GB2447017A - New use for inhibitors of glutaminyl peptide cyclotransferinase - Google Patents

New use for inhibitors of glutaminyl peptide cyclotransferinase Download PDF

Info

Publication number
GB2447017A
GB2447017A GB0704014A GB0704014A GB2447017A GB 2447017 A GB2447017 A GB 2447017A GB 0704014 A GB0704014 A GB 0704014A GB 0704014 A GB0704014 A GB 0704014A GB 2447017 A GB2447017 A GB 2447017A
Authority
GB
United Kingdom
Prior art keywords
disease
inhibitor
inhibitors
fibrosis
mcp
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
GB0704014A
Other versions
GB0704014D0 (en
Inventor
Stephan Schilling
Torsten Hoffmann
Holger Cynis
Hans-Ulrich Demuth
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Vivoryon Therapeutics AG
Original Assignee
Probiodrug AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Probiodrug AG filed Critical Probiodrug AG
Priority to GB0704014A priority Critical patent/GB2447017A/en
Publication of GB0704014D0 publication Critical patent/GB0704014D0/en
Publication of GB2447017A publication Critical patent/GB2447017A/en
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91045—Acyltransferases (2.3)
    • G01N2333/91074—Aminoacyltransferases (general) (2.3.2)
    • G01N2333/9108—Aminoacyltransferases (general) (2.3.2) with definite EC number (2.3.2.-)

Abstract

Use of an inhibitor of a glutaminyl peptide cyclotransferase in the preparation of a medicament for the treatment and/or prevention of a disease or disorder selected from <SL> <LI>a. neurodegenerative diseases, e.g. mild cognitive impairment (MCI), Alzheimer's disease, neurodegeneration in Down Syndrome, Familial British Dementia, Familial Danish Dementia, multiple sclerosis, <LI>b. chronic and acute inflammations, e.g. rheumatoid arthritis, atherosclerosis, restenosis, pancreatitis, <LI>c. fibrosis, e.g. lung fibrosis, liver fibrosis, renal fibrosis, <LI>d. cancer, e.g. cancer/hemangioendothelioma proliferation, gastric carcinomas, <LI>e. metabolic diseases, e.g. hypertension, <LI>f. and other inflammatory diseases, e.g. neuropathic pain, graft rejection/graft failure/graft vasculopathy, HIV infections/AIDS, gestosis, tuberous sclerosis. </SL> Further, the invention relates to a pharmaceutical composition comprising such compounds as well as a diagnostic method, assay and kit. The above use may also pertain to combinations with other biologically active compounds.

