering 3-[2-(3-
tert-butyl-ureido)-33-dieaithyl-butyryl]-6,6-dimnethyl--3-aza-bic\'clo[3.1.(l|hexane-2-carboxylic acid (2-
carbamoyl-l-cyclobutyiniethyl-2-oxo-ethyl)-amidc (SCH 503034) or a salt, ester, or prodrug thereof
with ritonavir or a salt, ester, or prodrug thereof.
5. A composition of claim 1 coadministered with one or more phannaceutical agents selected from the
group consisting of interferon alpha-2a, interferon alpha-2b, pegvlated interferon, pegylated
interferon alpha-2a, pegylated interferon alpha-2b, concensus interferon alpha, pegylated concensus-
inteferon alpha, interferon fused to a protein, ribavirin, and viramidine.
6. A composition of claim 3 coadministered with one or more phannaceutical agents selected from the group consisting of interferon alpha-2a, interferon alpha-2b, pegylated interferon, pegylated interferon alpha-2a, pegylated interferon alpha-2h, concensus interferon alpha, pegylated concensus-inteferon alpha, interferon fused to a protein, ribavirin, and viramidine.
7. A mediod for treating HCV in a mammal comprising coadministering 2-(2-{2-cyclohexyl-2[(pyrazine-2-carbonyl)-amino|-acety Liminej} -3,3-dimeth) l-butyr\l)-octahydro-cyclopenta[cjpyrrole-1-carboxylic acid(l-cyclopropylaminooxalyl-butvl)-amide (YX-950), or a salt, ester, or prodrug thereof and ritonavir or a salt, ester, or prodrug thereof.
8. A method ior treating HCV in a mammal comprising coadmistering 2-(2-{2-cyclohexyl-2[(pyrazine-
2-carbonyl)-aminoJ-acetylamino} -3,3-dimethyl-butyn l)-octahydro-cyclopenta[c]pyrrole-l -carboxylic
acid(l -cyclopropylaminooxalyl-buty l)-amide (VX-950), or a salt, ester, or prodrug thereof and
ritonavir or a salt, ester, or prodrug thereof, and further coadmistering with one or more
pharmaceutical agents selected from the group consisting of interferon alpha-2a, interferon alpha-2b,
pegylated interferon, pegylated interferon alpha-2a, pegylated interferon alpha-2b, concensus
interferon alpha, pegylated concensus-inteferon alpha, interferon fused to a protein, ribavirin, and
viramidine.
9. A method for treating HCV comprising coadminstering 3-[2-(3-tert-butyl-ureido)-3,3-diemdiyl-butyryl]-6,6-dimethyl-3-aza-bicyclo[3.1.0]hexane-2-carboxylic acid (2-carbamoyl-l-cyclobutylmethyl-2-oxo-ethyl)-amide (SCH 51)3034) or a salt, ester, or prodrug thereof comprising and ritonavir or a salt, ester, or prodrug thereof.
10. A method for treating HCV comprising coadminstering 3-[2-(3-tert-butyl-ureido)-3,3-diemdyl-butyryl|-6,6-dimethyl-3-aza-bicyclo[3.1.0]hexane-2-carboxylic acid (2-carbamoyl-l-cyclobutylmethyl-2-oxo-ethyl)-amide (SCH 503034) or a salt, ester, or prodrug thereof comprising and ritonavir or a salt, ester, or prodrug thereof, and further coadministering with one or more pharmaceutical agents selected from the group consisting oi interferon alpha-2a, interferon alpha-2b, pegylated interferon, pegylated interferon alpha-2a, pegylated interferon alpha-2b, concensus interferon alpha, pegylated concensus-inteferon alpha, interferon fused to a protein, ribavirin, and viramidine.
11. A method of inhibiting HCV in a mammal comprising coadministering 2-(2-{ 2-cyclohex\-l-
2[(pyrazine-2-carbonyl)-aniinoJ-acetylamino}-3,3-dimethyl-butyryl)-octahydro-cyctopenta[c]pyrrole-
1-carboxylic acid(l-cyclopropylaminooxal\l~butyl)-amide (VX-950), or a salt, ester, or prodrug
thereof and ritonavir or a salt, ester, or prodrug thereof.
12. A method of inliibiting HCV comprising coadminstering 3-[2-(3-tert-butyl-urddo)-3,3-diemthyl-
butyryl]-6,6-dimethyl-3-aza-bicyclo[3.1.()|hexane-2-carboxylic acid (2-carbamoyl-l-cyclobutylmcthyl-
2-oxo-ethyl)-amide (SCH 503034) or a salt, ester, or prodrug thereof and ritonavir or a salt, ester, or
prodrug thereof.