~··
`
`(19) l+I
`
`Canadian
`Intellectual Property
`Office
`
`Office de la Propriete
`lntellectuelle
`du Canada
`
`<11> CA 2 291 471
`(43) 04.06.2000
`
`<13> A1
`
`An Agency of
`lndusby Canada
`
`Un organisme
`d'lndustrie Canada
`
`(12)
`
`(21) 2 291 471
`
`(22) 02.12.1999
`
`(30)
`
`(71)
`
`60/111,100 us 04.12.1998
`
`PFIZER PRODUCTS INC.,
`Eastern Point Road, GROTON, XX (US).
`
`(51) Int. Cl.':
`
`A61K 031/4725, A61P 007/00,
`A61K 031/445, A61K 031/4523,
`A61K 031/47
`
`(72)
`
`(74)
`
`HAMANAKA, ERNEST SEllCHI (US).
`CHANG, GEORGE (US).
`
`SMART & BIGGAR
`
`(54)
`(54)
`
`DIMINUTION DES CONCENTRATIONS DE UPOPROTEINE(A) DANS LE SANG
`LOWERING BLOOD LEVELS OF LIPOPROTEIN(A)
`
`(57)
`
`The invention relates to methods of lowering
`levels of
`blood
`lipoprotein(a)
`In a mammal which
`comprises administering to a mammal in need thereof a
`lipoprotein(a)
`blood
`level-lowering amount of an
`triglyceride
`apolipoprotein
`B
`secretion/microsomal
`transfer
`protein
`inhibitor.
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`0 PI C
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`0PPICl DE 1"' PROPRliT~
`
`INTl!LUCTunLg ot.· CANADA
`
`\
`
`CI PO
`
`l'llOl'UtTY 0PPICg
`
`02H19)(CA) Demande-Application
`
`(21)(Al) 2,291,471
`(22) . 1999/12102
`(43) . 2000/06/04
`
`(72) CHANG, GEORGE, US
`[12) HAMANAKA. ERNEST SEIICHI, US
`[II) PFIZER PRODUCTS INC., US
`(51) Int.Cl.7 A61K 31/4725, A61K 31/47, A61K 31/4523, A61K 31/445,
`A61P 7/00
`(30) 1998/12/04 (60/111,100) us
`(54) DIMINUTION DES CONCENTRATIONS DE LIPOPROTEINE(a)
`DANS LE SANG
`(54) LOWERING BLOOD LEVELS OF LIPOPROTEIN(a)
`
`(57) The invention relates to methods of lowering blood levels of lipoprotein(a) in a mammal which comprises
`administering to a mammal in need thereof a lipoprotein(a) blood level-lowering amount of an apolipoprotein I3
`secretion/microsomal triglyceride transfer protein inhibitor.
`·
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`I+ I lndustrie Canada
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`Industry Canada
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`CA 02291471 1999-12-02
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`ABSTRACT
`
`The invention relates to methods of lowering blood levels of lipoprotein(a) in a
`
`mammal which comprises administering to a mammal in need thereof a lipoprotein(a)
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`blood level-lowering amount of an apolipoprotein 8 secretion/microsomal triglyceride
`
`transfer protein inhibitor.
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`PC10185CJG
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`CA 02291471 1999-12-02
`
`-1-
`
`LOWERING BLOOD LEVELS OF LIPOPROTEINfa l
`
`Background Of The Invention
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`The glycoprotein apolipoprotei~ (a) (apo(a)) is synthesized and seaeted from ·
`. hepatic cells and, in humans, dra.llates largely in assodaUon with .k)w density
`lipoprotein (LDL) in the fonn of a hybrid liP9Protein referred to as Lp(a). The
`the major protein moiety of LDL. namely
`.
`association between apo(a) and
`.
`apolipoprotein 8100 (apo 8100), is mediated through covalent linkage of a single
`unpaired cysteine residue in apo(a) to a complimentary unpaired cysteine residue in
`the extreme carboxyl terminus of apo 8100.
`Interest in the biology of this lipoprotein species is driven by 1he observation
`that elevated levels of Lp(a) In humans is associated with an lnaeased risk for
`
`atherosclerotic heart and vascular disease. The lowering or Lp(a) levels, however, has
`proven problematic since various conventional methods that are effective In reducing
`levels of LDL are not as efficacious or consistent In lowering levels of Lp(a). For
`example, it has been reported that neomycin, alone or in combination with niacin, is
`.
