US007645732B2
`
`(12) United States Patent
`Ye et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 7,645,732 B2
`Jan. 12, 2010
`
`(54)
`
`'l'REA'l'I.\'G HEPA'l'I'l'[S (: VIRUS [NFEC'l'l0N
`
`(56)
`
`References Cited
`
`(75)
`
`l11ve11Iors:
`
`‘
`,
`.Ii11Ye. Dallas. IX (US): Fang Sun.
`Chesire. CT (US); Hua Huang. Dallas,
`TX (US); Michael J. Gale. Dallas. TX
`(US)
`
`(73) Assignee: Board of Regents, The University of
`Texas System, Austin. TX (US)
`
`( ’* ) Notice:
`
`Subject to any disclaimer. tl1e term of this
`patent is extended or adiusted under 35
`U ‘S F 154 b b 324 d’
`‘
`"
`(
`l 3"
`‘W5’
`
`N0’:
`
`(22)
`
`Filed:
`
`Jan. 24, 2007
`
`(65)
`
`Pl'l0l' Pllbllcatfilfl Data
`
`U.S. PATENT DOCUMENTS
`
`2005.-"(E21464 Al *
`2009.-'0042s35 A1""
`
`10.-"2005 Andre el al.
`2.-"2009 Davis
`
`435.-‘"239
`514.63
`
`OTHER PUl3LlCATl0NS
`
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`potent:
`1
`itorso
`1oo1,__an1c
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`vol.
`|4.[ss11e2[l.[)c1. I8. 2004, pp. 5[Jt'1'?—5[}'1'(l.
`AC5 Registry x1o.2n2914—s4—o. Mar. 19. I998.
`AC5 Registry NO. 182431-12-5. UCL 30. 1996.
`
`3‘ cited by ‘“"“”"“““*’
`Pr1'mar_1-' E.\'arJ11'r.=cr—Sea11 R McCiar1'y
`(74) An‘0r11e_L'. Agem‘, or F1‘rm—Ricl1z1rd A1011 Os111aI1
`
`US 2(l()8a"()l7r'5864 Al
`
`Jul. 24, 2008
`
`(57)
`
`ABSTRA(:.[-
`
`(51)
`
`lnt.(Il.
`
`A_61K 31’/70
`C HQ ‘V68
`(-7 2N 5/00
`(52) U.S. Cl.
`
`(58)
`
`Field of Classification Search
`See application file for complete search history.
`
`(2U06'UU
`£20061”)
`(200611 ll
`51411: 43515; 5142: S14E44:
`4241229,]
`None
`
`Methods and compositions are provided to inhibit release of
`HCV from an HCV"—infected cell by contacting tl1e cell with a
`V] ,| )l ,asst:111bly ir1l1ibi1o1'_. and dt:1cc1i11g a ncsllltanl lI'll'lllZ)lllL)I'l
`of HVC release from the cell. The methods can be used to
`decrease serum viremia of an l-lCV"—i11fected person.
`
`2'1‘ Claims, No Drawings
`
`1 of 5
`
`PENN EX. 2266
`
`CFAD V. UPENN
`IPR20l5-01836
`
`

`
`US 7,645,?32 B2
`
`2
`prising: a] contacting the cell with a VLDL assembly inhibi-
`tor; and b) detecting a resultant in.l1ibitio11 of IICV release
`from the cell. In a particular embodiment, the cell is contacted
`with a submicromolar amount of the inhibitor. In various
`embodiments, the inhibitor is an MTP inhibitor or a small
`interfering RNA or antis ense oligonucleotide directed against
`ap-olip-oproteiri B. 111 particular embodiments, the contacting
`step further comprises contacting the cell with an antiviral
`agent selected from interferon a11d ribavirin.
`In another aspect, the invention is a method of decreasing
`serum viremia ofan HCV—intected person, the method com-
`prising: a] administering to the person a VLDL assembly
`inhibitor; and b) detecting a resultant decrease i11 serum vire-
`mia in the person. In a particular embodiment, the decrease in
`seru111 viremia is effected by a subniicromolar concentration
`o [the VI ,| )I , assembly inhibitor, such as an MTP inhibitor. In
`various other embodiments, the inhibitor is an M11’ inhibitor
`selected from the group consisting of BMS—200l50. BMS—
`2I2l22, HMS-201038 (AI".RCi-733]. HMS-201030, BMS-
`l97636,
`.l'l"I‘-I30, mitratapide (R-103757),
`implitapide
`(BAY—l 39952), CP—346086_. CI’-467688, and CP-319340. In
`another embodiment the inhibitor is a small interfering RNA
`or antisense oligonucleotide, such as ISIS 301012. directed
`against apolipoprotein I3. In particular embodiments, the con-
`tacting step further comprises contacting the cell with an
`antiviral agent selected from interferon and ribavirin.
