(13) United States Patent
`
`Wetterau, 11 et al.
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`US 6,492,365 B1
`Dec. 10, 2002
`
`US0064-92365131
`
`(54)
`
`MICRUSOMAI. TR[(}[.YClCR[[)E TR/\NSI*'I‘lR
`PROTEIN
`
`(75)
`
`Inventors:
`
`.Iohn R. Wcttcrau, II, Langhornc, PA
`(US); Darn Young Sharp, Perrineville;
`Richard E. Gregg, Pennington, both of
`NJ (US)
`
`(73)
`
`Assignee: Bristol-Myers Squihh Company, New
`York, NY (US)
`
`I“)
`
`Notice:
`
`Subject to any disclaimer, the term oi" this
`patent is extended or adjusted under 35
`USC. 154(1)) by 0 days.
`
`(31)
`
`Appl. No.: 08H-86,929
`
`(33)
`
`Filed:
`
`Jun. 7, 1995
`
`Related U.S. Application Data
`
`(60)
`
`(51)
`(52
`(58)
`
`(55)
`
`Division of application No. es.-*11?,3o'2, [ilcd on Sup. 3,
`1993, now Pat. No. 5,5GI5,8?2, which is a continuation—in—
`part of application No. USIULS,-’l49_. filed on Feb. 22, 1993,
`now abandoned, which is :1 conlinu:1tion—in—part of applica-
`tion No. 08,F34?,5U3, tiled on Mar. 6, 1992, now abandoned.
`
`Int. Cl.7
`US. Cl.
`
`
`C07D 26li'06
`51-H247; 51-‘U277
`514247, 277 Field of SI.-arcli
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`411994 Morris
`S_.3t]2_.ot3 A *
`5,321,031 A *
`(:'u‘l9FJ4 Dugar
`()'I'lll_-"R PUBl_IC.NI'I()NS
`
`514.’-151
`5143278
`
`llarrily et al., Circulation vol. 94(8): I-632, 1996.°"
`Wellerau el al., Science vol. 282: 751-754, 1998-.“
`Agrawal “Antisense Oligonueleotides: Towards Clinical
`'l'ria1s" Tibtech vol. 14, 376-387, Oct. 1996.*
`Branch “A Good Antisense is Ilard to Find" TIES vol.
`23:45-50. Feb. 1998.*
`
`* cited by examiner
`
`Priniary Exar:tinei‘—Sean McGarry
`(74) Atiorne -‘, Agent, or Firm—Christopher A. Klein
`
`(57)
`
`ABS'l‘RACl'
`
`Nucleic acid scqi1ences,particnlarly DNA sequences, cod-
`ing for all or part of the high molecular weight subunit of
`microsomal triglyceride transfer protein, expression vectors
`containing the DNA sequences, host cells containing the
`expression vectors, and methods utilizing these materials.
`The invention also concerns polypeptide molecules com-
`prising all or part ol‘ the high molecular weight subunit of
`microsomal triglyceride transfer protein, and methods for
`producing these polypeptide molecules. The invention addi-
`tionally concerns novel methods for preventing, stabilizing
`or causing regression of atherosclerosis and therapeutic
`agents having such activity. The invention concerns further
`novel methods for lowering serum liquid levels and thera-
`peutic agents having such activity.
`
`4_.?58_.5o‘J A "
`
`7x'l‘JS8 Swindell
`
`514.5222
`
`10 Claims, 8 Drawing Sheets
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`1of97
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`U.S. Patent
`
`Dec. 10, 2002
`
`Sheet 1 of 8
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`Us 6,492,365 B1
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`U.S. Patent
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`Dec. 10, 2002
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`Sheet 2 of 8
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`U.S. Patent
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`Dec. 10, 2002
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`Sheet 3 of 8
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`US 6,492,365 B1
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`FIG. 5
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`FIG; 6
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`4 Bf 97
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`Dec. 10, 2002
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`Sheet 4 of 8
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`US 6,492,365 B1
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`Dec. 10, 2002
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`Sheet 5 of 8
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`US 6,492,365 B1
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`Dec. 10, 2002
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`Sheet 6 of 8
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`Us 6,492,365 B1
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`100
`
`onD
`
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`
`
`
`PercentControl 88
`
`FIG. 10
`
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`
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`Dec. 10, 2002
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`Sheet 7 of 8
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`Us 6,492,365 B1
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`FIG. 12
`
`100
`
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`
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`
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`
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`
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`
`40
`
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`
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`
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`
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`
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`
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`
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`
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`
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`
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`
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`
`
`
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`
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`
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`
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`
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`Dec. 10, 2002
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`Sheet 8 of 8
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`160
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`1 40
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`1 20
`
`1 00
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`B0
`
`60
`
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`
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`
`FIG. 14
`
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`1
`MICROSONIAI. 'I‘RIGI.YCICRI[)IC TRANSl<'I£R
`PROTEIN
`
`CROSS—REFERENCE TO RELATED
`APPLICATIONS
`
`This is a divisional of US. Ser. No. 08,t1'l7,362, filed on
`Sep. 3, 1993 now U.S. Pat. No. 5,595,872.