Description

* 2447017
NEW USE OF GLUTANINYL CYCLIASE INHIBITORS
The present invention relates in general to an inhibitor of a glutaminyl peptide cyclotransferase, and use thereof for the treatment and/or prevention of a disease or disorder selected from the group consisting of rheumatoid arthritis, atherosclerosis, restenosis, lung fibrosis, liver fibrosis, renal fibrosis, pancreatitis, mild cognitive impairment, Alzheimer's disease, neurodegeneration in Down Syndrome, Familial British Dementia, Familial Danish Dementia, neuropathic pain, graft rejection/graft failure/graft vasculopathy, hypertension, HIV infections/AIDS, gestosis, cancer/hemangioendothelioma proliferation, taberous sclerosis, and gastric carcinomas.
Further, the present invention pertains to diagnostic kits and methods based on the use of a glutaminyl cyclase inhibitor.
Glutaminyl cyclase (QC, EC 2.3.2.5) catalyzes the intramolecular cyclization of N-terminal glutaminyl residues into pyroglutamic acid (5-oxo-proline, pGlu*) under liberation of ammonia and the intramolecular cyclization of N-terminal glutamyl residues into pyroglutamic acid under liberation of water.
-A QC was first isolated by Messer from the Latex of the tropical plant Carica papaya in 1963 (Messer, M. 1963 Nature 4874, 1299) . 24 years later, a corresponding enzymatic activity was discovered in animal pituitary (Busby, W. H. J. et al. 1987 J Biol Chem 262, 8532-8536; Fischer, W. H. and Spiess, 3.
1987 Proc Natl Acad Sci U S A 84, 3628-3632). For the mammalian QC5, the conversion of Gin into pGlu by QC could be shown f or the precursors of TRH and GnRH (Busby, W. H. J. et al. 1987 J Biol Chern 262, 8532- 8536; Fischer, W. H. and Spiess, J. 1987 Proc Nati Acad Sci U S A 84, 3628-3632) . In addition, initial localization experiments of QC revealed a co-localization with its putative products of catalysis in the bovine tractus hypothalamo-hypophysalis a QC inhibitor, in particular QCI in combination with interferones, preferably Aronex, for the prevention and/or treatment of multiple sclerosis, further improving the suggested function in peptide hormone maturation (Bockers, T. M. et al. 1995 J Neuroenclocrinol 7, 445-453). In contrast, the physiological function of the plant QC is less clear. In case of the enzyme from C. papaya, a role in the plant defence against pathogenic microorganisms was suggested (El Moussaoui, A. et al. 2001 Cell Mol Life Sci 58, 556-570). Putative QCs from other plants were identified by sequence comparisons recently (Dahi, S. W. et al. 2000 Protein Expr Purif 20, 27-36). The physiological function of these enzymes, however, is still ambiguous.
The QCs known from plants and animals show a strict specificity for L-Glutamine in the N-terminal position of the substrates and their kinetic behaviour was found to obey the Michaelis-Menten equation (Pohi, T. et al. 1991 Proc Natl Acad Sci U S A 88, 10059-10063; Consalvo, A. P. et al. 1988 Anal Biochem 175, 131-138; Gololobov, M. Y. et al. 1996 Biol Chern Hoppe Seyler 377, 395-398). A comparison of the primary structures of the QCs from C. papaya and that of the highly conserved QC from mammals, however, did not reveal any sequence homology (Dahl, S. W. et al. (2000) Protein Expr Purif 20, 27-36). Whereas the plant QCs appear to belong to a new enzyme family (Dahi, S. W. et al. (2000) Protein Expr Purif 20, 27-36), the mammalian QCs were found to have a pronounced sequence homology to bacterial aminopeptidases (Bateman, R. C. et al. 200]. Biochemistry 40, 11246-11250), leading to the conclusion that the QCS from plants and animals have different evolutionary origins.
EP 02 011 349.4 discloses polynucleotides encoding insect glutaminyl cyclase, as well as polypeptides encoded thereby.
This application further provides host cells comprising expression vectors comprising polynucleotides of the invention. Isolated polypeptides and host cells comprising insect QC are useful in methods of screening for agents that reduce glutaminyl cyclase activity. Such agents are described as useful as pesticides.
Chemotactic cytokines (chemokines) are proteins that attract and activate leukocytes and are thought to play a fundamental role in inflammation. Chemokines are divided into four groups categorized by the appearance of N-terminal cysteine residues ("C"-; "CC"-; "CXC"-and "CX3C"-chemokjnes). "CXC"-chemokines preferentially act on neutrophils. In contrast, "CC"-chemokines attract preferentially monocytes to sites of inflammation. Monocyte infiltration is considered to be a key event in a number of disease conditions (Gerard, C. and Rollins, B. J. (2001) Nat.Immunol 2, 108-115; Bhatia, M., et al.., (2005) Pancreatology. 5, 132-144; Kitamoto, S., Egashira, K., and Takeshita, A. (2003) J Pharmacol Sci. 91, 192-196). The MCP family, as one family of chemokines, consists of four members (MCP-1-4), displaying a preference for attracting monocytes but showing differences in their potential (Luini, W., et al., (1994) Cytokine 6, 28-31; Uguccioni, M., et al., (1995) Eur J Immunol 25, 64-68). In the following both cDNA as well as amino acid sequences of MCP-l-4 are indicated:
S
Human MCP-1 (CCL2) (GeneBank Accession: M24545) cDNA (300 bp) SEQ ID NO: 2 3. atgaaagtct ctgccgccct tctgtgcctg ctgctcatag cagccacctt cattccccaa 61 gggctcgctc agccagatgc aatcaatgcc ccagtcacct gctgttataa cttcaccaat 121 aggaagatct cagtgcagag gctcgcgagc tatagaagaa tcaccagcag caagtgtccc 181 aaaaagctg tgatcttcaa gaccattgtg gccaaggaga tctgtgctga ccccaagcag 241 aagtgggttc aggattccat ggaccacctg gacaagcaaa cccaaactcc gaagacttga Protein (Signal Sequence in bold: 23 aa; Mature MCP-1: 76 aa) SEQ ID NO: I
MKVSAALLCLLUAATFIPQGLAQPDAI NAPVTCCYN FTN RKISVQRLASYRRITSSKCP
KEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKT
Human MCP-2 (CCL8) (GeneBank Accession: Y10802) cDNA (300 bp) SEQ ID NO: 12 1 atgaaggttt ctgcagcgct tcttgcctg ctgctcatgg cagccacttt cagccctcag 61 ggacttgctc agccagattc agtttccatt ccaatcacct gctgctttaa cgtgatcaat 3.21 aggaaaattc ctatccagag gctggagagc tacacaagaa tcaccaacat ccaatgtccc 181 aaggaagctg tgatcttcaa gacccaacgg ggcaaggagg tctgtgctga ccccaaggag 241 agatgggtca gggattccat gaagcatctg gaccaaatat ttcaaaatct gaagccatga Protein (Signal Sequence in bold: 23 aa; Mature MCP-2: 76 aa) SEQ ID NO: 11
MKVSAALLCLLLMAATFSPQGLAQPDSVSI PITCCFNVI N RKI PIQRLESYTRITN IQCP
KEAVI FKTQRGKE VCADPKER WVRDSMKHLDQI FQNLKP
Human MCP-3 (CCL7) (GeneBank Accession: X71087) cDNA (300 bp) SEQ ID NO: 14 1 atgaaagcct ctgcagcact tctgtgtct ctgctcacag cagctgcttt cagcccccag 61 gggcttgctc agccagttgg gattaatact tcaactacct gctgctacag atttatcaat 121 aagaaaatcc ctaagcagag gctggagagc tacagaagga ccaccagtag ccactgtccc 181 cgggaagctg taatcttcaa gaccaaactg gacaaggaga tctgtgctga ccccacacag 241 aagtgggtcc aggactttat gaagcacctg gacaagaaaa cccaaactcc aaagctttga Protein (Signal Sequence in bold: 23 aa; Mature MCP-3: 76 aa) SEQ ID NO: 13 MKASAALLCLLLTAAAFSPQGLAQPVGINTSTTCCYRFINKKIPKQRLESYRRrrSSHCP
REAVIFKTKLDKEICADPTQKWVQDFMKHLDKKTQTPKL
Human MCP-4 (CCLI 3) (GeneBank Accession: U46767) cDNA (297 bp) SEQ ID NO: 16 1 atgaaagtct ctgcagtgct tctgtgcctg ctgctcatga cagcagcttt caacccccag 61 ggacttgctc agccagatgc actcaacgtc ccatctactt gctgcttcac atttagcagt 121 aagaagatct ccttgcagag gctgaagagc tatgtgatca ccaccagcag gtgtccccag 181 aaggctgtca tcttcagaac caaactgggc aaggagatct gtgctgaccc aaaggagaag 241 tgggtccaga attatatgaa acacctggc cggaaagctc acaccctgaa gacttga Protein (Signal Sequence in bold: 23 aa; Mature MCP-4: 75 aa) SEQ ID NO: 15
MKVSAVLLCLLLMTAAFNPQGLAQPDALNVPSTCCFTFSSKKISLQRLKSYVIUSRCPQ
KAVIFRTKLGKEICADPKEKWVQNYMKHLGRKAHTUçr A number of studies have underlined in particular the crucial role of MCP-l for the development of atherosclerosis (Gu, L., et al., (1998) Mol.Cell 2, 275-281; Gosling, J., et al., (1999) J Clin.Invest 103, 773-778); rheumatoid arthritis (Gong, J. H., et al., (1997) J Exp.Med 186, 131-137; Ogata, I-I., et al., (1997) J Pathol. 182, 106-114); pancreatitis (Bhatia, M., et al., (2005) Am.J Physiol Gastrointest.Liver Physiol 288, GJ.259-G1265); Alzheimer's disease (Yamamoto, M., et al., (2005) Am.J Pathol. 166, 1475-1485); lung fibrosis (Inoshima, I., et al., (2004) Am.J Physiol Lung Cell Mol.Physiol 286, L1038-L1044) ; renal fibrosis (Wada, T., et al., (2004) J Am.Soc.Nephrol. 15, 940-948), and graft rejection (Saiura, A., et al., (2004) Arterioscier. Thromb.
Vasc. Biol. 24, 1886-1890). Furthermore, MCP-l might also play a role in gestosis (Katabuchi, I-I., et al., (2003) Med Electron Microsc. 36, 253-262), as a paracrine factor in tumor development (Ohta, M., et al., (2003) Int.J Oncol. 22, 773-778; Li, S., et al., (2005) J Exp.Med 202, 617-624), neuropathic pain (White, F. A., et al., (2005) Proc. Nati.
Acad.Sci.U.S.A) and AIDS (Park, I. W., Wang, J. F., and Groopman, J. E. (2001) Blood 97, 352-358; Coil, B., et al., (2006) Cytokine 34, 51-55) t The mature form of human and rodent MCP-l is posttranslationally modified by Glutaminyl Cyclase (QC) to possess an N-terminal pyroglutarnyl (pGlu) residue. The N-terminal pGlu modification makes the protein resistant against N-terminal degradation by aminopeptidases, which is of importance, since chemotactic potency of MCP-1 is mediated by its N-terminus (Van Damme, J.,, et al., (1999) Chem Immunol 72, 42-56). Artificial elongation or degradation leads to a loss of function although MCP-1 still binds to its receptor (CCR2) (Proost, P., et al., (1998), J Immunol 160, 4034-4041; Zhang, Y. J., et al., 1994, J Biol.Chern 269, 15918-15924; Masure, S., et al., 1995, J Interferon Cytokine Res. 15, 955- 963; Hemmerich, S., et al., (1999) Biochemistry 38, 13013-13025) Due to the major role of MCP-1 in a number of disease conditions, an anti-MCP--l strategy is required. Therefore, small orally available compounds inhibiting the action of MCP-1 are promising candidates for a drug development.
Inhibitors of Glutaminyl Cyclase are small orally available compounds, which target the important step of pGlu-forTnation at the N-terminus of MCP-l (Cynis, H., et al., (2006) Biochirn.Biophys.Acta 1764, 1618-1625; Buchholz, M., et al., (2006) J Med Chern 49, 664-677). In consequence, caused by QC-inhibition, the N-terminus of MCP-l is not protected by a pGlu-residue. Instead, the N-terminus possesses a glutamine-proline motif, which is prone to cleavage by by dipeptidylpeptidases, e.g. dipeptidylpeptidase 4 and fibroblast activating protein (FAP, Seprase), which are abundant on the endothelium and within the blood circulation.
This cleavage results to the formation of N-terminal truncated MCP-l. These molecules unfold, in turn, an antagonistic action at the CCR2 and therefore, rnonocyte-related disease conditions are inhibited efficiently.
Atherosclerotic lesions, which limit or obstruct coronary blood flow, are the major cause of ischemic heart disease related mortality, resulting in 500,000-600,000 deaths annually. Percutaneous transluminal coronary angioplasty (PTCA) to open the obstructed artery was performed in over 550,000 patients in the TJ. S. and 945, 000+ patients worldwide in 1996 (Lemaitre et al., 1996) . A major limitation of this technique is the problem of post-PTCA closure of the vessel, both immediately after PTCA (acute occlusion) and in the long term (restenosis): 30% of patients with subtotal lesions and 50%-of patients with chronic total lesions will go on to restenosis after angioplasty. Additionally, restenosis is a significant problem in patients undergoing saphenous vein bypass graft. The mechanism of acute occlusion appears to involve several factors and may result from vascular recoil with resultant closure of the artery and/or deposition of blood platelets along the damaged length of the newly opened blood vessel followed by formation of a fibrin/red blood cell thrombus.
Restenosis after angioplasty is a more gradual process and involves initial formation of a subcritical thrombosis with release from adherent platelets of cell derived growth factors with subsequent proliferation of intimal smooth muscle cells and local infiltration of inflammatory cells contributing to vascular hyperplasia. It is important to note that multiple processes, among those thrombosis, cell proliferation, cell migration and inflammation each seem to contribute to the restenotic process.
In the U. S., a 30-50% restenosis rate translates to 120,000- 200,000 U. S. patients at risk from restenosis. If only 80%
I
of such patients elect repeat angioplasty (with the remaining 201 electing coronary artery bypass graft) and this is added to the cost of coronary artery bypass graft for the remaining 201, the total cost for restenosis easily reaches into billions of dollars. Thus, successful prevention of restenosis could result not only in significant therapeutic benefit but also in significant health care savings.
Monocyte chemoattractant protein 1 (MCP-l, CCL2) belongs to a family of potent chemotactic cytokines (CC chemokines), that regulate the trafficking of leukocytes, especially monocytes, macrophages and T-cells, to Sites of inflammation (Charo,I.F.
and Taubman,M.B. (2004) Circ.Res. 95, 858-866). Besides its role in, e.g. vascular disease, compelling evidence points to a role of MCP-]. in Alzheimer's disease (AD) (Xia,M.Q. and Hyman,B.T. (1999) J Neurovirol. 5, 32-41). The presence of MCP-l in senile plaques and in reactive microglia, the residential macrophages of the CNS, has been observed in brains of patients suffering from AD (Ishizuka,K., et al., (1997) Psychiatry Clin.Neurosci. 51, 135-138. Stimulation of monocytes and microglia with Arnyloid-f3 protein (Af3) induces chemokine secretion in vitro (Meda,L., et al., (1996) J Immunol 157, 1213-1218; Szczepanik,A.M., et al., (2001) J Neuroimmunol. 113, 49-62) and intracerebroventrjcular infusion of Af3(142) into murine hippocampus significantly increases MCP- l in vivo. Moreover, A13 deposits attract and activate microglial cells and force them to produce inflammatory mediators such as MCP-l, which in turn leads to a feed back to induce further chemotaxis, activation and tissue damage. At the site of AJ3 deposition, activated microglia also phagocytose A3 peptides leading to an amplified activation (Rogers,J. and Lue,L.F. (2001) Neurochem.Int. 39, 333-340).
Examination of chemokine expression in the 3xTg mouse model for AD revealed that neuronal inflammation precedes plaque formation and MP-l is upregulated by a factor of 11.
Furthermore, the upregulation of MCP-1 seems to correlate with the occurrence of first intracellular A deposits (Janelsins,M.C., et al., (2005) J Neuroinflammation. 2, 23).
Cross-breeding of the Tg2575 mouse model for AD with a MCP-l overexpressing mouse model has shown an increased microglia accumulation around A deposits and that this accumulation was accompanied by increased amount of diffuse plaques compared to single-transgenic Tg2576 littermates (Yamamoto,M., et al. (2005) Am.J Pathol. 166, 1475-1485).
MCP-l levels are increased in CSF of AD patients and patients showing mild cognitive impairment (MCI) (Galimberti,D., et al.., (2006) Arch.Neurol. 63, 538-543). Furthermore, MCP-l shows an increased level in serum of patients with MCI and early AD (Clerici,F., et al., (2006) Neurobiol.Aging 27, 1763-1768)
Summary of the invention
The present invention relates to an inhibitor of a glutaminyl peptide cyclotransferase and use thereof for the treatment and/or prevention of a disease or disorder selected from the group consisting of inflammatory diseases selected from a. neurodegenerative diseases, e.g. mild cognitive impairment (MCI), Alzheimer's disease, neurodegeneration in Down Syndrome, Familial British Dementia, Familial Danish Dementia, multiple sclerosis, b. chronic and acute inflammations, e.g. rheumatoid arthritis, atherosclerosis, restenosis, pancreatitis, c. fibrosis, e.g. lung fibrosis, liver fibrosis, renal fibrosis, d. cancer, e.g. cancer/hemangioendothelioma proliferation, gastric carcinomas, e. metabolic diseases, e.g. hypertension, f. and other inflammatory diseases, e.g. neuropathic pain, graft rejection/graft failure/graft vasculopathy, HIV infections/AIDS, gestosis, , tuberous sclerosis.
In particular the present invention pertains to the following items: 1. Inhibitor of a glutaminyl peptide cyclotransferase for the treatment and/or prevention of a disease or condition, selected from the group of inflammatory diseases selected from
S
a. neurodegenerative diseases, e.g. mild cognitive impairment (MCI), Alzheimer's disease, neurodegeneration in Down Syndrome, Familial British Dementia, Familial Danish Dementia, multiple sclerosis, b. chronic and acute inflammations, e.g. rheumatoid arthritis, atherosclerosis, restenosis, pancreatitis, c. fibrosis, e.g. lung fibrosis, liver fibrosis, renal fibrosis, d. cancer, e.g. cancer/hemangioendothelioma proliferation, gastric carcinomas, e. metabolic diseases, e.g. hypertension, f. and other inflammatory diseases, e.g. neuropathic pain, graft rejection/graft failure/graft vasculopathy, HIV infections/AIDS, gestosis, tuberous sclerosis.
2. Inhibitor according to item 1 which is l-(3-(1H-imidazol-l-yl) propyl) -3-(3, 4-dimethoxyphenyl) thiourea hydrochloride.
3. Inhibitor according to item 1 or 2 above, wherein the disease is a neurodegenerative disease, e.g. mild cognitive impairment (MCI), Alzheimer's disease, rieurodegeneration in Dowii Syndrome, Familial British Dementia, Familial Danish Dementia, multiple sclerosis.
4. Inhibitor according to any of items 1 to 3 above, wherein the inhibitor is administered in combination with a further agent, selected from the group consisting of nootropic agents, neuroprotectants, antiparkinsonian drugs, amyloid protein deposition inhibitors, beta amyloid synthesis inhibitors, antidepressants, an.xiolytic drugs, antipsychotic drugs and anti-multiple sclerosis drugs.
I
5. Use of a glutaminyl peptide cyclotransferase inhibitor for the treatment and/or prevention of a disease or condition selected from the group of inflammatory diseases selected from a. neurodegenerative diseases, e.g. mild cognitive impairment (MCI), Alzheimer's disease, neurodegeneration in Down Syndrome, Familial British Dementia, Familial Danish Dementia, multiple sclerosis, b. chronic and acute inflammations, e.g. rheumatoid arthritis, atherosclerosis, restenosis, pancreatitis, C. fibrosis, e.g. lung fibrosis, liver fibrosis, renal fibrosis, d. cancer, e.g. cancer/hemangioendothelioma proliferation, gastric carcinomas, e. metabolic diseases, e.g. hypertension, f. and other inflammatory diseases, e.g. neuropathic pain, graft rejection/graft failure/graft vasculopathy, HIV infections/AIDS, gestosis, tuberous sclerosis.
6. Use according to item 5, wherein said inhibitor is l-(3-(1H-imidazol-1-yl)propyl) -3-(3, 4-dimethoxyphenyl) thiourea hydrochloride.
7. Use according to item 5 or 6, wherein the disease is a neurodegenerative disease, e.g. mild cognitive impairment (MCI), Alzheimer's disease, neurodegeneratiori in Down Syndrome, Familial British Dementia, Familial Danish Dementia, multiple sclerosis.
S
8. Use according to any of items 5 to 7, wherein the inhibitor is administered in combination with a further agent, selected from the group consisting of nootropic agents, neuroprotectants, antiparkinsonian drugs, amyloid protein deposition inhibitors, beta amyloid synthesis inhibitors, antidepressants, anxiolytic drugs, antipsychotic drugs and anti-multiple sclerosis drugs.
9. Use of a glutaminyl peptide cyclotransferase inhibitor for the preparation of a medicament for treating and/or preventing a disease or conditions selected from the group of inflammatory diseases selected from a. neurodegenerative diseases, e.g. mild cognitive impairment (MCI), Alzheimer's disease, neurodegeneration in Down Syndrome, Familial British Dementia, Familial Danish Dementia, multiple sclerosis, b. chronic and acute inflammations, e.g. rheumatoid arthritis, atherosclerosis, restenosis, pancreatitis, c. fibrosis, e.g. lung fibrosis, liver fibrosis, renal fibrosis, d. cancer, e.g. cancer/hemangioendothelioma proliferation, gastric carcinomas, e. metabolic diseases, e.g. hypertension, f. and other inflammatory diseases, e.g. neuropathic pain, graft rejection/graft failure/graft vasculopathy, HIV infections/AIDS, gestosis, tuberous sclerosis.
10. Use according to item 9, wherein said inhibitor is l-(3-(lH-imidazol-1-yl)propyl) -3-(3, 4-dimethoxyphenyl) thiourea hydrochloride.
I
11. Use according to item 9 or 10, wherein the disease is a neurodegenerative disease, e.g. mild cognitive impairment (MCI), Alzheimer's disease, neurodegeneration in Down Syndrome, Familial British Dementia, Familial Danish Dementia, multiple sclerosis.
12. Use according to any of items 9 to 11, wherein the inhibitor is administered in combination with a further agent, selected from the group consisting of nootropic agents, neuroprotectants, antiparkinsonian drugs, amyloid protein deposition inhibitors, beta amyloid synthesis inhibitors, antidepressants, anxiolytic drugs, antipsychotic drugs and anti-multiple sclerosis drugs.
13. Method of treatment and/or prevention of a disease or condition, selected from the group of inflammatory diseases selected from a. neurodegenerative diseases, e.g. mild cognitive impairment (MCI), Alzheimer's disease, neurodegeneration in Down Syndrome, Familial British Dementia, Familial Danish Dementia, multiple sclerosis, b. chronic arid acute inflammations, e.g. rheumatoid arthritis, 1 restenosis, pancreatitis, c. fibrosis, e.g. lung fibrosis, liver fibrosis, renal fibrosis, d. cancer, e.g. cancer I hemangioendothelioma proliferation, gastric carcinomas, e. metabolic diseases, e.g. hypertension, f. and other inflammatory diseases, e.g. neuropathic pain, graft rejection/graft failure/graft vasculopathy, HIV infections/AIDS, gestosis, tuberous sclerosis, wherein an effective amount of a QC inhibitor is administered.
S
14. Method of treatment and/or prevention according to item 13, wherein said inhibitor is l-(3-(1H-imidazol-1-yl)propyl)- 3-(3,4 -dimethoxyphenyl) thiourea hydrochloride.
15. Method of treatment and/or prevention according to any of items 13 or 14, wherein the disease is a neurodegenerative disease, e.g. mild cognitive impairment (MCI), Alzheimer's disease, neurodegenerati.on in Down Syndrome, Familial British Dementia, Familial Danish Dementia, multiple sclerosis.
16. Method of treatment and/or prevention according to any of items 13 to 15, wherein the inhibitor is administered in combination with a further agent, selected from the group consisting of nootropic agents, neuroprotectants, antiparkinsonian drugs, amyloid protein deposition inhibitors, beta amyloid synthesis inhibitors, antidepressants, anxiolytic drugs, antipsychotic drugs and anti-multiple sclerosis drugs.
17. Use according to any of items 5 to 12 above, wherein the disease and/or condition afflict a human being.
18. The method of any of items 13 to 16, wherein the disease and/or condition afflicts a human being.
19. Diagnostic assay, comprising an inhibitor of a glutaminyl peptide cyclotransf erase.
20. Diagnostic assay according to item 19, wherein said inhibitor is 1-(3-(1H-imidazole-l-yl)propyl)-3_(3,4_ dimethoxy-phenyl) thiourea hydrochloride.
21. Method of diagnosing any one of the diseases and/or conditions as defined in item 1 above, comprising the steps of - collecting a sample from a subject who is suspected to be afflicted with said disease and/or condition, -contacting said sample with an inhibitor of a glutaminyl peptide cyclotransferase, and -determining whether or not said subject is afflicted by said disease and/or condition.