`Another aspect of the invention is a kit for decreasing
`serum viremia ofan HCV—in1'ected person. the kit compris-
`ing: a) a plurality of IVl'l‘I-’-inhibitor dosage forms: and a) a
`plurality ofribavirin dosage forms. In specific embodiments.
`the MTP-inhibitor is selected from the group consisting of
`HMS-200150, HMS-212122, HMS-201038 (AIERG-733),
`BMS—20l030_.
`BMS—l97636,
`JTT—l30.
`mitratapide
`(R-l03757],
`implitapide
`(BAY-139952).
`Cl-‘-34(:08(:,
`35 CP467688’ and CF31 9340'
`DETAILED DESCRIPTION OF SPECIFIC
`EMBODIMENTS OF THE INVENTION
`
`10
`
`15
`
`20
`
`25
`
`30
`
`1
`TREATING HEPATITIS (I VIRUS INFECTION
`
`This work was supported by National Institute of Health
`Grant No. 5 P01 I-IL209-48-30 titled Molecular Basis ofCho—
`
`lesterol Metabolism The U.S. government may have rights in
`any patent issuing on this application.
`I3A(TK(iROUNI) 01’ TI [Ii INVI €N'I'I()N
`
`Many viruses can be produced in large amount only in
`certain specialized cell types. A classic example is hepatitis (T
`virus (HCV), a single stranded positive RNA virus of the
`Iilaviviridae family (Appel et al., 2006). that can be secreted
`abundantly only by hepatocytes (Chisari. 2005). The factors
`responsible lor this restriction are largely unknown. In the
`case olil I(.'V’_. o11e clue derives from the demonstration that at
`least a portion of I-ICV circulates in plasma in complex with
`Very I .ow Density I.ipoproteins (VI .I)[ .) (Andre et al.. 2002;
`Nielsen et al._. 2006), a family of spherical particles that are
`produced only in liver (Gibbons et al., 2004) to export trig-
`lyceride and cholesterol ester into plasma (Gibbons et al..
`2000). Although I-ICV’ and VLDL circulate together. a role for
`VLDL in viral assembly or secretion l1as never been demon-
`stratcd.
`
`As for all positive—strand RNA viruses. HCV RNA repli-
`cation occurs in association with cytoplasmic membranes. In
`the case ofHCV these structures, called ‘membranous webs’.
`have been visualized in cultured human hepatoma Huh? cells
`that harbor a subgenomic replicon of HCV (Gosen et al.,
`2003; Moradpour et al._. 2004). These replicons are engi-
`neered I ICV RNA molecules that contain essential elements
`
`for RNA replication, including the coding sequence for the
`nonstructural (NS) proteins NS3. NS!-IA. NS4|3. NSSA and
`NSSB (Lohrnann et al._. 1999]. After transfection into I-Iuh7
`cells, the replicon RNA replicates but it does not produce
`in lectious viral panicles because it does not encode the struc-
`tural proteins that are required for assembly and secretion of
`the virus (I ,ohmann et al.. 1999). The membranous webs that
`harbor the I-ICV replication complex l1ave never been isolated
`40 We describe a method of inhibiting release of hepatitis C
`and lllcll cllmllllslllllll ls llllkllllwll‘
`' virus UICV] from an ”CV_in1bcIcd cc". the method c0mpriS_
`V] DI , assembly is currently believed to occur at two dill
`ing: a) contactino the cell with a VLDL assembly inhibitor:
`lierem Slages (Shelness and Sellers‘ 2001)‘ In the llrsl Slage’
`and b) detectingoa resultant inhibition of HCV release from
`Mlcmllllmal
`lllglycllllllc lmllslcr llmlclll (Mil?) llllllsllil-S
`the Ge”,
`lipid to nascent apolipoprotein B, a huge 540 Kda protein that
`The VLDL assemblv inhibitor preferably blocks the
`S‘l;.ll§llml ll[lIlllgllly1.llli (()llll§l:l llllll
`llllllfll’ 45
`-
`I -
`x
`v
`v
`-
`v
`-
`-
`-
`.
`ll.
