HK1093903A - A method for the prevention of malaria infection of humans by hepatocyte growth factor antagonists - Google Patents

Description

Method for preventing malaria infection in humans by hepatocyte growth factor antagonists

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based on and claims the right to U.S. provisional application No. 60/453,483 (Attorney Docket No.08907.6001), filed on 12/3/2003. The entire disclosure of this provisional application is hereby relied upon and incorporated herein by reference.

Technical Field

The present application relates to antagonists of hepatocyte growth factor receptors and to inhibitors of hepatocyte growth factor induced signals. More particularly, the application relates to the use of such compounds for the prevention of infection by plasmodium falciparum (plasmodium falciparum) and plasmodium vivax (plasmodium vivax).

Background

The pathogenesis of malaria has been extensively studied and described in numerous scientific publications and review articles [ recent examples are found in Miller et al, Nature 415: 673-679, (2002)]. The cause of the disease is Plasmodium falciparum, and less frequently Plasmodium vivax, Plasmodium malariae (Plasmodium malariae) and Plasmodium ovale (Plasmodium ovale). Malaria causes almost all deaths from plasmodium falciparum. The parasite is transmitted by the vector Anopheles gambiae, which preferentially feeds on humans and has a long life span. Sporozoites are injected into the skin due to mosquito bites. They move to the liver where they establish an infection and divide after passing through several hepatocytes. Each sporozoite develops into tens of thousands of merozoites that are released from the liver and invade the red blood cells. Plasmodium falciparum and plasmodium vivax reproduce asexually inside erythrocytes. Over a two-day period, approximately 20 merozoites were produced per merozoite. The red blood cells rupture and release merozoites that again invade the red blood cells. The disease begins with asexual reproduction of parasites within erythrocytes. A few merozoites develop into gametocytes, which do not cause disease but transmit the infection to others through the female anopheles mosquito. Plasmodium vivax develops into gametocytes shortly after release from the liver, whereas plasmodium falciparum gametocytes develop much later.

Malaria is an important health problem in some regions of asia and south america and especially in africa south of the sub-saharan desert. Almost 10% of the global population will develop malaria-6 billion clinical cases in any given year. According to recent estimates, at least 100 million malaria-caused deaths occur each year — one malaria-caused death every 30 seconds [ Greenwood and Mutabingwa, Nature 415: 670-672(2002)]. 1 of every 20 children before the age of five in africa died from malaria. Recently, malaria status has worsened as a result of many factors contributing to the aggravation of malaria burden, the most important being the emergence of plasmodium falciparum and plasmodium vivax varieties that are resistant to inexpensive and effective drugs, and the emergence of insecticide-resistant mosquitoes.

Summary of The Invention

The present invention helps fulfill these needs in the art. The avoidance of malaria has made definitive treatment difficult. Provided herein are agents and methods that can prevent the spread or acquisition of malaria infections and can help prevent and treat such infections.

More particularly, the present invention provides a method of inhibiting malaria activity in vivo, wherein the method comprises administering to a human host an antimalarial agent capable of exhibiting a protective effect by preventing the initial replication of malaria parasites in an infected host, such as the liver of a human. The antimalarial agent consists 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 hepatocyte infection by malaria in vivo or to prevent or at least inhibit replication or transmission of malaria parasite in vivo.

The present invention relates to the ability of hepatocytes to support the growth of parasites that cause malaria in humans. The Plasmodium parasites responsible for human diseases are Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale. More particularly the invention reveals Met activation and downstream signaling necessary for the establishment of plasmodium infection. It has previously been known that plasmodium sporozoites pass through several hepatocytes before they are able to form vesicles in and divide in hepatocytes. The penetration of sporozoites through hepatocytes has not previously been known to be associated with the well-known cytokine known as Hepatocyte Growth Factor (HGF). HGF is known to be released as an inactive single chain protein. It is activated by proteolytic cleavage, which forms disulfide-linked heterodimers. 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 different pathways. These signals lead to a variety of responses such as cell dispersion, proliferation, tubulogenesis (tubulogenesis) and invasive growth. The present invention discloses novel Met-mediated hepatocyte responses against HGF. HGF causes hepatocytes to allow invasion of sporozoites in a manner that allows the sporozoites to multiply inside the bleb.

The invention also provides a novel strategy for preventing plasmodium infection.

In a preferred embodiment, plasmodium infection of hepatocytes is prevented by a molecule that interferes with HGF production by damaged hepatocytes.

Also suitable for infection prevention are molecules that interfere with the cleavage of HGF protease into its active form, and molecules that sequester HGF and thus prevent its binding to hepatocytes via its receptor Met.

In another aspect, the invention discloses that Met is a drug target for the prevention of malaria infection.

In a preferred embodiment of the invention, malaria infection is prevented by a molecule that interferes with HGF binding to its receptor Met. Such molecules are antibodies specific for HGF, which block the binding site of HGF to 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) that 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 limited to NR 4.

In another aspect of the invention, plasmodium infection of hepatocytes is prevented by a drug that interferes 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 agents are selective inhibitors of the protein tyrosine kinase Met. In a preferred embodiment these inhibitors are small molecular weight compounds and are administered by oral route or as suppositories.

Brief Description of Drawings

FIG. 1 shows that sporozoites induce the release of "susceptibility to infection inducing factors" (ISIF) by cell migration and mechanical cell damage.

Fig. 2 shows that HGF secreted by host cells crossed by sporozoites is required for infection.

Fig. 3 shows the effect of HGF on plasmodium infection is mediated through its receptor MET.

Fig. 4 shows that HGF is expressed by cells crossed by plasmodium in vitro and in vivo and its signaling through MET is essential for malaria infection.

FIG. 5 shows that genistein affects liver infection caused by Plasmodium boidinii (Plasmodium berghei) sporozoites in vitro.

FIG. 6 shows that genistein affects liver infection caused by Plasmodium boidinii sporozoites in vivo.

Detailed Description

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 the group consisting of HGF receptor antagonists, inhibitors of HGF mediated signal transduction, and protein tyrosine kinase inhibitors. Inhibitors of HGF activity may be used alone or in combination with each other. Inhibitors of HGF activity may optionally be combined with one or more known antimalarial drugs to form the antimalarial agents of the present invention.

The present invention relates to the invasion of hepatocytes by malaria parasites. After transmission to a human host via mosquito bites, malaria sporozoites find their way into the liver where each sporozoite can produce up to 10000 merozoites that are released into the blood. The invasion of hepatocytes is an essential step in malaria infection. Sporozoites can invade hepatocytes by disrupting the plasma membrane and then the parasite migrates through the cell or, like intracellular bacteria or other parasites, by forming an internalizing vesicle around the invading pathogen. The initial sporozoites pass through the hepatocytes without forming internalized vesicles. Sporozoites break through the plasma membrane of hepatocytes, enter the hepatocytes, pass through the cytosol, and exit the host cell, which dies or successfully repairs the plasma membrane. The potential molecular mechanisms by which plasmodium passes through hepatocytes and subsequent parasitic bleb formation are well understood. Vesicle formation by P.yoelii (Plasmodium yoelii) and P.falciparum, but not by the rodent malaria parasite P.boidinii, is dependent on hepatocyte expression of the tetraspin protein CD 81. CD81 is known to be a receptor for hepatitis c virus, but it does not appear to interact with any ligands 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 cross the cytosol of several cells before invading hepatocytes by forming parasitic vesicles, which is essential for differentiation into the next infection stage [ Mota et al, Science 291: 440-42, 2001]]. This finding suggests that sporozoite-induced damage to hepatocytes releases one or more Infection Susceptibility Inducing Factors (ISIFs) that sensitize neighboring hepatocytes to infection. An important aspect of the present invention is the discovery that a protein known as hepatocyte growth factor is useful as ISIF in malaria infection.

HGF and its receptor Met

Hepatocyte growth factor is found as a mitogen for hepatocytes [ Michalopolous et al, Cancer res, 44: 441-4419 (1984); russel et al, Cell physiol, 119: 183-192 (1984); nakamura et al, biochem.biophysis.res.comm., 122: 1450-. For simplicity, this factor is referred to as HGF. HGF was first purified from liver-resected rat serum [ Nakamura et al, biochem.biophysis.res.comm., 122: 1450-: 6849 (1986) and human plasma purification [ Gohda et al, j.clin.invest.81: 414-419(1988)]. cDNA encoding rat HGF, human HGF and the naturally occurring variant known as "delta 5 HGF" was cloned [ Miyazawa et al, biochem, biophys, res, commun, 163: 967-973 (1989); nakamura et al Nature 342: 440-443 (1989); seki et al, biochem, biophysis, 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, 62kDa) and a beta-subunit of 234 amino acids (M, 34 kDa). It is produced biologically inactive (728 amino acids) and the pro-HGF is cleaved by the protease between Arg494 and Val495 to form a disulfide-linked heterodimer. The 62-kDa alpha-subunit comprises an N-terminal hairpin domain (approximately 27 amino acids) followed by 4 canonical kringle (canonical kringle) domains, which are 80 amino acid bi-cyclic structures stabilized by 3S-S bridges. The first loop domain binds to the protein tyrosine kinase receptor Met, as described in more detail below. The hairpin loop and second tricyclic domains bind membrane-bound heparan sulfate proteoglycan with low affinity. The 34kDa β -subunit comprises a serine protease-like domain that is very similar to the serine protease-like domain of the serine protease coagulation factor, but which is protease-free. HGF shows 38% total sequence identity to plasminogen and 45% identity to another cytokine called Macrophage Stimulating Protein (MSP). HGF binds to a protein tyrosine kinase receptor called Met, while its proximal MSP binds to another protein tyrosine kinase receptor called Ron.

HGF is secreted as a single chain pro-HGF. This HGF precursor binds to proteoglycans bound to the extracellular matrix or to the cell surface adjacent to the producer cells. Activation of the single-stranded precursor into the biologically active heterodimer by proteolytic cleavage between Arg494 and Val495 is a tightly controlled process (reviewed in Kataoka et al, Life XY 1: 1036-1042 (2001)).

The first enzymes involved in HGF activation are urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). Three additional HGF activating enzymes were subsequently identified, namely coagulation factor XIIa, membrane type serine protease-1 also known as matriptase (MT-SP1) and HGF activator (HGFA). Each of these enzymes is under the control of an endogenous inhibitor protein. HGFA is the most efficient HGF cleaving enzyme. Like HGF, HGFA is a heterodimer produced from single-chain pre-HGFA by post-Arg 407 cleavage. One of the HGFA cleaving enzymes is thrombin, an enzyme that is activated to the injured tissue by the coagulation cascade (clotting cascade). Active HGFA heterodimers are not inhibited by major serum protease inhibitors, but in two proteins: HGA inhibitor class I (HAI-1) and HGA inhibitor class II (HAI-2), the latter being equivalent to placental uropancreatin (PB). HAI-1 is upregulated in injured and regenerated tissue. It is expressed on the cell surface where it binds and inhibits HGFA. Cytokines such as IL-1 β induce the shedding of the HGFA/HAI-1 complex by TNF- α converting enzyme (TACE) and TACE-like metalloproteases of the ADAM (disintegrin and metalloprotease) protein family. HGFA dissociates from HAI-1 after shedding and is then able to activate HGF. Thus, HAI-1 is not only an inhibitor of mature HGFA but also a specific acceptor of HGFA, acting as a reservoir for this enzyme on the cell surface. HAI-1 is described in U.S. Pat. No. 6,465,622B2, published on 15/10/2002, wherein the use of HAI-1 as a control factor for HGF and HGFA is claimed.

The HGF receptor Met was originally discovered as a component of oncogenic fusion proteins produced in carcinogen-treated sarcoma cell lines [ Cooper et al, Nature, 311: 29-33(1984)]. In normal cells, the primary Met transcript produces a 150kDa polypeptide that is glycosylated and then cleaved to form S-S linked heterodimers. HGF and its receptor Met are the subject of U.S. patent No. 5,648,273, 7/15, which claims the use of ligand-receptors for the diagnosis of proliferative disorders and diseases such as hepatitis and liver carcinogenesis.

