(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World lntelledual Property
`Organization
`International Bureau
`
`I 0111111111Ellll01111111 llDI Imm ~111111llm11111111111~~ 1111 Ellll llHI DD
`
`(43) International Publication Date
`13 October 2005 (13.10.2005)
`
`PCT
`
`(10) InternalionaJ Publication Number
`WO 2005/094864 A2
`
`(81) Designated States (unless otherwise indicaJed, for every
`kind of national protection available): AE, AG, AL, AM,
`AT, AU, AZ. BA, BB, BO, BR, BW •. BY. BZ. CA, CH, CN,
`CO, CR, CU, CZ, DE, DK, DM, DZ. EC, BE, EG, ES, Fl,
`GB, GD, GB, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE,
`KG, KP, KR, 1CZ, LC, LK, LR, LS, LT, LU, LV, MA, MD,
`MG, MK, MN, MW, MX, MZ, NA, NI, NO, NZ, OM: PG,
`· PH, PL, PT, RO, RU, SC, SD, SE, SG, SK, SL, SM, SY. TJ,
`TM, TN, TR, TI, Tz, UA, UG, US, Uz, VC, VN, YU, ZA,
`ZM,'ZW.
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ. NA, SD, SL, SZ. Tz, UG, ZM,
`ZW), Eurasian (AM, AZ. BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BB, BG, CH, CY, CZ. DE, DK, EE, ES, FI,
`FR, GB, GR, HU, IB, IS, IT, CT, LU, MC, NL, PL, PT, RO,
`SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN,
`GQ, GW, ML, MR, NE, SN, ID, TG).
`
`Published:
`without international search repon and to be republished
`upon receipt of that report
`
`For two-lener codes and other abbreviations, .refer to tire nGuid(cid:173)
`ance Notes on Codes and Abb~iatlonsn appearing at the begin(cid:173)
`ning of each regulJJr issue of the PCT Ga~ne.
`
`(51) lotematlonal Patent CJasslDcat1on7:
`
`A61K38/00
`
`(21) lotematlonal AppUcatlon Number:
`PCflBP2005J003326
`
`(21} International Filing Date: 30 March 2005 (30.03.2005)
`
`(25) Flllng Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`04007732.3
`
`30 March 2004 (30.03.2004) EP
`
`(71) Applicant (for all designated Stales except US): MAX·
`PLANCK GESELLSCHAFT ZUR FORDERUNG
`DER WISSENSCHAFTEN E. V.. [DFJDE]; Berlin (DE).
`
`(71) Inventors; and
`(75) Inventors/Applicants (for US only): EATON, SUZBDne
`[US/DE]; Reger Str. 2, 01309 Dresden (DE). THIELE,
`Christoph [DFJDE]; Birkenstr. 32, 01474 Dresden (DE).
`PANAKOVA, Danlela [SK/DE]; Louisenstr. 12, 01099
`Dresden (DE). MAROIS, Eric [FRJDE); Neubenstr. 27,
`01307 Dresden (DE). SPRONG, Hein [NUNL]; Genii Jan
`van der Veenlaan I I, NL-3705 PE Zeist (NL).
`
`--
`=
`-= (74) Agent: VOSSIUS&Partner;Siebertstrasse4,81675Mu-
`-
`=
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`-
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`-
`
`nich(DE).
`
`--= = ----------
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`v
`\0
`QC
`V~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
`~ (54} 11tle: TREATMENT OF HEOOEHOG- AND WNT-SECRETING TUMORS WITH INHIBITORS OF LIPOPRCJfEIN PAR(cid:173)
`ln TICLE BIOGENESIS
`= Q
`N
`0 embodiment, growth and/or progression of the tumor are caused by one or more protein of the Wnt or Hedgehog family. Preferred
`::::;;.... tumors an: esophageal twnor, biliary tract tumor, gastric lumor, pancreatic tumor, malignant melanoma, colorectal tumor, squamous
`~ cell carcinoma and cervical tumor.
