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  Pharmaceutical Patents  

 

Title:  Method for the prevention of malaria infection of humans by hepatocyte growth factor antagonists
United States Patent: 
7,670,631
Issued: 
March 2, 2010

Inventors:
 Mota; Maria M. (Lisboa, PT), Rodriguez; Ana (Great Neck, NY), Giordano; Silvia (Turin, IT), Rodrigues; Margarida Cunha (Parede, PT)
Assignee:
  ALFAMA--Investigacao e Desenvolvimento de Produtos Farmac uticos, Lda. (Porto Salvo, PT)
Appl. No.:
 10/795,456
Filed:
 March 9, 2004


 

Covidien Pharmaceuticals Outsourcing


Abstract

A method for the prevention of infection of humans by plasmodium parasites is provided. The method consists of the application of compounds that interfere with the infection of hepatocytes by Plasmodium viax.

Description of the Invention

SUMMARY OF THE INVENTION

This invention aids in fulfilling these needs in the art. The evasiveness of malaria has made a definitive treatment difficult. Presented here is an agent and a method capable of preventing the spread or acquisition of malaria infection and of assisting in the prevention and treatment of such infection.

More particularly, this invention provides a method for inhibiting the activity of malaria in vivo, wherein the method comprises administering to a human host an antimalarial agent, which is capable of exhibiting a protective effect by preventing the initial replication of malaria parasites in the liver of an infected host such as humans. The antimalarial agent is comprised of at least one inhibitor of HGF activity, and optionally, an antimalarial drug, such as primaquine. The antimalarial agent is administered to the human in an amount sufficient to prevent or at least inhibit infection of hepatocytes by malaria in vivo or to prevent or at least inhibit replication or spread of a malaria parasites in vivo.

The present invention relates to the ability of hepatocytes to support the growth of parasites that cause the human disease malaria. Plasmodium parasites that cause human disease are Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale. More specifically the invention reveals Met activation and downstream signals to be essential for the establishment of plasmodium infection. It has previously been known that Plasmodium sporozoites pass through several hepatocytes before they are able to establish a vacuole in hepatocytes in which they divide. It has not previously been known that the passage of sporozoites through hepatocytes is associated with the production of a well known cytokine, referred to as hepatocyte growth factor (HGF). HGF is known to be released as an inactive, single chain protein. It is activated by proteolytic cleavage that forms a disulfide bridge linked heterodimer. The heterodimer binds to and activates the receptor protein tyrosine kinase Met. The cytoplasmic domain of activated Met recruits a variety of proteins that transmit signals through several distinct pathways. These signals result in a variety of responses such as cell scattering, proliferation, tubulogenesis and invasive growth. The present invention reveals a novel Met mediated response of hepatocytes to HGF. Hepatocytes are rendered permissive by HGF to the invasion by sporozoites in a manner that allows their proliferation within a vacuole.

The present invention also provides a novel strategy for the prevention of plasmodium infections.

In preferred embodiments plasmodium infection of hepatocytes is prevented by molecules which interfere with HGF production by wounded hepatocytes.

Also suitable for the prevention of infection are molecules, which interfere with the proteolytic cleavage of HGF into its active form and molecules which sequester HGF and thereby prevent it from binding to hepatocytes via its receptor Met.

In another aspect, the invention reveals Met to be a target for drugs that prevent malaria infection.

In a preferred embodiment of the invention, malaria infection is prevented by molecules, which interfere with the binding of HGF to its receptor Met. Such molecules are antibodies specific for HGF which block its binding site for Met. Also in a preferred embodiment of the invention such molecules are antibodies against Met, or fragments of such antibodies, which block HGF binding but do not activate Met. In another embodiment of the invention such molecules are oligonucleotides (aptamers) which bind to Met but do not activate Met. In yet another embodiment of the invention such molecules are HGF variants that interfere with Met activation by HGF. Such variants include, but are not restricted to NR4.

In another aspect of the invention, plasmodium infection of hepatocytes is prevented by drugs, which interfere with signal transduction by activated Met. In a preferred embodiment of the invention such drugs are protein tyrosine inhibitors. An example of such a drug is genistein.

In another preferred embodiment such drugs are selective inhibitors of the protein tyrosine kinase Met. In preferred embodiments these inhibitors are small molecular weight compounds and are administered by the oral route or as suppositories.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "antimalarial agent" means a composition comprising one or more inhibitors of HGF activity. The term "inhibitor of HGF activity" means one or more compounds independently selected from HGF receptor antagonists, inhibitors of HGF-mediated signal transduction, and protein tyrosine kinase inhibitors. The inhibitor of HGF activity can be employed alone or in combination with each other. The inhibitors of HGF activity optionally can be combined with one or more known antimalarial drugs to form the antimalarial agent of the invention.