`effective in reducing Lp(a) levels when administered over a period of several weeks to
`.
`years. See Spinier, et al., J. Ann. Pharmacother., ~ 343 (1994). 11le administration
`of high doses of niacin and neomycin, however, llmlts the desirabUity of this regimen
`due to many undesirable clinical .side~ffects. Alternatively, oral doses of foSlnopril; an
`angiotensin<e>nverting enzyme inhibitor, have been demonstrated to lower lp(a)
`levels after 12 weeks of treatment, however, Lp(a) reduction was significant on!}' in
`patients that showed Improvement In renal function and, therefore, the Lr:)(a) IOwering
`abifity of fosinopril may simply be attributable to the indirect consequence of improved
`kidney function. See Keilani, et al., Ann. Inter. Med., fil, 246 (1993). Additionally,
`certain steroidal hormones, estrogen for examPle, are known to down-regulate Lp(a)
`levels. See, for examPle, Frazer, et al., Nature Genet, a, 424 (1995). However,
`estrogen therapy alone is associated with an increased risk of endometrial cardnoma
`for this reason, estrogen is nonnally administered In combination with
`and,
`treatment with
`this estrogeillprogesterone
`progesterone. Although short-term
`combination Is an effective therapeutic strategy for reducing Lp(a) levels,; tong-term
`treatment, I.e. six months or more, does not result in the same degree of decreased
`inhibition as that observed for treatment with estrogen alone. See Soma, et al., Arch.
`
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`Internal. Med., lli. 1462 (1993) and Soma, et al., Chem. Phys. Lipids, 345, 67
`(1994). Furthermore, LOL apheresis has been shown to be an effective means for
`· lowering Lp{a) levels. See Koizumi, et al., Atherosclerosis, .l.QQ, 65 (1993); However,
`apheresis Is an invasive approach requiring weekly treatments and,. therefore, is not
`
`regarded as a current treatment of choice. Accordingly, improved methods of
`inhibiting Lp(a). or formation of the precur.;ors thereof, will have utility in the ~atment
`of conditions and diseases arising troin hypertipoproteinemia, including, for example,
`atherosclerosis, premature myocardial infarction: stroke, restenosis follQwlng coronary
`bypass surgery and so forth.
`Miaosomal triglyceride transfer protein (MTP) is known to ·mediate the.
`transport of triglyceride, cholesteryl ester and phospholipids and has been Implicated
`as a mediator In
`the assembly of apo6poproteln B containing
`lipoprotelns,
`chylomiaons and VLDL (very low density lipoprotein). Specifically, the subcellular
`{lumen of the miaosomal fraction) and tissue distribution (liver and Intestine) of MTP
`have led to speculation 1hat it plays a role in the assembly of plasma lipoproteins, as
`these are the sites of plasma lipoprotein assembly. The ability of MTP to catalyze the
`transport of triglyceride between membranes Is consistent with this specl.Jlation .·and
`
`suggests that MTP may catalyze the transport of triglyceride from its site of synthesis
`in the endoplasmic reticulum membrane to nascen~ lipoprotein particles Within 1he
`lumen of the endoplasmic reticulum. Accordingly, compounds that inhibit MTP and/or
`otherwise inhibit apo B seaetion are useful in the treatment of atherosclerosis• and
`other conditions related thereto. Such compounds are also useful in the treatment of
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`other diseases or conditions in which, _by inhibiting MTP and/or apo B secretion,
`serum cholesterol and triglyceride levels may be reduced. Such conditions may
`indude, for example, hypercholesterolemia, hypertriglyceridemia, panaeatitis, obesity
`
`25
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`
`and hypercholesterolemia, hypertriglyceridemia and hypertipidemia associated with
`pancreatitls, obesity and diabetes. For a detailed discussion see, for example,
`Wetterau et al., Science, ~. 999-1001 (1992} and Wetterau et al., Biochem;
`Biophys. Acta., 875, 61~17 (1986).
