Patent Application: US-201113194248-A

Abstract:
the present invention is based on the discovery of associations that exist between single nucleotide polymorphisms on chromosome 19 and virological outcomes in a diverse population of patients with hepatitis c virus who received interferon - free treatment .

Description:
to facilitate the understanding of this invention , a number of terms are defined below . terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention . terms such as “ a ”, “ an ” and “ the ” are not intended to refer to only a singular entity , but include the general class of which a specific example may be used for illustration . the terminology herein is used to describe specific embodiments of the invention , but their usage does not delimit the invention , except as outlined in the claims . the term “ response ” to treatment is a desirable response to the administration of an agent . “ interferon - free treatment ” refers to treatment of patients without the use of exogenous interferon or pegylated interferon as defined herein below . virological endpoints included “ early virological response ” ( evr ), defined as ≧ 2 - log drop in serum hcv rna (“ viral load ”) from baseline to week 12 ( by cobas amplicor hcv monitor test , v2 . 0 , limit of quantitation 600 iu / ml ), complete evr ( cevr ) defined as undetectable hcv rna in serum ( by cobas amplicor hcv test v2 . 0 , limit of detection 50 iu / ml ) or and “ sustained virological response ” ( svr ), defined as undetectable hcv rna (& lt ; 50 iu / ml ) at the end of a 24 - week untreated follow - up period . the terms “ sample ” or “ biological sample ” refers to a sample of tissue or fluid isolated from an individual , including , but not limited to , for example , tissue biopsy , plasma , serum , whole blood , spinal fluid , lymph fluid , the external sections of the skin , respiratory , intestinal and genitourinary tracts , tears , saliva , milk , blood cells , tumors , organs . also included are samples of in vitro cell culture constituents ( including , but not limited to , conditioned medium resulting from the growth of cells in culture medium , putatively virally infected cells , recombinant cells , and cell components ). the terms “ interferon ” and “ interferon - alpha ” are used herein interchangeably and refer to the family of highly homologous species - specific proteins that inhibit viral replication and cellular proliferation and modulate immune response . typical suitable interferons include , but are not limited to , recombinant interferon alpha - 2b such as intron ® a interferon available from schering corporation , kenilworth , n . j ., recombinant interferon alpha - 2a such as roferon ®- a interferon available from hoffmann - la roche , nutley , n . j ., recombinant interferon alpha - 2c such as berofor ® alpha 2 interferon available from boehringer ingelheim pharmaceutical , inc ., ridgefield , conn ., interferon alpha - nl , a purified blend of natural alpha interferons such as sumiferon ® available from sumitomo , japan or as wellferon ® interferon alpha - nl ( ins ) available from the glaxo - wellcome ltd ., london , great britain , or a consensus alpha interferon such as those described in u . s . pat . nos . 4 , 897 , 471 and 4 , 695 , 623 ( especially examples 7 , 8 or 9 thereof ) and the specific product available from amgen , inc ., newbury park , calif ., or interferon alpha - n3 a mixture of natural alpha interferons made by interferon sciences and available from the purdue frederick co ., norwalk , conn ., under the alferon tradename . the use of interferon alpha - 2a or alpha - 2b is preferred . interferons can include pegylated interferons as defined below . the terms “ pegylated interferon ”, “ pegylated interferon alpha ” and “ peginterferon ” are used herein interchangeably and means polyethylene glycol modified conjugates of interferon alpha , preferably interferon alfa - 2a and alfa - 2b . typical suitable pegylated interferon alpha include , but are not limited to , pegasys ® and peg - intron ®. the term “ ribavirin ” refers to the compound , 1 -(( 2r , 3r , 4s , 5r )- 3 , 4 - dihydroxy - 5 - hydroxymethyl - tetrahydro - furan - 2 - yl )- 1h -[ 1 , 2 , 4 ] triazole - 3 - carboxylic acid amide which is a synthetic , non - interferon - inducing , broad spectrum antiviral nucleoside analog and available under the names , virazole ® and copegus ®. “ direct acting antiviral agents ” exert specific antiviral effects independent of immune function . examples of direct acting antiviral agents for hcv include but are limited to protease inhibitors , polymerase inhibitors , ns5a inhibitors , ires inhibitors and helicase inhibitors . the terms “ rg7128 ” and “ r05024048 ” are used interchangeably and refer to the diisobutyl ester prodrug of the cytosine nucleoside analog b - d - 2 ′- deoxy - 2 ′- fluoro - 2 ′- c - methycytidine which is an inhibitor of the hcv ns5b rna polymerase . other hcv ns5b polymerase inhibitors include “ ana - 598 ” from anadys pharmaceuticals , “ abt - 333 ” from abbott , “ vx - 222 ” from vertex pharmaceuticals , “ bi - 207127 ” from boehringer ingelheim , and “ filibuvir ” from pfizer . the terms “ danoprevir ”, “ rg7227 ”, “ r05190591 ” and “ itmn - 191 ” are used interchangeably and refer to the macrocyclic peptidomimetic inhibitor of the hcv ns3 / 4a protease , 4 - fluoro - 1 , 3 - dihydro - isoindole - 2 - carboxylic acid ( z )-( 1s , 4r , 6s , 14s , 18r )- 14 - tert - butoxycarbonylamino - 4 - cyclopropanesulfonylaminocarbonyl - 2 , 15 - dioxo - 3 , 16 - diaza - tricyclo [ 14 . 