`This is a continuation-in-part of U.S. patent application
`Ser. No. 08f0'15.4-49 Filed Feb. 22, 1993 now abandoned,
`which is a continuation—in—part of U.S. patent application
`Ser. No. 082847.503, filed Mar. 6, 1992, now abandoned,
`each of which is hereby incorporated by reference.
`
`FIELD OF THE INVENTION
`
`This invention relates to microsomal triglyceride transfer
`protein, genes for the protein, expression vectors comprising
`the genes, host cells comprising the vectors, methods for
`producing the protein, methods for detecting inhibitors of
`the protein, and methods of using the protein andfor its
`inhibitors.
`
`BACKGROUND OF THE INVENTION
`
`The microsomal triglyceride transfer protein (MTP) cata-
`lyzes the transport of triglyceride (TG), cholesteryl ester
`[(_'l_-L), and phosphatidylcholine (PC) between small unila-
`mellar vesicles (SUV). Wetterau & Zilversmit, Cflmit. t"."r_vs.
`Lr'pid.5' 38, 205-22 (1985). When transfer rates are expressed
`as the percent of the donor lipid transferred per time, MTP
`expresses a distinct preference for neutral
`lipid transport
`("PG and CE), relative to phospholipid transport. The protein
`from bovine liver has been isolated and characterized.
`Wetterau & Zilversmit, Chem. Pltys. Lipids‘ 38, 205-22
`(1985). Polyacrylamide gel electrophoresis(PA(}I_-'.) analysis
`of the purifier] protein suggests that the transfer protein is a
`complex of two subunits of apparent molecular weights
`58,000 and 88,000, since a single band was present when
`purified MTP was electrophoresed under nondenaturing
`condition, while two bands of apparent molecular weights
`58,000 and 88,000 were identified when electrophoresis was
`performed in the presence of sodium dodecyl sulfate (SDS).
`l‘hese two polypeptides are hereinafter referred to as 58 kDa
`and 88 kDa, respectively, or the 58 kDa and the 88 kDa
`cotnponent of MTP, respectively, or
`the low molecular
`weight subunit and the high molecular weight subunit of
`MTP, respectively.
`Characterization of the 58,000 molecular weight compo-
`nent of bovine MTP indicates that
`it
`is the previously
`characterized multifunctional protein, protein disulfide
`isomerase (l’Dl). Wetterau et al.,J. Biol. Clieiir. 265, 9800-7
`(1990). The presence of PDI in the transfer protein is
`supported by evidence showing that (1) the amino terminal
`25 amino acids of the bovine 58,000 kDa component of
`MTP is identical to that of bovine PDI, and (2) disulfide
`isomerase activity was expressed by bovine MTP following
`the dissociation of the 58 k|)a—88 kl)a protein complex. In
`addition, antibodies raised against bovine PI)I, a protein
`which by itself has no TG transfer activity, were able to
`immunoprecipitate bovine TG transfer activity frotn a solu-
`tion containing purified bovine MTP.
`PDI normally plays a role in the folding and assembly of
`newly synthesized disulfide bonded proteins within the
`lumen of the endoplasmic reticulum. llulleid & lireedman,
`Nature 335, 649-51 (1988). It catalyzes the proper pairing
`of cysteine residues into disulfide bonds, thus catalyzing the
`proper folding, of disulfide bonded proteins. In addition, PD]
`
`10
`
`I5
`
`'
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`2
`to the beta subunit of
`has been reported to be identical
`human prolyl 4—hydroxylase. Koivu et al., J. Biol. Chem.
`262, 6447-9 (1987). The role of PDI in the bovine transfer
`protein is not clear.