`
`(57} Abstract: This invention relates to the use of an inhibitor of Microsomal Triglyceride Transfer Protein (MTP}, HMG-CoA
`reductase, DGAT and/or ACAT for the preparation of a pharmaceutical composition for the treatment of tumors. In a preferred
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`Treatment of Hedgehog- and Wnt-secreting
`Lipoprotein particle biogenesis
`
`tumors with
`
`inhibitors of
`
`This invention relates to the use of an inhibitor of Microsomal Triglyceride Transfer
`Protein (MTP), HMG-CoA reductase, DGAT and/or ACAT for the preparation of a
`pharmaceutical composition for the treatment of tumors. In a preferred embodiment,
`growth and/or progression of the tumor are caused by one or more protein of the Wnt
`or Hedgehog family. Preferred tumors are esophageal tumor, biliary tract tumor,
`gastric tumor, pancreatic tumor, malignant melanoma, colorectal tumor, squamous.
`cell carcinoma and cervical tumor.
`
`In this specification, a number of documents is cited. The disclosure of these
`documents, including manufacturer's manuals, is herewith incorporated by ,reference
`in its entirety.
`
`Despite intense investigations, the molecular mechanisms leading to tumor:formation
`and cancer are far from being completely understood. Chains of molecular events
`leading to malignant transformation are emerging, wherein certain mechanisms
`appear to be generic, while others are specific for certain tumors.
`
`Wnt and Hedgehog family proteins are secreted ligands that play multiple , critica.1
`roles in the development of multicellular organisms. Alterations in th~ cellular
`signaling pathways that respond to these Wnt and Hedgehog family ligands also play
`causative roles in the initiation and progression of a variety of tumors (Xie .and
`·Abbruzzese (2003)). Mutations in Hedgehog signal transduction components give
`rise to tumors of the skin, muscle and cerebellum.
`
`Alteration in Wingless signal transduction components is a critical step in . the
`development of colon cancer and is associated with a variety of other types of
`malignancies (reviewed in Giles et al. (2003}). While alterations in the downstream
`components of these pathways have been known for some time, recent studies have
`shown that tumorigenesis can depend on the unregulated production of the ligands
`themselves.
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`One of the earliest identified oncogenes is Wnt1, whose ectopic activation in mouse
`mammary cells is the basis for MMTV mediated tumorigenesis (Nusse and Varmus
`(1992)). Consistent with this, overproduction of a variety of Wnt family ligands has
`been observed in human breast cancers. Over-expression of · Wnt proteins in
`humans is also symptomatic of many gastric cancers, colorectal cancers, pancreatic
`cancers, esophageal cancers, squamous cell carcinoma!:!. cervical cancers a~d
`malignant melanomas. A causative role has been directly demonstrated for Wnt5a in
`promoting both cell motility and invasion of malignant melanoma cells (Weeraratna et
`al. (2002)). Although the ·extent to which . Wnt overproduction contributes to
`malignant phenotypes is not yet characterized iri all cases, the frequency With which
`mutations in Wnt signaling pathway components promote other neoplasias suggests
`that Wnt overproduction has tumorigenic consequences.
`
`Overproduction of Hedgehog ligands has been demonstrated to play a direG! causal
`role in promoting growth of tumors of the gastrointestinal tract, including those of the
`esophagus, stomach biliary tract and pancreas (Berman et al. (2003); Thayer et al.
`(2003). These tumors are very aggressive and some of the most resistant to current
`therapy.
`The relevance of Hedgehog signalling for cancer . formation and
`maintenance has been reviewed
`in Pasca di Magliano and Hebrok (2003)~
`Preventing secretion of Hedgehog and Wnt proteins should be an effective therapy
`for cancers that depend on the unregulated production of these ligands.
