# Patent

## Title
Tyrosine kinase inhibitors as anti-kinetoplastid agents

## Abstract

The present invention provides methods of killing, inhibiting the growth, and/or inhibiting the reproduction of kine-toplastid or apicomplexan protozoan with tyrosine kinase inhibitors.

## Background
CONTINUING APPLICATION DATA
This application is the §371 U.S. National Stage of International Application No. PCT/US2007/024319, filed 21 Nov. 2001, which claims the benefit of U.S. Provisional Application Ser. No. 60/860,717, filed Nov. 22, 2006, each of which are incorporated by reference herein in their entireties.


BACKGROUND
Human African trypanosomiasis (HAT) occurs in 36 countries in Sub-Saharan Africa, threatening an estimated 60 million people with debilitating disease. No vaccines are available for prevention of infection by Trypanosoma brucei, which causes trypanosomiasis. Drugs in use are toxic, and drug resistance can be an issue (for review, see Docampo and Moreno, 2003, Parasitol Res; 90 Supp 1:S10-3). As a result, new and safer drugs are needed for the treatment of trypanosomiasis (Cowman and Crabb, 2003, Trends Parasitol; 19(11):538-43; Pink et al., 2005, Nat Rev Drug Discov; 4(9):727-40; and Gelb and Hol, 2003, Science; 297(5580):343-4). In addition, additional new drugs must be developed, in order to prepare for possible emergence of drug resistance in the parasites (de Koning, 2001, Int J Parasitol; 31(5-6):512-22; Ouellette, 2001, Trop Med Int Health; 6(11):874-82; and Sinyangwe et al., 2004, Vet Parasitol; 119(2-3):12-35).
SUMMARY OF THE INVENTION
The present invention includes methods of killing, inhibiting the growth and/or inhibiting the reproduction of a kinetoplastid or apicomplexan protozoan, the method including contacting the protozoan with a tyrosine kinase inhibitor.
The present invention also includes methods of treating or preventing a kinetoplastid or apicomplexan protozoan infection in a subject, the method including administering to the subject an effective amount of a tyrosine kinase inhibitor.
The present invention includes methods of killing, inhibiting the growth and/or inhibiting the reproduction of a kinetoplastid or apicomplexan protozoan in a subject, the method including administering to the subject an effective amount of a tyrosine kinase inhibitor.
Also included in the present invention are compositions for treating or preventing a kinetoplastid or apicomplexan protozoan infection in a subject, the composition including an effective amount of two or more a tyrosine kinase inhibitors.
The present invention also includes compositions for treating or preventing a kinetoplastid or apicomplexan protozoan infection in a subject, the composition including an effective amount of one or more tyrosine kinase inhibitors and an effective amount of one or more conventional anti-kinetoplastid agents and/or one or more anti-apicomplexan therapeutic agents, wherein a conventional anti-kinetoplastid agent or anti-apicomplexan therapeutic agent is not a tyrosine kinase inhibitor.
With the methods and compositions of the present invention, the kinetoplastid protozoan may be of the genus Trypanosoma. 
With the methods and compositions of the present invention, the kinetoplastid protozoan may be selected from T. cruzi, T. brucei, T.b. gambiense, and T.b. rhodesiense. 
With the methods and compositions of the present invention, the kinetoplastid protozoan may be of the genus Leishmania. 
With the methods and compositions of the present invention, the apicomplexan protozoan may be selected from Falciparum, Toxoplasma gondii, cryptosporidia, Babesia microti, Babesia divergens, and Perkinsus marinus. 
With the methods and compositions of the present invention, the tyrosine kinase inhibitor may be selected from AG1478, A47, canertinib, sunitinib, axitinib, erlotinib, dasatinib, imatinib, nilotinib, sorafenib, lapatinib, and gefitinib.
With the methods and compositions of the present invention, the tyrosine kinase inhibitor may specifically inhibit only epidermal growth factor receptors (EGFR) tyrosine kinase activity. In some embodiments, the tyrosine kinase inhibitor may be selected from the group consisting of AG1478, A47, gefitinib, lapatinib, canertinib, and erlotinib.
With the methods and compositions of the present invention, the tyrosine kinase inhibitor may be a 4-anilinoquinazoline. In some embodiments, the 4-anilinoquinazoline is substituted at least one of the 2′, 3′, 4′ positions of the aniline ring, including, substituents selected from an alkyl, aryl, phenylalkylamino, amino, halide, hydroxide, carboxyl, acyl, alkoxide, aryloxide, or alkylsulphide. In some embodiments, the 4-anilinoquinazoline is substituted at least one of the 5, 6, 7, 8 positions of the quinazoline ring, including substituents selected from an alkyl, aryl, phenylalkylamino, amino, halide, hydroxide, carboxyl, acyl, alkoxide, aryloxide, or alkylsulphide. In some embodiments, the tyrosine kinase inhibitor is 1,4-anilinoquinazoline wherein the aniline hydrogen has been replaced by an alkyl, phenyl or acyl group.
With the methods and compositions of the present invention, the tyrosine kinase inhibitor may specifically inhibit only epidermal growth factor receptors (EGFR) tyrosine kinase activity and is a 4-anilinoquinazoline.
With the methods and compositions of the present invention, the tyrosine kinase inhibitor may be a tyrphostin.
With the methods and compositions of the present invention, the tyrosine kinase inhibitor may be a pyrrolopyridine.
With the methods and compositions of the present invention, the tyrosine kinase inhibitor may inhibit multiple protein tyrosine kinases (PTK).
With the methods and compositions of the present invention, the tyrosine kinase inhibitor may inhibit vascular endothelial growth factor receptor (VEGFR) kinase activity.
With the methods and compositions of the present invention, the tyrosine kinase inhibitor may inhibit platelet-derived growth factor receptor (PDGFR) kinase activity.
With the methods and compositions of the present invention, the tyrosine kinase inhibitor may inhibit c-Abl tyrosine kinase activity.
With the methods and compositions of the present invention, the tyrosine kinase inhibitor may inhibit c-Src tyrosine kinase activity.
With the methods and compositions of the present invention, the tyrosine kinase inhibitor may inhibit c-Kit protein (c-Kit) kinase activity.
With the methods and compositions of the present invention, the tyrosine kinase inhibitor may inhibit FMS-like tyrosine kinase 3 (FLT3) tyrosine kinase activity.
With the methods and compositions of the present invention, the tyrosine kinase inhibitor may inhibit multiple protein tyrosine kinases (PTK).
With the methods and compositions of the present invention, the tyrosine kinase inhibitor may be selected from a small molecule, an antibody, and a receptor ligand.
The terms “comprises” and variations thereof do not have a limiting meaning where these terms appear in the description and claims.
Unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. The effect of Tyrphostin A47 on blood stream T. brucei. 
FIG. 2. Tyrphostin AG1478 inhibits T. brucei growth.
FIG. 3. Carnetinib kills T. brucei. 
FIG. 4. Effect of TARCEVA (erlotinib) on T. brucei viability.
FIG. 5. SUTENT (sunitinib) kills T. brucei. 
FIG. 6. Axitinib kills T. brucei. 



DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
With the present invention it has been found that the genome of the protozoan parasite Trypanosoma brucei includes sequences encoding proteins whose kinase domains are similar in sequence to the kinase domains of various protein tyrosine kinases, including the epidermal growth factor receptor (EGFR), the vascular endothelial cell growth factor receptor (VEGFR), c-Src protein (c-Src), c-Abl protein tyrosine kinase (c-Abl), c-Kit protein (c-Kit), and FMS-like tyrosine kinase 3 (FLT3). The present invention provides for the use of tyrosine kinase inhibitors as anti-protozoan agents, provides methods of killing, inhibiting the growth, and/or inhibiting the reproduction of a protozoan by contacting the protozoan with one or more tyrosine kinase inhibitors. Such contact may be in vitro, ex vivo, and/or in vivo, provides methods of treating or preventing a protozoan infection in a subject by administering to the subject an effective amount of one or more tyrosine kinase inhibitors. The present invention provides methods of killing, inhibiting the growth, and/or inhibiting the reproduction of a protozoan in a subject by administering to the subject an effective amount of one or more tyrosine kinase inhibitors. The present invention also provides compositions including tyrosine kinase inhibitors.
The methods and compositions of the present invention are applicable for a variety of protozoa, including, but not limited to, kinetoplastid and apicomplexan protozoa. Kinetoplastids are a group of flagellate protozoa, including a number of parasites responsible for serious diseases in humans and other animals, including economically relevant livestock, as well as various forms found in soil and aquatic environments. They are included in the Euglenozoa, and are distinguished from other such forms mainly by the presence of a kinetoplast, a DNA-containing granule located within the single mitochondrion and associated with the flagellar bases. Kinetoplastids typically have complex life-cycles involving more than one host, and go through various morphological stages. The most distinctive of these is the trypomastigote stage, where the flagellum runs along the length of the cell and is connected to it by an undulating membrane. Kinetoplastid protozoa, include, for example, protozoa of the Blastocrithidia, Crithidia, Endotrypanum, Herpetomonas, Leishmania, Leptomonas, Phytomonas, Trypanosoma, and Wallaceina genera. Diseases caused by trypanosomes include African Sleeping Sickness and South American Chagas Disease, from species of Trypanosoma, and leishmaniasis, from species of Leishmania. 
Species of Trypanosoma to be treated by the present invention include, but are not limited to, T. avium, which causes trypanosomiasis in birds, T. boissoni, T. brucei, which causes sleeping sickness in humans and nagana in cattle, T. carassii, in freshwater teleosts, T. cruzi, which causes Chagas disease in humans, T. gambiense, T. rhodesiense, T. congolense, which causes nagana in cattle, horses, and camels, T. equinum, T. equiperdum, which causes dourine or covering sickness in horses, T. evansi, which causes one form of the disease surra in certain animals, T. lewisi, in rats, T. melophagium, T. percae in fish, T. rangeli, T. rotatorium in amphibian, T. simiae, T. suis, T. theileri, T. triglae, and T. vivax. In some embodiments of the present invention, species of Trypanosoma that infect humans or livestock are treated. In preferred embodiments, the protozoan is of the genus Trypanosoma, including, but not limited to, T. cruzi, T. brucei, Tb. gambiense, and T.b. rhodesiense. In one preferred embodiment of the present invention, the protozoan T. brucei is treated by the present invention.
Species of Leishmania to be treated by the present invention include, but are not limited to, L. aethiopica, L. amazonensis, L. arabica, L. archibaldi, L. aristedesi, L. braziliensis, L. chagasi, L. colombiensis, L. deanei, L. donovani, L. enriettii, L. equatorensis, L. forattinii, L. garnhami, L. gerbili, L. guyanensis, L. herreri, L. hertigi, L. infantum, L. killicki L. lainsoni, L. major, L. mexicana, L. naiffi, L. panamensis, L. peruviana, L. pifanoi, L. shawi, L. tarentolae, L. tropica, L. turanica, and L. venezuelensis. 
The apicomplexa are an extremely large and diverse group of protozoa, characterized by the presence of a unique organelle called an apical complex. They are single-celled, spore-forming, and exclusively parasites of animals. Seven species infect humans (Plasmodium, Babesia, Cryptosporidium, Isospora, Cyclospora, Sarcocystis, and Toxoplasma). Plasmodium, as the causative agent of malaria, has a great impact on human health. Several apicomplexan parasites are also important in terms of veterinary medicine and agriculture. Most notable are Babesia and Theileria in cattle and Eimeria in poultry. Apicomplexan protozoans include, for example, Babesia microti and Babesi divergens (causing babesiosis), Plasmodium (causing malaria), (Cryptosporidium parvum (causing cryptosporidiosis), Cyclospora cayetanensis (causing cyclosporiasis), and Toxoplasma gondii (causing toxoplasmosis). The present invention includes, but is not limited to, the administration of tyrosine kinase inhibitors for the treatment of any of these various apicomplexan protozoa.
Tyrosine kinases are a class of enzymes that catalyze the transfer of the terminal phosphate of adenosine triphosphate (ATP) to tyrosine residues in peptide and protein substrates. Tyrosine kinases, by way of substrate phosphorylation, play critical roles in signal transduction for a number of cell functions and are important in the regulation of cell signaling. Tyrosine kinases are important contributing factors in, for example, cell proliferation, cell differentiation, and carcinogenesis. Thus, tyrosine kinases are an important class of enzymes as targets for therapeutic interventions.
As used herein, a tyrosine kinase inhibitor is an agent that interferes with the ability of a tyrosine kinase to function. Tyrosine kinase inhibitors of the present invention include agents that inhibit protozoan tyrosine kinase activity. Such agents may reduce, decrease, inhibit, and/or block the functioning of a protozoan tyrosine kinase. Tyrosine kinase inhibitors of the present invention include, but are not limited to, agents that inhibit the enzymatic activity of a kinase, agents bind to the ATP binding site of a tyrosine kinase, agents that bind to the substrate binding site of the tyrosine kinase, and agents that block or destablize the dimerization of kinase enzyme subunits.
Tyrosine kinase inhibitors of the present invention inhibit the kinase activity of one or more protozoan tyrosine kinases. A tyrosine kinase inhibitor of the present invention may exhibit less toxicity than currently used chemotherapeutic agents when administered to a subject. Such inhibitors may be identified or characterized by their effect on the catalytic activity of other, more well-characterized tyrosine kinases, such as, for example, one or more of the receptor tyrosine kinases (RTK) EGF, HER2, HER3, HER4, IR, IGF-1R, IRR, PDGFRα, PDGFRβ, TrkA, TrkB, TrkC, HGF, CSFIR, C-Kit, C-fms, Flk-1R, Flk4, KDR/Flk-1, Flt-1, FGFR-1R, FGFR-1R, FGFR-3R and FGFR-4R and/or one or more of the cellular tyrosine kinases (CTK) Src, Frk, Btk, Csk, Abl, Zap70, Fes, Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk. A tyrosine kinase inhibitor of the present invention may demonstrate selective activity against a narrowed spectrum of protein tyrosine kinases. For example, a tyrosine kinase inhibitor of the present invention may inhibit only one of the following receptor tyrosine kinases: EGF, HER2, HER3, HER4, IR, IGF-1R, IRR, PDGFRα, PDGFRβ, TrkA, TrkB, TrkC, HGF, CSFIR, C-Kit, C-fm, Flk-1R, Flk4, KDR/Flk-1, Flt-1, FGFR-1R, FGFR-1R, FGFR-3R, or FGFR-4R; or only one of the following cellular tyrosine kinases: Src, Frk, Btk, Csk, Abl, Zap70, Fes, Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lek, Blk, Hck, Fgr or Yrk. In other aspects, a tyrosine kinase inhibitor of the present invention may inhibit only a subset of the above listed protein tyrosine kinases.
A tyrosine kinase inhibitor that inhibits the kinase activity of one or more protozoan tyrosine kinases can be identified and characterized by any of a variety of methods, including, but not limited to, any of a variety of known methods and any of those described herein. For example, the effect of a tyrosine kinase inhibitor on the catalytic activity of a protozoan tyrosine kinase may be evaluated in vitro, by incubating an in vitro culture of the protozoan with a tyrosine kinase inhibitor. Different concentrations of the inhibitor solubilized are added and living protozoan cells counted after exposure to the inhibitor. The mean and standard deviation of the cell count are plotted against inhibitor concentration. From the graphical plots, the concentration of the inhibitor that reduces protozoan cell density by 50% (IC50) is determined. Any of the various in vitro cultures systems for kinetoplastid and apicomplexan protozoa may be used. The effect of a tyrosine kinase inhibitor on the catalytic activity of a protozoan tyrosine kinase may also be evaluated using any of a variety of various in vivo animals models that are available as models for kinetoplastid and apicomplexan protozoan disease, including livestock and human disease.
