US 20190099429A1 ( 19 ) United States ( 12) Patent Application Publication ( 10 ) Pub . No. : US 2019 /0099429 A1 Lefebvre (43 ) Pub. Date : Apr. 4 , 2019
(54 ) CENICRIVIROC FOR THE TREATMENT OF A61K 45 / 06 (2006 .01 ) FIBROSIS A61K 9 /00 (2006 .01 ) A61K 4738 ( 2006 .01 ) (71 ) Applicant: Tobira Therapeutics, Inc ., South San A61K 38 /21 ( 2006 .01 ) Francisco , CA (US ) A61K 31 / 7072 ( 2006 . 01 ) A61K 38 / 13 ( 2006 . 01 ) ( 72 ) Inventor: Eric Lefebvre , South San Francisco , A61K 31 / 194 (2006 .01 ) CA (US ) A61K 31/ 427 ( 2006 .01 ) A61K 31 /513 (2006 . 01 ) ( 73 ) Assignee: Tobira Therapeutics , Inc ., South San A61K 31 /536 ( 2006 . 01 ) Francisco , CA (US ) ( 52) U . S . CI. CPC ...... A61K 31 /55 ( 2013 . 01 ) ; GOIN 33 /6893 ( 21 ) Appl. No. : 16 /148 , 749 ( 2013 .01 ) ; A61K 2300 / 00 ( 2013 .01 ); A61K 47 / 12 (2013 . 01 ) ; A61K 45 / 06 (2013 .01 ) ; A61K (22 ) Filed : Oct . 1 , 2018 9 /0053 (2013 .01 ) ; GOIN 2800 / 52 ( 2013 .01 ) ; Related U . S . Application Data GOIN 2800 / 7052 (2013 . 01 ) ; A6IK 47 / 38 (2013 .01 ) ; A61K 38 / 212 ( 2013 .01 ); A61K (63 ) Continuation of application No. 15 / 127, 720 , filed on 31 / 7072 (2013 . 01 ) ; A61K 38 / 13 (2013 . 01 ); Sep . 20 , 2016 , now abandoned , filed as application A61K 31/ 194 ( 2013 .01 ); A61K 31/ 427 No. PCT /US2015 / 021828 on Mar. 20 , 2015 . ( 2013 .01 ) ; A61K 31/ 513 ( 2013 .01 ) ; A61K ( 60 ) Provisional application No. 61/ 968, 829 , filed on Mar. 31/ 536 (2013 .01 ) ; A61K 38 / 12 ( 2013 .01 ) 21, 2014, provisional application No . 62 /024 ,713 , filed on Jul. 15 , 2014 , provisional application No . (57 ) ABSTRACT 62 / 114 , 304, filed on Feb . 10 , 2015 . or a fibrotic disease or condition in a subject in need thereof Publication Classification comprising administering to the subject a therapeutically (51 ) Int. CI. effective amount of cenicriviroc or a salt or solvent thereof. A61K 31/ 55 ( 2006 .01 ) The fibrosis or fibrotic disease may be liver fibrosis , renal GOIN 33 /68 ( 2006 . 01 ) fibrosis , non - cirrhotic hepatic fibrosis , associated with non A61K 38 / 12 ( 2006 .01 ) alcoholic steatohepatitis (NASH ) , non - alcoholic fatty liver A61K 47/ 12 ( 2006 . 01 ) disease (NAFLD ), or emerging cirrhosis .
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CENICRIVIROC FOR THE TREATMENT OF wherein the NASH or the livier fibrosis associated with FIBROSIS NASH is associated with type 2 diabetes mellitus ( T2DM ) . [0005 ] In one embodiment, the invention provides a CROSS REFERENCE TO RELATED method of treating NASH in a subject in need thereof APPLICATIONS comprising administering to the subject a therapeutically effective amount of cenicriviroc , or a salt or solvate thereof; [0001 ] This application is a Continuation of U .S . patent wherein the NASH or the livier fibrosis associated with application Ser . No . 15/ 127 ,720 , filed Sep . 20 , 2016 , which NASHis associated with metabolic syndrome (MS ) . claims priority to International Patent Application No. : 10006 ] In one embodiment, the invention provides a PCT /US2015 / 021828, filed Mar . 20, 2015 , which claims method of treating NASH in a subject in need thereof priority to U . S . Provisional Application No . 61/ 968 ,829 , comprising administering to the subject a therapeutically filed Mar. 21 , 2014 , U . S . Provisional Application No . effective amount of cenicriviroc , or a salt or solvate thereof; 62/ 024 ,713 , filed Jul. 15 , 2014 , and U . S . Provisional Appli wherein liver fibrosis is associated with HIV and HCV cation No . 62/ 114 , 304 , filed Feb . 10 , 2015 , which are hereby incorporated by reference herein in their entirety . [0007 ] In one embodiment, the invention provides a method of treating NASH in a subject in need thereof BACKGROUND comprising administering to the subject a therapeutically [0002 ] Cenicriviroc ( also known as CVC ) is the common effective amount of cenicriviroc , or a salt or solvate thereof; name of (S , E ) - 8 - ( 4 - ( 2 -Butoxyethoxyphenyl ) - 1 - ( 2 -methyl wherein liver fibrosis is associated with HCV infection propyl) - N - ( 4 - ( ( ( 1 - propyl- 1H - imidazol- 5 - yl) methyl ) sulfi [0008 ] In one embodiment, the invention provides a nyl) phenyl ) - 1 , 2 , 3 , 4 -tetrahydrobenzo [ b ]azocine - 5 -carbox method of treatment, wherein the cenicriviroc or a salt or amide . The chemical structure of cenicriviroc mesylate solvate thereof is formulated as an oral composition . appears in FIG . 1 . Cenicriviroc binds to and inhibits the 100091. In one embodiment, the invention provides a activity of the C - C chemokine receptor type 2 (CCR2 ) and method of treatment, wherein the cenicriviroc or a salt or C - C chemokine receptor type 5 (CCR5 ) receptors (24 ) . solvate thereof is administered once per day or twice per day . These receptors not only play a role in entry of viruses such [0010 ] In one embodiment, the invention provides a as Human Immunodeficiency Virus (HIV ) into the cell , but method of treatment, wherein the cenicriviroc or a salt or also are important for the recruitment of immune cells to solvate thereof is co - administered with one or more addi sites of injury . Inhibition of this receptor ' s activity may have tional active agents . In a further embodiment, the one or an anti- inflammatory effect. More recently , the role that more additional active agents are one or more antiretroviral inflammation plays in the development of fibrosis has been agents selected from the group consisting of entry inhibitors , examined [ 30 ] . It has been shown that C - C chemokine nucleoside reverse transcriptase inhibitors, nucleotide receptor type 2 (CCR2 ) and CCR5 may play a role in reverse transcriptase inhibitors , non -nucleoside reverse tran promoting hepatic fibrosis [ 3 , 4 , 5 , 31 32 ]. scriptase inhibitors , protease inhibitors , integrase strand transfer inhibitors , maturation inhibitors , and combinations thereof . In a further embodiment, the one or more additional SUMMARY OF THE INVENTION antiretroviral agents are selected from the group consisting [ 0003] In one embodiment, the invention provides a of lamivudine , efavirenz , raltegravir, vivecon , bevirimat, method of treating fibrosis or a fibrotic disease or condition alpha interferon , zidovudine, abacavir, lopinavir , ritonavir, in a subject in need thereof comprising administering to the tenofovir, tenofovir disoproxil , tenofovir prodrugs , emtric subject a therapeutically effective amount of cenicriviroc or itabine , elvitegravir , cobicistat, darunavir , atazanavir , rilpi a salt or solvate thereof. In another embodiment, the fibrosis virine , dolutegravir , and a combination thereof. or fibrotic disease or condition is liver fibrosis or renal [0011 ] In a further embodiment, the one or more addi fibrosis . In yet a further embodiment, the liver fibrosis is tional active agents are one or more immune system sup associated with non -alcoholic steatohepatitis (NASH ) . In yet pressing agents . In a further embodiment, the one or more a further embodiment, the liver fibrosis is associated with additional active agents are selected from the group con non - alcoholic fatty liver disease (NAFLD ). In yet a further sisting of cyclosporine, tacrolimus , prednisolone, hydrocor embodiment, the liver fibrosis is associated with emerging tisone , sirolimus, everolimus , azathioprine, mycophenolic cirrhosis . In another further embodiment, the liver fibrosis acid , methotrexate , basiliximab , daclizumab , rituximab , comprises non -cirrhotic hepatic fibrosis . In a further anti - thymocyte globulin , anti - lymphocyte globulin , and a embodiment, the subject is infected by human immunode combination thereof. ficiency virus ( HIV ) . In a further embodiment, the cenicri [ 0012 ] In a further embodiment , the one or more addi viroc or a salt or solvate thereof is formulated as a pharma tional active agents are one or more anit - fibrotic agents ceutical composition comprising cenicriviroc or a salt or including, but not limited to , agents such as N -acetyl - L solvate thereof and fumaric acid . In a further embodiment , cysteins (NAC ) as well as angiotensin - converting enzyme the subject has a disease or condition selected from the ( ACE ) inhibitors , AT II antagonists , obeticholic acid (OCA ) , group consisting of alcoholic liver disease , HIV and HCV GFT505 , simtuzumab , or a combination thereof. co - infection , HCV infection , type 2 diabetes mellitus [0013 ] In one embodiment, the invention provides a ( T2DM ) , metabolic syndrome (MS ) , and a combination method of treatment, comprising detecting a level of one or thereof. more biological molecules in the subject treated for fibrosis [0004 ] In one embodiment, the invention provides a or the fibrotic disease or condition , and determining a method of treating NASH in a subject in need thereof treatment regimen based on an increase or decrease in the comprising administering to the subject a therapeutically level of one or more biological molecules , wherein the effective amount of cenicriviroc , or a salt or solvate thereof; biological molecule is selected from the group consisting of US 2019 /0099429 A1 Apr . 4 , 2019 lipopolysaccharide ( LPS ) , LPs -binding protein (LBP ) , 16S post - infection . Error bars represent standard deviation . Two rDNA, SCD14 , intestinal fatty acid binding protein ( I- FABP ), zonulin - 1, Collagen lal and 3al , TGF - B , [0024 ] FIG . 9 shows the effects of CVC and MVC on fibronectin - 1 , and a combination thereof. R5 - tropic intracellular HIV DNA levels . Mean fold change [0014 ] In one embodiment, the invention provides a of intracellular strong - stop DNA levels of CVC or MVC method of treatment, comprising detecting a level of one or treated cells compared to a no drug control after 4 hrs. Error biologicalmolecules in the subject treated for fibrosis or the bars represent standard deviation . Fold change was calcu fibrotic disease or condition , wherein an increase or decrease lated using AACT in HIV /GAPDH multiplexed qPCR reac in the level of one or more biological molecules compared tions, with the no drug control at 4 hrs as a calibrator. Two to a predetermined standard level is predictive of the treat independent experiments are represented . ment efficacy of fibrosis or the fibrotic disease or condition , [0025 ] FIG . 10A and FIG . 10B show multiple binding wherein the biological molecule is selected from the group modes of CVC into CCR5 . Coordinates of CCR5 were consisting of lipopolysaccharide (LPS ) , LPs -binding protein generated from the CCR5 crystal structure bound to Mara (LBP ) , 16S rDNA , sCD14 , intestinal fatty acid binding viroc in the binding pocket (PDB ID : 4MBS ) . CVC binding protein ( I- FABP ) , zonulin - 1 , Collagen 1al and 3a1, TGF - B , sites were examined after docking of CVC . Docked poses of fibronectin - 1 , and a combination thereof. CVC are displayed as colored thin lines. The seven trans [0015 ]. In a further embodiment, the one or more biologi membrane ( 7TM ) a -helices are represented by helices and cal molecules are measured in a biological sample from a numbered ( 1 - 7 ) according to the order of amino acid subject treated for fibrosis or the fibrotic disease or condi sequences . ( 10A ) A top view from the extracellular side of tion . In yet a further embodiment, the biological sample is the receptorwith three potential binding sites that are circled selected from blood , skin , hair follicles , saliva , oral mucous, ( site 1 (white ) , site 2 (black ) and site 3 ( light pink ) ) . ( 10B ) vaginal mucous, sweat, tears, epithelial tissues, urine, A side view in the CCR5 transmembrane cavity . The extra semen , seminal fluid , seminal plasma, prostatic fluid , pre cellular loop 2 ( ECL2 ) is labeled . Secondary structures are ejaculatory fluid (Cowper 's fluid ), excreta , biopsy, ascites , represented as cartoon structures . All images were processed cerebrospinal fluid , lymph , brain , and tissue extract sample using PyMOL software . or biopsy sample . 100261. FIG . 11 shows a comparison of the ligand binding pocket between CCR5 /Maraviroc and CCR5 /Cenicriviroc . BRIEF DESCRIPTION OF THE DRAWINGS Top view of CCR5 displaying docked poses, colored thin [0016 ] FIG . 1 is the chemical formula of cenicriviroc lines, of CVC ( left ) and MVC , yellow stick , ( right) in the mesylate . ligand binding pocket . CCR5 is shown in a molecular [0017 ] FIG . 2 is a graph comparing the absolute bioavail surface representation . Key residues : Tyr37 , Trp86 , Trp94 , ability , in beagle dogs , of cenicriviroc mesylate compounded Leu104 , Tyr108 , Phe109 , Phe112 , Thr177 , Ile198 , Trp248 , as an oral solution with that of cenicriviroc mesylate pre Tyr251, Leu255 and Glu283 , that are involved in gp120 pared by wet granulation and mixed with various acid binding , are deep in the pocket and colored in red . solubilizer excipients . [0027 ] FIG . 12 shows the study schematic of the evalua [ 0018 ] FIG . 3 is a graph of the total impurity and degra tion of CVC in mouse UUO model of renal fibrosis . Vehicle dant content of different cenicriviroc formulations subjected control and CVC administered BID ; anti - TGF - B1 antibody , to accelerated stability testing at 40° C . and 75 % relative compound 1D11 (positive control) administered QD BID , humidity when packaged with a desiccant. twice daily ; CVC , cenicriviroc ; ip , intraperitoneal; PBS , [ 0019 ] FIG . 4 is a dynamic vapor sorption isotherm for phosphate buffered saline ; QD , once daily ; TGF, transform different cenicriviroc formulations. ing growth factor ; UUO , unilateral ureter occlusion [ 0020 ] FIG . 5 shows the absorption of cenicriviroc from [0028 ] FIG . 13 shows the change in body weight ( Day 5 ) different formulations at three pre - treatment states in beagle in each treatment group in mouse UUO model of renal dogs. fibrosis . [0021 ] FIG . 6 shows the beagle dog absolute bioavailabil [0029 ] FIG . 14 shows the Collagen Volume Fraction ity of cenicriviroc and lamivudine in combination tablets . (CVF ; % area ) score in each treatment group in mouse UUO [ 0022] FIG . 7A and FIG . 7B show intracellular HIV DNA model of renal fibrosis . Data presented exclude a single levels in the PBMCs of participants in Study 202 at 24 outlier from an animal in the CVC 20 mg /kg /day group , weeks. Scatter plot depicting fold change in intracellular which had a CVF value > 2 standard deviations higher than HIV DNA levels between baseline and 24 weeks, separated any other animal in the group . by treatment group . The lines and error bars represent mean 0030 ] FIG . 15A and FIG . 15B show the mRNA expres and standard error measurements , respectively . Fold change sion from renal cortical tissue of sham - surgery was calculated using AACT in HIV /GAPDH multiplexed [0031 ] FIG . 16 shows the change in body weight until qPCR reactions, with each patient' s baseline sample as a week 9 in animals treated with Cenicriviroc ( low or high calibrator. 7A ) Full - length HIV DNA ( late reverse tran dose ) . scripts ) , 7B ) strong - stop HIV DNA ( early reverse tran [0032 ] FIGS. 17A - 17C shows the change in liver and scripts ) body weight until week 9 in animals treated with Cenicri [0023 ] FIG . 8A and FIG . 8B show the effects of CVC and viroc ( low or high dose ) . Panel 17A shows the change in MVC on R5 -tropic viral RNA and p24 in culture fluids . 8A ) body weight, Panel 17B shows the change in liver weight, Viral load levels in culture fluids of controls or cells treated and Panel 17C shows the change in the liver -to body weight with CVC or MVC at 4 hrs post - infection . Error bars ratio . represent standard deviation . Two independent experiments [0033 ] FIGS . 18A - 18F shows the whole blood and bio are represented . 8B ) Mean p24 antigen levels in culture chemistry of animals treated with Cenicriviroc (low or high fluids of controls or cells treated with CVC or MVC 4 hrs at dose ) at week 9 . Panel 18A shows Whole blood glucose , US 2019 /0099429 A1 Apr . 4 , 2019
Panel 18B shows Plasma ALT, Panel 18C shows Plasma [0051 ] FIG . 36 shows the survival curve of animals treated MCP - 1 , Panel 18D shows Plasma MIP - 1ß , Panel 18E shows with Cenicriviroc ( low or high dose ) from 6 to 18 weeks . Liver triglyceride , and Panel 18F shows Liver hydroxypro [0052 ] FIG . 37A - 37C shows the body weight and liver line . weight at of animals treated with Cenicriviroc (low or high [0034 ] FIG . 19 shows the HE -stained liver sections of dose ) at week 18 . Panel 37A shows Body weight, Panel 37B animals treated with Cenicriviroc ( low or high dose ) at week shows Liver weight, and Panel 37C shows Liver- to -body weight ratio . [0035 ] FIG . 20 shows the NAFLD Activity score of ani 10053 ] FIG . 38A - 38C shows macroscopic appearance of mals treated with Cenicriviroc ( low or high dose ) at week 9 . livers of animals treated with Cenicriviroc ( low or high [0036 ] FIG . 21 shows representative photomicrographs of dose ) at week 18 . Panel 38A shows the livers of animals Sirius red - stained liver sections of animals treated with treated with vehicle only , Panel 38B shows the livers of Cenicriviroc ( low or high dose ) at week 9 . animals treated with low -dose Cenicriviroc , and Panel 38C [0037 ] FIG . 22 shows representative photomicrographs of shows the livers of animals treated with high - dose Cenicri F4 /80 - immunostained liver sections of animals treated with viroc . Cenicriviroc ( low or high dose ) at week 9 . [0054 ] FIG . 39 shows the number of visible tumor nodules [0038 ] FIG . 23 shows the percentages of inflammation of animals treated with Cenicriviroc ( low or high dose ) at area of animals treated with Cenicriviroc ( low or high dose ) week 18 . at week 9 . [0055 ] FIG . 40 shows the maximum diameter of visible 100391 FIG . 24 shows representative photomicrographs of tumor nodules of animals treated with Cenicriviroc ( low or F4 /80 and CD206 double - immunostained liver sections of high dose ) at week 18 . animals treated with Cenicriviroc ( low or high dose ) at week [0056 ] FIG . 41 shows representative photomicrographs of HE - stained liver sections of animals treated with Cenicrivi [0040 ] FIG . 25 shows the percentages of F4 /80 and roc ( low or high dose ) at week 18 . CD206 double positive cells of F4 /80 positive cells of [0057 ] FIG . 42 shows representative photomicrographs of animals treated with Cenicriviroc ( low or high dose ) at week GS - immunostained liver sections of animals treated with 9 . Cenicriviroc ( low or high dose ) at week 18 . [0041 ] FIG . 26 shows the representative photomicro [0058 ] FIG . 43 shows representative photomicrographs of graphs of F4 /80 and CD16 /32 double - immunostained liver CD31- immunostained liver sections of animals treated with sections of animals treated with Cenicriviroc ( low or high Cenicriviroc ( low or high dose ) at week 18 . dose ) at week 9 . [0059 ] FIG . 44 shows percentages of CD31- positive area [ 0042 ] FIG . 27 shows the percentages of F4 / 80 and CD16 / of animals treated with Cenicriviroc ( low or high dose ) at 32 double positive cells of F4 /80 positive cells of animals week 18 . treated with Cenicriviroc ( low or high dose ) at week 9 . [0060 ] FIG . 45 shows the median Changes in HIV - 1 RNA 100431 FIG . 28 shows the M1/M2 ratio of animals treated Levels from Baseline by Cohort and Study Day -Study 201 . with Cenicriviroc (low or high dose ) at week 9 . [ 0061 ] FIG . 46 Proportion of Subjects With HIV - 1 RNA [0044 ] FIG . 29 shows representative photomicrographs of < 50 Copies /mL Over Time up to Week 48 - Snapshot Algo oil red - stained liver sections of animals treated with Ceni rithm -ITT - Study 202 . criviroc ( low or high dose ) at week 9 . [0062 ] FIG . 47 shows the LS mean changes from baseline [ 0045 ] FIG . 30 shows the percentages of fat deposition in sCD14 levels (106 pg /mL ) over time up to Week 48 -ITT . area of animals treated with Cenicriviroc ( low or high dose ) [0063 ] FIG . 48 shows the CVC (Pooled Data )- and EFV at week 9 . treated subjects grouped according to APRI and FIB -4 [0046 ] FIG . 31 shows representative photomicrographs of fibrosis index scores at baseline , Week 24 , and Week 48 . TUNEL -positive cells in livers of animals treated with [0064 ] FIG . 49 shows the scatter plot of change from Cenicriviroc ( low or high dose ) at week 9 . baseline APRI versus change from baseline sCD14 - Week 48 [0047 ] FIG . 32 shows percentages of TUNEL - positive (ITT ). cells of animals treated with Cenicriviroc (low or high dose ) [ 0065 ] FIG . 50 shows a scatter plot of change from at week 9 . baseline FIB - 4 versus change from baseline sCD14 - Week [ 0048 ] FIG . 33A -33D show quantitative RT- PCR of ani 48 (ITT ) . mals treated with Cenicriviroc ( low or high dose ) at week 9 . [0066 ] FIG . 51 shows mean changes from baseline in FIG . 33A shows the measured levels of TNF - a ; FIG . 33B creatine phosphokinase (CPK ) over time up to Week shows the measured levels ofMCP - 1, FIGS . 33C shows the 48 -Safety Population . measured levels of Collagen Type 1 , and FIG . 33D shows [0067 ] FIG . 52 shows a dot density display of CPK the measured levels of TIMP - 1. elevations by severity grading vs. Cave ( ng /mL ) - Week 48 . [0049 ] FIG . 34A - 34F shows raw data for quantitative [0068 ] FIG . 53 shows a dot density display of ALT eleva RT- PCR of animals treated with Cenicriviroc ( low or high tions by severity grading versus cave (ng /mL ) -Week 48 . dose ) at week 9 . Panel 34A shows the levels of 36B4, Panel [0069 ] FIG . 54 shows a dot density display of AST eleva 34B shows the levels of TNF- a , Panel 34C shows the levels tions by severity grading versus Cave ( ng /mL ) - Week 48 . of TIMP- 1, Panel 34D shows the levels of collagen type 1 , [0070 ] FIG . 55 shows a dot density display of bilirubin Panel 34E shows the levels of 36B4 , and Panel 34F shows elevations by severity grading versus cave ( ng /mL ) - Week 48 . the levels of MCP - 1 . 10071 ] FIG . 56A and FIG . 56B show the mean changes [0050 ] FIG . 35 shows the body weight changes of animals from baseline in fasting total cholesterol, calculated LDL treated with Cenicriviroc ( low or high dose ) from 6 to 18 cholesterol, HDL cholesterol and triglycerides over time weeks . (mg /dL ) up to Week 48 US 2019 /0099429 A1 Apr . 4 , 2019