`
`Paradoxically, Wingless and Hedgehog are covalently modified by lipid, which is
`thought to mediate their interaction with the exoplasmic face of the plasnia
`membrane. This observation raises perplexing questions about how Wingless arid
`Hedgehog, having affinity for cell membranes, are released from the cells that make
`them and move through .adjacent tissue. As yet, it is unclear how lipid-modified
`proteins leave the plasma membrane and move over many cell diameters.
`
`The function of lipid modification of Wingless and Hedgehog is not yet understood.
`In Drosophila, mutant Hedgehog proteins that cannot be sterol-modified signal ·over
`inappropriately long distances when over-expressed (Porter et al. (1996), Burke et at
`(1999)). This would suggest that the role of lipid is to restrict the range of m6rphogen
`diffusion through the epithelial plane. On the other hand, mice that harbor this mutant
`form of Hedgehog in its normal chromosomal context are deficient in Jong-range
`Hedgehog signalling (Lewis et al. (2001 )). This suggests, in contrast, that lipld
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`modification may be necessary for movement of the protein. Mutations that ·prevent
`.
`the N-terminal palmitoylation of either Wingless or Hedgehog destroy their activity
`(Chamoun et al. (2001), Lee et al. (2001), Willert et al. (2003)); thus,. lipid
`modification is crucial to the function of these proteins.
`
`:
`
`:
`
`In summary, there is evidence for the involvement of .proteins of the Wnt or
`Hedgehog families in tumor formation and progression, however, a signifieant p~rt of
`the corresponding molecular events are still obscure, thereby impeding: a rational
`approach to therapy.
`
`The technical problem underlying the present. invention was to provide novel means
`and methods for the treatment of Hedgehog- and/or Wnt-secreting tumors. :
`
`Accordingly, this invention relates to the use of an inhibitor of Microsomal Triglyceride
`the
`Transfer Protein (MTP), HMG-CoA reductase, D~AT ~nd/or ACAT for
`preparation of a pharmaceutical composition for the treatment of tumors.
`
`The Microsomal Triglyceride Transfer Protein (MTP) is a heterodimeric lipid transfer
`protein
`that catalyzes
`the
`transport of
`triglyceride, cholesteryl ester an.d
`phosphatidylcholine between membranes. It is required for assembly and s~cretion
`of the lipoproteins containing apolipoprotein B (apoB), i.e., very low density
`lipoproteins (VLDL) and chylomicrons. VLDL
`in
`tum is converted :into LDL.
`.
`'
`Accordingly, inhibition of MTP function would affect the levels of . lipoproteins
`inhibitors of HMG-CoA
`comprising chylomicrons, VLDL and LDL. Similarly,
`reductase, DGAT or ACAT (which are enzymes involved in lipid biosynthesis) affect
`the levels of lipoproteins. The term "inhibitor" designates a compound lowering the
`activity of a target molecule, preferably by performing one or more of the following
`effects: (i) the transcription of the gene encoding the protein to be inhibited is
`lowered, (ii) the translation of the mRNA encoding the protein· to 'be inhibited is
`lowered, (iii) the protein performs its biochemical function with lowered efficiency in
`presence of the inhibitor, and (iv) the protein performs its cellular function with
`lowered efficiency in presence of the inhibitor. In one embodiment, in particular with
`regard to inhibition of HMG-CoA reductase, the inhibitor is a statin. Cpmpounds
`falling in class (i) include compounds interfering with the transcriptional machinery
`and/or its interaction with the pro~oter of said gene and/or with expression control
`elements remote from the promoter such as enhancers. Compounds. of class (ii)
`comprise antisense constructs and constructs for performing
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`RNA interference well known in the art (see, e.g. Zamora (2001} or Tuschl (2001}}.