Further, the effect of a tyrosine kinase inhibitor on the catalytic activity of a protozoan tyrosine kinase may be evaluated by a determination of whether an inhibitor blocks the phosphorylation of tyrosine residues on protozoan proteins. Phosphorylated tyrosine resides (pTyr) can be detected on polypeptides with specific antibodies (Johnson and Hunter, 2005, Nat Methods; 2(1):17-25; Ignatoski, 2001, Methods Mol Biol; 124:39-48). For example, such a determination may be employed as follows. To cultured T. brucei 427 blood stream cells (108/ml, 5 ml) in 10-ml culture flasks, the inhibitor (10 μM final concentration) is added, and the cells incubated at 37° C. for 20 minutes. As a control, parasites are incubated in solvent alone and handled similarly. Proteins from T. brucei (50 μg per lane) (Parsons et al. 1993, Proc Natl Acad Sci USA; 90(7):2656-60) are separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and pTyr residues on the polypeptides detected with a mixture of anti-pTyr antibodies (for example, 4G10, PY20, and FB2) (Zheng et al., 2005, Mol Cell Proteomics; 4(6):721-30; Kumar et al., 2005, Methods; 35(1):9-21; Johnson and Hunter, 2005, Nat Methods; 2(1):17-25; Ignatoski, 2001, Methods Mol Biol; 124:39-48; Bleasdale et al., 2001, Biochemistry; 40(19):5642-54; Parsons et al., 1995, Exp Parasitol; 80(3):507-14; Parsons et al. 1993, Proc Natl Acad Sci USA; 90(7):2656-60; Wheeler-Alm and Shapiro, 1993, Trop Med Parasitol; 44(4):281-4; Parsons et al., 1991, Mol Biochem Parasitol; 45:241-248). The specificity of the antibody for pTyr is ascertained in a negative control experiment by inclusion of Tyr (40 mM final concentration) in the buffer for antibody binding to the trypanosome proteins (Parsons et al. 1993, Proc Natl Acad Sci USA; 90(7):2656-60). Quantitative pTyr western blots (Chopard et al., 2000, Electrophoresis; 21(3):517-22; Fradelizi et al., 1999, Biotechniques; 26(3):484-6, 488, 490 passim; Rudolph et al., 1999, Anal Biochem; 269(1):66-71) are performed after lysing cells in buffer containing sodium orthovanadate (1 mM) which inhibits Tyr phosphatases (Johnson and Hunter, 2005, Nat Methods; 2(1):17-25; Mustafa et al., 1997, J Infect Dis; 175(3):669-73), to determine possible reduction in the intensity of pTyr protein bands with or without inhibitor treatment of T. brucei. 
Tyrosine kinase inhibitors include both protein and non-protein moieties. A tyrosine kinase inhibitor may be, for example, an antibody, a receptor ligand, or a small molecule inhibitor. A tyrosine kinase inhibitor may be in the form of a pharmaceutically acceptable salt, hydrate, solvate, crystal form, N-oxide(s), and/or individual diastereomer. In preferred embodiments, a tyrosine kinase inhibitor is a low molecular weight molecule, also referred to herein as a small molecule. The agent may, for example, be chemically synthesized, derived from a library of low molecular weight compounds, or derived from a library of extracts from plants or other organisms. Small molecule tyrosine kinase inhibitors may compete with the ATP-binding site of the catalytic domain of the target tyrosine kinase. Such inhibitors are generally orally active and have a favorable safety profile. A variety of small molecule tyrosine kinase inhibitors have been identified that possess antitumor activity and have been approved or are in clinical trials. These include, but are not limited to, gefitinib (IRESSA), sunitinib (SUTENT, SU11248), erlotinib (TARCEVA, OSI-1774), lapatinib (GW-572016), canertinib (CI 1033), semaxinib (SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006), imatinib mesylate (GLEEVEC, ST1571), and leflunomide (SU101).
Tyrosine kinase inhibitors of the present invention also include derivatives and analogs of tyrosine kinase inhibitors. Such derivatives and analogs thereof may be identified by methods well know to the skilled artisan, for example, using computer modeling or combinatorial chemistry.
Tyrosine kinase inhibitors of the present invention include tyrphostins. Tyrphostins are synthetic, low molecular weight protein tyrosine kinase inhibitors, also known as AG compounds, that inhibit tyrosine kinase activity by binding to the substrate binding site. The tyrphostins include a systematic series of molecules with a progressive increase in affinity toward the substrate site of the EGF receptor kinase domain. See, for example, Gazit et al., 1989, J Med Chem; 32:2344-52, Twaddle et al., 1999, J Surg Oncol; 70(2):83-90, and Levitzki and Mishani, 2006, Annu Rev Biochem; 75:93-109. Tyrphostin tyrosine kinase inhibitors of the present invention include, but are not limited to, Tyrphostin A8 (Gazit et al., 1989, J Med Chem; 32:2344; Wohlberg et al., 1994, J Biol Chem; 269:22470), Tyrphostin A9 (Gazit et al., 1989, J Med Chem; 32:2344; Bider et al., 1991, Am J Physiol; 260:C721; Levitzki and Gilon, 1991, Trends Pharmacol Sci; 12:171), Tyrphostin A23 (Bider et al., 1991, Am J Physiol; 260:C721; Levitzki and Gilon, 1991, Trends Pharmacol Sci; 12:171), Tyrphostin A30 (Wessely et al., 1997, Cell Growth Differen; 8:481; Schwartz et al., 1995, Oncol Res; 7:277), Tyrphostin A25 (Gazit et al., 1989, J Med Chem; 32:2344; Wohlberg et al., 1994, JBiol Chem; 269:22470; Bider et al., 1991, Am J Physiol; 260:C721), Tyrphostin A46 (Gazit et al., 1989, J Med Chem; 32:2344; Gazit et al., 1991, J Med Chem; 34:1896), Tyrphostin A48 (Gazit et al., 1989, J Med Chem; 32:2344), Tyrphostin AG 126 (Gazit et al., 1989, J Med Chem; 32:2344; Bider et al., 1991, Am J Physiol; 260:C721; Novogrodsky et al., 1994 Science; 264:1319), Tyrphostin A51 (Levitzki, 1990, Biochem Pharmacol; 40:913), Tyrphostin A47 (Levitzki, 1990, Biochem Pharmacol; 40:913; Szende et al., 1995, Cell Biol Int; 19:903), Tyrphostin AG 370 (Bryckaert et al., 1992, Nature Exp Cell Res; 199:255), Tyrphostin B42 (Gazit et al., 1991, J Med Chem; 34:1896; Levitzki, 1990, Biochem Pharmacol; 40:913; Meydan et al., 1996, Nature; 379:645), Tyrphostin B48 (Gazit et al., 1991, J Med Chem; 34:1896), Tyrphostin B44(−) (Gazit et al., 1991, J Med Chem; 34:1896), Tyrphostin B46 (Gazit et al., 1991, J Med Chem; 34:1896), Tyrphostin B56 (Gazit et al., 1991, J Med Chem; 34:1896), Tyrphostin AG 825 (Levitzki and Gazit, 1995, Science; 267:1782; Osherov et al., 1993, J Biol Chem; 268:11134), Tyrphostin B50 (Gazit et al., 1991, J Med Chem; 34:1896), Tyrphostin AG 879 (Ohmichi et al., 1993, Biochemistry; 32:4650; Konrad et al., 1996, J Biol Chem; 271:24179), Tyrphostin AG 957 (Anafi et al., 1992, J Biol Chem; 267:4518) Tyrphostin AG1288 (Novogrodsky et al., 1994, Science; 264:1319), Tyrphostin AG1295 ((Levitzki and Gazit, 1995, Science; 267:1782; Kovalenko et al., 1994, Cancer Res; 54:6106), Tyrphostin AG1296 (Kovalenko et al., 1994, Cancer Res; 54:6106), Tyrphostin AG1433 (Kroll and Waltenberger, 1987, J Biol Chem; 272:32521; Strawn et al., 1996, Cancer Res; 56:3540), Tyrphostin AG1478 ((Levitzki and Gazit, 1995, Science; 267:1782; Zhu et al., 2001, Cancer Lett; 169(1):27-32; and Ellis et al., 2006, Biochem Pharmacol; 71(10):1422-34), RG-1302 (Yoneda et al., 1991, Cancer Res; 51:4430), RG-14620 (Yoneda et al., 1991, Cancer Res; 51:4430), Bis-Tyrphostin (Levitzki and Gilon, 1991, Trends Pharmacol Sci; 12:171), and derivatives and analogs thereof. A variety of tyrphostins, including, but not limited to, A47 and AG1478 are commercially available, for example, from Calbiochem.
A tyrosine kinase inhibitor of the present invention includes an inhibitor that inhibits the catalytic activity of one or more receptor tyrosine kinases (RTK) selected from EGF, HER2, HER3, HER4, IR, IGF-1R, IRR, PDGFRα, PDGFRβ, TrkA, TrkB, TrkC, HGF, CSFIR, C-Kit, C-fms, Flk-1R, Flk4, KDR/Flk-1, Flt-1, FGFR-1R, FGFR-1R, FGFR-3R and/or FGFR-4R.
In addition to the receptor tyrosine kinases, there also exists a family of entirely intracellular PTKs called “non-receptor tyrosine kinases” or “cellular tyrosine kinases” (CTK). CTKs do not contain extracellular and transmembrane domains. Over twenty-four CTKs in eleven subfamilies (Src, Frk, Btk, Csk, Abl, Zap70, Fes, Fps, Fak, Jak and Ack) have been identified. The Src subfamily of CTKs includes Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk (Bolen, 1993, Oncogene; 8:2025-2031). A tyrosine kinase inhibitor of the present invention includes an inhibitor that reduces the catalytic activity of one or more cellular tyrosine kinases selected from Src, Frk, Btk, Csk, Abl, Zap70, Fes, Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and/or Yrk.
In some embodiments, a tyrosine kinase inhibitor of the present invention is an inhibitor that selectively inhibits the EGFR. In some embodiments, a tyrosine kinase inhibitor of the present invention inhibits only the EFGR. In some embodiments, a tyrosine kinase inhibitor of the present invention inhibits multiple tyrosine kinases. Examples of EGFR inhibitors include, for example, gefitinib, erlotinib, lapatinib, canertinib, sorafenib, and vandetanib. Examples of VEGFR inhibitors include, for example, axitinib, sunitinib, sematxinib, vatalanib, sorafenib, and vandetanib. Examples of PDGFR inhibitors include, for example, sunitinib, imatinib, sorafenib, and leflunomide. Examples of c-Kit inhibitors include, for example, sunitinib, imatinib, and semaxinib. Examples of FLT-3 inhibitors include, for example, sunitinib and semaxinib. Examples of BCR-ABL inhibitors include, for example, imatinib and dasatinib.