The present invention relates to the invasion of hepatocytes by malaria parasites. After transmission by a mosquito bite to a human host, malaria sporozoites find their way to the liver where each sporozoite can give rise to as many as 10,000 merozoites that are released into the blood. The invasion of hepatocytes is an obligatory step of malaria infection. Sporozoites can invade hepatocytes through disruption of the plasma membranes followed by parasite migration through the cells or, like intracellular bacteria and other parasites, through the formation of an internalization vacuole around the invading pathogen. Initially sporozoites pass through hepatocytes without forming an internalization vacuole. Sporozoites enter hepatocytes by breaching their plasma membranes, traverse the cytosol and leave the host cell which either dies or succeeds to repair the membrane. The molecular mechanisms underlying the passage of plasmodium through hepatocytes and the subsequent establishment of a parasitophorus vacuole are poorly understood. Vacuole formation by Plasmodium yoelii and Plasmodium falciparum, but not by the rodent malaria parasite Plasmodium berghei, is dependent on CD81, a tetraspin protein expressed by hepatocytes. CD81 is known to be a receptor for hepatitis C virus but it does not appear to interact with any ligand on the surface of sporozoites. Its role in hepatocyte invasion by certain plasmodium species remains to be elucidated [Silvie et al., Nature Medicine 9:93-96, (2003)]. Interestingly, sporozoites must traverse the cytosol of several cells before invading a hepatocyte by formation of a parasitophorous vacuole, which is indispensable for the differentiation into the next infective stage [Mota et al., Science 291:440-42, 2001)]. This finding suggests that hepatocytes wounded by sporozoites release one or more infection susceptibility inducing factors (ISIF) that render neighbouring hepatocytes susceptible to infection. An important aspect of the present invention is the discovery that a protein known as hepatocyte growth factor serves as ISIF in malaria infections.

I. HGF and its Receptor Met

Hepatocyte growth factor was discovered as a mitogen for hepatocytes [Michalopolous et al, Cancer Res., 44:441-4419 (1984); Rusasel et al J. Cell Physiol., 119:183-192 (1984); Nakamura et al., Biochem. Biophys. Res. Comm., 122:1450-1459 (1984)] and independently as a scatter factor that promotes the dissociation of epithelial cells and vascular endothelial cells [Stocker et al, Nature 327, 239-242 (1987)]. For simplicity the factor is referred to as HGF. HGF was first purified from the serum of hepatectomized rats [Nakamura et al., Biochem. Biophys. Res. Comm., 122:1450-1459 (1984)] and subsequently from rat platelets [Nakamura et al. Proc. Natl. Acad. Sci. USA, 83:6849-6493 (1986)] and from human plasma [Gohda et al., J. Clin. Invest. 81:414-419 (1988)]. The cDNAs encoding rat HGF, human HGF and a naturally occurring variant referred to as "delta5 HGF" were cloned [Miyazawa et al., Biochem. Biophys. Res. Commun., 163:967-973 (1989); Nakamura et al. Nature 342:440-443 (1989); Seki et al., Biochem. Biophys. Res. Commun., 172:321-327 (1990); Tashiro et al., Proc. Natl. Acad. Sci. USA, 87:3200-3204 (1990); Okajima et al., Eur. J. Biochem., 193:375-381 (1990)]. Human HGF consists of an .alpha.-subunit of 440 amino acids (M, 62 kDa) and a .beta.-subunit of 234 amino acids (M, 34 kDa). It is produced as biologically inactive pro-HGF (728 aa) that is cleaved by proteases between Arg494 and Val495 to form a disulfide-linked heterodimer. The 62-kDa .alpha.-subunit contains an N-terminal hairpin domain (about 27 aa) followed by four canonical kringle domains, which are 80-aa double-looped structures stabilized by three S-S bridges. The first kringle domain binds to a protein tyrosine kinase receptor, Met, which is described more in detail below. The hairpin loop and second kringle domain binds membrane-associated heparan sulfate proteoglycans with low-affinity. The 34 kDa .beta.-subunit contains a serine protease-like domain very similar to that of the serine protease blood clotting factors but which has no protease activity. HGF shows 38% overall sequence identity with plasminogen and 45% identity with another cytokine known as macrophage stimulating protein (MSP). HGF binds to a protein tyrosine kinase receptor referred to as Met, while its close relative, MSP, binds to another protein tyrosine kinase receptor known as Ron.

HGF is secreted as single chain Pro-HGF. This HGF precursor is bound to proteoglycans that are associated with the extracellular matrix or with cell surfaces in the vicinity of the producer cells. Activation of the single chain precursor into the biologically active heterodimer by proteolytic cleavage between Arg494 and Val495 is a tightly controlled process [(for a review see Kataoka et al., Life XY 1:1036-1042 (2001)].

The enzymes first implicated in HGF activation were urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). Subsequently three additional HGF activating enzymes have been identified, namely coagulation Factor XIIa, membrane type serine protease-1 (MT-SP1) also known as matriptase and HGF activator (HGFA). Each of these enzymes is under the control of endogenous inhibitor proteins. HGFA is the most effective HGF cleaving enzyme. Like HGF, HGFA is a heterodimer that is generated from a single chain Pro-HGFA by cleavage following Arg407. One of the HGFA cleaving enzymes is thrombin, an enzyme that is activated in injured tissues through the coagulation cascade. Active HGFA heterodimers are not inhibited by the major serum proteinase inhibitors, but are under the control of two proteins, HGA inhibitor type I (HAI-1) and HGA inhibitor type 2 (HAI-2), the latter being identical with placental bikunin (PB). HAI-1 is upregulated in injured and regenerating tissues. It is expressed at the cell surface where it binds and inhibits HGFA. Cytokines such as IL-1.beta. induce shedding of the HGFA/HAI-1 complex by TNF-.alpha. converting enzyme (TACE) and the TACE-like metalloproteinases of the ADAM (a disintegrin and metalloproteinase) family of proteins. After shedding HGFA dissociates from HAI-1 and is then able to activate HGF. Thus, HAI-1 is not only an inhibitor but also a specific acceptor of mature HGFA, acting as a reservoir of this enzyme on the cell surface. HAI-1 is described in U.S. Pat. No. 6,465,622B2, published in Oct. 15, 2002, wherein it is claimed for its use as control factor for HGF and HGFA.