DETAILED DESCRIPTION [0077 ] “ Substantially similar ” means a composition or [0072 ] It should be understood that singular forms such as formulation that resembles the reference composition or “ a ,” “ an , ” and “ the ” are used throughout this application for formulation to a great degree in both the identities and convenience , however, except where context or an explicit amounts of the composition or formulation . statement indicates otherwise , the singular forms are [0078 ] “ Pharmaceutically acceptable ” refers to a material intended to include the plural . Further , it should be under or method that can be used in medicine or pharmacy, stood that every journal article , patent, patent application , including for veterinary purposes , for example , in adminis publication , and the like that is mentioned herein is hereby tration to a subject . incorporated by reference in its entirety and for all purposes. [0079 ] “ Salt” and “ pharmaceutically acceptable salt ” All numerical ranges should be understood to include each includes both acid and base addition salts . “ Acid addition and every numerical point within the numerical range , and salt” refers to those salts that retain the biological effective should be interpreted as reciting each and every numerical point individually . The endpoints of all ranges directed to the cally or otherwise undesirable , and which are formed with same component or property are inclusive , and intended to inorganic acids and organic acids. “ Base addition salt ” refers to those salts that retain the biological effectiveness and be independently combinable . properties of the free acids , which are not biologically or Definitions: otherwise undesirable , and which are prepared from addition of an inorganic base or an organic base to the free acid . [0073 ] Except for the terms discussed below , all of the Examples of pharmaceutically acceptable salts include , but terms used in this Application are intended to have the are not limited to , mineral or organic acid addition salts of meanings that one of skill in the art at the time of the basic residues such as amines; alkali or organic addition salts invention would ascribe to them . of acidic residues ; and the like, or a combination comprising [ 0074 ] “ About” includes all values having substantially one or more of the foregoing salts . The pharmaceutically the same effect, or providing substantially the same result, as acceptable salts include salts and the quaternary ammonium the reference value. Thus , the range encompassed by the salts of the active agent. For example , acid salts include term “ about ” will vary depending on context in which the those derived from inorganic acids such as hydrochloric , term is used , for instance the parameter that the reference hydrobromic , sulfuric , sulfamic , phosphoric , nitric and the value is associated with . Thus , depending on context, like ; other acceptable inorganic salts include metal salts such " about” can mean , for example , + 15 % , 10 % , 15 % , + 4 % , as sodium salt, potassium salt , cesium salt , and the like; and 3 % , 12 % , + 1 % , or + less than 1 % . Importantly , all recita alkaline earth metal salts, such as calcium salt , magnesium tions of a reference value preceded by the term “ about” are salt, and the like, or a combination comprising one or more intended to also be a recitation of the reference value alone . of the foregoing salts . Pharmaceutically acceptable organic Notwithstanding the preceding , in this application the term salts includes salts prepared from organic acids such as “ about ” has a special meaning with regard to pharmacoki acetic , propionic , succinic , glycolic , stearic , lactic , malic , netic parameters , such as area under the curve ( including tartaric , citric , ascorbic , pamoic , maleic , hydroxymaleic , AUC , AUC , and AUC4) Cmax , Tmax , and the like . When phenylacetic , glutamic , benzoic , salicylic , mesylic , esylic , used in relationship to a value for a pharmacokinetic param besylic , sulfanilic , 2 - acetoxybenzoic , fumaric , toluenesulfo eter, the term “ about” means from 80 % to 125 % of the nic , methanesulfonic , ethane disulfonic , oxalic, isethionic , reference parameter. HOOC - (CH ) . – COOH where n is 0 - 4 , and the like ; [0075 ] “ Cenicriviroc” refers to the chemical compound organic amine salts such as triethylamine salt , pyridine salt , ( S ) - 8 - [ 4 - ( 2 - Butoxyethoxy )phenyl ] - 1 -isobutyl - N - ( 4 - { [ ( 1 picoline salt , ethanolamine salt , triethanolamine salt , dicy propyl- 1H - imidazol- 5 - yl) methyl ] sulfinyl } phenyl) - 1 , 2 , 3 , 4 clohexylamine salt, N , N -dibenzylethylenediamine salt , and tetrahydro - 1 - benzazocine - 5 - carboxamide ( structure shown the like ; and amino acid salts such as arginate , asparginate , below ) . Details of the composition of matter of cenicriviroc glutamate , and the like; or a combination comprising one or are disclosed in US Patent Application Publication No . more of the foregoing salts . 2012 /0232028 which is hereby incorporated by reference in [0080 ] In one embodiment, the acid addition salt of ceni its entirety for all purposes. Details of related formulations criviroc is cenicriviroc mesylate , e . g ., ( S ) - 8 - [ 4 - ( 2 - Butoxy are disclosed in U .S . Application No. 61/ 823, 766 which is ethoxy )phenyl ] - 1 - isobutyl- N - ( 4 - { [ ( 1 -propyl - 1H - imidazol hereby incorporated by reference in its entirety for all 5 -yl )methyl ] sulfinyl }phenyl ) - 1 , 2, 3 ,4 - tetrahydro - 1 purposes . benzazocine -5 -carboxamide monomethanesulfonoate . In one embodiment , the cenicriviroc mesylate is a crystalline material , such as a pale greenish - yellow crystalline powder. H3C In one embodiment, the cenicriviroc mesylate is freely soluble in glacial acetic acid , methanol, benzyl alcohol, CH3 H3C N dimethylsulfoxide, and N , N -dimethylformamide ; soluble in pyridine and acetic anhydride ; and sparingly soluble in 99 . 5 % ethanol; slightly soluble in acetonitrile , 1 - octanol, and tetrahydrofuran ; and practically insoluble in ethyl acetate and diethylether . In one embodiment, the cenicrivi ZTE roc mesylate is freely soluble in aqueous solution from pH ????? 1 to 2 ; sparingly soluble at pH 3 and practically insoluble [0076 ] “ Compound of the present invention ” or “ the pres from pH 4 to 13 and in water. ent compound ” refers to cenicriviroc or a salt or solvate 0081 ] “ Solvate " means a complex formed by solvation thereof. (the combination of solvent molecules with molecules or US 2019 /0099429 A1 Apr . 4 , 2019 ions of the active agent of the present invention ) , or an [0087 ] Fibrosis is similar to the process of scarring , in that aggregate that consists of a solute ion ormolecule ( the active both involve stimulated cells laying down connective tissue , agent of the present invention ) with one or more solvent including collagen and glycosaminoglycans . Cytokines molecules . In the present invention , the preferred solvate is which mediate many immune and inflammatory reactions hydrate . play a role in the development of fibrosis . Hepatocyte [0082 ] “ Pharmaceutical composition ” refers to a formula damage resulting from factors such as fat accumulation , tion of a compound of the disclosure and a medium gener viral agents , excessive alcohol consumption , hepatoxins, ally accepted in the art for the delivery of the biologically inevitably triggers an inflammatory immune response . The active compound to mammals , e . g . , humans. Such a medium increased production of cytokines and chemokines in the includes all pharmaceutically acceptable carriers , diluents or liver leads to recruitment of pro - inflammatory monocytes excipients therefor. ( precursor cells ) that subsequently mature into pro - inflam [0083 ] “ Treating ” includes ameliorating, mitigating , and matory macrophages . Pro -inflammatory macrophages are reducing the instances of a disease or condition , or the pro - fibrogenic in nature and ultimately lead to the activation symptoms of a disease or condition . of hepatic stellate cells (HSCs ) that are primarily responsible [0084 ] “ Administering ” includes any mode of administra for the deposition of extracellular matrix (ECM ) . tion , such as oral , subcutaneous, sublingual, transmucosal, [0088 ] Infiltration of various immune cell populations, parenteral, intravenous, intra - arterial, buccal, sublingual, resulting in inflammation , is a central pathogenic feature topical, vaginal, rectal , ophthalmic , otic , nasal, inhaled , and following acute - and chronic liver injury . Chronic liver transdermal. “ Administering " can also include prescribing inflammation leads to continuous hepatocyte injury which or filling a prescription for a dosage form comprising a can lead to fibrosis , cirrhosis , ESLD , and HCC . Interactions particular compound . “ Administering ” can also include pro between intra -hepatic immune cells lead to increased acti viding directions to carry out a method involving a particular vation and migration of Kupffer cells and HSCs and are compound or a dosage form comprising the compound . critical events for developing liver fibrosis . Additionally , [ 0085 ) “ Therapeutically effective amount" means the there is increasing evidence of the role of CCR2 and CCR5 amount of an active substance that , when administered to a in the pathogenesis of liver fibrosis [ 1 -7 ,9 , 31 ]. These subject for treating a disease, disorder, or other undesirable members of the C - C chemokine family are expressed by medical condition , is sufficient to have a beneficial effect pro - fibrogenic cells including pro - inflammatory monocytes with respect to that disease , disorder , or condition . The and macrophages, Kupffer cells , and HSCs [1 - 4 ]. CCR2 therapeutically effective amount will vary depending on the signaling plays an important role in the pathogenesis of renal chemical identity and formulation form of the active sub fibrosis through regulation of bone marrow -derived fibro stance , the disease or condition and its severity , and the age , blasts [ 8 ] . CCR2- and CCR5 -positive monocytes as well as weight, and other relevant characteristics of the patient to be CCR5 -positive T lymphocytes are attracted by locally treated . Determining the therapeutically effective amount of released MCP - 1 and RANTES , and can contribute to a given active substance is within the ordinary skill of the art chronic interstitial inflammation in the kidney [ 10 , 11 ] . In and typically requires no more than routine experimentation . rodents , CVC has high distribution in the liver, mesenteric lymph node , and intestine also described as the gut - liver Fibrosis: axis . Disruption of the intestinal microbiota and its down [0086 ] Fibrosis is the formation of excess fibrous connec stream effects on the gut- liver axis both play an important tive tissue in an organ or tissue in a reparative or reactive role in metabolic disorders such as obesity, non - alcoholic process . This can be a reactive , benign , or pathological state . fatty liver disease (NAFLD ) and non -alcoholic steatohepa The deposition of connective tissue in the organ and /or titis (NASH ) [ 16 , 23 ]. tissue can obliterate the architecture and function of the [0089 ] Table 1 lists chemokines expressed by liver cells underlying organ or tissue . Fibrosis is this pathological state [ 30 ] .
Cell type Chemokine Hepatocytes MCP - 1 ( CCL2 ) [ 38 ], MIP - 1a ( CCL3) [ 74 ] , RANTES ( CCL5 ) [ 16 , 74 ], MIP - 3B ( CCL19 ) [75 ] , SLC (CCL21 ) [ 75 ], Mig ( CXCL9 ) [64 ], IP - 10 (CXCL10 ) [64 ], CXCL16 [ 76 ], LEC (CCL16 ) [77 ], IL - 8 ( CXCLS ) [78 ] and Eotaxin ( CCL11) [41 ] Stellate cells MCP - 1 (CCL2 ) [52 , 60 ] ,, IP - la (CCL3 ) [60 ] , MIP - 1B ( CCL4 ) [60 ] , CX CL1 [59 ] , KC (CXCL1 ) [ 60 ] , MIP - 2 ( CXCL2) [60 ] , IP - 10 (CXCL10 ) [60 ] and SLC ( CCL21) [70 ] Kupffer cells MCP1 ( CCL2 ) 152 , 38 , 60 , 79 ) , MIP - 1a ( CCL3 ) [ 80 ] and MIP -3a ( CCL20 ) [ 56 ] Liver endothelial cells MCP - 1 ( CCL2) [52 ] , IL - 8 ( CXCL8 ) [81 , 76 ) , CXCL16 [ 75 ] , Mig (CXCL9 ) [69 ] , IP - 10 (CXCL10 ) [69 ] , CXCL16 [65 ], CXCL1 [82 ] , SLC ( CCL21) [83 ], Eotaxin (CCL11 ) [41 ] and TECK ( CCL25 ) [73 ] * Summarizes selected experimental data from humans and mice / rats regarding the expression of chemokines by different resident hepatic cell populations upon activation or following liver injury . P : Interferon - inducible protein ; KC : Kupffer cell ; LEC : Liver- expressed chemokine ; MCP : Monocyte chemoattractant protein ; MIP : Macrophage inflammatory protein ; SLC : Secondary lymphoid - organ chemokine; TECK : Thymus- expressed chemokine of excess deposition of fibrous tissue , as well as the process of connective tissue deposition in healing . plays an important role in the pathogenesis of hepatic US 2019 /0099429 A1 Apr . 4 , 2019 fibrosis . Following liver injury, hepatic stellate cells (HSCs ) [0094 ] Fibrosis can occur in many tissues within the body become activated and express a combination of matrix including but not limited to , the lungs, liver, bone marrow , metalloproteinases (MMPs ) and their specific tissue inhibi- joints , skin , digestive tract, lymph nodes , blood vessels , or tors ( TIMPs) [ 32 ]. In the early phases of liver injury , HSCs heart and typically is a result of inflammation or damage . transiently express MMP - 3 , MMP - 13 , and uroplasminogen Non - limiting examples include Pulmonary fibrosis , Idio activator (UPA ) and exhibit a matrix -degrading phenotype . pathic pulmonary fibrosis , Cystic fibrosis , Cirrhosis , Degradation of the extracellular matrix does not appear to be Endomyocardial fibrosis , myocardial infarction , Atrial CCR2 or CCR5 dependent. Fibrosis, Mediastinal fibrosis , Myelofibrosis , Retroperito [ 0091] Activated HSCs can amplify the inflammatory neal fibrosis , Progressive massive fibrosis , complications response by inducing infiltration of mono - and polymorpho from pneumoconiosis , Nephrogenic systemic fibrosis , nuclear leucocytes . Infiltrating monocytes and macrophages Crohn ' s Disease , Keloid , Scleroderma /systemic sclerosis , participate in the development of fibrosis via several mecha Arthrofibrosis , Peyronie ' s disease , Dupuytren ' s contracture , nisms, including increased secretion of cytokines and gen fibrosis associated with atherosclerosis , lymph node fibrosis , eration of oxidative stress - related products. Activated HSCS and adhesive capsulitis . can express CCR2 and CCR5 and produce chemokines that include MCP -1 , MIP - la , MIP - 1ß and RANTES . CCR2 Embodiments of Therapeutic Utilities : promotes HSC chemotaxis and the development of hepatic [0095 ] The present invention provides methods of treating fibrosis . In human liver diseases , increased MCP - 1 is asso fibrosis . Anti - fibrotic effects of CVC in animal studies were ciated with macrophage recruitment and severity of hepatic observed when CVC treatment was initiated at the onset of fibrosis and primary biliary cirrhosis . CCR5 stimulates HSC liver injury ( TAA ) or soon after ( TAA ; HFD ) but not once migration and proliferation . cirrhosis was established ( TAA ) . This suggests that anti [0092 ] In the later stages of liver injury and HSC activa fibrotic effects of CVC may be more pronounced in popu tion , the pattern changes and the cells express a combination lations with established liver fibrosis and at significant risk of MMPs that have the ability to degrade normal liver of disease progression . These include: Non -alcoholic hepa matrix , while inhibiting degradation of the fibrillar collagens tosteatosis (NASH ) associated with type 2 diabetes mellitus that accumulate in liver fibrosis . This pattern is characterized ( T2DM ) and metabolic syndrome (MS ) ; HIV and HCV by the combination of pro -MMP - 2 and membrane type 1 co - infection , or HCV infection . (MT1 ) -MMP expression , which drive pericellular genera tion of active MMP - 2 and local degradation of normal liver NASH matrix . In addition there is a marked increase in expression of TIMP- 1 leading to a more global inhibition of degradation [0096 ] The compositions of the invention may be used to of fibrillar liver collagens by interstitial collagenases (MMP treat liver fibrosis resulting from Nonalcoholic Steatohepa 1 /MMP - 13 ) . In liver injury associated with chronic alco titis (NASH ), a common liver disease that affects 2 to 5 holic liver disease , the production of TNF - a , IL - 1 , IL - 6 , as percent of Americans. Although liver damage due to NASH well as the chemokine IL - 8 /CXCL8 is increased . TNF - a is has some of the characteristics of alcoholic liver disease , it also an important mediator of non -alcoholic fatty liver occurs in people who drink little or no alcohol. The major disease . These pathways play a significant role in the pro feature in NASH is fat in the liver, along with inflammation gression of liver fibrosis. Inhibiting the activation of HSCS and hepatocyte damage (ballooning ) . NASH can be severe and accelerating the clearance of activated HSCs may be and can lead to cirrhosis , in which the liver is permanently effective strategies for resolution of hepatic fibrosis . damaged and scarred and no longer able to work properly . [0093 ] Chemokine families play important regulatory Nonalcoholic fatty liver disease (NAFLD ) is a common , roles in inflammation . Members of this family include , but often " silent” , liver disease associated with obesity related are not limited to CXC receptors and ligands including but disorders, such as type 2 diabetes and metabolic syndrome, not limited to CXCR1, CXCR2 , CXCR3 , CXCR4 , CXCR5 , occurring in people who drink little or no alcohol and is CXCR6 , CXCR7 , CXCR8 , CXCRO , CXCR10 , CXCL1, characterized by the accumulation of fat in the liver with no CXCL2 , CXCL3 , CXCL4 , CXCL5 , CXCL6 , CXCL7 , other apparent causes . [ 32 -43 ] At the beginning of the CXCLS, CXCL9, CXCL10 , CXCL11 , CXCL12 , CXCL13 , NAFLD spectrum is simple steatosis , which is characterized CXCL14 , CXCL15 , CXCL16 , and CXCL17 ; the CC by a build - up of fat within the liver . Liver steatosis without chemokines and receptors including but not limited to inflammation is usually benign and slow or non - progressive . CCL1, CCL2 , CCL3, CCL4 , CCL5 , CCL6 , CCL7 , CCL8 , NASH is a more advanced and severe subtype of NAFLD CCL9, CCL10 , CCL11, CCL12 , CCL13 , CCL14 , CCL15 , where steatosis is complicated by liver- cell injury and CCL16 , CCL17 , CCL18 , CCL19 , CCL20 , CCL21, CCR1, inflammation , with or without fibrosis . CCR2, CCR3, CCR4, CCR5, CCR6 , CCR7, CCR8, CCR , and CCR10 ; the C chemokines including but not limited to has contributed to a rapid increase in the prevalence of XCL1, XCL2 , and XCR1 ; and the CX3C chemokines NASH . Approximately 10 % to 20 % of subjects with including but not limited to CS3CL1 and CX3CR1. These NAFLD will progress to NASH [ 44 ] . molecules may be upregulated in fibrotic organs or tissues . [0098 ] NAFLD is the most common cause of chronic liver In further embodiments , these molecules may be downregu disease . [ 45 ] Most US studies report a 10 % to 35 % preva lated in fibrotic organs or tissues . In further embodiments , themolecules in the signaling pathways of these chemokines study population and the method of diagnosis . [ 46 ] Since may be upregulated in fibrotic organs or tissues. In further approximately one - third of the US population is considered embodiments , the molecules in the signaling pathways of obese , the prevalence of NAFLD in the US population is these chemokines may be downregulated in fibrotic organs likely to be about 30 % . [46 ] One study has found that or tissues . NAFLD affects approximately 27 % to 34 % of Americans , or US 2019 /0099429 A1 Apr . 