# Patent

## Title
Combination therapy and uses thereof for treatment and prevention of parasitic infection and disease

## Abstract

The invention relates to compounds, methods, uses, compositions, combinations, kits and packages for the prevention and/or treatment of parasite infection (e.g., Plasmodium parasites) and/or disease (e.g., malaria) based on uses of (a) cystamine, cysteamine, and analogs, derivatives, prodrugs, precursors thereof; an agent capable of inducing their production; and/or salts thereof, and (b) artemisinin and functional derivative, analog, conjugate, metabolite, prodrug or precursor thereof, and/or salts thereof.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to co-pending U.S. application Ser. No. 13/277,942, filed Oct. 20, 2011, and the benefit of U.S. provisional application Ser. No. 61/394,958, filed on Oct. 20, 2010; both of these prior applications are incorporated herein by reference in their entirety.


FIELD OF THE INVENTION
The present invention relates to the prevention and/or treatment of infectious diseases, and more particularly parasitic infection and disease, such as Plasmodium infection and associated disease such as malaria.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS AN ASCII TEXT FILE
A Sequence Listing is submitted herewith as an ASCII compliant text file named “Sequence_Listing.txt”, created on Jul. 14, 2014, and having a size of  ̃176 kilobytes, as permitted under 37 CFR 1.821(c). The material in the aforementioned file is hereby incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
Parasites are organisms that live on or within another organism (the host) and harm the host. Diseases caused by parasites such as protozoa and helminths are among the leading causes of death and disease in tropical and subtropical regions of the world.
Malaria is an infectious disease that causes severe morbidity and mortality with an estimated 300-500 million cases worldwide and more than 1 million deaths annually in sub-Saharan Africa alone. The disease is caused by protozoan parasites of the genus Plasmodium, transmitted by mosquitoes. The most serious forms of malaria are caused by Plasmodium falciparum and Plasmodium vivax, but other species (e.g., Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi) can also infect humans.
Among the murine malarial parasites, Plasmodium chabaudi (P. chabaudi) AS provides a unique experimental model to study the erythroid stage of the disease (Li, C. et al., 2001. Med. Microbiol. Immunol. (Berl) 189:115-126). P. chabaudi AS produces an infection in mice that shares many similarities with P. falciparum malaria in humans, including anemia, splenomegaly, hepatomegaly, renal alterations, hypoglycemia, and parasite sequestration (Cox, J. et al., 1987. Parasite Immunol. 9:543-561; Landau, I. and Gautret, P. 1998. Animal models: rodents. In Malaria, Parasite Biology, Pathogenesis, and Protection. I. W. Sherman, editor ASM Press, Washington D.C., pages 401-417). Among the murine malarial parasites, Plasmodium berghei (P. berghei) ANKA provides a unique model to study the cerebral stage of the disease (Hunt, N. H. et al., 2006 Int. J. Parasitol 36: 569-582). P. berghei ANKA produces an infection in mice that shares many similarities with cerebral malaria in humans, including sequestration of infected erythrocytes at the blood brain barrier, and appearance of cerebral symptoms such as fever, tremors, paralysis, coma and death.
In humans, malaria provides a clear example of host genetic factors influencing onset, progression, type of disease developed and ultimate outcome of infection (Hill, A. V. 1998. Annu. Rev. Immunol. 16: 593-617). Epidemiological data, together with linkage and association studies have shown that selection pressure from the parasite has caused retention of disease-associated but malaria-protective alleles in the human population, suggesting co-evolution of the host and parasite. Such otherwise deleterious alleles include those causing sickle cell anemia (Allison, A. C. 1954. Br. Med. J. 1(4857): 290-294; Willcox, M. A. et al., 1983. Ann. Trop. Med. Parasitol. 77: 239-246), thalassemias (Weatherall, D. J. 2001. Nat. Rev. Genet. 2: 245-255), and glucose-6-phosphate dehydrogenase deficiency (Ruwende, C. et al., 1995. Nature 376: 246-249). Polymorphisms in other erythroid proteins, including common variants of the Duffy antigen (Miller, L. H. et al., 1976. N. Engl. J. Med. 295: 302-304), the erythrocyte band 3 (anion exchanger) (Allen, S. J. et al., 1999. Am. J. Trop. Med. Hyg. 60: 1056-1060), and glycophorin C (Patel, S. S., et al., 2001. Blood 98:3489-3491), as well as variants in the TNF-α cytokine (McGuire, W. et al., 1994. Nature 371:508-510) and the CD36 scavenger receptor (Aitman, T. J. et al., 2000. Nature 405: 1015-1016) are also associated with protection against malaria. Additional linkage studies in Burkina Faso have suggested a complex genetic component of susceptibility showing blood parasitemia levels linked to the 5q31-q33 region (Rihet, P. et al., 1998. Am. J. Hum. Genet. 63: 498-505). The genetic component of malaria susceptibility is further modified by environmental factors (Kwiatkowski, D. 2000. Curr. Opin. Genet. Dev. 10: 320-324).
No efficacious vaccines are currently available to prevent or control the spread of parasitic diseases such as malaria, and most existing therapeutics are either not completely effective or toxic to the human host. Also, drugs often fail as a result of the selection and spread of drug resistant variants of the parasites. Notably, control of malaria has been hampered by the spread of drug resistance in both the Plasmodium parasites and the Anopheles insect vector, and by the lack of an efficacious vaccine (Moorthy, V. S. et al., 2004. Lancet 363: 150-156).
Therefore, there is a need to develop new approaches for the prevention and/or treatment of parasitic diseases such as malaria.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
SUMMARY OF THE INVENTION
The present invention relates to decreasing susceptibility to parasite infection or disease or to preventing or treating parasite infection or disease.
Accordingly, in a first aspect, the present invention provides a method for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease, in a subject, said method comprising administering to said subject an effective amount of (i) (a) cystamine; (b) cysteamine; (c) an agent capable of inducing the production of (a) or (b); (d) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of any of (a) to (c); (e) a pharmaceutically acceptable salt of any of (a) to (d); or (f) any combination of (a) to (e); and (ii) an artemisinin-related compound.
In another aspect, the present invention provides a method for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease, in a subject, said method comprising administering to said subject an effective amount of (i) (a) a compound of formula I: NH2—CH2—CH2—S—R (I), wherein R is H or S—CH2—CH2—NH2; (b) an agent capable of inducing the production of the compound of formula I; (c) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of (a) or (b); (d) a pharmaceutically acceptable salt of any of (a) to (c); or (e) any combination of (a) to (d); and (ii) an artemisinin-related compound.
In an embodiment, the above-mentioned method comprises administering to said subject an effective amount of (i) (a) cystamine; (b) cysteamine; (c) a pharmaceutically acceptable salt of (a) or (b); or (d) any combination of (a) to (c); and (ii) an artemisinin-related compound.
In an embodiment, the above-mentioned method comprises administering to said subject an effective amount of (i) cysteamine or a pharmaceutically acceptable salt thereof and (ii) (a) an artemisinin-related compound.
In various embodiments, the method results in reduced levels of parisitemia, delay in peak levels of parasitemia, or reduced severity of infection compared to treatment with cystamine, cysteamine, a derivative or pharmaceutically acceptable salt thereof or an artemisinin-related compound alone.
In another aspect, the present invention provides a use of (i) (a) cystamine; (b) cysteamine; (c) an agent capable of inducing the production of (a) or (b); (d) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of any of (a) to (c); (e) a pharmaceutically acceptable salt of any of (a) to (d); or (f) any combination of (a) to (e); and (ii) an artemisinin-related compound, for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease.
In another aspect, the present invention provides a use of (i) (a) cystamine; (b) cysteamine; (c) an agent capable of inducing the production of (a) or (b); (d) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of any of (a) to (c); (e) a salt of any of (a) to (d); or (f) any combination of (a) to (e); and (ii) an artemisinin-related compound, for the preparation of a medicament for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease.
In another aspect, the present invention provides a use of (i) (a) a compound of formula I: NH2—CH2—CH2—S—R (I), wherein R is H or S—CH2—CH2—NH2; (b) an agent capable of inducing the production of the compound of formula I; (c) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of (a) or (b); (d) a pharmaceutically acceptable salt of any of (a) to (c); or (e) any combination of (a) to (d); and (ii) an artemisinin-related compound, for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease.
In another aspect, the present invention provides a use of (i) (a) a compound of formula I: NH2—CH2—CH2—S—R (I), wherein R is H or S—CH2—CH2—NH2; (b) an agent capable of inducing the production of the compound of formula I; (c) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of (a) or (b); (d) a pharmaceutically acceptable salt of any of (a) to (c); or (e) any combination of (a) to (d); and (ii) an artemisinin-related compound, for the preparation of a medicament for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease.
In an embodiment, the above-mentioned use is of (i) (a) cystamine; (b) cysteamine; (c) a pharmaceutically acceptable salt of any of (a) or (b); or (d) any combination of (a) to (c); and (ii) (a) artemisinin, (b) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of artemisinin, (c) a pharmaceutically acceptable salt of (a) or (b), or (d) any combination of (a) to (c).
In another embodiment, the above-mentioned use is of (i) cysteamine or a pharmaceutically acceptable salt thereof; and (ii) an artemisinin-related compound.
In another aspect, the present invention provides a package for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease, said package comprising (i) (a) cystamine; (b) cysteamine; (c) an agent capable of inducing the production of (a) or (b); (d) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of any of (a) to (c); (e) a salt of any of (a) to (d); or (f) any combination of (a) to (e); and (ii) an artemisinin-related compound.
In another aspect, the present invention provides a package for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease, said package comprising (i) (a) a compound of formula I: NH2—CH2—CH2—S—R (I), wherein R is H or S—CH2—CH2—NH2; (b) an agent capable of inducing the production of the compound of formula I; (c) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of (a) or (b); (d) a pharmaceutically acceptable salt of any of (a) to (c); or (e) any combination of (a) to (d); and (ii) an artemisinin-related compound.
In another aspect, the present invention provides a package comprising (i) (a) a compound of formula I: NH2—CH2—CH2—S—R (I), wherein R is H or S—CH2—CH2—NH2; (b) an agent capable of inducing the production of the compound of formula I; (c) a functional derivative, analog, conjugate, prodrug or precursor of (a) or (b); (d) a pharmaceutically acceptable salt of any of (a) to (c); or (e) any combination of (a) to (d); and (ii) an artemisinin-related compound, for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease in a subject.
In an embodiment, the above-mentioned i) and ii) are packaged separately.
In another embodiment, the above-mentioned i) and ii) are packaged in the same formulation.
In an embodiment, the above-mentioned compound i) is present in a delayed release composition.
In another embodiment, the above-mentioned package further comprises labels and instructions for use.
In another aspect, the present invention provides a package comprising (i) a plurality of doses of a compound of formula I: NH2—CH2—CH2—S—R (I), wherein R is H or S—CH2—CH2—NH2; (b) an agent capable of inducing the production of the compound of formula I; (c) a functional derivative, analog, conjugate, prodrug or precursor of (a) or (b); (d) a pharmaceutically acceptable salt of any of (a) to (c); or (e) any combination of (a) to (d); and (ii) a plurality of doses of an artemisinin-related compound, for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease in a subject.
