1. Field of the Invention
The present invention relates generally to the field of molecular pharmacology of antiparasitics. More specifically, the present invention relates to inhibition of Toxoplasma gondii replication by pyridinylimidazoles.
2. Description of the Related Art
Infection with intracellular parasites such as Plasmodium species (agents of malaria), Leishmania species and Toxoplasma gondii are a major cause of morbidity and death worldwide. These parasites undergo complex life cycles that require replication within host cells and stage differentiation. Stage differentiation is the conversion from one life form to another. For example, T. gondii resides in a dormant tissue cyst form as a relatively metabolically inactive bradyzoite, but quickly reverts to a motile, destructive tachyzoite when specific external signals are sensed. Tachyzoites and bradyzoites are morphologically distinct and express gene products unique to their stage. However, host cell and parasite factors regulating intracellular replication and controlling stage differentiation are poorly understood.
Mitogen activated protein kinases (MAPKs) mediate enzymatic cascades that play diverse roles in mammalian cells including regulation of growth and differentiation, responses to inflammatory stimuli and control of apoptosis. They are highly conserved in evolution and are encoded by all eukaryotes. There are three major families of mitogen activated protein kinases: c-Jun-activated kinases (JNKs), extracellular signal-related kinases (ERKs) and p38s (1, 2).
Mitogen activated protein kinases are activated in a stereotypic fashion. A MAPK kinase (MKKK) is initially activated, which activates a specific MKK or MEK, which in turn activates the individual mitogen activated protein kinase. More than one MKK/MEK may activate a specific mitogen activated protein kinase, and one MKK/MEK may activate members of more than one mitogen activated protein kinase pathway. p38 MAPK is activated by cell stressors such as osmotic shock, heat and infection. p38 MAPK regulates proinflammatory cytokine and IL-10 secretion, apoptosis, proliferation and differentiation (3). Inhibition of p38 MAPK by potent, specific drugs is a novel method to inhibit inflammation in humans in vivo without inducing significant immunosuppression (4).
Pathogens have subverted mitogen activated protein kinase signaling to their own ends. For example, YopJ of Yersinia enterocolitica inhibits host cell p38 MAPK activation and interferes with host cell intracellular signaling upon invasion (5, 6). T. gondii infection induces host cell p38 MAPK activation (7). Inactivation of host cell mitogen activated protein kinase inhibits parasite entry (7), whereas bombesin and phorbol myristate acetate activate p38 MAPK and increase T. gondii infectivity (8). Listeria monocytogenes requires activated p42/44 MAPK for cell invasion (9). Leishmania donovani promastigotes fail to activate ERK, JNK or p38 following infection of macrophages (10), yet genetic deletion of an endogenous mitogen activated protein kinase in Leishmania demonstrates that it is required for intracellular replication (11). These studies underscore the importance of mitogen activated protein kinase signaling in pathogen infection, host anti-pathogen immunity, and pathogen differentiation and signaling.
Roisin et al. showed for the first time that the medically important, obligate intracellular parasite Toxoplasma gondii encodes homologues of human ERK-1 and ERK-2 (12). However, Roisin et al. failed to provide convincing evidence for a role of mitogen activated protein kinase in intracellular replication. Inhibition of T. gondii ERK using the specific drug PD098059 (13) slightly reduced intracellular replication that may have been due to defective cell entry, whereas the tyrosine kinase inhibitor genistein had no significant effect on replication (12).
T. gondii is a protozoan parasite in the order Coccidia. Cats are the only definitive hosts (14-16). Viable organisms may remain encysted within the host for extended periods of time, and possibly for life (17). With defective cell-mediated immunity, latent infection may recrudesce, producing encephalitis, chorioretinitis, disseminated disease or congenital infection. Populations at risk include recipients of organ allografts or cytotoxic chemotherapy, neonates with perinatally acquired disease, and persons infected with HIV (18-22). Cerebral toxoplasmic encephalitis is an important cause of morbidity and mortality in HIV disease (23-26). Up to 40% of T. gondii seropositive, HIV-infected individuals will develop this illness (25).