4 , 2019 an estimated 86 to 108 million patients. [ 44 ] NAFLD is not In a further embodiment, the subject has insulin resistance . unique to the US . Reports from the rest of the world , In further embodiments , the subject has increased blood including Brazil, China, India , Israel, Italy , Japan ,Korea , Sri glucose concentrations , high blood pressure, elevated cho Lanka , and Taiwan , suggest that the prevalence rate ranges lesterol levels , elevated triglyceride levels , or is obese . In a from 6 % to 35 % (median of 20 % ) . [46 ] A study by the further embodiment, the subject has Polycystic ovary syn Gastroenterological Society of Australia / Australian Liver drome. Association has found that NAFLD affects an estimated 5 . 5 million Australians, including 40 % of all adults aged 250 [0103 ] In one embodiment, the invention provides a years. [ 47 ] An Australian study of severely obese patients method of treatment, wherein the cenicriviroc or a salt or found that 25 % of these patients had NASH . [48 ] solvate thereof is coadministered with one or more addi [0099 ] Liver biopsy is required to make a definitive diag tional active agents . In a further embodiment, the one or nosis of NASH . In a US study ofmiddle -aged individuals , more additional active agents are one or more antiretroviral the prevalence of histologically confirmed NASH was agents selected from entry inhibitors , nucleoside reverse 12 . 2 % . [ 49 ] Current estimates place NASH prevalence at transcriptase inhibitors , nucleotide reverse transcriptase approximately 9 to 15 million in the US (3 % to 5 % of the inhibitors , non - nucleoside reverse transcriptase inhibitors , US population ) , with similar prevalence in the EU and protease inhibitors , integrase strand transfer inhibitors , China .[ 46 , 50 ] The prevalence of NASH in the obese maturation inhibitors , and combinations thereof. In a further embodiment, the one or more additional antiretroviral agents In an autopsy series of lean individuals from Canada , the are selected from the group consisting of lamivudine, efa prevalence of steatohepatitis and fibrosis was 3 % and 7 % , virenz, raltegravir, vivecon , bevirimat , alpha interferon , respectively . [ 46 ] The prevalence of NASH is also increasing zidovudine , abacavir , lopinavir , ritonavir , tenofovir , tenofo in developing regions, which has been attributed to people vir disoproxil , tenofovir prodrugs, emtricitabine , elvitegra in these regions starting to adopt a more sedentary lifestyle vir, cobicistat, darunavir , atazanavir , rilpivirine , dolutegra and westernized diet [51 ] consisting of processed food with vir, and a combination thereof. In a further embodiment, the high fat and sugar/ fructose content. [52 ] one or more additional active agents are one or more [0100 ) NASH is a serious chronic liver disease defined by immune system suppressing agents . In a further embodi the presence of hepatic steatosis and inflammation with ment, the one or more additional active agents are selected hepatocyte injury, with or without fibrosis . [ 34 ] Chronic from the group consisting of cyclosporine , tacrolimus, pred liver inflammation is a precursor to fibrosis , which can nisolone , hydrocortisone, sirolimus , everolimus, azathio progress to cirrhosis , end - stage liver disease and hepatocel prine , mycophenolic acid , methotrexate , basiliximab , dacli lular carcinoma. In addition to insulin resistance , altered zumab , rituximab , anti -thymocyte globulin , anti lipid storage and metabolism , accumulation of cholesterol lymphocyte globulin , and a combination thereof. within the liver, oxidative stress resulting in increased [0104 ] Certain embodiments include methods for moni hepatic injury , and bacterial translocation [ 34 , 53 - 56 ] second toring and /or predicting the treatment efficacy of the present ary to disruption of gut microbiota ( associated with high treatment as described herein . Such methods include detect fructose -containing diet) have all been implicated as impor ing the level of one or more biologicalmolecules , such as for tant co - factors contributing to progression of NASH . [ 57 -601 example , biomarkers, in a subject ( or in a biological sample Due to the growing epidemic of obesity and diabetes , NASH from the subject ) treated for fibrosis or a fibrotic disease or is projected to become the most common cause of advanced condition , wherein an increase or decrease in the level of one liver disease and the most common indication for liver or more biological molecules compared to a predetermined transplantation . [ 46 , 61 - 63 ] The burden of NASH , combined standard level indicates or is predictive of the treatment with a lack of any approved therapeutic interventions, rep efficacy of the present treatment. resents an unmet medical need . [0101 ] In further embodiments , liver fibrosis is associated [0105 ] In one embodiment, the invention provides a with emerging cirrhosis . In some embodiments, the cirrhosis method of treatment, comprising detecting the level of one is associated with alcohol damage . In further embodiments , or more biological molecules in the subject treated for the cirrhosis is associated with a hepatitis infection , includ fibrosis or the fibrotic disease or condition , and determining ing but not limited to hepatitis B and hepatitis C infections, a treatment regimen based on an increase or decrease in the primary biliary cirrhosis (PBC ) , primary sclerosing cholan level of one or more biological molecules , wherein the gitis , or fatty liver disease . In some embodiments , the biologicalmolecule is selected from the group consisting of present invention provides for methods of treating subjects lipopolysaccharide ( LPS ) , LPS -binding protein (LBP ) , 16S at risk of developing liver fibrosis or cirrhosis . rDNA , SCD14 , intestinal fatty acid binding protein [ 0102 ] In another embodiment, the fibrosis comprises non ( I -FABP ) , zonulin - 1 , Collagen lal and 3al, TGF- B , cirrhotic hepatic fibrosis . In another further embodiment, the fibronectin - 1 , hs -CRP , IL - 1B , IL - 6 , IL - 33 , fibrinogen , MCP subject is infected by human immunodeficiency virus (HIV ) . 1 , MIP - la and - 1B , RANTES , SCD163, TGF - B , TNF - a , a In yet a further embodiment, the subject is infected with a biomarker of hepatocyte apoptosis such as CK - 18 ( caspase hepatitis virus , including but not limited to HCV (hepatitis cleaved and total) , or biomarkers of bacterial translocation C virus ) . In further embodiment, the subject has diabetes. In such as LPS , LBP , sCD14 , and I- FABP, or a combination a further embodiment , the subject has type 2 diabetes . In a thereof. further embodiment, the subject has type 1 diabetes . In a [0106 ] In one embodiment, the invention provides a further embodiment, the subject has metabolic syndrome method of treatment, comprising detecting the level of one (MS ) . In further embodiments , the subject has one or more or biologicalmolecules in the subject treated for fibrosis or of these diseases or disorders . In a further embodiment, the the fibrotic disease or condition , wherein an increase or subject is at risk of developing one or more of these diseases. decrease in the level of one or more biological molecules US 2019 /0099429 A1 Apr . 4 , 2019 compared to a predetermined standard level is predictive of term " granules ” as used herein , refers to dry granular the treatment efficacy of fibrosis or the fibrotic disease or preparations which are prepared by mixing the compounds condition . and suitable auxiliary materials and have a certain particle 10107 ] In a further embodiment, the one or more biologi size. Granules can be divided into soluble granules (gener cal molecules are measured in a biological sample from a ally referred to as granules) , suspension granules, efferves subject treated for fibrosis or the fibrotic disease or condi cent granules, enteric - coated granules, sustained - release tion . In yet a further embodiment, the biological sample is granules , controlled -release granules and the like. The term selected from blood , skin , hair follicles , saliva , oral mucous , “ oral solutions ” as used herein , refers to a settled liquid vaginal mucous, sweat, tears, epithelial tissues, urine , preparation which is prepared by dissolving the compounds semen , seminal fluid , seminal plasma, prostatic fluid , pre in suitable solvents for oral administration . The term “ oral ejaculatory fluid (Cowper 's fluid ), excreta , biopsy, ascites, suspensions ” as used herein , refers to suspensions for oral cerebrospinal fluid , lymph , brain , and tissue extract sample administration , which are prepared by dispersing the or biopsy sample . insoluble compounds in liquid vehicles, also including dry suspension or concentrated suspension . The term “ syrups ” Dosages and Administration : as used herein , refers to a concentrated sucrose aqueous [0108 ] A dosage of a particular subject can be determined solution containing the compounds . The injectable dosage according to the subject 's age, weight , general health con form can be produced by the conventionalmethods in the art ditions , sex , meal, administration time, administration route , of formulations , and aqueous solvents or non - aqueous sol excretion rate and the degree of particular disease conditions vents may be selected . The most commonly used aqueous to be treated by taking into consideration of these and other solvent is water for injection , as well as 0 . 9 % sodium factors . chloride solution or other suitable aqueous solutions. The [0109 ] The present invention provides a method of treat commonly used non - aqueous solvent is vegetable oil , ment, wherein the cenicriviroc or a salt or solvate thereof is mainly soy bean oil for injection , and others aqueous solu formulated as an oral composition . tions of alcohol, propylene glycol, polyethylene glycol, and [0110 ] The present invention provides a method of treat etc . ment, wherein the cenicriviroc or a salt or solvate thereof is (0113 ] In one embodiment, a pharmaceutical composition administered , for example , once per day or twice per day . comprising cenicriviroc or a salt thereof and fumaric acid is The dosage form can be administered for a duration of time provided . In certain embodiments , the cenicriviroc or salt sufficient to treat the fibrotic disease or condition . thereof is cenicriviroc mesylate . 10111 ] In the case of oral administration , a daily dosage is [0114 ] In further embodiments , the weight ratio of ceni in a range of about 5 to 1000 mg, preferably about 10 to 600 criviroc or salt thereof to fumaric acid is from about 7 : 10 to mg , and more preferably about 10 to 300 mg, most prefer about 10 : 7 , such as from about 8 : 10 to about 10 : 8 , from ably about 15 to 200 mg as the active ingredient (i . e . as the about 9 : 10 to about 10 : 9 , or from about 95 : 100 to about compound of the invention ) per an adult of body weight of 100 : 95 . In other further embodiments , the fumaric acid is 50 kg, and the medicine may be administered , for example , present in an amount of from about 15 % to about 40 % , such once , or in 2 to 3 divided doses a day . as from about 20 % to about 30 % , or about 25 % , by weight [0112 ] The cenicriviroc or a salt or solvate thereofmay be of the composition . In other further embodiments, the ceni formulated into any dosage form suitable for oral or inject criviroc or salt thereof is present in an amount of from about able administration . When the compound is administered 15 % to about 40 % , such as from about 20 % to about 30 % , orally , it can be formulated into solid dosage forms for oral or about 25 % , by weight of the composition . administration , for example , tablets , capsules, pills , gran ules , and so on . It also can be formulated into liquid dosage cenicriviroc or a salt thereof and fumaric acid further com forms for oral administration , such as oral solutions, oral prises one ormore fillers . In more specific embodiments , the suspensions , syrups and the like. The term “ tablets ” as used one or more fillers are selected from microcrystalline cel herein , refers to those solid preparations which are prepared lulose , calcium phosphate dibasic , cellulose , lactose , by homogeneously mixing and pressing the compounds and sucrose, mannitol, sorbitol, starch , and calcium carbonate . suitable auxiliary materials into circular or irregular troches, For example , in certain embodiments , the one or more fillers mainly in common tablets for oral administration , including is microcrystalline cellulose . In particular embodiments , the also buccal tablets , sublingual tablets , buccal wafer , chew weight ratio of the one or more fillers to the cenicriviroc or able tablets , dispersible tablets , soluble tablets , effervescent salt thereof is from about 25 : 10 to about 10 : 8 , such as from tablets , sustained - release tablets , controlled - release tablets , about 20 : 10 to about 10 : 10 , or about 15 : 10 . In other par enteric - coated tablets and the like. The term " capsules” as ticular embodiments , the one or more fillers are present in an used herein , refers to those solid preparations which are amount of from about 25 % to about 55 % , such as from about prepared by filling the compounds, or the compounds 30 % to about 50 % or about 40 % , by weight of the compo together with suitable auxiliary materials into hollow cap sition . In other further embodiments , the composition further sules or sealing into soft capsule materials . According to the comprises one or more disintegrants . In more specific solubility and release property , capsules can be divided into embodiments , the one or more disintegrants are selected hard capsules ( regular capsules ) , soft capsules ( soft shell from cross -linked polyvinylpyrrolidone , cross - linked capsules ), sustained -release capsules , controlled - release sodium carboxymethyl cellulose , and sodium starch glyco capsules, enteric -coated capsules and the like . The term late . For example , in certain embodiments , the one or more " pills ” as used herein , refers to spherical or near- spherical disintegrants is cross - linked sodium carboxymethyl cellu solid preparations which are prepared by mixing the com lose . In particular embodiments , the weight ratio of the one pounds and suitable auxiliary materials via suitable meth - or more disintegrants to the cenicriviroc or salt thereof is ods , including dropping pills , dragee , pilule and the like . The from about 10 : 10 to about 30 : 100 , such as about 25 : 100 . In US 2019 /0099429 A1 Apr . 4 , 2019 other particular embodiments , the one or more disintegrants method further comprises admixing one or more disinte are present in an amount of from about 2 % to about 10 % , grants with the cenicriviroc or salt thereof and fumaric acid such as from about 4 % to about 8 % , or about 6 % , by weight to form the admixture . In other further embodiments , the of the composition . In other further embodiments , the com method further comprises admixing one or more lubricants position further comprises one or more lubricants . In more with the cenicriviroc or salt thereof and fumaric acid to form specific embodiments , the one or more lubricants are the admixture . In other further embodiments , the method selected from talc, silica , stearin , magnesium stearate , and further comprises compressing the dry granulated admixture stearic acid . For example , in certain embodiments , the one into a tablet . In other further embodiments , the method or more lubricants is magnesium stearate . In particular comprises filling a capsule with the dry granulated admix embodiments , the one or more lubricants are present in an amount of from about 0 . 25 % to about 5 % , such as from ture . about 0 .75 % to about 3 % , or about 1 . 25 % , by weight of the 10118 ]. Further, the compound of the invention can be composition . included or used in combination with blood for transfusion [0116 ] In other further embodiments , the composition of cenicriviroc or a salt thereof and fumaric acid is substan the invention can be included or used in combination with tially similar to that of Table 2 . In other further embodi one or more agents that purge latent HIV reservoirs and ments , the composition of cenicriviroc or a salt thereof and added to blood for transfusion or blood derivatives. Usually , fumaric acid is substantially similar to that of Tables 3 and blood for transfusion or blood derivatives are produced by 4 . In other further embodiments , any of the compositions of mixing blood obtained form plural persons and, in some cenicriviroc or a salt thereof and fumaric acid is produced by cases, uninfected cells are contaminated with cells infected a process involving dry granulation . In other further embodi ments , any of the compositions of cenicriviroc or a salt be infected with HIV virus . When the compound of the thereof and fumaric acid has a water content of no more than present invention is added to blood for transfusion or blood about 4 % by weight, such as no more than 2 % by weight, derivatives along with one or more agents that purge latent after six weeks exposure to about 40° C . at about 75 % HIV reservoirs , infection and proliferation of the virus can relative humidity when packaged with desiccant . In other be prevented or controlled . Especially, when blood deriva further embodiments , any of the above- mentioned compo tives are stored , infection and proliferation of the virus is sitions has a total impurity level of no more than about 2 .5 % , effectively prevented or controlled by addition of the com such as no more than 1 . 5 % , after 12 weeks of exposure to pound of the present invention . In addition , when blood for 40° C . at 75 % relative humidity when packaged with des transfusion or blood derivatives contaminated with HIV iccant . In other further embodiments , the cenicriviroc or salt virus are administered to a person , infection and prolifera thereof of any of the above -mentioned compositions has a tion of the virus in the person 's body can be prevented by mean absolute bioavailability after oral administration that is adding the compound of the invention to the blood or blood substantially similar to the bioavailability of the cenicriviroc derivatives in combination with one or more agents that or salt thereof in a solution after oral administration . In yet purge latent HIV reservoirs . For example , usually , for pre further embodiments , the cenicriviroc or salt thereof has an venting HIV infectious disease upon using blood or blood absolute bioavailability of about 10 % to about 50 % , such as derivatives by oral administration , a dosage is in a range of about 27 % , in beagle dogs. about 0 .02 to 50 mg/ kg , preferably about 0 .05 to 30 mg/ kg , [ 0117 ] In another embodiment, a pharmaceutical formu and more preferably about 0 . 