Tyrosine kinase inhibitors of the present invention include pyrimidine derivatives such as N-phenyl-2-pyrimidine-amine derivatives (U.S. Pat. No. 5,521,184 and WO 99/03854), indolinone derivatives and pyrrol-substituted indolinones (U.S. Pat. No. 5,792,783, EP 934 931, U.S. Pat. No. 5,834,504), U.S. Pat. Nos. 5,883,116, 5,883,113, 5,886,020, WO 96/40116 and WO 00/38519), as well as bis monocyclic, bicyclic aryl and heteroaryl compounds (EP 584 222, U.S. Pat. No. 5,656,643 and WO 92/20642), quinazoline derivatives (EP 602 851, EP 520 722, U.S. Pat. Nos. 3,772,295 and 4,343,940), 4-amino-substituted quinazolines (U.S. Pat. No. 3,470,182), 4-thienyl-2-(1H)-quinazolones, 6,7-dialkoxyquinazolines (U.S. Pat. No. 3,800,039), aryl and heteroaryl quinazoline (U.S. Pat. Nos. 5,721,237, 5,714,493, 5,710,158 and WO 95/15758), 4-anilinoquinazoline compounds (U.S. Pat. No. 4,464,375), and 4-thienyl-2-(1H)-quinazolones (U.S. Pat. No. 3,551,427).
4-anilinoquinazolines and 4-anilinoquinolines represent well known and important classes of protein kinase inhibitors, acting as ATP-competitive inhibitors of protein kinase enzymes. See, for example, Gosh et al., 1999, Anticancer Drug Des; 14(5):403-10; Denny, 2001, Farmaco; 56(1-2):51-6; Smaill et al., 2001, J Med Chem; 44(3):429-40; Assefa et al., 2003, J Comput Aided Mol Des; 17(8):475-93; Albuschat et al., 2004, Eur J Med Chem; 39(12):1001-11; Aparna et al., 2005, J Chem Inf Model; 45(3):725-38; Hennequin et al., 2006, Bioorg Med Chem Lett; 16(10):2672-6; Green et al., 2007, J Med Chem; 50(19):4728-45; and Liu et al., 2007, Bioorg Med Chem Lett; 17(22):6373-7. A tyrosine kinase inhibitor of the present invention may be a 4-anilinoquinazoline compound, a 4-anilinoquinoline compound, or a derivative thereof. Such compounds include, but are not limited to, 4-anilinoquinazoline compounds substituted at least one of the 2′, 3′, 4′ positions of the aniline ring, including substituents selected from an alkyl, aryl, phenylalkylamino, amino, halide, hydroxide, carboxyl, acyl, alkoxide, aryloxide, or alkylsulphide. Such compounds include, but are not limited to, 4-anilinoquinazoline compounds substituted at least one of the 5, 6, 7, 8 positions of the quinazoline ring, including substituents selected form an alkyl, aryl, phenylalkylamino, amino, halide, hydroxide, carboxyl, acyl, alkoxide, aryloxide, or alkylsulphide. Such compounds include, but are not limited to, 4-anilinoquinazoline compounds wherein the aniline hydrogen has been replaced by an alkyl, phenyl or acyl group.
A tyrosine kinase inhibitor of the present invention includes gefitinib (N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy) quinazolin-4-amine) (originally coded ZD1839). Gefitinib is a drug used in the treatment of certain types of cancer, acting in a similar manner to erlotinib (marketed as TARCEVA). It is marketed by AstraZeneca under the trade name IRESSA. Gefitinib is the first selective inhibitor of epidermal growth factor receptor's (EGFR) tyrosine kinase domain. The target protein (EGFR) is also sometimes referred to as Her1 or ErbB-1.
A tyrosine kinase inhibitor of the present invention includes dasatinib (N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl]-2-methyl-4-pyrimidinyl]amino]-5-thiazole carboxamide monohydrate) (Das et al., 2006, J Med Chem; 49: 6819-32). Dasatinib, also known as BMS-354825, is a drug produced by Bristol-Myers Squibb and sold under the trade name SPRYCEL. Dasatinib is an oral dual BCR/ABL and Src family tyrosine kinases inhibitor approved for use in patients with chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). It is also being assessed for use in metastatic melanoma.
A tyrosine kinase inhibitor of the present invention includes erlotinib (N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine). Erlotinib (trade name TARCEVA, produced by Genentech/OSIP, originally coded as OSI-774) is a drug used to treat non-small cell lung cancer, pancreatic cancer and several other types of cancer. Similar to gefitinib, erlotinib specifically targets the epidermal growth factor receptor (EGFR) tyrosine kinase. It binds in a reversible fashion to the adenosine triphosphate (ATP) binding site of the receptor.
A tyrosine kinase inhibitor of the present invention includes imatinib (4-[(4-methylpiperazin-1-yl)methyl]-N-[4-methyl-3-[(4-pyridin-3-ylpyrimidin-2-yl)amino]-phenyl]-benzamide). Imatinib is currently used to treat certain types of cancer. It is currently marketed by Novartis as GLEEVEC in the USA and GLIVEC in Europe and Australia as its mesylate salt, imatinib mesilate. It is also referred to as CGP57148B or STI571. It is used in treating chronic myelogenous leukemia (CML), gastrointestinal stromal tumors (GISTs) and a number of other malignancies. Imatinib is a 2-phenylaminopyrimidine derivative that functions as a specific inhibitor of a number of tyrosine kinase enzymes. It occupies the TK active site, leading to a decrease in activity. Imatinib is specific for the TK domain in abl (the Abelson proto-oncogene), c-kit and PDGF-R (platelet-derived growth factor receptor).
A tyrosine kinase inhibitor of the present invention includes lapatinib (N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6-[5-[(2-methylsulfonylethylamino) methyl]-2-furyl]quinazolin-4-amine). Lapatinib, also known as lapatinib ditosylate or GW572016, is an anti-cancer drug developed by GlaxoSmithKline (GSK) as a treatment for solid tumors such as breast and lung cancer. It is marketed by GSK in the U.S. as TYKER and will be marketed as TYVERB in Europe. Lapatinib is an epidermal growth factor receptor (EGFR) and HER2/neu (ErbB-2) dual tyrosine kinase inhibitor. It binds to the intracellular phosphorylation domain to prevent receptor autophosphorylation upon ligand binding.
A tyrosine kinase inhibitor of the present invention includes nilotinib (4-methyl-N-[3-(4-methylimidazol-1-yl)-5-(trifluoromethyl) phenyl]-3-[(4-pyridin-3-ylpyrimidin-2-yDamino]benzamide). Nilotinib, in the form of the hydrochloride monohydrate salt, is a tyrosine kinase inhibitor approved as TASIGNA in the USA for drug-resistant chronic myelogenous leukemia (CML) also known by its clinical code AMN107 (Kantarjian et al., 2006, N Engl J Med; 354(24):2542-51).
A tyrosine kinase inhibitor of the present invention includes sorafenib (4-[4-[[4-chloro-3-(trifluoromethyl)phenyl] carbamoylamino]phenoxy]-methyl-pyridine-2-carboxamide). Sorafenib, marketed as NEXAVAR by Bayer, is a drug approved for the treatment of advanced renal cell carcinoma (primary kidney cancer) and has received “Fast Track” designation by the FDA for the treatment of advanced hepatocellular carcinoma (primary liver cancer). It is a small molecular inhibitor of the Raf kinase, the PDGF kinase, the VEGF receptor 2 kinase, the VEGF receptor 3 kinase, and the c-Kit receptor for Stem cell factor.
A tyrosine kinase inhibitor of the present invention includes sunitinib (N-[2-(diethylamino)ethyl]-5-[(Z)-(5-fluoro-1,2-dihydro-2-oxo-3H-indol1-3-ylidine)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide). Sunitinib, marketed as SUTENT, and also known as SU11248, is an oral, small-molecule, multi-targeted receptor tyrosine kinase (RTK) inhibitor approved by the FDA for the treatment of renal cell carcinoma (RCC) and imatinib-resistant gastrointestinal stromal tumor (GIST). See, for example, Cabebe and Wakelee, 2006, Drugs Today; 42(6):387-98). Sunitinib has become the standard of care for both of these cancers, and is currently being studied for the treatment of many others. Sunitinib inhibits cellular signaling by targeting multiple RTKs. These include all platelet-derived growth factor receptors (PDGF-R) and vascular endothelial growth factor receptors (VEGF-R), which play a role in both tumor angiogenesis and tumor cell proliferation. The simultaneous inhibition of these targets therefore leads to both reduced tumor vascularization and cancer cell death, and ultimately tumor shrinkage. Sunitinib also inhibits KIT. (CD 117) and other RTKs including RET, CSF-1R, and flt3.
A tyrosine kinase inhibitor of the present invention includes canertinib (N-[-4-[(3-chloro-4-fluorophenyl)amino]-7[3-(4-morpholinyl) propoxy]-6-quinazolinyl]-2-propenamide). Canertinib, also known as CI-1033 or PD183805, is the hydrochloride salt of an orally bio-available quinazoline with antineoplastic activities. Canertinib binds to the intracellular domains of epidermal growth factor receptor tyrosine kinases (ErbB family), irreversibly inhibiting their signal transduction functions and resulting in tumor cell apoptosis and suppression of tumor cell proliferation. Canertinib has been well tolerated in a variety of phase I schedules with some disease stabilization in patients with refractory metastatic breast cancer.
A tyrosine kinase inhibitor of the present invention includes axitinib (N-methyl-2-[[3-[1E-2-(pyridin-2-yl)ethenyl]-1H-inadazol-6-yl]sulfanyl]benzaminde). Axitinib, also known as AG-01376, is marketed by Pfizer and has demonstrated effectiveness for the treatment of thyroid cancer and pancreatic cancer, Axitinib works by selectively inhibiting vascular, endothelial growth factor (VEGFR) 1, 2, and 3.
A tyrosine kinase inhibitor of the present invention includes SU5416, also called semaxanib. SU5416, a small molecule drug developed by Sugen, is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types. This agent can inhibit tumor growth in vivo in animal tumor models and is presently under evaluation in Phase I clinical studies for the treatment of human cancers (Fong et al., 1999, Cancer Res; 59(1):99-106).
Tyrosine kinase inhibitors of the present invention also include SU5614 (an inhibitor of VEGF (Flk-1) and PDGF receptor tyrosine kinases that does not have any effect on the EGF and IGF receptor tyrosine kinases), SU 11248 (an inhibitor FLT3) and ST1571 (an inhibitor c-Kit).