The HGF receptor Met was originally discovered as a component of an oncogenic fusion protein that was generated in a carcinogen treated sarcoma cell line [Cooper et al., Nature, 311:29-33 (1984)]. In normal cells the primary Met transcript produces a 150 kDa polypeptide that is glycosylated and then cleaved to form a S-S linked heterodimer. HGF and its receptor Met is subject of U.S. Pat. No. 5,648,273, published in July 15, which claims the use of the ligand-receptor for the diagnosis of proliferative disorders and diseases such as hepatitis and hepatocarcinogenesis.

The Met heterodimer consists of a .beta.-subunit, that is highly glycosylated and entirely extracellular and a .alpha.-subunit with a large extracellular region and an intracellular tyrosine kinase domain. Met is a member of a superfamily of receptor tyrosine kinases (RTKs). The superfamily is divided into at least 19 families including the Her family (EGFR, Her 2, Her 3, Her 4), the insulin receptor family (insulin receptor, IGF-1R, insulin-related receptor), the PDGF receptor family (PGFRa and b, CSF-R, kit, Flk2), the Flk family (Flk-1, Flt-1, Flk-4), the FGF receptor family (FGF-R1, 2, 3, and 4) and others. Met and its close relative Ron form a distinct family of receptors for the ligands HGF and macrophage stimulating protein (MSP), respectively.

Upon HGF binding, c-Met undergoes autophosphorylation of specific tyrosine residues. While phosphorylation of Tyr1234 and Tyr1235 located within the activation loop of the tyrosine kinase domain activates the intrinsic kinase activity of c-Met, phosphorylation of Tyr1349 and Tyr1356 in the C-terminus generates a multisubstrate docking site for signal transducing proteins such as phosphotidylinositol 3-kinase (PI3K), phospholipase C-.gamma. PLC-.gamma.), src, Stat3, Grb2 and the Grb2 associated docking protein Gab1. Grb2 also interacts with Met through the adaptor protein Shc. Grb2 recruits the Ras nucleotide exchange protein SOS which activates the Ras-MAPK signaling pathway. Thus, the docking of signal transducers to the activated Met receptor initiate signaling through a variety of pathways. The c-terminal 26 amino acids of Met provide not only docking sites for signal transducers, but also regulate the enzymatic activity of Met. A mutation (M1250T) in the kinase domain bypasses the regulatory role of the C-terminal amino acids [Gual et al., Oncogene 20:5493-502 (2001)].

A variety of responses to HGF have been described in different Met expressing target cells. These responses include proliferation, programmed cell death, dissociation of cells, mutual repulsion, movement of cells through the extracellular matrix and branching morphogenesis. During embryogenesis interactions between HGF producing, mesenchymal cells and Met expressing, epithelial cells appear to be involved in the formation of a neuronal tissues. HGF gene knock out mice as well as Met gene knock out mice exhibit defects in the development of the placenta, liver and muscles and die between E13.5 and 15.5 [(Schmidt et al., Nature 373:699-702 (1995); Uehara et al., Nature 373:702-705 (1995); Bladt et al., Nature 376:768-771 (1995)]. In adult life, HGF-Met interactions are involved in wound healing, angiogenesis, and tissue regeneration. Not surprisingly Met activation by HGF has been implicated in the growth, invasion and metastasis of tumors. The biology of HGF and of its receptor Met is well described in several review articles [Maulik et al., Cytokine & Growth Factor Reviews, 13: 41-59 (2002)] and in numerous publications referenced therein.

Based on their biological properties, both HGF and HGF antagonists have been proposed to be useful for the treatment of a variety of diseases. The production of HGF and its therapeutic applications have been claimed in several patents. HGF has been isolated from blood on the basis of its high affinity for heparin (U.S. Pat. No. 5,004,805 published Apr. 2, 1991). Pegylation of HGF prolongs its clearance, reduces the dose required, and is thought to ameliorate side effects of HGF therapy (U.S. Pat. No. 5,977,310, published in Nov. 2, 1999). HGF levels may be increased by HGF degradation inhibiting polysaccharides such as heparin, hyaluronic acid, dextran, dextran sulfate, heparan sulfate, dermatan sulfate, keratan sulfate, chodroitin, or chondroitin sulfate (U.S. Pat. No. 5,736,506, published in Apr. 17, 1998). A HGF activating protease has also been claimed (U.S. Pat. No. 5,677,164, published in Oct. 14, 1997). Applications of HGF therapy include the treatment of arterial occlusive disease (U.S. Pat. No. 6,133,231, published in Oct. 17, 2000), the occlusive disease (U.S. Pat. No. 6,133,231, published in Oct. 17, 2000), the treatment of inflammatory bowel diseases (U.S. Pat. No. 6,319,899B1, published in Nov. 20, 2001), the enhancement of re-surfacing of blood vessels traumatized or damaged, for instance by vascular surgery or angioplasty (U.S. Pat. No. 6,133,234, published in Oct. 17, 2000). HGF has also been claimed to ameliorate side effects caused by commonly used immunosuppressants (U.S. Pat. No. 5,776,464, published in Jul. 7, 1998). Finally, the topical application of HGF gene containing vectors to blood vessels or other target organs has been described for a variety of therapeutic purposes (U.S. Pat. No. 6,248,722B1, published in Jun. 19, 2001). Met and downstream signal transduction pathways have long been regarded as attractive targets for cancer therapy. First, studies with tumor cell lines and tumor models in animals have shown that Met plays an important role in the invasive growth and in metastasis of cancer cells. Second, Met gene amplification has been observed in liver metastasis of colorectal carcinomas. Third, Met is overexpressed in several types of human tumors such as thyroid and pancreatic carcinomas. Fourth, germ line mutations in the Met gene are found in hereditary, papillary renal carcinoma, and somatic Met gene mutations are found in sporadic papillary carcinomas.