3 . 0 . 0 * 4 , 6 *] nonadec - 7 - en - 18 - yl ester . other hcv ns3 and ns3 / 4a protease inhibitors include , “ boceprevir ” or “ sch - 503034 ”: ( 1r , 5s )- n -[ 3 - amino - 1 ( cyclobutylmethyl )- 2 , 3 - dioxopropyl ]- 3 -[ 2 ( s )-[[[( 1 , 1 - dimethylethyl ) amino ] carbonyl ] amino ]- 3 , 3 - dimethyl - 1 - oxobutyl ]- 6 , 6 - dimethyl - 3 - azabicyclo [ 3 . 1 . 0 ] hexan -( s )- carboxamide ; and “ telaprevir ” or “ vx - 950 ”: ( 1s , 3ar , 6as )- 2 -[( 2s )- 2 -[[( 2s )- cyclohexyl [( pyrazinylcarbonyeamino ] acetyl ] amino ]- 3 , 3 - dimethylbutanoyl ]- n — r1s )- 1 -[( cyclopropylamino ) oxoacetyl ] butyl ] octahydrocyclopenta [ c ] pyrrole - 1 - carboxamide ; “ vaniprevir ” or “ mk - 7009 ”: ( 1r , 21s , 24s )- 21 - tert - butyl - n -(( 1r , 2r )- 1 -{[( cyclopropylsulfonyl ) amino ] carbonyl }- 2 - ethylcyclopropyl )- 16 , 16 - dimethyl - 3 , 19 , 22 - trioxo - 2 , 18 - dioxa - 4 , 20 , 23 - triazatetracyclo [ 21 . 2 . 1 . 1 . 0 ] heptacosa - 6 , 8 , 10 - triene - 24 - carboxamide ; and “ narlaprevir ” or “ sch - 900518 ”: ( 1r , 2s , 5s )- 3 -(( s )- 2 -( 3 -( 1 -( tert - butylsulfonylmethyl ) cyclohexyl ) ureido )- 3 , 3 - dimethylbutanoyl )- n —(( s )- 1 -( cyclopropylamino )- 1 , 2 - dioxoheptan - 3 - yl )- 6 , 6 - dimethyl - 3 - azabicyclo [ 3 . 1 . 0 ] hexane - 2 - carboxamide . for patients with chronic hepatitis c ( chc ) the current recommended first line treatment , referred as standard of care or soc , is pegylated interferon alpha in combination with ribavirin for 48 weeks in patients carrying genotype 1 or 4 virus and for 24 weeks in patients carrying genotype 2 or 3 virus . combined treatment with ribavirin was found to be more effective than interferon alpha monotherapy in patients who relapsed after one or more courses of interferon alpha therapy , as well as in previously untreated patients . however , ribavirin exhibits significant side effects including teratogenicity and carcinogenicity . furthermore , ribavirin causes hemolytic anemia requiring dose reduction or discontinuation of ribavirin therapy in approximately 10 to 20 % of patients , which may be related to the accumulation of ribavirin triphosphate in erythrocytes . therefore , to reduce treatment cost and the incidence of adverse events , it is desirable to tailor the treatment to a shorter duration while not compromising efficacy . a shortened “ duration of treatment ” for genotype 1 patients with pegylated interferon alpha with ribovirin would be , for example , 24 weeks . a shortened duration of treatment for genotype 1 patients with a direct acting antiviral agent could be as short as 8 weeks , 12 weeks , or 16 weeks . as used herein , the terms “ allele ” and “ allelic variant ” refer to alternative forms of a gene including introns , exons , intron / exon junctions and 3 ′ and / or 5 ′ untranslated regions that are associated with a gene or portions thereof . generally , alleles occupy the same locus or position on homologous chromosomes . when a subject has two identical alleles of a gene , the subject is said to be homozygous for the gene or allele . when a subject has two different alleles of a gene , the subject is said to be heterozygous for the gene . alleles of a specific gene can differ from each other in a single nucleotide , or several nucleotides , and can include substitutions , deletions , and insertions of nucleotides . as used herein , the term “ polymorphism ” refers to the coexistence of more than one form of a nucleic acid , including exons and introns , or portion ( e . g ., allelic variant ) thereof . a portion of a gene of which there are at least two different forms , i . e ., two different nucleotide sequences , is referred to as a polymorphic region of a gene . a polymorphic region can be a single nucleotide , i . e . “ single nucleotide polymorphism ” or “ snp ”, the identity of which differs in different alleles . a polymorphic region can also be several nucleotides long . numerous methods for the detection of polymorphisms are known and may be used in conjunction with the present invention . generally , these include the identification of one or more mutations in the underlying nucleic acid sequence either directly ( e . g ., in situ hybridization ) or indirectly ( identifying changes to a secondary molecule , e . g ., protein sequence or protein binding ). one well - known method for detecting polymorphisms is allele specific hybridization using probes overlapping the mutation or polymorphic site and having about 5 , 10 , 20 , 25 , or 30 nucleotides around the mutation or