Tyrosine kinase inhibitors suitable for use in the methods of the present invention include any of those described herein. Examples of tyrosine kinase inhibitors suitable for use in the methods of the present invention include, but are not limited to, gefitinib, sunitinib, erlotinib, lapatinib, canertinib, semaxinib, vatalanib, sorafenib, imatinib, dasatinib, leflunomide, vandetanib, derivatives thereof, analogs thereof, and combinations thereof. Additional tyrosine kinase inhibitors suitable for use in the present invention are as described, for example, in U.S. Pat. Nos. 5,618,829, 5,639,757, 5,728,868, 5,804,396, 6,100,254, 6,127,374, 6,245,759, 6,306,874, 6,313,138, 6,316,444, 6,329,380, 6,344,459, 6,420,382, 6,479,512, 6,498,165, 6,544,988, 6,562,818, 6,586,423, 6,586,424, 6,740,665, 6,794,393, 6,875,767, 6,927,293, and 6,958,340. Inhibitors of the present invention may be selected from the group consisting of 2-(3-amino)arylamino-4-aryl-thiazoles, pyrimidine derivatives, pyrrolopyrimidine derivatives, quinazoline derivatives, quinoxaline derivatives, pyrazoles derivatives, bis monocyclic, bicyclic or heterocyclic aryl compounds, vinylene-azaindole derivatives and pyridyl-quinolones derivatives, styryl compounds, styryl-substituted pyridyl compounds, seleoindoles, selenides, tricyclic polyhydroxylic compounds and benzylphosphonic acid compounds.
The present invention includes methods of killing, inhibiting the growth, and/or inhibiting the reproduction of a kinetoplastid or apicomplexan protozoan by contacting the protozoan with one or more tyrosine kinase inhibitors. Such contact may be in vitro, ex vivo, and/or in vivo. As used herein in vitro is in cell culture, ex vivo is a cell that has been removed from the body of a subject, and in vivo is within the body of a subject. As used herein, the term “subject” represents an organism, including, for example, an animal. An animal includes, but is not limited to, a human, a non-human primate, a horse, a pig, a goat, a cow, a rodent, such as, but not limited to, a rat or a mouse, or a domestic pet, such as, but not limited to, a dog or a cat.
The present invention includes methods of killing, inhibiting the growth, and/or inhibiting the reproduction of a kinetoplastid or apicomplexan protozoan in a subject by comprising administering to the subject an effective amount of one or more tyrosine kinase inhibitors. An agent may be administered in an amount effective to inhibit replication and/or growth of the protozoan. Agents of the present invention may be administered in an amount effective to kill a protzoan in an infected individual. Inhibition of the growth and reproduction of a protozoan and killing of an a protozoan may be determined by any of various known methods, including, but not limited to, the methods described in the examples herein. An agent of the present invention may be administered in an amount effective to inhibit tyrosine kinase activity of a protozoan kinase.
The present invention includes methods of treating or preventing a protozoan infection, such as a kinetoplastid or apicomplexan protozoan infection, in a subject by administering to the subject an effective amount of one or more agents that inhibit protozoan tyrosine kinase activity. Such an agent may be identified by the methods described herein. As used herein “treating” or “treatment” includes both therapeutic and prophylactic treatments. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. An agent may be administered to a subject to reduce the severity of the symptoms associated with a protozoan infection. Agents of the present invention may be taken as a prophylactic to prevent the development of a protozoan infection. An agent of the present invention may be administered to a subject to prevent the infection of a subject with a protozoan. An agent of the present invention may be administered to a subject prior to and/or after exposure to a protozoan.
The present invention includes compositions including one or more tyrosine kinase inhibitors. In some aspects, the present invention includes compositions including two or more tyrosine kinase inhibitors. Such compositions may be administered in any of the methods of the present invention. A composition may be a pharmaceutical composition. Such compositions may be formulated in a variety of forms adapted to the chosen route of administration. The formulations may be conveniently presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. Formulations of the present invention include, for example, pharmaceutical compositions including a tyrosine kinase inhibitor and a pharmaceutically acceptable carrier. The phrase “pharmaceutically acceptable” refers to molecular entities and compositions suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, and the like. The preparation of such compositions is well understood in the art. The formulations of this invention may include one or more accessory ingredients including, but not limited to, diluents, buffers, binders, disintegrants, surface active agents, thickeners, lubricants, preservatives, including, for example, antioxidants, and the like. Pharmaceutically acceptable includes salts, amides and esters that are well known in the art. Representative acid addition salts include, for example, hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, toluenesulfonate, methanesulfonate, citrate, maleate, fumarate, succinate, tartrate, ascorbate, glucoheptonate, lactobionate, lauryl sulfate salts, and the like. Representative alkali or alkaline earth metal salts include, for example, aluminum, calcium, lithium, magnesium, potassium, sodium, or zinc salt, an ammonium salt such as a tertiary amine or quaternary ammonium salt, and an acid salt such as a succinate, tartarate, bitartarate, dihydrochloride, salicylate, hemisuccinate, citrate, isocitrate, malate, maleate, mesylate, hydrochloride, hydrobromide, phosphate, acetate, carbamate, sulfate, nitrate, formate, lactate, gluconate, glucuronate, pyruvate, oxalacetate, fumarate, propionate, aspartate, glutamate, or benzoate salt, and the like. Pharmaceutically acceptable carriers includes, for example, non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Examples of materials that may serve as pharmaceutically acceptable carriers include, but are not limited to, sugars, such as, for example, lactose, glucose and sucrose, starches such as, for example, corn starch and potato starch, cellulose and its derivatives such as, for example, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate, powdered tragacanth, malt, gelatin, talc, excipients such as, for example, cocoa butter and suppository waxes, oils such as, for example, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil, glycols, such as, for example, propylene glycol, polyols such as, for example, glycerin, sorbitol, mannitol and polyethylene glycol, esters such as, for example, ethyl oleate and ethyl laurate, agar, buffering agents such as, for example, magnesium hydroxide and aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants such as, for example, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present in the composition, according to the judgment of the formulator.
Tyrosine kinase inhibitors of the present invention may be administered as compositions including one or more isolated inhibitors. As used herein, the term isolated means a preparation that is either removed from its natural environment or synthetically derived, for instance by recombinant techniques, or chemically or enzymatically synthesized. In a preferred form, the isolated tyrosine kinase inhibitors is purified and substantially free of other agents. The present invention also includes compositions including two or more tyrosine kinase inhibitors.
The inhibitors of the present invention can be administered by any suitable means including, but not limited to, for example, oral, rectal, nasal, topical (including, for example, transdermal, aerosol, buccal and sublingual), vaginal, or parenteral (including, for example, subcutaneous, intramuscular, intravenous, intradermal, intraperitoneal, intrasternal, and intraarticular injections as well as various infusion techniques). For parenteral administration in an aqueous solution, the solution may be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
For enteral administration, the inhibitor may be administered in a tablet or capsule, which may be enteric coated, or in a formulation for controlled or sustained release. Many suitable formulations are known, including polymeric or protein microparticles encapsulating drug to be released, ointments, gels, or solutions which can be used topically or locally to administer drug, and even patches, which provide controlled release over a prolonged period of time. These can also take the form of implants.
Compositions for nasal administration may be formulated for aerosol or inhalation administration. Such compositions may include solutions in saline which may also contain, for example, benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and/or other solubilizing or dispersing agents such as those known in the art.
Compositions for rectal administration include, for example, suppositories which may contain a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquify and/or dissolve in the rectal cavity to release the drug.
Therapeutically effective concentrations and amounts may be determined for each application herein empirically by testing the compounds in known in vitro and in vivo systems, including, but not limited to, any of those described herein; dosages for humans or other animals may then be extrapolated therefrom. The efficacy of treatment may be assessed by any of various parameters well known in the art. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by the FDA.