The present invention identifies a previously unknown function of the Met receptor, the inhibition of which represents a novel therapeutic application of Met antagonists. Signaling through Met renders hepatocytes permissive to productive invasion by malaria sporozoites. Met signaling is essential for the entry of sporozoites into hepatocytes via the formation of an internalization vacuole and/or the proliferation of sporozoites within vacuoles that are formed by the plasma membranes of hepatocytes. The discovery of this function of Met is the basis of a novel approach to the prevention of malaria infection. A further embodiment of the present invention is the use of compounds that prevent the establishment of a malaria infection by interfering with HGF mediated activation of Met or signaling events downstream of Met, that are involved in rendering hepatocytes permissive to the infection by malaria parasites. Several Met antagonists have been described in the literature and some have been claimed in patents for applications in the treatment of diseases that are caused at least in part by the excessive or aberrant function of Met. The previously claimed indications of Met antagonists are the treatment of malignant tumors. The potential application of Met antagonists for the treatment of infectious disease, and in particular of infections by malaria parasites becomes apparent through the present invention. The claim of the present invention is the use of Met antagonist for the prevention of human infections by malaria parasites. Known HGF antagonists are described in the following sections.

II. HGF Receptor Antagonists

A. HGF Variants

Various forms of HGF--both occurring naturally and generated by genetic manipulation of HGF encoding cDNA antagonize some or all Met functions. Uncleaved pro-HGF binds to but cannot activate Met. Several HGF isoforms are generated by differential splicing of primary HGF transcripts. These include NK1 (consisting of an N domain and the first kringle domain of HGF) and NK2 (consisting of an N domain and the first two kringle domains of HGF). Two additional variants discovered in the macaque endometrium and placenta, namely dNK1 and dNK2 are similar to the NK1 and NK2 isoforms, except that they encode proteins with a five amino acid deletion in the first kringle domain [Lindsey and Brenner, Mol Human Reprod. 8:81-87 (2002)]. NK1, and NK2 bind with high affinity to the HGF receptor Met and have been reported to act as HGF antagonists [Lokker and P. J. Godowski, J. Biol. Chem. 268: 17145-17150 (1993); Chan et al., Science 254:1382-1387 (1991)]. However, subsequent studies have shown that these HGF variants may act either as partial HGF agonists or as HGF antagonists depending on the cell context, the presence or absence of heparin, and the HGF function analyzed. In vivo studies with mice overexpressing transgenic HGF, NK1, NK2, HGF+NK1, or HGF+NK2 have revealed potential in vivo functions of HGF isoforms. Transgenic expression of HGF has a variety of phenotypic consequences such as enhanced liver growth, progressive glomerulosclerosis, disruption of olfactory mucosa, aberrant localization of muscle cells in the central nervous system and of melanocytes in the dermis and epidermis, precocious mammary lobuloalveolar development and susceptibility to tumor induction. Transgenic expression of NK1 produces a similar phenotype, while transgenic expression of NK2 exhibits none of the HGF and NK1 induced phenotypic characteristics. In HGF+NK2 bitransgenic mice NK2 antagonizes the pathological consequences of HGF overexpression and downregulates the subcutaneous growth of transplanted, Met expressing tumor cells. However, transgenic overexpression of NK2 promotes metastasis of these same tumor cells. Thus, NK2 antagonizes many of the responses to HGF, but shares with HGF the ability to dissociate (scatter) cells, a response that facilitates metastasis [Otsuka et al., Molecular and Cellular Biology 20:2055-2065 (2000)].

NK4, another HGF variant, is generated by a single cut digestion of HGF with elastase. NK4 contains the N terminal hairpin structure and four kringle domains. In contrast to NK1 and NK2, NK4 is a pure HGF antagonist [Date et al., FEBS Letters 420:1-6 (1997)]. Like the isolated HGF a chain, NK4 binds to Met but does not induce its autophosphorylation unless an isolated HGF .beta.-chain is added. Because of its ability to antagonize HGF, administration of the NK4 protein or NK4 gene transfer [Hirao et al., Cancer Gene Ther 9:700-7 (2002); Maehara et al., Clin Exp Metastasis 19:417-26 (2002)] is being evaluated as a novel approach to the treatment of Met expressing cancers. Single chain HGF variants similar to NK4, which have been engineered to be resistant against proteolytic cleavage are described in U.S. Pat. No. 5,879,910, published in Mar. 9, 1999, and in U.S. Pat. No. 5,580,963 published in Dec. 3, 1996.