`It does appear
`to be an essential
`component of the transfer protein as dissociation of PDI
`from the 88 kDa component of bovine MTP by either low
`concentrations of a denaturant (guanidine IICI), a cliaotropic
`agent (sodium perchlorate}, or a nondenaturing detergent
`(octyl glucoside) results in a loss of transfer activity. Wet-
`terau et al., Bios.-'rrmti.5'tJ'y 30, 97%—35 (1991).
`Isolated
`bovine PD] has no apparent lipid transfer activity, suggest-
`ing that either the 88 kDa polypeptide is the transfer protein
`or that it confers transfer activity to the protein complex.
`The tissue and subcellular distribution of MTP activity in
`rats has been investigated. Wetterau & Zilversmit, Biocltem.
`Biripkys. Actrt. 875, 610-7 (1986). Lipid transfer activity
`was found in liver and intestine. Little or no transfer activity
`was found in plasma, brain, heart, or kidney. \Vithin the
`liver, MTP was a soluble protein located within the lumen of
`the microsomal fraction. Approximately equal concentra-
`tions were found in the smooth and rough microsomcs.
`Abetalipoproteineniia is an autesental recessive disease
`characterized by a virtual absence of plasma lipoproteins
`which contain apolipoprotein B (apoR)_ Kane & Havel
`in
`The t efnboiic Basis of Irthrzrircd Dismi-.99, Sixth edition,
`1139-64 (1989). Plasma TG levels may be as low as a few
`mgfdl., and they fail
`to rise after fat
`ingestion. Plasma
`cholesterol levels are often only 20-45 mg_.=’dI__ These abnor-
`malities are the result of a gene-tic defect in the assembly
`andfor secretion of very low density lipoproteins (VI.DI.) in
`the liver and chylomicrons in the intestine. The molecular
`basis for this defect has not been previously determined. In
`subjects examined, triglyceride, phospholipid, and choles-
`terol synthesis appear normal. At autopsy, subjects are free
`of atherosclerosis. Schaefer et al_, Chit. Ci'trmt_ 34, R9—l2
`(1988). A link between the apoll gene and abetalipopro-
`teinemia has been excluded in several families. Talmud et
`
`al., J. Chin. invest. 82, 1803-6 (1988) and Huang, et al.,.rtm.
`J. Hum. Genet. 46, 1141-8 (1990).
`Subjects with abetalipoproteinemia are aillicted with
`numerous maladies. Kane & Havel, supra. Subjects have fat
`malabsorption and TG accumulation in their entcrocytes and
`hepatocytes. Due to the absence of TG-rich plasma
`lipoproteins, there is a defect in the transport of fat-soluble
`vitamins such as vitamin E. This results in acanthocytosis of
`erythrocytes, spinocerebellar ataxia with degeneration of the
`fasciculus cuneatus and gracilis, peripheral neuropathy,
`degenerative pigmentary retinopalhy, and ceroid myopathy.
`Treatment of abetalipopmteinemic subjects includes dietary
`restriction of fat
`intake and dietary supplementation with
`vitamins A, 1:; and K.
`To date,
`the physiological role of MTP has not been
`demonstrated. In vitro, it catalyzes the transport of lipid
`molecules between phospholipid membranes. Presumably, it
`plays a similar role in vivo, and thus plays some role in lipid
`metabolism. The subeellular [lumen of the microsomal
`fraction) and tissue distribution (liver and intestine) of MTP
`have led to speculation that it plays a role in the assembly of
`plasma lipoproteins, as these are the sites of plasma lipo-
`protein assembly. Wetterau & Zilversmit, Bioeiiem. Br'opIty.s'.
`Actrr. 875, 610-7 {1 986). The ability of MTP to catalyze the
`transport of TG between membranes is consistent with this
`hypothesis, and suggests that MTP may catalyze the trans-
`port of TC: from its site of synthesis in the endoplasmic
`reticulum (ER) membrane to nascent lipoprotein particles
`within the lumen of the ER.
`
`Olofsson and colleagues have studied lipoprotein assem-
`bly in lIepG2 cells. Bostrom et al., J. Biol. Chem. 263,
`
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`3
`443442 (1988). Their results suggest small precursor lipo-
`proteins become larger with time. This would be consistent
`with the addition or transfer of lipid molecules to nascent
`lipoprotcins as they are assembled. MTP may play a role in
`this process.
`In support of this hypothesis, Howell and
`Palade, J’. Celt’ Biol’. 92, 833-45 ([982), isolated nasoent
`lipoproteins from the hepatic Golgi fraction of rat
`liver.