1 to 10 mg/ kg as the CCR5 / lation is provided that comprises a composition of cenicri CCR2 antagonist per an adult of body weight of about 60 kg, viroc or a salt thereof and fumaric acid . In further embodi and the medicine may be administered once or 2 to 3 doses ments , the composition in the formulation can be in the form a day . As a matter of course , although the dosage range can of a granulate . In other further embodiments , the composi be controlled on the basis of unit dosages necessary for tion in the formulation is disposed in a capsule shell. In other dividing the daily dosage , as described above , a dosage of a further embodiments , the composition of the formulation is particular subject can be determined according to the sub disposed in a sachet. In other further embodiments , the ject' s age , weight, general health conditions, sex , meal, composition of the formulation is a tablet or a component of administration time, administration route , excretion rate and a tablet . In still other further embodiments , the composition the degree of particular disease conditions to be treated by of the formulation is one or more layers of a multi -layered taking into consideration of these and other factors . In this tablet. In other further embodiments , the formulation com case , the administration route is also appropriately selected prises one or more additional pharmaceutically inactive and , the medicine for preventing HIV infectious disease of ingredients . In other further embodiments, the formulation is the present invention may be added directly to blood for substantially similar to that of Table 9 . In other further transfusion or blood derivatives before transfusion or using embodiments , a tablet having a composition substantially blood derivatives . In such a case , desirably , the medicine of similar to of Table 9 is provided . In other further embodi the present invention is mixed with blood or blood deriva ments , any of the above embodiments are coated substrates . tives immediately to 24 hours before, preferably immedi In another embodiment, methods for preparing any of the ately to 12 hours before, more preferably immediately to 6 above -mentioned embodiments are provided . In further hours before transfusion or using blood derivatives . embodiments , the method comprises admixing cenicriviroc [0119 ] Aside from blood for transfusion or blood deriva or a salt thereof and fumaric acid to form an admixture , and tives , when the compositions of the invention is adminis dry granulating the admixture . In other further embodi tered together with the blood for transfusion or blood ments, the method further comprises admixing one or more derivatives and / or other active agents, the medicine is fillers with the cenicriviroc or salt thereof and fumaric acid administered preferably at the same time of , to 1 hour before to form the admixture. In other further embodiments , the transfusion or using the blood derivatives . More preferably , US 2019 /0099429 A1 Apr . 4 , 2019 for example , the medicine is administered once to 3 times EXAMPLES per day and the administration is continued 4 weeks. Example 1 Combination Therapy : 10120 ] The compound of the invention may be used alone Cenicriviroc Mesylate Compositions or in combination with one or more additional active agents . The one or more additional active agents may be any [0124 ] A series of cenicriviroc mesylate compositions that compound , molecule , or substance which can exert thera were identical except for the identity of the acid solubilizer peutic effect to a subject in need thereof. The one or more were prepared by wet granulation in a Key 1L bowl granu additional active agents may be “ co - administered ” , i . e , lator, followed by tray drying, sieving, mixing and compres administered together in a coordinated fashion to a subject, sion into tablets on a Carver press . The composition of the either as separate pharmaceutical compositions or admixed formulations is shown in Table 2 . in a single pharmaceutical composition . By “ co - adminis tered " , the one or more additional active agents may also be TABLE 2 administered simultaneously with the present compound , or Unit Formula (mg / unit ) be administered separately with the present compound , including at different times and with different frequencies. Ex. 1b Ex . 1c Ex . ld The one or more additional active agents may be adminis Citric Fumaric Maleic Sodium tered by any known route, such as orally , intravenously, Components Acid Acid Acid Bisulfate intramuscularly , nasally , subcutaneously , intra -vaginally , Cenicriviroc Mesylate 28 . 45 28 .45 28 .45 28 . 45 intra -rectally , and the like ; and the therapeutic agent may Mannitol 7 . 88 7 . 88 7 . 88 7 .88 Hydroxypropyl 2 .62 2 .62 2 .62 2 .62 also be administered by any conventional route . In many Cellulose embodiments , at least one and optionally both of the one or Croscarmellose Sodium 1 .75 1 . 75 1 .75 1 . 75 more additional active agents may be administered orally . Croscarmellose Sodium 1 . 75 1 . 75 1 . 75 1 . 75 Citric Acid 43 . 75 — [ 0121] These one or more additional active agents include , Fumaric Acid 43 .75 but are not limited to , one or more anti- fibrotic agents , Maleic Acid 43. 75 antiretroviral agents , immune system suppressing agents , Sodium Bisulfate — 43. 75 and CCR2 and / or CCR5 inhibitors or treatments . When two Silicon Dioxide 0 .43 0 . 43 0 . 43 0 . 43 or more medicines are used in combination , dosage of each Magnesium Stearate 0 . 88 0 . 88 0 . 88 0 . 88 medicine is commonly identical to the dosage of the medi Total 87. 5 87 .5 87. 5 87 .5 cine when used independently , but when a medicine inter feres with metabolism of other medicines , the dosage of each medicine is properly adjusted . Each medicine may be 10125 ]. The tablets were administered to beagle dogs. An administered simultaneously or separately in a time interval oral solution was also administered as a control. The abso for example of less than 12 hours , 24 hours , 36 hours . A lute bioavailabilities of the formulations and of the oral dosage form as described herein , such as a capsule , can be solution were determined , and are shown in FIG . 2 . The administered at appropriate intervals . For example, once per result shows that the cenicriviroc mesylate with fumaric acid day, twice per day, three times per day, and the like . In has a significantly higher bioavailability than any of the particular, the dosage form is administered for example , other solubilizers tested . once or twice per day . Even more particularly, the dosage form is administered once per day . In one embodiment, the Example 2 one or more antiretroviral agents include , but are not limited to , entry inhibitors , nucleoside reverse transcriptase inhibi tors, nucleotide reverse transcriptase inhibitors , non -nucleo Cenicriviroc Mesylate Compositions side reverse transcriptase inhibitors , protease inhibitors, 10126 ] Cenicriviroc mesylate , fumaric acid , microcrystal integrase inhibitors , maturation inhibitors , and combinations line cellulose , cross - linked sodium carboxymethyl cellulose , thereof. In one embodiment, the one or more additional and magnesium stearate were admixed , dry granulated , antiretroviral agents include , but are not limited to , lamivu milled , blended with extragranular microcrystalline cellu dine, efavirenz, raltegravir , vivecon , bevirimat, alpha inter lose , cross - linked sodium carboxymethyl cellulose , and feron , zidovudine , abacavir , lopinavir , ritonavir , tenofovir , magnesium stearate and compressed into tablets having a tenofovir disoproxil , tenofovir prodrugs, emtricitabine , hardness greater than 10 kP and friability less than 0 .8 % elvitegravir , cobicistat, darunavir , atazanavir , rilpivirine , w / w . The resulting tablets had the composition shown in dolutegravir , and a combination thereof. Table 3 . [0122 ] In one embodiment, the one or more immune system suppressing agents include, but are not limited to , TABLE 3 cyclosporine, tacrolimus , prednisolone , hydrocortisone , sirolimus, everolimus, azathioprine , mycophenolic acid , Unit Formula (mg / unit ) methotrexate , basiliximab , daclizumab , rituximab , anti- thy mocyte globulin , anti - lymphocyte globulin , and a combina Components Ex . 2a Ex . 2b Ex . 20 Ex . 2d Ex . 2e Cenicriviroc Mesylate 170 .699 170 .699 170 .699 170 .699 170 .69a tion thereof Fumaric Acid 160 .00 160 .00 160 .00 160 .00 80 .00 [0123 ] The following Examples further illustrate the pres Microcrystalline 252 .68 272 . 18 272 . 18 272 . 18 66 . 35 ent invention in detail but are not to be construed to limit the Cellulose scope thereof. US 2019 /0099429 A1 Apr . 4 , 2019
TABLE 3 -continued Example 4 Unit Formula (mg /unit ) Reference Components Ex . 2a Ex . 2b Ex . 2c Ex . 2d Ex . 2e Crospovidone 19 . 50 [0130 ] The citric acid based formulation of Table 6 was Croscarmellose Sodium 58 . 50 39. 00 39 .00 19 .50 20 .70 prepared as follows. Cenicriviroc, hydroxypropyl cellulose , Magnesium Stearate 8 . 13 8 . 13 8 . 13 8 . 13 2 .55 mannitol, and cross - linked sodium carboxymethyl cellulose were admixed , wet granulated , dried , milled , and blended Total 650. 0 650 .0 650 .0 650 .0 340. 0 with microcrystalline cellulose , cross - linked sodium car " Equivalent to 150 mg cenicriviroc freebase . boxymethyl cellulose , citric acid , colloidal silicon dioxide , " Added in the extragranular portion of the powder blend talc , and magnesium stearate . The resulting blend was [0127 ] By way of illustration , the concentration percent compressed into tablets having a hardness greater than 10 kP age and mass per tablet of the components in Example 2b and friability less than 0 . 8 % w /w . The tablets were coated ( i. e ., Ex . 2b ) are given in Table 4 . with hydroxypropyl methylcellulose , polyethylene glycol 8000 , titanium dioxide, and yellow iron oxide . The coated TABLE 4 tablets thus produced were substantially identical to those disclosed in U .S . Patent Application Publication No . 2008 / Concentration Mass (mg ) Component % w / w ) per tablet 031942 ( see , e . g . , Table 3 ) . Cenicriviroc mesylate 26 . 26 170 .69a TABLE 6 Fumaric acid 24 .62 160 . 00 Microcrystalline cellulose 41 . 87 272 . 18 Component mg/ tablet % w / w Cross - linked sodium 6 . 00 39 . 00 carboxymethyl cellulose Cenicriviroc mesylate 28 .91 4 .68 Magnesium stearate 1 . 25 8 . 13 Mannitol 341. 09 Microcrystalline cellulose 80 . 00 12 . 94 Total 100. 0 650 . 0 Colloidal silicon dioxide 12 .00 2 . 00 equivalent to 150 mg cenicriviroc free base Citric acid anhydrous 75 . 00 12. 14 Hydroxypropyl cellulose 12 . 00 1 . 94 Cross - linked sodium 30 .00 4 . 85 carboxymethyl cellulose Example 3 Talc 12. 00 1 . 94 Magnesium stearate 9 .00 1 . 46 Cenicriviroc Mesylate Compositions Hydroxypropyl methylcellulose 11 .71 1 . 89 Polyethylene glycol 8000 2 .69 0 . 44 [0128 ] Cenicriviroc mesylate , microcrystalline cellulose , Titanium dioxide 3 . 03 0 .49 cross - linked sodium carboxymethyl cellulose , and magne 0 . 57 0 .09 sium stearate were admixed , dry granulated , dried , milled , blended with extragranular microcrystalline cellulose , cross linked sodium carboxymethyl cellulose , fumaric acid , col loidal silicon dioxide, and magnesium stearate and com Example 5 pressed into tablets having a hardness greater than 10 kPand friability less than 0 . 8 % w / w . The resulting tablets had the composition shown in Table 5 . Reference [0131 ] Cenicriviroc and hypromellose acetate succinate TABLE 5 were dissolved in methanol and spray dried into a fine Concentration Mass (mg ) powder containing 25 % cenicriviroc by weight (based on the Component % w /w ) per tablet weight of cenicriviroc free base ). The powder was admixed Cenicriviroc mesylate 26 . 26 28 .45a with colloidal silicon dioxide , microcrystalline cellulose , Fumaric acid 24 . 62 26 . 67 mannitol, sodium lauryl sulfate, cross - linked sodium car Microcrystalline cellulose 41. 87 45 . 36 boxymethyl cellulose , and magnesium stearate. The admix Cross - linked sodium 6 .00 39 . 00 carboxymethyl cellulose ture was compressed into tablets having a hardness greater Magnesium stearate 1 . 25 1 . 35 than 10 kP and friability less than 0 . 8 % w / w . The final composition of the tablets is shown in Table 7 . Total 100 . 0 108 . 3 " equivalent to 25 mg cenicriviroc free base TABLE 7 [0129 ] Notably , the formulation of Table 5 has the same Component Weight % Mass (mg ) ratio of components as that of Table 3b , and differs only in Cenicriviroc ( as mesylate 8 . 33 50 .00 the total amount of the components that are used for each salt ) tablet . Thus, Table 4 shows tablets with 150 mg cenicriviroc Hypromellose acetate 25 .00 150 .00 succinate ( based on free base ) , whereas Table CC - 1 shows tablets with Sodium lauryl sulfate 2 . 00 12 .00 25 mg cenicriviroc (based on free base ) with the same ratio Cross - linked sodium 6 . 00 36 .00 of components as the 150 mg tablets of Example 2b , shown carboxymethyl cellulose in Table 4 . US 2019 /0099429 A1 Apr . 4 , 2019 12
TABLE 7 - continued lation ) . These results are also surprising , because it was not previously known that the method of granulation could have Component Weight % Mass (mg ) an effect on both cenicriviroc bioavailability and stability . Microcrystalline cellulose 27 . 83 167 . 00 Mannitol 27 . 83 167 .00 Example 8 Colloidal silicon dioxide 1 . 00 6 .00 Magnesium stearate 2 . 00 12 . 00 Stability of CVC Formulation Total 100 . 0 600 . 0 [0135 ] The stability of the tablets of Examples 2 and 3 was tested by exposing the tablets to an environment of 75 % relative humidity at 40° C . for six weeks . All tablets were Example 6 packaged with a desiccant during the study. The results are shown in Table 9 , which shows that the tablets are very Bioavailibility of CVC Formulation stable under these conditions . [0132 ] The absolute bioavailability of the tablets of Example 3 in beagle dogs was compared to that of the tablets TABLE 9 of Examples 4 and 5 , as well as to both an oral solution of Time (Weeks ) Water content ( % ) Strength ( % ) Total Impurities ( % ) cenicriviroc mesylate and a gelatin capsule containing ceni 1 . 5 99 . 1 1 . 2 criviroc mesylate powder . The results are shown in Table 8 . 1 . 4 99 . 2 1 . 1 1 . 4 98 . 0 1 . 0 TABLE 8 98 . 6 1 . 0 Component Absolute bioavailability ( % ) Oral Solution 25 . 8 Example 9 Powder in capsule 6 . 4 Example 3 26 . 6 Example 4 21. 1 Stability of CVC Formulations Example 5 12 . 4 [0136 ] Dynamic vapor sorption isotherms at 25° C . cor relate to the stability of the tablets of Examples 3 and 4 with [0133 ] This example demonstrates that the bioavailability that of cenicriviroc mesylate . Sorption was performed from of cenicriviroc in dry granulated tablets with fumaric acid 0 % relative humidity to 90 % relative humidity at 5 % (Ex . 3 ) is substantially similar to that of an oral solution , and intervals . At each interval, each sample was equilibrated for is significantly higher than the bioavailability of cenicriviroc no less than 10 minutes and no longer than 30 minutes . in wet granulated tablets with fumaric ( Ex . 1b ) or citric acid Equilibration was stopped when the rate of mass increase (Ex . 4 ) , and over double that of cenicriviroc in tablets with was no more than 0 .03 % w / w per minute or after 30 minutes , amorphous cenicriviroc in a spray dried dispersion with whichever was shorter. The result, which appears in FIG . 4 , HPMC - AS ( Ex . 5 ) . These results are surprising , because shows that tablets of Example 2b are significantly more there was no reason to suspect that dry granulation of stable than those of Example 4 . This result is consistent with crystalline API provides a significant increase in bioavail Example 3 being significantly less hygroscopic than ability over wet granulation and amorphous spray dried Example 4 . The increased hygroscopicity of Example 4 , in dispersions. This is especially so because amorphous spray comparison to Examples 2b , can be associated with a higher dried dispersions are frequently used to increase the bio mobile water contentwhich can in turn cause partial gelation availability of poorly water soluble drugs . These results are and subsequent decreased stability of Example 4 . also surprising because fumaric acid has a slower dissolution time than citric acid and was used at a lower mass ratio of Example 10 acid relative to CVC API ( 3 : 1 for citric acid : API versus 1 .06 : 1 fumaric acid : API) . Hence it was therefore surprising Bioavailability of CVC Formulations that fumaric acid proved to be a more effective solubilizer [0137 ] The bioavailability of the tablets of Example 3 was than citric acid for CVC . compared to that of Example 5 and cenicriviroc mesylate powder in a gelatin capsule in different stomach states in Example 7 beagle dogs . The bioavailability was tested under different pre- treatment states, each of which alters the gastric pH . Accelerated Stability of CVC Formulation Specifically , pentagastric pretreatment provides the lowest [0134 ] The accelerated stability of the tablets of Example pH , no treatment provides an intermediate pH , and famoti 2b was compared to that of the tablets of Examples 1b , 4 , and dine treatment provides the highest pH . 5 via exposure to an environment of 75 % relative humidity [0138 ] The result , which appears in FIG . 5 , shows that the at 40° C . All tablets were packaged with a desiccant during tablets of Example 3 has a higher bioavailability under all the study. As shown in FIG . 3 , the tablets of Examples 2b are conditions that were tested . The bioavailability of Example surprisingly much more stable than the other wet granulated 3 varied less between pentagastrin treated and untreated tablets , and similarly stable as the spray dried dispersion dogs, whereas Example 4 showed a significant loss of tablets . This difference in stability between the tablets of bioavailability in fasted , non - treated dogs ( intermediate gas Examples 2b and Example 4 is particularly surprising since tric pH ) compared to that in pentagastrin treated dogs the only significant difference between the two is the method (lowest gastric pH ) . Pretreatment with famotidine, an H2 of making the formulations (dry granulation vs. wet granu receptor agonist that suppresses stomach acidity and raises US 2019 /0099429 A1 Apr . 4 , 2019 13 gastric pH decreased bioavailability for all samples , how TABLE 10 -continued ever, the reduction for Example 3 was much less than that Example 12a Example 12b Example 12c for Example 4 . 25 mg 150 mg [0139 ] These results demonstrate an additional unex cenicriviroc cenicriviroc cenicriviroc pected benefit of dry granulated cenicriviroc compositions and 300 mg and 300 mg and 300 mg with fumaric acid . Specifically , the pharmacokinetics of lamivudine lamivudine lamivudine such formulations do not vary as much as those of the spray % mg % mg % mg/ dried dispersion (Example 4 ) when administered across a the w / w tablet w / w tablet w / w tablet full range of potential human gastric pH conditions. This Extragranular Components result is unexpected and surprising , because the bioavail Lamivudine 60 .00 300 .00 31 . 58 600 .00 37. 50 300 .00 ability of other weakly basic antiretroviral drugs, such as (3TC ) atazanavir , is greatly effected by the gastric pH . For such Micro 16 .34 81. 71 6 . 3960 .75 3 . 78 30 . 21 drugs, changes in gastric pH , which can be caused by a crystalline disease or medical condition , such as achlorohydric patients , cellulose Cross - linked 5 .00 25 .00 5 . 26 50 .00 5 .00 40 .00 or by co - administration of drugs such as antacids , proton sodium pump inhibitors, or H2 receptor agonists , can lower the carboxy bioavailability to sub - therapeutic levels . These results show methyl cellulose ing that the dry granulated , fumaric acid based cenicriviroc Magnesium 1. 00 5 .00 1. 00 9 .50 1. 00 8 .00 mesylate formulation of Example 3 is less prone to bioavail stearate ability changes as the gastric pH changes shows that Example 3 is a more robust formulation that can be used in Total per 100 . 00 500 .00 100. 00 950 .00 100 .00 800 . 