22. The method of item 21, wherein said subject is a human being.
23. The method of item 21 or 22, wherein said inhibitor is 1-(3-(lH-imidazole-1-yl)propyl) -3-(3,4-dimethoxy-pheny1) thiourea hydrochloride.
24. The method of any of items 21 to 23, wherein said sample is a blood sample, a serum sample, a sample of cerebrospinal liquor or a urine sample.
25. Diagnostic kit for carrying out the method of items 21 to 24 comprising as detection means the diagnostic assay of item 19 or 20 and a determination means.
26. Pharmaceutical composition, comprising the inhibitor according to any of items 1 to 4.
Definitions Enzyme inhibitors, in particular inhibitors of QC Reversible enzyme inhibitors: comprise competitive inhibitors, non-competitive reversible inhibitors, slow-binding or tight-binding inhibitors, transition state analogues and multisubstrate analogues.
Competitive inhibitors show 1) non-covalent interactions with the enzyme, ii) compete with substrate for the enzyme active site, The principal mechanism of action of a reversible enzyme inhibitor and the definition of the dissociation constant can be visualized as follows: k0 E + I -E-I k0ff + ô ______ ______ E-S -E-P -E + p k KD=Kr on The formation of the enzyme-inhibitor EE-I] complex prevents binding of substrates, therefore the reaction cannot proceed to the normal physiological product, P. A larger inhibitor concentration [I] leads to larger [E-I], leaving less free enzyme to which the substrate can bind.
Non-competitive reversible inhibitors i) bind at a site other than active site (allosteric binding site) ii) cause a conformational change in the enzyme which decreases or stops catalytic activity.
Slow-binding or tight-binding inhibitors i) are competitive inhibitors where the equilibrium between inhibitor and enzyme is reached slowly, ii) (kon is slow), possibly due to conformational changes that must occur in the enzyme or inhibitor a) are often transition state analogues b) are effective at concentrations similar to the enzyme concentration (subnanornolar RD values) c) due to koff values being so low these types of inhibitors are "almost" irreversible.
Transition state analogues are competitive inhibitors which mimic the transition state of an enzyme catalyzed reaction. Enzyme catalysis occurs due to a lowering of the energy of the transition state, therefore, transition state binding is favored over substrate binding.
Multisubstrate analogues For a reaction involving two or more substrates, a competitive inhibitor or transition state analogue can be designed which contains structural characteristics resembling two or more of the substrates.
Irreversible enzyme inhibitors: drive the equilibrium between the unbound enzyme and inhibitor and enzyme inhibitor complex (E + I <---> E-I) all the way to the E-I-side with a covalent bond (-100 kcal/mole), making the inhibition irreversible.
Affinity labeling agents * Active-site directed irreversible inhibitors (competitive irreversible inhibitor) are recognized by the enzyme (reversible, specific binding) followed by covalent bond formation, and i) are structurally similar to substrate, transition state or product allowing for specific interaction between drug and target enzyme, ii) contain reactive functional group (e.g. a nucleophile, -COCH2Br) allowing for covalent bond formation.
The reaction scheme below describes an active-site directed reagent with its target enzyme where KD is the dissociation constant and k. is the rate of covalent bond 3flactivatjori formation.
E + I < > E * j > E I * Mechanism-based enzyme inactivators (also called suicide inhibitors) are active-site directed reagents (unreactive) which binds to the enzyme active site where it is transformed to a reactive form (activated) by the enzyme's catalytic capabilities. Once activated, a covalent bond between the inhibitor and the enzyme is formed.
The reaction scheme below shows the mechanism of action of a mechanism based enzyme inactivator, where is the dissociation complex, k2 is the rate of activation of the inhibitor once bound to the enzyme, k3 is the rate of dissociation of the activated inhibitor, P, from the enzyme (product can still be reactive) from the enzyme and k4 is the rate of covalent bond formation between the activated inhibitor and the enzyme.
KD k2 ________ E + I -E.I El E-l k3 E P Inactivation (covalent bond formation, k4) must occur prior to dissociation (k3) otherwise the now reactive inhibitor is 2]. released into the environment. The partition ratio, k3/k4: ratio of
released product to inactivation should be minimized for efficient inactivation of the system and minimal undesirable side reactions.
A large partition ratio (favors dissocation) leads to nonspecific reactions.
Uncompetitive enzyme inhibitors: As a definition of uncompetitive inhibitor (an inhibitor which binds only to ES complexes) the following equilibria equation can be assumed: Ks k2 E+S -ES WE+P
ESI
The ES complex dissociates the subtrate with a dissociation constant equal to KS, whereas the ESI complex does not dissociate it (i.e has a KS value equal to zero). The Km's of Michaslis-Menten type enzymes are expected to be reduced.
Increasing substrate concentration leads to increasing ESI concentration (a complex incapable of progressing to reaction products) therefore the inhibition cannot be removed.
Preferred according to the present invention are competitive enzyme inhibitors.
Most preferred are competitive reversible enzyme inhibitors.
The terms "k" or "K1" and "KD" are binding constants, which describe the binding of an inhibitor to and the subsequent release from an enzyme. Another measure is the "IC50t' value, which reflects the inhibitor concentration, which at a given substrate concentration results in 50 enzyme activity.
QC
The term "OC" as used herein comprises glutaminyl cyclase (QC) and QC-like enzymes. QC and QC-like enzymes have identical or similar enzymatic activity, further defined as QC activity. In this regard, QC-like enzymes can fundamentally differ in their molecular structure from QC.
The term "QC activity" as used herein is defined as intramolecular cyclization of N-terminal glutaminyl residues into pyroglutamic acid (pGlu*) or of N-terminal L- homoglutaminyl or L-beta-homoglutamjny]. to a cyclic pyro-homoglutamine derivative under liberation of ammonia. See schemes 1 and 2 in this regard.
Scheme 1: Cyclization of gluta.mine by QC peptide peide
NH I O2
Scheme 2: Cyclization of L-homoglutamine by QC peptide I peide
NH H2 a NH2
The term "EC" as used herein comprises the side activity of QC and QC-like enzymes as glutamate cyclase (EC), further defined as EC activity.
The term "EC activity" as used herein is defined as intramolecular cyclization of N-terminal glutamyl residues into pyroglutamic acid (pGlu*) by QC. See scheme 3 in this regard.
Scheme 3: N-terminal cyclization of uncharged glutarnyl peptides by QC (EC) peptide peptide peptide peptide o =eNH0:;:::oZNc,E; HOG QC The term "QC-inhibitor" "glutaminyl cyclase inhibitor" is generally known to a person skilled in the art and means
S
enzyme inhibitors as generally defined above, which inhibit the catalytic activity of glutaminyl cyclase (QC) or its glutamyl cyclase (EC) activity.
Potency of QC inhibition In light of the correlation with QC inhibition, in preferred embodiments, the subject method and medical use utilize an agent with a Ki for QC inhibition of 10 M or less, more preferably of 1 /LM or less, even more preferably of 0.1 M or less or 0. 01 jM or less, or most preferably 0.001 /LM or less.
Indeed, inhibitors with Ki values in the lower micromolar, preferably the nanomolar and even more preferably the picomolar range are contemplated. Thus, while the active agents are described herein, for convenience, as "QC inhibitors", it will be understood that such nomenclature is not intended to limit the subject matter of the invention in any way.
Molecular weight of QC inhibitors In general, the QC inhibitors of the subject method or medical use will be small molecules, e.g., with molecular weights of 1000 g/mole or less, 500 g/mole or less, preferably of 400 g/mole or less, and even more preferably of 350 g/mole or less and even of 300 g/mole or less.
The term "subject" as used herein, refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment and/or is suspected of being afflicted with a disease and/or condition as defined in the claims.
The term "therapeutically effective amount" as used herein, means that amount of an active compound or a pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human being sought by a researcher, veterinarian, medical doctor or other clinician, which
S
includes alleviation of the symptoms of the disease or disorder being treated.
As used herein, the term "pharmaceutically acceptable" embraces both human and veterinary use: for example the term "pharmaceutically acceptable" embraces a veterinary acceptable compound or a compound acceptable in human medicine and health care.
Pharmaceutically acceptable salts: In view of the close relationship between the free compounds and the compounds in the form of their salts or solvates, whenever a compound or inhibitor, respectively, is referred to in this context, a corresponding salt or solvate is also intended, provided such is possible or appropriate under the circumstances.
Salts and solvates of the inhibitors of the present invention and physiologically functional derivatives thereof which are suitable for use in medicine are those wherein the counter-ion or associated solvent is pharmaceutically acceptable.
However, salts and solvates having non-pharmaceutically acceptable counter-ions or associated solvents are within the scope of the present invention, for example, for use as intermediates in the preparation of other compounds and their pharmaceutically acceptable salts and solvates.
Suitable salts according to the invention include those formed with both organic and inorganic acids or bases.
Pharmaceutically acceptable acid addition salts include those formed from hydrochloric, hydrobrornic, sulphuric, nitric, citric, tartaric, phosphoric, lactic, pyruvic, acetic, trifluoroacetic, triphenylacetic, suiphamic, sulphanilic, succinjc, oxalic, fumaric, maleic, malic, rnandelic, glutamic, aspartic, oxaloacetic, methanesuiphonic, ethanesuiphonic, arylsuiphonic (for example p-toluenesulphonic, benzenesulphonic, naphthalenesulphonic or naphthalene-disuiphonic), salicylic, glutaric, gluconic, tricarballylic, cinnamic, substituted cinnamic (for example, phenyl, methyl, methoxy or halo substituted ciririamic, including 4-methyl and 4-methoxycinnamic acid), ascorbic, oleic, naphthoic, hydroxynaphthoic (f or example 1-or 3-hydroxy-2-naphthoic), naphthaleneacrylic (for example naphthalene-.2-acrylic), benzoic, 4 methoxybenzoic, 2-or 4-hydroxybenzoic, 4-chlorobenzoic, 4-phenylbenzoic, benzeneacrylic (for example l,4-benzenediacryj.jc), isethionic acids, perchioric, propionic, glycolic, hydroxyethanesulfonic, pamoic, cyclohexariesulfamic, salicylic, saccharinic and trifluoroacetic acid. Pharmaceutically acceptable base salts include ammonium salts, alkali metal salts such as those of sodium and potassium, alkaline earth metal salts such as those of calcium and magnesium and salts with organic bases such as dicyclohexylamine and N-methyl-D-glutamine.
All pharmaceutically acceptable acid addition salt forms of the inhibitors of the present invention are intended to be embraced by the scope of this invention.
Examples of solvates include hydrates.
Polymorph crystal forms: Furthermore, some of the crystalline forms of the inhibitors may exist as polymorphs and as such are intended to be included in the present invention. In addition, some of the compounds may form solvates with water (i.e. hydrates) or common organic solvents, and such solvates are also intended to be encompassed within the scope of this invention. The inhibitors, including their salts, can also be obtained in the form of their hydrates, or include other solvents used for their crystallization.
Prodrugs: The present invention further includes within its scope prodrugs of the inhibitors of this invention. In general, such prodrugs will be functional derivatives of the inhibitors, which are readily convertible in vivo into the desired therapeutically active inhibitors. Thus, in these cases, the methods of treatment of the present invention, the term "administering" shall encompass the treatment of the various disorders described with prodrug versions of one or more of the claimed inhibitors, but which converts to the above specified inhibitors in vivo after administration to the subject. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs", ed. H. Bundgaard, Elsevier, 1985 and the patent applications DE 198 28 113, DE 198 28 114, WO 99/67228 and WO 99/67279 which are fully incorporated herein by reference.
Protective Groups: During any of the processes for preparation of the inhibitors of the present invention, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This may be achieved by means of conventional protecting groups, such as those described in Protective Groups in Organic Chemistry, ed. J.F.W. McOmie, Plenum Press, 1973; and T.W. Greene & P.G.M. Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, fully incorporated herein by reference. The protecting groups may be removed at a convenient subsequent stage using methods known from the art.
As used herein, the term "composition" is intended to encompass a product comprising the claimed compounds in the therapeutically effective amounts, as well as any product, which results, directly or indirectly, from combinations of the claimed compounds.
Carriers and Additives for galenic formulations: Thus, for liquid oral preparations, such as for example, suspensions, elixirs and solutions, suitable carriers and additives may advantageously include water, glycols, oils, alcohols, flavouring agents, preservatives, colouring agents and the like; for solid oral preparations such as, for example, powders, capsules, gelcaps and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like.
Carriers, which can be added to the mixture, include necessary and inert pharmaceutical excipients, including, but not limited to, suitable binders, suspending agents, lubricants, flavorants, sweeteners, preservatives, coatings, disintegrating agents, dyes and colouring agents.
Soluble polymers as targetable drug carriers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmeth-acrylamidephenol, polyhydroxyethylaspartamidephenol, or polyethyleneoxidepolyllysine substituted with palmitoyl residue(s). Furthermore, the inhibitors of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled/sustained release of a drug, for example, polyactic acid, poly-epsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
Suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
S
Disintegrating agents include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.
In a preferred embodiment, the present invention provides a composition, preferably a pharmaceutical composition, comprising at least one QC inhibitor optionally in combination with at least one other agent selected from the group consisting of nootropic agents, neuroprotectants, antiparkinsonian drugs, amyloid protein deposition inhibitors, beta amyloid synthesis inhibitors, antidepressants, anxiolytic drugs, antipsychotic drugs and anti-multiple sclerosis drugs.
More specifically, the aforementioned other agent is selected from the group consisting of beta-amyloid antibodies, cysteine protease inhibitors, PEP-inhibitors, LiC1,, acetyicholinesterase (AChE) inhibitors, PIMT enhancers, inhibitors of beta secretases, inhibitors of gamma secretases, inhibitors of neutral endopeptidase, inhibitors of Phosphodiesterase-4 (PDE-4), TNFalpha inhibitors, muscarinic Ml receptor antagonists, N1DA receptor antagonists, sigma-i receptor inhibitors, histamine H3 antagonists, immunomodulatory agents, immunosuppressive agents, MCP-i antagonists or an agent selected from the group consisting of antegren (natalizumab), Neurelan (fampridine-SR), campath (alemtuzumab), IR 208, NBI 5788/MSP 77].
(tiplimotide), paclitaxel, Anergix.MS (AG 284), SH636, Differin (CD 271, adapalene), BAY 361677 (interleukin-4), matrix-metalloproteinase-inhjbjtors (e.g. BB 76163), interferon-tau (trophoblastin) and SAlK-MS.
Furthermore, the other agent may be, for example, an anti-anxiety drug or antidepressant selected from the group consisting of (a) Benzodiazepines, e.g. aiprazolam, chiordiazepoxide, clobazam, clonazepam, clorazepate, diazepam, fludiazepam, loflazepate, lorazepam, methaqualone, oxazepam, prazepam, tranxene, (b) Selective serotonin re-uptake inhibitors (SSRI's), e.g. citalopram, fluoxetine, fluvoxamine, escitalopram, sertraline, paroxetine, (c) Tricyclic antidepressants, e.g. amitryptiline, clomipramine, desipramine, doxepin, imipramine (d) Monoamine oxidase (MAO) inhibitors, Ce) Azapirones, e.g. buspirone, tandopsirone, (f) Serotonin-norepinephrine reuptake inhibitors (SNRI's), e.g. venlafaxine, duloxetine, (g) Mirtazapine, (h) Norepinephrine reuptake inhibitors (NRI's), e.g. reboxetine, (1) Bupropione, (j) Nefazodone, (k) beta-blockers, (1) NPY-receptor ligands: NPY agonists or antagonists.
In a further embodiment, the other agent may be, for example, an anti-multiple sclerosis drug selected from the group consisting of a) dihydroorotate dehydrogenase inhibitors, e.g. SC-12267, teriflunomide, MNA-715, HMR-1279 (syn. to HMR-l715, MNA-279) b) autoimmune suppressant, e.g. laquinimod, 3].
c) paclitaxel, d) antibodies, e.g. AGT-l, anti-granulocyte-.macrophage colony-stimulating factor (GM-CSF) monoclonal antibody, Nogo receptor modulators, ABT-874, alemtuzumab (CANPATH), anti-0X40 antibody, CNTO-1275, DN-1921, natalizumab (syn. to AN-l00226, Antegren, VLA-4 Nab), daclizumab (syn. to Zenepax, Ro-34-7375, SMART anti-Tac), J-695, priliximab (syn. to Centara, CEN-000029, cM-T412), MRA, Dantes, anti-IL-12-antibody, e) peptide nucleic acid (PNA) preparations, e.g. reticulose, f) interferon alpha, e.g. Alfaferone, human alpha interferon (syn. to Omniferon, Alpha Leukoferon), g) interferon beta, e.g. Frone, interferon beta-la like Avonex, Betron (Rebif), interferon beta analogs, interferon beta-transferrin fusion protein, recombinant interferon beta-lb like Betaseron, h) interferon tau, i) peptides, e.g. AT-008, AnergiX.MS, Immunokine (alpha-Immunokine-NNSO3), cyclic peptides like ZD-7349, j) therapeutic enzymes, e.g. soluble CD8 (sCD8), k) multiple sclerosis-specific autoantigen-encoding plasmid and cytokine-encoding plasmid, e.g. BHT-3009; 1) inhibitor of TNF-alpha, e.g. BLX-1002, thalidomide, SH- m) TNF antagonists, e.g. solimastat, lenercept (syn. to RO- 45-2081, Tenefuse), onercept (sTNFR1), CC-1069, n) TNF alpha, e.g. etanercept (syn. to Enbrel, TNR-0O1) o) CD28 antagonists, e.g. abatacept, p) Lck tyrosine kinase inhibitors, q) cathepsin K inhibitors,
S
r) analogs of the neuron-targeting membrane transporter protein taurine and the plant-derived calpain inhibitor leupeptin, e.g. Neurodur, s) chemokine receptor-i (CCR1) antagonist, e.g. BX-471, t) CCR2 antagonists, u) AMPA receptor antagonists, e.g. ER-167288-Ol and ER- 099487, E-2007, talampanel, v) potassium channel blockers, e.g. fampridine, w) tosyl-proline-phenyla].anine small-molecule antagonists of the VLP-4/VCAM interaction, e.g. TBC-3342, x) cell adhesion molecule inhibitors, e.g. TBC-772, y) antisense oligonucleotides, e.g. EN-lOl, z) antagonists of free immunoglobulin light chain (IgLC) binding to mast cell receptors, e.g. F-991, aa) apoptosis inducing antigens, e.g. Apogen MS, bb) alpha-2 adrenoceptor agonist, e.g. tizanidine (syn.
to Zanaflex, Ternelin, Sirdalvo, Sirdalud, Mionidine), cc) copolymer of L-tyrosine, L-lysine, L-glutamic acid and L-alanine, e.g. glatiramer acetate (syri. to Copaxone, COP-i, copolymer-l), dd) topoisomerase II modulators, e.g. mitoxantrone hydrochloride, ee) adenosine deaminase inhibitor, e.g. cladribine (syn. to Leustatin, Mylinax, RWJ-26251), ff) interleukin-lO, e.g. ilodecakin (syn. to Tenovil, Sch-52000, CSIF), gg) interleukin-12 antagonists, e.g. lisofylline (syn.
to CT-1501R, LSF, lysofylline), hh) Ethanaminum, e.g. SRI-62-834 (syn. to CRC-8605, NSC-614383), ii) immunomodulators, e.g. SAIK-MS, PNtJ-156804, alpha-fetoprotein peptide (AFP), IPDS, jj) retinoid receptor agonists, e.g. adapalene (syn. to Differin, CD-271) kk) TGF-beta, e.g. GDF-]. (growth and differentiation factor 1), 11) TGF-beta-2, e.g. BetaKine, mm) MMP inhibitors, e.g. glycomed, nn) phosphodiesterase 4 (PDE4) inhibitors, e.g. RPR- 00) purine nucleoside phosphorylase inhibitors, e.g. 9-(3-pyridylmethyl) -9-deazaguanine, peldesine (syn. to BCX-34, TO-200), pp) alpha-4/beta-]. integrin antagonists, e.g. ISIS-qq) antisense alpha4 integrin (CD49d) , e.g. ISIS-17044, ISIS-27 104 rr) cytokine-inducing agents, e.g. nucleosides, ICN-ss) cytokine inhibitors, tt) heat shock protein vaccines, e.g. HSPPC-96, uu) neuregulin growth factors, e.g. GGF-2 (syn. to neuregulin, glial growth factor 2), vv) cathepsin S -inhibitors, ww) bropirimine analogs, e.g. PNU-56169, PNtJ-63693, xx) Monocyte chemoattractant protein-i inhibitors, e.g. benzimidazoles like MCP-1 inhibitors, LKS-1456, PD- 064036, PD-064126, PD-084486, PD-172084, PD-172386.
Further, the present invention provides pharmaceutical compositions e.g. for parenteral, enteral or oral administration, comprising at least one QC inhibitor, optionally in combination with at least one of the other aforementioned agents.
These combinations provide a particularly beneficial effect.
Such combinations are therefore shown to be effective and useful for the treatment of the aforementioned diseases.
Accordingly, the invention provides a method for the treatment of these conditions.
The method comprises either co-administration of at least one OC inhibitor and at least one of the other agents or the sequential administration thereof.
Co-administration includes administration of a formulation, which comprises at least one QC inhibitor and at least one of the other agents or the essentially simultaneous administration of separate formulations of each agent.
Beta-amyloid antibodies and compositions containing the same are described, e.g. in Wa 2006/137354, Wa 2006/118959, Wa 2006/103116, WO 2006/095041, Wa 2006/08117]., WO 2006/066233, WO 2006/066171, WO 2006/066089, Wa 2006/066049, Wa 2006/055178, WO 2006/046644, WO 2006/039470, Wa 2006/036291, Wa 2006/026408, Wa 2006/016644, Wa 2006/014638, Wa 2006/014478, Wa 2006/008661, WO 2005/123775, WO 2005/120571, WO 2005/105998, wa 2005/081872, wa 2005/080435, wa 2005/028511, wa 2005/025616, WO 2005/025516, Wa 2005/023858, Wa 2005/018424, Wa 2005/011599, Wa 2005/000193, Wa 2004/108895, wa 2004/098631, Wa 2004/080419, Wa 2004/071408, Wa 2004/069182, WO 2004/067561, wa 2004/044204, wa 2004/032868, Wa 2004/031400, WO 2004/029630, wa 2004/029629, wa 2004/024770, Wa 2004/024090, WO 2003/104437, wa 2003/089460, wa 2003/086310, WO 2003/077858, wa 2003/074081, WO 2003/070760, Wa 2003/063760, WO 2003/055514, Wa 2003/051374, WO 2003/048204, W 2003/045128, WO 2003/040183, Wa 2003/039467, WO 2003/016466, WO 2003/015691, WO 2003/014162, WO 2003/012141, WO 2002/088307, WO 2002/088306, WO 2002/074240, Wa 2002/046237, WO 2002/046222, WO 2002/041842, WO 2001/062801, WO 2001/012598, WO 2000/077178, Wa 2000/072880, WO 2000/063250, Wa 1999/060024, WO 1999/027944, WO 1998/044955, WO 1996/025435, WO 1994/017197, WO 1990/014840, Wa 1990/012871, WO 1990/012870, Wa 1989/006242.