As used herein, a “subject” or an “individual” is an organism, including, for example, a mammal. A mammal may include, for example, a rat, mouse, a primate, a domestic pet (such as, but not limited to, a dog or a cat), livestock (such as, but not limited to, a cow, a horse, and a pig), or a human.
A tyrosine kinase inhibitor may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions and methods.
In accordance with the present invention, a tyrosine kinase inhibitor may be administered in combination with the administration of one or more previously known treatment modalities. As used herein, the term “additional therapeutic agent” represents one or more agents previously known to be effective for the treatment of a protozoan disease. Such an additional therapeutic agent is not a tyrosine kinase inhibitor. The administration of the tyrosine kinase may take place before, during, and/or after the administration of the other mode of therapy.
In some embodiments of the present invention, the administration of an inhibitor of tyrosine kinase in combination with additional therapeutic agents may demonstrate therapeutic synergy. Likewise, the administration of two or more tyrosine kinase inhibitors may demonstrate therapeutic synergy. As used herein, a combination may demonstrate therapeutic synergy if it is therapeutically superior to one or other of the constituents used at its optimum dose (Corbett et al., 1982, Cancer Treatment Reports; 66:1187. In some embodiments, a combination demonstrates therapeutic synergy if the efficacy of a combination is characterized as more than additive actions of each constituent.
By a “therapeutically effective amount” of a tyrosine kinase inhibitor is meant a sufficient amount of the compound to treat the subject at a reasonable benefit/risk ratio applicable to obtain a desired therapeutic response. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including, for example, the disorder being treated and the severity of the disorder, activity of the specific compound employed, the specific composition employed, the age, body weight, general health, sex and diet of the patient, the time of administration, route of administration, and rate of excretion of the specific compound employed, the duration of the treatment, drugs used in combination or coincidentally with the specific compound employed, and like factors well known in the medical arts. Total daily dose of the compounds of this invention administered to a host in single or divided doses may be in amounts as determined by the attending physician, typically, for example, in amounts of from about 0.001 to 100 mg/kg body weight daily and preferably 0.01 to 10 mg/kg/day. As used herein, the terms “administration of a tyrosine kinase inhibitor” or “administering a tyrosine kinase inhibitor” refer to the act of providing a tyrosine kinase inhibitor or pharmaceutical composition thereof to the subject in need of treatment.
The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein. For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
EXAMPLES
Example 1
Bioinformatic Analysis of the Protein Tyrosine Kinases in the T. brucei Genome
New drugs are needed for treatment of human African trypanosomiasis (HAT), which is caused by the protozoan parasite Trypanosoma brucei. “Alternative Use Drug Discovery,” whereby drugs that are approved for control of other ailments are tested as anti-parasite agents is a cost effective route for finding new anti-parasite drugs. In this example, the “Alternative Use” approach was applied to find new lead compounds that may be developed into drugs for treatment of HAT. To determine which class of drugs to focus on, a bioinformatic analysis was performed of the T. brucei genome sequence. This analysis revealed that the parasite contained proteins whose kinase domains are similar in sequence to that of the epidermal growth factor receptor (EGFR). This analysis also identified kinase domains in the T. brucei genome that are homologous to the kinase domains of the vascular endothelial cell growth factor receptor (VEGFR), c-Src protein (c-Src), c-Abl protein tyrosine kinase (c-Abl), c-Kit protein (c-Kit), and FMS-like tyrosine kinase 3 (FLT3).
Further, as presented in Example 2, it was determined that the 4-anilinoquinazoline, AG1478, which specifically inhibits EGFR kinase, killed T. brucei. A focused screen of other protein tyrosine kinase (PTK) specific anti-cancer drugs for trypanocidal activity was carried out in Examples 3 and 4, using cultured bloodstream T. brucei. As presented in Example 5, promising anti-trypanosome drugs will be evaluated for curing T. brucei infections in a murine model of HAT. This bioinformatics-informed drug discovery process will be extended to VEGFR, c-Src, and c-Abl because T. brucei encodes proteins with related kinase domains, and anti-cancer drugs that inhibit those enzymes are available. Many inhibitors of PTKs are being developed as drugs against cancer. The present invention will produce new lead compounds for anti-trypanosome drug discovery.
Example 2
Tyrosine Kinase Inhibitors Arrest Growth of Bloodstream T. brucei 
New drugs are needed for treatment of infections by the trypanosomatid protozoans Leishmania and Trypanosoma brucei. However, the pathway for identifying new biological targets and developing new compounds that are specifically directed at the new targets can be tedious, drawn-out, expensive and frustrating (reviewed in Gelb and Hol, 2002, Science; 297:343-344 and Barrett and Barrett, 2000, Parasitol Today; 16:7-9). One possible shortcut to this traditional path for drug discovery involves “piggy-backing” on compounds that are already approved for treatments of other ailments or diseases and have gone through at least some phases of clinical trials.
Protein tyrosine kinases (PTKs) are important signaling molecules in eukaryotes (Robinson et al., 2000, Oncogene; 19:5548-5557; Wilks, 1993, Adv Cancer Res; 60:43-73; and Hubbard and Till, 2000, Annu Rev Biochem; 69:373-398). Recently, several inhibitors of PTKs, including the tyrphostins, have proven effective against certain cancers, and are currently in clinical trials as anti-cancer agents (Ellis et al., 2006, Biochem Pharmacol; 71:1422-1434; Zhu et al., 2001, Cancer Lett; 169:27-32; Twaddle et al., 1999, J Surg Oncol; 70:83-90; and Roskoski, 2003, Biochem Biophys Res Commun; 309:709-717). To explore the effectiveness of these anti-cancer drugs as anti-trypanosomal agents, two tyrphostins were tested using Trypanosoma brucei in an in vitro cell culture system. The PTK inhibitors, Tyrphostin A47 and Tyrphostin AG1478, were selected for initial evaluation. Tyrphostin A47 inhibits multiple PTKs, including EGFR (Turbov et al., 2002, J Surg Oncol; 79:17-29; Levitzki and Gazit, Science, 1995; 267:1782-8) and stops growth of HER14 transformed cells (Merkel et al., 1993, Biochem Biophys Res Commun; 192:1319-26; Lyall et al., 1989, J Biol Chem; 264:14503-9). Tyrphostin AG1478 is an anilinoquinazoline that inhibits EGFR kinase (Shushan et al., 2004, Hum Reprod; 19:1957-67; Zhu et al., 2001, Cancer Lett; 169:27-32). Several drugs whose structures similar to AG 1478 are used to treat human cancer. The results, summarized below indicate that inhibitors of trypanosomal PTKs can be developed as anti-trypanosome compounds.
Cultured trypanosomes (seeded at 104/ml) in 100 μl of a 96-well plate received varying amounts of Tyrphostin A47 (Sigma). Parasites were counted with a haemocytometer after 48 hours in culture. Data are means of triplicate determinations, they are representative of three independent trials. Blood stream form of T. brucei were used for these studies since that stage of the parasite causes disease in humans. Inhibitory concentration 50 (IC50) is the drug concentration which reduces the growth rate by 50%. Minimum inhibitory concentration (MIC) is the lowest concentration which killed all the parasites.
As shown in FIG. 1, Tyrphostin A47 kills T. brucei, with an IC50 between 1 to 5 μM. Note that the y-axis in FIG. 1 uses a logarithmic scale.
T. brucei strain 427 was cultured axenically and seeded at 104 cells/ml in 200 μl of medium a 96-well plate. Tyrphostin AG1478 (Sigma) (Zhu et al., 2001, Cancer Lett; 169:27-32; Shushan et al., 2004, Hum Reprod; 19:1957-1967) was added to the stated final concentrations, cells were incubated at 37° C. for 40 hours, and parasites were counted with a haemocytometer. Data are mean values representative of two independent trials each of which was performed in triplicate. T. brucei replicates every 8-10 hours.
As shown in FIG. 2, Tyrphostin AG1478 inhibited growth of T. brucei bloodstream form. The IC50 was between 2.5 to 5 μM.
The IC50 of Tyrphostin A47 on T. brucei is in the range that arrests replication of transformed NIH3T3/HER14 cells (Merkel et al., 1993, Biochem Biophys Res Commun; 192:1319-26; Lyall et al., 1989, J Biol Chem; 264:14503-9). AG1478, which is highly specific for EGFR kinase, also inhibited replication of T. brucei (FIG. 2). The IC50 of AG1478 on T. brucei was between 2.5 to 5 μM, in the same range as the amount needed to arrest growth of human leiomyoma cells (Shushan et al., 2004, Hum Reprod; 19(9):1957-67).
