METHOD FOR THE INHIBITION OF PLASMODIUM IN A PLASMODIUM-

INFECTED PATIENT

Introduction

[0001] Malaria has been recognized as an important parasitic disease of humans for centuries, having been described by the early Egyptians in the third millennium B.C. Despite the introduction of control programs in many parts of the world over the past few decades, the impact of malaria on human populations continues to increase. Recent estimates suggest (1) that 1.5 billion persons live in areas of the world where malaria is an endemic disease, (2) that the number of infected humans exceeds 500,000,000, and (3) that 1-2 million persons die each year of this disease.

[0002] Four species of Plasmodium infect humans and cause malaria. Among them, Plasmodium falciperum causes the most lethal form of malaria. All species cause are vector borne disease, being spread by anopheline mosquitoes, and the disease is distributed throughout much of the world (view distribution). In the human host the parasite is found primarily inside of the red blood cells (RBC). The parasite reproduces asexually inside of the RBC, and following this the RBC breaks open releasing many new parasites (merozoites). These parasites then infect more RBCs, and this ultimately leads to the destruction of massive numbers of RBCs. The characteristic "chill and fever" (paroxysm) associated with malaria occurs when the parasites are released from the RBCs, and since the release of parasites is periodic, the paroxysms are periodic. For examples, the paroxysms associated with a tertian malaria (e.g., Plasmodium vivax) occur about every48 hours, and those associated with a quarten malaria (e.g., Plasmodium malariae) occur about every 72 hours. The main lethal effect of the malaria is the tendency of infected RBC to form rosettes (clumps) and adhere to endothelial cells in the walls of blood vessels. This clumping and adherence leads to blockage of the vessels, particularly in the brain, which prevents blood supply to essential nerve centers consequently starving them of oxygen and nutrients. This leads to irreversible damage to the brain resulting in death in a high proportion of infected children under five and to a lesser extent in older

individuals. Many survivors suffer debilitating brain damage. In infected pregnant women malaria leads to blockage of oxygen to the fetuses leading to abortion and at times death to the mothers.

The clumping of the infected RBC to non-infected RBC also helps concealing them from the immune system of the infected person which otherwise would sequester them and remove them from the circulation.

[0003] Babesia is a plasmodium-related tick-transmitted protozoan parasite that also resides in RBC and causes malaria-like symptoms and hemolytic anemia in humans and animals.

[0004] CTC-96 is a molecule that binds covalently to the imidazole group of histidine (see attached brochure). This compound is disclosed in U.S. Patent No. 5,756,491, the contents of which are hereby incorporated by reference. Its interaction with specific histidines in proteins depends on the tertiary structure of the proteins and the amino acid residues adjacent to the histidines. This interaction with the histidines results in conformational changes in protein molecules and can in many cases inactivate the protein. We have shown this for the gp41 protein of HIV in which binding of CTC-96 to a specific histidine inactivates the protein. The gp41 protein is involved in the fusion of the HIV coat protein and the cell membrane, an essential function in the process of viral entry into the infected cell. We have shown that viral entry into cells is abolished by CTC-96.

[0005] In addition, it was shown that CTC-96 binds to histidines in Zinc fingers and causes the ejection of the Zinc thus inactivating some zinc finger proteins such as cellular SP1 and HIVs NCP 7. In addition, we have shown that CTC 96 binds the histidine in the active site of proteases rendering them inactive.

See Louie, A. Y.; Meade, T. J.; A Cobalt Complex That Selectively Disrupts The Structure And Function Of Zinc Fingers, Proc. Natl. Acad. Sci. USA Vol. 95, 6663- 6668, June 1998; See also, Takeuchi, T.; Bottcher, A.; Quezada, CM.; Meade, TJ. , Gray, H. B.; Inhibition Of Thermolysin And Human α-Thrombin By Cobalt(lll) Schiff Base Complexes. Biorganic & Medicinal Chemistry, 7 (1999) 815-819.

Summary of the Invention

[0006] I have discovered that Plasmodium infection or Babesia sp. infections in a subject may be inhibited by the administration of an anti-Plasmodium effective amount or anti-Babesia effective amount of CTC-96 to the infected subject. Accordingly, the present invention includes a method for preventing the development of malaria in a subject who has been infected with either Plasmodium or Babesia by the administration of an anti-Plasmodium effective amount of CTC-96 to the subject. Plasmodium has at least 4 proteins with unusually high contents of histidines. KAHRP (HRP-I), HRP-II, HRP-III and MAHRP. With the exception HRP-III, the activity of these proteins in the life cycle of the Plasmodium is known although their precise mode of action at the molecular level is not completely resolved. All of these (perhaps with the exception of HRPIII) are essential for the pathogenic effect of the Plasmodium.

KAHRP:

[0007] This 80-110KD protein contains a highly histidine-rich domain (residues

61-106 contain 27 histidines) \ It is associated with knob formation through binding PfEMPI protein by electrostatic interactions, the latter is involved in cytoadherence ^2i3>4 . There is evidence that it may be connected with trafficking of PfEMP to the erythrocyte surface. Targeted disruption of the KAHRP protein by binding of CTC 96 to its abundant histidines will result in lack of formation of knobs and, under flow conditions, alter the ability of the infected erythrocyte to bind to endothelial cells via CD 36 5. Thus, CTC-96 will interrupt the adherence to blood vessel walls and their clumping and as a consequence will, therefore, reduce or completely abolish the blockage of blood vessels. Lack of clumping will also expose the infected RBC to cells of the immune system which in turn sequester them and remove them from the circulation. This will considerably reduce the number of infectious parasites in the blood system.