`There was a spectrum of sizes of particles present with
`varying lipid and protein compositions. Particles of high
`density lipoprotein (IIDL) density, yet containing apoB,
`were found. Higgins and llutson, J. Lipid Res. 25
`1295-1305 (1984),
`reported lipoproteins isolated from
`Golgi were consistently larger than those from the endo-
`plasmic reticulnm, again suggesting the assembly of lipo-
`protcins is a progressive event. Ilowever, there is no direct
`evidence in the prior art demonstrating that MTP plays a role
`in lipid metabolism or the assembly of plasma lipoprotein.
`
`SUMMARY OF THE INVENTION
`
`The present invention concerns an isolated nucleic acid
`molecule comprising a nucleic acid sequence coding for all
`or part of the high molecular weight subunit of MTP andfor
`intron, 5', or 3' flanking regions thereof. Preferably,
`the
`nucleic acid molecule is a DNA (deoxyribonucleic acid)
`molecule, and the nucleic acid sequence is a DNA sequence.
`l-‘urther preferred is a nucleic acid having all or part of the
`nucleotide sequence as shown in SEQ. ID. NOS. 1, 3, 5, 7,
`8, 1 together with 5, 2 together with 7, the first 108 bases of
`2 together with 8, the first 108 bases of 2 together with 7 and
`8, or 8 together with 31 and 32.
`The present invention also concenrs a nucleic acid mol-
`ecule having a sequence complementary to the above
`sequences andfor intron, 5', or 3' flanking regions thereof.
`The present invention further concerns expression vectors
`comprising a DNA sequence coding for all or part of the
`high molecular weight subunit of M’l'P.
`The present invention additionally concerns prokaryotic
`or eukaryotic host cells containing an expression vector that
`comprises a DNA sequence coding for all or part of the high
`molecular weight subunit of MTP.
`
`The present invention additionally concerns polypeptides
`molecules comprising all or part of the high molecular
`weight subunit of MTP. Preferably, the polypeptide is the
`high molecular weight subunit of human MTP or the recom-
`binantly produced high molecular weight subunit of bovine
`MTP.
`
`The present invention also concerns methods for detecting
`nucleic acid sequences coding for all or part of the high
`molecular weight subunit of MTP or related nucleic acid
`sequences.
`
`invention further concerns methods for
`The present
`detecting inhibitors of MTP and, hence, anti-atherosclerotic
`and lipid lowering agents.
`
`The present invention further concerns a novel method for
`treatment of atherosclerosis, or for lowering the level of
`serum lipids such as serum cholesterol, TG, PC, or CE in a
`mammalian species comprising administration of a thera-
`peutically effective amount of an agent that decreases the
`activity or amount of MTP. Such agents would also be useful
`for treatment of diseases associated or affected by serum
`lipid levels, such as pancreatitis, hyperglycemia, obesity and
`the like. In particular, this invention concerns a method of
`treatment wherein the agent that decreases the activity of
`MTP is a compound of the formula
`
`1U
`
`I5
`
`30
`
`35
`
`4U
`
`45
`
`SU
`
`55
`
`oil
`
`65
`
`wherein:
`
`l{' is alkyl, alkcnyl, alkynyl, aryl, hcteroaryl, arylalky.,
`heleroarylalkyl, cycloalkyl, cycloalkylalkyl (all option-
`ally substituted through available carbon atoms with I,
`2, or 3 groups selected from halo, alkyl, alkcny,
`alkoxy, aryloxy, aryl, arylalkyl, alkylmercapto,
`arylmereapto, eycloalkyl, cycloalkylalkyl, heteroary.,
`heteroarylalkyl);
`
`R2, R3, R4 are independently hydrogen, halo, alky.,
`alkenyl, alkoxy, aryloxy, aryl, arylalkyl, alkylmercapto,
`arylmercapto, cycloalkyl, cyeloal.kylal.kyl, heteroary.,
`heteroarylalkyl;
`
`RS and R" are independently hydrogen, alkyl, alkeny.,
`aryl, hctcroary], arylalkyl, heleroarylalkyl, eyeloa1ky-,
`eycloalkylalkyl
`(all optionally substituted through
`available carbon atoms with 1, 2, or 3 groups selected
`from hydrogen, halo, alkyl, alkenyl, alkoxy, aryloxy,
`aryl, arylalkyl, alkylmercapto, arylmercapto,
`eycloalkyl, cyeloalkylalkyl, hetcroaryl, heteroaryla—
`lkyl; with the proviso that when R5 is CII3, R5 is not
`hydrogen.