B. Soluble Met receptors

A soluble form of Met is released from cultured endothelial cells, smooth muscle cells, and various tumor cell lines. The soluble receptor is thought to counteract the activation of cell surface associated Met by HGF. Met-IgG fusion proteins have been generated which retain the ability to bind HGF with high affinity and thus are able to neutralize HGF activity.

C. Angiostatin

Angiostatin, an inhibitor of angiogenesis, is a fragment of plasminogen that contains 3-4 kringles domains. The anti-angiogenic effects of angiostatin are thought to be based on its ability to inhibit ATPase on the endothelial cell surface, and to interfere with integrin functions and with pericelluar proteolysis. Recent research indicates that the anti-angiogenic activity of angiostatin is at least in part due to its ability to neutralize the effects of HGF [Wajih and Sane, prepublished online in Blood, Oct. 24, (2002)].

Angiostatin, which has 47% sequence homology with HGF, binds to Met and prevents HGF mediated signaling in endothelial cells and smooth muscle cells. It inhibits the proliferation of these cells in response to HGF but not in response to other growth factors such as vascular endothelial cell growth factor (VEGF) or basic fibroblast growth factor (BFGF), which act through protein tyrosine kinase receptors other than Met. Thus angiostatin functions as a selective Met antagonist.

D. Anti-HGF Receptor Antibodies

While some anti-Met antibodies are receptor agonists others block ligand mediated receptor activation. Met blocking monoclonal antibodies and various derivatives of such antibodies have been developed by the company Genentech and are described in U.S. Pat. No. 6,468,529 B1 (published in Oct. 22, 2002), U.S. Pat. No. 6,214,344B1 (published in Apr. 10, 2001), U.S. Pat. No. 6,207,152B1 (published in May 1996) and of U.S. Pat. No. 5,686,292 (published in June 1995). These antibodies or derivatives of such antibodies are claimed to be useful for the treatment of cancer.

E. Met Selective Aptamers

Single stranded oligonucleotides with random sequences can form a large variety of structures. Oligonucleotides which bind to a particular target can be selected from large random oligonucleotide libraries by a method known as the SELEX process. Oligonucleotide ligands that selectively bind to Met and block ligand mediated Met activation have been identified by the company Gilead using the SELEX method. These HGF antagonists are described in U.S. Pat. No. 6,344,321 B1 (published in Feb. 2, 2002), in U.S. Pat. No. 5,843,653 (published in June 1995) and in U.S. Pat. No. 5,475,096 (published in June 1991).

III. Inhibitors of HGF-Mediated Signal Transduction

A. Met c-Tail Peptide

Modeling of the cytoplasmic domain of Met suggests that the c-terminal tail gets into contact with the catalytic pocket and thereby acts as an intramolecular modulator of the receptor. Bardelli et al designed peptides that correspond to sequences in the c-tail of Met. The peptides were rendered cell-permeable by extending them with sequences corresponding to internalization mediating sequences of the Antennapedia homeodomain. A Met tail peptide blocked ligand induced autophosphorylation as well as downstream Met signaling. The peptide also blocked signal transduction by Ron, a close relative of Met, but did not affect signaling by EGF, PDFG or VEGF through other protein tyrosine kinase receptors. Thus, the Met c-tail peptide is a selective Met/Ron antagonist.

B. Grb2 Antagonists

SH2 domains recognize phosphotyrosine residues (Tyr-P) with additional secondary binding interactions within two or three amino acids C-proximal to the Tyr-P residue. Differences in residues adjacent to Tyr-P generate differential affinity toward SH2 domain subfamilies. Thus, SH2 domains of particular sets of signal transducers can be selectively blocked by Tyr-P containing tripeptides. Inhibitors of SH2 domain interactions with phosphorylated tyrosine are described in U.S. Pat. No. 5,922,697, published in Jul. 13, 1999. Compounds in which the Tyr-P residue is replaced by phosphonomethyl phenylalanine or related structures, are resistant to degradation phosphatases. A variety of other modifications of the peptides increase the affinity for particular SH2 domains or increase the ability of the compounds to pass through plasma membranes to reach their intracellular targets [Yao et al J. Med. Chem., 42:25-35 (1999)]. Tripeptide based inhibitors of the Grb2 SH2 domain have been reported to block HGF mediated cell motility, matrix invasion, and branching morphogenesis. These same inhibitors have only a minor effect on HGF mediated cell proliferation. Inhibitors with particularly high affinity for the SH2 domain of Grb2 are described in U.S. Pat. No. 6,254,742B1, published in Jun. 12, 2001 as compounds that are useful for the treatment of cancer, metastasis, psoriasis as well as allergic, autoimmune, viral and cardiovascular diseases.

C. Inducers of Gab1 Phosphorylation

Phosphorylation of serine/threonine residues of the Grb2 associated binder 1 (Gab1) by PKC-.alpha. and PKC-.beta.1 provides a mechanism for the downregulation of Met signals. Inhibition of serine/threonine phosphatases PP1 and PP2A by okadaic acid results in the activation of serine/threonine kinases such as PKCs, and in the hyperphosphorylation of the serine/threonine residues of gab1. The concomitant hypophosphorylation of tyrosine residues prevents Gab1 from recruiting PI 3 kinase to Met [Gual et al., Oncogene 20:156-166 (2001].