`01.11. SL1
`ICICIII
`I
`I
`III
`In
`El 0
`CC[Jl'l'lCS U l
`-
`c:i:;“lu_10I]_1£1ll:;1I;(l1::l1l"L)l:~):‘llggblfifliifile activity of
`uitinzited and degraded duiing transl%tio[h (Avramoglu ahld
`In one embfdinielit
`the VLDL Izissetiibly inhibitor is a
`Adell’ 2004)‘ The apolgicolllallllllg lllllll plllllcles pmclllced
`small interfering RNA For antisense oligonucleotide directed
`ll] lllc llrsl Slagll or V,l‘l)l’ assembly cllllllllll only llmllcd
`amollllls olilrlglycelilde lcllsamva el al“ 2003)‘ ln the Second so against apoB As one example ISIS 301012 is an antisense
`llllllgc’
`lllllll3'Clllllllllllll‘l-l prllcllrslll
`llllrllclcs are lllllcd wllll
`in oligonucleotide in clinical devielopment that targets human
`triglyceride droplets in the luminal compartment (Shelness
`Apflmlm (Human (Mn, Opin M0] Thur (2006) $46 L7)
`and Sellers’ 20m l’ a Step probably facllllaled by apnllpnplw
`I
`rt’ hr etiibbditnerits the V"I_DI_i'1ssembl
`-inhibitor
`lain E lapolal’ anotller major protein Component ln VLDL
`is aliii)tillL'i;liilti that binds to arid inhibits lVl'l‘l-’ actimllit ' and is
`(Mensenkamp et al.. 2001). Although not essential for the $5
`‘refenbl
`a S mhefic 6 e nUn_nauu_a1l‘ Uccumnjj nylljleculé
`dimcl fusion Wlml' MTP is mqulmd lo lmmllcr lrlglyccrldc _
`l:l'IEll irihib}its l\)ll'I‘I-’ activiti at submicrohiolar contentrations
`from the cytosol to the luminal compartment (Shelness and
`g mhclic h‘“.P inhibi1013:
`wc"_kmwn in ‘he an “ch
`Sellers, 2001). I11 htunan and mice, a genetic defect in MTP
`i3)£AS_200l so (See e
`Jfimil e,[ '11 Pmc NaI1 Acad SC} Us
`severely reduces VLDL secretion (Sharp et al.. 1993; Raabe
`([996) 9.H‘l99l_5) ']g3'M‘g_2]21‘2é (“:66 g R‘0b1ma1 J
`etaI., l998).While the first stage otV’LDL assembly is known 60 chem (idol) 44_85’l_6)‘ BMS_201‘0,’8 (‘alder develé mem
`to occur at the endoplasmic reticulum (ER) (Gusarova et al.,
`' as M|.i{G_.n3_
`C g glllqky ct al
`Igiomg Med Chem? [ml
`2003.)-' Th‘: "”“‘°l l°°“.l.l°" °l' lhe secml Stage ’°‘“"l“S °°“”°'
`(20()ct) t4:5ii6r-7()).'13i\i1s-it 976°: 5 see c.g.Wal1getal. J Iiiol
`"'°““*‘l lmhcr and C’”“l’°"c" 2002)‘
`(Iheni (1999) 274:2i793—s(x)), J'l"l‘- 130 (see eg. Aggarwal ct
`SUMMARY OI’ 'I‘I Ili lNVIiN'I‘I()N
`al. I3M(.'(.'ardiovasc I)isord (2005) 5:30; and Burnett. l|)rugs
`(2006) 9:495—9), mitratapide (also known as R—l03757; see
`e.g. Verreck et al, J Pharm Sci (2004) 9321217528).
`impli-
`tapidc (also known as BAY-I 39952; see eg. Ueshima ct al.
`PENN EX. 2266
`
`In one aspect, the invention is a method of inhibiting
`release of IICV from an IICV-infected cell. the method co1n-
`
`GS
`
`2 of 5
`
`CFAD V. UPENN
`IPR20l5-01836
`
`

`
`US 7,645,?32 B2
`
`4
`3
`29:528-35]. In a particular eiiibodiiiient_. a resultant decrease
`Biol Pharm Bull (2005) 281247-52), CP-346086 [see e.g.
`in serum virernia is detected by demonstrating a significant
`Chaiidleret al_. J Lipid Res. (2003) 44: 1887-901 ), CP-467688
`decrease in seruni HCV RNA titer (e. g. using NASBA® test.
`and CP—3l 9340 (see e.g. U.S. Pat. No. 5,919,795), and others.