Met heterodimers consist of a highly glycosylated and fully extracellular β -subunit and an α -subunit with a large extracellular region and an intracellular tyrosine kinase domain. Met is a member of the superfamily of Receptor Tyrosine Kinases (RTKs). The superfamily is divided into at least 19 families including the Her family (EGFR, Her2, Her3, Her4), the insulin receptor family (insulin receptor, IGF-1R, insulin related receptor), the PDGF receptor family (PGFRa and b, CSF-R, kit, FIk2), the FIk family (F1k-1, F1t-1, F1k-4), the FGF receptor family (FGF-R1, 2, 3 and 4) and others. Met and its close Ron form different receptor families for the ligands HGF and Macrophage Stimulating Protein (MSP), respectively.

Upon HGF binding, c-Met undergoes autophosphorylation of specific tyrosine residues. Phosphorylation of the C-terminal tyrs 1349 and Tyr1356 generates signal transduction proteins, such as phosphatidylinositol 3-kinase (PI3K), phospholipase C- γ (PLC- γ), src, Stat3, Grb2 and Grb2 binding the multi-substrate docking site of the docking protein Gab1, when phosphorylation of Tyr1234 and Tyr1235 located within the activation loop of the tyrosine kinase domain activates the intrinsic kinase activity of C-Met. Grb2 also interacts with Met by the adapter protein Shc. Grb2 recruits the Ras nucleoside exchange protein SOS, which activates the Ras-MAPK signaling pathway. Thus, docking of the activated Met receptor by a signal transduction protein initiates signal transduction through multiple pathways. The C-terminal 26 amino acids of Met not only provide docking sites for signal transduction proteins, but also regulate the enzymatic activity of Met. Mutations in the kinase domain (M1250T) circumvent the regulation of the C-terminal amino acid [ Gual et al, Oncogene 20: 5493-502(2001)].

Various responses against HGF in different Met expressing target cells have been described. These responses include proliferation, programmed cell death, dissociation (dissociation) of cells, mutual repulsion, movement of cells through the extracellular matrix, and branched morphogenesis. During embryogenesis, the interaction between HGF-producing mesenchymal cells and Met-expressing epithelial cells appears to be involved in the formation of various organs such as placenta, breast, liver, muscle and neuronal tissue. HGF and Met knockout mice show defects in placental, liver and muscle development and die between E13.5 and 15.5 [ (Schmidt et al, Nature 373: 699-) (1995); Uehara et al, Nature 373: 702-) (1995); Bladt et al, Nature 376: 768-) (1995) ]. in adult life, HGF-Met interaction is involved in wound healing, angiogenesis and tissue regeneration. not surprisingly, the activation of Met by HGF has been involved in tumor Growth, invasion and metastasis.

Based on their biological properties, both HGF and HGF antagonists have been suggested to be useful for the treatment of various diseases. The production of HGF and its therapeutic use have been claimed in several patents. HGF has been isolated from blood based on its high affinity for heparin (disclosed in US 5,004,805 on 4/2 of 1991). Pegylation of HGF prolongs its clearance, reduces the required dose, and is believed to improve the side effects of HGF treatment (disclosed in 1999 in U.S. patent No. 5,977,310 on day 11/2). HGF degradation inhibitory polysaccharides such as heparin, hyaluronic acid, dextran sulfate, heparan sulfate, dermatan sulfate, keratan sulfate, chondroitin, or chondroitin sulfate (disclosed in U.S. patent No. 5,736,506 at 17.4.1998) can increase HGF levels. HGF activating proteases have been claimed (U.S. Pat. No. 5,677,164, 10/14/1997). Applications of HGF therapy include treatment of arterial occlusive disease (disclosed in us patent No. 6,133,231, 10/17/2001), treatment of inflammatory bowel disease (disclosed in us patent No. 6,319,899B1, 11/20/2001), increased resurfacing of damaged and damaged blood vessels (resurfacing), for example, by vascular surgery or angioplasty (disclosed in us patent No. 6,133,234, 10/17/2000). HGF is also claimed to ameliorate side effects caused by commonly used immunosuppressants (disclosed in U.S. patent No. 5,776,464 at 7.7.1998). Finally, topical application of vectors containing the HGF gene to blood vessels or other target organs has been described for a variety of therapeutic purposes (disclosed in U.S. patent No. 6,248,722B1, 6/19 of 2001). Met and downstream signaling pathways have long been recognized as attractive targets for cancer therapy. First, studies using tumor cell lines and tumor models in animals have shown that Met plays an important role in the invasive growth and metastasis of cancer cells. Second, amplification of Met gene was observed in liver metastasis of colorectal cancer. Third, Met is overexpressed in several types of human tumors, such as thyroid and pancreatic cancers. Fourth, germline (germ line) mutations in the Met gene are found in hereditary papillary renal cancers and somatic Met gene mutations are found in sporadic papillary cancers.

The present invention identifies previously unknown functions of the Met receptor, and inhibition of this receptor represents a novel therapeutic application for Met antagonists. Signaling through Met allows the hepatocytes to productive invasion of malaria sporozoites. Met signaling is necessary for sporozoites to enter hepatocytes by endocytosis vesicle formation and/or sporozoites to proliferate inside vesicles formed by the cytoplasmic membrane of the liver. The discovery of this function of Met is the basis of a new approach to prevent malaria infection. A further embodiment of the invention is the use of a compound to prevent the development of malaria infection by interfering with HGF-mediated Met activation or signaling events downstream of Met that are involved in causing hepatocytes to permissive to infection by malaria parasites. Several Met antagonists have been described in the literature and some have been patented for the treatment of diseases caused at least in part by an excessive or abnormal function of Met. The previously claimed indication for Met antagonists is the treatment of malignancies. The potential use of Met antagonists for the treatment of infectious diseases and in particular malaria parasite infection is made apparent by the present invention. The invention claims the use of Met antagonists for the prevention of human infections by malaria parasites. Known HGF antagonists are described in the following sections.

HGF receptor antagonists

HGF variants

Various forms of HGF, both naturally occurring and produced by genetic manipulation of HGF-encoding cDNA, antagonize some or all of the Met function. Uncleaved pro-HGF binds but does not activate Met. Several isoforms of HGF are produced by the initial differential splicing of HGF transcripts. These isoforms include NK1 (consisting of the N domain of HGF and the first tricyclic domain) and NK2 (consisting of the N domain of HGF and the first two tricyclic domains). The other two variants found in the endometrium and placenta of macaques, dNK1 and dNK2, are similar to the NK1 and NK2 isoforms except that dNK1 and dNK2 encode proteins with a5 amino acid deletion in the first three-loop 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 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 depending on the cellular environment, the presence or absence of heparin, and the function of HGF being analyzed, these HGF variants act as both partial HGF agonists and HGF antagonists. In vivo studies using mice overexpressing transgenic HGF, NK1, NK2, HGF + NK1, or HGF + NK2 revealed potential in vivo functions of HGF isoforms. Transgenic expression of HGF has a variety of phenotypic outcomes, such as enhanced liver growth, progressive glomerulosclerosis, destruction of the olfactory mucosa, aberrant localization of myocytes in the central nervous system and melanocytes in the dermis and epidermis, premature mammary lobular alveolar development (lobular vehicle) and susceptibility to tumor induction. Transgenic expression of NK1 produced a similar phenotype, whereas transgenic expression of NK2 did not show HGF and NK1 induced phenotypic features. NK2 antagonizes the pathological consequences of HGF overexpression and down-regulates the subcutaneous growth of transplanted Met-expressing tumor cells in HGF + NK2 dual transgenic mice. However, transgenic overexpression of NK2 promoted metastasis of these same tumor cells. Thus, NK2 antagonizes many responses to HGF, but both have the ability to dissociate (scatter) cells with HGF-a response that promotes metastasis [ Otsuka et al, Molecular and cellular Biology 20: 2055-2065(2000)].

Another NGF variant, NK4, was produced by single cleavage digestion of HGF with elastase. NK4 comprises an N-terminal hairpin structure and 4 tricyclic domains. NK4 is a pure HGF antagonist compared to NK1 and NK 2[ Date et al, FEBS Letters 420: 1-6(1997)]. Like the isolated HGF α chain, NK4 binds to Met but does not induce autophosphorylation thereof unless the HGF β chain is added. Administration of NK4 protein or NK4 Gene transfer due to its ability to antagonize HGF [ Hirao et al, Cancer Gene Ther 9: 700-7 (2002); maehara et al, Clin ExpMetastasis 19: 417-26(2002) is estimated as a novel approach to the treatment of Met-expressing cancers. Single chain HGF variants similar to NK4 that have been engineered to be resistant to proteolytic cleavage are described in U.S. patent No. 5,879,910, published at 9.3.1999 and in 3.3. japanese patent No. 5,580,963, published at 12.1996.

B. Soluble Met receptor

Soluble forms of Met are released from cultured endothelial cells, smooth muscle cells and various tumor cell lines. Soluble receptors are thought to block HGF activation of cell surface bound Met. Met-IgG fusion proteins have been produced that retain the ability to bind HGF with high affinity and are therefore capable of neutralizing HGF activity.

C. Angiostatin

Angiostatin, an inhibitor of angiogenesis, is a fragment of plasminogen that contains 3-4 tricyclic domains. The anti-angiogenic effect of angiostatin is thought to be based on its ability to inhibit atpase on the endothelial cell surface, and to interfere with integrin function and peripheral protein hydrolysis. Recent studies have indicated that the anti-angiogenic activity of angiostatin is due at least in part to its ability to neutralize HGF effects [ Wajih and san, previously published online in Blood, October 24, (2002) ].

Angiostatin has 47% sequence homology with HGF, binds to Met and prevents HGF-mediated signal transduction in endothelial cells and smooth muscle cells. It inhibits proliferation of these cells in response to HGF, but does not inhibit proliferation of these cells in response to other growth factors that act through protein tyrosine kinase receptors other than Met, such as Vascular Endothelial Growth Factor (VEGF) or Basic Fibroblast Growth Factor (BFGF). Angiostatin thus acts 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 derivatives of various such antibodies have been developed by Genentech and described in US6,468,529B 1 (published at 10/22/2002), US6,214,344B 1 (published at 4/10/2001), US6,207,152B 1 (published at 5/1996) and US 5,686,292 (published at 6/1995). These antibodies or derivatives of such antibodies are claimed to be useful in cancer therapy.

Met-selective aptamers

Single stranded oligonucleotides containing random sequences are capable of forming a wide variety of structures. Oligonucleotides that bind to specific targets can be screened from large random oligonucleotide libraries by a method known as the SELEX method. Oligonucleotide ligands that selectively bind to Met and block ligand-mediated Met activation have been identified by Gilead using the SELEX method. These HGF antagonists are described in US6,344,321B1 (published in 2.2.2002), US 5,843,653 (published in 6.1995) and US 5,475,096 (published in 6.1991).

Inhibitors of HGF-mediated signal transduction

Met c-tail peptides

Modeling of Met cytoplasmic domains suggests that the c-terminal tail is in contact with the catalytic pocket and thus acts as an intramolecular modulator of the receptor. Bardelli et al designed a peptide corresponding to the sequence in the c-tail of Met. These peptides are made cell permeable by extending the peptides with a sequence corresponding to an internalization mediating sequence of the antennapedia homeodomain. The Met tail peptide blocks ligand-induced autophosphorylation and downstream Met signaling. This peptide also blocks signal transduction by the next-to-parent Ron of Met, but does not affect signal transduction by EGF, PDGF or VEGF through other protein tyrosine kinase receptors. Thus, the Met c-tail peptide is a selective Met/Ron antagonist.