`
`R7 is alkyl (optionally substituted with oxo), aryl, or
`arylalkyl (wherein the alkyl portion is optionally sub-
`stituted with oxo). Examples of such oxo-sustitutecl
`groups are described in Cortizo, I... J’. Med. Chem. 34,
`2242-224? (1991).
`Also in accordance with the present invention are novel
`compounds of formula I, wherein R1 is alkyl, alkenyl, aryl,
`heteroaryl, arylalkyl (wherein the alkyl comprises at
`least
`two carbon atoms), heteroarylalkyl (wherein the alkyl com-
`prises at
`least
`two carbon atoms), cycloalkyl, or
`cyeloalkylalkyl, all optionally substituted as described
`above.
`
`The present invention further concerns novel compounds
`of formula II wherein R'
`is arylalkyl or heteroarylalkyl,
`wherein the alkyl portion of each comprises at least two
`carbon atoms and wherein each is optionally substituted as
`described above.
`
`Further still in accordance with the present invention are
`novel compounds of the formula
`
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`N
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIG.
`1 shows bovine eD.\lA clones. The live bovine
`cDNAinserts are illustrated. The continuous line at the top
`of the figure represents the total c[)N/\ sequence isolated.
`Small, labeled bars above this line map peptide and probe
`sequences. The open reading frame is indicated by the
`second line, followed by ““‘ corresponding to 3' noncoding
`sequences. Clone number and length are indicated to the left
`of each line representing the corresponding region of the
`composite sequence. Clones 64 and '.-'6 were isolated with
`probe 2A, clones 22 and 23 with probe 37Aand clone 2 with
`probe 19/\. Eco RI linkers added during the cDNA library
`construction contribute the Eco RI restriction sites at the 5'
`and 3' ends of each insert. The internal Eco RI site in inserts
`22 and 76 is encoded by the cDNA sequence. The Nhe I
`restriction site was utilized in preparing probes for isolation
`of human cI)N[\ clones (below). The arrows under each
`insert line indicate individual sequencing reactions.
`FIG. 2 shows TG transfer activity in normal subjects.
`Protein-stimulated transfer of “(T-'l'G from donor SUV to
`acceptor SUV was measured in homogenized intestinal
`biopsies obtained from five normal subjects. The results are
`expressed as the percentage of donor TG transferred per
`hour as a function of homogenized intestinal biopsy protein.
`FIG. 3 shows TG transfer activity in abetalipoprotcinemic
`subjects. Protein-stimulated transfer of "'C—TG from donor
`SUV to acceptor SUV was measured in homogenized intes-
`tinal biopsies obtained from four abetalipoproteinemic sub-
`jects. The results are expressed as the percentage of donor
`TG transferredfhour as a function of homogenized intestinal
`biopsy protein.
`FIG. 4 shows TG transfer activity in control stlbjects.
`Protein stimulated transfer of “C-TG from donor SUV to
`acceptor SUV in homogenized intestinal biopsies were
`obtained from three control subjects, one with chylomicron
`retention disease (open circles), one with homozygous hypo-
`betalipoproteinemia (solid circles), and one non-fasted (x).
`The results are expressed as the percentage of donor 'l'G
`transferredfhour as a function of homogenized intestinal
`biopsy protein.
`FIG. 5 shows western blot analysis of MTP in normal
`subjects. An aliquot of purified bovine MTP (lane 1) or the
`post
`l03,00fl><g proteins following deoxycholate treatment
`of 23 gig of homogenized intestinal biopsies from 3 normal
`subjects (lanes 2-4) were fractionated by SDS—l-’AGl_i and
`then transferred to nitrocellulose. The blots were probed
`with anti—88 kDa.
`
`FIG. 6 shows western blot analysis of MTP in control
`subjects. An aliquot of purilied bovine MTP (lane 1) or the
`post 'lf)3,000xg proteins following dcoxyeholate treatment
`of 15 gig, 25 gig, and 25 gig homogenized intestinal biopsies
`from a subject with chylomicron retention disease (lane 2),
`a subject with homozygous hypobetalipoproteinemia (lane
`3), and a non—fasted subject (lane 4), respectively, were
`fractionated by SDS-PAGE and then transferred to nitrocel-
`lulose. 'lhe blots were probed with anti-88 kDa.