`Orgaiion Teknika, Boxtel, The Netherlands) compared to pre-
`The invention encompasses methods useful for screening
`treatiiieiittiter. In particular embodiments. the method results
`VLDL assembly inhibitors for their inhibition of HCV 5
`in at least a 25%, 50%, 75%, 80%, or greater decrease in
`release from a cell, which may be in vitro or in situ in chim-
`serum l-1C V RNA titer. l11 other embodiments. a decrease in
`panzee, an animal model for HCV infection. In these embodi—
`serum viremia is detected inferentially,
`for example by
`ments_. the contacting step is effected using any method suit—
`observing a reduction of l l(.'V" symptoms in the patient, or
`ableto achieve uptake of the VI.l)[.assembly inhibitor by the
`cell. For example, siRNA can be transfected into the cells in 10 indirectly, such as by showing an improvement in some other
`vitro with 0ligofectAMlNETM reagent (Invitrogen). Small
`indicator of HCV infection (e.g. normalization of aiiii-
`iiiolecule inhibitors, such as the above-nientioned MTP
`iiotransferase levels compared to pre-treatiiieiit levels).
`inhibitors, can be simply added to the medium of cells in
`The invention provides combination therapies for treating
`culture. Additional MTP inhibitors for use in the method can
`[[(.'V'
`infection in a person comprising administering the
`be identified using an MTP inhibition assay (see e.g. Chaii-
`15 patient a VLDL assembly inhibitor in combination with one
`dler et al_. J I.ipid Res. (2003) 44: 1887-901). and optionally
`or more additional antiviral agents that act by a mechanism
`P
`P
`Y
`Y
`P
`further validated in cliini anzee. liia articular embodiment.
`other than b VLDL asseinbl
`inhibition. hi a
`articular
`the cell is contacted with a subiiiicroiiiolar amount of the
`embodiment. tlieVLDL assemblyinhibitortargets and iiiliib-
`Vl ,l)l . assembly inhibitor. l-‘or example, the cell may be in a
`its activity of MT]-’ or production of Apol3 protein. and the
`culture medium to which is added an amount of the V[.l)[. 20 additional antiviral agent is interferon andfor ribavirin. Kits
`asseinbly inhibitor to achieve a concentration in the medium
`for decreasing serum viremia of an HC\v"-infected person can
`o f less than 1000 nM, and preferably less than 500. 250. l0'0
`comprise the combined antiviral agents. For example. in one
`or 10 iiM. A resultant inhibition of HCV release is detected
`embodiment. the kit comprises a plurality ofV'LDL assembly
`using any suitable method. such as the HCV release assay
`inhibitor dosage fomis, preferably orally administered cap-
`described in lixample 2. The method can be used to assess the 25 sules or tablets, and a plurality of ribavirin dosage forms.
`additive or synergi stie effects a VI Di. assembly inhibitor has
`Alternatively the two or more antiviral agents may be formu-
`with other antiviral agents such as ribavirin andilor interferon.
`lated in a single dosage liirrn. The kit may comprise the
`Accordingly, the contacting step of the method may further
`dosage forms packaged in a blister pack to facilitate proper
`comprise contacting the cell with an antiviral agent selected
`daily dosing. In a specific embodiment. the kit comprises a
`from interferon andfor ribavirin.
`30 plurality of MTP-inhibitor dosage forms wherein the MTP-
`The invention encompasses methods to decrease serum
`iiihihitor is selected from the group consisting of BMS-
`viremia in an IICV-infected person. the method comprising:
`200l50, HMS-2l2l22, HMS-201038 (AIERG-7'33), HMS-
`a) administering to the person a VLDL assembly inhibitor;
`197636,
`JTT-130, mitratapide (R—l03757).
`implitapide
`and b) detecting a resultant decrease in serum viremia in the
`(HAY-l39952),
`(IP-346()86_. Cl-’-467688, and C13-3l934().
`person. Prior to the contacting step, the patient is preferably 35 The kit further comprises a plurality of orally administrable
`diagnosed as having an HCV" infection, which may be by any
`ribavirin andfor interferon (see e.g. Bernard. Curr Opiii Inves-
`iiiedically-acceptable method. The Vi .l)[. assembly inhibitor
`tig Drugs (2002) 3:693-7) dosage forms.
`may be a -kl‘iO\Vl‘l‘dl'11g used in or in development for treatment
`EXAMPLE 1
`ol hyperlipidemia.
`Decreased Secretion of In feetioiis I i(‘V’ Particles
`. Applicable protocols for administering a VLDL. aSSembl.y 40
`from Cells Treated with ,iRN A Ta
`etino a 0B
`inhibitor to a person are known in the art and routinely opti-
`rg
`'
`b
`‘
`° 13
`mized. For example_.