In an embodiment, the above-mentioned (i) and (ii) are packaged separately.
In another embodiment, the above-mentioned (i) and (ii) are packaged together.
In an embodiment, the above-mentioned package comprises (i) (a) cystamine; (b) cysteamine; (c) a pharmaceutically acceptable salt of (a) or (b); or (d) any combination of (a) to (c) and (ii) an artemisinin-related compound.
In a further embodiment, the above-mentioned package comprises (i) cysteamine or a pharmaceutically acceptable salt thereof; and (ii) an artemisinin-related compound.
In another embodiment, the above-mentioned package further comprises instructions for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease.
In another aspect, the present invention provides a package for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease, said package comprising (i) (a) cystamine; (b) cysteamine; (c) an agent capable of inducing the production of (a) or (b); (d) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of any of (a) to (c); (e) a salt of any of (a) to (d); or (f) any combination of (a) to (e); and (ii) instructions for using (i) in combination with an artemisinin-related compound, for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease.
In another aspect, the present invention provides a package for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease, said package comprising (i) (a) a compound of formula I: NH2—CH2—CH2—S—R (I), wherein R is H or S—CH2—CH2—NH2; (b) an agent capable of inducing the production of the compound of formula I; (c) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of (a) or (b); (d) a pharmaceutically acceptable salt of any of (a) to (c); or (e) any combination of (a) to (d); and (ii) instructions for using (i) in combination with an artemisinin-related compound, for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease.
In another aspect, the present invention provides a package for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease, said package comprising (i) an artemisinin-related compound; and (ii) instructions for using (i) in combination with (a) cystamine; (b) cysteamine; (c) an agent capable of inducing the production of (a) or (b); (d) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of any of (a) to (c); (e) a salt of any of (a) to (d); or (f) any combination of (a) to (e); for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease.
In another aspect, the present invention provides a package for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease, said package comprising (i) an artemisinin-related compound; and (ii) instructions for using (i) in combination with (a) a compound of formula I: NH2—CH2—CH2—S—R (I), wherein R is H or S—CH2—CH2—NH2; (b) an agent capable of inducing the production of the compound of formula I; (c) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of (a) or (b); (d) a pharmaceutically acceptable salt of any of (a) to (c); or (e) any combination of (a) to (d); for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease.
In another aspect, the present invention provides a composition for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease, said composition comprising: (i) (a) cystamine; (b) cysteamine; (c) an agent capable of inducing the production of cysteamine; (d) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of any of (a) to (c); (e) a pharmaceutically acceptable salt of any of (a) to (d); or (f) any combination of (a) to (e); and (ii) an artemisinin-related compound.
In another aspect, the present invention provides a composition for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease, in a subject, said composition comprising (i) (a) a compound of formula I: NH2—CH2—CH2—S—R (I), wherein R is H or S—CH2—CH2—NH2; (b) an agent capable of inducing the production of the compound of formula I; (c) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of (a) or (b); (d) a pharmaceutically acceptable salt of any of (a) to (c); or (e) any combination of (a) to (d); and (ii) an artemisinin-related compound.
In an embodiment, the above-mentioned composition comprises (i) (a) cystamine; (b) cysteamine; (c) a pharmaceutically acceptable salt of (a) or (b); or (d) any combination of (a) to (c); and (ii) an artemisinin-related compound.
In a further embodiment, the above-mentioned composition comprises (i) cysteamine or a pharmaceutically acceptable salt thereof; and (ii) an artemisinin-related compound.
In an embodiment, the above-mentioned composition further comprises a pharmaceutically acceptable carrier or excipient.
In another aspect, the present invention provides a combination comprising: (i) (a) cystamine; (b) cysteamine; (c) an agent capable of inducing the production of cysteamine; (d) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of any of (a) to (c); (e) a pharmaceutically acceptable salt of any of (a) to (d); or (f) any combination of (a) to (e); and (ii) (a) an artemisinin-related compound; for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease.
In another aspect, the present invention provides a combination comprising (i) (a) a compound of formula I: NH2—CH2—CH2—S—R (I), wherein R is H or S—CH2—CH2—NH2; (b) an agent capable of inducing the production of the compound of formula I; (c) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of (a) or (b); (d) a pharmaceutically acceptable salt of any of (a) to (c); or (e) any combination of (a) to (d); and (ii) an artemisinin-related compound; for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease.
In another aspect, the present invention provides (i) (a) cystamine; (b) cysteamine; (c) an agent capable of inducing the production of cysteamine; (d) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of any of (a) to (c); (e) a pharmaceutically acceptable salt of any of (a) to (d); or (f) any combination of (a) to (e); or (ii) a composition comprising (i) and a pharmaceutically acceptable carrier; for use in combination with (iii) an artemisinin-related compound; or (iv) a composition comprising (iii) and a pharmaceutically acceptable carrier; for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease.
In another aspect, the present invention provides (i) (a) a compound of formula I: NH2—CH2—CH2—S—R (I), wherein R is H or S—CH2—CH2—NH2; (b) an agent capable of inducing the production of the compound of formula I; (c) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of (a) or (b); (d) a pharmaceutically acceptable salt of any of (a) to (c); or (e) any combination of (a) to (d); or (ii) a composition comprising (i) and a pharmaceutically acceptable carrier; for use in combination with (iii) an artemisinin-related compound; or (iv) a composition comprising (iii) and a pharmaceutically acceptable carrier; for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease.
In another aspect, the present invention provides (i) an artemisinin-related compound; or (ii) a composition comprising (i) and a pharmaceutically acceptable carrier; for use in combination with (iii) (a) cystamine; (b) cysteamine; (c) an agent capable of inducing the production of cysteamine; (d) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of any of (a) to (c); (e) a pharmaceutically acceptable salt of any of (a) to (d); or (f) any combination of (a) to (e) or (iv) a composition comprising (iii) and a pharmaceutically acceptable carrier; for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease.
In another aspect, the present invention provides (i) an artemisinin-related compound; or (ii) a composition comprising (i) and a pharmaceutically acceptable carrier; for use in combination with (iii) (a) a compound of formula I: NH2—CH2—CH2—S—R (I), wherein R is H or S—CH2—CH2—NH2; (b) an agent capable of inducing the production of the compound of formula I; (c) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of (a) or (b); (d) a pharmaceutically acceptable salt of any of (a) to (c); or (e) any combination of (a) to (d); or (iv) a composition comprising (iii) and a pharmaceutically acceptable carrier; for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease.
The present invention further provides a combination for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease, said combination comprising: (i) (a) cystamine; (b) cysteamine; (c) an agent capable of inducing the production of cysteamine; (d) a functional derivative, analog, conjugate, prodrug or precursor of any of (i) to (iii); (e) a pharmaceutically acceptable salt of any of (a) to (d); or (f) any combination of (a) to (e); and (ii) an artemisinin-related compound.
The present invention further provides a combination for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease, in a subject, said combination comprising (i) (a) a compound of formula I: NH2—CH2—CH2—S—R (I), wherein R is H or S—CH2—CH2—NH2; (b) an agent capable of inducing the production of the compound of formula I; (c) a functional derivative, analog, conjugate, prodrug or precursor of (a) or (b); (d) a pharmaceutically acceptable salt of any of (a) to (c); or (e) any combination of (a) to (d); and (ii) (a) an artemisinin-related compound.
In embodiments, the above-mentioned combination comprises: (i) (a) cystamine; (b) cysteamine; (c) a pharmaceutically acceptable salt of (a) or (b); or (d) any combination of (a) to (c); and (ii) an artemisinin-related compound.
In embodiments, the above-mentioned combination comprises: (a) cysteamine or a pharmaceutically acceptable salt thereof; and (b) an artemisinin-related compound.
In an embodiment, the above-mentioned artemisinin-related compound is (a) artemisinin, (b) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of artemisinin, (c) a pharmaceutically acceptable salt of (a) or (b), or (d) any combination of (a) to (c).
In an embodiment, the above-mentioned artemisinin derivative is artesunate. In another embodiment, the above-mentioned artemisinin metabolite is dihydroartemisinin.
In another embodiment, the above-mentioned agent capable of inducing the production of cystamine or cysteamine is (a) a pantetheinase polypeptide, (b) a fragment or variant of (a) having pantetheinase activity; (c) a nucleic acid encoding the polypeptide of (a) or (b), (d) an agent capable of increasing pantetheinase activity or expression, or (e) any combination of (a) to (d).
In an embodiment, the above-mentioned polypeptide comprises the amino acid sequence of SEQ ID NO: 6, 8, 10, 12, 14 or 16.
In another embodiment, the above-mentioned nucleic acid comprises a nucleotide sequence which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 6, 8, 10, 12, 14 or 16. In a further embodiment, the above-mentioned nucleic acid comprises the coding sequence of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 18 or 19.
In an embodiment, the above-mentioned (i) and (ii) are packaged separately.
In another embodiment, the above-mentioned (i) and (ii) are packaged together.
In an embodiment, the above-mentioned compounds i) and ii) act synergistically.
In an embodiment, the above-mentioned synergy results in use of effective doses of compound i) and/or ii) that are lower than doses administered when the compounds are administered in the absence of the other composition.
In an embodiment, the above-mentioned effective dose of compound (i) is lower than a dose of (i) administered in the absence of compound (ii).
In an embodiment, the above-mentioned effective dose of compound (ii) is lower than a dose of (ii) administered in the absence of compound (i).
In an embodiment, the above-mentioned effective dose of (i) and (ii) are lower than a dose of compound (i) or compound (ii) administered in the absence of the other composition.
In an embodiment, the dose of compound (i) and/or (ii) is suboptimal.
In an embodiment, the above-mentioned effective dose of compound (i) is in the range of 1 to 500 mg/kg.
In an embodiment, the above-mentioned compound (i) is present in a delayed release composition.
In an embodiment, the peak level of parisitemia is reduced.
In an embodiment, the above-mentioned administering prevents parisitemia.
In an embodiment, the above-mentioned compound (i) is administered less than four times a day.
In an embodiment, the above-mentioned compound (i) is administered twice daily.
In an embodiment, the above-mentioned compounds (i) and (ii) are administered coextensively.
In an embodiment, the above-mentioned parasite is of the genus Plasmodium. In a further embodiment, the above-mentioned parasite is Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi. 
In another embodiment, the above-mentioned disease is malaria. In a further embodiment, the above-mentioned malaria is blood-stage malaria or cerebral malaria.
In an embodiment, the above-mentioned subject is a mammal. In a further embodiment, the above-mentioned mammal is a human.
Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.