Suitable examples of beta-amyloid antibodies are ACIJ-5A5, huCO9l (Acumen/Merck); PF-4360365, RI-1014, RI-1219, RI-409, RN-1219 (Rinat Neuroscience Corp (Pfizer mc)); the nanobody therapeutics of Ablynx/Boehringer Ingelheim; beta-amyloid-specific humanized monoclonal antibodies of Intellect Neurosciences/IBL; m266, m266.2 (Eli Lilly & Co.); AAB-02 (Elan); bapineuzumab (Elan); BAN-2401 (Bioarctic Neuroscience AB); ABP-102 (Abiogen Pharma SpA); BA-27, BC-05 (Takeda); R- 1450 (Roche); ESBA-212 (ESBATech AG); AZD-3 102 (AstraZeneca) and beta-amyloid antibodies of Mindset BioPharmaceuticals Inc. Suitable cysteine protease inhibitors are inhibitors of cathepsin B. Inhibitors of cathepsin B and compositions containing such inhibitors are described, e.g. in WO 2006/060473, WO 2006/042103, WO 2006/039807, WO 2006/021413, Wa 2006/021409, WO 2005/097103, Wa 2005/007199, W02004/084830, Wa 2004/078908, Wa 2004/026851, wa 2002/094881, Wa 2002/027418, WO 2002/021509, Wa 1998/046559, Wa 1996/021655.
Examples of suitable PIMT enhancers are lO-aminoaliphatyl-dibenz[b, fi oxepines described in WO 98/15647 and WO 03/057204, respectively. Further useful according to the
S
present invention are modulators of PIMT activity described in WO 2004/039773.
Inhibitors of beta secretase and compositions containing such inhibitors are described, e.g. in W003/059346, W02006/099352, W02006/078576, W02006/060].09, W02006/057983, W02006/057945, W02006/055434, W02006/044497, W02006/034296, W02006/034277, W02006/029850, W02006/026204, W02006/014944, W02006/014762, W02006/002004, US 7,109,217, W02005/l13484, W02005/103043, W02005/103020, WQ2005/065195, W02005/051914, W02005/044830, W02005/032471, W02005/018545, W02005/004803, W02005/004802, W02004/062625, W02004/0439l6, W02004/013098, W003/099202, W003/043987, W003/039454, US 6,562,783, W002/098849 and Suitable examples of beta secretase inhibitors for the purpose of the present invention are WY25105 (Wyeth); Posiphen, (+)-phenserine (TorreyPines I NIH); LSN-2434074, LY-2070275, LY-2070273, LY-2070102 (Eli Lilly & Co.); PNU- 159775A, PNtJ-178025A, PNU-17820A, PNU-33312, PNU-38773, PNU- 90530 (Elan / Pfizer); KMI-370, KMI-358, kmi-008 (Kyoto University); OM-99-2, OM-003 (Athenagen Inc.); AZ-12304146 (AstraZeneca I Astex); GW-840736X (GlaxoSmithKljne plc.) and DNP-004 089 (De Novo Pharmaceuticals Ltd.).
Inhibitors of gamma secretase and compositions containing such inhibitors are described, e.g. in W02005/008250, W02006/004880, Us 7,122,675, US 7,030,239, US 6,992,081, Us 6,982,264, W02005/097768, W02005/028440, W02004/10l562, US 6,756,511, Us 6,683,091, W003/066592, W003/014075, W003/013527, W002/36555, WO01/53255, Us 7,109,217, Us 7,101,895, US 7,049,296, Us 7,034,182, Us 6,984,626,
S
W02005/040126, W02005/030731, W02005/014553, US 6,890,956, EP 1334085, EP 1263774, W02004/101538, W02004/00958, W02004/089911, W02004/073630, W02004/069826, W02004/039370, W02004/031139, W02004/031137, US 6,713,276, US 6,686,449, W003/091278, US 6,649,196, US 6,448,229, WO01/77144 and Suitable gamma secretase inhibitors for the purpose of the present invention are GSI-953, WAY-GSI-A, WAY-GSI-B (Wyeth); MK-0752, MRK-560, L-852505, L-685-458, L-852631, L-852646 (Merck & Co. Inc.); LY-450139, LY-411575, AN-37124 (Eli Lilly & Co.); BMS-299897, BMS-433796 (Bristol-Myers Squibb Co.); E-2012 (Eisai Co. Ltd.); EHT-0206, EHT-206 (ExonHit Therapeutics SA); and NGX-555 (TorreyPines Therapeutics Inc.) Suitable beta amyloid synthesis inhibitors for the purpose of the present invention are for example Bisnorcymserine (Axonyx Inc.); (R) -flurbiprofen (MCP-7869; Flurizan) (Myriad Genetics); nitroflurbiprofen (NicOx); BGC-20-0406 (Sankyo Co. Ltd.) and BGC-20-0466 (BTG plc.).
Suitable amyloid protein deposition inhibitors for the purpose of the present invention are for example SP-233 (Samaritan Pharmaceuticals); AZD-103 (Ellipsis Neurotherapeutics Inc.); AAB-00i (Bapineuzumab), AAB-002, ACC-00l (Elan Corp plc.); Colostrinin (ReGen Therapeutics plc.); Tramiprosate (Neurochem); AdPEDI-(amyloid-betal-6) 11) (Vaxin Inc.); MPI-l27585, MPI-423948 (Mayo Foundation); SP-08 (Georgetown University); ACU-5A5 (Acumen I Merck); Transthyretin (State University of New York); PTI-777, DP-74, DP 68, Exebryl (ProteoTech Inc.); m266 (Eli Lilly & Co.); EGb-761 (Dr. Wilimar Schwabe GrnbH); SPI-014 (Satori Pharmaceuticals Inc.); ALS-633, ALS-499 (Advanced Life Sciences Inc.); AGT-l60 (ArmaGen Technologies Inc.); TAK-070 (Takeda Pharmaceutical Co. Ltd.); CHF-5022, CHF-5074, CHF- 5096 and CHF-5105 (Chiesi Farmaceutjcj SpA.).
Suitable PDE-4 inhibitors for the purpose of the present invention are for example Doxofylline (Instituto Biologico Chemioterapica ABC SpA.); idudilast eye drops, tipelukast, ibudilast (Kyorin Pharmaceutical Co. Ltd.); theophylline (Elan Corp.); cilomilast (GlaxoSmithKline plc.); Atopik (Barrier Therapeutics Inc.); tofimilast, CI-1044, PD-l89659, CP-220629, PDE 4d inhibitor BHN (Pfizer Inc.); arofylline, LJS-37779 (Almirall Prodesfarma SA.); roflumilast, hydroxypumafentrine (Altana AG), tetomilast (Otska Pharmaceutical Co. Ltd.); tipelukast, ibudilast (Kyorin Pharmaceutical), CC-10004 (Celgene Corp.); HT-0712, IPL-4088 (Inflazyme Pharmaceuticals Ltd.); MEM-1414, MEM-19].7 (Memory Pharmaceuticals Corp.); oglemilast, GRC-4039 (Glenmark Pharmaceuticals Ltd.); AWD-l2-281, ELB-353, ELB-526 (Elbion AG); EHT-0202 (ExonHit Therapeutics SA.); ND-l251 (Neuro3d SA.); 4AZA-PDE4 (4 AZA Bioscience NV.); AVE-8ll2 (Sanof 1-Aventis); CR-3465 (Rottapharm SpA.); GP-0203, NCS-6l3 (Centre National de la. Recherche Scientifique); KF- 19514 (Kyowa Hakko Kogyo Co. Ltd.); ONO-6126 (Ono Pharmaceutical Co. Ltd.); OS- 0217 (Dainippon Pharmaceutical Co. Ltd.); IBFB-130011, IBFB- 150007, IBFB-130020, IBFB-l4030l (IBFB Pharma GmbH); IC-485 (ICOS Corp.); RBx-14 016 and RBx-11082 (Ranbaxy Laboratories Ltd.). A preferred PDE-4- inhibitor is Rolipram.
MAO inhibitors and compositions containing such inhibitors are described, e.g. in W02006/091988, W02005/007614, W02004/089351, WOO1/26656, WOOl/12176, W099/57120, W099/57119, W099/13878, W098/40102, W098/01157, W096/20946, Suitable MAO-inhibitors for the purpose of the present invention are for example Linezolid (Pharmacia Corp.); RWJ- 416457 (RW Johnson Pharmaceutical Research Institute); budipine (Altana AG); GPX-325 (BioResearch Ireland); isocarboxazid; pheneizine; tranylcypromine; indantadol (Chiesi Farmaceutici SpA.); mociobemide (Roche Holding AG); SL-25.1131 (Sanofi-Synthelabo); CX-1370 (Burroughs Welicome Co.); CX-157 (Krenitsky Pharmaceuticals Inc.); desoxypeganine (HF Arzneimittelforschung GmbH & Co. KG); bifemelane (Mitsubishi-Tokyo Pharmaceuticals Inc.); RS-l636 (Sankyo Co. Ltd.); esuprone (BASF AG); rasagiline (Teva Pharmaceutical Industries Ltd.); ladostigil (Hebrew University of Jerusalem); safinamide (Pfizer) and NW-1048 (Newron Pharmaceuticals SpA.).
Suitable histamine H3 antagonists for the purpose of the present invention are, e.g. ABT-239, ABT-834 (Abbott Laboratories); 3874-Hi (Aventis Pharma); UCL-2173 (Berlin Free University), UCL-1470 (BioProjet, Societe Civile de Recherche); DWP-302 (Daewoong Pharmaceutical Co Ltd); GSK- l89254A, GSK-207040A (GlaxoSmithKline Inc.); cipralisant, GT- 2203 (Gliatech Inc.); Ciproxifan (INSERN), 1S,2S)-2-(2-Aminoethyi) -i-(1H-imidazol-4-yl) cyclopropane (Hokkaido University); JNJ-l7216498, JNJ-5207852 (Johnson & Johnson); NNC-0038-0000-1049 (Novo Nordisk A/S); and Sch-79687 (Schering-Piough).
PEP inhibitors and compositions containing such inhibitors are described, e.g. in JP 01042465, JP 03031298, JP 04208299, WO 00/71144, US 5,847,155; JP 09040693, JP 10077300, JP 05331072, JP 05015314, WO 95/15310, Wa 93/00361, EP 0556482, JP 06234693, JP 01068396, EP 0709373, Us 5,965,556, US 5,756,763, Us 6,121,311, JP 63264454, JP 64000069, JP 63162672, EP 0268190, EP 0277588, EP 0275482, US 4,977,180, Us 5,091,406, Us 4,983,624, Us 5,112,847, US 5,100,904, Us 5,254,550, US 5,262,431, US 5,340,832, US 4,956,380, EP 0303434, JP 03056486, JP 01143897, JP 1226880, EP 0280956, US 4,857,537, EP 0461677, EP 0345428, JP 02275858, US5,506,256, JP 06192298, EP 0618193, JP 03255080, EP 0468469, US 5,118,811, JP 05025125, Wa 9313065, JP 05201970, WO 9412474, EP 0670309, EP 0451547, JP 06339390, US 5,073,549, Us 4,999,349, EP 0268281, US 4,743,616, EP 0232849, EP 0224272, JP 62114978, JP 62114957, Us 4,757,083, Us 4,810,721, US 5,198,458, US 4,826,870, EP 0201742, EP 0201741, US 4,873,342, EP 0172458, JP 61037764, EP 0201743, US 4,772,587, EP 0372484, US 5,028,604, WO 91/18877, JP 04009367, JP 04235162, US 5,407,950, WO 95/01352, JP 01250370, JP 02207070, US 5,221,752, EP 0468339, JP 04211648, WO 99/46272, Wa 2006/058720 and PCT/EP2006/06J.428.
Suitable prolyl endopeptidase inhibitors for the purpose of the present invention are, e.g. Fmoc-Ala-Pyrr-CN, Z-Phe-Pro-Benzothiazole (Probiodrug), Z-321 (Zeria Pharmaceutical Co Ltd.); ONO-1603 (Ono Pharmaceutical Co Ltd); JTP-4819 (Japan Tobacco Inc.) and S-17092 (Servier).
Other suitable compounds that can be used according to the present invention in combination with QC-inhibitors are NPY,
I
an NPY mimetic or an NPY agonist or antagonist or a ligand of the NPY receptors.
Preferred according to the present invention are antagonists of the NPY receptors.
Suitable ligands or antagonists of the NPY receptors are 3a, 4,59b-tetrahydro-1h-benz[e]indo1-2-y1 amine-derived compounds as disclosed in WO 00/68197.
NPY receptor antagonists which may be mentioned include those disclosed in European patent applications EP 0 614 911, EP 0 747 357, EP 0 747 356 and EP 0 747 378; international patent applications WO 94/17035, Wa 97/19911, WO 97/19913, WO 96/12489, WO 97/19914, WO 96/22305, WO 96/40660, WO 96/12490, Wa 97/09308, WO 97/20820, WO 97/20821, WO 97/20822, WO 97/20823, WO 97/19682, WO 97/25041, wa 97/34843, wa 97/46250, WO 98/03492, WO 98/03493, WO 98/03494 and wa 98/07420; WO 00/30674, US patents Nos. 5,552,411, 5,663,192 and 5,567,714; 6,114,336, Japanese patent application JP 09157253; international patent applications WO 94/00486, WO 93/12139, Wa 95/00161 and wa 99/15498; US Patent No. 5,328,899; German patent application DE 393 97 97; European patent applications EP 355 794 and EP 355 793; and Japanese patent applications JP 06116284 and JP 07267988. Preferred NPY antagonists include those compounds that are specifically disclosed in these patent documents. More preferred compounds include amino acid and non-peptide-based NPY antagonists. Amino acid and non-peptide-based NPY antagonists which may be mentioned include those disclosed in European patent applications EP 0 614 911, EP 0 747 357, EP 0 747 356 and EP 0 747 378; international patent applications Wa 94/17035, WO 97/19911, WO 97/19913, WO 96/12489, WO 97/19914, WO 96/22305, WO 96/40660, WO 96/12490, WO 97/09308, WO 97/20820, WO 97/20821, WO 97/20822, WO 97/20823, WO 97/19682, WO 97/25041, WO 97/34843, WO 97/46250, WO 98/03492, WO 98/03493, WO 98/03494, WO 98/07420 and WO 99/15498; US patents Nos. 5,552,411, 5,663,192 and 5,567,714; and Japanese patent application JP 09157253. Preferred amino acid and non-peptide-based NPY antagonists include those compounds that are specifically disclosed in these patent documents.
Particularly preferred compounds include amino acid-based NPY antagonists. Amino acid-based compounds, which may be mentioned include those disclosed in international patent applications WO 94/17035, WO 97/19911, WO 97/19913, WO 97/19914 or, preferably, WO 99/15498. Preferred amino acid-based NPY antagonists include those that are specifically disclosed in these patent documents, for example B1BP3226 and, especially, (R) -N2-(diphenylacetyl)-(R) -N-[1-(4-hydroxy-phenyl) ethyl] arginine atnide (Example 4 of international patent application Wa 99/15498).
Ml receptor agonists and compositions containing such inhibitors are described, e.g. in W02004/087158, W091/10664.
Suitable Ml receptor antagonists for the purpose of the present invention are for example CDD-0102 (Cognitive Pharmaceuticals); Cevimeline (Evoxac) (Snow Brand Milk Products Co. Ltd.); NGX-267 (TorreyPines Therapeutics); sabcomeline (GlaxoSmithKljne); alvarneline (H Lundbeck A/S); LY-593093 (Eli Lilly & Co.); VRTX-3 (Vertex Pharmaceuticals Inc.); WAY-132983 (Wyeth) and Cl-b]. 7/ (PD-151832) (Pfizer Inc.)
I
Acetyicholinesterase inhibitors and compositions containing such inhibitors are described, e.g. in W02006/071274, W02006/070394, W02006/040688, W02005/092009, W02005/079789, W02005/039580, W02005/027975, W02004/084884, W02004/037234 W02004/032929, W003/101458, W003/091220, W003/082820, W003/020289, W002/32412, wOOl/85145, WOOl/78728, WOOl/66096, W000/02549, WOOl/00215, W000/15205, W000/23057, W000/33840, W000/30446, W000/23057, W000/15205, W000/09483, W000/07500, W000/02549, W099/47131, W099/07359, W098/30243, W097/38993, W097/13754, W094/29255, W094/20476, W094/19356, W093/03034 and W092/19238.
Suitable acetyicholinesterase inhibitors for the purpose of the present invention are for example Donepezil (Eisai Co. Ltd.); rivastigmine (Novartis AG); (-)-phenserine (TorreyPines Therapeutics); ladostigil (Hebrew University of Jerusalem); huperzine A (Mayo Foundation); galantamine (Johnson & Johnson); Memoquin (Universita di Bologna); SP-004 (Samaritan Pharmaceuticals Inc.); BGC-20-1259 (Sankyo Co. Ltd.); physostigmine (Forest Laboratories Inc.); NP-0361 (Neuropharma SA); ZT-l (Debiopharm); tacrine (Warner-Lambert Co.); metrifonate (Bayer Corp.) and INM-176 (Whanln).
NMDA receptor antagonists and compositions containing such inhibitors are described, e.g. in W02006/094674, W02006/058236, W02006/058059, W02006/010965, W02005/000216, W02005/102390, W02005/079779, W02005/079756, W02005/072705, W02005/070429, W02005/055996, W02005/035522, W02005/009421, W02005/000216, W02004/092189, W02004/03937l, W02004/028522, W02004/009062, W003/010159, W002/072542, W002/34718, WOO]./98262, WOOl/9432]., WOOl/92204, WOOl/81295, WOOl/32640, WOOl/10833, WOOl/10831, W000/56711, W000/29023, W000/00197, W099/53922, W099/48891, W099/45963, W099/01416, W099/07413, W099/01416, W098/50075, W098/50044, W098/l0757, W098/05337, W097/32873, W097/23216, W097/23215, W097/23214, W096/i4318, W096/08485, WQ95/31986, W095/26352, W095/26350, W095/26349, W095/26342, W095/12594, W095/02602, W095/02601, W094/20109, W094/13641, W094/090l6 and W093/25534.
Suitable NMDA receptor antagonists for the purpose of the present invention are for example Mernantine (Merz & Co. GmbH); topiramate (Johnson & Johnson); AVP-923 (Neurodex) (Center for Neurologic Study); EN-3231 (Endo Pharmaceuticals Holdings Inc.); neramexane (MRz-2/579) (Merz and Forest); CNS-5161 (CeNeS Pharmaceuticals Inc.); dexanabinol (HtJ-211; Sinnabidol; PA-50211) (Pharmos); EpiCept NP- i (Daihousie University); indantadol (V-3381; CNP-3381) (Vernalis); perzinfotel (EAA-090, WAY-126090, EAk-129) (Wyeth) ; RGH-896 (Gedeon Richter Ltd.); traxoprodil (CP-101606), besonprodil (PD-196860, CI-1041) (Pfizer Inc.); CGX-1007 (Cognetix Inc.); delucemine (NPS-1506) (NPS Pharmaceuticals Inc.); EVT-lO].
(Roche Holding AG); acamprosate (Synchroneuron LLC.); CR- 3991, CR-2249, CR-3394 (Rottapharm SpA.); AV-lOl (4-Cl-kynurenine (4-Ci-KYN)), 7-chloro-kynurenic acid (7-Ci-KYNA) (VistaGen); NPS-l407 (NPS Pharmaceuticals Inc.); YT-1006 (Yaupon Therapeutics Inc.); ED-1812 (Sosei R&D Ltd.); himantane (hydrochloride N-2-(adamantly) -hexamethylen-imine) (RAMS); Lancicemine (AR-R-l5896) (AstraZeneca); EVT-102, Ro- 25-6981 and Ro-63-1908 (Hoffmann-La Roche AG I Evotec).
Furthermore, the present invention relates to combination therapies useful for the treatment of atherosclerosis, restenosjs or arthritis, administering a QC inhibitor in
I
combination with another therapeutic agent selected from the group consisting of inhibitors of the angiotensin converting enzyme (ACE); angiotensin II receptor blockers; diuretics; calcium channel blockers (CCB); beta-blockers; platelet aggregation inhibitors; cholesterol absorption modulators; HMG-Co-A reductase inhibitors; high density lipoprotein (HDL) increasing compounds; renin inhibitors; IL-6 inhibitors; antiinflammatory corticosteroids; antiproliferative agents; nitric oxide donors; inhibitors of extracellular matrix synthesis; growth factor or cytokine signal transduction inhibitors; MCP-l antagonists and tyrosine kinase inhibitors providing beneficial or synergistic therapeutic effects over each moriotherapy component alone.
Arigiotensin II receptor blockers are understood to be those active agents that bind to the AT]. -receptor subtype of angiotensin II receptor but do not result in activation of the receptor. As a consequence of the blockade of the AT].
receptor, these antagonists can, e.g. be employed as antihypertensive agents.
Suitable angiotensin II receptor blockers which may be employed in the combination of the present invention include AT1 receptor antagonists having differing structural features, preferred are those with non-peptidic structures.
For example, mention may be made of the compounds that are selected from the group consisting of valsartan (EP 443983), losartan (EP 253310), candesartan (EP 459136), eprosartan (EP 403159), irbesartan (EP 454511), olmesartan (EP 503785), tasosartan (EP 539086), telmisartan (EP 522314), the compound with the designation E-41 77 of the formula
I
the compound with the designation SC-52458 of the following formula / and the compound with the designation the compound ZD-8731 of the formula or, in each case, a pharmaceutically acceptable salt thereof.
Preferred AT1-receptor antagonists are those agents that have been approved and reached the market, most preferred is valsartan, or a pharmaceutically acceptable salt thereof.
I
The interruption of the enzymatic degradation of angiotensin to angiotensin II with ACE inhibitors is a successful variant for the regulation of blood pressure and thus also makes available a therapeutic method for the treatment of hypertension.
A suitable ACE inhibitor to be employed in the combination of the present invention is, e.g. a compound selected from the group consisting alacepril, benazepril, benazeprilat; captopril, ceronapril, cilazapril, delapril, enalapril, enaprilat, fosinopril, imidapril, lisinopril, moveltopril, perindopril, quinapril, ramipril, spirapril, temocapril and trandolapril, or in each case, a pharmaceutically acceptable salt thereof.
Preferred ACE inhibitors are those agents that have been marketed, most preferred are benazepril and enalapril.
A diuretic is, for example, a thiazide derivative selected from the group consisting of chiorothiazide, hydrochiorothiazide, methyiclothiazide, and chiorothalidon.
The most preferred diuretic is hydrochiorothiazide. A diuretic furthermore comprises a potassium sparing diuretic such as amiloride or triameterine, or a pharmaceutically acceptable salt thereof.
The class of CCBs essentially comprises dihydropyridines (DHPs) and non-DHPs, such as diltiazem-type and veraparnil-type CCBs.
S
A CCB useful in said combination is preferably a DHP representative selected from the group consisting of amlodipine, felodipine, ryosidine, isradipine, lacidipine, nicardipine, nifedipine, niguldipine, niludipine, nimodipine, nisoldipine, nitrendipine and nivaldipine, and is preferably a non-DHP representative selected from the group consisting of flunarizine, prenylamine, diltiazem, fendiline, gallopamil, mibefradil, anipamil, tiapamil and verapamil, and in each case, a pharmaceutically acceptable salt thereof. All these CCBs are therapeutically used, e.g. as anti-hypertensive, anti-angina pectoris or anti-arrhythmic drugs.
Preferred CCBs comprise amlodipine, diltiazem, isradipine, nicardipine, nifedipirie, nimodipine, nisoldipine, nitrendipine and verapamil or, e.g. dependent on the specific CCB, a pharmaceutically acceptable salt thereof. Especially preferred as DHP is amlodipine or a pharmaceutically acceptable salt thereof, especially the besylate. An especially preferred representative of non-DHPs is verapamil or a pharmaceutically acceptable salt, especially the hydrochloride, thereof.
Beta-blockers suitable for use in the present invention include beta-adrenergic blocking agents (beta-blockers), which compete with epinephrine for beta-adrenergic receptors and interfere with the action of epinephrine. Preferably, the beta-blockers are selective for the beta-adrenergic receptor as compared to the alpha-adrenergic receptors, and so do not have a significant alpha-blocking effect. Suitable beta-blockers include compounds selected from acebutolol, atenolol, betaxolol, bisoprolol, carteolol, carvedilol, esmolol, labetalol, metoprolol, nadolol, oxprenolol, perthutolol, pindolol, propranolol, sotalol and timolol. Where the beta-blocker is an acid or base or otherwise capable of forming pharmaceutically acceptable salts or prodrugs, these forms are considered to be encompassed herein, and it is understood that the compounds may be administered in free form or in the form of a pharmaceutically cceptable salt or a prodrug, such as a physiologically hydrolyzable and acceptable ester. For example, metoprolol is suitably administered as its tartrate salt, propranolol is suitably administered as the hydrochloride salt, and so forth.
Platelet aggregation inhibitors include PLAVIX (clopidogrel bisulf ate), PLIETAL (cilostazol) and aspirin.
Cholesterol absorption modulators include ZETIA (ezetimibe) and KT6-971 (Kotobuki Pharmaceutical Co. Japan).
HMG-Co-A reductase inhibitors (also called beta-hydroxy-beta-methylglutaryl-co-enzyme-A reductase inhibitors or statins) are understood to be those active agents which may be used to lower lipid levels including cholesterol in blood.
The class of HMG-Co-A reductase inhibitors comprises compounds having differing structural features. For example, mention may be made of the compounds, which are selected from the group consisting of atorvastatin, cerivastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatjn and simvastatin, or in each case, a pharmaceutically acceptable salt thereof.
Preferred HIiG-Co-A reductase inhibitors are those agents, which have been marketed, most preferred is atorvastatin,
I
pitavastatin or simvastatin, or a pharmaceutically acceptable salt thereof.
HDL-increasing compounds include, but are not limited to, cholesterol ester transfer protein (CETP) inhibitors.
Examples of CETP inhibitors include JTT7O5 disclosed in Example 26 of U.S. Patent No. 6,426,365 issued July 30, 2002, and pharmaceutically acceptable salts thereof.
Inhibition of interleukin 6 mediated inflammation may be achieved indirectly through regulation of endogenous cholesterol synthesis and isoprenoid depletion or by direct inhibition of the signal transduction pathway utilizing interleukin-6 inhibitor/antibody, interleukin-6 receptor inhibitor/antibody, interleukin-6 antisense oligonucleotide (ASON), gpi3O protein inhibitor/antibody, tyrosine kinase F serine/threonine kinase inhibitors/antibodies, mitogen-activated protein (MAP) kinase inhibitors/antibodies, phosphatidylinositol 3-kinase (P13K) inhibitors/antibodies, Nuclear factor kappaB (NF-KB) inhibitors/antibodies, licE kinase (IKK) inhibitors/antibodies, activator protein-i (AP-1) inhibitors/antibodies, STAT transcription factors inhibitors/antibodies, altered IL-6, partial peptides of IL-6 or IL-6 receptor, or SOCS (suppressors of cytokine signaling) protein, PPAR gamma and/or PPAP. beta/delta activators/ligands or a functional fragment thereof.