Grb2 antagonists

The SH2 domain recognizes a phosphotyrosine residue (Tyr-P) using an additional secondary binding interaction within 2 or 3 amino acids C-proximal to the Tyr-P residue. The difference in the proximity of residues of Tyr-P gives rise to different affinities for the SH2 domain subfamily. Thus, the SH2 domain of a particular group of signal transduction proteins can be selectively blocked by a Tyr-P-containing tripeptide. Inhibitors of the interaction of the SH2 domain with phosphotyrosine are described in U.S. patent No. 5,922,697, published at 13.7.1999. Compounds in which the Tyr-P residue is replaced with phosphonomethylphenylalanine or a related structure are resistant to degrading phosphatases. Various other modifications of the peptide increase the affinity for the specific SH2 domain or enhance the ability of compounds to reach their intracellular targets through the plasma membrane [ 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 minor effects on HGF-mediated cell proliferation. Inhibitors with specific high affinity for the SH2 domain of Grb2 are described in U.S. patent No. 6,254,742B1, published on 12.7.2001, as compounds useful for the treatment of cancer, metastasis, psoriasis, as well as allergic, autoimmune, viral and cardiovascular diseases.

Inducers of gab1 phosphorylation

The phosphorylation of serine/threonine residues of Grb 2-related adhesive 1(Gab1) by PKC- α and PKC- β provides a mechanism for Met signal downregulation. Inhibition of the serine/threonine phosphatases PP1 and PP2A by okadaic acid results in activation of serine/threonine kinases such as PKCs, as well as hyperphosphorylation of the serine/threonine residue of gab 1. The concomitant hyperphosphorylation of tyrosine residues prevents Gab1 from recruiting PI3 kinase to Met [ Gual et al, Oncogene 20: 156-166(2001)].

D. Dominant negative src variants

Src binds via its SH2 domain to the phosphorylated tyrosine residue of ligand-activated Met. The mutant receptor METM1268T constitutively binds src, 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 prevent their growth and down-regulate phosphorylation of Focal Adhesion Kinase (FAK) and paxilillin, 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 include the canonical motif YXXM but rather the novel motif YVXV. Although the new motif has low affinity for the N-and C-terminal SH2 domains of the p85 subunit of PI3K, the two closely spaced YVXV motifs of the Met C-tail represent the docking sites for PI 3K. This binding is inhibited by the synthetic phosphopeptide. PI 3K-mediated signaling appears to be essential for HGF-induced cell dispersion (cytoskeletal reorganization, loss of intercellular junctions, cell migration) and morphogenesis. Wortmannin, an inhibitor of PI3K, inhibits Met-induced branching of renal cells on collagen matrices. The PI3K signal appears to be not essential for cell transformation, but aids in metastasis.

NFkB inhibitors

In hepatocytes, HGF stimulates NF-kappaB DNA binding and transcriptional activation via the canonical ikppab phosphorylation degradation cycle and via the extracellular signal-regulated kinase 1/2 and the p38 mitogen-activated protein kinase cascade. Studies with NFkB inhibitors indicate that HGF-induced activation of NGkB is required for proliferation and tubulogenesis, but not for the dispersing and anti-apoptotic functions of HGF [ (Muller et al, Mol Cell Biol 22: 1060-72, (2002) ].

G. Inhibitors of small GTP-binding proteins

Inhibition of Ras interferes with epithelial cell spreading, actin reorganization, and dispersion. Dominant negative Rac eliminates HGF-induced diffusion and actin reorganization in non-small cell lung cancer cells. Microinjection of Rho inhibited HGF-induced diffusion and dispersion but not motility.

Hsp90 antagonists

The chaperone Hsp90 stabilizes many proteins involved in signal transduction. This chaperone protein 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. Client proteins for Hsp90 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 (herbimycin) and the structurally unrelated radicicol (radicol) block the nucleoside binding pocket at the end of Hsp90N and cause degradation of Hsp90 client proteins, many of which are involved in tumor development. 17-allylaminogeldanamycin (17AAG), an inhibitor of Hsp90, is currently in phase I clinical trials, and a novel oxime derivative of radicicol (KF58333) is being evaluated preclinically (Soga et al, Cancer Chemother Pharmacol 48: 435-45, (2001)).

Recent studies have shown that Met is a client of Hsp90, which is particularly sensitive to geldanamycin or related compounds. At nanomolar concentrations, geldanamycin down-regulates Met protein expression, inhibits HGF-mediated cell motility and invasion and reverts to the cell transformation phenotype of HGF and Met or constitutively activated Met mutants. Downstream of the Met signaling pathway appears to be more sensitive to Hsp90 inhibitors. Geldanamycin inhibits HGF-mediated plasmin activation at femtomolar (femtolar) concentrations that are 9 orders of magnitude below their growth inhibitory concentration. Interestingly, radicicol has been reported to have appropriate activity against P.boidinii in mice. [ Tanaka et al, J.Antibiott.51: 153-60(1998)]. However, this activity may not be associated with Met inhibition [ Tanaka et al, J antisiot 10: 880-8(1999)].

Inhibitors of protein tyrosine kinases

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

1. Universal protein kinase inhibitors

Inthiocarbazole K252a was originally isolated from a culture of Actinomadura (Actinomardra) and then incubated in the presence of Ca²⁺Screening for antagonists of mediated signal transduction was isolated from Nocardiopsis (Nocardiopsis). K252a inhibits multiple isoforms of serine/threonine protein kinases such as protein kinase c (pkcs), cAMP and cGMP dependent kinases, as well as protein tyrosine kinases, especially those of the Trk and Met families. K252a inhibited Met-mediated signaling at nanomolar concentrations. The compounds inhibit Met autophosphorylation and prevent activation of its downstream effector MAPK kinase and Akt. It prevents HGF-mediated dispersion in MLP-29 cells, reduces Met-driven proliferation in GTL-16 gastric cancer cells, and reverses Met-mediated transformation of NIH3T3 fibroblasts. K252a and related compounds are a promising drug paradigm for the treatment of Trk and Met driven cancers [ Morotti et al, Oncogene 21: 4885-4893, (2002)]. Conceivably, K252a could serve as a template in the development of Met-specific inhibitors.

2. Inhibitors selective for protein tyrosine kinases

Several classes of compounds are known as protein tyrosine kinase inhibitors. Several such compounds have been isolated from plants or microorganisms and have been widely used for research purposes. Most notably genistein, fumonisin A, tyrphostin 47, herbimycin, staurosporine and radicicol. Herbimycin a is a benzoquinone-type ansamycin antibiotic that inhibits a broad spectrum of protein tyrosine kinases by covalently interacting with their kinase domains. Staurosporine is an indolocarbazole antibiotic that inhibits a variety of kinases, including scr family members, and serine/threonine kinases. A number of protein tyrosine kinase inhibitors have recently been synthesized and claimed in several patent applications. 1) Bi-monocyclic, bicyclic or heterocyclic aryl compounds (PCT WO 92/20642); 2)1, 2-etheno-azaindole derivatives (PCT WO 94/14808); 3) 1-cyclopropyl-4-pyridinyl-quinolones (U.S. Pat. No. 5,330,992). 4) Styryl compounds (U.S. patent No. 5,217,999); 2) styryl-substituted pyridyl compounds (U.S. patent No. 5,302,606); 5) quinazoline derivatives (EP application No. 0566266 a1 and U.S. patent No. 6,103,728); 6) selenoindoles and selenides (PCT WO 94/03427); 7) tricyclic polyols (PCT WO 92/21660); 8) benzylphosphonic acid compounds (PCT WO 91/15495); 9) tyrphostin-like compounds (U.S. Pat. No. 6,225,346B 1); 10) thienyl compounds (U.S. patent No. 5,886,195); 11) benzodiazepine * -based compounds selective for src and FGF-r tyrosine kinases (disclosed in U.S. Pat. No. 6,100,254 at 8.8.2000). Tyrosine kinase inhibitors from various classes are claimed for the treatment of cancers driven by tyrosine kinases such as Met and HER2, EGFR, IGFR, PDGFR, scr and KDR/FLK-1. None of the known tyrosine kinase inhibitors are selective for Met. However, it is conceivable that inhibitors specific for Met could 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 has been approved for cancer therapy and several of which are in clinical development. These compounds include: 1) pyrazolopyrimidine PP1 shows selectivity for lck and src kinases over ZAP-70, JAK2 and EGF receptor kinases. 2) STI-571(GLEEVEC *) inhibits all forms of abl, PDGF receptors and c-kit tyrosine kinase. 3) ZD1839 is a synthetic anilinoquinazoline which is selective for the EGF receptor. 4) OSI-774 is another orally active quinazoline derivative with some selectivity for the EGF receptor. 5) 4-anilinoquinazoline derivatives exhibit selectivity for VEGF-R (U.S. Pat. No. 6,291,455B1, published at 9/18/2001). 6) SU101 shows selectivity for the PDGF receptor, but its antiproliferative effect is due in part to the ring opening metabolite, which inhibits dihydroorotate dehydrogenase, a mitochondrial enzyme essential for pyrimidine biosynthesis. 7) Aryl and heteroaryl quinazoline compounds exhibit selectivity for CSF-R (U.S. Pat. No. RE37,650E, published at 9.4.2002), and VEGF receptor (Flk1/KDR) antagonists SU 5416 were designed based on crystallographic studies of indolin-2-one pharmacophores and the FGF receptor tyrosine kinase domain. 9) Bi-monocyclic and bicyclic aryl and heteroaryl compounds show selectivity for EGFR and PDGFR (U.S. patent No. 5,409,930). 10) Piceatannol (3, 4,3, 5V-tetrahydroxy-trans-stilbene) showed selectivity for syk and lck, but also inhibited serine/threonine kinase and adenosine triphosphatase. 11) Several benzodiazepine-based compounds show some selectivity for the non-receptor type tyrosine kinase src and for the FGF-R tyrosine kinase receptor family. These examples show that compounds can be produced which are selective for one or more tyrosine kinases.

Antimalarial effect of protein kinase inhibitors

Like plants, the relevant apicomplexan parasites, such as plasmodium, appear not to produce protein tyrosine kinases. A few reports indicate protein tyrosine phosphorylation in plasmodium (see section a below). However, homology searches failed to detect any sequences related to the known protein tyrosine kinase family. It is therefore conceivable that the antimalarial effect of protein tyrosine kinase inhibitors is due to the inhibition of these enzymes produced by the human host. Several 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-dichlorobenzyl-nitrosoamino ] -quinazoline (CI-679) [ Schmidt and Rossan, am. J. Trop. Med. Hyg.28: 78192, (1979) ], several other 2, 4-diamino-6-substituted quinazoline derivatives synthesized by Elslager and colleagues [ Elsager et al, J.Med. Chem.21: 1059-70, (1978) ] and Chinese scientists [ Gy et al, Xao Xue Bao 19: 108-18, (1984), Yao et al, Yao Xue Bao 19: 76-8, (1984) ], several other 2, 4-diamino-6-substituted quinazoline derivatives, 2, 4-diamino-5-693, U.S. Methylanilino. 3,4,376,858, published in 1984 As described. One possible mode of action of quinazoline derivatives against plasmodium is the inhibition of tyrosine kinases (disclosed in us patent No. 6,103,728 at 8/15/2000).

A) Inhibition of plasmodium protein kinases

1) Dluzeski and Garda reported that several protein kinase inhibitors (staurosporine, genistein, methyl 2, 5-dihydroxycinnamate, tyrphostin B44 and B46, fumonisin a and RO3) inhibited the erythrocytic cycle of plasmodium falciparum [ Dluzewski and Garda, Experientia 52: 621-623, (1996)]. In addition to staurosporine, a strong serine/threonine kinase inhibitor, these compounds preferably inhibit protein tyrosine kinases. These inhibitors prevent the development and/or invasion of parasites inside erythrocytes. Because of the broad spectrum of activity of these inhibitors, it is unclear whether inhibition of protein tyrosine kinases plays any role in the observed effects, nor whether the target protein is derived from erythrocytes or parasites.