`Compounds of class (iii) interfere with molecular function of the protein to be
`inhibited, in case of MTP with its enzymatic activity, in parti~lar with the protein
`disulfide isomerase activity. Accordingly, active site binding compounds, in particular
`compounds capable of binding to the active site of any protein disulfide isomerase,
`are envisaged. More preferred are compounds specifically binding to an active site of
`MTP. Also envisaged are compounds binding to or blocking substrate binding. sites of
`MTP as are compounds binding to or blocking binding sites of MTP~ for other
`interaction partner would be
`interaction partners. An example for such an
`apolipoproteinB (apoB}. The latter group of compounds blocking binding sites of MTP
`may be fragments or modified fragments with improved pharmacological ·properties
`of the naturally occurring binding partners. Class (iv} includes compounds which do
`not necessarily directly bind to MTP, but still interfere with MTP activity, for example
`by binding to and/or inhibiting the function or inhibiting expression of members of a
`pathway which comprises MTP. These members may be either upstream. or
`downstream of MTP within said pathway.
`
`In a preferred embodiment, the inhibitor is a low molecular weight compound. ·Low
`molecular weight compounds are compounds of natural origin or chemically
`synthesized compounds, preferably with a molecular weight between 100 and 1"000,
`more preferred between 200 and 750, and even more preferred between 300 · and
`600.
`
`The efficiency of the inhibitor can be quantitized by comparing the level of activity in
`the presence of the inhibitor to that in the absence of the inhibitor. For example, as
`an activity measure may be used: the change in amount of mRNA fo'nned: the
`change in amount of protein formed, the change in amount of substrate converted or
`product formed, and/or the charige in the cellular phenotype or in the phenotype of
`an organism.
`
`In a preferred embodiment, the level of activity is less than 90%, more preferred.less
`than 80%, 70%, 60% or 50% of the activity in absence of the inhibitor. Yet more
`preferred are inhibitors lowering the level down to less than 25%, Jess than 10%, less
`than 5% or less than 1 % of the activity in absence of the inhibitor.
`
`Using Drosophila as a model organism, the inventors have surprisingly shoym that
`Hedgehog and Wingless are released from cells on Lipoprotein particles. It has been
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`speculated that these particles (which were termed "Argosomesj represent a new
`type of particles. It further has been shown that these particles are required for the
`signaling activity of Wingless and Hedgehog (Panakova et al, in preparation}. The
`single insect lipoprotein organized by Apolipophorin I /If is similar in biosYrithesis,
`structure and function to vertebrate lipoprotein. particles like LDL and VLDL; it
`transports lipid and cholesterol from the gut to the fat body (an organ analogous to
`the liver and adipose tissue}, and distributes lipid and sterol to peripheral tissues.
`These similarities are evident at the sequence level as well; Apolipophorin is highly
`homologous to ApolipoproteinB. Therefore, drugs that inhibit the formation of
`lipoprotein particles in humans are expected to be useful for the treatment: of tumors
`that secrete proteins of the Hedgehog and Wnt families.
`
`In this regard, the Microsomal Triglyceride Transfer Protein (MTP} appears of
`particular interest. MTP is critical for the transfer of lipid to Apo~ipoproteinB and for
`the biosynthesls of LDL and VLDL. Compounds inhibiting MTP are known in the art
`and disclosed herein below. Further compounds can be identified in a straightforward
`manner using MTP assays described in the art. Wetterau et al. (1992} provide such
`an assay and a modified form has been published in Jamil et al. (1995}. A new,
`comparably simply assay is provided in Athar et al. (2004}. A further variant can be
`found in Gordon et al. {1996).
`
`The data reported herein suggest a novel role for Lipoprotein particles: the transport
`of lipid-modified proteins.
`Lipid modifications like those present in ·Wingless,
`Hedgehog and gpi-anchored proteins, which target proteins to the· exoplasmic
`membrane leaflet, would be of the correct length and topology to insert ·into the
`phospholipid monolayer of a Lipoprotein particle. The present inventors have shown
`. that Wingless, Hedgehog and gpi-anchored proteins co-purify anp co(cid:173)
`immunoprecipitate with Drosophila Lipophorin. Futhermore, Wingless and Hedghog
`co-localize extensively with Lipoprotein particles in tissue. Therefore, the ·particles
`previously termed "argosomesn are actually morphogen-bearing Lipoprotein particles.