`While the precise mechanisms governing blood levels of l.p{a) are presentlY
`unknown, ~re is evidence to suggest that Lp(a} levels are regulated at the level of
`synthesis rainer than catabolism. Accordingly, because it is known thatJnhlbltion. of
`hepatic secretion of VLOL and apolipoprotein B (apo B) results in lhe pre-secretorY
`degradation of apo B and concomitant deaease in hepatic apo B levels and because
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`CA 02291471 1999-12-02
`
`-3-
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`each Lp(a) particle contains one copy of apo(a) bound to apo
`B, it is believed that decreasing the concentration of
`hepatic apo B, by the administration of an apo B secretion/(cid:173)
`MTP inhibitor, will result in a lowering of Lp(a) seereted
`and, thereby, a lowering of blood Lp(a) levels.
`Summary of the Invention
`The instant invention is directed to pharmaceutical
`compositions for lowering blood levels of lipoprotein(a)
`(Lp(a)) in a manunal, which comprise (i) an effective Lp(a)
`blood level-lowering amount of an apolipoprotein B secretion/(cid:173)
`microsomal triglyceride transfer protein inhibitor and (ii) a
`pharmaceutically acceptable carrier or diluent.
`Another aspect of the present invention provides
`commercial packages comprising the above-described pharma-
`ceutical compositions and written matters including
`indications that the pharmaceutical compositions can or should
`be used for lowering blood levels of Lp(a) in a mammal.
`.
`In a preferred embodiment of the invention, the apo
`B secretion/MTP inhibitor is a compound selected from the
`group consisting of 4'-trifluoromethylbiphenyl-2-carboxylic
`acid- [2- (lH".'" [l, 2 ,4] triazol-3-ylmethyl )-1, 2, 3 ,4-tetrahydro~ _
`isoguinolin-6-yl]-amide,· 4' -trifluoromethylbiphenyl-2-
`carboxylic acid-[2-(2-acetylaminoethyl)-1,2,3,4-tetrahydro(cid:173)
`isoquinolin-6-yl]-amide, 9H-{4-{4-[4'-trifluoromethyibiphenyl-
`2-carbonyl)amino]-piperidin-l-yl}butyl)-9H-fluorene-9-
`carboxylic acid-(2,2,2-trifluoroethyl)-amide 9-{4-[4-(2-
`benzothiazol-2-ylbenzoylamino)piperidin-l-yl]butyl}-9H;..
`fluorene-9-carboxylic acid-(2,2,2-trifluoroethyl)amide,
`[lla-RJ-8-[(4-cyanophenyl)methoxy]-2-cyclopentyl-7-(prop-2-
`enylT;..2,3,ll,lla-tetrahydro-6H-'pyrazino[l,2b]isoquinoline-
`l,4-dione, [lla-R]-cyclopentyl-7-(prop-2-enyl)-8-[(pyridin-
`2-yl)methoxy]-2,3,11,1la-tetrahydro-6H-pyrazino[l,2b]iso(cid:173)
`guinoline-1,4-dione, 2-cyclopentyl-2-[4-(2,4-dimethylpyrido-'
`[2,3b]indol-9-ylmethyl)phenyl]-N-(2-hydroxy-l-phenylethyl)-
`acetamide and 2-cyclopentyl-N- (2-hydroxy-1-phenylethy·l )-2-
`[4- (quinolin-2-ylmethoxy)-phenyl Jacetamide, a hydrate or
`
`.
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`72222-394
`
`-4-
`
`stereoisomer thereof, or a pharmaceutically acceptable salt
`of the compound, hydrate or stereoisomer.
`Especially preferred apo B secretion/MTP inhibitors
`useful in the practice of invention are the compounds
`4 '-trifluoromethylbiphenyl-2-carboxylic acid_. [2- (lH- [l, 2 ,4 ]-:(cid:173)
`triazol-3-ylmethyl)-l; 2, 3,4-tetrahydroisoquinolin-6-yl]-amide
`and 4'-trifluoromethylbiphenyl-2-carboxylic acid-[2-(2-
`acetylaminoethyl)-1,2,3,4-tetrahydroisoquinolin-6-:--ylJ-amide,
`hydrates thereof or the pharmaceutically acceptable salts of
`the compounds or hydrates.
`Detailed Description of the Invention
`This invention provides pharmaceutical compositions
`for lowering blood levels of lipoprotein(a) (Lp(a)) in a
`mammal, which comprise an effective Lp(a) blood level-lowering
`amount of an apolipoprotein B secretion/microsomal triglyceride
`transfer protein inhibitor.