`
`6
`FIG. 7 shows western blot analysis of MTP in normal
`subjects with affinity—purified antibodies. An aliquot ofpuri—
`lied bovine MTP (lane '1) or the post 'lO3,000><g proteins
`following deoxycholate treatment of 34 pg (lane 2) or 25 gig
`(lane 3) of homogenized intestinal biopsies from 2 normal
`subjects were fractionated by SDS-PAGE and then trans-
`ferred to nitrocellulose. The blots were probed with aflinity
`purified anti-88 kDa.
`FIG. 8 shows western blot analysis of MTP in abetalipo-
`proteincmic subjects. An aliquot of purified bovine MTP
`(lane 1] or post 1{)3,0UUxg proteins following deoxycholale
`treatment of 18 Jug (lane 2), 23 pg (lane 3], 33 pg (lane 4),
`23;ig (lane 5) of homogenized intestinal biopsies from four
`different abetalipoproteinemic subjects were fractionated by
`SIJS-P.«'\(il_" and then transferred to nitrocellulose. In lanes 6
`and 7, ‘I00 ,ug of the whole intestinal homogenate (subjects
`corresponding to lane 4 and 5) was fractionated by SD8-
`PAGE and transferred to nitrocellulose. The blots were
`
`probed with anti-88 k[)a.
`FIG. 9 shows a Southern blot analysis of a gene defect in
`an abetalipoprotcinemic subject. Ten gig of genomic DNA
`from a control, the abetalipoproteinemic subject {proband),
`and front
`the subject’s mother and father were cut
`to
`completion with 'laq I, electrophorescd on 1% agarosc and
`transferred to nitrocellulose. Southem hybridization was
`performed using exon '13 cDNA as a probe. Two hybridizing
`bands in the normal lane indicated the presence of a Taq I
`site in the normal exon 13. One hybridizing band in the
`abctalipoproteinemic subject lane demonstrated the absence
`of this restriction sequence in both alleles in exon 13,
`conftmting a homozygous mutation in this subject. The
`heterozygous state in the mother and father is shown by the
`three hybridizing bands, corresponding to both the normal
`and the mutant restriction patterns.
`FIG. 10 shows inhibition in MTP-catalyzed transport of
`TG from donor SUV to acceptor SUV by compound A
`described hereinafter. Compound A was dissolved in DMSD
`and then diluted into l5;'40 buffer. Aliquots were added to a
`lipid transfer assay to bring the compound to the indicated
`final concentrations. DMSL) concentration in the assay never
`exceeded 2 ,rrI.f6flU ;rI., a concentration that was indepen-
`dently determined to have minimal efleet on the assay.
`MTP-catalyzed lipid transport was measured for 30 minutes
`at 37° C. 'l'(_i
`transfer was calculated and compared to a
`control assay without
`inhibitor. Three independent assay
`conditions were used to demonstrate MTP inhibition by
`compound A. Assay conditions were: 8 nmol donor PC, 48
`nmol acceptor PC, and 75 rig MTP [open circles]; 24 nmol
`donor PC, 144 nmol acceptor PC, and 100 ng MTP (solid
`circles); 72 nmol donor PC, 432 nmol acceptor PC, and 125
`ng MTP (open squares).
`FIG. II shows the dose response of Compound A on
`ApoB, ApoAI and HSAse-eretion from HepG2 cells. HepG2
`cells were treated with compound A at the irtdieated doses
`for "l 6 hours. The concentration in the cell culture media of
`
`apol-I, apoAI and HSA after the incubation period was
`measured with the appropriate Lil.ISA assay and normalized
`to total cell protein. The data shown are expressed as a
`percentage of the control (DMSO only).
`FIG. "[2 shows the elfect of compound /\on '|‘G secretion
`from HepG2 cells. HepG2 cells were treated with Com-
`pound A at the indicated doses for 18 hours, the last two
`hours of which were in the presence of 5 ,rrCi.imI.
`3lI—glycerol. The concentration of radiolabelled triglycer-
`ides in the cell culture media was measured by quantitative
`extraction, followed by thin layer chromatography analysis
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`US 6,492,365 B1
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`7
`and normalization to total cell protein. The data shown are
`expressed as a percentage of the control (DMSO only).
`FIG. 13 shows inhibition in MTP-catalyzed transport of
`TC} from donor SUV to acceptor SUV by compound I}
`described hereinafter. Compound B was dissolved in DMSO
`and then diluted into 15;‘4-U buffer. Aliquots were added to a
`lipid transfer assay to bring the compound to the indicated
`final concentrations. DMSO concentration in the assay never
`exceeded 2 ;rL;‘6UU gtL, a concentration that was indepen-
`dently determined to have minimal effect on the assay.