D. Dominant Negative Src Variants

Src binds via its SH2 domain to phosphorylated tyrosine residues of ligand activated Met. The mutant receptor MET M1268T binds src constitutively and NIH3T3 cells expressing the mutant receptor gene form tumors in nude mice. Transfection of dominant negative src constructs into these cells was reported to retard their growth, and to downregulate the phosphorylation of the focal adhesion kinase (FAK) and of paxicillin, but had no effect on Grb2 binding or PLC-.gamma. phosphorylation [Nakaigawa et al., Oncogene 19:2996-3002 (2000)].

E. PI3K Inhibitors

The binding of PI3K to Met is unusual in that it does not involve the canonical motif YXXM but a novel motif YVXV. Although the novel motif has low affinity for the N- and C-terminal SH2 domains of the p85 subunit of PI3K, two closely spaced YVXV motifs in the c-tail of Met represent a docking site for PI3K. The binding is inhibited by synthetic phosphopeptides. The PI3K-mediated signal appears to be essential for HGF induced cell scattering (cytoskeletal reorganization, loss of intercellular junction, cell migration) and morphogenesis. Wortmannin, an inhibitor of PI3K, inhibits Met induced branching of renal cells on a collagen matrix. PI3K signals appear not to be essential for cell transformation, but do contribute to metastasis.

F. NFkB Inhibitors

In liver cells HGF stimulates NF-kappaB DNA binding and transcriptional activation via the canonical IkappaB phosphorylation-degradation cycle and via the extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase cascades. Studies with NFkB inhibitors indicate that HGF induced NFkB activation is required for proliferation and tubulogenesis, but not for scattering nor for the antiapoptotic function of HGF [(Muller et al., Mol Cell Biol 22:1060-72, (2002)].

G. Inhibitors of Small GTP-Binding Proteins

Inhibition of Ras interferes with the spreading, actin reorganization, and scattering of epithelial cells. Dominant negative Rac abolishes HGF induced spreading and actin reorganization in non-small cell lung cancer cells. Microinjection of Rho inhibits HGF induced spreading and scattering but not motility.

H. Hsp90 Antagonists

The chaperone Hsp90 stabilizes many proteins involved in signal transduction. The chaperone appears to be required for the stability and function of a variety of mutated or aberrantly expressed signaling proteins that promote the growth and/or survival of cancer cells. Hsp90 client proteins include mutated p53, Bcr-Abl, src, Raf-1, Akt, ErbB2 and hypoxia-inducible factor 1.alpha. (HIF-1.alpha.). The benzoquinone ansamycin compounds geldanamycin and herbimycin and the structurally unrelated radicicol block the N-terminal nucleotide binding pocket of HsP90 and cause the degradation of Hsp90 client proteins, many of which are involved in tumor progression. One Hsp90 inhibitor, 17-allylaminogeldanamycin (17AAG), is currently in phase I clinical trial, and a novel oxime derivative of radicicol (KF58333) is in preclinical evaluation [(Soga et al., Cancer Chemother Pharmacol 48: 435-45, (2001)].

Recent research has shown that Met is a Hsp90 client that is particularly sensitive to geldanamycin or related compounds. At nanomolar concentrations, geldanamycins downregulates Met protein expression, inhibit HGF-mediated cell motility and invasion and revert the transformed phenotype of cells expressing HGF and Met or constitutively activated Met mutants. Signaling pathways downstream of Met appear to be even more sensitive to Hsp90 inhibitors. Geldanamycins inhibited HGF-mediated plasmin activation at femtomolar concentrations which is nine orders of magnitude below their growth inhibitory concentrations. Interestingly, radicicol has been reported to be moderately active against Plasmodium berghei in mice [Tanaka et al., J. Antibiot. 51:153-60 (1998)]. However, this activity is likely not related to Met inhibition [Tanaka et al., J Antibiot 10:880-8 (1999).

IV. Protein Tyrosine Kinase Inhibitors

The reversible phosphorylation of tyrosine residues on proteins is an important mechanism of signal transduction. A large variety of natural and synthetic compounds are known to be tyrosine kinase inhibitors. Almost all of these inhibitors block protein kinases by blocking the ATP pocket of the enzymes. Therefore, many have a broad spectrum of activity not only against tyrosine kinases but also against serine/threonine kinases and/or other ATP utilizing proteins.

1. General Protein Kinase Inhibitors

The Indrocarbazole K252a was first isolated from the culture broth of Actinomadura and later from Nocardiopsis in a screen for antagonists of Ca2+-mediated signaling. K252a inhibits serine/threonine protein kinases such as various isoforms of protein kinase C (PKCs), cAMP and cGMP dependent kinases as well as protein tyrosine kinases, in particular those of the Trk and Met families. K252a inhibits Met mediated signals at nanomolar concentrations. The compound inhibits Met autophosphorylation and prevents activation of its downstream effectors MAPKinase and Akt. It prevents HGF-mediated scattering in MLP-29 cells, reduces Met-driven proliferation in GTL-16 gastric carcinoma cells, and reverses Met mediated transformation of NIH3T3 fibroblasts. K252a and related compounds are promising leads of drugs that may be used against Trk and Met driven cancers [Morotti et al., Oncogene 21:4885-4893, (2002)]. Conceivably, K525a may serve as a lead in the development of Met specific inhibitors.