BRIEF DESCRIPTION OF DRAWINGS
In the appended drawings:
FIGS. 1A and 1B show the effect of cysteamine on replication of Plasmodium chabaudi in vivo. FIG. 1A: plasma levels of cysteamine-free base (measured by HPLC) following either intraperitoneal (i.p.) or subcutaneous (s.c.) injections (120 mg/kg) were measured in 3 mice and used to calculate Cmax and AUC pharmacokinetic parameters (see text). Error bars indicate standard deviation from the mean. FIG. 1B: A/J female mice were infected with P. chabaudi (105 pRBC i.v.) and treated daily (either s.c. or i.p.) with cysteamine (120 mg/kg) starting at day 1 to day 10. Blood parasitemia was monitored on days 5, 6, and 7 and is plotted. The % inhibition of parasite replication was calculated by comparison to the blood parasitemia measured in PBS-treated controls and is indicated below the graphs. Each dot represents a mouse. Levels of statistical significance are represented by asterisks; ***, P<0.01; **, P<0.05 (compared to PBS control group);
FIGS. 2A and 2B show the effect of cysteamine dosing used for treatment of cystinosis on replication of Plasmodium chabaudi in vivo. FIG. 2A: the plasma levels of cysteamine-free base (measured by HPLC) following subcutaneous (s.c.) injection (50 mg/kg) were measured in 3 mice and used to calculate Cmax and AUC pharmacokinetic parameters (see text). Error bars indicate standard deviation from the mean. FIG. 2B: A/J female mice were infected with P. chabaudi (105 pRBC i.v.) and treated daily with cysteamine (s.c.) from day 1 to day 10, with the indicated dosing: 1×150 mg/kg, 3×50 mg/kg, or 4×50 mg/kg, given at 1 or 2 h intervals. Blood parasitemia was monitored on days 5, 6, and 7 and is plotted. The % inhibition of parasite replication was calculated by comparison to the blood parasitemia measured in PBS-treated controls and is indicated below the graphs. Each dot represents a mouse. Levels of statistical significance are represented by asterisks; ***, P<0.01 (compared to PBS control group);
FIGS. 3A to 3C show the synergistic effect of cysteamine on artemisinin efficacy against replication of Plasmodium chabaudi in vivo. Groups (n=6) of female A/J (FIGS. 3A and B) or C57BL/6 (FIG. 3C) mice were infected with P. chabaudi (107 pRBC, i.v.) and treated for 4 days (days 0, 1, 2, and 3) with indicated doses (in mg/kg) of artesunate (FIGS. 3A and C) or dihydroartemisinin (DHA) (FIG. 3B) and/or cysteamine (170 mg/kg, i.p.), and blood parasitemia (expressed as percentage of parasitized erythrocytes) was determined at days 4 (left) and 5 (right) post infection. In all experiments, control groups were treated with PBS. The presence or absence of cysteamine is indicated by a plus or a minus, respectively, and doses of artemisinin derivatives in mg/kg are indicated below the plots. Each dot represents a mouse and bars indicate the mean of the group;
FIGS. 4A to 4C show the dose-dependent synergistic effect of cysteamine on artemisinin efficacy against replication of Plasmodium chabaudi in vivo. Groups (n=6) of female A/J mice were infected with P. chabaudi (107 pRBC, i.v.) and treated for 4 days (days 0, 1, 2, and 3) with increasing doses (indicated) of artesunate (FIG. 4C) and/or cysteamine (FIGS. 4A and B) given i.p. Blood parasitemia was determined at days 4 and 5 post-infection, and the inhibitory effects of the different drug treatments on blood-stage P. chabaudi replication were calculated for each animal compared to the mean of PBS-treated controls (expressed as a percentage). The presence or absence of drug is indicated by a plus or minus, respectively, and all doses are in mg/kg. Error bars represent standard error of the mean;
FIGS. 5A to 5D show the effect of cysteamine and artesunate combinations on progression and resolution of P. chabaudi infection in vivo. Groups (n=6) of female A/J mice were infected with P. chabaudi (106 pRBC, i.v.) and treated for 4 days (days 0, 1, 2, and 3) with PBS (FIG. 5A), cysteamine (60 mg/kg, FIG. 5A), or cysteamine (60 mg/kg) combined with increasing doses of artesunate (0.5, 1.0, 5, or 10 mg/kg, FIG. 5B), all given i.p. Blood parasitemia was measured daily up to day 20 (expressed as percentage of pRBC), and death was recorded (indicated by a cross). Solid and dashed lines represent mice receiving artesunate doses alone or in combination with cysteamine, respectively; artesunate doses are depicted by the abbreviations “Art0.5”, “Art1”, “Art5”, and “Art10”, as indicated. Error bars represent standard deviation of the mean, and arrows represent drug treatment days. FIG. 5C: Kaplan-Meier survival plot for experimental treatment groups for which lethality was observed. Depiction of artesunate doses and dashed versus solid lines are as described for FIG. 5B. FIG. 5D: Parasitemia levels at day 6 post-infection for all experimental groups are shown, with each dot representing a mouse. Mean levels are shown as bars;
FIGS. 6A and 6B show the effect of cysteamine and artesunate combinations on progression of P. chabaudi in pantetheinase-sufficient B6 mice. Groups (n=6) of female B6 mice were infected with P. chabaudi (106 pRBC, i.v.) and treated for 4 days (days 0, 1, 2, and 3) with either PBS or artesunate (1.0 or 30 mg/kg) combined with, or without, cysteamine (60 mg/kg, FIG. 6A), all given i.p. Blood parasitemia was measured daily up to day 22 (expressed as percentage of pRBC). Solid and dashed lines represent mice receiving artesunate doses alone or in combination with cysteamine, respectively. Error bars represent standard deviation of the mean, and arrows represent drug treatment days. FIG. 6B: Parasitemia levels at day 6 post-infection for all experimental groups are shown, with each dot representing a mouse. Mean levels are shown as bars;
FIGS. 7A to 7Z show the nucleotide and amino acid sequences of murine and human pantetheinase (Vanin, Vnn) genes and polypeptides;
FIGS. 8A and 8B show the results of two independent experiments on the effect of cysteamine and artesunate combinations on the progression of Plasmodium berghei ANKA infection (parasitemia). Groups of 5 adult 18-20 g C57BL/6J males and females were infected intravenously with 1×106 erythrocytes parasitized with P. berghei ANKA at time “0”. Two hours later, mice were injected i.p with either Artemisinin alone or with Artemisinin/Cysteamine combinations (at the indicated concentrations in mg/kg body weight). In the case of the latter, Artemisinin was injected first in one quadrant, and cysteamine was injected second, 10-15 minutes later in another quadrant. The drug treatment was further repeated at days 1, 2 and 3 post-infection to emulate the standard 4-day test used in anti-malarial drug discovery. Starting at day 5, blood was collected, thin blood smears were prepared, and parasitemia was determined (400 erythrocytes counted, expressed as percentage parasitized erythrocytes). Error bars show standard deviations on the mean; and
FIGS. 9A and 9B show the results of two independent experiments on the effect of cysteamine and artesunate combinations on the survival of Plasmodium berghei ANKA-infected mice. Infection and drug administration were performed as described above for FIGS. 8A and 8B. Animals were monitored for the appearance and severity of cerebral symptoms (CM phase, shown as rectangle in graph), and moribund animals were euthanized, and time of death was recorded.



DISCLOSURE OF THE INVENTION
Described herein are studies using the mouse model system of Plasmodium infection which show that treatment of mice with a combination of cysteamine and artemisinin-related compounds (e.g., the artemisinin derivative artesunate and the artemisinin metabolite dihydroartemisinin) leads to a synergistic reduction in parasitemia in these mice and to an increase in survival.
Cysteamine (C2H7NS, CAS#60-23-1) has the following chemical formula: NH2—CH2—CH2—SH
It is often used as a salt, such as the hydrochloride salt, C2H8ClNS (CAS#156-57-0), which has the following formula: −Cl+NH3—CH2—CH2—SH
Cystamine (C4H12N2S2) is the oxidized form of cysteamine (i.e., a dimer of cysteamine) and has the following chemical formula: NH2—CH2—CH2—S—S—CH2—CH2—NH2 
Cystamine may also be in the form of a salt, such as a dihydrochloride salt (CAS #56-17-7) or phosphate salt (CAS#3724-89-8).
As such, a compound of formula I: NH2—CH2—CH2—S—R (I) wherein R is H or S—CH2—CH2—NH2; an agent capable of inducing the production of the compound of formula I; a functional derivative, analog, conjugate, prodrug or precursor of the compound of formula I; or salts (e.g., pharmaceutically acceptable salts) thereof, are also useful in the methods, uses, and compositions of the present invention.
Cysteamine, and more particularly the bitartrate salt thereof (commercialized under the trade name CystagonTM) has been approved for the pharmacological management of cystinosis, an autosomal recessive disorder caused by mutations in the lysosomal cystine carrier cystinosin (encoded by the CTNS gene), whose absence leads to intracellular cystine crystals, widespread cellular destruction, renal Fanconi syndrome in infancy, renal glomerular failure in later childhood and other systemic complications (Kleta R. and Gahl W. A., 2004. Expert Opin. Pharmacother. 5(11): 2255-2262).
Cysteamine is a metabolite (product) generated by pantetheinase enzymatic activity. Pantetheinase (EC 3.5.1.92) is a ubiquitous enzyme encoded by the Vanin genes (FIG. 3, SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, and 17-19); 2 genes in mice (Vanin-1 and -3) and 3 genes in human (Vanin-1, -2 and -3). It is an amidohydrolase that hydrolyzes pantetheine (which is a metabolic product of Coenzyme A (CoA) degradation) to pantothenic acid (also called pantothenate or vitamin B5) and cysteamine.
Artemisinin (CAS#63968-64-9) is a sesquiterpene lactone which was first isolated from the plant Artemisia annua, and has the following formula:




An active metabolite of artemisinin and artemisinin-related compounds is dihydroartemisinin (CAS#71939-50-9), which has the following formula:




Accordingly, in an aspect, the present invention provides a method for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease, in a subject, said method comprising administering to said subject an effective amount of (i) (a) cystamine; (b) cysteamine; (c) an agent capable of inducing the production of (a) or (b); (d) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of any of (a) to (c); (e) a salt of any of (a) to (d); or (f) any combination of (a) to (e); and (ii) an artemisinin-related compound.
The present invention further provides a method for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease in a subject (an animal such as a mammal, in a further embodiment a human), said method comprising administering to said subject an effective amount of (i) (a) a compound of formula I:NH2—CH2—CH2—S—R (I) wherein R is H or S—CH2—CH2—NH2; (b) an agent capable of inducing the production of the compound of formula I; (c) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of (a) or (b); (d) a pharmaceutically acceptable salt of any of (a) to (c); or (e) any combination of (a) to (d) and (ii) an artemisinin-related compound.
In another aspect, the invention provides a use of (i) (a) cystamine; (b) cysteamine; (c) an agent capable of inducing the production of cysteamine and/or cystamine; (d) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of (a) to (c); (e) a pharmaceutically acceptable salt of any of (a) to (d); or (f) any combination of (a) to (e); and (ii) an artemisinin-related compound, for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease; or for the preparation of a medicament for decreasing susceptibility to parasitic infection or disease or for preventing or treating parasite infection or disease.
In another aspect, the invention provides a combination of (i) (a) cystamine; (b) cysteamine; (c) an agent capable of inducing the production of cysteamine and/or cystamine; (d) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of (a) to (c); (e) a pharmaceutically acceptable salt of any of (a) to (d); or (f) any combination of (a) to (e); and (ii) an artemisinin-related compound, for use in decreasing susceptibility to parasite infection or disease or for use in preventing or treating parasite infection or disease; or for use in the preparation of a medicament for decreasing susceptibility to parasitic infection or disease or for use in preventing or treating parasite infection or disease.
In an embodiment, the above-mentioned parasite infection is an infection of a parasite of the genus Plasmodium. In an embodiment, the above-mentioned Plasmodium parasite is an artemisinin-resistant human Plasmodium parasite.
In an embodiment, the above-mentioned disease is malaria. In a further embodiment, the above-mentioned malaria is blood-stage malaria. In another embodiment, the above-mentioned malaria is cerebral malaria.
Accordingly, the invention further provides a method for treating or preventing malaria in an animal, comprising administering to the animal (i) a cysteamine-related compound and (ii) an artemisinin-related compound.
As used herein, the term “cysteamine-related compound” refers to cysteamine and functional derivatives, analogs, conjugates, prodrugs or precursors of cysteamine and various cysteamine salts (such as cysteamine hydrochloride, cysteamine salicylate, cysteamine phosphate and cysteamine bitartrate [CystagonTM]). Also included within the scope of the subject invention are analogs, derivatives, conjugates, metabolites, prodrugs and precursors of cysteamine (such as cystamine, the oxidized form of cysteamine, cysteine, and the like), which have the ability, as described herein, to prevent and/or treat and/or decrease the susceptibility to parasite infections, such as infection by a Plasmodium parasite (e.g., P. falciparum infection) and/or to prevent and/or treat associated disease (e.g., malaria), and more particularly to act synergistically with artemisinin and artemisinin-related compounds. Various analogs, derivatives, conjugates, prodrugs and metabolites of cysteamine are known and include, for example, compounds, compositions, formulations and methods of delivery as set forth in U.S. Pat. Nos. 6,521,266; 6,468,522; 6,340,746; 5,714,519 and 5,554,655 and PCT publication No. WO 2007/089670.
As used herein, the term “artemisinin-related compound” refers to artemisinin and to functional derivatives, analogs, conjugates, metabolites, prodrugs or precursors of artemisinin, as well as salts thereof, and includes the artemisinin derivatives/analogs artesunate, artemether, arteether, artelinic acid, artenimol and artemotil, the artemisinin precursor artemisinic acid (Ro D K, et al., Nature 440:940-943), as well as the artemisinin metabolite dihydroartemisinin. Also included within the scope of the subject invention are analogs, derivatives, conjugates, metabolites, prodrugs and precursors of artemisinin which have the ability, as described herein, to prevent and/or treat and/or decrease the susceptibility to parasite infections, such as infection by a Plasmodium parasite (e.g., P. falciparum infection) and/or to prevent and/or treat associated disease (e.g., malaria), and more particularly to act synergistically with cysteamine and cysteamine-related compounds. Also included within the scope of the subject invention are analogs, derivatives, conjugates, metabolites, prodrugs and precursors of artemisinin which have the ability which may be metabolized into a biologically active metabolite of artemisinin (e.g., dihydroartemisinin), as well as synthetic trioxolanes (mimicking the trioxolane structure of artemisinin) such as those described in Vennerstrom et al., 2004, Nature 430, 900-904 (Arterolane) and O'Neill et al., Angewandte Chemie International Edition, 2010, 49(33): 5693-97. Various functional analogs, derivatives, conjugates, prodrugs and metabolites of artemisinin, as well as methods to produce them, are described, for example, in Posner et al., 1999, J. Med. Chem. 42(2): 300-304, Li et al., 2000, J. Med. Chem. 43(8): 1635-1640, Li et al., 2003, Bioorganic & Medicinal Chemistry 11(20): 4363-4368, Ploypradith P, 2004. Acta Trop 89:329-342, PCT publications No. WO/2008/127381, WO/2008/046109, WO/2007/116135, WO/2007/009388, WO/2003/076446, WO/2000/042046, WO/2000/004025, WO/2000/004024, WO/1999/065914 and WO/1991/014689. Artemisinin-related compounds have been shown to be active against a variety of parasites including Plasmodium parasites, Toxoplasma parasites, Schistosoma parasites and helminths and (Dunay I R, et al., 2009, Antimicrob Agents Chemother 53:4450-4456; Keiser J, Utzinger J (2007) Curr Opin Infect Dis 20:605-612; Sissoko M S et al., (2009) PLoS One 4:e6732).
Methods to isolate and/or produce artemisinin and/or artemisinin-related compounds are well known in the art. Methods to produce/isolate artemisinin from tissue culture or whole plant of Artemisia annua are described, for example, in Liu et al., 2006, Appl Microbiol Biotechnol. 72(1):11-20, Epub 2006 Jun. 3. The synthesis of artemisinin may also be performed using basic organic reagents, for example using the methods described in Schmid and Hofheinz, J. Am. Chem. Soc. (1983) 105(3): 624-625. The precursor of artemisinin, artemisinic acid, may for example be produced at high levels in an engineered Saccharomyces cerevisiae system (Ro D K et al., 2006, Nature 440(7086): 940-943). Methods to produce/synthesize various functional analogs, derivatives, conjugates, prodrugs and metabolites of artemisinin, are described, for example, in Posner et al., 1999, J. Med. Chem. 42(2): 300-304, Li et al., 2000, J. Med. Chem. 43(8): 1635-1640, Li et al., 2003, Bioorganic & Medicinal Chemistry 11(20): 4363-4368, PCT publications No. WO/2008/127381, WO/2008/046109, WO/2007/116135, WO/2007/009388, WO/2003/076446, WO/2000/042046, WO/2000/004025, WO/2000/004024, WO/1999/065914 and WO/1991/014689.
In an embodiment, the above-mentioned agent capable of inducing the production of cystamine, cysteamine, or a compound of formula I is (a) a pantetheinase polypeptide, (b) a fragment or variant of (a) having pantetheinase activity; (c) a nucleic acid encoding the polypeptide of (a) or (b), (d) an agent capable of increasing pantetheinase activity or expression, or (e) any combination of (a) to (d).
In an embodiment, the above-mentioned pantetheinase polypeptide comprises the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16 (FIG. 7), or a variant/fragment thereof having pantetheinase activity.
In an embodiment, the above-mentioned pantetheinase nucleic acid comprises (a) the coding sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 18 or 19 (FIG. 7); (b) a nucleotide sequence which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16; or (c) a fragment, variant or complement of (a) or (b) encoding a pantotheinase polypeptide.
The above-mentioned coding sequences correspond to: (a) nucleotides 22 to 1560 for SEQ ID NO: 1, (b) nucleotides 113-1615 for SEQ ID NO: 3, (c) nucleotides 15-1556 for SEQ ID NO: 5, (d) nucleotides 12-1574 for SEQ ID NO: 7, (e) nucleotides 113-1516 for SEQ ID NO: 9, (f) nucleotides 73-897 for SEQ ID NO: 11, (g) nucleotides 73-516 for SEQ ID NO: 13, (h) nucleotides 73-426 for SEQ ID NO: 15, (i) the junction of nucleotides 1959-2168, 4155-4278, 21806-22005, 22680-22971, 23411-23772, 31490-31660 and 32673-32855 for SEQ ID NO: 17, (j) the junction of nucleotides 2009-2221, 2346-2476, 3857-4049, 7144-7432, 8375-8748, 10028-10198 and 15403-15594 for SEQ ID NO: 18, and (k) the junction of nucleotides 1814-2026, 2123-2253, 7573-7765 and 9494-9781 for SEQ ID NO: 19 (FIGS. 7A to Z).
In another embodiment, the above-mentioned nucleic acid fragment or variant encodes a polypeptide having pantetheinase activity.
In an embodiment, the above-mentioned pantetheinase nucleic acid comprises the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 18 or 19 (FIGS. 7A to Z).
The increase of expression of a pantetheinase nucleic acid or encoded polypeptide or pantetheinase activity in cell or tissue of said subject may be achieved, for example, by administrating to a subject: (a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16, or a variant or fragment thereof having pantetheinase activity; (b) a nucleic acid molecule encoding pantetheinase or a functional variant thereof (e.g., a nucleic acid which encodes the polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16, or a variant or fragment thereof having pantetheinase activity) or (c) a composition (e.g., a pharmaceutical composition) comprising the above-mentioned polypeptide or nucleic acid and, for example, a pharmaceutically acceptable carrier/excipient.
A variant and/or fragment of pantetheinase which retains activity (e.g., having a domain conferring pantetheinase activity) may also be used in the uses and methods of the invention. Variants or homologs include protein sequences, which are substantially identical to the amino acid sequence of a pantetheinase (e.g., SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16), sharing significant structural and functional homology with a pantetheinase (e.g., SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16). Variants include, but are not limited to, proteins or peptides, which differ from a pantetheinase (e.g., SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16) by any modifications, and/or amino acid substitutions, deletions or additions. Modifications can occur anywhere including the polypeptide backbone, (i.e. the amino acid sequence), the amino acid side chains and the amino or carboxy termini. Such substitutions, deletions or additions may involve one or more amino acids. Fragments include a fragment or a portion of a pantetheinase or a fragment or a portion of a homolog or variant of a pantetheinase which retains pantetheinase activity. The pantetheinase polypeptide (or a variant or fragment thereof having pantetheinase activity) may also be fused with another polypeptide or conjugated to one or more molecules.