`the aiitiseiise oligoiiucleotide ISIS
`We transfected Huh?-GL cells, a line of Huh? cells that
`301012 fiem0.n§.n'atef' bmafmlablhty by .oral and parental
`.
`.
`.
`.
`routes of adniimstratioii [Isis Pharmaceuticals 2005 Annual
`contain a chrornosoiiially integrated genotype 2a II(.\/ cl)N/\
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`,
`.
`Report). Smallmolecule MTP inhibitors are routinely adniiii- 45
`and constitutively produce infectious virus [(.ai et al.. 2005),
`.
`.
`.
`.
`.
`.
`.
`,
`.
`.
`.
`istered in oral dosage forms. Suitable protocols for adiiiinis-
`with a duplex siRNA targeting apoB or OFF as a control.
`.
`.
`_,
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`tration of the V I.l)I. assembly inhibitor to a patient can be
`Followingincubation in serum-free medium. culture medium
`.
`.
`.
`.
`.
`.
`.
`‘
`_
`‘
`.
`,
`.
`readily derived from the extensive clinical trials and pre-
`was harvested and the amount of apol3 and II(.\/
`in the
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`clinical pliariiiacokiiietic studies that have been conducted on
`so medwm was analyzed" Transiecnon of cells wlth me apoB
`VLDL assembly inhibitors for the treatment of l1yper]ipi-
`'
`' siRN/\ reduced the amount of apol3 inRl\l_/X by about 80“:i
`demh
`without affecting intracellular HCV RNA. The apoB siRNA
`‘
`'
`.
`.
`markedl decreased the amount of a QB Secreted mm the
`ln a preferred embodiment, a submiciiomolar serum con-
`.
`y
`.
`i
`.
`. P
`.
`.
`.
`centratioii of the \-''LDL assembly inhibitor. particularly an
`medium, but it did not affect secretion of or.l—antitrypsin. In
`.
`.
`.
`.
`.
`.
`.
`_
`.
`.
`.
`_.
`M I
`l-’ inhibitor. effects the decrease in serum viremia. ln a
`_ control cells transfected with the GFP siRNA. the HCV copy
`.
`.
`V.
`.
`particular embodiment, l mg per kilogram ofbody weight per 5:
`.
`.
`_
`‘
`.
`dd
`.
`.
`.
`.
`.
`.
`.
`.
`number and titer increased by more than 10 fold during the
`' y or less of the M 11-’ inhibitor is administered to the person
`.
`.
`.
`.
`.
`.
`.
`‘
`.
`period of 4-hr incubation. In cells receiving the apoB siRNA.
`_
`.
`I
`.
`‘
`_
`_
`.
`to achieve an active submicromolar concentration of the
`.
`.
`,
`.
`.
`this increase was reduced by about 50% as assayed by viral
`.
`.
`.
`.
`.
`.
`.
`.
`.
`inhibitor for a duration sufiicient to decrease serum viremia in
`co
`number md 700/, 15 fig“ _ed b the viral titer
`the patient. l-‘or example. the MTP inhibitor may be formu-
`’ ‘
`D ‘
`‘
`(3
`y
`lated in oral dosage forms of 0.03. 0.1. 0.3 and 1.0 mg per 60
`l:‘.XAMl’l_l:‘ 2
`kilogram ofbody weight per day, delivered 1-4 times daily to
`achieve a subiiiicromolar serum concentration of the MT]-’
`
`py
`
`'
`
`Decreased Secretion of Infectious l l(.'V’ Particles
`from (Iells Treated with the MTP liiliibitor
`HMS-2101038
`
`inhibitor. The duration of treatment is typically in the range of
`about 4 weeks-4 months. depending on the tolerance of the
`drugs by patients. The resultant decrease in serum viremia
`may be detected quantitatively using a suitable method
`known in the an (see e.g. Lunel et al, llepatology (1999)
`
`G5
`
`l liili7-(ii. cells were incubated in the absence or presence
`of the MTP iriliibitcr BMS-210138. 1-‘ollewing incubation in
`PENN EX. 2266
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`3of5
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`CFAD\KU?ENN
`IPIl20l5-01836
`
`

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`US 7,645,?32 B2
`
`5
`serum—free medium, culture medium was harvested and the
`amount of11CV RNA, l1CV titer. and apoB in the medium