2. Inhibitors with Selectivity for Protein Tyrosine Kinases

Several classes of compounds are known protein tyrosine kinase inhibitors. Several such compounds have been isolated from plants or microorganisms and have been extensively used for research purposes. The best known are genistein, lavendustin A, tyrphostin 47, herbimycin, staurosporin and radicicol. Herbimycin A is a benzoquinoid ansamycin antibiotic that inhibits a broad spectrum of protein tyrosine kinases by covalently interacting with their kinase domains. Staurosporin is an indole carbazole antibiotic which inhibits a broad spectrum of kinases including scr family members, and serine/threonine kinases. More recently a large number of protein tyrosine kinase inhibitors have been synthesized and are claimed in several patent applications. 1) bis monocyclic, bicyclic or heterocyclic aryl compounds (PCT WO 92/20642); 2) vinylene-azaindole derivatives (PCT WWO 94/14808); 3) 1-cyclopropyl-4-pyridyl-quinolines (U.S. Pat. No. 5,330,992). 4) styryl compounds (U.S. Pat. No. 5,217,999); 2) styryl-substituted pridyl compounds (U.S. Pat. No. 5,302,606); 5) quinazoline derivatives (EP Application No. 0 566 266A1 and U.S. Pat. No. 6,103,728); 6) selenoindoles and selenides (PCT WO 94/03427); 7) tricyclic polyhydroxylic compounds (PCT WO 92/21660); 8) benzylphosphonic acid compounds (PCT WO 91/15495); 9) tyrphostin like compounds (U.S. Pat. No. 6,225,346B1); 10) thienyl compounds (U.S. Pat. No. 5,886,195). 11) benzodiazepine based compounds with some selectivity for src and FGF-r tyrosine kinases (U.S. Pat. No. 6,100,254, published in Aug. 8, 2000). Tyrosine kinase inhibitors from various classes are claimed for the treatment of cancers that are driven by tyrosine kinases such as Met as well as HER2, EGFR, IGFR, PDGFR, src and KDR/FLK-1. None of the known tyrosine kinase inhibitors are selective for Met. However, it is conceivable that a Met specific inhibitor can be developed in the future. This optimism is based on the fact that several compounds have been synthesized which inhibit a limited set of protein tyrosine kinases one of which is approved for cancer therapy and several of which are in clinical development. These compounds include: 1) The pyrazole pyrimidine PP1 shows selectivity for lck and src kinases over ZAP-70, JAK2 and EGF receptor kinases. 2) STI-571 (GLEEVEC.RTM.) inhibits all forms of abl, PDGF receptor, and c-kit tyrosine kinases. 3) ZD1839 is a synthetic anilinoquinazoline with some selectivity for the EGF receptor. 4) OSI-774 is another orally active quinazoline derivative with some selectivity for the EGF receptor. 5) 4-anilinoquinazoline derivatives show selectivity for the VEGF-R (U.S. Pat. No. 6,291,455B1, published in Sep. 18, 2001). 6) SU101 shows selectivity for the PDGF receptor, but its antiproliferative effects are in part due to an ring-opened metabolite which inhibits dihydro-orotate dehydrogenase, a mitochondrial enzyme crucial to pyrimidine biosynthesis. 7) Aryl and heteroaryl quinazoline compounds show selectivity for CSF-R (U.S. Pat. No. RE 37,650 E, published in Apr. 9, 2002.8) SU 5416, a VEGF receptor (Flk1/KDR) antagonist was designed on the basis of crystallographic studies of the indolin-2-one pharmacophore and the FGF receptor tyrosine kinase domain. 9) Bis mono- and bicyclic aryl and hetero aryl compounds show selectivity for EGFR and PDGFR (U.S. Pat. No. 5,409,930). 10) Piceatannol (3,4,3,5V-tetrahydroxy-trans-stilbene) shows selectivity for syk and lck, but also inhibits serine/threonine kinases and ATPase. 11) Several compounds that are based on benzodiazepines show some selectivity for the non-receptor tyrosine kinase src and for the FGF-R tyrosine kinase receptor family. These examples show that compounds with selectivity for one or a few tyrosine kinases can be generated.

V. Anti-Malarial Effects of Protein Kinase Inhibitors

Like plants the related apicomplexan parasites such as plasmodium appear not to produce protein tyrosine kinases. A few reports suggest that protein tyrosine phosphorylation occurs in plasmodium (see section A below). However homology searches have failed to detect any sequences related to the known protein tyrosine kinase families. Therefore it is conceivable that antimalarial effects of protein tyrosine kinase inhibitors are due to the inhibition of the enzymes that are produced by the human host. A variety of quinazoline derivatives have been reported to have antimalarial activity. These compounds include 2,4-diamino-6(3,4-dichlorobenzylamine quinazoline (PAM1392 [Thompson et al. Exp. Parasitol 25:32-49, 1969)], 2,4-diamino-6-[93,4-dichlorobenzyl0-nitrosoamino]-quinazoline (CI-679) [Schmidt and Rossan, Am. J. Trop. Med. Hyg. 28:781-92, (1979)], several other 2,4-diamine-6-substituted quinazoline derivatives Elslager and colleagues [Elsager et al., J. Med. Chem. 21:1059-70, (1978)] and by Chinese scientists [Gy et al., Xao Xue Bao 19:108-18, (1984), Yao et al., Yao Xue Bao 19:76-8, (1984)]. The antimalarial activity of 2,4-diamino-5-methyl-693,4,5-trimethoxyanilinomethyl) quinazoline salts is described in U.S. Pat. No. 4,376,858, published in Mar. 15, 183. One possible mode of action of quinazoline derivatives against plasmodium are inhibition is the inhibition of tyrosine kinases (U.S. Pat. No. 6,103,728, published in Aug. 15, 2000).