“Homology”, “homologous” and “homolog” refer to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing each position in the aligned sequences. A degree of homology between nucleic acid or between amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at positions shared by the sequences. As the term is used herein, a nucleic acid sequence is “homologous” to or is a “homolog” of another sequence if the two sequences are substantially identical and the functional activity of the sequences is conserved (as used herein, the term ‘homologous’ does not infer evolutionary relatedness). Two nucleic acids or amino acid sequences are considered “substantially identical” if, when optimally aligned (with gaps permitted), they share at least about 50% sequence similarity or identity, or if the sequences share defined functional motifs. In alternative embodiments, sequence similarity in optimally aligned substantially identical sequences may be at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, e.g., with any of SEQ ID NOs: 1-19. As used herein, a given percentage of homology between sequences denotes the degree of sequence identity in optimally aligned sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, though preferably less than about 25% identity, with any of SEQ ID NOs: 1-19.
Substantially complementary nucleic acids are nucleic acids in which the complement of one molecule is substantially identical to the other molecule. Two nucleic acid or protein sequences are considered substantially identical if, when optimally aligned, they share at least about 70% sequence identity. In alternative embodiments, sequence identity may for example be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, e.g., with any of SEQ ID NOs: 1-19. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, and the computerised implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis., U.S.A.). Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al., 1990, J. Mol. Biol. 215:403-10 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information. The BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold. Initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program may use as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10 (or 1 or 0.1 or 0.01 or 0.001 or 0.0001), M=5, N=4, and a comparison of both strands. One measure of the statistical similarity between two sequences using the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. In alternative embodiments of the invention, nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
An alternative indication that two nucleic acid sequences are substantially complementary is that the two sequences hybridize to each other under moderately stringent, or preferably stringent, more preferably highly stringent conditions. Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1% SDS at 42° C. (see Ausubel, et al. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3). Alternatively, hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (see Ausubel, et al. (eds), 1989, supra). Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, New York). Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
The above-mentioned nucleic acid may be delivered to cells in vivo using methods well known in the art such as direct injection of DNA, receptor-mediated DNA uptake, viral-mediated transfection or non-viral transfection and lipid based transfection, all of which may involve the use of gene therapy vectors. Direct injection has been used to introduce naked DNA into cells in vivo (see e.g., Acsadi et al. (1991) Nature 332: 815-818; Wolff et al. (1990) Science 247: 1465-1468). A delivery apparatus (e.g., a “gene gun”) for injecting DNA into cells in vivo may be used. Such an apparatus may be commercially available (e.g., from BioRad). Naked DNA may also be introduced into cells by complexing the DNA to a cation, such as polylysine, which is coupled to a ligand for a cell-surface receptor (see for example Wu, G. and Wu, C. H. (1988) J. Biol. Chem. 263: 14621; Wilson et al. (1992) J. Biol. Chem. 267: 963-967; and U.S. Pat. No. 5,166,320). Binding of the DNA-ligand complex to the receptor may facilitate uptake of the DNA by receptor-mediated endocytosis. A DNA-ligand complex linked to adenovirus capsids which disrupt endosomes, thereby releasing material into the cytoplasm, may be used to avoid degradation of the complex by intracellular lysosomes (see for example Curiel et al. (1991) Proc. Natl. Acad. Sci. USA 88: 8850; Cristiano et al. (1993) Proc. Natl. Acad. Sci. USA 90: 2122-2126).
Defective retroviruses are well characterized for use as gene therapy vectors (for a review see Miller, A. D. (1990) Blood 76: 271). Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art. Examples of suitable packaging virus lines include psiCrip, psiCre, psi2 and psiAm. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85: 6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85: 3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87: 6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88: 8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88: 8377-8381; Chowdhury et al. (1991) Science 254: 1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89: 7640-7644; Kay et al. (1992) Human Gene Therapy 3: 641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89: 10892-10895; Hwu et al. (1993) J. Immunol. 150: 4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573).
For use as a gene therapy vector, the genome of an adenovirus may be manipulated so that it encodes and expresses a nucleic acid compound of the invention (e.g., a pantetheinase nucleic acid), but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See for example Berkner et al. (1988) BioTechniques 6: 616; Rosenfeld et al. (1991) Science 252: 431-434; and Rosenfeld et al. (1992) Cell 68: 143-155. Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art. Recombinant adenoviruses are advantageous in that they do not require dividing cells to be effective gene delivery vehicles and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al. (1992) cited supra), endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad. Sci. USA 89: 6482-6486), hepatocytes (Herz and Gerard (1993) Proc. Natl. Acad. Sci. USA 90: 2812-2816) and muscle cells (Quantin et al. (1992) Proc. Natl. Acad. Sci. USA 89: 2581-2584).
Adeno-associated virus (AAV) may be used as a gene therapy vector for delivery of DNA for gene therapy purposes. AAV is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle (Muzyczka et al. Curr. Topics in Micro. and Immunol. (1992) 158:97-129). AAV may be used to integrate DNA into non-dividing cells (see for example Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7: 349-356; Samulski et al. (1989) J. Virol. 63: 3822-3828; and McLaughlin et al. (1989) J. Virol. 62: 1963-1973). An AAV vector such as that described in Tratschin et al. (1985) Mol. Cell. Biol. 5: 3251-3260 may be used to introduce DNA into cells (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81: 6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4: 2072-2081; Wondisford et al. (1988) Mol. Endocrinol. 2: 32-39; Tratschin et al. (1984) J. Virol. 51: 611-619; and Flotte et al. (1993) J. Biol. Chem. 268: 3781-3790). Lentiviral gene therapy vectors may also be adapted for use in the invention.
General methods for gene therapy are known in the art. See for example, U.S. Pat. No. 5,399,346 by Anderson et al. A biocompatible capsule for delivering genetic material is described in PCT Publication WO 95/05452 by Baetge et al. Methods of gene transfer into hematopoietic cells have also previously been reported (see Clapp, D. W., et al., Blood 78: 1132-1139 (1991); Anderson, Science 288: 627-9 (2000); and Cavazzana-Calvo et al., Science 288: 669-72 (2000)).
The present invention relates to the administration of (i) (a) cystamine; (b) cysteamine; (c) an agent capable of inducing the production of cysteamine and/or cystamine; (d) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of (a) to (c); (e) a pharmaceutically acceptable salt of any of (a) to (d); or (f) any combination of (a) to (e); and (ii) an artemisinin-related compound, to elicit any of the effects discussed above. The (a) cystamine; (b) cysteamine; (c) an agent capable of inducing the production of cysteamine and/or cystamine; (d) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of (a) to (c); (e) a pharmaceutically acceptable salt of any of (a) to (d); or (f) any combination of (a) to (e); and the artemisinin-related compound may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The (a) cystamine; (b) cysteamine; (c) an agent capable of inducing the production of cysteamine and/or cystamine; (d) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of (a) to (c); (e) a pharmaceutically acceptable salt of any of (a) to (d); or (f) any combination of (a) to (e); and the artemisinin-related compound may be administered alone or in combination with other agents, drugs or hormones. The (a) cystamine; (b) cysteamine; (c) an agent capable of inducing the production of cysteamine and/or cystamine; (d) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of (a) to (c); (e) a pharmaceutically acceptable salt of any of (a) to (d); or (f) any combination of (a) to (e); and the artemisinin-related compound utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal means. The (a) cystamine; (b) cysteamine; (c) an agent capable of inducing the production of cysteamine and/or cystamine; (d) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of (a) to (c); (e) a pharmaceutically acceptable salt of any of (a) to (d); or (f) any combination of (a) to (e); and artemisinin-related compound may be administered separately or together (e.g., together in a composition). The combination of therapeutic agents and compositions of the present invention may be administered or co-administered in any conventional dosage form. Co-administration in the context of the present invention refers to the administration of more than one therapeutic in the course of a coordinated treatment to achieve an improved clinical outcome. Such co-administration may also be coextensive, that is, occurring during overlapping periods of time. For example, the (a) cystamine; (b) cysteamine; (c) an agent capable of inducing the production of cysteamine and/or cystamine; (d) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of (a) to (c); (e) a pharmaceutically acceptable salt of any of (a) to (d); or (f) any combination of (a) to (e) may be administered to a patient before, concomitantly, before and after, or after the artemisinin-related compound is administered.
As such, in embodiments, the invention further provides:
(1) a composition (e.g., a pharmaceutical composition or medicament) comprising (a) cystamine; (b) cysteamine; (c) an agent capable of inducing the production of cysteamine and/or cystamine; (d) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of (a) to (c); (e) a pharmaceutically acceptable salt of any of (a) to (d); or (f) any combination of (a) to (e)) and a pharmaceutically acceptable diluent or carrier;
(2) a composition comprising (a) an artemisinin-related compound and a pharmaceutically acceptable diluent or carrier;
(3) a composition comprising (i) (a) cystamine; (b) cysteamine; (c) an agent capable of inducing the production of cysteamine and/or cystamine; (d) a functional derivative, analog, conjugate, prodrug or precursor of (a) to (c); (e) a pharmaceutically acceptable salt of any of (a) to (d); or (f) any combination of (a) to (e); and (ii) an artemisinin-related compound; or
(4) a composition comprising (i) (a) cystamine; (b) cysteamine; (c) an agent capable of inducing the production of cysteamine and/or cystamine; (d) a functional derivative, analog, conjugate, prodrug or precursor of (a) to (c); (e) a pharmaceutically acceptable salt of any of (a) to (d); or (f) any combination of (a) to (e); and (ii) an artemisinin-related compound; and (iii) a pharmaceutically acceptable diluent or carrier.