`was measured. Incubation of cells with the MTP inhibitor
`
`blocked the secretion ofapoB but not oL1—antitrypsin. Treat-
`ment ofthe cells with the MTP inhibitor reduced the amount
`
`ofHCV RNA in the medium and viral titer by about 80%. The
`decreased amount of HCV in the medium is not due to in.lii—
`
`bition of HCV RNA synthesis because intracellular HCV
`RNA remained the same in the absence or presence of the
`MTP inhibitor. We did not observe an accumulation of intra-
`cellular HCV RNA in cells treated with the MTP inhibitor
`because even in cells that were not incubated with the inhibi-
`tor, the amount ofHCV RNA detected in the medium was less
`than 1% of that found in cells
`
`l".XAMPl_.l-'. 3
`
`Various MTP Inhibitors Decrease Cellular Release of
`l ICV
`
`I-1uh7—GL cells are cultured as described in Example 2. On
`day l, the cells are treated with 11M. l() nM. 100 iiM, and 500
`1iM of the following MTP inhibitors: BMS—20l038 (positive
`control), BMS-2(l(}l5(), HMS-212122. HMS-197636. .l'l'l‘-
`130, Iniplitapide, mitratapide, and C‘P—346086. 16 hr later on
`day 2, cells are switched to seruni—free medium in the absence
`or presence of the same amount of the MTP inhibitor. A fter
`incubation for 4 hours, decreases in cellular release of virus
`for each tested inhibitor is demonstrated by redttctioiis in
`HCV RNA copy numbers and titers in the media as deter-
`mined above.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`EXAMPLE 4
`
`MTP lnhibitors Decrease Sertmi HCV Vireniia
`
`35
`
`6
`weeks afier the final dose by using a qualitative multicycle
`reverse transcription polymerase chain reaction method.
`Titers are calculated up to 5 million copiesfniL: if a titer is
`greater than 5 million copiesfmL_. it is simply reported as such
`and the exact value is not given. Efficacy of BMS—20l038 is
`demonstrated by a decrease in serum viremia as demonstrated
`by a significant decrease in viral load and normalization of
`elevated aminotransferase levels.
`
`References cited above are incorporated herein by refer-
`ence for their disclosure ofthe structures and synthesis ofthe
`VLDL assembly inhibitors referenced therein.
`
`REFERENCES
`
`Appel, N., et al. (2006) J. Biol. Chem. 281, 9833-9836.
`Avramoglu, R. K. and Adeli, K. (2004) Rev. 1-'.ndocr.
`Metab. Disord. 5, 293-301.
`Cai, Z._. et al. (2005) J. Virol. 79, 13963-13973.
`Cliaiidler, (7. 1i., et al. (2003) J. I.ipid Res. 44. 1887-1901.
`Cliisari, I’. V. (2005) Nature 436, 930-932.
`Fisher, E. A. and (jiiisberg, H. N. (2002) J. Biol. Chem.
`277, 17377-17380.
`Gibbons, G. 15., etal. (2004) Biochem. soc. trans. 32, 59-64.
`Gibbons, G. F._. et al. (2000)Biochi1iiica et Biophysica Acta
`(I313./\) Molecular and (Tell Biology ofl.ipids 1483, 37-57.
`Gosert, R._. et al. (2003) J.Virol. 77, 5487-5492.
`(iusarova, V., et al. (2003) J. Biol. Chem. 278, 48051-
`48058.
`
`Higaslii,Y., et al. (2003) J. Biol. Chem. 278, 21450-21458.
`l,ohmann, V., et al. (1999) Science 285, 110-113.
`Mensenkamp_.A. R., et al. (2001 ) E. Journal ofHepatology
`35. 816-822.
`
`Moradpour, D._. et al. (2004). J. Virol. 78, 1400-7409.
`Nielsen, S. U., et al. (2006) J. Virol. 80, 2418-2428.
`O1ofsso1i,S.O.and Boren, J. (2005) J. oflnterrial Medicine
`258, 395-410.
`Raabe, M._. et al (1998) Proc. Natl. Acad. Sci. 95. 8686-
`8691.
`
`A randomized, doub1e—bli1id. placebo—controlled trial
`(Raymond et al, Ann lntern Med. (1998) November l5;l29
`(l0):797—800) is used to evaluate the efficacy and safety of
`Randhawa, et al. (2000) Mol. Biol. Cell 1 1, 2403-2417.
`BMS20l 038 in patients with chronic HCV infection who did 40
`Rowe, T., et al (1996) J . Cell Biol. 135. 895-91 1.