00 patients who have or are likely to have varying gastric pH tablet levels . Examples 11a -11c Example 12 Anti- Fibrotic And Anti- Inflammatory Activity Of Preparation of Cenicriviroc Mesylate and The Dual CCR2 And CCR5 Antagonist Lamivudine Formulations Cenicriviroc in a Mouse Model of NASH [0140 ] The formulations of cenicriviroc mesylate and [0141 ] Background : Non - alcoholic steatohepatitis lamivudine of Table 10 were prepared as follows. First, the (NASH ) is characterized by fat accumulation , chronic intragranular components were admixed and dry granulated inflammation ( including pro - inflammatory monocytes and to form a composition as a dry granulated admixture . This macrophages ) and when fibrosis is present , it can lead to dry granulated admixture was then further admixed with the cirrhosis or hepatocellular carcinoma. There are currently no extragranular components to form a mixture . The mixture approved therapies for NASH . Evidence suggests that C - C was compressed into tablets . The absolute bioavailability of chemokine receptor (CCR ) type 2 and its main ligand , the cenicriviroc (CVC ) and lamivudine ( 3TC ) in beagle dogs monocyte chemotactic protein - 1 , contribute to pro - inflam in the 150 mg CVC strength tablets (Examples 11b and 11c ) matory monocyte recruitment in the liver . Cenicriviroc were measured . The results are shown in FIG . 6 . (CVC ) is an oral, potent, dual CCR2/ CCR5 antagonist that showed favorable safety and tolerability in a 48 -week Phase 2b study in 143 HIV - 1 - infected adults (NCT01338883 ) . TABLE 10 CVC was evaluated in a mouse model of diet- induced Example 12a Example 12b Example 12c NASH that leads to hepatocellular carcinoma ; data from the 25 mg 150 mg 150 mg first, fibrotic stage of the model are presented . cenicriviroc cenicriviroc cenicriviroc and 300 mg and 300 mg and 300 mg (0142 ] Methods: NASH was induced in male mice by a lamivudine lamivudine lamivudine single injection of 200 ug streptozotocin 2 days after birth (causing impaired glucose control) , followed by a high fat % mg % mg % mg / diet from 4 weeks of age . From 6 to 9 weeks of age , 3 groups w / w tablet w / w tablet w / w tablet of animals (n = 6 /group ) were administered CVC doses of 0 Intragranular Components (vehicle ) , 20 (low dose ) or 100 (high dose ) mg/ kg /day , via twice daily oral gavage . Animals were sacrificed at 9 weeks Cenicriviroc 5 .69 28 .45 17 . 97 170 .69 21. 34 170 .69 mesylate of age , and biochemical, gene expression , and histologic Fumaric 5 . 33 26 .67 16 .84 160 .00 20 .00 160 .00 evaluations of the liver were conducted . Acid [0143 ] Results: CVC treatment had no effect on body or Micro 5 .82 29 .11 18. 39 19 .50 2 .64 21. 10 crystalline liver weight, whole blood glucose , or liver triglycerides . cellulose Mean ( USD ) alanine aminotransferase levels were signifi Cross - linked 0 .65 3 . 25 2 . 05 19 .50 2 .64 21. 10 cantly decreased in both CVC treatment groups compared to sodium control (58 + 12 , 51 + 13 and 133 + 80 U / L for low dose , high carboxy methyl dose and vehicle , respectively ; p < 0 . 05 ) and liver hydroxy cellulose proline tended to decrease in treated groups. By real- time Magnesium 0 .16 0 .81 0. 51 4 .88 0. 53 4. 20 RT- PCR , collagen type 1 mRNA in whole liver lysates stearate decreased by 27 - 37 % with CVC treatment. The percentage of fibrosis area (by Sirius red staining ) was significantly US 2019 /0099429 A1 Apr . 4 , 2019 14 decreased by CVC treatment relative to control ( p < 0 .01 ) : [0148 ] Conclusions: CVC is a potent anti- fibrotic agent in 0 .66 % + 0 . 16 , 0 .64 % + 0 . 19 and 1 . 10 % + 0 . 31 for 20 mg/ kg non - cirrhotic hepatic fibrosis due to TAA . The drug was day , 100 mg/ kg /day and control, respectively , when perivas effective in early intervention (Group 1 ) and in emerging cular space was included ; 0 .29 % + 0 .14 , 0 . 20 % + 0 .06 , and fibrosis (Group 2a ), but not when cirrhosis was already 0 .61 % + 0 .23 , respectively , when perivascular space was sub established (Group 3 ) . tracted . Importantly , the histologic non -alcoholic fatty liver disease activity score ( score is O for untreated mice in this Example 14 model ) was significantly decreased with CVC treatment ( 4 . 0 + 0 .6 , 3 . 7 + 0 . 8 and 5 . 3 + 0 . 5 for low dose , high dose and vehicle , respectively ; p < 0 .05 ), primarily due to reduced Cenicriviroc Achieves High CCR5 Receptor inflammation and ballooning scores . As previously shown in Occupancy at Low Nanomolar Concentrations humans, a CVC dose - related compensatory increase in [0149 ] Background : Cenicriviroc (CVC ) is a novel , once plasmamonocyte chemotactic protein - 1 levels was observed daily , potent, CCR5 and CCR2 antagonist that has com in mice ( 1 . 1 - and 1 . 5 - fold increase for low and high dose , pleted Phase 2b evaluation for the treatment of HIV - 1 respectively ) , consistent with antagonism of CCR2 . infection in treatment- naïve adults (NCT01338883 ). The [0144 ] Conclusions: These data suggest that CVC , an aims of this study were to evaluate in vitro receptor occu investigational agent currently in human trials for HIV - 1 , pancy and biology after treatment with CVC , BMS- 22 has anti - fibrotic and anti - inflammatory activity in a mouse ( TOCRIS , a CCR2 antagonist ) and an approved CCR5 model of NASH , warranting clinical investigation . These antagonist , Maraviroc (MVC ). findings provide further evidence that disrupting the CCR2 / [0150 ] Methodology : PBMCs from 5 HIV + and 5 HIV monocyte chemotactic protein - 1 axis may be a novel treat subjects were incubated with CVC , BMS - 22 or MVC , ment approach for NASH . followed by either no treatment or treatment with a RANTES (CCR5 ligand ) or MCP - 1 (CCR2 ligand ). The Example 13 capacity of each drug to inhibit CCR5 or CCR2 internal ization was evaluated . Cell - surface expression of CCR5 and Significant Anti - fibrotic Activity of Cenicriviroc , a CCR2 was assessed by flow cytometry , and fluorescence Dual CCR2/ CCR5 Antagonist, in a Rat Model of values were converted into molecules of equivalent soluble Thioacetamide - Induced Liver Fibrosis and Cirrhosis fluorescence (MESF ) . [0145 ] Background : C - C chemokine receptor (CCR ) types [0151 ] Results : Both CVC and MVC , in the absence of 2 and 5 are expressed on pro - inflammatory monocytes and RANTES , increased cell -surface expression of CCR5 . This macrophages, Kupffer cells and hepatic stellate cells effect was seen to a much greater degree in HIV -negative (HSCs ) , which contribute to inflammation and fibrogenesis subjects (CD4 + and CD8 + T cells ). CVC prevented in the liver. Cenicriviroc (CVC ; novel, potent, oral, dual RANTES - induced CCR5 internalization at lower effective CCR2 /CCR5 antagonist) had favorable safety / tolerability in concentrations than MVC . The effective concentration at a 48 -week Phase 2b study in 143 HIV - 1 -infected adults which saturation of CCR5 was reached for CVC was 3 . 1 nM ( NCT01338883 ) . This study evaluates the in vivo anti for CD4 + and 2 . 3 nM for CD8 + T cells ( ~ 91 % and - 90 % fibrotic effect of CVC , and timing of treatment intervention receptor occupancy , respectively ). MVC reached saturation relative to disease onset, in rats with emerging hepatic at 12 . 5 nM for both CD4 + and CD8 + T cells , representing fibrosis due to thioacetamide ( TAA ) - induced injury . - 86 % and ~ 87 % receptor occupancy , respectively . CVC and [0146 ] Methods: Fibrosis was induced in male Sprague MVC achieved high but incomplete saturation of CCR5 , an Dawley rats by intraperitoneal administration of TAA 150 effect that may be amplified by the observation of increased mg/ kg 3 times/ week for 8 weeks. Rats ( n = 4 - 8 / group ) CCR5 expression with both agents in the absence of received CVC 30 mg/ kg / day ( a ) , CVC 100 mg/ kg /day ( b ) or RANTES . In the absence of MCP - 1 , CVC induced CCR2 vehicle control ( c ) , concurrently with TAA for the first 8 internalization and decreased cell- surface expression on weeks (Group 1 ; early intervention ) , during Weeks 4 -8 (Group 2 ; emerging fibrosis ) or during Weeks 8 - 12 follow expression . CVC prevented MCP - 1 - induced CCR2 internal ing completion of TAA administration (Group 3 ; cirrhosis ization at lower concentrations than BMS - 22 . Saturation of reversal) . Biochemical, gene expression and histologic monocyte CCR2 was reached at 6 nM of CVC , representing evaluations of the liver were conducted . 98 % CCR2 occupancy . To reach > 80 % receptor occu [0147 ] Results : When started concurrently with TAA pancy, an average of 18 nM of BMS - 22 was required , (Group 1 ), CVC at 30 mg (Group la ) and 100 mg (Group compared to 1 . 8 nM of CVC . 1b ) significantly reduced fibrosis ( by 49 % and 38 % , respec [0152 ] Conclusions: CVC more readily prevented tively ; p < 0 .001 ) , as assessed by collagen morphometry . RANTES - induced CCR5 internalization ( at lower concen Protein levels for collagen type 1 were reduced by 30 % and tration ) than MVC in vitro , indicating CVC more be more 12 % for Groups la and lb , respectively , while a - SMA was effective at preventing cellular activation by RANTES than reduced by 17 % and 22 % , respectively . When treatment MVC in vivo . Baseline CCR5 expression levels in treated started 4 weeks after TAA - induced injury (Group 2 ) , a subjects may be a determinant of CCR5 antagonist activity statistically significant anti - fibrotic effect was observed for in vivo . CVC achieved - 98 % receptor occupancy of CCR2 CVC 30 mg (Group 2a, 36 % reduction in collagen ; p < 0 . on monocytes at low nanomolar concentrations in vitro , and 001 ) , but not for CVC 100 mg (Group 2b ) . When treatment reduced CCR2 expression on monocytes in the absence of was started at Week 8 (cirrhosis present) and continued for MCP - 1 . High saturation of CCR2 by CVC paired with 4 weeks (Group 3 ), there was no significant effect of CVC reduced expression may explain the potent CCR2 blockade on fibrogenic gene expression or fibrosis . observed with CVC in the clinic . CVC has potent immuno US 2019 /0099429 A1 Apr . 4 , 2019 15 modulatory activities in vitro , and may be an important 500 ng of p24 Ag of HIV - 1 Bal were added per 5x105 PM - 1 combined immunotherapeutic and anti -retroviral in chronic cells that were pre - incubated at 37C for 1 hr in the absence HIV infection . of drug treatment. Each drug treatment and control was performed in duplicate . Viral RNA was extracted from 140 Example 15 ul of supernatant fluid using the QIAamp Viral RNA mini kit according to manufacturer ' s instructions . Samples were CVC Blocks HIV Entry but Does Not Lead to stored at - 80C until analysis . Supernatant viral loads were Redistribution of HIV into Extracellular Space Like measured using quantitative real- time reverse transcription MVC PCR (QRT - PCR ) with the primers USISSF (5 ' [0153 ] Background : In vivo , CVC has shown efficacy AACTAGGGAACCCACTGCTTAA - 3 ') , US1SSR ( 5 ' during monotherapy of treatment- experienced individuals harbouring CCR5 -tropic virus 7 . In the phase IIb clinical probe ( 5 ' - ( FAM ) CCTCAATAAAGCTTGCCTTGAGT study (652 - 2 - 202 ; NCT01338883 ) , CVC demonstrated GCTTCAA ) and the Invitrogen qRT- PCR Supermix Kit . similar efficacy at 24 weeks (primary analysis ) to the non Cycling parameters were: 50° C . for 15 minutes , 95° C . for nucleoside reverse transcriptase inhibitor (NNRTI ) efa 10 minutes , followed by 50 cycles of 95° C . for 15 seconds , virenz (EFV ) , and a superior toxicity profile than the non and 60° C . for 1 minute . All values are the result of replicate nucleoside reverse transcriptase inhibitor (NNRTI ) testing over 2 independent experiments. RNA copy number efavirenz ( EFV ) , each when both were administered in com was quantified by use of 10 - fold serial dilutions of pBaL /wt bination with emtricitabine ( FTC ) and tenofovir ( TDF ), with to generate standard curves for each assay and calibrated favorable safety and tolerability . We hypothesized that the against samples with known copy numbers from previous antiretroviral efficacy of CVC in Study 202 ( Example 22 ) studies . might have been underestimated as a result of the rebound [0157 ] Patient Samples : Peripheral blood mononuclear phenomenon observed with MVC . Accordingly we con cells (PBMC ) samples were obtained from 30 patients ( 10 , ducted an ex vivo sub -analysis of Study 202 ( Example 22 ) 13 and 7 on CVC 100 mg, CVC 200 mg and EFV , respec by measuring intracellular HIV DNA declines in stored tively ) who achieved virologic success at week 24 in Study PBMCs from 30 subjects who achieved virologic success at 202 a phase IIb clinical trial comparing the efficacy , safety , week 24 of the study . We also performed in vitro assays to and tolerability of CVC ( 100 mg or 200 mg ) or EFV in determine and compare the extent of any cell - free virion combination with emtricitabine / tenofovir disproxil fumarate redistribution that CVC or MVC might cause . (FTC / TDF ) in HIV - 1 infected , treatment - naive patients har [0154 ] We now show that CVC does not trigger viral boring CCR5- tropic virus. Samples at baseline and 24 weeks particle rebound . Indeed , comparable declines in intracellu were taken from participants possessing baseline viral loads lar DNA were seen in individuals treated with either CVC or of < 100 ,000 but > 1 , 000 viral RNA copies/ ml , with CD4 EFV , suggesting that plasma viral load is an accurate mea counts 2200 cells / ul that were randomly assigned to receive sure of CVC treatment success . Structural modeling pro either CVC or EFV . vides a potential explanation for differences between results [0158 ] Intracellular DNA qPCR . Total DNA was obtained with MVC and CVC . extracted , quantified , and stored at - 80C . Intracellular [0155 ] Methods: Cells . PM - 1 cells that express CD4, strong- stop DNA levels were quantified with the USISS CCR5, and CXCR4 were maintained in RPMI- 1640 primer/ probe set described above . Intracellular full - length medium containing 10 % fetal bovine serum (R10 medium ) DNA levels were quantified using the US1FL primer/ probe at 37C , 5 % CO2. 293T cells used for transfection were set ( Forward : 5 -AACTAGGGAACCCACTGCTTAA ; maintained in DMEM at 10 % FBS , L - glutamine , and anti Reverse : 5 '- CGAGTCCTGCGTCGAGAGA; Probe : 5 biotics (D10 medium ) at 37C , 5 % CO2. Virus Stocks. HIV - 1 [FAM ]- CCTCAATAAAGCTTGCCTTGAGTGCTTCAA ). BaL virus was produced by transfecting 293T cells with the Both DNA levels were multiplexed with a GAPDH primer / plasmid pWT/BaL . Lipofectamine 2000 was used as a probe set ( Forward : 5 ' - ACCGGGAAGGAAATGAATGG ; transfection agent. Culture supernatants were collected at 48 Reverse : 5 -GCAGGAGCGCAGGGTTAGT ; Probe : 5 ' hrs post- transfection , filtered through a 0 .45 um pore filter, (VIC ) - ACCGGCAGGCTTTCCTAACGGCT ) to normalize and treated with 50 units of benzonase per ml of virus stock DNA inputs and verify sample integrity . for 20 minutes at 37C to remove contaminating plasmid [0159 ] Statistical Analysis : The Mann - Whitney test was DNA . Virus stocks were frozen at - 80C to halt benzonase used to analyze in vitro intracellular HIV DNA levels for all activity . Benzonase - treated virus stocks were propagated in three treatment groups. All data were analyzed using Prism cord blood mononuclear cells (CBMCs ) . CBMCs were 5 software . stimulated for 72 h with phytohemagluttinin (PHA - M ) in R10 medium prior to infection with HIV - 1 BaL . The viral Molecular Docking of Cenicriviroc in CCR5 amplification culture was subsequently grown in R10 [0160 ] The crystal structure of the CCR5 chemokine supplemented with interleukin 2 ( IL - 2 ) and incubated at receptor (Protein Data Bank identificationNo . [PDB ID ] 37C , 5 % CO2 . 4MBS ) was obtained through the Research Collaboratory 10156 ) Infections : We exposed PM - 1 cells to HIV - 1 Bal in for Structural Bioinformatics (RCSB ) Protein Data Bank the presence of inhibitory concentrations of CVC (20 nM ) and used as a docking target . The structure of the CCR5 and MVC (50 nM ) . Both drugs were incubated with PM - 1 receptor antagonist, cenicriviroc, ( formerly TAK - 652 / TBR cells for 1 hr at 37C prior to the addition of virus . 500 ng of 652 ) was obtained from PubChem and used as a ligand . p24 antigen of HIV - 1 Bal were incubated per 5x105 cells in Minimization of ligand - docked structures was facilitated by 1 ml of R10 media . Virus only controls , described as “ no the use of a UCSF Chimera , that prepared CCR5 and CVC cell” in the text, were used to measure viral decay . Viral as inputs for DOCK calculations , that predict the orientation adsorption was measured in the no -drug controls , whereby of the ligand in the CCR5 seven - transmembrane (7TM ) US 2019 /0099429 A1 Apr . 4 , 2019 16
a -helix receptor cavity . Docking calculations were per whereas CVC - treated cells exhibited a relative intracellular formed and a maximum sized grid box was used to include DNA level of only 0 . 1 . The difference between relative DNA all possible docking sites into CCR5 . The binding site levels of MVC and CVC - treated cells was significant. consists of all residues less than 15 A from the 7TM cavity 0165 ). A crystal structure exists of the CCR5 7TM com ( around residues Glu283 and Tyr10 ) . Docking results were plexed with MVC (PDB ID 4 MBS ) and this was used to processed to identify inter -molecular interactions . The test generate a model of CCR5 with CVC docked into the nine poses were kept for further analysis . In order to validate binding pocket. We predicted docked poses that were also the accuracy of the docking system , MVC was docked to assessed by re - docking MRV into CCR5 ; the top poses with CCR5 using the same method and its orientation with the most favorable energies had the proper orientation and respect to the crystal structure was determined . The root overlap with the conformation in the crystal structure mean square deviation (RMSD ) , calculated using PyMOL , (RMSD < 0 . 3 A ) . In silico CCR5 docking simulations indi between the observed crystal structure and the predicted cated that CVC binds only at the hydrophobic pocket in the conformation obtained from AutoDock Vina was 0 .275 Å , CCR5 structure, also known as the ligand -binding pocket indicating that the protocol was sound . ( FIG . 10 ) . Only the top 9 poses were kept for further analysis . There are three different conformations that CVC Results exhibits post- docking into CCR5 and they are clustered into [0161 ] First we quantified HIV intracellular DNA in order three sites (FIG . 10A , B ) . The first site ( site 1 ) spans deep to validate measures of viral load that were obtained during into the hydrophobic pocket and fills a large volume (FIG . the Study 202 clinical trial. Ex vivo analyses of full - length 10A ) . The second site ( site 2 ) is partially positioned in the intracellular HIV DNA levels ( indicative of early reverse middle of the pocket but also bulges outward from the CCR5 transcription ) in PBMCs isolated from participants in this between TM1 and TM7 ( FIG . 10A ) . At the third site ( site 3 ) , clinical trial were similar across all groups (CVC 100 mg, few CVC poses are located near the entrance of the receptor CVC 200 mg, EFV 600 mg) at week 24 (FIG . 7A ) . The mean cavity . fold -changes from baseline were 0 .643 and 0 .787 for the [0166 ] Site - directed mutagenesis of residues within the CVC groups 100 mg ( n = 10 ) and CVC 200 mg ( n = 11 ) , extracellular loops and transmembrane domain in CCR5 respectively . The EFV 600 mg group ( n = 7 ) had a mean fold have identified key residues that are involved in gp120 change from baseline of 0 .825 at 24 weeks. The differences binding ;mutations at the different positions either abolished , were not statistically significant. compromised or affected gp120 binding to CCR5 . The [0162 ] Next, strong - stop intracellular HIV DNA levels thirteen key residues that were identified to be important for ( indicative of late reverse transcription ) were measured gp120 binding within CCR5 are Tyr37 , Trp86 , Trp94 , concomitantly with full - length levels at week 24 ( FIG . 7B ) . Leu104 , Tyr108 , Phe109 , Phe112 , Thr177 , Ile198 , Trp248 , The mean fold - change from baseline was 0 .49 for the CVC Tyr251, Leu255 and Glu283. FIG . 11 shows a molecular 100 mg group , 0 .63 for the CVC 200 mg group , and 1 . 01 for surface representation of CCR5 with docked poses of CVC the EFV 600 mg group . The means were not statistically ( left ) and MVC ( right) in the binding pocket. CVC and significant MVC have molecular surface areas of - 1285 and 1790 Å ? [ 0163] In vitro experiments measuring extracellular viral ( calculated using PyMOL ) , respectively . MVC occupies the levels following CVC and MVC exposure were also per middle of the binding pocket. All thirteen residues that were formed . Levels of virus in culture fluids were measured by determined to be important for gp120 binding are within 4 qRT- PCR and P24 ELISA at 4 hrs following infection of Å from MVC , as measured by PyMOL (cut off distance used entry - inhibitor exposed cells . After 4 hrs , culture fluids from in this study for electrostatic and / or hydrophobic interac the MVC - treated cells exhibited higher RNA levels com tions ) . In contrast, the docked CVC poses occupy the same pared to baseline (baseline : 1. 