There are two implications from the data presented in FIG. 1 and FIG. 2. First, it appears that T. brucei contains an EGFR-like kinase activity, although the parasite lacks a “classic” EGFR. This observation is not entirely surprising because the ligand-binding region of EGFR is distinct from the kinase domain (reviewed in Linggi and Carpenter, 2006, Trends Cell Biol; 16:649-56). It is also interesting to note that many human cancer cells (e.g., glioblastomas and some breast cancer) express high levels of a mutant EGFR (named EGFRvIII) that lacks a ligand-binding domain. Remarkably, the PTK activity of EGFRvIII is constitutively activated in the absence of ligand (Okamoto, 2003, Cancer Sci; 94:50-6; Chu et al., 1997, Biochem J; 324(Pt 3):855-61; Wikstrand et al., 1997, Cancer Res; 57:4130-40; Batra et al., 1995, Cell Growth Differ; 6:1251-9). Thus, EGFRvIII is effectively a non-receptor Tyr kinase variant of EGFR. This example demonstrates that T. brucei has a Tyr kinase with an ATP binding site that is similar to that of EGFR kinase. Consistent with this theory, six TbPTKs have kinase domains with homology to that of the human EGFR, the target of AG1478. One or all of these TbPTKs could be inhibited by AG1478. Second, AG1478 is a 4-anilinoquinazoline. Since it kills T. brucei, the effect of other 4-anilinoquinazoline drugs, such as, for example, gefitinib, erlotinib, lapatinib, and canertinib, will be assayed for their trypanocidal effect (see Example 3).
These data from the two inhibitors tested in this example indicate that compounds directed against PTK's can be effective against trypanosome infection. Future experiments will test additional inhibitors of PTKs, including inhibitors known to be more potent against this class of enzymes, as anti-trypanosomal compounds.
Example 3
Effect of Canertinib and Erlotinib on T. brucei Viability
The 4-anilinoquinazoline canertinib (also referred to as CI-1033) is an inhibitor of EGFR kinase (Dewji, 2004, J Chemotherapy; 16(Suppl 4):44-48; Hamid, 2004, J Am Pharm Assoc; 44:52-58). To test the trypanocidal activity of canertinib, culture of blood stream T. brucei strain 427 was initiated in 24-well plates at 104 cells/ml (in 500 μl of medium (Hirumi and Hirumi, 1991, Parasitology; 102(Pt 2):225-236)). Canertinib (Heymach et al., 2006, Clin Cancer Res; 12:4441s) (provided by Pfizer) was added to the stated final concentrations, and cells were incubated at 37° C. for 40 hours. Control cells received an equal volume of solvent (DMSO). Parasites were counted with a haemocytometer. Data plotted are mean values of quadruplicate determinations from one of three independent trials that produced similar results.
As show in FIG. 3, T. brucei are killed by canertinib (IC50 of 1 μM). Above 4 μM of canertinib, all parasites were killed. Along with Example 2, these data further demonstrate that 4-anilinoquinazoline drugs can kill T. brucei. Inhibitors of protein tyrosine kinases are typically effective in the micromolar range when used on cells where ATP concentration is in the millimolar range (Knight and Shokat, 2005, Chem Biol; 12:621-637). Therefore, the concentration of canertinib needed to kill blood stream T. brucei is consistent with that expected for a susceptible cell. In vitro, when the drugs are used to inhibit purified enzymes, only micromolar amounts of ATP are used in the assays. Consequently the drugs, which compete for ATP binding sites of PTKs, are effective at nanomolar concentrations (Carter et al., 2005, Proc Natl Acad Sci USA; 102:11011-11016; reviewed in Knight and Shokat, 2005, Chem Biol; 12:621-637). Finally, it is noted that the plasma concentration of a 4-anilinoquinazoline can reach 10 μM (Ellis et al., 2006, Biochem Pharmacol; 71:1422-1434), a level that exceeds the amount required to kill all T. brucei in culture (FIG. 3). From these observations it is likely that canertinib will be effective against T. brucei infection of mice in an animal model of human African trypanosomiasis (HAT).
This example also determined that erlotinib (TARCEVA), a 4-anilinoquinazoline tyrosine kinase inhibitor is trypanocidal. erlotinib (TARCEVA), like canertinib, is an inhibitor of EGFR kinase (Bulgaru et al., 2003, Expert Rev Anticancer Ther; 3(3):269-79; Akita and Sliwkowski, 2003, Semin Oncol; 30(3 Suppl 7):15-24). The culture of blood stream T. brucei strain 427 was initiated in 24-well plates at 104 cells/ml in 500 μl of medium (Hirumi and Hirumi, 1991, Parasitology; 102 Pt 2:225-236). Erlotinib (TARCEVA) (provided by Genentech/OSI), was added to the stated final concentrations shown in FIG. 4, and cells were incubated at 37° C. for 40 hours. Control cells received an equal volume of solvent (DMSO. Parasites were counted with a haemocytometer. Data plotted are mean values of quadruplicate determinations from one of three independent trials that produced similar results.
As shown in FIG. 4, erlotinib (TARCEVA) kills T. brucei. No parasites survived exposure to 10 μM of the drug. The IC50 of TARCEVA against T. brucei is approximately 1.7 μM (FIG. 4). Thus, TARCEVA kills blood stream T. brucei. In human patients receiving a cycle of 150 mg/day TARCEVA, the plasma concentration of TARCEVA can reaches 60 μM (Broniscer et al., 2007, Clin Cancer Res; 13(5):1511-5), a level that exceeds the single does amount required to kill all T. brucei in this example (FIG. 4). A single dose of the drug yields a plasma concentration of 2 μM (Ling et al., 2006, Drug Metab Dispos; 34(3):420-6).
The trypanocidal effect of canertinib and erlotinib (TARCEVA), and any of the other inhibitors described herein, will be tested in animal models of human African trypanosomiasis (HAT), including mice infected with T. brucei, as described in Example 5 below.
Example 4
Sunitinib and Axitinib are Trypanocidal
This example presents data showing sunitinib and axitinib (both provided by Pfizer) are trypanocidal. Sunitinib (also known as SU11248 and marketed under the trade name SUTENT) inhibits VEGFR, PDGF, and c-Kit kinases (reviewed in Steeghs et al., 2007, Ann Surg Oncol; 14(2):942-53) and is approved by the Food and Drug Administration for treatment of renal cell carcinoma. The bioinformatics analysis of Example 1 indicated that the T. brucei genome encodes “VEGFR-like” kinase domains. Therefore, this example tests whether sunitinib (SUTENT) compromised T. brucei viability. To blood stream form T. brucei 427 (seeded at 104 cells/ml in 500 μl of medium) sunitinib (SUTENT) (Abrams et al., 2003, Mol Cancer Ther; 2:471-478; Mendel et al., 2003, Clin Cancer Res; 9:327-337) was added to the stated final concentrations and cells were incubated at 37° C. for 40 hours. Parasites were counted with a haemocytometer. Data plotted are mean values of quadruplicate determinations from a representative experiment. FIG. 5 shows that SUTENT kills T. brucei. SUTENT kills T. brucei at low micromolar levels, indicating that VEGFR, PDGF, and c-Kit kinase domains may be targeted for anti-trypanosome drug discovery. Using the procedures described above, as shown in FIG. 6, axitinib (also known as AG-013736) also kills T. brucei. 
Example 5
Use of T. brucei rhodesiense for Mouse Infection Studies
Following guidelines of the World Health Organization (WHO) parasite drug discovery initiative (see Nwaka and Hudson, 2006, Nat Rev Drug Discov; 5:941-955 and Nwaka and Ridley, 2003, Nat Rev Drug Discov; 2:919-928 for reviews) the candidate trypanocidal drugs of the present invention will be tested in animal models for both acute and late stage HAT. Work with T. brucei 427 will predict effectiveness of drugs against acute stage disease. For a model of late stage disease, T. brucei rhodesiense STIB 900 infection of mice will be studied. T. b. rhodesiense STIB 900 infection in mice is thought to mimic late stage disease (Dardonville et al., 2006, J Med Chem; 49:3748-3752) because it does not respond to drugs used in treating acute stage (e.g., pentamidine) (Docampo and Moreno, 2003, Parasitol Res; 90(Supp 1):S10-13); instead the infection is cured with melarsoprol, a toxic arsenical, which is used in treatment of late stage HAT. (Stewart et al., 2004, Antimicrob Agents Chemother; 48:1733-1738). Thus, a cure of T. b. rhodesiense STIB 900 infection may signal that the drugs that we are studying could cure late stage HAT. (Nwaka and Hudson, 2006, Nat Rev Drug Discov; 5:941-955; Nwaka and Ridley, 2003, Nat Rev Drug Discov; 2:919-928). Drugs will be administered to mice by gavage (i.e., forced feeding) because all of them are orally bioavailable (Rugo et al., 2005, J Clin Oncol; 23:5474-5483; Kim et al., 2006, J Clin Endocrin Metab; 91:4070-4076; Simon et al., 2006, Clin Cancer Res; 12:4645-4651; and Gridelli et al., 2007, The Oncologist; 12:191-200; reviewed in Steeghs et al., 2007, Ann Surg Oncol; 14(2):942-53).
PTK Inhibitor anti-trypanosome drugs are likely to be very valuable in the field. The urgent need for new anti-trypanosome drugs cannot be overstated (reviewed in Nwaka and Hudson, 2006, Nat Rev Drug Discov; 5:941-955; Nwaka and Ridley, 2003, Nat Rev Drug Discov; 2:919-928; and Gelb and Hol, 2002, Science; 297:343-344). Current anti-trypanosome drugs are toxic, and must be injected intramuscularly in a clinic (reviewed in Docampo and Moreno, 2003, Parasitol Res; 90(Supp 1):S10-13 and Fairlamb, 2003, Trends Parasitol; 19:488-494). Unfortunately, in those areas of rural Africa where HAT. is endemic, clinics are not commonplace. Fortunately, the drugs of the present invention are well-tolerated, with minimal side effects in human clinical trials, and can be administered by swallowing (Rugo et al., 2005, J Clin Oncol; 23:5474-5483; Kim et al., 2006, J Clin Endocrin and Metab; 91:4070-4076; Simon et al., 2006, Clin Cancer Res; 12:4645-4651; and Gridelli et al., 2007, The Oncologist; 12:191-200).
The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.
All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