`
`Given the data presented herein, the skilled person envisages the following further
`findings: (i) Reducing lipophorin abrogates Hedgehog and/or Wnt signalling; {ii) MTP
`activity is required for the biosynthesis of Drosophila lipophorin low-density particles;
`(iii) MTP is required for the release of Wingless and Hedgehog proteins on
`Lipoprotein particles; Qv) Drosophila MTP mutants are defective in Wingless and
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`Hedgehog signalling; (v) treatment with MTP inhibitors in mouse models reduces the
`growth of cancers, for example of the gastrointestinal tract and melanoma; and (vi)
`retrospective studies of patients treated for high cholesterol with MTP inhibitors show
`a protective effect for cancers, for example of the gastrointestinal• tract and
`melanoma.
`
`In supplementary experiments. the inventors have further supported their teaching
`that lipid-linked proteins of the exoplasmic face of the membrane asso~ate with
`Lipoproteins. These include many gpi-linked proteins with diverse functions, as well
`as the lipid-linked morphogens Wingless and Hedgehog. The mechanism allowing
`long-range dispersal of lipid-linked proteins is not yet. understood. The finding that
`these proteins exist in both membrane-associated and Lipoprotein-associated forms
`suggests reversible binding to Lipoprotein particle·s as a plausible mechanism for
`intercellular transfer, and the consequences of lowering lipoprotein levels in
`Drosophila larvae supports this idea.
`
`Lipophorin knock down narrows the range of both Wingless and Hedgehog
`signalling. Hedgehog accumulates to an abnormally high level in ·cells near the
`source of production and long-range signaling is inhibited; short-range target genes,
`however, are expressed normally. These data suggest that Hedgehog does not
`move as far when Lipophorin levels are low. The range over which Hedgehog moves
`is nonnally restricted by Patched-mediated endocytosis.
`In discs from Upophorin
`RNAi larvae, accumulated Hedgehog co-localizes with Patched in endosomes;
`suggesting that it is more efficiently sequestered by Patched.
`
`The disclosed data is consistent with the idea that Lipophorin is continuously needed
`for movement, rather than required only for the release of morphogens. If Lipophorin
`were important only for Hedgehog secretion, one would expect Lipophorin RNAi to
`decrease the amount of Hedgehog found in receiving tissue; this seems ndt to be the
`case. Furthermore, altered Hedgehog trafficking in receiving tissue is consistent with
`a model in which Lipophorin is required at each step of intercellular transfer. Without
`being bound by a theory, the inventors favour the idea that reversible association of
`Hedgehog with Lipophorin particles facilitates its transfer from the plasma membrane
`of one cell to that of the next. This model predicts that lowering Lipophorin levels
`should increase the length of time that Hedgehog spends in the plasma membrane
`before becoming associated with Lipophorin. This would slow its rate ot:transfer and
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`increase the probability of Patched endocytosing Hedgehog before it moved to the
`next cell. Hedgehog would then signal e~cientry in the short range, ·but be so
`efficiently sequestered by Patched that very little protein would travel far:enough to
`activate long-range target genes. These predictions are completely consistent with
`the disclosed observations. .
`
`This model differs significantly from the original concept of argosome function. It was
`initially speculated that argosomes were exosome-like particles with · an intact
`membrane bilayer,' and that lipid-linked morphogens needed to be assembled on
`these particles to be secreted by producing cells.