As such, in an embodiment, the present invention further provides a combination of compositions (1) and (2) mentioned above for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease (e.g., malaria). The present invention further provides composition (3) or composition (4) mentioned above for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease (e.g., malaria). In an embodiment, components (i) and (ii) of the composition of (3) are formulated together. In an embodiment, components (i) and (ii) of the composition of (3) are formulated separately.
As used herein “pharmaceutically acceptable carrier” or “excipient” includes any and all solvents, buffers, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier can be suitable, for example, for intravenous, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, epidural, intracisternal, intraperitoneal, intranasal or pulmonary (e.g., aerosol) administration. Formulations may be in the form of liquid solutions or suspension; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of active agent(s)/composition(s) suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin (e.g., unit dose); (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, e.g., sucrose, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art. The oral formulation may further contain one or more coatings, such as an enteric coating. Enterically coated formulations of cystamine, cysteamine and derivatives thereof are described, for example, in PCT publication No. WO 2007/089670.
Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for compounds/compositions of the invention include ethylenevinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, (e.g., lactose) or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
For preparing pharmaceutical compositions from the compound(s)/composition(s) of the present invention, pharmaceutically acceptable carriers are either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substance, which may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
In powders, the carrier is a finely divided solid, which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired. The powders and tablets may typically contain from 5% or 10% to 70% of the active compound/composition. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term “preparation” is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.
Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions. For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.
Aqueous solutions suitable for oral use are prepared by dissolving the active compound(s)/composition(s) in water and adding suitable colorants, flavors, stabilizers, and thickening agents as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.
Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art (Rowe et al., Handbook of pharmaceutical excipients, 2003, 4th edition, Pharmaceutical Press, London UK). Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated.
It is further contemplated that the cystamine, cysteamine, a derivative or pharmaceutically acceptable salt thereof can be administered orally in a delayed release formulation. Exemplary delayed release formulations are disclosed in U.S. Pat. No. 8,026,284.
The composition may also contain a combination of active compounds for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. It may be desirable to use the above-mentioned composition in addition to one or more agents currently used to prevent or treat the disorder in question (e.g., an antimalarial such as sulfadoxine-pyrimethamine [Fansidar®], mefloquine [Lariam®], atovaquone, proguanil, atovaquone-proguanil [Malarone®], quinine, doxycycline, primaquine), Lumefantrine (or benflumetol). The above-mentioned agents may be formulated in a single composition or in several individual compositions which may be co-administered in the course of the treatment.
Formulations to be used for in vivo administration are preferably sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes.
The amount of the pharmaceutical composition which is effective in the prevention and/or treatment of a particular disease, disorder or condition (e.g., parasite infection and/or parasite-related disease) will depend on the nature and severity of the disease, the chosen prophylactic/therapeutic regimen, the target site of action, the patient's weight, special diets being followed by the patient, concurrent medications being used, the administration route and other factors that will be recognized by those skilled in the art. The dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease and different parameters from the patient. Typically, 0.001 to 1000 mg/kg of body weight/day will be administered to the subject. In an embodiment, a daily dose range of about 0.01 mg/kg to about 500 mg/kg, in a further embodiment of about 0.1 mg/kg to about 200 mg/kg, in a further embodiment of about 1 mg/kg to about 100 mg/kg, in a further embodiment of about 10 mg/kg to about 50 mg/kg, may be used. The dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial prophylactic and/or therapeutic response in the patient over time. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration. Effective doses may be extrapolated from dose response curves derived from in vitro or animal model test systems. For example, in order to obtain an effective mg/kg dose for humans based on data generated from rat studies, the effective mg/kg dosage in rat may be divided by six.
The cystamine, cysteamine, a derivative or pharmaceutically acceptable salt thereof or any combination thereof may be administered one, two or three or four times per day. In various embodiments, an effective dosage of cystamine, cysteamine, or derivative of a pharmaceutically acceptable salt thereof may be within the range of 0.01 mg to 1000 mg per kg (mg/kg) of body weight per day. Further, the effective dose may be 0.5 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg/25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, and may increase by 25 mg/kg increments up to 1000 mg/kg, or may range between any two of the foregoing values. In some embodiments, the cystamine, cysteamine, a derivative or pharmaceutically acceptable salt thereof is administered at a total daily dose of from approximately 0.25 g/m2 to 4.0 g/m2 body surface area, e.g., at least about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 g/m2, or up to about 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.5, 2.7, 3.0, or 3.5 g/m2. In some embodiments, the cystamine, cysteamine, a derivative or pharmaceutically acceptable salt thereof may be administered at a total daily dose of about 1-1.5 g/m2 body surface area, or 0.5-1 g/m2 body surface area, or about 0.7-0.8 g/m2 body surface area, or about 1.35 g/m2 body surface area.
Examples of treatment regimens for artemisinin, artesunate and artemether recommended by the World Health Organization (WHO) (The use of Artemisinin and its derivatives as anti-malarial drugs, Report of a Joint CTD/DMP/TDR Informal Consultation, Geneva, 10-12 Jun. 1998) for the treatment of parasitic disease (malaria) are provided below:
Artemisinin may be administered at 20 mg/kg in a divided dose (loading dose) on the first day, followed by 10 mg/kg once a day for 6 days. Artesunate may be administered at 4 mg/kg in a divided dose on the first day, followed by 2 mg/kg once a day for 6 days. Artemether may be administered at 4 mg/kg in a divided dose on the first day, followed by 2 mg/kg once a day for 6 days.
In an embodiment, the dose of (a) cystamine; (b) cysteamine; (c) an agent capable of inducing the production of cysteamine and/or cystamine; (d) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of (a) to (c); (e) a pharmaceutically acceptable salt of any of (a) to (d); or (f) any combination of (a) to (e) and/or (a) an artemisinin-related compound that is used/administered in the methods, uses, compositions, packages and combinations of the invention is a suboptimal dose. “Suboptimal dose” as used herein refers to a dose of one of the compounds of the combination described herein, which, when used in the absence of another compound of the combination, results in a biological effect of 50% or less (e.g., inhibition of parasitemia of 50% or less), in an embodiment of 40% or less, in a further embodiment of 30% or less, in a further embodiment of 20% or less, in a further embodiment of 10% or less. As such, use of a combination of the compounds described herein, where one or more compounds in the combination is used at a suboptimal dose, may achieve increased efficacy/biological effect (e.g., inhibition of parasitemia) relative to using the compound(s) in the absence of the other(s), at a comparable suboptimal dose.
The terms “treat/treating/treatment” and “prevent/preventing/prevention” as used herein, refers to eliciting the desired biological response, i.e., a therapeutic and prophylactic effect, respectively. In accordance with the subject invention, the therapeutic effect comprises one or more of a decrease/reduction in parasite load (parasitemia), an amelioration of symptoms and parasite-related effects, and increased survival time of the affected host animal, following administration of (a) cysteamine, cystamine, a compound of formula I, an agent capable of increasing expression of pantetheinase or pantetheinase activity, an agent capable of inducing the production of cysteamine, a functional derivative, analog, metabolite, prodrug or precursor thereof, or salts thereof, and (b) an artemisinin-related compound. In embodiments, the decrease in parasite load or parasitemia induced by the treatment may be, for example, a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% (i.e., complete elimination of the parasite) decrease in parasitemia. In accordance with the invention, a prophylactic effect may comprise a decrease in the onset of or of the severity of one or more of parasite load or parasitemia, symptoms and parasite-related effects, and increased survival time of the affected host animal, following administration of (a) cysteamine, cystamine, a compound of formula I, an agent capable of increasing expression of pantetheinase or pantetheinase activity, an agent capable of inducing the production of cysteamine, a functional derivative, analog, prodrug or precursor thereof, or salts thereof and (b) an artemisinin-related compound.
As such, a “therapeutically effective” or “prophylactically effective” amount of (a) cysteamine, cystamine, a compound of formula I, an agent capable of inducing expression of pantetheinase, an agent capable of inducing the production of cysteamine, a functional derivative, analog or precursor thereof, or salts thereof, or any combinations thereof, and (b) an artemisinin-related compound, may be administered to an animal, in the context of the methods of treatment and prevention, respectively, described herein.
In an embodiment, the above-mentioned subject is a mammal. A mammal, including for purposes of treatment and prevention, refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports or pet animals such as dogs, horses, cats, cows etc. In an embodiment, the mammal is human.
Parasitic or parasite infection refers to an infection by an organism that lives on or inside another organism (host) and typically causes harm to the host. Parasite disease or parasitic disease refers to a disease or condition associated with parasite infection of a host. In an embodiment, the above-mentioned parasite is a protozoa. In an embodiment, the above-mentioned parasite is of the Plasmodium genus. In a further embodiment, the parasite is Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, or Plasmodium malariae. 
The invention further provides kits or packages (e.g., commercial packages) comprising the above-mentioned compositions or agents together with instructions for their use for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease such as malaria (e.g., blood-stage malaria or cerebral malaria).