`As employed throughout the instant specification
`including appendant claims, the term nblood", when employed
`in relation to the term "blood levels", includes whole blood,
`plasma, i. e. the fluidic portion of the blood in which
`particulate components are suspended and serum, i. e. the
`fluidic portion of the blood obtained following removal of
`the fibrin clot and corpuscles.
`As employed throughout the instant specification
`including appendant claims, the term "apo B secretion/MTP
`inhibitor" means a compound having the ability to inhibit
`the secretion of apolipoprotein B and/or the ability to
`inhibit the transfer action of microsomal triglyceride
`transfer protein.
`A variety of apo B secretion/MTP inhibitors are,
`or will be, known to one of ordinary skill in the art.
`Although any apo B secretion/MTP inhibitor may be used in
`the practice of the instant invention, generally preferred
`apo B secretion/MTP inhibitors include those compounds,
`hydrates or stereoisomers thereof, or the pharmaceutically
`acceptable salts of the compounds, hydrates or stereoisomers
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`CA 02291471 1999-12-02
`
`-4a-
`
`which are disclosed in, for example, European Patent
`Publication Nos. EP 643057, EP 719763, EP 753,517, EP 764647,
`EP 765878, EP 779276, EP 779279, BP 799828, EP 799829, EP
`802186, EP 802188, EP 802192 and EP 802197; PCT Patent
`Publication Nos. WO 96/13499, WO 96/33193, WO 96/40640, WO
`97/26240, WO 97/43255, WO 97/43257, WO 98/16526 and WO
`98/23593; and u. s. Pat. Nos. 5,595,872; S,646,162,
`5,684,014; 5,712,279; 5,739,135 and 5,789,197.
`Especially preferred apo B secretion/MTP inhibitors
`are those biphenyl-2-carboxylic acid-tetrahydroisoquinolin-6-
`yl amide derivatives, and the stereoisomers, hydrates,
`prodrugs and pharmaceutically acceptable salts thereof
`disclosed in commonly assigned PCT Patent Publication Nos.
`WO 96/40640 and WO 98/23593, each of which designate, inter
`alia, the United States. Especially preferred apo B
`secretion/MTP inhibitors disclosed in PCT Paterit Publication
`Nos. WO 96/40640 and WO 98/23593, and useful in the present
`invention, are
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`-5-
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`the compounds illustrated hereinbelow: 4'-trifluoromethyl-blphenyl-2-carboxylic acid(cid:173)
`(2-(1 H-{1,2,4)triazol-3-ylmethyl)-1.2,3,4-letrahydroisoquin-6-yf)-amide.
`and
`4'(cid:173)
`
`acld-{2-(acetylaminoethyl)-1,2,3,4-
`tri0uoromethyl-biphen~2-carboxylic
`tetrahydroisoquloolin-6-yf)-amide, or the hydrates thereof, or the pharmaceuticaliy
`acceptable salts of the oompounds or the hydrates.
`CFs
`
`5
`
`o roN~NY.
`I
`o •
`N
`H
`.
`.
`
`H
`
`. .
`
`.
`
`.
`
`Another especially preferred class of apo 8 seaetiorVMTP Inhibitors is
`disclosed in U.S. Pal Nos. 5.595,872; 5,712,279; 5,739,135 and 5,789,197 and
`includes compounds having the structural fonnula
`
`!'
`
`a.'
`
`··?-D-·· .. />-0
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`20
`
`R'
`Especially preferred apo B secretionlMTP inhibito!S disclosed in U.S. Pat. Nos;
`5,595,872; 5,712,279; 5,739,135 and 5,789,197, and useful in the methods of the
`invention, are
`present
`9H-{4-{4-[4 'trifluoromethyl-biphenyl-2-carbonyl)-amlno](cid:173)
`piperidin-1-yl}-butyl-9H-tluorene-9-carboxylic acld-{2,2,2-lrifluoroethyl)-amide and 9-
`
`{4-{4-(2-benzothiazol-2-yl-benzoylamino)-pipericlin-1-yl}butyl}-9H-fluorene-9-
`carboxylic acid-{2,2,2-trifluoroethyl)-amide.