`Instead, the present inventi~n ·
`discloses that argosomes are exogenously derived lipoproteins that facilitate· that
`movement of.morphogens through the epithelium. Many questions remain as to how
`morphogens become associated with argosomes, and how the spread and cell(cid:173)
`interactions of these particles are regulated. Clearly, heparan sulfate proteoglycans
`are essential for the movement of Hedgehog and Wingless into receiving tissue 35
`36
`•
`•
`38
`Because heparan sulfate binds to vertebrate Lipoprotein particles 37
`, :one might
`•
`speculate that HSPG's facilitate morphogen movement through Lipoprotein binding.
`Conyersely, the inventors find many gpi-linked proteins, including the HSPG's Dally
`and Dally-like (unpublished data}, on Lipoprotein particles themselves. These
`associated proteins have the potential to modulate the cellular affinities or trafficking
`properties of Lipoprotein~ and the morphogens they carry.
`
`The disclosed data suggests that Lipophorin particles not only medlate intercellulat
`transfer of Hedgehog, but may also be endocytosed together with the morphogen.
`Interestingly, LDL receptor related proteins Arrow and Megalin have demonstrated
`roles in Wingless signaling and Hedgehog endocytosis, respectively 3M 1.:
`It is
`intriguing to speculate that these receptors might be important for interaction with the
`Lipoprotein-associated form of the morphogen.
`
`Cholesterol has the potential to modulate the activity of the HedgehoQ pathway at
`4244
`many different points 3•
`• Whether changes in the level of cellular cholesterol
`normally play a role in regulating the activity of the pathway is unclear. Here it is
`shown that Hedgehog interacts with the particle that delivers sterol to cells. This
`observation raises th.e possibility that internalization of Hedgehog is linke'd to sterol
`uptake, and suggests new mechanisms to link nutrition, growth and signalling during
`development. All known Hedgehog signal transduction pathways in vertebrates arid
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`invertebrate act by regulating the processing of GU-family transcription factors, and
`ectopic activation of GU proteins is a common feature of many tumors {Ruiz i Altaba,
`2002 #674}.
`Inhibiting the production of Lipophorin alters the processing of the
`drosophila GU protein Cubitus
`lnterruptus, causing the accumulation· of an
`unprocessed, inactive form of the protein (Figure 21 ). Thus, lowering Lipophorin
`levels in drosophila perturbs the Hedgehog signal transduction pathway through a
`protein that is conserved in humans, and whose activation is characteristic of a wide
`variety of tumors. The present invention therefore teaches to utilize drugs ~hat lower
`LDL levels in humans to treat tumors that secrete proteins of the Hedgehog and .Wnt
`families.
`
`Lowering Lipophorin levels also inhibits the growth of imaginal discs by mechanisms
`that do ~ot depend on morphogen signalling. The inventors have shown that sterol
`delivery itself is essential for growth; lowering Lipophorin levels, or removing sterol
`from the diet blocks growth primarily at the level of cell division. Like insect cells,
`· many human cell types do not. synthesize their own sterol but rely on 'LDL-mediated
`uptake. The existence of a sterol-dependent growth control checkpoint indicates· that
`LDL-lowering drugs should block the proliferation of tumors derived from such
`tissues. The inventors therefore propose to utilize drugs that lower LDL levels in
`humans to treat tumors derived from neurons and steroidogenic cells.
`
`In view of the above, all steps in the life cycle of lipoproteins are envisaged as targets
`for therapeutic intervention. The life cycle of lipoproteins · comprises formation,
`secretion, transport and association with a target cell. These steps are ·well
`understood and known to the skilled person. Accordingly, an alternative embodiment
`relates to the use of an inhibitor of lipoprotein secretion for the preparation of a
`pharmaceutical composition for the treatment of tumors. Another alternative
`embodiment relates to the use of an inhibitor of lipoprotein formation for: the
`preparation of a pharmaceutical composition for the treatment of tumors.
`
`Yet another alternative embodiment relates to the use of an inhibitor of iipoproteih
`transport for the preparation of a pharmaceutical composition for the treatment of
`tumors.