A suitable antiinflammatory corticosteroid is dexamethasone.
Suitable antiproliferative agents are cladribine, rapamycin, vincristirie and taxol.
I
A suitable inhibitor of extracellular matrix synthesis is halofuginone.
A suitable growth factor or cytokine signal transduction inhibitor is, e.g. the rae inhibitor R115777.
A suitable tyrosine kinase inhibitor is tyrphostin.
Suitable renin inhibitors are described, e.g. in WO 2006/116435. A preferred renin inhibitor is aliskiren, preferably in the form of the hemi-fumarate salt thereof.
MCP-1 antagonists may, e.g. be selected from anti-MCP-l antibodies, preferably monoclonal or humanized monoclonal antibodies, MCP-1 expression inhibitors, CCR2-antagonists, TNF-alpha inhibitors, VCAM-l gene expression inhibitors and anti-C5a monoclonal antibodies.
MCP 1 -antagonists and compositions containing such inhibitors are described, e.g. in W002/070509, W002/081463, W002/060900, US2006/670364, US2006/677365, W02006/097624, tJS2006/316449, W02004/056727, W003/053368, W000/198289, W000/157226, W000/046195, W000/046196, W000/046199, W000/04 6198, W000/046197, W099/046991, W099/007351, W098/006703, W097/012615, W02005/105133, W003/037376, W02006/125202, W02006/085961, W02004/024921, W02006/074265.
Suitable MCP 1 -antagonists are, for instance, C-243 (Telik Inc.); NQX-E36 (Noxxon Pharma AG); AP-761 (Actimis Pharmaceuticals Inc.); ABN-912, NIBR-177 (Novartis AG); CC- 11006 (Celgene Corp.); SSR-150106 (Sanofi-Aventis); MLN-1202 (Mjllenjum Pharmaceuticals Inc.); AGI-1067, AGIX-4207, AGI- 1096 (AtherioGenics Inc.); PRS-211095, PRS-211092 (Pharmos Corp.); anti-CSa monoclonal antibodies, e.g. neutrazumab (G2 Therapies Ltd.); AZD-6942 (AstraZeneca plc.); 2-mercaptoimidazoles (Johnson & Johnson); TEI-E00526, TEI-6122 (Deltagen); RS-504393 (Roche Holding AG); SB-282241, SB- 380732, ADR-7 (Gla.xoSmithKline); anti-MCP-l monoclonal antibodies(Johnson & Johnson).
Combinations of QC-inhibitors with MCP-1 antagonists may be useful for the treatment of inflammatory diseases in general, including neurodegenerative diseases.
Combinations of QC-inhibitors with MCP-1 antagonists are preferred for the treatment of Alzheimer's disease.
Most preferably the QC inhibitor is combined with one or more compounds selected from the following group: PF-4360365, m266, bapineuzumab, R-1450, Posiphen, (+)-phenserine, MK-0752, LY-450139, E-2012, (R)-flurbiprofen, AZD-103, A.PB-00l (Bapineuzumab), Tramiprosate, EGb-761, TAK- 070, Doxofylline, theophylline, cilomilast, tofimilast, roflumilast, tetomilast, tipelukast, ibudilast, HT-0712, MEM- 1414, oglemilast, Linezolid, budipine, isocarboxazid, pheneizine, tranylcypromine, indantadol, moclobernide, rasagiline, ladostigil, safinamide, ABT-239, ABT-834, GSK- l89254A, Ciproxifan, JNJ-172l6498, Fmoc-Ala-Pyrr-CN, Z-Phe-Pro-Benzothiazole, Z-321, ONO-1603, JTP-4819, S-17092, B1BP3226; (R) -N2-(diphenylacetyl) -(R) -N-[1-(4-hydroxyphenyl) ethyl] arginine amide, Cevimeline, sabcomeline, (PD-l51832), Donepezil, rivastigmine, (-) -phenserine, ladostigil, galantamine, tacrine, metrifonate, Memantine, topiramate, AVP-923, EN-3231, neramexane, valsartan, benazepril, enalapril, hydrochiorothiazide, amlodipine, diltiazem, isradipine, nicardipine, nifedipine, nimodipine, nisoldipine, nitrendipine, veraparnil, amlodipine, acebutolol, atenolol, betaxolol, bisoprolol, carteolol, carvedilol, esmolol, labetalol, metoprolol, nadolol, oxprenolol, perthutolol, pindolol, propranolol, sotalol, timolol, PIAVIX (clopidogrel bisulfate), PLETAL (cilostazol), aspirin, ZETIA (ezetimibe) and KT6-97l, statins, atorvastatin, pitavastatin or simvastatin; dexamethasone, cladribine, rapainycin, vincristine, taxol, aliskiren, C-243, ABN-912, SSR-150106, MLN-l202 and betaferon.
In particular, the following combinations are considered: -a QC inhibitor, in particular QCI, in combination with Atorvastatin for the treatment and/or prevention of artheroscierosis -a QC inhibitor, in particular QCI in combination with immunosuppressive agents, preferably rapamycin for the prevention and/or treatment of restenosis -a QC inhibitor, in particular QCI in combination with immunosuppressive agents, preferably paclitaxel for the prevention and/or treatment of restenosis a QC inhibitor, in particular QCI in combination with AChE inhibitors, preferably Donepezil, for the prevention and/or treatment of Alzheimer's disease -a QC inhibitor, in particular QCI in combination with interferones, preferably Aronex, for the prevention and/or treatment of multiple sclerosis -a QC inhibitor, in particular QCI in combination with interferones, preferably betaferon, for the prevention and/or treatment of multiple sclerosis -a QC inhibitor, in particular QCI in combination with interferones, preferably Rebif, for the prevention and/or treatment of multiple sclerosis
S
-a QC inhibitor, in particular QCI in combination with Copaxone, for the prevention and/or treatment of multiple sclerosis.
Such a combination therapy is in particular useful for AD, FAD, FDD and neurodegeneration in Down syndrome as well as atherosclerosis, rheumatoid arthritis, restenosis and pancreatitis.
Such combination therapies might result in a better therapeutic effect (less proliferation as well as less inflammation, a stimulus for proliferation) than would occur with either agent alone.
With regard to the specific combination of inhibitors of QC and further compounds it is referred in particular to Wa 2004/098625 in this regard, which is incorporated herein by reference.
In a further embodiment the present invention provides a method for preventing or treating a disease or condition, selected from a group consisting of inflammatory diseases selected from a. neurodegenerative diseases, e.g. mild cognitive impairment (MCI), Alzheimer's disease, neurodegeneration in Down Syndrome, Familial British Dementia, Familial Danish Dementia, multiple sclerosis, b. chronic and acute inflammations, e.g. rheumatoid arthritis, atherosclerosis, restenosis, pancreatitis, c. fibrosis, e.g. lung fibrosis, liver fibrosis, renal fibrosis, d. cancer, e.g. cancer/hemangioendothelioma proliferation, gastric carcinomas, e. metabolic diseases, e.g. hypertension, f. and other inflammatory diseases, e.g. neuropathic pain, graft rejection/graft failure/graft vasculopathy, HIV infections/AIDS, gestosis, , tuberous sclerosis.
Additionally, the present invention includes the use of the compounds of this invention and their corresponding pharmaceutically acceptable acid salt forms for the preparation of a medicament for the prevention or treatment of any of the above diseases or conditions.
Most preferably, the present QC inhibitor is used for the treatment of the above-mentioned neurodegenerative diseases.
The compound may be administered to a patient by any conventional route of administration, including, but not limited to, intravenous, oral, subcutaneous, intramuscular, intradermal, parenteral and combinations thereof.
In a further preferred form of implementation, the invention relates to pharmaceutical compositions, that is to say, medicaments, that contain at least one compound of the invention or salts thereof, optionally in combination with one or more pharmaceutically acceptable carriers and/or solvents.
The pharmaceutical compositions may, for example, be in the form of parenteral or enteral formulations and contain appropriate carriers, or they may be in the form of oral formulations that may contain appropriate carriers suitable for oral administration. Preferably, they are in the form of oral formulations.
The inhibitors of QC activity administered according to the invention may be employed in pharmaceutically administrable formulations or formulation complexes as inhibitors or in combination with inhibitors, substrates, pseudosubstrates, inhibitors of QC expression, binding proteins or antibodies of those enzyme proteins that reduce the QC protein concentration in mammals. The compounds of the invention make it possible to adjust treatment individually to patients and diseases, it being possible, in particular, to avoid individual intolerances, allergies and side-effects.
The compounds also exhibit differing degrees of activity as a function of time. The physician providing treatment is thereby given the opportunity to respond differently to the individual situation of patients: he is able to adjust precisely, on the one hand, the speed of the onset of action and, on the other hand, the duration of action and especially the intensity of action.
The compounds may be advantageously administered, for example, in the form of pharmaceutical preparations that contain the active ingredient in combination with customary additives like diluents, excipients and/or carriers known from the prior art. For example, they can be administered parenterally (for example i.v. in physiological saline solution) or enterally (for example orally, formulated with customary carriers) Depending on their endogenous stability and their bioavailability, one or more doses of the compounds can be given per day in order to achieve the desired reduction of MCP activity. For example, such a dosage range in humans may be in the range of from about 0.01 rug to 250.0 mg per day, preferably in the range of about 0.01 to 100 mg of compound per kilogram of body weight per day.
The compounds used according to the invention can accordingly be converted in a manner known per se into conventional formulations, such as, for example, tablets, (bitable) capsules, dragées, pills, suppositories, granules, aerosols,
I
syrups, drops, liquid, solid and cream-like emulsions and suspensions and/or also as suppositories or as nasal sprays solutions, using inert, non-toxic, pharmaceutically suitable carriers and additives or solvents. In each of those formulations, the therapeutically effective compounds are preferably present in a concentration of approximately from 0.1 to 80% by weight, more preferably from 1 to 50% by weight, of the total mixture, that is to say, in amounts sufficient for the mentioned dosage latitude to be obtained.
The formulations may be advantageously prepared, for example, by extending the active ingredient with solvents and/or carriers, optionally with the use of emulsifiers and/or dispersants, it being possible, for example, in the case where water is used as diluent, for organic solvents to be optionally used as auxiliary solvents.
Examples of excipients useful in connection with the present invention include: water, non-toxic organic solvents, such as paraff ins (for example natural oil fractions), vegetable oils (for example rapeseed oil, groundnut oil, sesame oil), alcohols (for example ethyl alcohol, glycerol), glycols (for example propylene glycol, polyethylene glycol); solid carriers, such as, for example, natural powdered minerals (for example highly dispersed silica, silicates), sugars (for example raw sugar, lactose and dextrose); ernulsifiers, such as non-ionic and anionic emulsifiers (for example polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, alkylsulphonates and arylsulphonates), dispersants (for example lignin, sulphite liquors, methylcellulose, starch and polyvinylpyrrolidone) and lu.bricants (for example magnesium stearate, talcum, stearic acid and sodium lauryl sulphate) and optionally flavourings.
Administration may be carried out in the usual manner, preferably enterally or parenterally, especially orally. In the case of enteral administration, tablets may contain in addition to the mentioned carriers further additives such as sodium citrate, calcium carbonate and calcium phosphate, together with various additives, such as starch, preferably potato starch, gelatin and the like. Furthermore, lubricants, such as magnesium stearate, sodium lauryl sulphate and talcum, can be used concomitantly for tabletting. In the case of aqueous suspensions and/or elixirs intended for oral administration, various taste correctives or colourings can be added to the active ingredients in addition to the above-mentioned excipients.
In the case of parenteral administration, solutions of the active ingredients using suitable liquid carriers can be employed. In general, it has been found advantageous to administer, in the case of intravenous administration, amounts of approximately from 0.01 to 2.0 mg/kg, preferably approximately from 0.01 to 1.0 mg/kg, of body weight per day to obtain effective results and, in the case of enteral administration, the dosage is approximately from 0.01 to 2 mg/kg, preferably approximately from 0.01 to 1 mg/kg, of body weight per day. It may nevertheless be necessary in some cases to deviate from the
stated amounts, depending upon the body weight of the experimental animal or the patient or upon the type of administration route, but also on the basis of the species of animal and its individual response to the medicament or the interval at which administration is carried out. Accordingly, it may be sufficient in some cases to use less than the above-mentioned minimum amount, while, in other cases, the mentioned upper limit will have to be exceeded. In cases where relatively large amounts are being administered, it may be advisable to divide those amounts into several single doses over the day. For administration in human medicine, the same dosage latitude is provided. The above remarks apply analogously in that case.
The above disclosure describes the present invention in general. A more complete understanding can be obtained by reference to the following figures and examples. These examples are described solely for purposes of illustration and are not intended to limit the scope of the invention.
Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.
S
Brief description of the Drawings
Figure 1 shows the incubation of MCP-1(1..76) bearing an N-terminal glutaminyl (A) or Pyroglutamyl (5-oxo-L-Prolyl) residue (B) with recombinant human DP4 for 24 h. For cyclization of N-terminal glutamine into pyroglutamate MCP-l was incubated with recombinant human QC 3 h prior to assay start. The DP4 cleavage products were analyzed after 0 rain, mm, 30 mm, lh, 4h and 24 h using Maldi-TOF mass spectrometry.
Figure 2 shows the incubation of MCP-1(176) bearing an N-terminal glutaminyl (A) or Pyroglutamyl (5-oxo-L-Prolyl) residue with human synovial fibroblast MMP-l for 24 h. For cyclization of N-terminal glutamine into pyroglutarnate MCP-1 was incubated with recombinant human QC 3 h prior to assay start. The MNP-1 cleavage products were analyzed after 0 rain, mm, 30 mm, lh, 2h, 4h and 24 h using Maldi-TOF mass spectrometry.
Figure 3 shows the incubation of MCP-1(1...76) carrying an N-terminal glutaminyl (A) or Pyroglutamyl (5-oxo-L-Prolyl) with human synovial fibroblast MMP-l and recombinant human DP4 for 24 h. For cyclization of N-terminal glutamine into pyroglutamate, MCP-l was incubated with recombinant human QC 3 Ii prior to assay start. Resulting MMP-1 cleavage products were analyzed after 0 mm, 15 mm, 30 mm, lh, 2h, 4h and 24 h using Maldi-TOF mass spectrometry Figure 4 shows the isolation of human MCP-1 from human neuroblastoma cell line SH-SY5Y. (M:DNA standard in bp; 1: full length human MCP-1 isolated from SH-SY5Y) Figure 5 shows the nucleotide (A) and amino acid (B) alignment of human MCP-l isolated from SH-SY5Y (upper lane) and human MCP-l genebank accession M24545 (lower lane).
Single nucleotide polymorphism is depicted in bold.
Figure 5 C: shows the concentration of human MCP-l(176) (WT) and mutant human MCP-]. lacking the N-terminal pGlu residue (LiQl) in the supernatant of transfected HEK293 cells in comparison to vector transfected control (pcDNA). (n.s.: not significant, Student's t-test; n=6) D: Migration of THP-1 monocytes towards the generated supernatant of transfected HEK293 cells in dilutions 1:1, 1:3, 1:10 and 1:30. (*, P<D.05; **, P< 0.01; P<0.001; Student's t-test, n=3).
Figure 6 A: shows the concentration of human MCP-1(1.76) (WT) and mutant human MCP-1 lacking the two N-terminal amino acids (QlP2) in the supernatant of transfected HEK293 cells in comparison to vector transfected control (pcDNA).
P<O.01; Student's t-test; n=6) B: Migration of THP-1 monocytes towards the generated supernatant of transfected HEK293 cells in dilutions 1:1, 1:3, 1:10 and 1:30. (*, P<O.05; *, P< 0.01; P<0.001; Student's t-test, n=3).
Figure 7 A: shows the concentration of human MCP-1(l-'76) (WT) in the supernatant of transfected HEK293 cells in absence and presence of 10 j.M 1-(3-(1H-imidazol-1-yl)propyl) -3-(3,4-dimethoxyphenyl)thiourea hydrochloride in comparison to vector transfected control (pcDNA). (n.s.: not significant; Student's t-test; n=6) B: Migration of THP-l monocytes towards the generated supernatant of transfected HEK293 cells in absence or presence of 10 /.LM l-(3-(1H-imidazol-1-yl)propyl) -3-(3,4 -dimethoxyphenyl) thiourea hydrochloride in dilutions of 1:1, 1:3, 1:10 and 1:30. (**, P< 0.01; Student's t-test, n=3) Figure 8 shows the quantification of the vascular remodeling of the cuffed vessel wall segments of untreated ApoE3 Leiden mice (black bars) and mice, which were treated (open bars) with 1-(3-(1H-imidazol-].-yl) propyl) -3-(3, 4-dimethoxyphenyl) thiourea hydrochloride. Mice were sacrificed 14 days after cuff placement. Expressed is the vascular circumference (A) i.e. the total area within the outer diameter of the vessel segment and the remaining lumen (B) in m2.
Figure 9 shows the quantification of the vascular remodeling of the cuffed vessel wall segments of untreated ApoE3 Leiden mice (black bars) or mice treated with (open bars) 1-(3-(J.H- imidazol-1-yl)propyl) -3-(3, 4-dimethoxyphenyl)thiourea hydro-chloride. Mice were sacrificed 14 days after cuff placement.
Expressed is the lumen stenosis A in and the area of neointima B in m2. (*, P<O.05, Student's t-test): Figure 10 shows the quantification of the vascular remodeling of the cuffed vessel wall segments of untreated ApoE3 Leiden mice (black bars) or mice, which were treated with (open bars) of 1-(3-(1H-imidazol-1-yl)propyl)-3-(3,4-dimethoxyphenyl)thiourea hydrochloride. Mice were sacrificed 14 days after cuff placement. Expressed is the area of the media A in /.zm2 and the intima / media ratio B. (*, P<O.05, Student's t-test) Figure 11 shows adhering and infiltrating cells per cross section in absence (black bars) or presence (open bars) of 1- (3-(1H-imidazol-1-yl)propyl) -3-(3,4 -dimethoxyphenyl) thiourea hydrochloride treatment. Total number of adhering cells per cross section was counted in the cross section of the cuffed femoral arteries harvested two days after cuff placement.
Within the total population of adhering cells a specific staining for monocytes/macrophages was used to identify the adhering and infiltrating monocytes. (*, P<O.O5, Student's t-test) Figure 12 shows examples of MCP-1 staining by immunohistochemistry of lesions at the early time point (2 days) and the late time point (14 days) in untreated mice (control) and mice, which were treated with l-(3-(1H-imidazol-1-yl)propyl) -3-(3, 4-dimethoxyphenyl) thiourea hydrochloride.
Figure 13 shows the quantification of MCP-1 staining in cross sections of mice sacrificed after 2 days (early time point) A or after 14 days (late time point) B within the media and neointima in absence (black bars) and presence (open bars) of 1-(3-(].H-imidazol-l-yl)propyl) -3-(3,4-dimethoxyphenyl) thiourea hydrochloride treatment. (*, P<O.05; Student's t-test) Figure 14 shows the relative amount of MCP-1 staining () in cross sections of mice sacrificed after 2 days (early time point) (A) or after 14 days (late time point) (B) within the media and neointima in absence (black bars) and presence (open bars) of 1-(3-(1H-imidazol-1-yl)propyl) -3-(3,4-dimethoxyphenyl)thiourea hydrochloride treatment. (*, P<O.05; Student's t-test) Figure 15 shows the quantification of the accelerated atherosclerosis in the vessel wall based on the quantification of monocyte/macrophage staining using marker A1A31240. Presented are cross sections of mice sacrificed at the late time point (14 days) treated in absence (black bars) and presence (open bars) of 1-(3-(1H-imidazol-1-yl)propyl)-3- (3, 4-dimethoxyphenyl) thiourea hydrochloride. Foam cell accumulation is illustrated as (A) foam cell positive area / cross section in and (B) foam cell positive area / cross section in jm2.
Reference Example 1: Preparation of Human QC Host strains and media Pichia pastoris strain X33 (AOX]., A0X2), used for the expression of human QC was grown, transformed and analyzed according to the manufacturer's instructions (Invitrogen).
The media required for P. pastoris, i.e. buffered glycerol (BMGY) complex or methanol (BMMY) complex medium, and the fermentation basal salts medium were prepared according to the manufacturer' s recommendations.
Molecular cloning of plasmid vectors encoding the human OC All cloning procedures were done applying standard molecular biology techniques. For expression in yeast, the vector pPICZaB (Invitrogen) was used. The pQE-31 vector (Qiagen) was used to express the human QC in E. coli. The cDNA of the mature QC starting with codon 38 was fused in frame with the plasmid encoded 6xhistidine tag. After amplification utilizing the primers pOCyc-]. and pQCyc-2 (WO 2004/098625) and subcloning, the fragment was inserted into the expression vector employing the restriction sites of SphI and Hindill.
Transformation of P. pastoris and mini-scale expression Plasmid DNA was amplified in E. coli JM1O9 and purified according to the recommendations of the manufacturer (Qiagen). In the expression plasmid used, pPICZaB, three restriction sites are provided for linearization. Since Sad and BstXI cut within the QC cDNA, PmeI was chosen for linearization. 20-30 p.g plasmid DNA was linearized with PmeI, precipitated by ethanol, and dissolved in sterile, deionized water. 10,ug of the DNA was then applied for transformation of competent P. pastoris cells by electroporation according to the manufacturer's instructions (BioRad). Selection was done using plates containing 150 j.g/ml Zeocin. One transformation using the linearized plasmid yielded several hundred transformants.
In order to test the recombinant yeast clones for QC expression, recombinants were grown for 24 h in 10 ml conical tubes containing 2 ml BMGY. Afterwards, the yeast was centrifuged and resuspended in 2 ml BMNY containing 0.5 % methanol. This concentration was maintained by addition of methanol every 24 h up to 72 h. Subsequently, QC activity in the supernatant was determined. The presence of the fusion protein was confirmed by western blot analysis using an antibody directed against the 6xhistidine tag (Qiagen).
Clones that displayed the highest QC activity were chosen for further experiments and fermentation.