polymorphic region . for use in a kit , e . g ., several probes capable of hybridizing specifically to allelic variants , such as single nucleotide polymorphisms , are provided for the user or even attached to a solid phase support , e . g ., a bead or chip . the single nucleotide polymorphisms , “ rs28416813 ”, “ rs12979860 ”, and “ rs810314 ” refer to snps identified by accession number in the database of snps ( dbsnp , www . ncbi . nlm . nih . gov / snp /) and are located on human chromosome 19 within the il28b gene and its promoter region . utilization of baseline genetic predictors for interferon responsiveness allows tailoring of direct acting antiviral therapies , as well as standard of care with interferon . patients who are defined as poorly interferon responsive , i . e . two t alleles in rs12979860 ( or two c alleles of rs8103142 or 2 g alleles of rs2841683 ), may be poor candidates for small molecule therapeutics that rely on additive or synergistic effects of endogenous or exogenous interferon mediated pathways — particularly if these are provided as a single agent in addition to soc . the concern would be that these patients would have an increased risk of developing drug resistant mutations as a result of effective monotherapy . conversely , patients with an interferon responsive phenotype , i . e . two c alleles in rs12979860 ( or two t alleles of rs8103142 or two c alleles of rs2841683 ), would make superior candidates for shorter courses of therapy with soc alone , or in combination with small molecules . additional considerations that genetic predisposition to interferon responsiveness allow is “ tuning ” of combination direct acting antiviral agents . patients predicted to have acceptable endogenous interferon responsiveness may be excellent candidates for drugs that target viral functions - such as protease inhibitors which also have an inhibitory role on endogenous interferon response — and poorer candidates for drugs that decrease the amounts of viral pamp ( pathogen associated molecular pattern , e . g . polymerase inhibitors ) as these may serve to impair the patients capacity to facilitate their own cure via their endogenous interferon responsiveness . similarly , patients predicted to have poor interferon responsiveness might be candidates for “ quad ” therapy as a first line of therapy ( 2 direct acting antiviral agents , added to peginterferon with ribavirin ), as compared to a direct acting antiviral agent alone , or triple therapy ( soc with one daa ). the results of analysis of the inform - 1 trial ( described in the examples section ), suggest that interferon responsive and poorly interferon responsive phenotypes display early differences to 2 combined direct acting antiviral agents for the treatment of chc in the absence of soc . as early responses are known to correlate with sustained virological response for chc patients treated with soc , markers of interferon responsiveness may potentially be used for guidance of interferon - free treatments . for example patient populations with two c alleles in rs12979860 ( or two t alleles of rs8103142 or two c alleles of rs2841683 ), indicative of an interferon responsive phenotype , may achieve svr with a shorter duration or reduced dosages of 2 or more combined direct acting antiviral agents without soc compared with patient populations two t alleles in rs12979860 ( or two c alleles of rs8103142 or 2 g alleles of rs2841683 ) indicative of a poorly interferon responsive phenotype . il28b genotype in combination with clinical and other laboratory parameters may also be used to select the optimal treatment regimen for an individual patient among interferon - containing and interferon free treatments . by minimizing where possible the expected duration and intensity of treatment needed to achieve svr , drug safety , in terms of adverse event rates , and efficacy , in terms of treatment compliance rates , may be improved . genotype analysis for il28b polymorphisms was performed on patients with chronic hepatitis c ( chc ) enrolled in the inform - 1 trial . the aim of the inform - 1 trial was to demonstrate that a combination of two experimental direct acting antivirals ( daas ) without pegylated - interferon or ribavirin , currently standard of care ( soc ) for chc , could be safely administered and provide significant antiviral activity without the emergence of resistance . the study medications consisted of rg7128 ( also known as r05024048 ), the diisobutyl ester prodrug of the cytosine nucleoside analog β - d - 2 ′- deoxy - 2 ′- fluoro - 2 ′- c - methylcytidine which is an inhibitor of the hcv ns5b rna polymerase and danoprevir ( also known as rg7227 , r05190591 and itmn - 191 ), the macrocyclic peptidomimetic inhibitor of the hcv ns3 / 4a protease . both have potent in vitro and in vivo activity against hcv , and at the time of this study both compounds were in phase i development . inform - 1 was a phase 1b , randomized , double - blind , placebo - controlled , dose - escalating trial . eligible patients were males and females of