HRP-II:

[0008] This protein is expressed in trophozoite and early gametocyte stages of the Plasmodium life cycle ⁶ : It is a protein of 327 amino acids with -30% histidine residues. Its functions are: a) facilitating hemozoin formation (and it thus contributes to heme detoxification) by binding to a large number of heme molecules and aiding in their dimerization ⁷ ' This detoxification promotes the viability of the paprasite; b) interaction with erythrocyte cytoskeleton (actin) thus aiding in export of proteins from the parasitophorous vacuole to the host cytoplasm ¹ . Because of the unusually high proportion of histidine residues in HRP-II, CTC-96 will interfere with the detoxification of heme and also the transport of essential parasite proteins into the host cytoplasm. Among these is PfEMP which is essential in cytoadherence. The inhibition of HRP Il will thus kill the parasite and reduce blood vessel blockage.

MAHRP:

[0009] This protein contains 249 amino acids ~13% of which are histidines (34 his) clustered between residues 64-219 ⁸ . This protein has been implicated in the binding of ferriprotoporphyrin (FP), the toxic product of hemoglobin degradation. The bound FP is 10 fold more susceptible to H ₂ O ₂ - induced degradation than the unbound form. Thus MAHRP most likely promotes oxidative stress and thus enhances the removal of the toxic FP. CTC 96 will inactivate MAHRP and will thus remove the oxidative damage to FP and will promote its accumulation in the parasite and subsequently causing its killing. MAHRP is located in Maurer's clefts and is involved in promoting the trafficking of exported essential proteins from the parasite to the erythrocyte membrane. The inhibition of this protein by CTC-96 will considerably reduce the infectious ability of the parasite.

CTC-96 can also target the following:

[0010] a. Some of the proteases involved in hemoglobin degradation, which are essential for the nutrition of the parasite, are susceptible to CTC-96. Their inactivation will cause starvation of the parasite.

[0011] b. There are at least 8 Zinc finger Pf proteins in Plasmodium ⁹ , some of which contain one essential histidine. These are involved in post- transcriptional regulation of protein levels and are thus very important in cellular growth and metabolism. These proteins are not present in the host erythrocyte in which no protein synthesis takes place (after maturation of the reticulocyte) and thus, if the drug penetrates into the infected cell, it will interrupt essential protein synthesis exclusively in the parasite.

[0012] c. Differential uptake of CTC-96 into infected erythrocytes.

In order for the drug to be effective it must be taken up by the Infected RBC. Pf produces two specific ion channels that are incorporated into the erythrocyte membrane. These increase the uptake of parasite-required nutrients ^{10- 11} . One of the two channels enables uptake of organic cations of up to the size of 1400 Da ¹² . Our drug is an organic cation with molecular size of 550 Da. Thus, the drug will most likely be taken up into infected cells much more efficiently than into uninfected cells that do not possess the special membrane channel.

Perspective

[0013] CTC-96 is a drug that can penetrate into Plasmodium infected erythrocytes and interact with a variety of histidine-rich proteins, Zn finger proteins and proteases that are parasite specific. The parasite-infected cells will serve as "sinks" for the CTC-96, which will then function as an anti-Plasmodium drug. This sink effect is hypothesized to be due to the preferential entrance of the drug into parasite- infected RBC; because the parasite in the infected cells contains histidine-rich and Zinc-finger proteins the drug will reach relatively high concentrations in these and not in uninfected RBC. (See Bδttcher, A.; Takeuchi, T.; Hardcastle, K.I.; Meade, TJ.,; Gray, H. B.,; Cwiker, D.,; Capon, M.; Dory, Z.; Spectroscopy and Electrochemistry of Cobalt(lll) Schiff Base Complexes, Inorganic Chemistry 1997, 36, 12; 2498-2504.;

Blum, O.; Haiek, A.; Wagner, J.D.; Cwikel, D.; Dori, Z.; Meade, T.J.; Gray, H.B.; An Anti-Herpes Metallodrug That Unfolds Proteins, 217™ ACS National Meeting Anaheim CA, March 1999; Blum, O.; Haiek, A.;Wagner, J.D,; Cwikel, D.; Dori, Z.; Meade, T.J.; Gray, H.B.; Isolation of a Myoglobin Molten Globule by Selective Cobalt(fll)-lnduced Unfolding; Pore. Natl. Acad. Sci. USA, Vol. 95. 6659-6662, June 1998.)

[0014] Very importantly, drug resistance of Plasmodium, Babesia and other related parasites is cardinal problem that renders malaria control at present very problematic. The resistance in the parasites develops through mutations in single proteins against which currently used drugs act. Because CTC 96 has a different mode of action and acts against multiple target proteins inside the parasite the development of resistance to this drug through mutations in all of the target proteins in a single parasite cell will be extremely unlikely. In addition, because the mode of action of CTC-96 is unique, it is expected to act against existing strains of Plasmodium which are refractory to currently used drugs and will help eradicate malaria.

[0015] Alt that was described above applies to genera of parasites belonging to the phylum Apicomplexa. These include Babesia, Theileria, Plasmodium, Toxoplasma, Eimeria, Neopora and Cryptosporidium. All of these are related protozoan parasites that cause serious, and in some cases, deadly diseases in humans and animals.

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