Large-scale expression in a fermenter Expression of the QC was performed in a 5 1 reactor (Biostat B, B. Braun biotech), essentially as described in the "Pichia fermentation process guidelines" (Invitrogen). Briefly, the cells were grown in the fermentation basal salts medium supplemented with trace salts, and with glycerol as the sole carbon source (pH 5.5). During an initial batch phase for about 24 h and a subsequent fed-batch phase for about 5 h, cell mass was accumulated. Once a cell wet weight of 200 g/l was achieved, induction of QC expression was performed using methanol applying a three-step feeding profile for an entire fermentation time of approximately 60 h. Subsequently, cells were removed from the QC-containing supernatant by centrifugation at 6000xg, 4 C for 15 mm. The pH was adjusted to 6.8 by addition of NaOH, and the resultant turbid solution was centrifuged again at 37000xg, 4 C for 40 mm. In cases of continued turbidity, an additional filtration step was applied using a cellulose membrane (pore width 0.45 jm).
Purification of 6 x histidine tagged QC expressed in P. pastoris The His-tagged QC was first purified by immobilized metal affinity chromatography (IMAC). In a typical purification,
S
1000 ml of culture supernatant were applied to a Ni2-1oaded Chelating Sepharose FF column (1.6 x 20 cm, Pharmacia), that.
was equilibrated with 50 mM phosphate buffer, pH 6.8, containing 750 mM NaC1, at a flow rate of 5 mi/mm. After washing with 10 column volumes of equilibration buffer and 5 column volumes of equilibration buffer containing 5 mM histidine, the bound protein was eluted by a shift to 50 mM phosphate buffer, pH 6.8, containing 150 mM NaC1 and 100 mM histidine. The resulting eluate was dialyzed against 20 mM Bis-Tris/IICI, pH 6.8, at 4 C overnight. Subsequently, the QC was further purified by anion exchange chromatography an a Mono Q6 column (BioRad), equilibrated with dialysis buffer.
The QC-containing fraction was loaded onto the column using a flow rate of 4 mi/mm. The column was then washed with equilibration buffer containing 100 mN NaC1. The elution was performed by two gradients resulting in equilibration buffer containing 240 mM and 360 mM NaCl in 30 or 5 column volumes, respectively. Fractions of 6 ml were collected and the purity was analyzed by SDS-PAGE. Fractions containing homogenous QC were pooled and concentrated by ultrafiltration. For long-term storage (-20 C), glycerol was added to a final concentration of 50. Protein was quantified according to the methods of Bradford or Gill and von Hippel (Bradford, M. M. 1976 Anal Biochem 72, 248-254; Gill, S.C. and von Hippel, P.H. 1989 Anal Biochem 182, 319-326.).
Expression and purification of QC in E. coil The construct encoding the QC was transformed into M15 cells (Qiagen) and grown an selective LB agar plates at 37 C.
Protein expression was carried out in LB medium containing 1% glucose and l ethanol at room temperature. When the culture reached an 0D600 of approximately 0.8, expression was induced with 0,1 mM IPTG overnight. After one cycle of freezing and thawing, cells were lysed at 4 C by addition of 2.5 mg/mi lysozyme in 50 mM phosphate buffer, pH 8.0, containing 300 mM NaC1 and 2 mM histidine for approximately 30 mm. The solution was clarified by centrifugation at 37000xg, 4 C for mm, followed by a filtration applying a glass frit (DNA separation) and two additional filtration steps applying cellulose filters for crude and fine precipitates. The supernatant (approx. 500 ml) was applied onto a Ni2-affinity column (1.6 x 20 cm) at a flow rate of 1 mi/mm. Elution of QC was carried out with 50 mM phosphate buffer containing 150 mM NaC1 and 100 mM histidine. The QC-containing fraction was concentrated by ultrafiltration.
Reference Example 2: MALDI-TOF mass spectrometry Matrix-assisted laser desorption/ionization mass spectrometry was carried out using the Voyager De-Pro (Applied Biosystems, Darmstadt) with a linear time of flight analyzer. The instrument was equipped with a 337 nm nitrogen laser, a potential acceleration source and a 1.4 m flight tube.
Detector operation was in the positive-ion mode. Samples (5 jil) were mixed with equal volumes of the matrix solution. For matrix solution we used sinapinic acid, prepared by solving mg sinapinic acid (Sigma-Aldrich) in 1 ml acetonjtrjle/O.1% TFA in water (1/1, v/v). A small volume ( 1 l) of the matrix-analyte-mixture was transferred to a probe tip.
For long-term testing of G1u1-cyclization, Ai-derived peptides were incubated in 100 /.Ll 0.1 M sodium acetate buffer, pH 5.2 or 0.1 M Bis-Tris buffer, pH 6.5 at 30 C.
Peptides were applied in 0.5 mM [A3-l1 a] or 0.15 mM [Ai3- 21a] concentrations, and 0.2 U QC was added all 24 hours. In case of AE3-21a, the assays contained 1 % DMSO. At different times, samples were removed from the assay tube, peptides extracted using ZipTips (Millipore) according to the manufacturer's recommendations, mixed with matrix solution (1:1 v/v) and subsequently the mass spectra recorded.
Negative controls contained either no QC or heat deactivated enzyme. For the inhibitor studies the sample composition was the same as described above, with exception of the inhibitory compound added (5 mM benzimidazole or 2 mM 1,10-phenanthroline).
Example 1: Preparation and Expression of human MCP-l in mammalian cel]. culture Cell lines and media Human neuroblastoma cell line SH-SY5Y, human embryonic kidney cell line HEK293 and human monocyte cell line THP-l were cultured in appropriate cell culture media (DMEM, 10% FBS for SH-SY5Y and HEK293), (RPMI164O, 10 % FBS for THP-l), in a humidified atmosphere of 5% CO2 (HEK293, THP-l) or 10% CO2 (SH-SY5Y) at 37 C.
Isolation of human MCP-l Full-length cDNA of human MCP-1 was isolated from SH-SY5Y cells using RT-PCR. Total RNA of SH-SYSY cells was reversely transcribed by SuperScript II (Invitrogen) and subsequently, human MCP-1 was amplified on a 1:12,5 dilution of generated cDNA product in a 25 l reaction with Pfu-DNA-Polymerase (Promega) using primers hNCP-l-l (sense) and hMCP-1-2 (antisense) (Table 1). The resulting PCR-product was cloned into vector pcDNA 3.1 using the Hindlil and NotI restriction sites and the sequence confirmed by DNA-sequencing.
Site-directed mutagenesis of human MCP-1 Deletions of the first (Q1) and first and second (LQlP2) amino acids of the mature human MCP-1 were generated by site-directed mutagenesis using primer iQl-1 and IXQ1-2 for f.Q1 (Table 1) and primers QlP2-l and LQ1P2-2 for LQ1P2 (Table 1). Parental DNA was digested with Dpn I. The pCDNA 3.1 plasmids with the deletions LxQl and LQlP2 of the mature human MCP-1 were transformed into E. coli JM1O9. Ampicillin-resistant clones were confirmed by sequencing and subsequently isolated for cell culture purposes using the EndoFree Maxi Kit (Qiagen).
Expression of N-terminal variants of human MCP-l in HEK293 cells For expression of N-terminal variants of human MCP-l, HEK293 cells were cultured in collagen I coated 6-well dishes and grown until 80% confluency, transfected using Lipofectamin2000 (Invitrogen) according to manufacturer's manual and incubated in the transfection solution for 5 hours. Afterwards, cells were allowed to recover in normal growth media over night. The next day, cells were incubated another 24 h in growth media. For analysis of efficacy of QC-inhibition, cells were incubated for 24 h in absence or presence of the specific inhibitor. After 24 h, the media containing the human MCP-1 variants were collected and investigated in a migration assay for chemotactic potency.
Furthermore, an aliquot of cell culture supernatant was stored at -80 C for quantification of human MCP-l concentration using a human MCP-l-ELISA (Pierce).
TransWell chemotaxis assay The chemotaxis assay was performed using 24 well Transwell plates with a pore size of 5 tm (Corning). Media containing the human MCP-l variants expressed in HEK293 were used as chemoattractant. To this avail, 600 l of the culture media of N-terminal human MCP-l variants was applied undiluted or in dilutions 1:3, 1:10 and 1:30 in RPMI164O to the lower chamber of the TransWell plate. Furthermore, undiluted media of HEK293 cells transfected with vector control were applied as negative control to the lower chamber. THP-l cells were harvested and resuspended in RPMI164O in a concentration of i*i06 cells / 100 zl and applied in 100,ul aliquots to the upper chamber. Cells were allowed to migrate towards the chemoattractant for 2 h at 37 C. Subsequently, cells from the upper chamber were discarded and the lower chamber was mixed with 50 l 70 mM EDTA in PBS and incubated for 15 mm at 37 C to release cells attached to the membrane. Afterwards, cells migrated to the lower chamber were counted using a cell counter system (Schärfe System). The chemotactic index was calculated by dividing cells migrated to the stimulus from cells migrated to the negative control.
Example 2: Investigations on the proteolytic degradation of human MCP-1(1..76) Methods N-terminal degradation by recombinant human DP4 Full length recombinant human MCP-l(176) (SEQ ID NO: 1) encoded by the nucleic acid sequence as shown in SEQ ID NO: 2, obtained in Example 1 above, starting with an N-terminal glutamine (Peprotech) was dissolved in 25 mM Tris/HC1 pH 7.6 in a concentration of 10 g/ml. The MCP-1 solution was either pre-incubated with recombinant human QC (0.0006 mg/mi) (obtained according to Reference Example 1 above, SEQ ID No: 3 for nucleic acid sequence and SEQ ID No: 4 for amino acid sequence) for 3 h at 30 C and subsequently incubated with recombinant human DP4 (0.0012 mg/mi) at 30 C (see Fig. 1) or incubated with DP4 without prior QC application. Resulting DP4 cleavage products were analyzed after 0 mm, 15 mm, 30 mm, lh, 4h and 24 h using Maldi-TOF mass spectrometry.
N-terminal degradation by human rheumatoid synovial fibroblast MMP-1 Human recombinant MCP-1 carrying an N-terminal glutaminyl instead of a pyroglutamyl residue (Peprotech) was dissolved in 25 mM Tris/HC1, pH 7.6, in a concentration of 10 jg/ml.
The MMP-1 proenzyme from human rheumatoid synovial fibroblasts (Calbiochem) was activated using 25 mM p-aminophenylmercuric acetate (APMA), dissolved in 0.1 N NaOH at 37 C for 3h in a APMA:enzyme-mixture of 10:1. MCP-1 was either pre-incubated with recombinant human QC (0.0006 mg/mi) for 3 h at 30 C and subsequently incubated with MMP-1 at 30 C or incubated with MMP-]. without prior QC application.
Resulting MMP-3. cleavage products were analyzed after 0 mm, mm, 30 mm, lii, 2h, 4h and 24 h using Maldi-TOF mass spectrome try.
N-terminal degradation by human rheumatoid synovial fibroblast MMP-1 and recombinant human DP4 Human recombinant MCP-]. starting with a N-terminal glutamine (Peprotech) was dissolved in 25 mM Tris/HC1, pH 7.6, in a concentration of 10 g/m1. MMP-1 proenzyme from human rheumatoid synovial fibroblasts (Calbiochem) was activated using 25 mM p-aminophenylmercuric acetate (APMA) dissolved in 0.1 N NaOH. The APMA:enzyme-mixture of 10:1 was incubated at 37 C for 3h. MCP-l solution was either pre-incubated with recombinant human QC (0.0006 mg/mi) for 3 h at 30 C and subsequently incubated with MMP-1 and DP4 at 30 C or incubated with MNP-l and DP4 without QC application.
Resulting MMP-l cleavage products were analyzed after 0 mm, mm, 30 mm, lh, 2h, 4h and 24 h using Maldi-TOF mass spectrometry.
Example 3: Effect of QC specific inhibitor 1-(3-(1H-imidazo].-l-yl)propyl) -3-(3, 4-dimethoxyphenyl) thiourea hydrochloride (in the following also designated as QCI) on cuff-induced accelerated atherosclerosis in ApoE3*L,ejden mice Time line male ApoE3*Leiden mice (age 12 weeks) were fed a mildly hypercholesterolemic diet for 3 weeks prior to surgical cuff placement.
After 3 weeks, the mice underwent surgical non-constricting cuff placement (day 0) and were divided into 2 groups, matched for plasma cholesterol levels. The mice either received control (acidified) drinking water or drinking water containing the QC specific inhibitor 1-(3-(1H-imidazol-l-yl)propyl) -3-(3,4 -dimethoxyphenyl) thiourea hydrochloride in a concentration of 2.4-mg/mi. 7 days after start of treatment, the inhibitor concentration was reduced to 1.2 mg/mi. 5 Mice of each group were sacrificed after 2 days for analysis of monocyte adhesion and infiltration, and 10 mice were sacrificed after 2 weeks for histomorphometric analysis to quantify the inhibition of accelerated atherosclerotic lesions and neointima formation.
Surgical procedure of cuff placement At the time of surgery, mice were anaesthetized with an intraperitoneal injection of 5 mg/kg Dormicum, 0.5 mg/kg Domitor and 0.05 mg/kg Fentanyl. This cocktail gives complete narcosis for at least one hour and can be quickly antagonized with Antisedan 2.5 mg/kg and Anexate 0.5 mg/kg.
A longitudinal 1 cm incision is made in the internal side of the leg and the femoral artery is dissected for 3 mm length from the femoral nerve and femoral vein. The femoral artery is looped with a ligature and a non-constrictive fine bore polyethylene tubing (0.4 mm inner diameter, 0. 8 mm outer diameter, length 2 mm) is longitudinally opened and sleeved loosely around the femoral artery. The cuff is closed up with two ligature knots. The skin is closed with a continued suture.
After surgery, the animals were antagonized and placed in a clean cage on top of a heating pad for a few hours.
Sacrifice of the animals For histological analysis, animals were sacrificed either 2 days or 14 days after cuff placement. After anaesthesia, the thorax was opened and a mild pressure-perfusion (100 mm}lg) with 4% formaldehyde was performed for 3 minutes by cardiac puncture. After perfusion, a longitudinal 2 cm incision was made in the internal side of the leg and the cuffed fernoral artery was harvested as a whole and fixed overnight in 4 formaldehyde and processed to paraffin.
Analysis of monocyte adhesion and MCP-l expression Adhesion of leukocytes in general and monocytes/rnacrophages in particular to the activated endothelium of the cuffed vessel wall was analyzed by microscopic analysis of cross sections harvested 2 days after cuff placement. The number of adhering and/or infiltrating leukocytes in general, identified as adhering cells at the luminal side of the vessel segment, and monocytes/macrophages in particular was counted and illustrated as cells per cross-section or as defined areas per cross section. Monocytes were identified by specific immunohistochernical staining by the polyclonal rabbit A1A31240 antibody, recognizing monocytes and macrophages. In addition on these sections a specific immunohistochemical staining for MCP-1 was performed.
Analysis of vascular remodeling and accelerated athero sclerosis Vessel wall remodeling, accelerated atherosclerosis and neoinitima formation were analyzed morphometrically in all mice sacrificed after 14 days. A full comparison between the two groups was performed for all relevant vessel wall parameters (neointima formation, vascular circumference (i.e. outward remodelling), media thickness, lumen stenosis).
Accelerated atherosclerosis was analyzed by immunohistochemical staining for macrophages and foam cells in the lesion area by AIA3124O antibody. Furthermore, these sections were also stained for MCP-l.
Results Preparation and Expression of human MCP-]. in mammalian cell culture Amplification of human MCP-l from human neuroblastoma cell line SH-SY5Y RNA resulted in a PCR-product of 300 bp.
Sequencing of the isolated cDNA revealed a silent single nucleotide polymorphism of codon 105 coding for cysteine 35.
Expression of human MCP-1 variants in HEK293 leads to elevated levels within cell culture supernatant as monitored by human MCP-l ELISA. Thereby, the level between the expressions of MCP-1 (WT) and MCP-l (1Ql) (Figure 5C), and MCP-l (WT) in absence or presence of 10 /M l-(3-(lH-imidazol-l-yl)propyl) -3-(3, 4-dimethoxyphenyl) thiourea hydrochloride (Figure 7A) are not significantly changed. However, the expression of MCP-l (rQlP2) is reduced by 28 % compared to MCP-a. (WT). The supernatant was collected and applied in TransWell migration assays (see Figures 4 and 5 C and D in this regard).
TransWell chemotaxis assay Purified human MCP-1 displays a bell-shaped chemotactic dose response curve, when attracting, e.g. monocytes, showing an optimum at approx. 1-50 ng/ml. Therefore, the generated cell culture supernatants containing MCP 1 variants were sequentially diluted in order to achieve the optimal working concentration of MCP-1 for chernotaxis assay attracting THP-l monocytes.
After expression of MCP-l (WT) and MCP-1 (Ql), the concentrations of MCP-1 variants did not significantly differ (Figure 5C). Application of MCP-l (WT) to the chemotaxis assay led to a chemotactic response of THP-l cells (Figure SD), implied by the elevated chemotactic index. However, MCP- 1 (LQl) failed to induce chemotaxis of THP-l (Figure SD) suggested by a chemotactic index of approx. 1. These results support previous results, that N-truncated MCP-l is inactive.
This finding is further substantiated by the inability of MCP-l (tiQlP2) to induce chemotaxis of THP-l cells (Figure 6B). Expression of MCP-1 (WT) in HEK293 cells has no influence on MCP-l concentration in absence or presence of chemotactic cytokines (chemokines). However, the application of chemokines leads to significantly lower chetnotaxis of THP 1 cells at dilutions 1:3 and 1:10 (Figure 7B) . This suggests a prevention of N-terminal pGlu-formation of MCP-l (WT) by QC-specific inhibitor l-(3-(1H-imidazol-1-yl)propyl)-3-(3,4-dimethoxyphenyl)thiourea hydrochloride and, therefore, an inactivation of MCP-l (WT), either by N-terminal proteolytic degradation or by the sole prevention of pGlu formation.
Investigations on the proteolytic degradation of human MCP 1(1-76)Within the circulation, MCP-1 is protected by a N-terminal pGlu-residue, which confers resistance against N-terminal cleavage by aminopeptidases, e.g. DP4. As a result of QC inhibitor administration, the unprotected N-terminus is readily cleaved by DP4. The N-terminal truncation, in turn, leads to inactivation of human MCP-1 (Figure 5 and 6). MMP-1 inactivates mature MCP-1 by cleavage of the 4 N-terminal amino acids (pE/Q-P-D-A). The reaction is independent from the presence of a N-terminal pGlu residue. This process reflects the situation of MCP-1 inactivation within the circulation. The resulting cleavage product MCP 1(5..76) has been shown to be present within plasma and resembles a naturally occurring CCR2 receptor antagonist. The present experiments point to the finding that MrIP-1 cleavage is slightly faster in case of a N-terminal glutamine residue (Figure 2A: 2h, 4h vs. 2B: 2h, 4h). Furthermore, incubation of human MCP-1 carrying an N-terminal Gln residue (Figure 3A) with human DP4 and human MMP-1 shows an accelerated degradation in comparison to pGlu-MCP-l (Figure 3E).
S
Taken together, the results imply that the N-terminal pGlu formation represent a mechanism of protection, conferring resistance against N-terminal degradation by post-proline cleaving enzymes, e.g. DP4, aminopeptidases and, as implied by the results with MMP-l, to a certain extent also endoproteases. Prevention of N-terminal pGlu formation by QC inhibitor application leads results in a faster inactivation human MCP-l.
Analysis of vascular remodeling and accelerated atherosclerosis in ApoE3*Leiden mice Treatment of cuff-induced accelerated atherosclerosis in ApoE3*Leiden mice had no effect on the total area within the outer diameter of the vessel segment (Figure 8A) and no statistically significant effect on the remaining lumen (Figure 8 B), although a slight increase in the remaining lumen can be observed. However, 1-(3-(1H-imidazol-l-yl)propyl) -3-(3,4 -dimethoxyphenyl) thiourea hydrochloride shows a profound reduction of 40 on the percentage of lumen stenosis (Figure 9A) and 45 reduction of the area of neointima formation (Figure 9B). Both values are statistically significant. Furthermore, the inhibitor also reduced the area of the media (Figure 10 A) and the intima / media ratio (Figure lOB), although the reduction in intima / media ration lacks statistically significance (P<O.l02) The analysis of the cellular composition in the specific vessel wall layers shows no differences in relative contribution of smooth muscle cells and macrophages/foam cells to the composition of both the media and the adventitia after 2 days and 14 days (Figure 15). Although one could expect a more specific effect on Tnonocyte/macrophage content in the vessel wall due to the effect of 1-(3-(1H-imidazol-1-yl)propyl) -3-(3,4 -dimethoxyphenyl) thiourea hydrochloride on MCP-1, and therefore on monocyte attraction, it should be noted that MCP-]. also has a direct effect on smooth muscle cell proliferation as recently has been discovered and published.
Analysis of monocyte adhesion and MCP-1 expression Treatment of the mildly hypercholesterolemic ApoE3*Leiden mice (plasma cholesterol levels 12-15 mM) with l-(3-(1H-imidazol-l-yl)propyl) -3-(3, 4-dimethoxyphenyl) thiourea hydrochloride resulted in a profound reduction of total adhering cells by 45, (p < 0.05) after 2 days. Specific analysis of adhering monocytes revealed an even stronger reduction of 67 (p < 0.05) to the treated cuffed vessel segments (Figure 11).
MCP-l expression was reduced in the vessel segments of l-(3-(1H-imidazol-1-yl)propyl) -3-(3, 4-dimethoxyphenyl)thiourea hydrochloride treated mice 2 days after surgery, the moment of the highest elevation of MCP-1 expression in the model used (Figure 12, 13A, 14A). These results indicate that early after vascular injury within the lesions a reduction of MCP-i expression can be detected in both the media and the intima (i.e inside the Larnina elastica interna) of the vessel wall segment, when 1-(3-(1H-imidazol-1-yl)propyl)-3-(3,4-dimeth-oxyphenyl)thiourea hydrochloride is administered. Analysis of the relative area of the cross sections positive for MCP-1 revealed a 52 (P=0.01) reduction of MCP 1 expression in the media and a 36 (P=0.00l) reduction in the intima (Figure 14A). Analysis of the absolute area positive for MCP-1 (expressed in im2 positive per cross section) reveals a similar reduction of MCP-3. expression in the media (4l6 reduction, p=O.O9) and the intima (40 reduction, p=0.05), although the reduction within the media is statistically not significant (Student's T-test) (Figure 13A).
At the later time point of 14 days, when the neointima formation / accelerated atherosclerosis has progressed, the overall MCP-l expression is lower than observed for the early time point and in contrast, no reduction of MCP-l expression can be monitored, in the media or in the neointima (Figure l3B, 14B) suggesting an effect of l-(3-(1H--imidazol-1-yl)propyl) -3-(3,4 -dimethoxyphenyl) thiourea hydrochloride only for the time of strong induction of MCP-l.
Taken together, these data indicate that oral dosing of l-(3-(lH-imidazol-l--yl)propyl) -3-(3, 4-dimethoxyphenyl) thiourea hydrochloride has a beneficial effect on post interventional vascular remodelling and accelerated atherosclerosis in the ApoE3*Lejden cuff model.
Table 1: Utilized primers Primer Sequence (5'43') Application SEQ ID NO Isolation of hMCP-1-1 ATAT AAGCU ATGAAAGTCTCTGCCGCCCTC human MCP-1 5 Isolation of hMCP-1-2 ATAT GCGGCCGC TCAAGTCTrCGGAGITTGGG human MCP-1 6 Site-directed iQ1-1 CAUCCCCAAGGGCTCGCTCCAGATGCAATCAATGCC mutagenesis AQI 7 Site-directed Q1-2 GGCATrGATrGCATCTGGAGCGAGCCCTTGGGGAATG mutagenesis AQI 8 Site-directed Q1 P2-I CATTCCCCAAGGGCTCGCTGATGCAATCAATGCCCCAG mutagenesis 9 zQ1P2 Site-directed Q1 P2-2 CTGGGGCAUGATrGCATCAGCGAGCCCTTGGGGAATG mutagenesis 10 zQ1P2 1-(3-(1H-imidazol-1-yl)propyl) -3-(3,4-dimethoxyphenyl)thiourea hydrochloride 0" SL%J
H H
N H-Cl