`not respond to or were intolerant of ititerferon tiiotiotlicrapy.
`Sharp, D., et al. (1993) Nature 365, 65-69.
`Patients are recruited and are eligible for enrollment if they
`Wetteraue, J. R. al (1998) Science 282, 751-754.
`are positive for HCV’ RNA on serologic testing after at least 3
`ha .
`months of interferon therapy. Patients who can not tolerate
`_
`_l_ .
`_d . U
`,
`,
`.
`.
`.
`interferon because of severe side effects. such as fatigue. 45 W 1 [5 L ‘mm .13‘.
`lleuropsyclliatfic dismrbances‘ or thmmbocympenja’ are also
`I
`l. A method of mhibitmg release of II(.V' from an 1 1(_ V-
`iiicluded. Patients are excluded if they are taking lipid-low-
`Infected Cell} the method _""mI”‘3“‘E3
`‘
`‘
`_
`ering medications, are pregllalm are Currently abusing drugs
`a] contacting the cell with aV’l.D1. assembly inhibitor. that
`or alcohol, have liepatoriia. are seropositive fiir [ [[V, have an
`'5 an
`I P mhlblmr-3
`_
`_
`absolute granulocyte count less than 1000 cel1st‘ni.1n3, or have 50
`b) dc"-'C““3 3 “-'5”h"‘m lllhlblmm "F 1 {CV 11319359 [mm ‘he
`cell.
`a coexistent cause of liver disease.
`2. The method o 1‘ claim 1 wherein the inhibitor is art MTP
`
`Patients are randomly assigned in a double-blitided tiiatiner
`to receive a 12-week course of either BMS201038 at 0.15 mg
`per kilogram of bodyweight twice daily or a placebo that is
`identical in shape, color, and packaging to the active drug.
`Physicians and patients are blinded to treattiietit assignments.
`Patients are evaluated every 3 weeks for compliance. which is
`assessed by pill count. and for the development of adverse
`reactions. Adverse reactions are not expected at these doses
`and the duration of the treatment given the high tolerability of
`HMS-201038 as demonstrated in previous clinical trials (see
`e.g. (Iuchel et al, N. ling. J Med (2007) 356: 148-56). At each
`evaluation visit, a complete blood count is dotie and serum
`levels of electrolytes, blood urea nitrogen. creatitiiiie, aspar-
`tate aminotransferase, alanine aniinotransferase. albumin.
`and total bilirubin are measured. Titers ofserum 1 l(fV RNA
`
`are assessed before en.rolln1ent. at each evaluation visit, and 6
`
`inhibitor selected l1'(}]']'] the group consisting o1'l3-MS-200150,
`HMS-212122, HMS-201038 (Al {RC}-733). 13-MS-201030,
`BMS—l 97636, JTT— 1 30, mitratapide (R—lO3757). iniplitapide
`(HAY-139952), (JP-346()86_. (71-’-467688. and C13-319340.
`3. The method o 1‘ claim 1 wherein the inhibitor is art MTP
`
`G0
`
`65
`
`inhibitor that is HMS-20l038(AlERG-733).
`4. The method of claim 1 wherein the cell is contacted with
`a submicromolar amount of the inhibitor.
`5. The method of claim 2 wherein the cell is contacted with
`a submicromolar amount of the inhibitor.
`6. The method of claim 3 wherein the cell is contacted with
`a submicromolar amount of the inhibitor.
`7. '11ie method of clairii 1 wherein the cell is contacted with
`a sttbmicroiiiolar amount o ftlie inhibitor, wlierein the amount
`is less than 100 nM.
`
`4of5
`
`PENN EX. 2266
`
`CFAD V. UPENN
`IPR20l5—0l836
`
`

`
`US 7,645,?32 B2
`
`7
`8. The method ofclaim 2 wherein the cell is contacted with
`a submicromolar amount of the inhibitor. wherein tl1e amount
`is less than 100 11M.
`9. The method ofclaim 3 wherein the cell is contacted with
`
`8
`VLDL assembly inhibitor. achieved by administering to the
`person 1
`l11gi1i\'g body weight per day or less of the MTP
`inhibitor for a duration sufficient to decrease the viremia.
`20. The method ofclaim 14 wherein the decrease in serum
`
`a submicromolar amount ofthe inhibitor. wherein the amount
`is less than 100 nM.