## Claims

1. A method of killing and/or inhibiting the growth of the bloodstream form of a kinetoplastid protozoan of the genus Trypanosoma, the method comprising contacting the protozoan with a tyrosine kinase inhibitor; wherein the tyrosine kinase inhibitor is a 4-anilinoquinazoline or tyrphostin selected from the group consisting of canertinib, sunitinib, axitinib, dasatinib, and nilotinib.

2. A method of treating a protozoan infection in a subject, the method comprising administering to the subject an effective amount of a tyrosine kinase inhibitor; wherein the tyrosine kinase inhibitor is a 4-anilinoquinazoline or tyrphostin selected from the group consisting of canertinib, sunitinib, axitinib, dasatinib, and nilotinib.

3. A method of killing and/or inhibiting the growth of the blood stream form of a kinetoplastid protozoan of the genus Trypanosoma in a subject, the method comprising administering to the subject an effective amount of a tyrosine kinase inhibitor; wherein the tyrosine kinase inhibitor is a 4-anilinoquinazoline or tyrphostin selected from the group consisting of canertinib, sunitinib, axitinib, dasatinib, and nilotinib.

4. The method of claim 1, wherein the kinetoplastid protozoan is selected from the group consisting of T. cruzi, T. brucei, T.b. gambiense, and T.b. rhodesiense.

5. The method of claim 1, wherein the tyrosine kinase inhibitor specifically inhibits epidermal growth factor receptors (EGFR) tyrosine kinase activity.

6. The method of claim 1, wherein the tyrosine kinase inhibitor inhibits vascular endothelial growth factor receptor (VEGFR) kinase activity.

7. The method of claim 2, wherein the protozoan is selected from the group consisting of T. cruzi, T. brucei, T.b. gambiense, and T.b. rhodesiense.

8. The method of claim 3, wherein the kinetoplastid protozoan is selected from the group consisting of T. cruzi, T. brucei, T.b. gambiense, and T.b. rhodesiense.

9. The method of claim 2, wherein the tyrosine kinase inhibitor specifically inhibits epidermal growth factor receptors (EGFR) tyrosine kinase activity.

10. The method of claim 3, wherein the tyrosine kinase inhibitor specifically inhibits epidermal growth factor receptors (EGFR) tyrosine kinase activity.

11. The method of claim 2, wherein the tyrosine kinase inhibitor inhibits vascular endothelial growth factor receptor (VEGFR) kinase activity.