Claims (26)

S CLAIMS
1. Inhibitor of a glutaminyl peptide cyclotransferase f or the treatment and/or prevention of a disease or condition, selected from the group of inflammatory diseases selected from a. neurodegenerative diseases, e.g. mild cognitive impairment (MCI), Alzheimerts disease, neurodegeneration in Down Syndrome, Familial British Dementia, Familial Danish Dementia, multiple sclerosis, b. chronic and acute inflammations, e.g. rheumatoid arthritis, atherosclerosis, restenosis, pancreatitis, c. fibrosis, e.g. lung fibrosis, liver fibrosis, renal fibrosis, d. cancer, e.g. cancer/hemangioendothelioma proliferation, gastric carcinomas, e. metabolic diseases, e.g. hypertension, f. and other inflammatory diseases, e.g. neuropathic pain, graft rejection/graft failure/graft vasculopathy, HIV infections/AIDS, gestosis, tuberous sclerosis.
2. Inhibitor according to claim 1 which is l-(3-(1H-imidazol-l-yl)propyl) -3-(3, 4-dimethoxyphenyl) thiourea hydrochloride.
3. Inhibitor according to claim 1 or 2 above, wherein the disease is a neurodegenerative disease, e.g. mild cognitive impairment (MCI), Alzheimer's disease, neurodegeneration in Down Syndrome, Familial British Dementia, Familial Danish Dementia, multiple sclerosis.
S
4. Inhibitor according to any of claims 1 to 3 above, wherein the inhibitor is administered in combination with a further agent, selected from the group consisting of nootropic agents, neuroprotectants, antiparkinsonian drugs, amyloid protein deposition inhibitors, beta amyloid synthesis inhibitors, antidepressants, anxiolytic drugs, antipsychotic drugs and anti-multiple sclerosis drugs.
5. Use of a glutaminyl peptide cyclotransferase inhibitor for the treatment and/or prevention of a disease or condition selected from the group of inflammatory diseases selected from a. neurodegenerative diseases, e.g. mild cognitive impairment (MCI), Alzheimer's disease, neurodegeneration in Down Syndrome, Familial British Dementia, Familial Danish Dementia, multiple sclerosis, b. chronic and acute inflammations, e.g. rheumatoid arthritis, I restenosis, pancreatitis, c. fibrosis, e.g. lung fibrosis, liver fibrosis, renal fibrosis, d. cancer, e.g. cancer/hemangioendothelioma proliferation, gastric carcinomas, e. metabolic diseases, e.g. hypertension, f. and other inflammatory diseases, e.g. neuropathic pain, graft rejection/graft failure/graft vasculopathy, lily infections/AIDS, gestosis, tuberous sclerosis.
S
6. Use according to claim 5, wherein said inhibitor is 1-(3-(1}i-imidazol-l-yl)propyl)-3-(3,4-dimethoxyphenyl) thiourea hydrochloride.
7. Use according to claim 5 or 6, wherein the disease is a neurodegenerative disease, e.g. mild cognitive impairment (MCI), Alzheimer's disease, neurodegeneration in Down Syndrome, Familial British Dementia, Familial Danish Dementia, multiple sclerosis.
8. Use according to any of claims 5 to 7, wherein the inhibitor is administered in combination with a further agent, selected from the group consisting of nootropic agents, neuroptrotectants, antiparkinsonian drugs, amyloid protein deposition inhibitors, beta arnyloid synthesis inhibitors, antidepressants, anxiolytic drugs, antipsychotic drugs and anti-multiple sclerosis drugs.
9. Use of a glutaminyl peptide cyclotransferase inhibitor for the preparation of a medicament for treating and/or preventing a disease or conditions selected from the group of inflammatory diseases selected from a. neurodegenerative diseases, e.g. mild cognitive impairment (MCI), Alzheimer's disease, neurodegeneration in Down Syndrome, Familial British Dementia, Familial Danish Dementia, multiple sclerosis, b. chronic and acute inflammations, e.g. rheumatoid arthritis, atherosclerosis, restenosis, pancreatitis, c. fibrosis, e.g. lung fibrosis, liver fibrosis, renal fibrosis, d. cancer, e.g. cancer/hemangloendothelioma proliferation, gastric carcinomas, e. metabolic diseases, e.g. hypertension, f. and other inflammatory diseases, e.g. neuropathic pain, graft rejection/graft failure/graft vasculopathy, HIV infections/AIDS, gestosis, tuberous sclerosis.
10. Use according to claim 9, wherein said inhibitor is 1-(3-(1H-imidazol-l-yl)propyl) -3-(3,4-dimethoxyphenyl) thiourea hydrochloride.
11. Use according to claim 9 or 10, wherein the disease is a neurodegenerative disease, e.g. mild cognitive impairment (MCI), Alzheimer's disease, neurodegeneration in Down Syndrome, Familial British Dementia, Familial Danish Dementia, multiple sclerosis.
12. Use according to any of claims 9 to 11, wherein the inhibitor is administered in combination with a further agent, selected from the group consisting of nootropic agents, neuroprotectants, antiparkinsonian drugs, amyloid protein deposition inhibitors, beta amyloid synthesis inhibitors, antidepressants, anxiolytic drugs, antipsychotic drugs and anti-multiple sclerosis drugs.
13. Method of treatment and/or prevention of a disease or condition, selected from the group of inflammatory diseases selected from a. neurodegenerative diseases, e.g. mild cognitive impairment (MCI), Alzheimer's disease, neurodegeneration in Down Syndrome, Familial British Dementia, Familial Danish Dementia, multiple sclerosis, b. chronic and acute inflammations, e.g. rheumatoid arthritis, 1 restenosis, pancreatitis, c. fibrosis, e.g. lung fibrosis, liver fibrosis, renal fibrosis, d. cancer, e.g. cancer / hemangioendothelioma proliferation, gastric carcinomas, e. metabolic diseases, e.g. hypertension, f. and other inflammatory diseases, e.g. neuropathic pain, graft rejection/graft failure/graft vasculopathy, HIV infections/AIDS, gestosis, tuberous sclerosis, wherein an effective amount of a QC inhibitor is administered.
14. Method of treatment and/or prevention according to claim 13, wherein said inhibitor is l-(3-(1H-imidazol-l-yl)propyl) -3-(3, 4-dimethoxyphenyl) thiourea hydrochloride.
15. Method of treatment and/or prevention according to claim 13 or 14, wherein the disease is a neurodegenerative disease, e.g. mild cognitive impairment (MCI), Alzheimer's disease, neurodegeneration in Down Syndrome, Familial British Dementia, Familial Danish Dementia, multiple sclerosis.
16. Method of treatment and/or prevention according to any of claims 13 to 15, wherein the inhibitor is administered in combination with a further agent, selected from the group consisting of nootropic agents, neuroprotectants, antiparkinsonian drugs, amyloid protein deposition inhibitors, beta amyloid synthesis inhibitors, antidepressants, anxiolytic drugs, antipsychotic drugs and anti-multiple sclerosis drugs.
17. Use according to any of claims 5 to 12 above, wherein the disease and/or condition afflict a human being.
18. The method of any of claims 13 to 16, wherein the disease and/or condition afflicts a human being.
19. Diagnostic assay, comprising an inhibitor of a glutaminyl peptide cyclotransferase.
20. Diagnostic assay according to claim 19, wherein said inhibitor is 1-(3-(1H-imidazole-1-yl)propyl)-3-(3,4-dimethoxy-phenyl) thiourea hydrochloride.
21. Method of diagnosing any one of the diseases and/or conditions as defined in item 1 above, comprising the steps of - collecting a sample from a subject who is suspected to be afflicted with said disease and/or condition, -contacting said sample with an inhibitor of a glutaminyl peptide cyclotransferase, and -determining whether or not said subject is afflicted by said disease and/or condition.
22. The method of claim 21, wherein said subject is a human being.
23. The method of claim 21 or 22, wherein said inhibitor is 1-(3-(1H-imidazole-1-yl)propyl) -3-(3,4--dimethoxy-phenyl) thiourea hydrochloride.
24. The method of any of claims 21 to 23, wherein said sample is a blood sample, a serum sample, a sample of cerebrospinal liquor or a urine sample.
25. Diagnostic kit for carrying out the method of claims 21 to 24 comprising as detection means the diagnostic assay of item 19 or 20 and a determination means.
26. Pharmaceutical composition, comprising the inhibitor according to any of items 1 to 4.
GB0704014A 2007-03-01 2007-03-01 New use for inhibitors of glutaminyl peptide cyclotransferinase Withdrawn GB2447017A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
GB0704014A GB2447017A (en) 2007-03-01 2007-03-01 New use for inhibitors of glutaminyl peptide cyclotransferinase