`Another class of especially preferred apo B secreUon/MlP inhibitors is
`disclosed in PCT Application Publication No. WO 98/16526 and includes oompounds
`having the structural fonnula
`
`Especially preferred Apo-8 secretion/MTP inhibitors disclosed In PCT Application
`Publication No. WO 98/16526, and useful in the methods of the present invention, are
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`-6-
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`[11a-R]-8-{(4-cyanophenyl)methoxy)-2.;cyclopentyl-7-(prop-2-enyl}-2 ,3, 11, 11a(cid:173)
`
`telrahydro-6H-pyrazino[1,2b]isoquinoline-1,4-dione and [11 a-R]-cyclopentyl-7-(prop-
`
`2-enyl)-8-[(pyridin-2-yl)methoxy]-2,3, 11, 11 a-tetrahydro-6H-pyrazino[1,2b]isoquinoline~
`
`5
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`1,4-dione.
`Another especially preferred class of apo B secretion/MTP inhibitors is
`disdosed in U.S. Pal No. 5,684,014 and includes compounds having the structural
`
`formula
`
`~
`
`Rt
`
`~~~
`J~ ..
`'~~(''+~
`
`10
`
`D
`An especially preferred apo B secretion/MTP inhibitor disclosed in U.S. Pal No.
`5,684,014. and useful in the methods of the present invention, is 2-cyclopentyJ-2-[4-
`(2.4-dimethyl-pyrido[2,3-b)indol-9-ylmethyl}-phenyl]-N-(2-hydroxy-1-phenyl-ethyl)(cid:173)
`acetamide.
`Yet another dass of especially preferred apo B secretion/MTP inhibitors is
`disclosed in U.S. Pat No. 5,646,162 and indudes compounds having the structural
`
`15
`
`formula
`
`A~~E I
`I ~ 4~~
`
`t.:
`
`N
`H
`
`OH
`
`20
`
`An especially preferred apo B secretion/MTP inhibitor disdosed in U.S. Pal No.
`5,646, 162, and useful in the methods or the present invention, is 2-cyclopentyJ-N-(2-
`hydroxy-1-phenyl-ethyl)-2-{4-(quinolin-2-yfmethoxy)-phenyl]-acetamide.
`The apo B seaetlon!MTP inhibitors, including the preferred and especially
`preferred erTibodiments and the pharmaceutically acceptable salts, bydrates .or
`stereoisomers thereof, or the pharmaceutically acceptable salts of the hydrates· or
`stereoisomers employed in the methods of the instant invention may be prepared
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`according to the disclosures of the aforementioned European, PCT and U.S. patents
`and application publication documents.
`The apo B secretion/MT? inhibitors. the phannaceutically acceptable Salts,· ·
`hydrates or stereoisomers thereof, or the pharmaceutically acceptable Salts of the.
`hydrates or stereoisomers employed in the methods of the invention, are preferably
`form of a pharmaceutical oomposition oomprising a
`..
`administered
`in
`the
`pharmaceutically acceptable carrier or diluent Accordingly, the apo B secretion/MlP
`
`Inhibitors, the pharmaceutically acceptable salts! hydrates or stereoisomers thereof, or .
`the pharmaceutically acceptable salts of the hydrates or stereolsomers of this .
`Invention, can be administered indMdually or together in any conventional oral,
`parenteral or transdennal dosage form.
`
`Suitable pharmaceutically-acceptable carriers include inert solid fillers or
`diluents and sterile aqueous or organic solutions. The apo B secretion/MTP inhibitors,
`the pharmaceutically acceptable salts, hydrates or stereoisomers thereof, or the
`phannaceutically acceptable salts of the hydrates or stereoisomers of this Invention
`will be present in such pharmaceutical compositions in amounts sufficient to provide
`the desired dosage amount in the range described hereinbelovi. Thu&, for oral
`administration, the compounds can be combined with a suitable solid or liquid carrier
`or diluent to form capsules, tablets, powders, syrup~. solutions, suspension$ and the ·
`like. The pharmaceutical compositions may, if desired, oontain additional components·
`such as flavorants, sweeteners, excipients and the like.
`The tablets, pills, capsules, and the like may also oontain a binder such as .
`gum tragacanth, acacia, oom starch or gelatin; excipients such ·. as dicalcium
`
`· phosphate; a disintegrating agent such as com starch, potato stardi, alglnlc acid; a ·
`lubricant such es magnesium stearate; and a sweetening agent such as· sucrose,
`lactose or saccharin. When a dosage unit form is a capsule, it may contain, in .