19x1010 copies/ ml , 4 hrs : pocket but not at the center as seen for MVC ( FIG . 11 ) . 1 .67x1010 copies /ml ) (FIG . 8A ) than did CVC -treated cells . Rather , CVC shifts to one side of the pocket (FIG . 12A / B ) ( baseline: 506 ng /ml , 4 hrs : 520 ng /ml ) ( FIG . 8B ) . Viral and a consensus of residues in CCR5 within 4 Å ofCVC was RNA in culture fluids from CVC - treated cells did not change determined . Even though CVC occupies a larger surface significantly after 4 hrs ( baseline : 1 . 19x1010 copies /ml , 4 area than MVC , only seven of the thirteen residues that are hrs : 1 . 26x101° copies/ ml ) (FIG . 8A ) . P24 levels declined important for gp120 binding are within 4 Å of CVC i .e . from baseline after 4 hrs with CVC treatment ( baseline : 506 Tyr37 , Trp86 , Tyr108 , Phe109 , Ile198 , Leu255 and Glu283 . ng /ml , 4 hrs : 192 ng /ml ) (FIG . 8B ) the viral RNA declines Overall , these simulations suggest that CVC occupies a for the no cell and no drug controls were similar after 4 hrs , region similar to MVC in the binding pocket of CCR5 . 1 . 14x1010 copies /ml , and 1 . 1x1010 copies /ml respectively [0167 ] Discussion : In this study , we observe that CVC and ( FIG . 8A ) . Following a baseline p24 level of 506 ng/ ml , the MVC , both CCR5 antagonists preventing HIV entry , have a p24 antigen level for the no cell control after 4 hrs was 138 differential effect on extracellular virus levels. ng/ ml . The p24 no drug control level was 244 ng /ml (FIG . [0168 ] In a phase IIb double - blind , double - dummy study 8B ) . comparing CVC with EFV , both with FTC / TDF in treat 101641. These differences in extracellular virus levels fol ment- naive subjects , 76 % of patients receiving CVC 100mg lowing CVC and MVC treatment prompted us to examine intracellular strong - stop HIV DNA levels in PM - 1 cells weeks compared to 73 % of patients receiving CVC 200 mg exposed to either CVC or MVC for 1 hr before being and 71 % of patients receiving EFV . We previously showed infected with HIV - 1 BaL . Total DNA was extracted from that MVC might artificially increase viral load , because cell pellets after 4 hrs . Intracellular strong - stop HIV DNA cell - free virions can be repelled from the target cell follow levels of CVC or MVC - treated cells were compared to no ing a failed attempt at entry in the presence of MVC . The drug controls ( FIG . 9 ) . We observed a relative DNA level of current study was designed to address whether the same 0 .02 in MVC - treated cells compared to the no drug control effect might occur for CVC and whether intracellular DNA US 2019 /0099429 A1 Apr . 4 , 2019 measurements might be a more accurate representation of been shown to be important for CCR5 activation , explaining antiviral efficacy when comparing entry and reverse tran the inactivation of the chemokine receptor. A second hypoth scriptase inhibitors . esis is that the binding of MVC to CCR5, may cause CCR5 [0169 ] In fact, intracellular DNA levels across Study 202 to undergo a global conformational change , that may be less treatment arms were similar at 24 weeks ( FIG . 7 ) in selected altered in the presence of CVC . [0174 ] Based on site -directed mutagenesis experiments by treat (ITT ) analysis . Full - length HIV -DNA levels were also other groups and the tissue culture experiments and docking similar for all groups at week 24 , suggesting similar antiviral simulations presented in this study, we hypothesize that efficacy for both CVC and EFV . Differences in strong - stop MVC occupies the middle of the hydrophobic pocket , poten HIV DNA levels were observed between the CVC and EFV tially leading to an inaccessibility of some of residues in groups , whereas both CVC groups exhibited steeper CCR5 that are important for gp120 binding . These residues declines in viral load compared to EFV . As strong - stop HIV may also be important for gp120 binding through direct DNA levels are directly impacted by entry inhibitors , this electrostatic , or hydrophobic interactions and/ or water -me result was expected . The similarities between EFV and CVC diated hydrogen bonds. In contrast, CVC occupies the in terms of virologic success and intracellular HIV DNA binding site , and it may be that gp120 can still access some levels suggest that the antiviral potency of this dual CCR5 of the residues important for CCR5 binding even in the and CCR2 inhibitor is not masked by viral load measure presence of docked CVC . This hypothesis is supported by ments . site - directed mutagenesis studies that suggest that gp120 [0170 ] We also asked whether CVC can result in virus partly fills the receptor cavity while occupying the entirety repulsion as seen forMVC in vitro . Two separate measure of ECL2 . However , the degree to which the V3 loop of ments of virus quantitation , qRT- PCR and p24 ELISA , gp120 penetrates the CCR5 7TM remains unknown . It has showed that MVC treatment maintained extracellular viral also been reported that dissociation rates of gp120 from levels up to 4 hrs post - infection . In contrast , treatment with CCR5 are accelerated in the presence of MVC , since the CVC resulted in a decline in viral levels decline at 4 hrs, latter hinders the tight association between ECL2 and the V3 comparable to that of the no drug or no cell controls (FIG . loop . Based on these studies , CVC may have a different 8 ) . Despite an ostensibly similar antiviral mechanism , there effect on the ECL2 /V3 interaction than does MVC . Disso appear to be differences between CVC and MVC in regard ciation and surface plasma resonance studies as well as to interactions between cell - free virus and CCR5 . crystallization of CCR5 in complex with CVC will provide [0171 ] A further examination of intracellular strong -stop valuable information on this topic . DNA in vitro showed that CVC caused a slight albeit 10175 ] Site -directed mutagenesis and biochemical studies significant increase in levels compared to MVC (FIG . 9 ) . are required to elucidate the residues that are important for This may be due to the differential effect of both inhibitors CCR5 interaction with CVC . Determining the proximal on CCR5, which , in turn , affects the rate of dissociation location of the N terminus of CCR5 is also of interest. between virus and receptor. This raises the possibility that 0176 ] In this study, we have demonstrated that, viral load gp120 may associate more durably with CVC - bound CCR5 quantification is an accurate measurement of the antiviral compared to MVC . efficacy of CVC , and that inhibition of viral entry by CVC [0172 ] We also aimed to understand how CVC inhibits does not lead to the rebound of viral particles from the cell HIV entry into target cells by examining the binding site of surface to the extracellular environment. Our in silico struc CCR5 . An engineered human CCR5 construct has been ture modeling provides a potential explanation for functional previously crystalized in complex with MVC at a resolution differences between CVC and MVC . Further studies are of 2 .7 A . Although this is not a full -length crystal structure required to understand how CVC affects gp120 binding to of CCR5, it was utilized to better understand CVC interac CCR5. tions with CCR5 in in silico docking assays. All purported docking models for CVC imply a deep penetration of the Example 16 drug into the 7TM cavity of CCR5 , as is also seen for MVC . However, the CVC docked poses were not in close prox Anti -Fibrotic Activity of Dual CCR5 /CCR2 imity to extracellular loop 2 , ECL2 remained accessible Antagonist Cenicriviroc in a Mouse Model of post -docking . Other groups have reported that the CCR5 Renal Fibrosis N -terminus and ECL2 domains both play a critical role in [0177 ] Background : Cenicriviroc (CVC ) is a novel , oral, the interaction of HIV - 1 with CCR5. In addition , the stem once -daily , dual CCR5/ CCR2 antagonist that has completed region of the V3 loop of gp120 is reported to bind to the Phase 2b HIV development (Study 202 ; NCT01338883 ) . CCR5 N - terminus while the V3 crown interacts with ECL2 CVC has a favorable safety profile with 555 subjects having and with residues inside the binding pocket . Based on our been treated with at least one dose , including 115 HIV - 1 model, we can assume that CVC does not interfere directly infected adults treated with CVC over a 48 -week duration . with the gp120 V3 loop interaction with ECL2 , since ECL2 Recently , CVC demonstrated significant anti - fibrotic activ appears to be exposed in the model . ity in a mouse model of diet - induced , non - alcoholic steato 0173 ] It is conceivable that CVC can block CCR5 acti hepatitis (NASH ) and a rat model of thioacetamide - induced vation if CCR5 remains in an inactive state . Two residues, fibrosis . Here , we evaluated CVC in a well -established Tyr37 and Trp248 , in the 7TM region have been shown to mouse model of renal fibrosis induced by unilateral ureter be important for CCR5 activation upon binding chemokine occlusion (UUO ). ligands, and this has also been shown to be important for [0178 ] Methodology : Test animals were allocated to MVC binding . Similar to MVC , different docked poses of weight- matched treatment groups on the day prior to the CVC are buried in the hydrophobic binding site . Our model surgical procedure (Day - 1) . Male CD -1 mice (N = 51 ; age , shows that access to Trp248 is blocked by CVC ; Trp248 has 7 - 8 weeks ) underwent either sham surgery or total ligation US 2019 /0099429 A1 Apr . 4 , 2019 of the right ureter, i. e . UUO , via aseptic laparotomy (FIG . fumarate /emtricitabine ( TDF/ FTC ) when co -administered 12 ) . From Days 0 to 5 : mice undergoing sham surgery with guideline - preferred third agents . received vehicle control ( 0 . 5 % methylcellulose + 1 % Tween [0182 ] Results : Body weight and obstructed kidney 80 ) via twice -daily oral gavage ; mice with permanent UUO weight : CVC 7 mg/ kg /day and compound 1D11 (positive received either vehicle control, CVC 7 mg/ kg / day or CVC control) had no effect on body weight, whereas CVC 20 20 mg/ kg /day via twice - daily oral gavage . Another group mg/ kg / day led to a modest, but significant, decrease ( 5 % ) in received the anti- transforming growth factor TGF -B1 anti body weight, relative to that of the UUO + vehicle - control body , compound 1D11 (positive control) at 3 mg/ kg /day group at Day 5 ( p < 0 .05 ) (FIG . 13 ; change in body weight from Days - 1 to 4 , injected intraperitoneally once daily , and shown in grams [ g ] ). No significant treatment effects (CVC vehicle control from Days 0 to 5 . A CVC 100 mg/ kg / day or compound 1011 [ positive control] ) were observed on group ( N = 9 ) was initially included in the study but was obstructed or contralateral kidney weight or kidney weight terminated early due to moribundity (no analyses were index versus the UVO + vehicle - control group ( data not conducted because no animal reached Day 5 ) . CVC doses up shown ). Histology : The composite measure of CVF ( % area to 2000 mg/ kg / day were well tolerated in mouse toxicity averaged across three depths [ SEM ] ) was significantly studies that did not involve surgical procedures. On Day 5 , higher in the UUO + vehicle -control group compared with animals were anaesthetised , blood and tissues were collected that in the sham - surgery group ( 11. 4 - 1 . 0 - fold ; p < 0 .05 ) prior to sacrifice . (FIG . 14 ) . CVC 7 and 20 mg/kg /day and compound 1D11 [ 0179 ] Study endpoints : Study endpoints included : a ) (positive control) significantly attenuated UUO - induced body and kidney weights ; b ) fibrosis in obstructed kidney increases in the composite measure of CVF (averaged across evaluated via histological quantitative image analysis of three depths ( USEM ]) relative to that of the UUO + vehicle picrosirius red staining ( ten images /depth /kidney obtained control group ( 28 .628 . 8 % , 31 . 8 + 6 . 8 % and 50 . 3 + 7 .3 % and assessed in a blinded fashion using light microscopy [at reduction , respectively ; p < 0 . 05 ) . 200x ] to enable sampling of 60 -70 % of the renal cortical [0183 ] Hydroxyproline content: Hydroxyproline content area ) and quantified by a composite Collagen Volume Frac ( % of protein ) in obstructed kidneys from the UUO + vehicle tion ( CVF [ % total area imaged ]) score expressed as the control group increased significantly relative to the sham average positive stain across three anatomically distinct surgery group ( 0 . 72 % vs 0 .27 % ; p < 0 .05 ) ( data not shown ). ( 200 - 250 uM apart ) tissue sections, or depths , from the Neither dose of CVC tested affected UUO - induced increases obstructed kidney ; c ) hydroxyproline content of frozen renal in obstructed kidney hydroxyproline content relative to the cortical tissue biopsies as assessed by biochemical analyses ; UUO + vehicle - control group ; however , the compound 1D11 d ) mRNA expression of profibrotic and inflammatory bio ( positive control) group had significantly lower levels markers ( including MCP - 1 , Collagen lal , Collagen 3al , ( 0 .55 % vs 0 .72 % ; p < 0 . 05) ( data not shown ). TGF -B1 , Fibronectin - 1 , a -smooth muscle actin ( a - SMA ) [0184 ] Profibrotic and inflammatory biomarker mRNA and connective tissue growth factor - 1 (CTGF - 1 ) ; assessed expression : For each of the biomarkers evaluated (MCP - 1 , via Luminex® (Life TechnologiesTM , Carlsbad , Calif . , USA ) Collagen lal, Collagen 3al , TGF - B1, Fibronectin - 1 , assay with relative expression normalised to HPRT (hypox a - SMA and CTGF - 1 ) , expression ofmRNA in the UUO + anthine phosphoribosyltransferase ). vehicle - control group increased significantly compared with that in the shamsurgery group (p < 0 .05 ) (FIG . 15 ) . CVC 7 [0180 ] Statistical analysis : Data are expressed as and 20 mg/ kg / day attenuated UVO - induced increases in mean - standard error of mean (SEM ) . Statistical analyses Collagen lal, Collagen 3al, TGF- B1 and Fibronectin - 1 were performed using GraphPad Prism® (GraphPad Soft mRNA expression . However , these reductions, compared ware , Inc . , San Diego , Calif ., USA ) . Treatment differences with the UUO + vehicle -control group , did not reach statis between sham - surgery + vehiclecontrol and UUO + vehicle tical significance . Compound 1D11 (positive control) sig control groups , and between UUO + vehicle - control and nificantly reduced UUO - induced increases in mRNA expres UU + compound - 1D11 (positive control) groups, were ana sion of Collagen lal, Collagen 3al , TGF- B1 and lysed by unpaired t- Test . Treatment differences between Fibronectin - 1 relative to the UUO + vehicle -control group UUO + vehicle - control and CVC - dose groups were analysed ( p < 0 .05 ) . CVC 7 and 20 mg/ kg /day and compound 1D11 by one- way ANOVA ( analysis of variance ) with Dunnett ' s (positive control) did not have significant effects on UUO test (post -hoc ) . induced increases in obstructed kidney cortical MCP - 1 , [0181 ] Methods: CVC demonstrated significant antifi Q -SMA and CTGF - 1 mRNA expression , compared with the brotic effects , as defined by reductions in Collagen Volume UUO + vehicle - control group (data not shown for a -SMA Fraction or CVF ( % area stained positively for collagen in and CTGF - 1 mRNA ). histological obstructed -kidney sections) , in a well -estab [0185 ] Conclusions : CVC demonstrated significant antifi lished mouse UUO model of renal fibrosis . Trends were brotic effects , as defined by reductions in Collagen Volume observed for decreases in Collagen lal , Collagen 3al , Fraction or CVF ( % area stained positively for collagen in TGF -B1 and Fibronectin - 1 mRNA expression in the histological obstructed -kidney sections) , in a well -estab obstructed kidney , but these did not achieve statistical sig lished mouse UUO model of renal fibrosis . Trends were nificance . Taken together, CVC ' s mode of action , antifi observed for decreases in Collagen lal, Collagen 3al, brotic activity in animal models (kidney and liver) , and TGF -B1 and Fibronectin - 1 mRNA expression in the extensive safety database support further evaluation in obstructed kidney , but these did not achieve statistical sig fibrotic diseases. A proof- of - concept study in non -HIV nificance . Taken together, CVC ' s mode of action , antifi infected patients with NASH and liver fibrosis is planned . brotic activity in animal models (kidney and liver ) , and extensive safety database support further evaluation in evaluate a fixed dose combination of CVC / lamivudine fibrotic diseases . A proof - of - concept study in non -HIV ( 3TC ) as a novel “backbone versus tenofovir disoproxil infected patients with NASH and liver fibrosis is planned . US 2019 /0099429 A1 Apr . 4 , 2019 19
Phase III trials in HIV - 1 -infected patients are also planned to Example 18 evaluate a fixed dose combination of CVC / lamivudine (3TC ) as a novel ‘ backbone versus tenofovir disoproxil In Vivo Efficacy Study of Cenicriviroc in STAM fumarate / emtricitabine ( TDF /FTC ) when co - administered Model of Non - alcoholic Steatohepatitis with guideline- preferred third agents . [0190 ] This in vivo efficacy study was performed to exam ine the effects of Cenicriviroc in the STAMTM mouse model Example 17 of Non - alcoholic Steatohepatitis . Improvements in APRI and FIB - 4 Fibrosis Scores Correlate with Decreases in sCD14 in HIV - 1 Materials and Methods Infected Adults Receiving Cenicriviroc Over 48 Experimental Design and Treatment Weeks [0186 ] Background and aims: Cenicriviroc (CVC ) , a Study Groups novel, oral, once - daily CCR2/ CCRS antagonist, has dem [0191 ] Group 1 – Vehicle : Eighteen NASH mice were onstrated favorable safety and anti - HIV activity in clinical orally administered vehicle at a volume of 10 mL /kg twice trials . CVC demonstrated antifibrotic activity in two animal daily ( 9: 00 and 19: 00 ) from 6 weeks of age . models of liver disease . Post - hoc analyses were conducted [0192 ] Group 2 – Cenicriviroc 20 mg/ kg (CVC - low ) : on APRI and FIB - 4 scores in Study 202 (NCT01338883 ) . Eighteen NASH mice were orally administered vehicle [0187 ] Methods: 143 adults with CCR5 tropic HIV - 1 , supplemented with Cenicriviroc at a dose of 10 mg/ kg twice BMI< 35 kg/ m2 and no apparent liver disease ( ie , ALT /AST daily ( 20 mg/ kg / day ) ( 9 : 00 and 19 :00 ) from 6 weeks of age . Grades2 , total bilirubinSULN , no HBV, HCV, active or f0193 ] Group 3 – Cenicriviroc 100 mg/ kg (CVC -high ) : chronic liver disease , or cirrhosis ) were randomized 4 : 1 to Eighteen NASH mice were orally administered vehicle CVC or efavirenz (EFV ) . APRI and FIB - 4 scores were supplemented with Cenicriviroc at a dose of 50 mg/ kg twice calculated . Change in score category from baseline ( BL ) to daily ( 100 mg/ kg /day ) ( 9 : 00 and 19 : 00 ) from 6 weeks of Weeks 24 and 48 was assessed in patients with non -missing age . data . Correlations between changes from BL in APRI and [0194 ] Table 12 summarizes the treatment schedule : FIB - 4 scores, and MCP - 1 (CCR2 ligand ) and sCD14 ( in flammatory biomarker ) levels were evaluated . TABLE 12 10188 ] Results : At BL , more patients on CVC than EFV Sacri had APRI20 . 5 and FIB - 421. 45 ; proportion of CVC patients No . Test Dose Volume fice above these thresholds decreased at Weeks 24 and 48 Group mice Mice substance (mg /kg ) (mL / kg ) Regimen (wks ) ( Table ) . Significant correlations were observed at Week 24 1 18 STAM Vehicle 10 Oral , twice 9 and between changes in APRI score and MCP - 1 levels (p = 0 . daily , 18 014 ) , and between FIB - 4 score and sCD14 levels ( p = 0 .011 ) , 6 - 9 wks , and at Week 48 , between changes in APRI ( p = 0 . 028 ) and 6 -18 wks FIB - 4 scores (p = 0 . 007 ) and sCD14 levels . ( Table 11 ). TABLE 11 CVC EFV Baseline Week 24 Week 48 Baseline Week 24 Week 48 Fibrosis index ( n = 113 ) ( n = 92 ) ( n = 80 ) ( n = 26 ) ( n = 20 ) ( n = 17 ) APRI < 0 . 5 84 % 93 % 91 % 98 % 100 % 100 % category 0 .5 - 1. 5 14 % 7 % 8 % 4 % > 1 . 5 2 % 1 % Decreased 1 N / A 14 % 10 % N / A 5 % 8 % category from baseline FIB - 4 < 1 . 45 82 % 93 % 94 % 100 % 100 % 94 % category 1. 45 - 3 . 25 17 % 7 % 5 % 8 % > 3 . 25 1 % Decreased 1 N / A 13 % 14 % N / A category from baseline