2) Upon screening artemisinin-like compounds from microorganisms, Tanaka and colleagues identified several fungal metabolites with antimalarial activity. One of these compounds, the broad-spectrum protein kinase inhibitor radicicol, is moderately active against plasmodium boidinii in mice [ Tanaka et al, j.antitoot 51: 153-60, (1998)].

3) More recently Sharma reported that membrane-bound PTK activity increased during maturation from the ring phase to the vegetative phase. Inhibition of PTK activity by chloroquine has been suggested to represent one possible mechanism of action of this drug against plasmodium [ Sharma and Mishra, Indian j.biochem.biophy.36: 299-304 (1999); sharma, Indian j.exp.biol.38: 1222-6(2000)].

B) Inhibition of human protein tyrosine kinases

The various pathogenic effects of plasmodium are mediated by protein tyrosine kinases in human hosts and can therefore be inhibited by protein tyrosine kinase inhibitors. Several examples have been reported in the literature.

1) Adhesion of infected erythrocytes to the endothelium of blood vessels involves binding of plasmodium falciparum membrane protein 1(PfEMP1) to CD36 expressed by endothelial cells of the host. CD 36-mediated signaling is essential for adhesion. Selective inhibitors of Src and lck kinases pyrazolopyrimidine PP1 inhibits this signal and prevents adhesion [ Yipp et al, Blood online, (2002) ].

2) CD36 and CD36 mediated protein kinase dependent signaling is also implicated in the non-opsonic clearance of plasmodium 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 the major toxin of plasmodium falciparum. Malarial GPI induced rapid initiation of tyrosine phosphorylation of multiple intracellular substrates within 1 minute of cell addition. These signals are involved in the upregulation of parasite adhesion and in the induction of Nitric Oxide (NO) release by macrophages and endothelial cells. Both adhesion and NO release can be inhibited by tyrosine kinase antagonists, inhibitors of tyrosine phosphorylation and genistein [ Tachado et al, J Immunol 156: 1897-; schofield et al, j.immunol.156: 1886-96].

Protein tyrosine kinase receptor Met has not been implicated in malaria infection in previous work. The present invention identifies the protein tyrosine kinase Met as an extremely important mediator of liver cells' susceptibility to infection by malaria sporozoites.

HGF-related antimalarial agents

A. Sulfated polysaccharides

As described above, the level of HGF can be increased by HGF degradation inhibiting polysaccharides including dextran sulfate, heparan sulfate, dermatan sulfate, keratan sulfate, chondroitin and chondroitin sulfate. The use of sulfated polysaccharides such as sulfated curdlan, dextrin sulfate, chondroitin sulfate, heparin, carageenan in combination with quinine for the treatment of malaria is described in U.S. patent No. 5,780,452, published 7, 14, 1998. The proposed strategy is based on the ability of sulfated polysaccharides to inhibit the invasion of malaria parasites into human erythrocytes. The present invention raises concerns about this strategy because sulfated polysaccharides can increase HGF levels by inhibiting HGF degradation, a fact described in U.S. patent No. 5,736,506, published 4-17 1998. Sulfated polysaccharides are therefore excluded from the antimalarial agents of the invention.

Examples

Example 1

Hepatocytes incubated with Plasmodium sporozoites release 'infection susceptibility inducing factors' (ISIF)

Supernatants were generated from cultures containing mouse hepatoma cells, Hepa1-6, and plasmodium yoelii (p.yoeliii) sporozoites (mH/Py conditioned medium). To test for ISIF activity, fresh hepatoma cells were incubated with mH/Py conditioned medium for various periods of time. The cells were then washed and incubated with plasmodium yoelii sporozoites. Infection was checked after 24 hours by staining the parasites in their infrared forms (EEFs). As a control, we preincubated Hepa1-6 cells for the same period of time with fresh medium before addition of Plasmodium yoelii sporozoites. Pretreatment of hepatoma cells with mH/Py conditioned medium increased the level of infection (fig. 1 a). The greatest enhancement in susceptibility to infection was observed in hepatoma cells pretreated with mH/Py conditioned medium for 1 hour (fig. 1 a). The mH/Py conditioned medium obtained with heat-inactivated sporozoites was not effective (fig. 1 b). Since sporozoites were obtained by dissection of the salivary glands of infected mosquitoes, we also tested conditioned media obtained from cultures containing hepatoma cells and salivary gland material of uninfected mosquitoes. The conditioned medium thus obtained was not effective (FIG. 1 b).

Example 2

ISIF release from damaged hepatocytes

To investigate the source of ISIF (sporozoites or hepatocytes) and the requirements for its release, Hepa1-6 cells were damaged using mechanical stress. Damaged cells were placed in tissue culture wells and supernatants were collected after 1 hour. Fresh Hepa1-6 cells were pre-incubated with this supernatant prior to addition of Plasmodium yoelii sporozoites. Preincubation of the supernatant resulted in an enhancement of infection similar to that observed with mH/Py conditioned medium (fig. 1 c). This finding suggests that ISIF is not derived from sporozoites but is released from hepatocytes as a result of injury.

Example 3

ISIF is Hepatocyte Growth Factor (HGF)

To test whether ISIF activity is mediated by known growth factors, two well-characterized growth factors known to be released after injury were tested: basic fibroblast growth factor (bFGF) and Hepatocyte Growth Factor (HGF). HepG2 cells were pre-incubated with human HGF or bFGF prior to addition of plasmodium boidinii sporozoites. As positive and negative controls, cells were incubated with hH/Pb conditioned medium or fresh medium, respectively. HGF enhanced infection to a greater extent than hH/Pb conditioned media (FIG. 2 a). bFGF was ineffective. To determine whether ISIF in hH/Pb conditioned media was HGF, neutralizing monoclonal anti-HGF antibody was added to this media before incubation with new HepG2 cells and sporozoites. The antibody not only abolished the effect of hH/Pb conditioned media, but also reduced infection below the basal level observed in control cultures (fig. 2 a). The addition of monoclonal anti-bFGF antibody had no effect (fig. 2 a). These results indicate that ISIF in conditioned media is HGF and indicate that HGF release is a prerequisite for sporozoite infection of hepatocytes.

Example 4

ISIF/HGF is secreted by hepatocytes after injury

HepG2 cells were treated with brefeldin-a (bfa), an inhibitor of protein transport to golgi vesicles, which blocks intrinsic secretion in eukaryotic cells. BFA-treated cells and untreated control cells were then washed and incubated with plasmodium boidinii sporozoites to produce conditioned media. Testing of this conditioned medium showed that BFA treatment inhibited ISIF/HGF secretion (fig. 2 b). The effects of BFA are dose-dependent. Supernatants of mechanically injured HepG2 cells were collected after incubation for different periods of time. ISIF/HGF levels increased over time as determined by infection susceptibility testing (fig. 2c) and western blot analysis (fig. 2 d). ISIF activity was abolished by the addition of neutralizing anti-HGF antibody (fig. 2 c).

Example 5

Correlation of ISIF/HGF Activity with infection levels

Despite the lower efficiency, p.berghei was able to infect the non-hepatic epithelial cell line HeLa (fig. 2 e). A series of experiments were performed to compare the responsiveness of HepG2 cells and HeLa cells to parasite-induced production and to ISIF/HGF. Conditioned media were generated from cultures of parasites with HepG2 cells (hH/Pb conditioned media) and with HeLa cells (HeLa/Pb conditioned media). Both conditioned media contained ISIF/HGF, although at different levels. ISIF activity was significantly correlated with HGF levels as determined by western blot and ELISA. HeLa cells are sensitive to ISIF/HGF, like HepG2 cells. HeLa cell infection was enhanced by hH/Pb conditioned medium and HepG2 infection was enhanced by HeLa/Pb conditioned medium. The ISIF activity in hH/Pb conditioned medium was always higher than that in HeLa/Pb conditioned medium (FIG. 2 e). These data indicate that HeLa cells respond to ISIF/HGF and the extent of infection increases with HGF dose.

Example 6

The effects of HGF on infection are mediated by Met

Various experimental protocols were applied to demonstrate the effect of HGF via its receptor Met. First, incubation of the plasmodium boidinii sporozoites with Hepa1-6 cells for 1 hour resulted in activation of Met kinase as evidenced by tyrosine phosphorylation of the receptor (fig. 3 a). Second, in the presence of constitutively activated MET tyrosine kinase (tpr-Met)¹⁴Transfected HepG2 cells (FIG. 3b), and in the use against MET¹⁵The infection of P.boidinii was enhanced in the anti-monoclonal antibody treated HepG2 cells of the extracellular domain (FIG. 3 c). These results show that activation of MET enhances the susceptibility of hepatocytes to infection by sporozoites. In addition, since the tpr sequence replaces the extracellular domain of Met in tpr-Met, this precludes the possibility that plasmodium sporozoites, as occur in Listeria (Listeria) infections, can utilize Met as a receptor into hepatocytes.

Two protocols were used to down-regulate MET. First, HepG2 was transfected with a chimeric construct containing the extracellular and transmembrane domains of met fused to gfp sequence. The product of this construct is expressed on the plasma membrane and binds to HGF but is unable to transduce signals into the cell because it lacks the tyrosine for the kinase domain and serving as a docking site for intracellular transducers; this chimera functions as a dominant interference protein, preventing its activation due to its dimer formation with endogenous MET. Due to the transfection efficiency of only 54.3 ± 2.1%, plasmodium boidinii sporozoite infection was reduced by approximately 60% in the total cell population (fig. 3 d). Transfected individual cells were completely resistant to infection as shown by GFP expression. Similar experiments with dominant interfering constructs of FGF receptors did not affect the susceptibility of HepG2 to plasmodium boidinii infection. In a second method, MET is down-regulated using interfering RNA. Transfection of two independent populations of HepG2 cells with specific MET oligos caused a decrease in MET expression as detected by western blot (fig. 3 e). The infection rate of these cells was reduced by 90% compared to mock transfected cells (FIG. 3 f). These results demonstrate that HGF signaling through its receptor MET is a prerequisite for plasmodium sporozoite infection of hepatocytes.

Example 7

In vivo correlation of HGF/MET in malaria infection

Primary hepatocytes were obtained by liver perfusion. The media conditioned by these cells had similar ISIF activity to the media conditioned by the liver cell line (fig. 4 a). The specific HGF receptor inhibitor K252a abolished ISIF activity (fig. 4 a). Cells that have been crossed by sporozoites can be detected using cell-impermeable fluorescent tracer macromolecules that permeate only into the injured cells. Before HGF staining, plasmodium yoelii sporozoites were incubated with Hepa1-6 cells in the presence of fluorescently labeled dextran. To detect cells that are crossed by sporozoites in vivo, standard assays for the detection of damaged cells in mice are utilized. Liver tissue sections were obtained and stained for HGF. Neither in vitro nor in vivo dextran negative cells expressed HGF, while most dextran positive cells were also positive for HGF staining (fig. 4 b). The results indicate that hepatocytes crossed by sporozoites express HGF during hepatocyte infection, presumably due to stress caused by injury, and that signaling of HGF through its receptor MET is required for infection. To demonstrate that MET signaling is required for the course of hepatocyte infection in natural infection with malaria, a group of 3 mice was injected with a lentivirus expressing a dominant interference protein against MET (MET-GFP, example 7). As a control, a group of 3 mice was injected with a similar virus but expressing only GFP. Two days later, both groups of mice were stimulated with 300000 plasmodium sporozoites. And examined for parasitemia after 2 or 3 days. Each liver section was obtained to determine the level of viral infection (fig. 4 c). The results show that expression of MET-GFP in liver is required for natural infection (fig. 4 d).