A) Inhibition of Plasmodium Protein Kinases

1) Dluzeski and Garda reported that several protein kinase inhibitors (staurosporin, genistein, methyl 2,5-dihydroxycinnamate, tyrphostin B44 and B46, lavendustin A and RO3) inhibited the erythrocytic cycle of plasmodium falciparum [Dluzewski and Garda, Experientia 52:621-623, (1996)]. With the exception of staurosporin, a strong serine/threonine kinase inhibitor, these compounds preferentially inhibit protein tyrosine kinases. These inhibitors prevented the development of the parasites within erythrocytes and/or invasion. Because of the broad spectrum of activities of these inhibitors it is not clear whether inhibition of a protein tyrosine kinase played any role in the observed effects, nor is it clear whether the target proteins were derived from the erythrocytes or from the parasites.

2) While screening artemisinin like compounds from microorganisms, Tanaka and colleagues identified seven fungal metabolites with antimalarial activity. One of these compounds, radicicol, a broad spectrum protein kinase inhibitor was moderately active against Plasmodium berghei in mice [Tanaka et al., J. Antibiot 51:153-60, (1998)].

3) More recently Sharma reported that a membrane bound PTK activity was increased during maturation from the ring stage to the trophozoite stage. Inhibition of the PTK activity by chloroquine was proposed to represent one possible mechanism of action of this drug against the parasite [Sharma and Mishra, Indian J. Biochem. Biophys. 36:299-304 (1999); Sharma, Indian J. Exp. Biol. 38:1222-6 (2000)].

B) Inhibition of Human Protein Tyrosine Kinases

A variety of pathogenic effects of plasmodium are mediated by protein tyrosine kinases of the human host and thus can be inhibited by protein tyrosine kinase inhibitors. Several examples have been reported in the literature.

1) Adhesion of infected erythrocytes to vascular endothelium involves the binding of P. falciparum membrane protein 1 (PfEMP1) to CD36 that is expressed by endothelial cells of the host. A signal mediated by CD36 is essential for adhesion. The pyrazolopyrimidine PP1, a selective inhibitor of src and lck kinases, inhibits this signal and prevents adhesion [Yipp et al., Blood online, (2002)].

2) CD36 and CD36 mediated, protein kinase dependent signals are also involved in the nonopsonic clearance of P. falciparum infected erythrocytes by monocytes and macrophages. Both genistein and selective ERK and p38 MAPK inhibitors (PD98059 and SB203580, respectively) reduced the uptake of infected erythrocytes to almost the same extent as CD36 blockade [McGilvray et al., Blood 96:3231-40, (2000)].

3) Glycosylphosphatidylinositol (GPI) is a major toxin of Plasmodium falciparum. Malarial GPI induces rapid onset tyrosine phosphorylation of multiple intracellular substrates within 1 min of addition to cells. These signals are involved in the upregulation of parasite adherence and in the induction of nitric oxide (NO) release by macrophages and endothelial cells. Both adherence and NO release are prevented by the tyrosine kinase antagonists tyrphostin and genistein [Tachado et al., J Immunol 156:1897-1907, (1996); Schofield et al., J. Immunol. 156:1886-96].

In previous work the protein tyrosine kinase receptor Met has not been implicated in malaria infections. The present invention identifies the protein tyrosine kinase Met as a crucial mediator of hepatocyte susceptibility to infection by malaria sporozoites.

VI. HGF Related Anti-Malarials

A. Sulfated Polysaccharides

As mentioned above, HGF levels may be increased by HGF degradation inhibiting polysaccharides including dextran sulfate, heparan sulfate, dermatan sulfate, keratan sulfate, chondroitin, or chondroitin sulfate. The combination of sulfated polysaccharides, such as sulfated curdlan, dextrin sulfate, chondroitin sulfate, heparin, carageenan) with quinine for the treatment of malaria is described in U.S. Pat. No. 5,780,452, published in Jul. 14, 1998. The proposed strategy is based on the ability of sulfated polysaccharides to inhibit the invasion of human erythrocytes by malarial parasites. The present invention raises concerns regarding this strategy, since sulfated polysaccharides may increase HGF levels by inhibiting its degradation, a fact described in U.S. Pat. No. 5,736,506, published in Apr. 17, 1998. Therefore sulfated polysaccharides are excluded from the antimalarial agent of this invention.
 

Claim 1 of 5 Claims

1. A method of inhibiting malaria infection, wherein the method comprises administering genistein to a human in need thereof in an amount sufficient to inhibit infection of the human by malaria parasites.
 

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