[ 0189 ] Conclusions: In this population with no apparent TABLE 12 -continued liver disease , CVC treatment was associated with improve Sacri ments in APRI and FIB -4 scores, and correlations were No . Test Dose Volume fice observed between changes in APRI and FIB -4 scores and Group mice Mice substance (mg / kg ) (mL / kg ) Regimen (wks ) sCD14 levels at Week 48 . Proven CCR2/ CCR5 antagonism , 2 18 STAM CVC 20 1 0 Oral, twice 9 and antifibrotic effects in animal models and extensive clinical low daily , 18 6 - 9 wks, safety data all support clinical studies of CVC in liver 6 - 18 wks fibrosis . US 2019 /0099429 A1 Apr . 4 , 2019
TABLE 12 -continued TABLE 14 Sacri No . Test Dose Volume fice Blood and Liver Biochemistry at Week 9 Group mice Mice substance (mg / kg ) (mL /kg ) Regimen (wks ) 3 18 STAM CVC - 100 10 Oral, twice 9 and Cenicriviroc- Cenicriviroc high daily , 18 Parameter 6 - 9 wks, Vehicle low high 6 - 18 wks (Mean - SD ) ( n = 6 ) ( n = 6 ) (n = 6 ) [0195 ] Results Whole blood glucose (mg /dL ) 590 = 108 585 + 91 585 + 91 Plasma ALT ( U / L ) 133 I+ 80 58 12 52 + + 13 Part 1 : Study for Assessing the Anti -NASH / Fibrosis Effects Plasma MCP - 1 (pg /mL ) 60 1 4 68 16 91 = 14 of CVC Plasma MIP - 1B (pg /mL ) 18 It+ 5 18 + 2 20 + 4 [0196 ] Body weight changes and general condition until Liver triglyceride (mg / g liver ) 40 . 8 + 20 .4 48. 5 16 . 1 51. 7 - 14 . 1 Week 9 (FIG . 16 ) Liver hydroxyproline 0 . 75 + 0 . 18 0 .63 + 0 . 05 0 .62 + 0 .09 [0197 ] Body weight gradually increased during the treat (ug /mg total protein ) ment period . There were no significant differences in mean body weight between the Vehicle group and either the CVC - low or the CVC -high groups during the treatment [ 0205 ] Plasma MCP - 1 data are shown in FIG . 18C and period . None of the animals in the present study showed Table 14 . The CVC - high group showed a significant deterioration in general condition throughout the treatment increase in plasma MCP - 1 levels compared with the Vehicle period . group . There were no significant differences in plasma [0198 ] Body weight at the day of sacrifice at Week 9 ( FIG . MCP - 1 levels between the Vehicle group and the CVC - low 17A and Table 13 ) group (Vehicle : 60 + 4 pg /mL , CVC - low : 68 : 16 pg /mL , [0199 ). There were no significant differences in mean body weight between the Vehicle group and either the CVC - low CVC - high : 91 : 14 pg /mL ) . or the CVC -high groups (Vehicle : 18 . 9 + 3 . 3 g , CVC - low : [0206 ] Plasma MIP - 1ß data are shown in FIG . 18D , Table 19 . 5 + 2 .0 g , CVC - high : 18 . 7 + 0 . 9 g ) . 14 . There were no significant differences in plasma MIP - 1B levels between the Vehicle group and either the CVC - low or TABLE 13 the CVC -high groups ( Vehicle : 1805 pg/ mL , CVC - low : Body Weight and Liver Weight at Week 9 18 + 2 pg /mL , CVC - high : 20 + 4 pg /mL ) . Parameter Vehicle Cenicriviroc -low Cenicriviroc - high Liver Biochemistry at Week 9 (Mean SD ) ( n = 6 ) ( n = 6 ) ( n = 6 ) Body weight ( g ) 18 . 9 + 3 . 3 19 . 5 = 2 . 0 18 . 7 + 0 . 9 [0207 ] Liver triglyceride content data are shown in FIG . Liver weight (mg ) 1270 326 1334 + 99 1307 + 119 18D and Table 14 . There were no significant differences in Liver - to -body 6 . 6 – 0 . 8 6 . 9 - 1 . 0 7 . 0 + 0 . 8 liver triglyceride content between the Vehicle group and weight ratio ( % ) either the CVC - low or the CVC -high groups ( Vehicle : 40 . 8 + 20 . 4 mg /g liver , CVC -low : 48 .5 - 16 .1 mg/ g liver , [0200 ] Liver weight and liver- to -body weight ratio at CVC -high : 51. 7 + 14 . 1 mg/ g liver ). week 9 (FIGS . 17 B & C and Table 13 ) [0201 ] There were no significant differences in mean liver [0208 ] Liver hydroxyproline content data are shown in weight between the Vehicle group and either the CVC - low FIG . 18E and Table 14 . The liver hydroxyproline content or the CVC - high groups (Vehicle : 1270 + 326 mg, CVC - low : tended to decease in the CVC - low and the CVC - high groups 1334 + 99 mg, CVC - high : 1307 : 119 mg ) . compared with the Vehicle group (Vehicle : 0 .75 + 0 . 18 [ 0202 ] There were no significant differences in mean ug/ mg , CVC - low : 0 .63 + 0 .05 ug /mg , CVC -high : 0 .62 + 0 .09 liver - to -body weight ratio between the Vehicle group and ug/ mg ) . either the CVC - low or the CVC - high groups ( Vehicle : 6 . 6 + 0 . 8 % , CVC - low : 6 . 9 - 1. 0 % , CVC -high : 7 . 0 + 0 . 8 % ). Histological Analyses at Week 9 Whole blood and Biochemistry at Week 9 [ 0203] Whole blood glucose data are shown in FIGS . [0209 ] HE staining and NAFLD Activity score data are shown in FIGS. 19 and 20 , and Table 15 . Liver sections from [0204 ] There were no significant differences in blood the Vehicle group exhibited severe micro - and macrovesicu glucose levels between the Vehicle group and either the lar fat deposition , hepatocellular ballooning and inflamma CVC - low or the CVC -high groups ( Vehicle : 590 - 108 tory cell infiltration . The CVC - low and the CVC -high mg/ dL , CVC - low : 585 + 91 mg /dL , CVC -high : 585 + 91 groups showed moderate improvements in inflammatory mg/ dL ) . 4 . 4 . 2 . Plasma ALT (FIG . 18B , Table 14 ). The cell infiltration and hepatocellular ballooning , with a sig CVC - low and the CVC -high groups showed significant nificant reduction in NAS compared with the Vehicle group decreased in plasma ALT levels compared with Vehicle (Vehicle : 5. 3 + 0 .5 , CVC - low : 4 .0 + 0 .6 , CVC -high : 3 . 7 + 0 .8 ). group ( Vehicle : 133 = 80 U / L , CVC - low : 58 12 U /L , CVC Representative photomicrographs of the HE - stained sections high : 52 : 13 U /L ). are shown in FIG . 19 . US 2019 /0099429 A1 Apr . 4 , 2019
TABLE 15 NAFLD ACActivity Score at Week 9 Score Steatosis Lobular inflammationHepatocyte ballooning NAS Group n 0 1 2 3 0 1 2 3 0 1 2 (Mean + SD ) Vehicle 6 - 4 2 5 . 3 + 0 . 5 Cenicriviroc - low 3 ?? - ?????? 4 . 0 + 0 . 6 Cenicriviroc -high 6 1 5 - - - ??3 - NWT 3 . 7 + 0 . 8 Definition of NAS Components Item Score Extent Steatosis < 5 % 5 - 33 % > 33 -66 % > 66 % Hepatocyte None ballooning Few balloon cells Many cells/ prominent ballooning Lobular WNONOWNEO No foci inflammation < 2 foc / 200x 2 - 4 foci /200x > 4 foci/ 200x
[0210 ] Sirius red staining data are shown in FIGS. 21, 22 , CVC - low and the CVC - high groups compared with the 23 and Table 16 . Liver sections from the Vehicle group Vehicle group ( Vehicle : 1 . 10 + 0 .31 % , CVC -low : 0 . 66 + 0 . showed collagen deposition in the pericentral region of the 16 % , CVC - high : 0 .64 + 0 . 19 % ) . The modified fibrosis areas liver lobule . Compared with the Vehicle group , collagen were also significantly reduced in the CVC - low and the deposition in the pericentral region was markedly reduced in CVC -high groups compared with the Vehicle group (Ve the CVC - low and the CVC -high groups . The fibrosis area hicle: 0 .6120 .23 % , CVC - low : 0 .29 + 0 . 14 % , CVC -high : (Sirius red -positive area ) significantly decreased in the 0 .20 + 0 .06 % ) . TABLE 16 Histological Analyses at Week 9 Cenicriviroc - Cenicriviroc Parameter Vehicle low high (Mean - SD ) ( n = 6 ) ( n = 6 ) (n = 6 ) Sirius red - positive area ( % ) 1 . 10 + 0 . 31 0 .66 + 0 . 16 0 .64 + 0 . 19 Modified Sirius red -positive area 0 .61 + 0 .23 0 .29 + 0 . 14 0 .20 + 0 .06 F4 / 80 -positive area ( % ) 4 . 99 = 1 . 10 4 .77 1 .02 4 . 96 + 0 .60 F4 / 80 and CD206 - positive cells ( % ) 34 . 3 + 4 . 2 34 . 7 + 6 . 3 33 . 1 + 3 . 0 F4 / 80 and CD16 / 32 - positive cells ( % ) 33 . 5 = 3 . 7 38 . 7 + 7 . 6 41 . 5 = 8 . 2 M1/M2 ratio ( % ) 99 . 6 = 20 . 2 112 . 3 + 17. 0 125 . 1 + 21. 9 Oil red - positive area ( % ) 9 .66 + 5 .02 6 .51 3 .88 7 .23 + 3 . 59 TUNEL -positive cells ( % ) 36 . 0 + 3 . 7 43. 3 + 2 . 9 39 . 0 + 5 . 3 Cenicriviroc -high Total Positive Modified Modified Modified Mouse Photo Total positive perivascular positive positive ID No. area ( pix ) area (pix ) area (pix ) area (pix ) area ( % ) area ( % ) 301 1264424 9749 6409 3340 0 .26 0 . 18 ????? 1291238 3234 2491 743 0 . 06 1289200 4737 3491 1246 0 . 10 1252731 17225 5180 0 . 41 ? 12045 1277575 6253 5119 1134 0 . 09 302 ? 1217885 16038 13242 2796 0 . 23 0 . 20 1248706 4876 2134 0 . 17 ????? 7010 1253036 14194 10634 3560 0 . 28 ? 1301898 4914 2070 2844 0 . 22 ? 1268269 7439 6404 1035 0 .08 303 1 1285828 4306 3322 984 0 .08 0 . 12 2 1297994 2159 1550 609 0. 05 ???? 1279156 3201 1176 0 .09 US 2019 /0099429 A1 Apr . 4 , 2019
TABLE 16 - continued Histological Analyses at Week 9 1285026 12648 8537 4111 0 . 32 1285009 4011 3119 892 0 . 07 304 1294810 3685 1677 2008 0 . 16 0 .26 1274697 2221 1222 999 0 . 08 1286001 11356 8814 2542 0 . 20 1236232 10705 8252 2453 0 . 20 1217017 18761 10537 8224 0 .68 305 1287425 5774 2832 2942 0 . 23 0 . 17 1278985 2638 1733 905 0 .07 0. 17 1272127 7654 4214 3440 0 . 27 1289371 5726 3563 2163 0 . 17 1200639 3654 2171 1483 0 . 12 306 1236260 6253 2852 3401 0 . 28 0 . 27 1270484 12655 11196 1459 0 . 11 1144610 20504 12793 7711 0 . 67 1292425 7266 4401 2865 0 . 22 5 1295488 1921 976 945 0 . 07
[ 0211 ] Representative photomicrographs of Sirius red [0216 ] Oil red staining data are shown in FIGS. 29, 30 , stained sections of livers are shown in FIG . 21 . and Table 16 . There were no significant differences in the fat 0212 ] F4 /80 immunohistochemistry data are shown deposition between the Vehicle group and either the CVC FIGS . 22 and 23 , and Table 16 . F4 / 80 immunostaining of low or the CVC -high groups, as well as in the percentage of liver sections form the Vehicle group demonstrated accu fat deposition area (oil - positive area ) ( Vehicle : 9 .66 + 5 .02 % , mulation of F4 /80 + cells in the liver lobule . There were no CVC - low : 6 .513 . 88 % , CVC - high : 7 . 23 + 3 .59 % ) . significant differences in the number and size of F4 / 80 + cells between the Vehicle group and either the CVC - low or the [0217 ] Representative photomicrographs of the oil red CVC -high groups , as well as in the percentage of inflam stained sections are shown in FIG . 29 . mation area ( F4 / 80 -positive area ) ( Vehicle : 4 . 99 + 1 . 10 % , [0218 ] TUNEL staining data are shown in FIGS. 31, 32 CVC - low : 4 .77 - 1 .02 % , CVC - high : 4 . 96 + 0 .60 % ) . and Table 16 . The percentages of TUNEL -positive cells 10213 ] Representative photomicrographs of the F4 / 80 - im significantly increased in the CVC - low group compared munostained sections are shown in FIG . 22 . with the Vehicle group . There was no significant difference (0214 ] F4 /80 + CD206 + and F4 / 80 + CD16 / 32 + immunohis in percentages of TUNEL - positive cells between the Vehicle tochemistry data are shown in FIGS. 24 , 25 , 26 , 27, 28 , and group and the CVC -high group ( Vehicle : 36 . 0 + 3 . 7 % , CVC Table 16 ) . There were no significant differences in the percentages of F4 /80 + CD206 + cells in macrophages low : 43. 3 + 2 .9 % , CVC -high : 39. 0 - 5. 3 % ) . between the Vehicle group and either the CVC- low or the [0219 ] Representative photomicrographs of TUNEL -posi CVC -high groups (Vehicle : 34 . 3 + 4 . 2 % , CVC -low : 34 .746 . tive cells in livers are shown in FIG . 31. 3 % , CVC - high : 33 . 1 + 3 . 0 % ) . There was no significant dif [0220 ] Gene Expression Analysis at Week 9 data are ference in the percentages of F4 / 80 + CD16 /32 + cells in shown in FIG . 33 and Tables 17 - 18 . macrophages between the Vehicle group and the CVC - low group . The percentages of F4 /80 + CD16 /32 + cells tended to TABLE 17 ( Vehicle : 33 . 5 + 3 . 7 % , CVC - low : 38 . 7 + 7 .6 % , CVC - high : Gene Expression Analysis at Week 9 41 . 5 - 8 . 2 % ) . There was no significant difference in the M1/M2 ratio between the Vehicle group and the CVC - low Parameter Vehicle Cenicriviroc - low Cenicriviroc- high group . In the CVC -high group , the M1/M2 ratio tended to (Mean - SD ) ( n = 6 ) ( n = 6 ) ( n = 6 ) TNF - a 1 . 00 + 0 . 24 1 . 16 + 0 . 39 1 .09 + 0 . 23 increase compared with the Vehicle ( Vehicle: 99 .6 + 20 . 2 % , MCP - 1 1 . 00 – 0 . 31 1 . 05 + 0 . 50 1 .00 + 0 . 53 CVC - low : 112 . 3 – 17 . 0 % , CVC -high : 125 . 1 - 21. 9 % ) . Collagen Type 1 1 . 00 + 0 .42 0 .63 + 0 . 10 0 . 73 + 0 .04 [0215 ] Representative photomicrographs of the F4 /80 and TIMP- 1 1 .00 + 0 .46 0 . 75 + 0 . 32 0 . 80 + 0 . 20 CD206 , F4 / 80 and CD16 / 32 double - immunostained sec tions are shown in FIGS. 24 and 26 . TABLE 18 P values at Week 9 Liver P values Body Liver to -body (Student ' s t - test , one- tailed ) weight weight weight ratio Vehicle v .s . Cenicriviroc- low 0 .3517 0 .3265 0. 2732 V . S . Cenicriviroc- high 0 . 4487 0 .3993 0 . 1929 US 2019 /0099429 A1 Apr . 4 , 2019 23
TABLE 18 -continued P values at Week 9 Whole P values blood Plasma Plasma Plasma Liver Liver (Student ' s t -test , one -tailed ) glucose ALT MCP - 1 MIP - 1ß triglyceride hydroxyproline Vehicle vs . Cenicriviroc - low 0 . 4629 0 .0239 0 . 1329 0 . 3861 0 . 2421 0 .0794 v. s . Cenicriviroc - high 0 .4651 0 . 0177 0 .0003 0 . 1587 0 . 1545 0 .0661 P values Collagen ( Studentstudents' s t- very test , oneone- - tailedtuled ) ) TNF- - a MCPMcP: - 1 coelestintype 1 TIMPTIMP- - 1 Vehicle vs . Cenicriviroc - low 0 . 2054 0 .4149 0 .0312 0 . 1473 V. S . Cenicriviroc -high 0 .2611 0 .4982 0 .0738 0 . 173 Modified F4 / 80 Sirius Sirius F4 / 80 and and CD Oil NAFLD red red F4 / 80 CD206 16 / 32 red TUNEL P values Activity positive positive positive positive positive M1/M2 positive positive ( Student ' s t- test, one- tailed ) score area area area cells cells ratio area cells Vehicle v .s . Cenicriviroc - low 0 . 0013 0 .0058 0 .0067 0 . 3633 0 .4525 0 .0818 0 .1333 0 . 1261 0 .0017 v . s . Cenicriviroc -high 0 . 0009 0 .0054 0 . 0008 0 .481 0 .292 0 . 0273 0 .0311 0 . 1791 0 . 1416
TNFa (ID208 ) in the CVC - low group . Five out of twelve mice died at day 62 ( ID317 ) , day 65 ( ID312 ) , day 70 (ID316 ), day 78 [0221 ] There were no significant differences in TNFa (1D314 ) and day 85 ( ID309 ) in the CVC -high group . There mRNA expression levels between the Vehicle group and were no abnormal necropsy findings in the dead animals either the CVC - low or the CVC -high groups ( Vehicle : except for the typical hepatic lesions of NASH . There were 1 .00 + 0 . 24 , CVC - low : 1 . 16 + 0 .39 , CVC - high : 1 . 09 + 0 . 23 ) . no significant differences in survival rate between the MCP - 1 Vehicle group and either the CVC -low or the CVC - high [ 0222] There were no significant differences in MCP - 1 groups . By consigner instruction , the rest of the animals mRNA between the Vehicle group and either the CVC - low were sacrificed earlier than scheduled at 18 weeks of age or the CVC -high groups (Vehicle : 1 .00 + 0 .31 , CVC - low : ( scheduled sacrificed at 20 weeks of age ) . 1 .05 + 0 .50 , CVC -high : 1 . 00 + 0 .53 ) . [0228 ) Body Weight at the Day of Sacrifice at Week 18 data are shown in FIG . 37A and Table 19 . The body weight Collagen Type 1 tended to decrease in the CVC -high group compared with [ 0223] Collagen Type 1 mRNA expression levels were the Vehicle group . There was no significant difference in significantly down - regulated in the CVC - low group com mean body weight between the Vehicle group and the pared with the Vehicle group . Collagen Type 1 mRNA CVC - low group (Vehicle : 23 .0 + 2 . 3 g , CVC - low : 22. 9 + 3 . 5 g , expression levels tended to be down -regulated in the CVC CVC -high : 20 . 8 + 2 . 7 g ) . high group compared with the Vehicle group . (Vehicle : 1 . 00 + 0 .42 , CVC - low : 0 .63 + 0 . 10 , CVC -high : 0 .73 + 0 .04 ) . TABLE 19 TIMP- 1 Body Weight and Liver Weight at Week 18 [ 0224 ] There were no significant differences in TIMP - 1 Parameter Vehicle Cenicriviroc- low Cenicriviroc -high mRNA expression levels between the Vehicle group and (Mean - SD ) ( n = 8 ) ( n = 6 ) ( n = 7 ) either the CVC - low and the CVC -high groups (Vehicle : Body weight ( g ) 23. 0 + 2 . 3 20 . 8 + 2 . 7 Liver weight (mg ) 1782 + 558 1837 + 410 1817 + 446 1 . 00 + 0 .46 , CVC - low : 0 . 75 + 0 . 32 , CVC - high : 0 . 80 + 0 . 20 ) . Liver - to -body 7 . 7 + 2 . 2 8 . 3 + 2 . 8 8 . 8 + 2 . 3 Part 2 : Study for Assessing the Anti -HCC Effects of CVC weight ratio ( % ) [ 0225 ) Body Weight Changes until Week 18 (FIG . 35 ) 102261 Body weight gradually increased during the treat [0229 ] Liver Weight and Liver - to - Body Weight Ratio at ment period . There were no significant differences in mean Week 18 data are shown in FIGS. 37B & C and Table 19 . body weight between the Vehicle group and either the There were no significant differences in mean liver weight CVC -low or the CVC -high groups during the treatment between the Vehicle group and either the CVC - low or the period . CVC -high groups (Vehicle : 1782 558 mg , CVC - low : [ 0227] Survival analysis data are shown in FIG . 36 . Four 1837 + 410 mg, CVC -high : 1817 + 446 mg ). There were no out of twelve mice died at day 59 ( ID112 ) , day 75 ( ID113 , significant differences in mean liver- to -body weight ratio 115 ) and day 84 ( ID116 ) in the Vehicle group ( The first day between the Vehicle group and either the CVC -low or the of administration was designed as day 0 ) . Six out of twelve CVC -high groups (Vehicle : 7 .7 + 2 .2 % , CVC - low : 8 .312 .8 % , mice died at day 62 ( ID209 ) , day 64 ( ID217 ) , day 75 CVC -high : 8 . 8 + 2. 3 % ) . Macroscopic Analyses of Liver at ( ID212 ), day76 ( ID213 ), day 84 ( ID215 ) and day 86 Week 18 US 2019 /0099429 A1 Apr . 4 , 2019 24
[0230 ] Macroscopic appearance of livers is shown in group. No obvious differences were found between the FIGS . 38A - C . Vehicle group and either the CVC - low or the CVC -high [0231 ] Number of visible tumor nodules formed on liver groups. surface are shown in FIG . 39 and Table 20 . There were no significant differences in the number of hepatic tumor nod [0234 ] Representative photomicrographs of the ules per individual mouse between the Vehicle group and HE - stained sections are shown in FIG . 41. either the CVC - low or the CVC -high groups (Vehicle : [0235 ] GS immunohistochemistry data are shown in FIG . 2. 4 + 4 .1 , CVC -low : 1. 5 - 1 .9 , CVC -high : 3 .6 + 2 .5 ). 42 . GS -positive nodules in the sections were detected in six out of eight mice in the Vehicle group , five out of six mice TABLE 20 in the CVC - low group and seven out of seven mice in the Macroscopic Analyses of Liver at Week 18 CVC -high group , respectively . Parameter Vehicle Cenicriviroc -low Cenicriviroc - high [0236 ] Representative photomicrographs of the (Mean - SD ) ( n = 8 ) ( n = 6 ) ( n = 7 ) GS -stained sections are shown in FIG . 42 . Number of visible 2 . 4 + 4 . 1 1 . 5 + 1 . 9 3 . 6 + 2 . 5 [ 0237 ] CD31 immunohistochemistry data are shown in tumor nodules Maximum diameter of 4 . 0 + 4 . 7 4 . 8 + 5 . 4 5 . 3 + 5 . 1 FIGS. 43 and 44 and Table 21 . The CD31 -positive area visible tumor nodules tended to decrease in the CVC - low group compared with the (mm ) Vehicle group . The CD31- positive area tended to increase in the CVC -high group compared with the Vehicle group [ 0232] Maximum diameters of visible tumor nodules (Vehicle : 2 . 71 - 1. 36 % , CVC -low : 1 . 47 + 1 . 10 % , CVC - high : formed on liver surface are shown in FIG . 40 and Table 20 . 3 .68 - 1 . 37 % ). There were no significant differences in maximum diameter of tumor between the Vehicle group and either the CVC - low stained sections are shown in FIG . 43 . or the CVC -high groups ( Vehicle : 4 . 0 + 4 . 7 mm , CVC - low : 4 . 8 + 5 . 4 mm , CVC -high : 5 . 3 - 5 . 1 mm ) . TABLE 21 Histological Analyses at Week 18 Histological Analyses at Week 18 [0233 ] HE staining data are shown in FIG . 41. HE staining Parameter Vehicle Cenicriviroc - low Cenicriviroc- high revealed infiltration of inflammatory cells , macro - and (Mean - SD ) (n = 8 ) (n = 6 ) ( n = 7 ) microvesicular fat deposition , hepatocellular ballooning , CD31- positive area 2 .71 + 1 . 36 1 .47 = 1 . 10 3 .68 + 1 .37 altered foci and nodular lesions in the Vehicle group . Six out ( % ) of eight mice in the Vehicle group exhibited HCC lesions . HCC lesions were detected in five out of six mice in the CVC - low group and six out of seven mice in the CVC -high US 2019 /0099429 A1 Apr . 4 , 2019 25