Example 8

Effect of Genistein on in vitro liver infection by Plasmodium boidinii sporozoites

HepG2 cells were cultured in DMEM 10% FCS, 1mM glutamine. Plasmodium boidinii sporozoites were obtained from dissection of the salivary glands of infected Anopheles hindensis (Anopheles stephensi). The sporozoites of Plasmodium boidinii (5X 10)⁴) Adding into 2X 10⁵Monolayers of HepG2 cells (in the presence or absence of genistein) for 24 hours before fixation and subsequent staining with mAb against EEF (2E6) followed by anti-mouse IgG-FITC antibody. Infection was quantified by counting the number of EEFs per coverslip. The results are shown in fig. 5. The results show the number of infected cells. Genistein at 25 μ M has been shown to reduce infection by about 75%.

Example 9

Effect of Genistein on liver infection by Plasmodium boidinii sporozoites in vivo

Again, plasmodium boidinii sporozoites were obtained from the dissection of the salivary glands of infected anopheles indica indiae. Malaria of BoehmeriaProtozoan sporozoites (5X 10)⁴) Two groups of 5 mice were injected intravenously. One group was injected 6 hours ago with DMSO containing 4mg of genistein, while the other group was injected with DMSO alone (control). Liver infection was quantified after 42 hours by real-time RT-PCR using parasite-specific primers. The results are shown in fig. 6. Infection was reduced by approximately 80%.

I. Currently used antimalarial drugs

As previously mentioned, the antimalarial agents of the present invention may optionally comprise currently used antimalarial drugs, in combination with inhibitors of HGF activity.

Although antimalarial drugs have long been known to be present in the flowering plants Artemisia annua (artemisia annua) and cinchona bark, there are currently only a few drugs available for treating or preventing this disease. Currently used antimalarial drugs are described in recent review articles [ Ridley, Nature 415: 686-. The most widely studied antimalarial drugs are quinolines, antifolates, artemisinin, electron transfer inhibitors such as atoquinone (atoquavone), and antibiotics such as tetracycline. To eliminate the development of resistance, some drugs are used in fixed combinations, and several new drug combinations are currently under investigation.

A. Quinolines

In south america, powders derived from the bark of cinchona have long been used to treat fever. Cinchona powder was introduced into europe in the 17 th century and quinine, an anti-pyretic component, was isolated in 1820 by Pelletier and Caventou. Quinine is currently used in the treatment of severe malaria, multi-drug resistant malaria, and malaria during the first three months of pregnancy. Quinidine is the dextrorotatory diastereomer of quinine, is more active than quinine, but is also more cardiotoxic and more expensive. Due to its broad effectiveness as an antiarrhythmic agent, parenteral quinidine is used in the united states for the treatment of severe malaria. Quinine and quinidine, when administered by bolus injection, can cause hypotension, as well as hypoglycemia, which is a particular problem in pregnant women. In order to identify more effective and safer antimalarial drugs, a number of related compounds were synthesized based on quinine structure. Chloroquine was first synthesized in 1934 in germany and was independently identified as the most promising first-choice drug in a series of 4-aminoquinolines synthesized in the united states during world war ii. Chloroquine, having a variety of trade names such as Nivaquine, malaquine and Aralen, has been the mainstay of plasmodium falciparum chemotherapy for decades. It is inexpensive, safe when applied in the correct dosage, efficient and feasible for outpatient use. Chloroquine is generally well tolerated, although it may cause itching, especially in black skin patients, nausea, and rare neuropsychiatric symptoms or cerebellar dysfunction. Chloroquine may be administered intramuscularly or subcutaneously or by intravenous infusion. Resistance to chloroquine has been slowly developed, but is now widespread, not only in south-east asia, but also in many areas of africa. It is currently used for the treatment of non-malignant infections and for the treatment and prevention of plasmodium falciparum malaria in areas where resistance has not yet developed.

Chloroquine is 4-aminoquinoline. In order to identify new active drugs against chloroquine-resistant plasmodium strains, a large number of 4-aminoquinolines were synthesized. This effort led to the discovery of amonoquine (amonoquine) currently used for the treatment of chloroquine-resistant malaria. However, the use of amonoquine is limited by the fact that it shows some cross-resistance with chloroquine and by side effects when used for prophylaxis, such as hepatitis and agranulocytosis. Despite the extensive efforts of the past thirty years, scientists have failed to produce inexpensive and effective 4-aminoquinolines as alternatives to chloroquine [ reviewed by O' Neill et al, pharmacol, ther.77: 29-58, (1998)].

In the 60's of the 20 th century, two antimalarial drugs mefloquine and halofantrine emerged from quinine-related structural testing in the U.S. Walter Reed Institute of Medical Research. Mefloquine, developed by Hoffmann La Roche under the trade name Lariam, was first applied for prophylaxis in 1985, and has been used later for prophylaxis in 1450 million people and treatment in 160 million people. It is currently used in the treatment and prevention of areas with chloroquine resistance. Mefloquine has an elimination half-life of 2 to 3 weeks. The course of treatment includes 2 or 3 doses and side effects include digestive tract disorders and neuropsychiatric effects. Like mefloquine, the closely related halofantrines are expensive. Intravenous formulations have been developed because their absorption varies from patient to patient. Halofantrine is used in the treatment of malaria caused by plasmodium falciparum suspected of being chloroquine resistant. Its use is limited by fatal cardiotoxicity.

Primaquine is an 8-aminoquinoline which was developed after Paul Ehrlich 1891 discovered that methyl blue has weak anti-plasmodium activity. From a large series of methoxy and 8-aminoquinoline derivatives, pamaquine was first identified as the lead and the drug was introduced in 1926. The search for less toxic, more potent compounds of this class has led to the emergence of pentameraquine, isomeraquine, and primaquine. Primary amine quines are extensively tested during the freshest war and are now used in specific indications. Primaquine is structurally related to chloroquine, but differs in mode of action. Unlike other quinine antimalarial drugs, primaquine acts against the hepatic stage of plasmodium falciparum and destroys the late hepatic stage and latent forms of plasmodium vivax and plasmodium ovale. The latter activity is unique among currently used antimalarial drugs and makes primaquine the drug of choice for preventing malaria recurrence, which may not occur until 40 weeks after the initial challenge with plasmodium vivax and plasmodium ovale. Although primaquine acts against the erythroid forms of p. In contrast to the erythrocytic forms of plasmodium vivax and plasmodium ovale, those forms of plasmodium falciparum are insensitive to primaquine. Therefore, primaquine is not used for the treatment of malaria caused by plasmodium falciparum. Primaquine has a short half-life and must be administered daily. Gastrointestinal side effects are generally mild, but more severe oxidative hemolysis may occur, especially in patients with glucose-6-phosphate dehydrogenase deficiency. The related compound, tafenoquine (tafenoquine), is much slower in clearance, with a terminal half-life of about 14 days. The novel compounds may have a greater therapeutic index than primaquine, but their therapeutic effects remain to be determined.

B. Artemisinin

Artemisinin is an active ingredient of the chinese flowering plant Artemisia annua (Artemisia annua) which chinese herbal physicians have used before the last 2000 years. The ether extract artemisinin of artemisia annua was found to be effective against malaria in mice in the 60's of the 20 th century. In 1972, Chinese scientists isolated their active ingredients. China produced water-based artesunate formulations and was safely used in the treatment of over 100 million malaria patients. In the united states, Klayman discovered the artemisia species artemisia apiacea, and developed an oil-based extract for severe malaria treatment testing. Oil-based formulations are not approved in the western world due to neurotoxic effects in animals. However, due to the emergence of quinoline-based antimalarial drug resistance, interest in artemisinin has been enhanced and several semi-synthetic derivatives have been produced. In addition to artemisinin, which has been obtained by extraction from the plant Artemisia annua, several semisynthetic derivatives are currently used. They include artemether, artesunate and dihydroartemisinin. The latter compound is a metabolite of all other artemisinin-based drugs and the main active agent in the body. Artemisinin has a broad spectrum of activity against all parasite stages within erythrocytes, especially the juvenile toroids. Artemisinin reduces parasitemia and inhibits gametocyte transmission more rapidly than other antimalarial drugs. A disadvantage of semi-synthetic artemisinin derivatives is that they are more expensive than the maternal drug. The short half-life of artemisinin derivatives and their active metabolite dihydroartemisinin requires 5-7 days of treatment when these compounds are applied alone. Artemether was originally used to treat severe malaria. However, intramuscular use of this drug proved not to be better than intravenous use of quinine. Artemisinin and its derivatives are currently used in combination with other antimalarial drugs for the treatment of non-complicated malaria.

B. Mode of action of quinolines and artemisinin

Understanding the role of known antimalarial drugs and the molecular mechanisms underlying resistance of plasmodium to the development of these drugs is important for future drug development. Quinoline and artemisinin concentrate in lysosomal food vesicles where they appear to exert their antimalarial activity through interaction with heme. Heme is produced by the degradation of hemoglobin abundant in host red blood cells. Heme (FeII) is oxidized to hematin (FeIII) and sequestered in the cytoplasm as an inert pigment called hemozoin. Hemozoin comprises a structural lattice of aggregated heme dimers. The sequestration of hemoglobin protects the parasite from lipid peroxidation of free heme or other toxic effects. The primary targets of quinoline are the older trophozoites, which produce large amounts of heme in their food vacuoles. Chloroquine and other antimalarial quinolines are believed to inhibit dimerization of heme or prevent its processing from the food vacuole to the cytoplasm, which is where hemozoin forms. Artemisinin also depends on heme for its antimalarial effect. These drugs are believed to kill parasites via free radicals generated as a result of oxidative cleavage of the drug's peroxide bond in the presence of heme. However, quinoline antimalarials [ Sullivan et al, j. biol Chem 273: 31103-: the precise mode of action of 122-126 (2001) remains to be elucidated.

The resistance of plasmodium falciparum to chloroquine and possibly other quinolines appears to be due to a reduction in drug transport to the food vacuole. Defects in drug transport may be caused by mutations in the putative chloroquine-resistant transporter gene (PFCRT) and the P-glycoprotein-encoding gene (Pfmdr 1). Although artemisinin transport appears to be affected by mutations in the Pfmdr1 gene, no clinical resistance to artemisinin and its derivatives was observed.

D. Antifolate

The most important antimalarial drugs, besides chloroquine, are compounds designed to inhibit the synthesis of the folate co-factor, which is essential for nucleotide synthesis and is involved in amino acid metabolism. The most commonly used antifolates are 2, 4-diaminopyrimidine, pyrimethamine, proguanil (proguanil or albedone) and the sulphur drugs sulfadoxine, sulfamethoxypyrazine or dapsone. Pyrimethamine inhibits dihydrofolate reductase (DHFR) which is present in plasmodium as a fusion protein with Thymidylate Synthase (TS). Sulfadoxine inhibits dihydropteroate synthase (DHPS), another enzyme in the folate pathway. The success of anti-folate therapy against plasmodium falciparum has been attributed to differences in drug binding of the corresponding enzymes of the host-parasite involved in folate cofactor synthesis. Pyrimethamine has a higher affinity for plasmodium DHFR-TS than for human DHFR. However, other DHFR-TS inhibitors are also selectively toxic to parasites, and do not bind the plasmodium enzyme more strongly. The increased susceptibility of parasites to antifolates compared to mammalian cells appears to be due, at least in part, to differences in the regulation of DHFR translation between malaria parasites and human hosts [ Zhang and ratho, Science 296: 545-7, (2002)].