`M'l‘P—eatalyzed lipid transport was measured for 30 minutes
`at 37° C. TG transfer was calculated and compared to a
`control assay without
`inhibitor. Two independent assay
`conditions were used to demonstrate MTP inhibition by
`compound B. Assay conditions were: 24 nmol donor PC,
`144 nmol acceptor PC, and 100 rig MTP (open circles); 72
`nmol donor PC, 432 nmol acceptor PC, and [25 ng MTP
`(solid circles).
`l"I(}. 14 shows the dose response of compound B on
`ApoB, ApoAI and I-ISA secretion from I-IepG2 cells. HepG2
`cells were treated with compound B at the indicated doses
`for 16 hours. Tlie cortcentration in the cell culture media of
`apoli, apoAl and HSA after the incubation period was
`measured with the appropriate ELISA assay and normalized
`to total cell protein. The data shown are expressed as a
`percentage of the control (DMSO only).
`DETAILED DESCRIPTION OF TIIE
`INVENTION
`Dlzil-‘INITION OF 'I'I:‘RMS
`
`The following definitions apply to the terms as used
`throughout
`this specification, unless otherwise limited in
`specific instances.
`The term "MTP” refers to a polypeptide or protein
`complex that (1) if obtained from an organism (e.g., cows,
`llumans, etc.), car] be isolated front tlle nlicrosornal fraction
`of homogenized tissue; and (2) stimulates the transport of
`triglycerides, cholesterol esters, or phospholipids from syn-
`thetic phospholipid vesicles, membranes or lipoproteins to
`synthetic vesicles, membranes, or lipoproteins and which is
`distinct from the cholesterol ester transfer protein [Drayna et
`al., Nnmrc 327, 632-634 (1987)] which may have similar
`catalytic properties. Ilowever, the MTP molecules of the
`present invention do not necessarily need to be catalytically
`active. For example, catalytically inactive MTP or fragments
`thereof may be useful in raising antibodies to the protein.
`The term "modified", when referring to a nucleotide or
`polypeptide sequence, means a nucleotide or polypeptide
`sequence which differs from the wild—type sequence found
`in nature.
`
`The term “related”, when referring to a nucleotide
`sequence, means a nucleic acid sequence which is able to
`hybridize to an oligonucleotide probe based on the nucle-
`otide sequence ofthe high molecular weight subunit ofMTP.
`The phrase “control regions” refers to nucleotide
`sequences that regulate expression of MTP or any subunit
`thereof, including but not limited to any promoter, silencer,
`enhancer elements, splice sites,
`transcriptional
`initiation
`elements, transcriptional termination elements, polyadeny-
`lation signals, translational control elements, translational
`start site, translational termination site, and message stability
`elements. Such control regions may be located in sequences
`5' or 3' to the coding region or in introns interrupting the
`coding region.
`The phrase "stabilizing” atherosclerosis as used in the
`present application refers to slowing down the development
`of andlor inhibiting the formation of new atherosclcrotic
`lesions.
`
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`The phrase “causing the regression of” atherosclerosis as
`used in the present application refers to reducing andfor
`eliminating atherosclerotic lesions.
`The terms “alkyl” and “alk" refer to straight and branched
`chain hydrocarbon radicals of up to 20 carbon atoms, with
`1 to 12 carbon atoms preferred and 1 to 8 carbon atoms most
`preferred. Exemplary alkyl groups are methyl, ethyl, propyl,
`isopropyl, butyl,
`t—butyl, isobutyl, pentyl, hexyl, isohcxyl,
`hcptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl,
`nonyl, decyl, undecyl, dodccyl, the various branched chain
`isomers thereof, and the like.
`The term “alkylene"' refers to alkyl groups having single
`bonds for attachment to other groups at two different carbon
`atorus. Exerriplary alkylene groups are —CH—
`CH3CH3CH—,—CH—Cl-l(CH3)—CH._:—CH—, and the
`like.
`The term “alkenyl” refers to both straight and branched
`chain hydrocarbon groups of up to 20 carbon atoms, with 1
`to 12 carbon atoms preferred and l
`to 8 carbon atoms most
`preferred, having at least one double bond. The term "cis-
`alkenyl” refers to alkenyl groups having a cis double bond
`orientation.
`
`The term "alkenylene” refers to alkenyl groups having
`single bonds for attachment at two different carbon atoms.