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
GB0704014A GB2447017A (en) 2007-03-01 2007-03-01 New use for inhibitors of glutaminyl peptide cyclotransferinase

Publications (2)

Publication Number Publication Date
GB0704014D0 GB0704014D0 (en) 2007-04-11
GB2447017A true GB2447017A (en) 2008-09-03

Family

ID=37965769

Family Applications (1)

Application Number Title Priority Date Filing Date
GB0704014A Withdrawn GB2447017A (en) 2007-03-01 2007-03-01 New use for inhibitors of glutaminyl peptide cyclotransferinase

Country Status (1)

Country Link
GB (1) GB2447017A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3632433A4 (en) * 2017-05-26 2021-03-03 Obschestvo S Ogranichennoi Otvetstvennostiyu "Pharmenterprises" Novel glutaminyl cyclase inhibitors and the use thereof in treatment of various diseases
RU2791703C2 (en) * 2018-08-09 2023-03-13 Общество С Ограниченной Ответственностью "Фарминтерпрайсез" New glutamine cyclase inhibitor and its application

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040224875A1 (en) * 2003-05-05 2004-11-11 Stephan Schilling Inhibitors of glutaminyl cyclase
WO2005039548A2 (en) * 2003-10-15 2005-05-06 Probiodrug Ag Use of effectors of glutaminyl and glutamate cyclases
WO2005049027A2 (en) * 2003-11-03 2005-06-02 Probiodrug Ag Combinations useful for the treatment of neuronal disorders
WO2005075436A2 (en) * 2004-02-05 2005-08-18 Probiodrug Ag Novel inhibitors of glutaminyl cyclase

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040224875A1 (en) * 2003-05-05 2004-11-11 Stephan Schilling Inhibitors of glutaminyl cyclase
WO2005039548A2 (en) * 2003-10-15 2005-05-06 Probiodrug Ag Use of effectors of glutaminyl and glutamate cyclases
WO2005049027A2 (en) * 2003-11-03 2005-06-02 Probiodrug Ag Combinations useful for the treatment of neuronal disorders
WO2005075436A2 (en) * 2004-02-05 2005-08-18 Probiodrug Ag Novel inhibitors of glutaminyl cyclase

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
"The first potent inhibitors for human glutaminyl cyclase: synthesis and structure-activity relationships" *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3632433A4 (en) * 2017-05-26 2021-03-03 Obschestvo S Ogranichennoi Otvetstvennostiyu "Pharmenterprises" Novel glutaminyl cyclase inhibitors and the use thereof in treatment of various diseases
US11845740B2 (en) 2017-05-26 2023-12-19 LTD “Valenta-Intellekt” Glutaminyl cyclase inhibitors and the use thereof in treatment of various diseases
RU2791703C2 (en) * 2018-08-09 2023-03-13 Общество С Ограниченной Ответственностью "Фарминтерпрайсез" New glutamine cyclase inhibitor and its application

Also Published As

Publication number Publication date
GB0704014D0 (en) 2007-04-11

Similar Documents

Publication Publication Date Title
US8338120B2 (en) Method of treating inflammation with glutaminyl cyclase inhibitors
CA2679446C (en) New use of glutaminyl cyclase inhibitors
US8809010B2 (en) Method for prophylactic treatment of alzheimer&#39;s disease using inhibitors of glutaminyl cyclase and glutamate cyclases
US7803810B2 (en) Inhibitors
EP2142513B1 (en) Nitrovinyl-diamine derivatives as glutaminyl cyclase inhibitors
CA2789440C (en) Heterocyclic inhibitors of glutaminyl cyclase (qc, ec 2.3.2.5)
US9034907B2 (en) Inhibitors of glutaminyl cyclase
KR101694556B1 (en) Novel inhibitors
EP2142515B1 (en) Nitrovinyl-diamine derivatives as glutaminyl cyclase inhibitors
US20080260688A1 (en) Novel inhibitors of glutaminyl cyclase
US8530670B2 (en) Inhibitors
KR20150128928A (en) Novel inhibitors
US20120184518A1 (en) Methods of treating or preventing an inflammatory disease or condition using glutaminyl cyclase inhibitors
GB2447017A (en) New use for inhibitors of glutaminyl peptide cyclotransferinase
JP2014148508A (en) Novel use of glutaminyl cyclase inhibitor
Figure PatentApplication Publication Mar. 12, 2009 Sheet 1 of 49 US 2009/0068699 A l

Legal Events

Date Code Title Description
WAP Application withdrawn, taken to be withdrawn or refused ** after publication under section 16(1)