Table 22 : P Values at Week 18 P value (Student ' s t - test , Body Liver Liver- to The number Maximum CD31 one - tailed ) Weight | Weight body weight of visible diameter of positive area ratio tumor nodules visible tumor nodules Vehicle vs Cenicriviroc - low | 0 .4758 0 . 4215 0 . 341 0 .3191 0 . 3812 0 .0456 Vehicle vs Cenicriviroc- 0. 0574 0. 4476 0 . 184 0 . 2578 0 . 3096 0 .0972 high
P values (Logrank - test) Survival Curve Vehicle vs Cenicriviroc - low 0 . 7513 Vehicle vs Cenicriviroc 0 .5701 high US 2019 /0099429 A1 Apr . 4 , 2019
SUMMARY AND DISCUSSION major component detected in the plasma of rats and dogs , [0239 ] In the analyses at week 9 , treatment with low and the AUCO - 24 ratio of CVC to total 14C being 58 . 9 % and high dose of CVC significantly reduced fibrosis area in a 47 . 4 % , respectively [ 44 ] . In monkeys , this ratio was only dose dependentmanner , demonstrating anti - fibrotic effect of 12 . 9 % , whereas a relatively large amount ofmetabolite M - II CVC in the present study . Treatment with low and high dose was detected , the AUCO - 24 ratio of M - II to total 14C being of CVC also reduced the mRNA expression levels of Col 34 . 3 % . Especially in dogs and monkeys , the amounts of lagen Type 1 and liver hydroxyproline content, supporting M - II were significantly greater after oral administration than its anti - fibrotic property . CVC treatment groups significantly after IV administration . These results suggest that CVC can decreased plasma ALT levels and NAS compared with the be metabolized to M -II before reaching the systemic circu Vehicle group in a dose dependent manner. The improve lation . Minor metabolites, including M - I , T - 1184803 , and ment in NAS was attributable to the reduction in lobular T - 1169518 , were also detected in the plasma of rats , dogs, inflammation and hepatocyte ballooning. Since hepatocyte and monkeys. It is postulated that the metabolite M - I is ballooning is derived from oxidative stress - induced hepato formed by oxidation of the sulfinyl moiety of CVC and that cellular damage and is associated with disease progression M - II is formed by the subsequent reduction of the sulfinyl of NASH [ 26 ; 27 ), it is strongly suggested that CVC moiety with cleavage of the C - S bond of the [ ( 1 - propyl- 1H improved NASH pathology by inhibiting hepatocyte dam imidazol- 5 - yl) methyl ] sulfinyl group , followed by S -methyl age and ballooning . Together , CVC have potential anti ation . NASH and hepatoprotective effects in this study. [0240 ] As shown in humans, plasma MCP - 1 levels Clinical Trials increased by the treatment with CVC in the present study, indicating dose - dependent antagonism of CCR2 by CVC , Example 21 but plasma MIP - 1ß levels did not show any significant changes by the treatment. To investigate the mechanism of Short -Term Efficacy Data in HIV - 1 Infected Adult action of CVC , we evaluated the effect of CVC on popula Subjects Methods [ 0245 ] A Phase 2a double - blind , randomized , placebo that CVC showed the tendency of high M1/M2 ratio com controlled , dose - escalating study evaluating the antiviral pared with Vehicle group , suggesting that CVC might inhibit activity , PK , safety, and tolerability ofmonotherapy of CVC the fibrogenesis by regulating the balance of macrophage for 10 days in subjects with CCR5 - tropic HIV - 1 infection . subpopulation in the inflamed liver . This will be further Participants were required to be antiretroviral treatment investigated in the future . experienced , CCR5 antagonist - naive , with HIV - 1 RNA lev [ 0241 ] In the analyses at week 18, the effect on NASH els of at least 5000 copies /mL and CD4 + cell counts of at derived HCC was not observed in the CVC treatment least 250 cells /mm was performed . Groups of 10 subjects groups . In conclusion , CVC showed anti -NASH , hepatopro were sequentially enrolled in a ratio of 4 : 1 subjects per tective and anti- fibrotic effects in the present study. cohort to receive CVC (25 , 50 , 75 , 100 , or 150 mg ) or Example 19: Receptor - Binding Properties of CVC matching placebo . All subjects received once -daily doses of and Metabolites CVC or placebo for 10 days and were followed to Day 40 . [0242 ] CVC has the unique property in vitro of being a Demographics and Other Baseline Characteristics CCR2 antagonist with 50 % inhibitory concentrations ( IC50 ) of 5 . 9 nmol/ L . CVC dose - dependently inhibited the binding [0246 ] A total of 54 subjects were enrolled into this study. of RANTES , MIP - 1a , and MIP - 1ß to CCR5- expressing Demographics were generally similar across the dose Chinese hamster ovary (CHO ) cells with an IC50 of 3 . 1 , 2 . 3 , groups . A majority of the subjects in each dose group were and 2 . 3 nmol/ L , respectively . CVC achieved 90 % receptor male (66 .7 % to 100 % ), and median age ranged from 33 .5 occupancy for CCR5 at concentrations of 3 . 1 nM for CD4 + years (placebo group ) to 45 . 0 years (150 -mg group ) . Most and 2 . 3 nM for CD8 + T - cells ex vivo in humans [ 4 ) . CVC subjects were Caucasian or African American . Median BMI inhibited the binding of MCP - 1 to CCR2b with an IC50 of ranged from 22 . 9 kg /m2 ( 100 -mg group ) to 27 . 4 kg /m2 5 . 9 nmol/ L . CVC achieved 98 % receptor occupancy for ( 25 -mg group ) . Median HIV - 1 RNA values ranged from CCR2 on monocytes at 6 nM ex vivo in humans and reduced 4 .00 log10 copies/ mL (150 -mg group ) to 4 .60 log10 copies/ CCR2 expression on monocytes in the absence of MCP - 1 . mL (75 -mg group ) . Median CD4 + cell count was highest in CVC only weakly inhibited ligand binding to CCR3 and the 150 -mg group ( 508 . 0 cells /mm ) and ranged from 402 . 0 CCR4 . CVC did not inhibit ligand binding to CCR1 or to 460 . 0 cells/ mm3 across the remaining groups . CCR7 . CVC blocked RANTES - induced Ca2 + mobilization . [ 0243] Two metabolites of CVC (M -I and M - II ) were Efficacy and Safety Results detected in animal studies (see Example 20 ); M - II was a [0247 ] CVC showed a potent effect on HIV - 1 RNA levels major metabolite in monkeys and dogs , M - I was a minor that persisted after completion of treatment. The median metabolite in all species. M - I inhibited the binding of nadir changes from baseline for the 25 -, 50 - , 75 -, and RANTES to CCR5 - expressing cells with an IC50 of 6 . 5 150 -mg doses were - 0 . 7 , - 1 . 6 , - 1 .8 , and - 1 . 7 loglº copies / nmol/ L , which is approximately 2- fold the IC50 of CVC . mL , respectively, in CCRS -antagonist naive , treatment- ex M - II had no effect on binding of RANTES . perienced HIV - 1 infected subjects. These results demon strate the potent antagonistic CCRS activity of CVC . The Example 20 mean changes in HIV - 1 RNA levels are shown in FIG . 45 . Identification of Metabolites [0248 ] Exploratory assessment of changes in MCP - 1 (a [0244 ] After single -dose , oral administration of [ 14C ] ligand of CCR2 , which is a chemokine co -receptor CVC at 3 mg/ kg to fed animals , unchanged CVC was the expressed on pro - inflammatory monocytes, also known as US 2019 /0099429 A1 Apr . 4 , 2019
CCL2 ), hs -CRP , and IL - 6 were performed and significant elevation were in the 50 mg dose group , and one of these dose - dependent increases in MCP - 1 were observed ( Table subjects had a Grade 1 elevated AST at baseline . The AST 23 ). elevations observed in subjects in the 100 mg and 150 mg [0249 ] On Day 10 , least square mean MCP - 1 levels were dose groups during treatment ( observed in 1 subject in each 56 . 3, 94 . 2 , 34 . 4 , and 334 . 3 pg /mL higher than at Baseline in dose group ) , returned to normal values during continuation the 25 -, 50 -, 75 -, and 150 -mg dose groups, respectively , of treatment. No Grade 2 - 4 elevations in ALT or AST compared to a slight decline in the placebo group . At the 50 and 150 -mg doses, these results were statistically significant occurred during the study. ( p = 0 .024 and p < 0 . 001, respectively ). These results demon [0252 ] The only Grade 3 or higher laboratory abnormali strate the potent antagonistic CCR2 activity of CVC . CVC ties were a Grade 3 hypophosphatemia in the 25 mg dose had no effect on hs -CRP or IL - 6 levels overall in this 10 - day group that was present before dosing , a Grade 4 elevated study . triglyceride in the 50 mg dose group in a subject who had a TABLE 23 Parameter Placebo CVC 25 mg CVC 50 mg cVC 75 mg CVC 150 mg Baseline, pg /mL n = 10 n = 9 n = 7 n = 7 n = 8 Mean 22. 4 20 . 0 12. 6 26 . 6 31 . 6 Median 18 . 5 16 . 0 6 . 0 8 . 0 19 . 5 6 - 50 7 - 44 5 - 37 5 - 92 8 -82 Day 10 , pg /mL n = 10 n = 9 n = 7 n = 7 n = 8 Mean 21 . 0 75 . 3 101. 3 59 . 1 372 . 0 Median 12 . 5 39 . 0 65 . 0 43 . 5 368 . 0 Range 5 - 52 10 - 287 21 - 266 70 - 128 79 -605 Change from n = 10 n = 9 n = 7 n = 7 n = 8 Baseline to Day 10 LS mean - 1 . 9 + 56 . 3 + 94 . 2 + 34 . 4 + 333 . 4 P - valuea 0 .095 0 . 024 0 . 222 < 0 .001 Median 0 .0 + 25 .0 + 56 . 0 + 36 . 0 + 322 . 0 Abbreviation : LS, least squares a P - values were one- sided and based on comparison of each dose of CVC with placebo without multiple comparisons adjustment.
Adverse Events Grade 3 triglyceride at baseline , and Grade 3 and 4 amylase and lipase , respectively , in a subject with a prior history of [0250 ] Cenicriviroc was generally well tolerated at the pancreatitis . doses studied and no safety concerns were identified . There were no deaths, SAEs, or other significant AEs , and there Cardiovascular Safety and Physical Examinations were no discontinuations because of an AE . Most treatment emergent AEs were mild or moderate in severity . Subjects [ 0253 ] A Grade 3 systolic hypertension was observed in a who received 150 mg of CVC ( ie , the highest dose studied ) subject in the 150 -mg dose group who had a Grade 2 had more Aes compared to subjects in the other dose groups, elevation in systolic blood pressure at baseline. There were although the severity of AEs was comparable across all dose no clinically relevant physical examination or ECG findings . groups. The most common (210 % ) treatment -emergent AEs [0254 ) As previously described , CVC has a dual activity in this study were nausea ( 18 . 5 % ), diarrhea ( 16 .7 % ), head as a CCR5 and CCR2 antagonist . Exploratory assessment of ache ( 14 . 8 % ) , and fatigue ( 11 . 0 % ) . changes in MCP - 1 ( the ligand of CCR2, also known as CCL2 ) , hs -CRP , and IL - 6 were performed and significant Laboratory Safety dose - dependent increases in MCP - 1 were observed ( see Table 24 ) . On Day 10 , least square mean MCP - 1 levels were [ 0251 ] There were 6 subjects with ALT and / or AST eleva 56 . 3 , 94. 2 , 34 . 4 , and 334 .3 pg /mL higher than at Baseline in tions in the 25 mg ( 2 subjects ) , 50 mg ( 2 subjects ), 100 mg the 25 , 50 , 75 , and 150 mg dose groups, respectively , ( 1 subject) , and 150 mg ( 1 subject ) dose groups, and 1 compared to a slight decline in the placebo group . At the 50 subject with an AST elevation in the placebo group during and 150 mg doses , these results were statistically significant the observation period . All elevations were Grade 1 , were ( p = 0 . 024 and p < 0 .001 , respectively ) . These results demon isolated except in 2 subjects (both in the 50 -mg dose group ) strate the potent antagonistic CCR2 activity of CVC . CVC who had more than a single elevation , and resolved without had no effect on hs - CRP or IL - 6 levels overall in this 10 -day sequelae . The 2 subjects who had more than a single study . TABLE 24 Summary of MCP - 1 Levels by Cohort - Study 201 Parameter Placebo CVC 25 mg CVC 50 mg cVC 75 mg CVC 150 mg Baseline n = 10 n = 9 n = 7 n = 7 n = 8 Mean 22 . 4 20 . 0 12 . 6 26 . 6 31 . 6 Median 18 . 5 16 . 0 6 . 0 8 . 0 19 . 5 US 2019 /0099429 A1 Apr . 4 , 2019 28
TABLE 24 - continued Summary of MCP- 1 Levels by Cohort - Study 201 Parameter Placebo CVC 25 mg CVC 50 mg CVC 75 mg CVC 150 mg Range 6 - 50 7 -44 5 - 37 5 - 92 8 - 82 Day 10 , pg /mL n = 10 n = 9 n = 7 n = 8 n = 8 Mean 21 . 0 75 . 3 101. 3 59 . 1 372 . 0 Median 12 . 5 39 . 0 65 . 0 43 . 5 368 . 0 Range 5 - 52 10 - 287 21- 266 20 - 128 79 -605 Change from n = 10 n = 9 n = 7 n = 7 n = 8 Baseline to Day 10 LS mean - 1 . 9 + 56 . 3 + 94 . 2 + 334 . 3 P value - 0 . 095 0 . 024 0 . 222 < 0 .001 Median 0 . 0 + 25 . 0 + 56 . 0 + 36 . 0 + 322 . 0 Abbreviation : LS, least squares " P - values were one - sided and based on comparison of each dose of CVC with placebo without multiple comparisons adjustment. Resistance Data inhibitory effect of the drug . Thus, dose levels of 75 and 150 [0255 ] In Study 201 , drug resistance testing was per mg QD displayed potent antiviral activity , with PD effects formed at Baseline , Day 7, and Day 40 (or at the “ Early greater than 80 % of the Emax of CVC in HIV - 1 - infected Termination ” visit , if applicable ). All subjects with evalu subjects . able samples remained fully susceptible to CVC . Example 22 Viral Tropism Long - Term Efficacy Data in HIV - 1 Infected Adult [ 0256 ] All subjects in Study 201 were tested for viral Subjects tropism to exclude that their virus was CXCR4 tropic or dual/ mixed . All subjects had CCR5 -tropic virus at screening Efficacy Results of Study 202 (based on the enhanced sensitivity profile assay ) . A total of 39 subjects on CVC had evaluable samples following treat Study Design and Objectives ment, and one of these subjects ( in the CVC 150 mg dose group ) was found to have dual/ mixed - tropic virus on Day [0260 ] This was a randomized , double- blind , double 10 . Further testing ( at another laboratory using a different dummy, 48 -week comparative study evaluating efficacy and assay ) revealed that this subject had mainly CXCR4- tropic safety of CVC 100 mg and CVC 200 mg compared to virus at Baseline , therefore, this subject should not have approved antiretroviral agent efavirenz (EFV , Sustiva® ), all been enrolled in the study according to the inclusion criteria . administered in combination with approved antiretroviral agents emtricitabine/ tenofovir disoproxil fumarate (FTC / This subject did not respond to CVC treatment ; the largest TDF ) , in HIV - 1 infected , antiretroviral treatment- naive adult decrease in HIV - 1 RNA of this subject was 0 . 13 logio subjects with only CCR5 - tropic virus . Subjects with a his copies /mL below the baseline value . tory of HIV - 2 , hepatitis B and /or C , cirrhosis of the liver or any known active or chronic active liver disease were excluded from the study. [0257 ] For all doses tested in Study 201, a more than dose [0261 ] Approximately 150 subjects were planned to be proportional increase in exposure was observed for “ For randomized ( 143 subjects were actuall randomized ) in a mulation Fl ” , which was used for all but the 100 mg dose 2 : 2 : 1 ratio to CVC 100 mg + placebo , CVC 200 mg + placebo cohort . or the approved antiviral agent efavirenz (EFV ) + placebo , all [0258 ] Drug response was characterized using the follow in combination with approved antiviral agents emtricitabine/ ing maximum effect (Emar ) model: tenofovir disoproxil fumarate (FTC / TDF) provided as open label study drug in a fixed dose combination formulation ( TRUVADA® ) . A pharmacokinetic assessment was con E = Ep + (Imax – Eo ). CY ductedin the first 25 study subjects to confirm that adequate IC :o + CY CVC plasma exposures were achieved at the selected doses of CVC 100 mg and CVC 200 mg prior to enrolling the remainder of the study population . where E is effect, E0 is the baseline effect ( fixed to 0 ), Imamax is the maximum inhibition , C denotes the PK variable ( AUC0- 24 , Cmax, or steady - state concentration [ C ]) , IC5 , is Demographic and Baseline Characteristics the value of the PK variable which corresponds to 50 % of [0262 ] Most subjects were male ( 94 % ) and white (62 % ), themaximum inhibition and y is the shape parameter which with a mean age of 35 years and a mean body mass index of describes the degree of sigmoidicity . 26 . 2 kg /m2 . In total , 32 % of subjects were Black / African [0259 ] The Emax of CVC in the PK /PD model was - 1. 43 American . In addition , 24 % of the randomized subjects were log10 copies/ mL . Based on the Emax model, average Css of of Hispanic ethnicity . CVC for the 25, 50 , 75 , and 150 mg doses were expected to [0263 ] At Baseline , the median duration of HIV - 1 infec result in 54. 9 % , 79 . 8 % , 85 . 9 % , and 95 . 9 % of the maximum tion ( ie , time [months ] since first positive HIV - 1 test to US 2019 /0099429 A1 Apr . 4 , 2019
informed consent date ) was 8 months, the mean HIV - 1 RNA [0265 ] The Week 48 data were consistent with the data was 4 .50 loglº copies /mL . (80 % of subjects had viral load observed at Week 24 . The percentage of subjects with < 100 ,000 copies /mL ), and the mean CD4 + cell count was virologic success over time was generally comparable 402 cells /mm (58 % of subjects had CD4 + cell counts2350 among the 3 treatment arms, although higher in the CVC cells /mm3 ) . arms compared to the EFV arm at Week 48 (68 % with CVC 100 mg, 64 % with CVC 200 mg , and 50 % with EFV ) . Primary Efficacy Results [0264 ] The primary efficacy endpoint was virologic Secondary and Exploratory Analyses response at Week 24 , defined as HIV - 1 RNA < 50 copies /mL using the FDA Snapshot Algorithm . The percentage of Biomarkers of Inflammation subjects with virologic success (response ) was comparable [0266 ] As an exploratory analysis , levels of inflammation among the 3 treatment arms (76 % with CVC 100 mg, 73 % biomarkers MCP - 1 , sCD14 , high sensitivity C - reactive pro with CVC 200 mg, and 71 % with EFV ) . More subjects in the tein [ hs -CRP ] , interleukin - 6 ( IL - 6 ) , D - dimer, and fibrino EFV arm prematurely discontinued the study ( 11 out of 28 gen ) were measured . Baseline values and changes from subjects , 39 % ) than in the CVC 100 mg arm ( 17 out of 59 baseline at Week 24 and Week 48 of MCP - 1 , SCD14 , subjects , 29 % ) and the CVC 200 mg arm (15 out of 56 hs -CRP , IL - 6 , D - dimer , and fibrinogen are summarized in subjects , 27 % ) . Table 25 . TABLE 25 CVC CVC EFV 100 mg 200 mg 600 mg Mean (SE ) Mean (SE ) Mean (SE ) Parameter N Median (min ; max ) N Median (min ; max ) N Median (min ; max ) MCP- 1 (pg / mL ) Baseline value 55 128 ( 8 . 3 ) 54 153 ( 8 . 4 ) 139 ( 19 . 2 ) 110 ( 57 ; 337 ) 137 (68 ; 393 ) 122 (57 ; 608 ) Changes from baseline 48 493 ( 46 . 2 ) * 753 (50 .2 )* - 44 (24 . 1 ) at Week 24 429 ( 184 ; 2352 ) 695 (48 ; 1557) - 17 ( -471 ; 77 ) Changes from baseline 41 636 (63 . 8 ) * 39 900 (90 . 9 ) * 4 . 2 ( 29 . 49 ) at Week 48 523 (220 ; 2616 ) 756 (121 ; 3259 ) 33. 6 ( - 437; 175 ) sCD14 (x10 pg /mL ) ( original values) Baseline value 55 1 .80 ( 0 . 062 ) 54 1 .88 (0 . 069 ) 28 2 .00 (0 .105 ) 1 .73 ( 1 .07 ; 3. 77 ) 1 . 86 ( 1 .05 ; 3 .76 ) 2 .02 (0 . 93 ; 3 .95 ) Changes from baseline 48 - 0 . 19 ( 0 .064 ) * 44 - 0 . 23 ( 0 .066 ) * 0 .23 (0 .143 ) at Week 24 - 0 . 18 (- 1. 33 ; 0 .95 ) - 0 . 19 ( - 1 .78 ; 0 . 80 ) 0 . 13 (- 1 .60 ; 1. 33 ) Changes from baseline 41 0 . 10 ( 0 .070 ) * 39 - 0 . 04 (0 .081 ) * 19 0 .64 ( 0 . 178 ) at Week 48 - 0 . 04 ( - 1 . 24 ; 1 . 15 ) 0 .46 ( - 0 .50 ; 2 .51 ) hs - CRP (mg /dL ) Baseline value 57 0 . 39 ( 0 . 128 ) 54 0 . 46 (0 . 149 ) 28 0 .81 ( 0 .374 ) 0 .15 ( 0 .01 ; 6 .48 ) 0 . 15 (0 .02 ; 6 .81 ) 0 . 14 0 .02 ; 9 .81 ) Changes from baseline 52 - 0 . 16 0 .121 ) 49 - 0 .04 ( 0 . 138 ) - 0 .46 (0 . 529 ) at Week 24 - 0 .03 ( - 6 .07 ; 0 .86 ) - 0 .04 (- 4 .03 ; 4 .72 ) - 0 .01 ( - 9 .26 ; 4 . 12 ) Changes from baseline 44 - 0 .08 ( 0 . 161) 40 - 0 . 18 (0 . 144 ) 20 - 0 .71 ( 0 .484 ) at Week 48 - 0. 01 (- 6. 22 ; 2. 72 ) - 0. 04 ( -4 . 13 ; 0. 67 ) - 0 .03 ( - 8 . 93 ; 0 . 17 ) IL - 6 (pg /mL ) Baseline value 57 2. 51 (0 . 306 ) 52 3. 34 (0 . 561) 28 13 .81 ( 9 .418 ) 1 .90 ( 1 . 90 ; 18 .00 ) 1 . 90 ( 1 . 90 ; 21 .50 1 . 90 ( 1 . 90 ; 264 . 00 ) Changes from baseline 52 0 .42 (0 .375 ) 47 0. 81 (0 .877 ) 21 - 8. 72 ( 7 .518 ) at Week 24 0 .00 ( - 4 . 80 ; 12 .80 ) 0 . 00 ( - 12. 10 ; 33. 80 ) 0 .00 ( - 149 .00 ; 29 .70 ) Changes from baseline 44 0 . 29 ( 0 . 362) 38 -0 .04 (0 .471 ) 20 - 13 . 11 ( 10 .320 ) at Week 48 0 .00 ( - 5 . 20 ; 10 . 90 ) 0 .00 ( - 12. 10 ; 7 .70 ) 0 . 00 ( - 204. 10 ; 5 .00 ) D - dimer ( ng/ mL ) Baseline value 56 187 (21 . 1 ) 54 184 ( 19 . 0 ) 163 ( 19 . 0 ) 150 (49 ; 800 ) 125 (49 ; 750 ) 150 (49 ; 450 ) Changes from baseline 51 - 32 (25 . 4 ) - 64 (16 . 2 ) - 53 ( 24 . 7 ) - 1 . 0 ( - 550 ; 801 ) - 50 (- 500 ; 100 ) - 26 ( - 350 ; 150 ) Changes from baseline 42 - 41 ( 23 . 1 ) 40 - 70 (21 . 3 ) - 34 (25 . 7 ) at Week 48 - 1 .0 ( - 650 ; 250 ) - 50 ( - 701; 100 ) 0 .0 (- 300 ; 150 )