When antifolate drugs are used alone, resistance to their effect rapidly develops due to mutations in the target enzymes, dihydrofolate reductase (DHFR) for pyrimethamine and dihydropteroate (dhps) for sulfadoxine and related sulfur drugs. Thus, antifolates are used in combination. Pyrimethamine is formulated in fixed combination with other antifolate compounds such as sulfadoxine, sulfamethoxypyrazine or dapsone. The fixed combination of pyrimethamine and sulfadoxine, known under the trade name Fansidar, represents the most important antifolate treatment of malaria. Sulfadoxine/pyrimethamine or sulfamethoxypyrazine/pyrimethamine are used for the treatment of severe plasmodium falciparum infections considered to be chloroquine resistant. These combinations have proven useful for intermittent treatment during pregnancy. Occasional hypersensitivity to sulfur components can cause painful blistering of the skin. This side effect prevents the prophylactic use of sulfadoxine/pyrimethamine. A combination of two compounds acting independently of each other on two different enzymes in the folate pathway is designed to reduce the risk of resistance development. Unfortunately, however, strains of plasmodium falciparum have emerged due to widespread combinatorial use.

More recently, antifolates have been combined in fixed combinations with drugs that combat malaria parasites through mechanisms unrelated to folate synthesis. Atopoquinone, a drug originally developed to combat pneumocystis infection in AIDS patients, proved to be effective against malaria, presumably by interfering with electron transport in mitochondria. To counteract the rapid development of resistance, atopoquinone was combined with proguanil (proguanil, albedone). The antimalarial activity of proguanil is due to its cyclic triazine metabolite, cycloproguanil, which selectively inhibits the plasmodium bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS). The atoquinone-proguanil combination sold under the trade name Malarone by GlaxoSmithkline ltd is a safe and effective novel antimalarial drug. However, due to its complex synthesis, atopoquinone is expensive. Manufacturers have initiated drug donation programs for africa, but the number of treatments donated may not be sufficient for their first line use.

E. Antibiotic

Plasmodium and other parasites possess a plastid organelle called the acrosome (apicoplast) which contains 35kb circular DNA. Plastids incorporate elements that resemble prokaryotic transcription and translation systems. This system is sensitive to compounds known to inhibit bacterial protein synthesis such as tetracycline, doxycycline and clindamycin. Because of their slow mode of action, these antibiotics are used mainly in combination with other fast acting drugs. The use of tetracycline and doxycycline is limited to patients over the age of 8 years and is contraindicated in pregnant and lactating women. The two antibiotics are used in combination with quinine. Clindamycin (7-chloro-lincomycin), a semi-synthetic derivative of lincomycin, was introduced as an antibiotic in the 60 s of the 20 th century. Clindamycin is safe for children and pregnant women. Several general formulations of clindamycin are available. A three day treatment course spent higher than sulfadoxine/pyrimethamine but less than either atoquarone-chloroguanidine or halofantrine. Clindamycin has been used in several trials for monotherapy of malaria, but is most effective in combination with fast acting drugs [ Lell and Kremsner, antimicrobial agents and chemitherapy 46: 3215-2320, (2002)].

F. Treatment to inhibit intrahepatic development of malaria parasites

Only a few of the currently used antimalarial drugs act against the development of plasmodium in hepatocytes. These drugs include primaquine and the antifolate combination pyrimethamine/sulfadoxine. Although the mechanism of action of primaquine against the hepatic form of plasmodium is unknown, antifolate combinations may inhibit the synthesis of plasmodium DNA required for sporozoite proliferation. The present invention provides novel drug targets and drug target candidates that interfere with intrahepatic development of malaria parasites.

Current efforts in antimalarial drug development

A. Drug resistance reversal agents

The major problem with malaria is the production of drug resistant plasmodium strains. Resistance to a drug can be counteracted by combining the drug with a compound that reverses resistance. Resistance of plasmodium falciparum to chloroquine was reduced in vitro by a variety of compounds [ Singh and Puri, Acta tropica 77: 185-193, (2000)]. However, only cyproheptadine has been shown to be effective against chloroquine resistant lines of the yoelii niger plasmodium in a mouse model, while others such as verapamil and antihistaminic chlorpheniramine show moderate activity. Chlorpheniramine is often used to treat pruritus caused by chloroquine. In clinical studies, the chloroquine/chlorpheniramine combination produced higher therapeutic efficacy than chloroquine alone [ Sowumi et al, clinical Mecicine and International Health 3: 177-185, (1998) ], whereas the combination of chloroquine and desipramine had no clinical benefit in previous studies [ Warsame et al, Transactions of the Royal Society of clinical Medicine Hygiene 86: 235-236, (1992)]. Antisense oligonucleotides designed to reduce the expression of proteins involved in drug trafficking are described in U.S. patent No. 6,440,660B1, 8/27/2002.

B. Novel antimalarial drug combinations

The most important strategy to prevent resistance is to use drug combinations. As already mentioned above, this strategy has been applied in the past by using fixed combinations such as pyrimethamine and sulfoximine (fansidar) or a combination of atoquinone and proguanil (Mlalarone). More recently, a variety of new strategies have been implemented and several others are under investigation [ reviewed in the latest World Health Organization publication (WHO/CDS/RBM/2001.35) ]. Quinoline compounds such as chloroquine, amonoquine, mefloquine and quinine are combined with the antifolate combination sulfadoxine/pyrimethamine. Based on the observation that these compounds have additive antimalarial activity, a combination of mefloquine and sulfadoxine/pyrimethamine (Fansimef, Roche) was developed. However, unexpectedly, the use of this combination as a first-line treatment for non-concurrent malaria results in a rapid development of resistance to mefloquine. Therefore, the combination is not recommended for use in prophylaxis or therapy. Artemisinin is combined with a longer half-life drug to reduce treatment time and increase compliance. It is believed that rapid clearance of the parasite from artemisinin reduces the chance of developing resistance to the chaperone drug. Combinations based on artemisinin include artesunate plus chloroquine, or amonoquine, or mefloquine, or sulfadoxine/pyrimethamine and combinations of artemether and lumefantrine. The latter combination, known under the trade name Coartem and rimat (Novartis), is available as a fixed combination and represents the most promising combination therapy currently available. The combination has recently been approved by regulatory agencies. Combinations under investigation include various piperaquine-dihydroartemisinin-trimethoprim (arteom), arteom plus primaquine (CV8), artesunate plus bisquine, naphthoquine phosphate (naphtoquine) plus dihydroartemisinin, and chlorogenanil-dapsone plus artesunate (CDA or lapap plus). The antibiotics tetracycline and doxycycline are often used in combination with quinine, and clindamycin in combination with quinine, chloroquine, and more recently with the novel antimalarial drug fosmidomycin (fosmidomycin), which inhibits the 1-deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase, a key enzyme of the non-mevalonate pathway of isoprenoid biosynthesis.

C. Novel antimalarial drug

As the mechanisms underlying drug action and drug resistance are elucidated and the understanding of the biochemical pathways utilized by plasmodium diseases is increased, the development of antimalarial drugs is expected to advance faster than in the past. An overview of the metabolic pathways in plasmodium is available on the internet (http:// sites. huii. ac. il/malaria /). The implementation of plasmodium genome projects, the development of improved transfection techniques, and the application of RNA interference techniques have accelerated the learning process. New approaches to antimalarial drug development have been reviewed in recent publications [ Winstanley, Parasitology Today 16: 146-; mechanisms of action, resistance, and new directions in drug discovery, Humana, Totowa, new jersey, edited by p.rosenthal, (2001); ridley, Nature 415: 686-693 (2002); robert and McConkey, Molecular & Biochemical Parasitology 119: 273-278(2002)]). In order to contrast these efforts with the strategy that is the subject of the present invention, a short description of these efforts is provided in the following section.

Current approaches to malaria treatment can be divided into three categories: improved classes of known drugs, drugs directed to newly identified targets, and drugs with unknown or poorly defined targets.

1) Improved types of known medicaments

Chloroquine remains an attractive guide for the development of new drugs. Examples of novel lead compounds include short-chain chloroquine analogs (bisquinolines), analogs of amonoquine lacking the ability to form toxic metabolites, and bisquinapine, a 4-aminoquinoline originally developed by china. High throughput screening of large compound libraries has been used to identify new structures that bind heme in a similar manner to the binding of quinoline. Artemisinin-related trioxanes are described in U.S. patent 6,136,847, published at 10 months and 24 days in 2000. The novel drug candidates also include biguanides directed to DHFR, a target of pyrimethamine, and other inhibitors of purine and pyrimidine metabolism in plasmodium (disclosed in U.S. patent No. 5,663,155, 9/2/1997). Novel inhibitors of cytochrome c reductase-atoquarone targets include beta-methoxy acrylates.

2) Drugs against newly discovered targets

2.1 protease inhibitors. Protease inhibitors for the treatment of parasitic infections in metazoan animals are described in U.S. patent No. 5,739,170, published at 14.4.1998 and in U.S. patent No. 6,194,421B 1, published at 27.2.2001.

2.1.2 proteases involved in hemoglobin degradation. The red blood cell form of plasmodium degrades up to 80% of the host cell hemoglobin in the food vacuole. Hemoglobin is degraded in the food vacuole into peptides, which are then exported to the cytoplasm and eventually degraded into amino acids. Enzymes involved in hemoglobin degradation include the aspartic protease (plasmepsins), the cysteine protease falcipain, a metallopeptidase and other peptidases. The best known hemoglobin degrading protease is aspartic protease [ Coombs et al, Trends in Parasitology 17: 532-7, (2001)]. Drugs that inhibit the catalytic activity of Human Immunodeficiency Virus (HIV) aspartic proteases have been previously developed and leaders for inhibition of human aspartic proteases such as renin and aspartic acids from a variety of pathogenic microorganisms such as Aspergillus (Aspergillus) and Candida (Candida) have been identified. Homology searches of plasmodium genomes revealed 8 plasmepsins in addition to the two previously known plasmepsins I and II. Inhibitors of plasmodium plasmepsins are found in a large number of compounds that have been synthesized for screening drugs against human aspartic proteases. Homology modeling revealed inhibitors of the cysteine protease falcipain-2 (vinylsulfone), which inhibited malaria parasite growth in vitro [ Sabnis et al, j.biomol. struct. dyn.19, 765-74, (2002) ]. The challenge is to find inhibitors that are active against plasmodium enzymes but inactive or much less active against homologous human proteases.

2.1.3. Proteases involved in erythrocyte invasion. The entry of merozoites into erythrocytes requires proteolytic cleavage of several proteins on the parasite and on the surface of the erythrocytes. Two proteases expressed by merozoites have been studied: plasmodium falciparum subtilisin-like proteases-1 and-2 (PfSUB-1 and PfSUB-2). These and several other potential drug Targets for proteases [ Blackman, curr. drug Targets 1: 59-83, (2000)].

2.2. And (4) synthesizing fatty acid. Fatty acid synthesis occurs by repeated elongation of the acyl chain using the 2-carbon donor malonyl-coa (coa). This pathway in bacteria (termed the class II pathway) includes several fatty acid synthase enzymes (FAS). This pathway (termed the class I pathway) is catalyzed in animals by a large multifunctional protein. The class II pathway for de novo fatty acid synthesis is found not only in bacteria but also in plants and in the acrosomes of certain parasites including plasmodium. The plasmodium class II pathway includes Acyl Carrier Protein (ACP), β -ketoacyl-ACP-synthetases iii (fabh) and I/II (FabB/F), and enoyl-ACP reductase (FabI). The antibiotics triclosan and thiolactam and their derivatives are lead compounds in the study of new antimalarial drugs that inhibit fatty acid synthesis [ Waller et al, Antimicrobial Agents and chemotherapy 47: 297-; prigge et al, Biochemistry 42: 1160-69, (2003)].

2.3. A non-mevalonate pathway for isoprenoid synthesis. Although isoprenoids are synthesized in humans via the mevalonate pathway, in plasmodium they are synthesized via the non-mevalonate pathway, also known as the MEP pathway. This pathway is known to play a role in certain bacteria and plants. In plasmodium it includes the enzyme encoded by the circular DNA in the acrosome. 1-deoxy-D-xylulose 5-phosphate [ DOXP ] reductoisomerase, a key enzyme in the non-mevalonate pathway, is inhibited by fosmidomycin. The antibiotic was originally isolated from Streptomyces lavendulae (Streptomyces lavendulae). Fosmidomycin has strong antimalarial activity in vitro and in murine malaria. Initial clinical trials showed that the drug was well tolerated. Unfortunately, however, this drug results in the rapid development of resistance. It must therefore be used in combination with other drugs. Preclinical studies have shown that fosmidomycin may be useful in combination with lincomycin and clindamycin.