`addition to materials of the above type, a liquid carrier such as a fatty oil.
`Various other materials may be present as coatings or to modify the physical
`fonn of the dosage unit. For instance. tablets may be coated with shellac, sugar or
`both. A syrup or elixir may contain, in addition to the active ingredient, sucrose as a
`sweetening a.gent, methyl and propylparabens as preservatives, a dye and a flavoring
`such as cherTy or orange flavor.
`The pharmaceutical compositions of this invention may also be administered
`parenterally. For parenteral administration the pharmaceutical comj>ositions can be
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`oombined with sterile aqueous or organic media to form injectable solutions or
`
`suspensions. Solutions or suspensions of these pharmaceutical compositions can be
`
`prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose:
`Dispersions can also be prepared in sesame or peanut oil, ethanol, water, polyol (e.g.,
`glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof,
`vegetable oils, N-methyl glucamine, polyvinylpyrrolidone and mixtures thereof in oils
`"
`as weD as aqueous solutions of water-soluble pharmaceutically acceptable salts of the
`compounds. Under ordinary conditions of s!<>rage and use, these preparations.
`• contain a preservative to prevent the growth of microorganisms. The Injectable
`solutions prepared
`in
`this manner can
`i0traven0usly,
`then be administered
`intraperitoneally, subcutaneously, or intramuscular1y, with intramuscular administration
`
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`being the preferred parenteral route in humans. Solutions prepared fer Intravenous
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`administration are preferably rendered isotonic prior to usage.
`
`The phannaceutical fonns suitable for Injectable use include sterile aqueous
`solutions or dispersions and sterile powders for the preparation of stenle injectable
`solutions or dispersions. In all cases, the fonn must be sterile and must be fluid to Ule
`It must be stable under the conditions' of
`extent that easy syringability exists.
`manufacture and storage and must be preserved against contamination by
`microorganisms such as bacteria and fungi.
`The pharmaceutical compositions may also be administered transdennally.
`Suitable fonnulations for transdermal application include an effective lipoprotein(a)
`blood level-lowering amount of a compound or pharmaceutical comp0sition. of the
`invention with a suitable transderrnal carrier. Preferred transdermal carriers include
`
`absorbable pharmaoologically acceptable solvents to promote and assist passage
`through the skin of the subject being treated. Characteristically. transdermal devices
`comprise the form of a bandage having a backing member, a reservoir containing the
`oornpound, optionally with carriers, optionally a rate-controlling barrier tO deliver· the
`compound to the skin of the subject being treated at a controlled and predetermined•
`rate over a prolonged period of time and means to secure the device to Ule skin of the
`subject being treated.
`Methc;><fs of preparing the various pharmaceutical compositions with a desired,
`amount of an active ingredient are known, or will be apparent In light of this.. disclosure, ·
`to one of ordinary skill in the art. See, for example, Remington's Pharmaceutical•
`Sciences, Mack Publishing Company, Easton, PA, 15th Edition (1975).
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`The dosage of the ape B secretion/MTP inhibitor or the pharmaceutically
`
`acceptable salt. hydrate or stereo isomer thereof, or the pharmaceutically acceptable
`
`salt of the hydrate or stereoisomer necessa,Y to achieve the desired therapeutic effect
`
`according to this invention is within the ordinary skill of those who practice in the art of
`
`5
`
`having the benefit of the disclosure herein.
`In general, dosage· ranges for certain apo B sec:retion/MTP inhibitors have
`.,
`been reported with representative effective ranges being from·about 0.01 .mgJkglday
`to about 15.0 mg/kg/day. Generally preferable dC?S8Q05 range from about 0.1 to about
`5.0 mg/kg/day and especially preferred dosages range from about 0.1 to about 1.0
`1 O mg/kg/day. Dosage ranges for the partirular compounds desaibed hereinabove are
`set forth in the above-referenced patents and patent application publications.
`However, some variability in the general dosage range may be required depending
`
`upon the age and weight of the patient. the inlended route of administration, and the
`
`progress and degree of severity of the illness being treated.
`
`15
`
`It will be appreciated that, when treating a mammal according to the methods
`
`of the instant invention, the actual preferred route of administration and optimum
`dosage utilized will be at the sound professional discretion of the person responsible
`for the treatment and may vary according to the severity of the condition to be treated,·
`the intended route of ·administration and patient cha~cteristics such as age, weight.