The mainstay of treatment is combination therapy with pyrimethamine plus sulfadiazine (26). However, treatments are toxic and may interfere with specific antiretroviral therapies in HIV infected individuals. Pyrimethamine is marrow toxic and associated with cytopenias. It also causes rash and is hepatotoxic. Sulfadiazine is also marrow toxic and induces neutropenia. Rash and drug fevers are additional prominent side effects. Marrow toxicity is often dose limiting for both drugs in the setting of advanced HIV disease (27). Neither agent is very active against cysts, and therapy must be given indefinitely in HIV infected individuals (26). Newer agents include Atovaquone and Clindamycin are sometimes substituted for sulfadiazine in the setting of drug related toxicity (27), although none of these agents is very effective. Thus, alternatives or adjuncts allowing for equal efficacy with lower dosing would be of great therapeutic value.
Thus, the prior art is deficient in method and compositions for safely and effectively inhibiting Toxoplasma gondii replication in humans. The present invention fulfills this long-standing need and desire in the art.
Toxoplasma gondii is a medically important, obligate intracellular parasite. Little is known regarding factors that regulate its replication within cells. Such knowledge would further understanding of T. gondii pathogenesis, and might lead to novel therapeutic strategies. Mitogen activated protein kinases (MAPKs) govern diverse cellular processes including proliferation and differentiation. The present study shows that treatment of T. gondii infected cells with SB203580 and SB202190, substituted pyridinylimidazoles that are potent inhibitors of human p38 MAPK, inhibits intracellular T. gondii replication. Toxoplasma gondii encodes two known functional mitogen activated protein kinase homologues related to human ERK-1 and ERK-2. However, neither the specific ERK inhibitor U0126 nor other general kinase inhibitors affected intracellular T. gondii replication. Pyridinylimidazole-treated tachyzoites downregulated tachyzoite specific SAG2, upregulated bradyzoite specific p21 and p36, and formed cyst-like structures, suggesting stage conversion from active tachyzoites to relatively dormant bradyzoites. SB203580 also significantly enhanced in vitro inhibition of T. gondii replication by the approved anti-Toxoplasma drug pyrimethamine.
Several independent experimental approaches suggest that the antiproliferative effects of pyridinylimidazoles depend on direct action on tachyzoites, not the host cell. Selective inhibition of host p38 MAPK using recombinant adenoviruses had little effect on tachyzoite replication. Pyridinylimidazole-treated tachyzoites developed abnormal morphology suggesting defective parasite division and pyridinylimidazole-resistant mutant tachyzoites were developed through culture in progressively higher drug concentrations. T. gondii encodes no known p38 MAPK homologue, but its genome is incompletely characterized. It is proposed that pyridinylimidazoles target a human p38 MAPK homologue in tachyzoites that regulates their replication. As all eukaryotic pathogens, including agents of malaria, Leishmaniasis and Trypanosomiasis encode endogenous mitogen activated protein kinases, drugs inhibiting endogenous mitogen activated protein kinase activation may represent a novel, potentially broadly-acting class of antiparasitic agents. Pyridinylimidazoles also represent tools to elucidate factors governing intracellular tachyzoite replication.
In one aspect of the present invention, there are provided methods of inhibiting intracellular replication of a parasite that possesses endogenous mitogen activated protein kinases. The methods involve treating infected cells with a halogenated imidazole drug that specifically inhibit a human p38 mitogen activated protein kinase homologue expressed in the parasite, or treating infected cells with substituted pyridinylimidazole such as SB203580 or SB202190. In general, the parasite can be a species of Plasmodium, Leishmania or Toxoplasma.
In another aspect of the present invention, there is provided a method of inhibiting intracellular replication of parasite that possesses endogenous mitogen activated protein kinases by treating infected cells with a halogenated imidazole drug that specifically inhibits mitogen activated protein kinases endogenous to said parasite. In general, the parasite can be a species of Plasmodium, Leishmania or Toxoplasma.
In yet another aspect of the present invention, there is provided a method of inhibiting intracellular replication of Toxoplasma gondii by treating infected cells with substituted pyridinylimidazole agent SB203580 or SB202190.
The present invention is also directed to improved methods of inhibiting intracellular replication of Toxoplasma gondii by treating infected cells with an anti-Toxoplasma drug pyrimethamine in combination with a halogenated imidazole drug or with a substituted pyridinylimidazole drug SB203580 or SB202190. The combined treatments are more effective in inhibiting intracellular replication of Toxoplasma gondii as compared to treatment with either drug alone.
The present invention also provides methods of treating an individual infected with Toxoplasma gondii using anti-Toxoplasma drug pyrimethamine in combination with a halogenated imidazole drug or with a substituted pyridinylimidazole drug SB203580 or SB202190.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.
USPTO Backgrounds
USPTO Backgrounds