`
`A further embodiment relates to the use of an inhibitor of lipoprotein association:with
`a target cell for the preparation of a pharmaceutical composition for the treatment of
`tumors. Targets that affect interaction of Lipoproteins with target cells _are e.g. LDL
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`r~ceptors and LDL receptor family proteins, preferably LRPS and 6, and the LRP.
`Megalin which have been shown to be involved in wingless and hedgehog: signalling:
`Other targets affecting interaction of Upoproteins with target cells are Heparan
`sulfate proteoglycans
`
`A further embodiment relates to the use of an inhibitor of _the association of a protein
`of the Wnt or Hedgehog family with lipoproteins for the preparation of a
`pharmaceutical composition for the treabnent of tumors. This class of inhibitors
`.
`.
`prevent association of Wingless or Hedgehog with Lipoproteins include any inhibitors
`of the acyl-transferases that acylate Wingless and Hedgehog {these are called
`Porcupine and Skinny Hedgehog, respectively). Another inhibitor which is in
`accordance with the teaching of the present invention is the enzyme acyl protein
`thioesterase (APT-1), which will cleave the palmitate from the Wingless protein,
`preventing its association with Upoproteins (Willert et al., Nature 423, p 448).
`
`The Wnt pathway and the Hedgehog pathways are similar, evolutionary conserved
`signal transduction pathways playing a role, inter a/ia, in embryogenesis and
`tumorigenesis. Despite identity or similarity of several components of the' two
`pathways, th~ Wnt and Hedgehog protein, although located at correspondin:g
`positions of the two pathways, are unrelated proteins. The Hedgehog family of
`proteins includes Hedgehog (in Drosophila) and, in humans, Desert Hedgehog;
`Indian Hedgehog, and Sonic Hedgehog. The Wnt family of proteins appears to be
`larger, comprising the Drosphila proteins Wingless, DWnt2, DWnt3/5, · DWnt4,
`DWnt6, DWntB and DWnt10. There are more than a dozen vertebrate Wnt proteins,
`from Wnt1 to Wnt16, however, not strictly sequentially numbered. Vertebrate Wnt1 is
`the orthologue of Drosophila Wingless.
`
`In a preferred embodiment, said association of a protein of the Wnt or Hedgehog
`family with lipoproteins is inhibited by inhibiting the covalent lipid modifieation of said
`protein of the Wnt or Hedgehog family, thereby reducing its affinttY for said
`lipoprotein. The
`lipid modifications
`include palmitoylation and cholesterol
`modification. Hedgehog proteins are palmitoylated and cholesterol modified; Wnt
`proteins are palmitoylated. Any inhibitor of the lipid modification of Wnt or Hedgehog
`proteins is an inhibitor useful for the purpose of the present invention. Accordingly,
`use of such inhibitors for the preparation of the pharmaceutical . composition
`according to the invention is envisaged.
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`In a further preferred embodiment, said lipoprotein is very low density lipoprotein
`(VLDL} or low density lipoprotein (LDL}.
`
`Lipoprotein particles comprise a phospholipid monolayer surrounding .a core of
`esterified sterol and triglycerides, and are organized by different Apolipoproteins. In
`vertebrates, Lipoprotein particles of different densities and apolipoprotein
`composition transport lipid, sterols and fat-soluble. vitamins between the gut, liver a~d
`peripheral tissues.
`Insects have only one apolipoprotein, Apolipophoriri, which is
`most similar to vertebrate ApoB. Physiological studies indicate that it plays a rol~
`analogous to that of vertebrate Lipoproteins, transporting sterol and diglyceride
`between tissues.
`
`In a further preferred embodiment, said tumor is a malignant tumor. Malignant tumors
`are also referred to as cancers herein.
`
`In a further preferred embodiment, growth and/or progression of the tumor are
`caused by one or more protein of the Wnt or Hedgehog family. The person skilled in
`the art can identify which tumors fall under this definition without undue burden.