`rate of exaetion, concurrently administered medications and general physical
`
`condition of the subject. Normally, the optimum dosage for the subject being treated
`will be determined by generally administering smaller doses initially and thereafter .
`incrementally modifying the regimen, if required, to determine the most suitable
`- dosage. This- may vary according to the particular-compound employed and with the.
`nature of the subject being treated.
`
`EXPERIMENTAL
`
`_ The utility of apo B seaetion/MTP inhibitors in the lowering of blood levels of
`lipoprotein (a) according to the practice of the methods of the invention may be
`demonstrated. according to protocols disdosed in Nassir, et al., J. Biological Chem.,
`m. 1ns3-hsoo (1998), which protocols are summarized hereinbelow. -
`.
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`CA 02291471 1999-12-02
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`-10-
`
`Pulse-Chase Studies
`
`s
`
`Transfected HepG2 and McA-RH7777 cells are grown to 90% C90fluence ·in
`T-25 flasks. On the day of the experiment, the cells are washed twice with phosphate(cid:173)
`buffered saline, prelncubated In methionine-- and cysteine-free DMEM for 1 hr without
`serum, pulse-labeled for 4 hr in the same medium containing 250 µCVml Tran35~1abel
`,
`.
`and the apo B seaetion/MTP Inhibitor and then chased in complete medium.
`containing 3 mM cysteine and 10 mM methionin~ for a predetermined time~ The apo B
`secretion/MTP inhibitor is dissolved in dimethyl sulfoxlde at a concentration of 100
`1 O mg/ml and diluted to an appropriate concentration in media just prior ti> in'cubation With
`the cells. Dimethyl sulfoxide, at identical final concentrations, Is added alone to control
`cells. At pre-detennined times following radiolabeling, media are collected on ice and
`adjusted to a final concentration of the following protease inhibitors (100 mM
`leupeptin, 450 mM apoprotin, 2 mM pepstatin, 1 mM phenytmethylsulfonyt fluoride
`and 1 mM benzamidine). The cells are washed three times with Ice-cold, phosphate(cid:173)
`buffered saline and subsequently lysed in cold lysis buffer (100 mM Tris, pH 8.0, 100
`mM NaCl, 10 mM EOTA. 1% Triton X-100, 0.1% SOS) containing protease inhibitors
`and, for HepG2 cells, 100 mM E-Sminocaproic add. cell lysates and media are
`darified by centrifugation at 10,000 rpm at 4•c for 5 min to remove cellular debris and
`
`15
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`20
`
`immunopredpitations are then conducted as desaibed hereinbelow. Incorporation of·
`radioactivity Into total protein is determined by trichloroacetic add precipitation of c:eli •
`lysates, in all cases demonstrating comparable values between c0ntror and ·
`
`experimental groups.
`
`25
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`lmmunoprecipitations
`Both medium and lysates are predeared by incubation with· protein G-agarose
`
`30
`
`for 2-3 hr at 4•c. Aliquots are lmmunoprecipitated with saturating quantities of anti(cid:173)
`
`apo(a), a·nti-apo B, and apoA-1 or anti-albumin antisera. After overnight incubation at
`4•c. protein G-agarose beads are added and the incubation continuedfor another 2-3
`hr at 4•c. The final pellet is washed four times in invnunopreclpl1ation wash buffer (50
`.
`mM Tris, pH 7.4, 0.65 M NaCl. 10 mM EOTA, 1% Triton X-100, 1% Sodium
`deoxycholate, 01.% SOS), two times in water and boHed for 10 min in SOS ~rni:>le
`buffer (4o/o SOS, 20% gycerol, 0.001% bromphenol blue, 125 mM Tris, pH 6.8 and
`
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`100 mM dithiothreitol). After centrifugation, the supernatant is analyzed by SOS-PAGE
`and fluorography. Quantitation is then effected by standard techniques.
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`CA 02291471 1999-12-02
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`-12-
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`. 72222-394
`
`CLAIMS:
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`A pharmaceutical composition for lowering blood
`1.
`levels of lipoprotein(a) in a mammal, which comprises (i) a
`lipoprotein(a) blood level-lowering amount of an apolipo-
`protein B secretion/microsomal triglyceride transfer protein
`inhibitor and (ii) a pharmaceutically acceptable carrier or
`diluent.