The arrangement and construction of such kits is conventionally known to one of skill in the art. Such kits may include, for example, container(s) (e.g., syringe and/or vial and/or ampoule) for containing the agent or combination of agents or compositions, other apparatus for administering the therapeutic agent(s) and/or composition(s) and/or diluent(s). The kit may optionally further include instructions. The instructions may describe how the agent(s) and the diluent should be mixed to form a pharmaceutical formulation. The instructions may also describe how to administer the resulting pharmaceutical formulation to a subject.
As used herein, a synergistic effect (e.g., reduction in parasitemia, increase in survival time) is achieved when the effect of the combined drugs is greater than the theoretical sum of the effect of each agent in the absence of the other. One potential advantage of combination therapy with a synergistic effect is that lower dosages (e.g., a suboptimal dose) of one or both of the drugs or therapies may be used in order to achieve high therapeutic activity with low toxicity. In an embodiment, the combination therapy results in at least a 5% increase in the effect as compared to the predicted theoretical additive effect of the agents. In a further embodiment, the combination therapy results in at least a 10% increase in the effect as compared to the predicted theoretical additive effect of the agents. In a further embodiment, the combination therapy results in at least a 20% increase in the effect as compared to the predicted theoretical additive effect of the agents. In a further embodiment, the combination therapy results in at least a 30% increase in the effect as compared to the predicted theoretical additive effect of the agents. In a further embodiment, the combination therapy results in at least a 50% increase in the effect as compared to the predicted theoretical additive effect of the agents.
A further advantage of using the drugs in combination is that efficacy may be achieved in situations where either drug alone would not have an effect. For example, in a case where the parasite is resistant to a drug when used alone but is affected by the drugs when used in combination.
Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. In the claims, the word “comprising” is used as an open-ended term, substantially equivalent to the phrase “including, but not limited to”. The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. The term “such as” is used herein to mean, and is used interchangeably, with the phrase “such as but not limited to”. The following examples are illustrative of various aspects of the invention, and do not limit the broad aspects of the invention as disclosed herein.
MODE(S) FOR CARRYING OUT THE INVENTION
The present invention is illustrated in further details by the following non-limiting examples.
Example 1
Materials and Methods
Mice.
A/J and C57BL/6 (B6) mice were purchased from the Jackson Laboratories (Bar Harbor, Me.) and were housed at McGill University according to the guidelines of the Canadian Council on Animal Care. An LDH virus-free isolate of P. chabaudi AS was maintained by weekly passage in A/J mice. Mice were infected intravenously into the tail vein (i.v.) with 106 or 107 pRBC suspended in pyrogen-free saline. Following infection, the percentage of pRBC was determined daily on thin blood smears stained with Dif-QuikTM (Dade Behring, Newark, Del.), as described (Fortin A, et al. (2001) Proc Natl Acad Sci USA 98: 10793-10798).
Pharmacokinetic Studies of Cysteamine Hydrochloride In Vivo.
Cysteamine was detected in plasma by high performance liquid chromatography analysis with ultraviolet detection (Dias V C, et al. (1998) Clin Chem 44: 2199-2201). Briefly, blood was collected in EDTA/heparin-containing tubes, and plasma was obtained by centrifugation. Plasma thiols were reduced by treatment with Tris(2-carboxyethyl)phosphine (0.05M final concentration, 20 min. at 20° C.), and proteins were precipitated with tri-chloroacetic acid (TCA, 10% final concentration). Free thiols from the protein-free supernatant were derivatized using SBD-F (7-benzo-2-oxa-1,3-diazole-4-sulfonic acid), used at a final concentration of 0.2 mg/ml (1 hr at 60° C.) in 0.05 M borate buffer (pH 9.5). The mixture was then analyzed by HPLC: the mobile phase consisted of an aqueous solvent (0.1M acetic acid, 0.1 sodium acetate, pH 4.3) running on a SupelcoTM LC-8 column, and elution of plasma analytes was with a 0-10% acetonitrile gradient. Detection of SBD-F derivatized analytes was by reading fluorescence at 515 nm (excitation at 385 nm). Cysteamine elution peaks were quantified (surface area), and plasma concentrations were calculated using a set of internal cysteamine standards processed at the same time. Area under the curve (AUCt0-tlast) was calculated using the trapezoid approximation method.
Cysteamine, Chloroquine, Artesunate and Dihydroartemisinin Administration In Vivo.
Cysteamine hydrochloride (Sigma, Burlington ON) was prepared in PBS. Chloroquine hydrochloride, artesunate and dihydroartemisinin were provided by Dafra Pharmaceuticals; chloroquine was prepared in PBS, artesunate and DHA were prepared in 5% sodium bicarbonate and diluted in water to appropriate concentrations. All solutions were prepared fresh daily, filter sterilized and injections were performed intra-peritoneally (i.p) or subcutaneously (s.c.) for 4 days or according to treatment regimen. Mice were weighed prior to treatment to determine appropriate doses and injection volumes ranged from 100-400 μL per mouse. In the case of animals treated with two drugs, artemisinin derivatives were administered first (due to the short half life of cysteamine), followed by cysteamine 5-10 minutes later on alternate sides. Untreated control animals were injected with PBS alone.
Statistical Tests.
Groups with normally distributed data points were compared using parametric unpaired t-tests, while groups with non-Gaussian distributions were compared using non-parametric Mann-Whitney tests. Survival differences were analyzed using the Log-Rank test. Synergistic effects were defined as: the percent inhibition of the combination therapy was >10% greater than the sum of the percent inhibition of the individual mice. Standard error of percent inhibition was calculated from individual mice compared to the mean parasitemia level of the control group.
Example 2
Characteristics of Cysteamine Activity Against Plasmodium chabaudi Infection In Vivo
To gain more insight into the anti-malarial effect of cysteamine (Cys) in vivo, the pharmacokinetic characteristics (plasma level) of Cys administered through the sub-cutaneous (s.c.) and intra-peritoneal (i.p) routes was compared. Peak plasma concentration (Cmax) and total bioavailability (area under the curve, AUC) after administration of a single dose of 120 mg/kg of Cys hydrochloride (FIG. 1A) was measured. The Cmax was higher (665 μM) and reached more rapidly (Tmax<5 min) following i.p injection, compared to the s.c. route, where a Cmax of 250 μM was attained with a Tmax of 30 min. On the other hand, total Cys bioavailability (AUCT0-Tlast) was comparable for both routes (24282 vs 15277 min×μM for i.p. and s.c., respectively). To determine which pharmacokinetic parameter (AUC vs. Cmax) is important for efficacy against Plasmodium, the i.p. and s.c. routes of injection were compared in a continuous treatment regimen, starting one day prior to infection (105 pRBC of P. chabaudi, i.v.) and continuing daily for 7 days. Parasitemia was monitored on thin blood smears at days 5, 6 and 7 following infection (FIG. 1B). Treatment of infected animals with 120 mg/kg of Cys administered either s.c. or i.p. caused a highly significant (p<0.01) 50% reduction in parasitemia at day 5 and 6, relative to saline injected controls. These results suggest that total Cys exposure (AUCT0-Tlast) is a pharmacokinetic parameter influencing the anti-malarial effect of Cys.
Example 3
Cysteamine Dosing Used in the Treatment of Cystinosis Reduces Parasitemia During P. chabaudi Infection In Vivo
It was next determined whether Cys at equivalent dosing to that used in the clinical treatment of nephropathic cystinosis in humans has an effect on the course and severity of P. chabaudi infection in mice. In cystinosis patients, Cys is given orally as Cys bitartrate (Cystagon®). The PK profile of an oral dose of 1475 mg of Cys bitartrate (500 mg cysteamine base), includes a peak plasma concentration of 39 μM (Cmax) with a concomitant AUCT0-Tlast of 3613 min×(Fidler M C, et al. (2007) Br J Clin Pharmacol 63: 36-40). Results depicted in FIG. 2A show that a single s.c. injection of 50 mg/kg Cys hydrochloride in mice has a PK profile comparable to that of one oral dose of Cystagon® in humans, including a Cmax of  ̃80 μM and an AUC of 2845 min×μM. The efficacy of different regimens of 50 mg/kg Cys s.c. (number of injections, interval between injections) on replication of P. chabaudi in vivo was evaluated. P. chabaudi-infected mice were treated daily, starting at day −1 and continuing to day 10, with either 1×150 mg/kg, 3×50 mg/kg given at 2 hr intervals, 4×50 mg/kg given at 2 hr intervals or 3×50 mg/kg given at 1 hr intervals of Cys, and blood parasitemia was monitored at days 5, 6 and 7 (FIG. 2B). Significant reduction (40-67%) of blood parasitemia was seen for all treatment regimens, with the strongest effect achieved with 3×50 mg/kg given at 1 hr intervals. All 50 mg/kg repeated dosing regimens (s.c.) showed inhibitory effects on parasitemia that were similar to that produced by a single s.c. injection of 150 mg/kg Cys, in agreement with data from FIGS. 1A and 1B showing that is a pharmacokinetic parameter influencing the anti-malarial effect of Cys. These results suggest that multiple Cys treatments at doses similar to those used in humans for cystinosis, can significantly reduce blood-stage replication of Plasmodium parasites in mice.
Example 4
Cysteamine and Artemisinin Derivatives Show Synergistic Effects Against Plasmodium In Vivo
The effect of Cys on the potency and efficacy of the anti-malarial artemisinin derivatives was tested. In these studies, artemisinin derivatives were given at sub-optimal concentrations to distinguish between the lack of an effect and additive or synergistic effects of Cys addition. Synergy (Tallarida R J (2001) J Pharmacol Exp Ther 298: 865-872) is defined as a total anti-malarial activity (reduction in blood parasitemia compared to untreated controls in a standard 4-day test) of the two compounds administered together being greater than the sum of the independent activities of the two compounds given alone. We tested combinations of Cys and either artesunate (ART) or dihydroartemisinin (DHA), the bioactive form of artemisinin. Pantetheinase-deficient mice were infected with P. chabaudi (107 pRBC, i.v.) and treated with Cys (170 mg/kg) and/or sub-optimal doses of ART (0.2 or 0.5 mg/kg) (FIG. 3A) or DHA (0.15 or 0.3 mg/kg) (FIG. 3B) from day 0-3 and parasitemia was monitored on days 4 and 5. Sub-optimal doses of the artemisinin derivatives alone resulted in parasitemia inhibition ranging from 20-30%, while higher doses of these drugs could inhibit parasitemia 40-60%, compared to controls (FIG. 3A/B; TABLE 1). However, addition of Cys to either ART or DHA resulted in stronger inhibition of parasitemia than the additive effect of the two compounds, indicating a synergistic effect (TABLE 1; stars). Synergy was observed at all concentrations of ART and DHA tested. Mice receiving both Cys and ART/DHA also showed fewer symptoms of disease (ruffled fur, lethargy), compared to mice receiving either PBS or only one compound. To assess whether the synergistic effect between Cys and ART was restricted to A/J mice deficient in pantetheinase, the experiment were repeated in pantetheinase sufficient and malaria-resistant C57BL/6 mice (FIG. 3C). Potentiation of the anti-malarial activity of ART (0.5 mg/kg) by Cys was also clearly evident in these C57BL/6 mice at both days 4 and 5 post-infection, with combined treatment causing a 65-71% reduction in parasitemia compared to PBS controls, greater than either compound tested alone (13-29%) (TABLE 1).