non - child - bearing potential between 18 and 65 years , with chronic genotype 1 hcv infection , without cirrhosis , and with a minimum baseline hcv rna of 10 5 iu / ml ( roche taqman assay ). the study included soc treatment - naïve , treatment failure ( tf ), and null responder patients . treatment - naïve was defined as never having received an interferon - based treatment regimen for chc ; treatment failure ( non null ) was defined as either relapsers ( patients whose hcv rna fell below the limit of detection while receiving soc , but relapsed following discontinuation of therapy ) or partial - responders ( patients who had a hcv rna reduction of at least 2 log 10 units after 12 weeks of treatment while on prior soc but whose hcv rna always remained detectable in blood ). null responders demonstrated less than 1 log 10 unit reduction in hcv rna with one month and / or less than 2 log 10 reduction following 12 weeks of prior soc therapy . enrolled patients were randomized to either study treatment or placebo according to table 1 . cohorts b - g received 13 days of experimental therapy . while all cohorts showed significant viral load reduction , cohorts c , d , f , and g received the highest doses of study drug with and had comparable exposures . these cohorts also demonstrated similar reductions in viral load . plasma hcv rna levels were measured with the cobas taqman hcv assay , version 2 , ( roche molecular systems ), with a lower limit of quantification of 43 iu / ml and a lower limit of detection of 15 iu / ml hcv rna levels were measured at the time of screening , at baseline , and on treatment at multiple times through the end of study drug administration and over 90 days of follow - up . additional details of the study design , inclusion and exclusion criteria and primary results of these trials are published elsewhere . blood samples for genetic analysis were collected from patients who consented to participate in genetic analyses were stored in the roche sample repository . dna was extracted at the roche sample repository and normalized to 50 ng / μl . 53 patients ( 37 treatment naive , 8 prior treatment failures ( tfs ) who were partial responders / relapsers to soc , and 8 null responders to soc ) who received combination therapy at different doses for 14 days were studied . naive patients were randomized to 4 treatment cohorts which differed in dosage and scheduling of treatment medications . genotypes at the rs28416813 , rs12979860 , and rs8103142 loci were determined by direct sequencing of portions of the il28b gene and upstream regions . viral kinetic profiles during 13 days of interferon free therapy as well as after 4 and 12 weeks of subsequent soc were compared by genotype . for analysis purposes , the lowest dosage cohort ( b ) was not considered . overall frequencies of rs12979860 genotypes for all 53 inform 1 patients were cc — 28 . 3 %, ct — 58 . 5 %, and tt — 13 . 2 %. the cc genotype frequency was similar across the high dose cohorts ( n = 45 ) ( table 2 ). genotype results for rs28416813 and rs8103142 were essentially identical to those for rs12979860 . differences ( non - statistically significant owing to the small number of patients studied ) in viral kinetics were observed over 13 days of ifn - free treatment when patients were stratified by rs12979860 genotype . for patients receiving comparable doses of study drugs ( fig1 , table 3 ), those carrying the cc genotype of rs12979860 have a mean viral load reduction approximately 0 . 5 log 10 unit greater than other genotypes after 14 days of therapy and the distribution of responses is shifted compared to those with non - cc genotypes . these data suggest that il28b genotype may influence early viral kinetics in patients receiving interferon - free treatments for hepatitis c , but appear to do so to a lesser extent than for interferon - containing treatments . this observation is consistent with the hypothesis that il28b genotype information reflects endogenous interferon responsiveness . il28b genotype information could inform selection of chc treatment in terms of medications , dosage , or duration for both interferon free and interferon - containing regimens . all of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure . while the compositions and methods of this invention have been described in terms of preferred embodiments , it will be apparent to those of skill in the art that variations may be applied to the compositions and / or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept , spirit and scope of the invention . all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit , scope and concept of the invention as defined by the appended claims . 1 . ghany m g , strader d b , thomas d l , seeff l b . diagnosis , management , and treatment of hepatitis c : an update . hepatology 2009 ; 49 : 1335 - 1374 . 2 . manns m p , mchutchison j g , gordon s c , rustgi v k , shiffman m , reindollar r , goodman z d , koury k , ling m , albrecht j k . peginterferon alfa - 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