`
`composition according to claim 1,
`A pharmaceutical
`2.
`B secretion/microsomal triglyceride
`wherein the apolipoprotein
`is a compound selected from the
`transfer protein inhibitor
`group consisting of 4'-trifluoromethylbiphenyl-2-carboxylic
`acid-(2-(1H-(l,2,4Jtriazol-3-ylmethyl)-l,2,3,4-tetrahydro(cid:173)
`isoquinolin-6-yl]-amide, 4'-trifluorornethylbiphenyl-2-
`carboxylic acid-[2-(2-acetylaminoethyl)-1,2,3,4-tetrahydro-
`isoquinolin-6-yl]-amide, 9H-(4-{4-[4'-trifluorornethylbiphenyl-
`2-carbonyl)amino]-piperidim-l-yl}butyl)-9H-fluorene-9-
`carboxylic acid-(2,2,2-trifluoroethyl)-amide, 9-{4-(4-(2-
`benzothiazol-2-ylbenzoylamino)piperidin-l-yl]butyll-9H(cid:173)
`fluorene-9-carboxylic acid-(2,2,2-trifluoroethyl)amide,
`[lla-R]-8-{(4-cyanophenyl)methoxy]-2-cyclopentyl-7-(prop-2-
`enyl)-2,3,ll,lla-tetrahydro-6H-pyrazino[l,2b]isoquinoline-
`1,4-dione, (lla-R]-cyclopentyl-7-(prop-2-enyl)-8-f(pyridin-
`2-yl)methoxy]-2,3,ll,lla-tetrahydro-6H-pyrazino(l,2b]iso(cid:173)
`quinoline-1,4-dione, 2-cyclopentyl-2-[4-(2,4-dirnethylpyrido-
`12, 3b] indol-9-ylrnethyl) phenyl]-N- {2-hydroxy-1-phenylethyi)(cid:173)
`acetamide and 2-cyclopentyl-N-(2-hydroxy-l-phenylethyl)-2-
`[4-(quinolin-2-ylmethoxy)-phenyl]acetamide, a hydrate thereof,
`a- stereoisomer-thereof-, or- a--pha-rmaceut-ically--acceptable salt
`of the compound, hydrate or stereoisomer.
`
`A pharmaceutical composition according to claim 2,
`3.
`wherein the apolipoprotein B secretion/microsomal triglyceride
`transfer protein inhibitor is the compound 4'-trifluoromethyl(cid:173)
`biphenyl-2-carboxylic acid-[2-(1H-[l,2,4]triazol-3-ylmethyl)-
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`. 72222-394
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`CA 02291471 1999-12-02
`
`-13-
`
`1,2,3,4-tetrahydroisoquinolin-6-yl]-amide or a pharmaceuti-cally
`acceptable salt thereof.
`
`A pharmaceutical composition according to claim 2,
`4.
`wherein the apolipoprotein B secretion/microsomal triglyceride
`transfer protein inhibitor is the compo~nd 4'-trifluoromethyl(cid:173)
`biphenyl-2-carboxylic acid-[2-(2-acetylaminoethyi)-l,2,3;4-
`tetrahydroisoquinolin-6-yl]-amide, a hydrate thereof or a
`pharmaceutically acceptable salt of said compound or said
`hydrate.
`
`A pharmaceutical composition according to any one
`5.
`of claims l to 4, wherein the apolipoprotein B secretion/(cid:173)
`microsomal triglyceride transfer protein inhibitor is
`contained at a dosage 0.1 to 5.0 mg/kg/day.
`
`A pharmaceutical composition according to any one
`6.
`of claims 1 to 4, wherein the apolipoprotein B secretion/(cid:173)
`microsomal triglyceride transfer protein inhibitor is
`contained at a dosage 0.1 to 1.0 mg/kg/day.
`
`5
`
`10
`
`15
`
`1.
`A commercial package comprising the pharmaceutical
`composition as defined in any one of claims l to 6 and a
`20 written matter which includes an indication that the pharma(cid:173)
`ceutical composition can or should be used for lowering blood
`levels of lipoprotein(a) in a mammal.
`
`a.
`A commercial package according to claim 7, wherein
`the mammal is a human.
`
`SMART & BIGGAR
`OTTAWA, CANADA
`PATENT AGENTS
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