2.4 protein prenyltransferase. Various proteins include small G proteins such as Ras, Rac, Rap, Rho, Rab, heterotrimeric G protein gamma-subunits, nuclear lamin, protein kinases, and protein tyrosine phosphatases are prenylated post-translationally at the carboxy terminus with farnesyl (C15) or busulfanyl (C20). The attachment of farnesyl or geranylgeranyl is catalyzed by isopentenyl transferase. Inhibitors of these enzymes are widely studied candidates for anticancer drugs. Two prenyltransferases, PET and PGGT-1, have been identified in Plasmodium falciparum. Several peptidomimetics and monoterpenes, limonene inhibit prenylation and parasite growth [ Chakrabarti et al, j.biol.chem.277: 42066-73, (2002)]. Farnesyl transferase inhibitors (phenylsiquerpene) are described in U.S. patent 6,429,203, 8/2/2002.

2.5. Lactate Dehydrogenase (LDH). Plasmodium ldh (pldh) is essential for anaerobic production of ATP in the sexual and asexual stages of plasmodium. Different isomers of pLDH were found to be present in different plasmodium species. Their detection is used as a diagnostic test and for monitoring the antimalarial efficacy of drugs. pLDH is considered a promising drug target due to structural differences from human LDH [ Dunn et al, nat. struct. biol.3: 912-5, (1996)].

2.6. A phospholipid biosynthesis inhibitor. The development and proliferation of plasmodium erythrocyti requires large amounts of phospholipids. Phosphatidylcholine (PC), the major phospholipid present in infected erythrocytes, is synthesized from plasma-derived choline primarily by the enzymes of the parasite. A large number of choline-like compounds have been synthesized and some have antimalarial activity. Lead compound: g25 and its analogs, VB5-T, VB5-T inhibit the growth of Plasmodium falciparum and Plasmodium vivax in vitro at concentrations that are non-toxic to mammalian cell lines. Very low dose G25 treatment cured monkeys infected with plasmodium falciparum and plasmodium cynomolgus (p. These choline-based drugs appear to interfere with choline absorption and thus PC synthesis [ Wengelnik et al, Science 295: 1311-14, (2002)].

2.7. Glycosylphosphatidylinositol (GPI) synthesis. GPIs are ubiquitous in eukaryotic cells. They are synthesized in the Endoplasmic Reticulum (ER) by the action of glycosyltransferases by the sequential addition of sugar residues to Phosphatidylinositol (PI). Mature GPI translocates across the membrane from the cytoplasm into the luminal surface of the ER. After synthesis is complete, the GPI glycolipid is exported to the cell surface, either free or covalently bound to a protein. GPIs are important inflammation-inducing compounds of plasmodium and other parasites. Two GPI-anchored proteins circumsporozoite protein (CS) and the merozoite surface proteins MSP-1 and MSP-2, as well as GPI, are vaccine candidates per se. Because of the differences between plasmodium GPIs and mammalian GPIs, synthesis of GPIs in plasmodium is an attractive drug target [ Delorenzi et al, Infection and Immunity 70: 4510-4522, (2002)].

Evidence for the principle of this method has been obtained in Trypanosoma brueckii (Trypanosoma brucei): blocking GPI synthesis by disruption of the PIG-B gene renders the blood stage of Trypanosoma brucei non-viable.

2.8. A protein kinase. Plasmodium protein kinases can be divided into several groups and families [ kappa et al, Parasitology Today 15: 449-454(1999)]. Most of these kinases show 40 to 60% homology at the amino acid level with their mammalian counterpart kinases. Of particular importance for drug development are calcium-dependent protein kinases (CDPKs) which have been found to be present in plants and some protozoan species but not in mammals, and several kinases with large insertions in the catalytic domain, such as PfPK1, PfPK4 and FEST. Pfnek-1 is an example of a potential drug target in this class. It shows homology to a protein kinase involved in eukaryotic cell division, which never exists in the mitosis/aspergillus (NIMA)/NIMA-like kinase (Nek) family. Similar to other Plasmodium falciparum protein kinases and the NIMA/Nek family, Pfnek-1 has a large C-terminal extension in addition to the catalytic domain. One of its substrates is Pfmap-2, an atypical Plasmodium falciparum MAPK homolog. The bacterially expressed recombinant Pfnek-1 protein can be used in inhibition assays to screen for inhibitors [ Dorin et al, Eur J Biochem 268: 2600-8, (2001)]. No protein with homology to known protein tyrosine kinases has been found in plasmodium, although protein tyrosine phosphorylation has been reported to occur.

2.9. A polyamine. Like all eukaryotes, plasmodium contains three polyamines: diamineputrescine and derivatives thereof, spermidine and spermine. The compounds have pleiotropic functions in cell proliferation and differentiation. Strategies to interfere with polyamine function include inhibition of polyamine synthesis, polyamine inversion (back conversion) and polyamine transport, or deregulation of polyamine metabolism by structural analogs. Combinations of polyamine synthesis inhibitors with polyamine structural analogs are under investigation for the treatment of malaria and diseases caused by other pathogenic protists. This approach benefits from the large compound libraries generated in the study of novel anticancer drugs [ Muller et al Trends in Parasitology 17: 242-9, (2001)].

2.10. Histone deacetylase

Histones are nuclear proteins involved in transcriptional regulation via continuous acetylation/deacetylation of specific lysine residues. In plasmodium falciparum, histones are abundant and at least one histone deacetylase has been identified. Apicidin, an isolated cyclic tetrapeptide of Fusarium (Fusarium), inhibits mammalian cell proliferation and in vitro development of Apicomplexan parasites including plasmodium species, possibly by interfering with the sequential acetylation/deacetylation process [ Darkin-Rattray et al, Proc Natl Acad Sci USA 93: 13143-7, (1996)]. This finding led to the search for selective histone deacetylase inhibitors of plasmodium. Lead compounds include trichostatin a (tsa), sodium n-butyrate, hexamethylene bis-acetamide (HBMA) and newly developed HMBA analogs such as azelaic acid bis-hydroxamic acid (ABHA) and suberic acid bis-dimethylamide [ Andrews et al, International Journal For Parasitology 30: 761-768, (2000)].

2.11. A shikimic acid pathway. The shikimic acid pathway is present in the plastids of prokaryotes, fungi, and plants and algae, but not in vertebrates. This pathway generates the necessary substrates for the synthesis of mycolic acids, para-aminobenzoic acid (PABA) and folic acid. It is also required for the synthesis of ubiquinone, aromatic amino acids and almost all other aromatic compounds. Mammals do not have a shikimic acid pathway and rely on exogenous folic acid. The mycolic acid synthase (CS) has been identified as a useful drug target using recently developed RNA interference techniques [ Robert and McConkey, Molecular & Biochemical parasitism 119: 273-278(2002)]. A guide to drugs that inhibit this pathway has been obtained. The herbicide glyphosate (more well known by its trade name of RoundUp, Zero or Tumbleweed) is an inhibitor of 5-enopyruvyl shikimate-3-phosphate synthase, which inhibits the growth of plasmodium in vitro.

2.12. Cyclophilin. Cyclophilins are present in all living organisms. Human cyclophilin a (hcypa) was originally identified as a cytoplasmic target for the immunosuppressive drug cyclosporin a (csa). Attempts to suppress mouse immunity with CSA revealed the unexpected fact that CSA inhibited malaria growth in rodents. CSA and several non-immunosuppressive CSA analogs have subsequently been shown to have anti-malarial activity in vitro. Early erythrocytic ring stage parasites appear to be particularly sensitive. Among the three cloned plasmodium cyclophilins, PfCyP19 is the closest homolog of human CypA. Like other cyclophilins, PfCyP19 has peptidyl proline cis-trans isomerase (PPIase or rotamase) activity. It binds CSA with high affinity. Its ability to inhibit parasite growth does not appear to be related to inhibition of rotamase activity, but rather to inhibition of unidentified target proteins by the PfCyP19-CSA complex.

2.13. A transport system. Parasite invasion of erythrocytes is associated with changes in the erythrocyte membrane transport system not found in uninfected erythrocytes and the appearance of New Permeability Pathways (NPPs) [ identified by Kirk, Physiological Reviews 81: 495-537 (reviewed in 2001). Parasite and/or erythrocyte derived transport proteins are localized to the vesicle membrane and the parasite surface. Some transporters on the surface of parasites, such as ATP/ADP exchanger, V-type H1-ATPase, H1-PPase, are commonly found on the membranes of intracellular organelles. Transport proteins are important for new approaches to malaria chemotherapy. In one aspect, drugs can be designed that block nutrient uptake by the parasite. On the other hand, the delivery system can be used as a way of targeting cytotoxic agents to intracellular parasites. Drug target candidates under investigation include voltage-dependent channels localized to the surface of infected erythrocytes, which play a role in nutrient uptake [ Desai et al, Nature 406: 949-51, (2000) ], proteins involved in the uptake of delta-aminolevulinic acid dehydratase (ALAD) and other host enzymes that may be used by Plasmodium for heme synthesis [ Bonday et al, NatureMedicine 6: 898-903, (2000) ], and a parasite-encoded hexose transporter localized to the plasma membrane of the parasite inside infected erythrocytes [ Woodrow et al, j.biol.chem.274: 7272-7, (1999)].

3. Antimalarial drug with unclear action mechanism

3.1 tryptanthrin. After its synthesis in 1963, indolo [2, 1-b ] -quinazoline-6, 12-dione (tryptanthrin) was isolated from Isatis tinctoria, an ancient European and Chinese dye plant and herb. This compound is readily synthesized and produced by Candida lipolytica (Candida lipolytica) when grown in media containing excess tryptophan and anthranilic acid, hence the name tryptanthrin. Tryptanthrin is active against a variety of microorganisms, especially intracellular microorganisms such as mycobacteria (mycobacteria), leishmania donovani (leishmania donovani), Trypanosoma cruzi (Trypanosoma cruzi) and plasmodium [ Bhattacharjee et al, Bioorganic & Medicinal Chemistry 10: 1979-; scovill et al, Antimicrobial Agents And Chemotherapy 46: 882-883, (2002)]. The compounds are agonists of the aryl hydrogen receptor and induce the expression of cytochrome P4501a1 in hepatocytes [ Schrenk et al, biochem. pharmacol.54: 165-71, (1997) ] and inhibit cyclooxygenase-2 and 5-lipoxygenase [ Danz et al, Planta Med.68: 152-7, (2002)]. The mechanism of action against intracellular microorganisms is not known. A series of derivatives developed to obtain optimal activity against malaria parasites is described in US6284772 published at 9, 4.2001.

3.2. Dichroine is used. At the end of the 40 s and early 50 s of the 20 th century, dichroine and halofuginine were found as antimalarial agents in extracts of dichroa febrifuga (dichrafebrifuga) or Hydrangea umbellata (Hydrangea umbellate). Although dichroine shows structural similarity to chloroquine [ Chang, j.Theor.biol.59: 497-501, 1976], but its antimalarial activity does not appear to be associated with hemoglobin degradation. This compound enhances nitric oxide production, a possible mode of action against plasmodium [ Murata et al, Biochemical Pharmacology 58: 1593-1601, (1999)]. The synthesis of dichroine and its antimalarial activity is described in us patent No. 6,420,372B 1.