TABLE 1







Effect of cysteamine and artemisinin derivative


combinations on blood-stage replication of


Plasmodium chabaudi in vivo













Inhibition of





parasitemia



Dose
Cysteamine
(% PBS control)a











Mouse type and drug
(mg/kg)
(170 mg/kg)
Day 4
Day 5














Pantetheinase-deficient A/J






Artesunate
0.2
−
30
20


Artesunate
0.2
+
 65*
 56*


Artesunate
0.5
−
65
43


Artesunate
0.5
+
93
 80*


DHA
0.15
−
22
13


DHA
0.15
+
 56*
 46*


DHA
0.3
−
50
40


DHA
0.3
+
80
 71*


NAb
0
+
28
23


Pantetheinase-sufficient


C57BL/6


Artesunate
0.5
−
29
21


Artesunate
0.5
+
 71*
 65*


NA
0
+
13
25





a* indicates synergy between the compounds.


bNA, no drug administered.






Example 5
Synergistic Inhibition of Plasmodium Replication by Artesunate and Cysteamine is Dose-Dependent
It was subsequently examined whether Cys potentiation of ART was dose dependent. Initially, Cys doses of 60, 100, 140 and 180 mg/kg were tested with a sub-optimal ART dose of 0.2 mg/kg. The drugs were administered from day 0-3 post infection, parasitemia was counted at day 4 and day 5 and the percent inhibition was calculated compared to PBS-treated controls (FIG. 4A). At 0.2 mg/kg, ART alone inhibits parasitemia by  ̃20% (day 4) and 40% (day 5) while inhibition by Cys alone was partially dose dependent (varying between 10% and 25%). Synergy was observed for all Cys doses tested (varying between 50% and 75% reduction in parasitemia), although without a clear dose-dependent effect in this Cys dosing range. Testing a lower Cys dose range (20, 40 and 60 mg/kg) revealed a clear dose-dependent effect on synergistic inhibition of parasitemia, with doses as low as 20-40 mg/kg showing potentiation of the ART effect (FIG. 4B). It was also assessed whether Cys could potentiate low doses of artesunate which, given alone, have no significant effect on parasitemia. In this experiment, Cys (170 mg/kg) was administered in combination, or not, with increasing doses of ART (0.05, 0.1, 0.2, 0.4 mg/kg) in the same 4-day experimental protocol. In these experiments, a strong potentiation (minimum of 3-fold) of low-dose artesunate by Cys was detected, with 60-75% inhibition of parasitemia replication for combinations containing low dose ART at 0.1 and 0.2 mg/kg, compared to <10% for these doses of ART used alone (FIG. 4C).
Example 6
The Impact of Cysteamine and Artesunate in Combination on the Resolution of P. chabaudi Infection
It was investigated if low dose Cys could potentiate standard doses of ART that show therapeutic activity in vivo and concurrently determined possible long-term effects on patent parasitemia, resolution of infection and survival in a lethal infection model. In this protocol, mice were infected with 106 pRBC P. chabaudi (i.v.) and treated with Cys (60 mg/kg) and/or ART (0.5, 1, 2, 5 and 10 mg/kg) for 4 days (days 0-3), while blood parasitemia and survival were followed for 22 days. Control animals treated with either PBS or Cys alone (60 mg/kg) developed high parasite burdens, which peaked at day 6, and all mice succumbed to the infection by day 7 (FIGS. 5A and 5C). In animals receiving ART alone, there was a dose-dependent effect on infection, which manifested as a delay in the onset of parasitemia and a reduction of peak parasitemia. Strikingly, the addition of Cys (60 mg/kg) to all ART doses tested had a beneficial effect on infection kinetics, causing both a further delay in onset (by 2 to 3 days), and a reduction of peak levels of parasitemia relative to mice receiving only the corresponding dose of ART (FIG. 5B). Notably, the addition of Cys to 0.5 mg/kg or 1 mg/kg of ART caused a strong potentiation of the ART effect, with a further 60-70% reduction in parasitemia at day 6 (FIG. 5D). Likewise, although all mice treated with 0.5 mg/kg ART succumbed to the infection early (day 8), mice additionally receiving Cys survived until day 9; moreover, addition of Cys to 1.0 mg/kg ART completely rescued animals from lethality of infection, with 100% survival in this group (FIGS. 5B, 5C). These results indicate that the synergistic effect of low doses of Cys on artemisinin derivatives not only impacts early parasite burdens, but also significantly improves ultimate outcome to infection.
Example 7
Effect of Cysteamine and Artesunate Combinations on Progression of P. chabaudi in Pantetheinase-Sufficient B6 Mice
To investigate whether the effects on parasite burden over the course of infection would also be observed with a pantetheinase-sufficient mouse strain, a similar experiment was performed using female B6 mice. Groups of mice were infected with 106 P. chabaudi pRBC i.v. and treated with PBS, 1 mg/kg or 30 mg/kg of Art, or 1 mg/kg or 30 mg/kg of Art plus 60 mg/kg of Cys for 4 days. A reduction in parasite levels and a delay in the peak were observed when Cys and Art are given in combination, compared to results with Art administered alone, at both high and low doses (FIG. 6A). As in A/J mice, the effect of Cys addition to 1 mg/kg of Art has a clear effect on early parasite replication at day 6 (FIG. 6B). Although a “curative” dose combination was not achieved with a 4-day treatment regimen, parasite levels remained under 12% pRBC in the 30 mg/kg Art-plus-Cys group, and mice did not display any outward symptoms of disease such as lethargy or ruffled fur. B6 mice were able to completely clear parasite burdens and survive the infection, even in the control PBS-treated group. However, the addition of Cys eliminated the appearance of recrudescent parasitemia around day 14, as seen with the control group (FIG. 6A). These results indicate that the synergistic effect of low doses of Cys on artemisinin derivatives not only impacts early parasite burdens but can also significantly improve ultimate outcome to infection.
Example 8
Effect of Cysteamine and Artesunate Combinations on Progression of Plasmodium berghei ANKA Infection
Intravenous infection with Plasmodium berghei ANKA is an accepted mouse model of cerebral malaria (CM) (Hunt, N. H. et al. 2006, Int. J. Parasitol. 36: 569-582). The infection causes the following pathology. First, there is appearance of blood parasitemia, starting at days 3 or 4, which can go up to 10% by day 7-8. Starting at day 5-6, there is emergence of cerebral symptoms caused by permeability of the blood brain barrier, concomitant to trapping of parasitized red cells in the microvasculature and acute pathological host inflammatory response in situ. This cerebral phase quickly progresses from tremors, to paralysis, to coma, and is uniformly lethal in mice by days 8-10. In this model, progress of infection and possibly drug effects may be monitored by a) appearance and intensity of blood parasitemia (between days 5-8), b) appearance of cerebral symptoms, and c) lethality.
The results depicted in FIGS. 8A and 8B show that addition of Cysteamine to either of the two Artemisinin dosings (5 or 10 mg/kg) causes a delay in the rise of parasitemia and seems to cause a reduction in absolute levels measured at days 6-8 over what is detected in animals treated with Artemisinin alone. Second, the addition of cysteamine to Artemisinin causes an effect which is comparable (FIG. 8A) or superior (FIG. 8B) to that of doubling the dose of Artemisinin.
With respect to survival (FIGS. 9A and 9B), Cysteamine alone had a minor positive effect on survival of P. berghei-infected animals. Adding cysteamine to Artemisinin prolonged survival of P. berghei-infected animals over that measured in animals treated with Artemisinin alone, and this by a factor of 1-2 days.
Although the present invention has been described herein above by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

## Claims

1. A method for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease, in a subject in need thereof, said method comprising administering to said subject an effective amount of
(i) (a) a compound of formula I:


NH2—CH2—CH2—S—R  (I)


wherein R is H or S—CH2—CH2—NH2; (b) an agent capable of inducing the production of the compound of formula I; (c) a functional derivative, analog, conjugate, prodrug or precursor of (a) or (b); (d) a pharmaceutically acceptable salt of any of (a) to (c); or (e) any combination of (a) to (d); and
(ii) an artemisinin-related compound selected from the group consisting of artemether, arteether, artelinic acid, artenimol, artemotil, and artemisinic acid.

2. The method of claim 1, wherein said method comprises administering to said subject an effective amount of
(i) (a) cystamine; (b) cysteamine; (c) a pharmaceutically acceptable salt of (a) or (b); or (d) any combination of (a) to (c); and
(ii) an artemisinin-related compound selected from the group consisting of artemether, arteether, artelinic acid, artenimol, artemotil, and artemisinic acid.

3. The method of claim 2, wherein said method comprises administering to said subject an effective amount of
(i) cysteamine or a pharmaceutically acceptable salt thereof; and
(ii) an artemisinin-related compound selected from the group consisting of artemether, arteether, artelinic acid, artenimol, artemotil, and artemisinic acid.

4. The method according to claim 1, wherein said parasite is of the genus Plasmodium.

5. The method according to claim 1, wherein said disease is malaria.

6. The method according to claim 5, wherein said malaria is blood-stage malaria.

7. The method according to claim 5, wherein said malaria is cerebral malaria.

8. The method according to claim 1, wherein said subject is a human.

9. The method according to claim 1 wherein compounds (i) and (ii) act synergistically.

10. The method of claim 9, wherein the synergy results in use of effective doses of compound (i) and/or (ii) that are lower than doses administered when the compounds are administered in the absence of the other compound.

11. The method of claim 10, wherein the dose of compound (i) and/or (ii) is suboptimal.

12. The method of claim 1, wherein the effective dose of compound (i) is in the range of 1 to 500 mg/kg.

13. The method of claim 1, wherein compound (i) is present in a delayed release composition.

14. The method according to claim 1, wherein the peak level of parisitemia is reduced.

15. The method according to claim 1, wherein the administering prevents parisitemia.

16. The method according to claim 1, wherein compound (i) is administered less than four times a day.

17. The method according to claim 1, wherein compound (i) is administered twice daily.

18. The method of claim 1, wherein compounds (i) and (ii) are administered coextensively.

19. A composition for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease, in a subject, said composition comprising
(i) (a) a compound of formula I:


NH2—CH2—CH2—S—R  (I)


wherein R is H or S—CH2—CH2—NH2; (b) an agent capable of inducing the production of the compound of formula I; (c) a functional derivative, analog, conjugate, prodrug or precursor of (a) or (b); (d) a pharmaceutically acceptable salt of any of (a) to (c); or (e) any combination of (a) to (d); and
(ii) an artemisinin-related compound selected from the group consisting of artemether, arteether, artelinic acid, artenimol, artemotil, and artemisinic acid, (b) a functional derivative, analog, conjugate, metabolite, prodrug or precursor of artemisinin, (c) a pharmaceutically acceptable salt of (a) or (b), or (d) any combination of (a) to (c).

20. The composition according to claim 19, wherein said composition comprises
(i) (a) cystamine; (b) cysteamine; (c) a pharmaceutically acceptable salt of (a) or (b); or (d) any combination of (a) to (c); and
(ii) an artemisinin-related compound selected from the group consisting of artemether, arteether, artelinic acid, artenimol, artemotil, and artemisinic acid.

21. The composition according to claim 20, wherein said composition comprises
(a) cysteamine or a pharmaceutically acceptable salt thereof; and
(b) an artemisinin-related compound selected from the group consisting of artemether, arteether, artelinic acid, artenimol, artemotil, and artemisinic acid.

22. The composition according to claim 19, further comprising a pharmaceutically acceptable carrier or excipient.

23. A package comprising
(i) (a) a compound of formula I:


NH2—CH2—CH2—S—R  (I)


wherein R is H or S—CH2—CH2—NH2; (b) an agent capable of inducing the production of the compound of formula I; (c) a functional derivative, analog, conjugate, prodrug or precursor of (a) or (b); (d) a pharmaceutically acceptable salt of any of (a) to (c); or (e) any combination of (a) to (d); and
(ii) an artemisinin-related compound selected from the group consisting of artemether, arteether, artelinic acid, artenimol, artemotil, and artemisinic acid;

for decreasing susceptibility to parasite infection or disease or for preventing or treating parasite infection or disease in a subject.

24. The package of claim 23, wherein (i) and (ii) are packaged separately.

25. The package of claim 23, wherein (i) and (ii) are packaged in the same formulation.

26. The package of claim 23 wherein compound M is present in a delayed release composition.

27. The package of claim 23, further comprising labels and instructions for use.

28. The method according to claim 4, wherein the Plasmodium is an artemisinin-resistant human Plasmodium parasite.

29. A method for decreasing susceptibility to parasite infection or disease or treating parasite infection or disease wherein the parasite is an artemisinin-resistant human Plasmodium parasite, in a subject in need thereof, said method comprising administering to said subject an effective amount of
(i) (a) a compound of formula I:


NH2—CH2—CH2—S—R  (I)


wherein R is H or S—CH2—CH2—NH2; (b) an agent capable of inducing the production of the compound of formula I; (c) a functional derivative, analog, conjugate, prodrug or precursor of (a) or (b); (d) a pharmaceutically acceptable salt of any of (a) to (c); or (e) any combination of (a) to (d); and
(ii) an artemisinin-related compound.

30. The method of claim 29, wherein said method comprises administering to said subject an effective amount of
(i) (a) cystamine; (b) cysteamine; (c) a pharmaceutically acceptable salt of (a) or (b); or (d) any combination of (a) to (c); and
(ii) an artemisinin-related compound.

31. The method according to claim 29, wherein said disease is malaria.

32. The method according to claim 29, wherein said malaria is blood-stage malaria.

33. The method according to claim 29, wherein said malaria is cerebral malaria.

34. The method according to claim 29, wherein compounds (i) and (ii) act synergistically.

35. The method of claim 34, wherein the synergy results in use of effective doses of compound i) and/or ii) that are lower than doses administered when the compounds are administered in the absence of the other composition.

36. The method of claim 35, wherein the dose of compound (i) and/or (ii) is suboptimal.

37. The method of claim 29, wherein the effective dose of compound (i) is in the range of 1 to 500 mg/kg.

38. The method of claim 29, wherein compound (i) is present in a delayed release composition.

39. The method according to claim 29, wherein the peak level of parisitemia is reduced.

40. The method according to claim 29, wherein the administering prevents parisitemia.

41. The method according to claim 29, wherein compound (i) is administered less than four times a day.

42. The method according to claim 29, wherein compound (i) is administered twice daily.

43. The method of claim 29, wherein compounds (i) and (ii) are administered coextensively.