`10. The method of claim 4 wherein the contacting step
`further comprises contacting the cell with an antiviral agent
`111119911111 1111111 1111911911111 111111 1111111111111‘
`11' 1-1119 1119111051 91' 91111111 5 W11919111 1119 9511115191111E 51913
`111111191 9111111911595 9911111911118 1119 9911 W1111 3111 31111V11'?11 339111
`selected from interferon and ribavirin.
`12' 1-1119 1119111051 91' 91111111 9 W11919111 1119 9511115191111E 51911
`FuI1her comprises contacting the cell with an antiviral agent
`591991951 119111 111191191911 111151 111151111111‘
`11191111“ 111 *1“ 11111/'
`_ 13- A m°111‘1‘1 111 11131131151113
`infected person, the method comprising:
`5'1 9‘111111115191111g 10 1119 13915911 11 VLDL 355911111131 1111111111013
`1119113 an M11) 11111111111111 and
`111 dclccling 9 msuham decrease in serum Vimmia 1" 111‘:
`11615011‘
`
`5 viremia is effected by a submicromolar concentration of" the
`VLDL assembly inhibitor. achieved by administering to the
`person 1 mgfkg body weight per day or less of the Mn;
`inhibitor for a duration Sufi-icieiit to decrease the vitemia_
`21 .The method ofclaim '15 wherein the decrease in serum
`1131 viremia is effected by a submicromolar concentration of" the
`VLDL assembly inhibitor. achieved by administering to the
`1
`"
`‘
`person mgilkg body weight per day or less of the MTP
`inhibitor for a duration sufficient to decrease the viremia.
`22_ The method 01-claim -13 wherein the decrease in Serum
`15 viremia is effected by a submicromolar concentration of" the
`VLDL assembly inhibitor. achieved by administering to the
`person 01 myko body weight per day or less of me M-_-P
`inhibitor for a dufation sufficient to decrease the viremia.
`23 method of claim 15 wherein the decrease in serum
`211 vireniia is effected by a submieromolar concentration oftie
`VLDL
`bl
`'ih'b‘t
`( 1‘
`d b ( d
`1
`‘
`t
`'
`tl
`
`'
`
`'
`
`‘If“‘
`
`d
`
`'ih'b‘ 1"-d
`“
`25 vireniia is effected b aisubmieromolar concentration oftie
`201030" HMS-197636’ In‘-130’ mimuapidc 11111113757)’
`VLDL assembly inhibitor achieved by '1dministering to the
`implimpide (BAY'139952)’ CPJ46086‘ CP467688‘ and
`"
`1.
`1
`.
`-_
`(I1-1-319340.
`§:gr:;s:e5:eoV1i;2:1£lA‘P
`15. The method of claim 13 wherein the inhibitor is an
`,
`,
`_
`_
`'
`M'l‘l-’ inhibitor that is HMS-201038 [A|il{("r-733).
`1
`1
`,
`,
`16. The method ofclaim '13 wherein the decrease in sertmi 30 F 215 "T113 me11_11’f1 111d°1a_111_1‘1? _w11e1e111h111e a,d1111111r51e1111$ S131;
`viremia is effected by a submicromolar concentration of" the
`11” “'11 (l’‘1111§C1l'1;‘'’‘’
`‘1_ m111f11"1911111%£11,£ 5 ,l1‘91‘—*1111 ‘111 ‘11111"11"1
`V] D] J assembly mhibimn
`agent se ect
`rom inter eron an
`avirin.
`‘
`-
`-
`17. The method ofclaim '14 wherein the decrease in sertmi
`26' T119 1119111011 01 °1a_111_1 211 _“111919111 1119 ad111111151e1111$ 51911
`viremia is effected by a submieromolar concentration oftlie
`1111111311 °0111p1-15'_'eS 511_1n11111§1er111g 1O_111e_He15011 an 311111111111
`VLDL assembly inhibitor,
`35 agent selected from interferon and ribavirin.
`‘
`_
`_
`18. The method ofclaim '15 wherein the decrease in sentni
`_ 27- T119 1119111011 0191111111 21 _W1191'91111119 31111111115-1911113 51911
`viremia is effected by a submieromolar concentration ofthe
`111111191 5-151111P115'_'95 91_111111115191111B 10'1119'P‘91"5011 311 5111111111111
`VLDL assembly iiihibitei-_
`agent selected from interferon and ribavirin.
`19. The method of claim 13 wherein the decrease in serum
`
`vireinia is effected by a submicroinolar concentration of" the
`
`=k
`
`=><
`
`*
`
`*
`
`=t=
`
`5 01 5
`
`PENN EX. 2266
`
`CFAD V. UPENN
`IPR20l5-01836