3.3. Hybrid peptides. Peptides consisting of naturally occurring cyclic peptides such as cecropin, magrocoxin, sapecin, bovine leukocyte antimicrobial peptide, alamethicidin, defensin and PGLA2 and toxins such as streptolysin, melittin, barbatalysin, paradoxin and delta hemolysin are described as antimalarial compounds in us patent 5,714,467, published on 3.2.1998.

Antimalarial Effect of cytokines

Interleukin-1 (IL-1) inhibits intrahepatic development of plasmodium falciparum sporozoites in rhesus monkeys, but is effective only when applied prior to sporozoite inoculation [ Maheshwari, fill. world Health organ.68: 138-44, 1990]]. The protective effect of IL-1 may be due to its ability to induce acute phase proteins such as IL-6[ Vreden et al, eur.j. immunol.22: 2271-5, (1992) ] or c-reactive protein (CRP). CRP is likely to bind to sporozoites of plasmodium falciparum and plasmodium yoelii via the phosphocholine binding site and thus inhibit infection of hepatocytes in vitro and in vivo. Injection of turpentine also induced CRP production and protected rats from plasmodium infection. This protective effect can be transferred by serum of turpentine injected rats and this protection can be abrogated by antibodies against CRP [ j.immunol 139: 4192, (1987)]. Interferon gamma (IFN- γ) appears to interfere with the development of the erythropoiesis (EEFs) of plasmodium inside hepatocytes [ j.immunol 138: 4447]. IFN- γ was in vitro at low doses [ j.immunol 139: 2020, (1987) ] and in vivo [ Ferreira et al, Science 232: 881-; masheshwari et al, inf. immunity 53: 628-630, 1986] inhibits the development of EEF inside hepatocytes. Five doses of human IFN- γ given on days-2, 0 and +2 protected macaques from infection by cynomolgus sporozoites on day 0. No protection against trophoblast-induced infection was observed [ Maheshwari, bull.world Health organisn.68: 138-44, 1990]]. The use of IFN- γ for malaria treatment is described in U.S. patent No. 5,270,037, published at 12/14/1993 and U.S. patent No. 4,915,941, published at 4/10/1990. Tumor Necrosis Factor (TNF) administration failed to protect against infection by plasmodium venorum (p. vinckei) [ Acta Tropica 45: 289, (1988), but prolonged administration by micropump reduces parasitemia following infection with sporozoites from plasmodium chafer (p. chabaudi) [ j.immunol.139: 3493, 1987)].

Malaria vaccine

Another important strategy against malaria is vaccination. The induction of protective immunity by immunization with attenuated microorganisms or non-pathogenic components is a great medical success. Vaccination has virtually eliminated morbidity and mortality from several acute infectious diseases. Unfortunately, vaccination has been less successful in preventing chronic infections such as tuberculosis and malaria. Vaccines against the three main developmental stages of plasmodium falciparum and plasmodium vivax have been vigorously developed in the second half of the 20 th century. Vaccines against the pre-erythrocytic stage aim to prevent infection into the blood of a human host. Vaccines against asexual blood stage parasites aim to combat these disease-causing stages once the infection has entered the blood. Vaccines against the sexual stage of parasites in the blood and mosquito midgut are aimed at preventing the parasite from infecting the mosquito vector and thus cutting off the spread of malaria in humans and mosquito populations. At present, multicomponent vaccines are under development. These vaccines are aimed at inducing humoral and cell-mediated immunity against a variety of antigens expressed at different stages of plasmodium development [ recent examples are found in Kumar et al, Trends in Parasitology 18: 129(2002)]. None of the vaccines tested in the past have proven to be effective.

Targeting host components

The targets of vaccines and most antimalarial drugs are the components of parasites. Many of these targets are involved in host parasite interactions necessary for parasite survival and growth and/or in pathologies caused by infection. A possible alternative strategy to the current strategy is to modulate host components known to interact with molecules produced by the parasite. At first sight this policy seems counterintuitive. In fact, an important requirement of traditional antimicrobial chemotherapy is not to interfere with host function. However, targeting of host components has the advantage that changes in drug targets do not cause drug resistance. Two important classes of antimalarial drugs, quinoline and artemisinin, are very good because they target the host's component heme. Resistance to quinoline develops slowly because it requires selection of variants that affect drug transport. Resistance is even less likely to occur if the targeted host component remains outside the microorganism. In rodent models of malaria, disease can be prevented or alleviated by blocking host cell proteins on the surface of hepatocytes or erythrocytes, as well as by various signal transduction inhibitors (see below). These findings help in understanding the pathogenesis of the disease and in vaccine design, but fail to facilitate any plan for the development of antimalarial drugs. The present invention relates to proteins produced by a host and required by malaria parasites to form infections. The host protein is not enclosed within the parasite membrane. It is a protein tyrosine kinase called Met, which acts as a receptor for another host protein called hepatocyte growth factor.

It will also be appreciated that the methods of the invention may be practiced with compounds that change in vivo to antimalarial agents, as well as compounds that produce in vivo metabolites similar to those produced by antimalarial agents.

Combinations of one or more antimalarial drugs may be used to practice the methods of the invention. Thus, for example, inhibitors of HGF activity may be used with other antimalarial drugs such as chloroquine or in combination with antimalarial drugs such as sulfadoxine/pyrimethamine. The antimalarial drug may be used in the form of the free base or in the form of a pharmaceutically acceptable acid salt. Examples of suitable salts are chloride, hydrochloride, sulfate, phosphate and diphosphate. Other water soluble, non-toxic, inorganic or organic salts may also be used.

In practicing the methods of the invention, the antimalarial agent is administered to the human host by the oral route, since its mode of action is primarily in the liver. For oral administration, the antimalarial active agents of the present invention may be prepared in solid forms, such as capsules, tablets, pills, powders, lozenges and granules, or in liquid forms, such as emulsions, solutions, suspensions, syrups and elixirs, containing inert diluents commonly used in the art, such as water. Other modes of administration may also be used.

The antimalarial agent is used in the methods of the invention in a sufficient amount to provide a sufficient concentration of the agent to prevent or at least inhibit infection of the malaria vector in vivo, or to prevent or at least inhibit spread of malaria in vivo. The amount of active agent is thus dependent on absorption, partitioning and clearance by the human host. Of course, the effect of antimalarial agents is dose-related. The dose of antimalarial agent should be sufficient to produce the least detectable effect, but should be at least 10-fold lower than the identified lethal dose. The dose of antimalarial agent administered to the host may vary within wide limits. The active agent can be administered in a minimum amount that is therapeutically effective and the dose can be increased as necessary to the maximum dose that the patient can tolerate. The antimalarial agent may be administered at a relatively high loading dose, followed by a lower maintenance dose, or may be administered at a uniform dose.

The dosage and frequency of administration will vary with the antimalarial agent used in the method of the invention. For example, genistein may be used in an amount of from 5mg per day to about 5000mg per day, preferably from about 50mg per day to about 500mg per day by the oral route. Typically, the dose does not exceed about 500mg per day, and most typically does not exceed about 50mg per day. The dose of antimalarial agent is specified for an average size adult. Thus, it will be appreciated that the dose may be adjusted by 20-25% for patients with lighter or heavier weight. Similarly, the dosage for a child may be adjusted using well known calculation formulas.

The amount of antimalarial drug used in combination with an inhibitor of HGF activity to form an antimalarial active agent of the present invention does not generally exceed the amount found to be safe and effective for malaria treatment. Thus, as an example, primaquine diphosphate can be administered orally in the form of a tablet containing 5mg-7.5mg of drug, at a rate of 2-3 tablets per day. The dose of primaquine for adults is about 15 mg/day of base (26 mg/day salt) or about 45 mg/week of base (79 mg/week salt) per mouth. For children, the dosage is about 0.3mg/kg per day of base (0.5 mg/kg per day of salt) or about 0.9mg/kg per week of base (1.5 mg/kg per week of salt).

The efficacy of the antimalarial agents of the invention in preventing and inhibiting cellular infections is demonstrated using standard in vitro assays. Thus, the inhibitory effect of an antimalarial agent on malaria infection or replication can be demonstrated by adding malaria sporozoites to a culture of hepatocytes with or without an antimalarial agent and then testing the proliferation of the sporozoites within the hepatocytes by standard methods. The effect of an antimalarial agent in preventing or inhibiting malaria infection or replication can be demonstrated in vivo in a mammalian model of malaria infection. The malaria required to perform these tests can be obtained from conventional sources using conventional techniques.

Antimalarial agents and their pharmaceutically acceptable salts are useful in the prophylaxis or treatment of mammals, including but not limited to humans, in the form of pills, tablets, lozenges, troches (troche), capsules, suppositories, injectable or absorbable solutions, and the like.

To prepare a pharmaceutical composition for a pathological condition in a mammal, a suitable pharmaceutically acceptable carrier, diluent, adjuvant may be combined with the antimalarial agent described herein. The pharmaceutical compositions of the present invention comprise an active agent and a solid or liquid pharmaceutically acceptable non-toxic carrier. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Examples of suitable liquids are peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Physiological saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium carbonate, magnesium stearate, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk, glycerol, propylene glycol, water, ethanol and the like. These compositions may take the form of solutions, suspensions, tablets, pills, capsules, powders, sustained release formulations and the like. Suitable Pharmaceutical carriers are described by e.w. martin in "Remington's Pharmaceutical Sciences". The pharmaceutical composition comprises a therapeutically effective amount of the active agent and a suitable amount of carrier so as to provide a form of correct administration to the host.

In conclusion, the antimalarial agent is useful as an agent for preventing human malaria infection. It shows activity against malarial vectors, which is very unusual and unexpected. The antimalarial agents showed significant inhibition of malaria sporozoite proliferation in hepatocyte cultures and in liver of mice injected with malaria sporozoites. Antimalarial agents reduce mortality and morbidity phenomena in humans, particularly by reducing the incidence of infection.

Claims (16)

1. A method of preventing or inhibiting the activity of malaria in vivo, wherein the method comprises administering to a human in need thereof an antimalarial agent in an amount sufficient to prevent or inhibit infection of the human by a malaria parasite or to prevent or inhibit spread of malaria in vivo.

2. The method of claim 1, wherein the antimalarial agent is administered to the human in admixture with a pharmaceutically acceptable carrier.

3. A treatment for a human that prevents the development of an infection by a strain of plasmodium that is pathogenic to the human, wherein such plasmodium strain is plasmodium falciparum, plasmodium vivax, plasmodium malariae, and plasmodium ovale.

4. The treatment of claim 3, wherein the treatment comprises a molecule that interferes with the activation of the protein tyrosine kinase Met by passage of Plasmodium across hepatocytes.

5. The molecule of claim 4, wherein said molecule interferes with HGF activation.

6. The molecule of claim 4, wherein the molecule sequesters HGF and thereby prevents its binding to Met.

7. The molecule of claim 4, wherein the molecule is a Met antagonist.

8. The molecule of claim 7, wherein the molecule is an antibody directed against Met or a fragment of such an antibody.

9. The molecule of claim 7, wherein the molecule is an oligonucleotide (aptamer).

10. The molecule of claim 7, wherein the molecule is an HGF variant that binds to, but does not activate, Met.

11. The molecule of claim 10, wherein said molecule is an NK4 protein.

12. The molecule of claim 5, wherein the molecule is a small molecular weight protein tyrosine kinase inhibitor.

13. The molecule of claim 11, wherein the molecule is genistein.

14. The molecule of claim 12, wherein the molecule is a selective Met antagonist.

15. A method for preventing or inhibiting the activity of malaria in vivo, wherein the method comprises administering an antimalarial agent to a human in need thereof in an amount sufficient to prevent or inhibit infection of the human by a malaria parasite or to prevent or inhibit spread of malaria in vivo.

16. A method for preventing or inhibiting the activity of malaria in vivo, wherein the method comprises administering an antimalarial agent to a human in need thereof in an amount sufficient to prevent or inhibit infection of the human by a malaria parasite or to prevent or inhibit spread of malaria in vivo.