`Exemplary alkenylene groups are —CII,:—CII=CII—
`(TI[:—, —{.‘lI:—(fll[(fll3)—(II[=(Tll—(7ll2—(TlI2—,
`and the like. The term “cis-alkenylene" refers to alkenylene
`groups having a cis double bond orientation.
`The term “alkynyl" refers to both straight and branched
`chain hydrocarbon groups of up to 20 carbon atoms, with l
`to 12 carbon atoms preferred and '1 to 8 carbon atoms most
`preferred, having at least one triple bond.
`The term “cycloal1<y1" refers to saturated cyclic hydro-
`carbon groups containing 3 to 20 carbons, preferably 3 to 12
`carbons. Excnrplary cycloalkyl groups include cyclopropyl,
`cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
`cyclooctyl, eyelodecyl, and eyelododecyl
`The terms "aryl" or ‘‘air''' as employed herein refer to
`monocyclic or bicyclic aromatic groups containing from 6 to
`"[0 carbon atoms in the ring portion, such as phenyl or
`napthyl, may be optionally substituted.
`The term "heteroaryl" refers to (1) 5- or 6-membered
`aromatic rings having 1 or 2 heteroatoms in the ring wherein
`the heteruatoms are selected from nitrogen, oxygen, and
`sulfur, {2} such rings fused to an aryl [e_g., benzothiophenyl,
`indolyl). Exemplary heteroaryl groups include pyrrolyl,
`furanyl,
`thiuphenyl,
`irnidazolyl, oxazolyl,
`tlriazolyl,
`pyramlyl, pyridyl, pyrimidinyl, and the like, and may be
`optionally substituted andfor fused to an aryl as in indolyl
`and benzothioplieuyl.
`Preferred Moities
`lior methods of use and novel compounds in accordance
`with the present invention, the following moities of formu-
`lae l and II are preferred:
`Rl is —R"— "' or
`
`R" and R” are each independently alkylene or cis-
`alkenylene of up to 6 carbon atoms;
`R” is aryl or heteroaryl; and
`R" and R: are each independently alkyl, alkenyl, aryl,
`arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl, or
`eycloalkylalkyl.
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`US 6,492,365 B1
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`EMBODIMENTS
`
`10
`9
`For the methods ofuse and novel compounds of formulae
`buffer, antibacterial, bulking agent [such as mannitol), anti-
`oxidants (ascorbic acid or sodium bisulfite) or the like. Oral
`I and II, R“ and R‘ are most preferred to be independently
`dosage forms are preferred, although parenteral forms are
`aryl, arylalkyl, heteroaryl, or heteroarylalkyl.
`quite satisfactory as well.
`Use and Utility
`The nucleic acids of the present invention can be used in
`The dose adiiiinistered must be carefully adjusted accord-
`a variety ol‘ ways in accordance with the present invention.
`ing to the age, weight, and condition ol‘ the patient, as well
`l-‘or example, they can be used as DNA probes to screen
`as the route of administration, dosage form and regimen, and
`other CDNA and genomic DNA libraries so as to select by
`the desired result. In general, the dosage forms described
`hybridizatioii other DNA sequences that code for proteins
`above may be administered in single or divided doses of one
`related to the high molecular weight subunit of M'l'l’._In 1U 10 four tirrlcg Ci,-,j1y_
`addition, the nucleic acids of the present invention coding
`l.‘II_)1' ("Ill DI part
`Il'lC
`lTl0lCClll£l1' Wtllglll Sl.llJlJl']ll OT l']Ul'l1‘c1l'l
`or bovine M ll’ can be used as DNA probes to screen other
`,
`,
`cDNA and genomic DNAIrbraries to select by l]yh['lCl1Zall(')I'l
`NuclcicAcids
`DNA_,
`,
`h
`,
`_
`H,
`_,
`__
`_
`I
`_
`_
`_
`_
`sequenees t at eode for Mll molecules from other
`l'_“_'Crlllr’r‘ Cr’rl_C°rrl§ all l5r‘l3rr"‘3l llurlelf 3‘31rl
`The llr'-5-"*‘-“ll
`organisms. The nucleic acids may also be used to generate
`molecule oomprising a nucleic acid sequence corling tor all
`1,,-[mm-5 to ampfify ¢r)NA or gcmmfc DNA Ming p(,|y_
`or l-“lrl Ul
`lrle lllgll
`rrlr_3l3‘-'“lar Wlilglll 5‘-lburlll Ol MTP-
`mcrase chain reaction (PCR)
`techniques. The DNA
`Prererahllttlh