`
`In a further preferred embodiment, said tumor over-expresses one or more protein of
`the Wnt or Hedgehog family. The skilled person is aware of concrete tumors. and
`malignant diseases comprised in this definition. Furthennore, means and method,~
`are described below enabling the person skilled in the art to identify which tumors or
`malignant diseases are encompassed by the embodiment recited above.
`
`The tenn "over-expressionn denotes an expression level of an mRNA encoding a
`protein of the Wnt or Hedgehog family and/or of a protein of the Wnt or Hedgehog
`family, which is elevated in comparison to normal expression. The term "normal
`.expression" refers to a reference expressioi:i level determined in one or more
`samples from healthy individuals. These samples are preferably from healthy tissue
`corresponding to the tissue affected by the tumor under consideration. Samples may
`be drawn from a mixed population, from a fraction of the population, wherein the
`population has previously been stratified according to one or more parameters, or
`from healthy regions of the tissue affected by the tumor from the same patient.
`Statistical methods known in the art may be used in order to assign significance
`values and confidence intervals to the measured expression and over-expression
`data.
`
`In a more preferred embodiment, the expression level to be determined is the mRNA
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`expression level. Methods for the determination of mRNA expression levels are
`known in the art and comprise Real Time PCR, Northern blotting and hybridization on
`microarrays or DNA chips equipped with one or more probes or probe sets specific
`for transcripts encoding proteins of the Wnt or Hedgehog family.
`
`In another more preferred embodiment, the expression level to be determine:d is t~e
`protein expression level. The skilled person is aware of methods for the quantitation
`of proteins. Amounts of purified protein in solution can be determined by physical
`methods, e.g. photometry. Methods of quantifying a particular protein in a mixture
`rely on specific binding, e.g of antibodies. Specific detection and quanti~tion
`methods exploiting the specificity of antibodies comprise immunohistochemistry (in
`situ) and suif~ce · plasmon resonance. Western blotting combines separation of a
`mixture of proteins by electrophoresis and specific detection with antibodies.
`
`In a further preferred embodiment, said protein of the Wnt family is Wnt1, Wnt2,
`Wnt2B, Wnt3, Wnt3A, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt8B,
`Wn9A, Wnt98, Wnt10A, Wnt108, Wnt11 and/or Wnt16 and/or said protein of the
`Hedgehog family is Desert Hedeghog, Indian Hedgehog and/or Sonic Hedgehog. A
`list of Wnt · genes and pertinent information is maintained by Roel · Nusse at
`http://www.stanford.edu/-musse/wntwindow.html.
`
`In a further preferred embodiment; said tumor is selected from the group consisting
`.
`.
`.
`of esophageal tumor, biliary tract tumor, gasbic tumor, pancreatic furnor and
`malignant melanoma. For these tumors, Wnt and/or Hedgehog family proteins have
`been shown to play a causative role in tumor growth or progression.
`
`Yet a further preferred embodiment relates to a tumor selected from _the group
`consisting of gastric tumor, colorectal tumor, pancreatic tumor, esophageal tumor,_
`squamous cell carcinoma, ceivical tumor and malignant melanoma .. The~e tumors
`have been shown to over-express one or more members of the Wnt and/or
`Hedgehog families of proteins.
`
`In a further preferred embodiment, the tumor or cancer is a tumor or . cancer of
`muscle, cerebellum or breast.
`
`One class of drugs known to reduce Lipoprotein secretion acts by inhibiting the
`activity of Microsomal Triglyceride Transfer Protein (MTP). MTP is critical . for the
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`transfer of lipid to ApolipoproteinB and for the biosynthesis of LDL and VLDL. The
`Drosophila MTP homologue has a conserved enzymatic activity and can support tt~e
`secretion of ApoB-containing Lipoprotein. A spectrum of small mole.cuie MTP
`inhibitors is already u~ed in patients to treat elevated serum cholesterot MTP
`inh