WO2013071365A1

WO2013071365A1 - A method of treatment and prophylaxis and compositions useful therefor

Info

Publication number: WO2013071365A1
Application number: PCT/AU2012/001422
Authority: WO
Inventors: Simon James Foote; Brendan John MCMORRAN; Clare Margaret SMITH
Original assignee: Macquarie University
Priority date: 2011-11-16
Filing date: 2012-11-16
Publication date: 2013-05-23

Abstract

A method for the treatment or prophylaxis of a subject infected with or who has or had exposure to a member of Phylum Apicomplexa, said method comprising administering to said subject an effective amount of griseofulvin or N-methyl protoporphyrin (NMPP).

Description

A METHOD OF TREATMENT AND PROPHYLAXIS AND

COMPOSITIONS USEFUL THEREFOR

FIELD

[0001] The present disclosure teaches the treatment and prophylaxis of infection of subjects by members of Phylum Apicomplexa, including species of Plasmodium and compositions useful therefore.

BACKGROUND

[0002] Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.

[0003] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge.

(0004J Infection by members of Phylum Apicomplexa can cause serious and debilitating diseases in a range of animals including higher order primates such as humans. Of note is infection by Plasmodium species and, in particular, leading to malaria in humans.

[0005| According to the World Health Organisation, approximately hal f of the world's population is at risk of contracting or suffering from malaria. New infections are diagnosed at a rate of about 250 million cases each year. Nearly one million people die annually from malaria. People living in the poorest countries are the most vulnerable. Malaria is a particular problem in Africa, where one in every five chi ldhood deaths is due to the effects of the disease. An African child has on average between 1 .6 and 5.4 episodes of malaria fever each year and every 30 seconds a child dies from malaria.

[0006] Humans are the definitive host for malarial parasites and transmission from host to host is carried out by mosquitoes thus malaria occurs wherever human and the transmitting species of mosquito coexist. In humans, four species i Plasmodium sp. cause malaria. Severe malaria is a medical emergency and is usually caused by Plasmodium falciparum. [0007] Members of Phylum Apicomplexa have complex life cycles with many life cycle stages (forms) in one or more hosts. Several genera have life cycle stages that invade and proliferate in red blood cells. Malaria, for example, develops as a result of multiplication of Plasmodium sp. in cells of the liver (hepatocytes) and red blood cells. Sporozoites are transmitted to a human host by infected mosquitoes when they take a blood meal. Sporozoites pass rapidly in the blood stream to the liver where they invade hepatocytes and proliferate and differentiate into merozoites. Merozoites break out of hepatocytes into the blood stream where they invade red blood cells. There, they differentiate through various forms and proliferate clonally to produce more merozoites which emerge and invade uninfected red cells establishing an unremitting cycle of escalating infection. Sexual forms (gametocytes) are also produced and if these are taken up by a mosquito they will undergo genetic exchange. The waves of fever that are characteristic of malarial infection are caused by the cycle of intraerythrocytic merozoites bursting out of old red blood cells and invading new red blood cells. If this prol i ferative cycle continues, the infected subject may rapidly (within days) become comatose and die.

[0008] Vector control is one form of malaria prevention and insecticide-treated bed nets (mostly using pyrethroidal insecticides) have been useful, in reducing deaths in endemic countries. However, there are concerns regarding the development of insecticide resistance. There is, however, currently no effective vaccine against malaria. Chemotherapy has also been successfully used. Chloroquine was the mainstay of antimalarial chemotherapy for fifty years but chloroquine resistance strains are now widespread. Currently, artemisinin-based combination therapy is recommended although artemisinin resistance has already been observed. Resistance appears to occur through (i ) mutations that reduce the ability of the drug to bind to its parasite target: or ( ii ) the development of parasite membrane transporters able to transport drugs away from the parasite. The established ability of Plasmodium sp. to develop drug resistance means that current anti-plasmodium drugs have to be considered as relatively short-term solutions.

[0009) There is an urgent need to develop new therapeutic agents and protocols to treat and prevent or othemise reduce infection by members of Phylum Apicomplexa, including species of Plasmodium. SUMMARY

(0010] The present disclosure is instructional for a method for the treatment or prophylaxis of infection in a subject by a member of Phylum Apicomplexa, the method comprising administering to the subject an effective amount of griseofulvin or N-methyl protoporphyrin or a salt, homolog, analog, isoform or enantiomer thereof. The subject may have an infection, or has had exposure to infection or may have exposure to infection. In an embodiment, the Phylum Apicomplexa member is a species of Plasmodium.

[001 11 An example of a Plasmodium species is Plasmodium falciparum.

[0012] Taught herein is the treatment or prevention of infection by a species of Plasmodium in a subject by providing the subject with griseofulvin or N-methyl protoporphyrin or a salt, homolog, analog, isoform or enantiomer thereof prior to, during or following infection or exposure to the Plasmodium sp. Griseofulvin is able to inhibit the erythrocytic heme biosynthetic enzyme errochelatase (FECH) which is shown to be an essential host factor for malarial parasite growth. FECH catalyses the conversion of its substrate protoporphyrin IX (PPIX) and is the terminal enzyme in the heme biosynthetic pathway which pathway is present in mammalian hosts of apicomplaxal parasites and in apicomplexal parasites such as plasmodium. The anti-FECH activities of both the clinically approved antifungal agent, griseofulvin, and N-methyl protoporphyrin which is a metabolic product of griseofulvin (via a cytochrome-dependent process) and also a FECH substrate analogue, are demonstrated herein to reduce the growth and development of intraerythrocytic (red blood cell) stages of Plasmodium sp. Importantly. FECH inhibition was shown to be effective in reducing parasite growth in chloroquine resistant and multidrug resistant strains as well as chloroquine sensitive strain of . falciparium (which causes the most acute forms of malaria). IC50 values for griseofulvin about Ι Ομηι to about 50μιη (depending upon parasite strain) while NMPP was effective in the nM range, showing an IC50 of 25nM or about l OOnM to about l OOOnM (depending upon parasite strain).

[0013) In an embodiment, the subject or population of subjects is (i) tested for infection with a member of Phylum Apicomplexa and then (ii) administered a composition comprising griseofulvin or N-methyl protoporphyrin or a salt, homolog. analog, isoform or enantiomer thereof if the subject or population of subjects tests positive for infection by or prior exposure to or potential exposure to an Apicomplexa member. In an example, the subject is (i) tested for infection with Plasmodium sp. and (ii) administered griseofulvin or N-methyl protoporphyrin or a salt, homolog. analog, isoform or enantiomer thereof if the subject tests positive for infection by or prior exposure to or potential exposure to Plasmodium sp.

[0014] Further taught herein is the testing of a subject for infection or infection levels including minimal residual disease (MRD) levels with a member of Phylum Apicomplexa such as a Plasmodium sp. after administration of griseofulvin or N-methyl protoporphyrin or a salt, homolog. analog, isoform or enantiomer thereof.

[0015] Enabled herein is the composition comprising griseofulvin or N-methyl protoporphyrin or a salt, homolog, analog, isoform or enantiomer thereof. Optional ly, the composition comprises a pharmaceutically or physiologically acceptable carrier, diluent or excipient. The composition may also comprise another active agent which directly or indirectly targets the Apicomplexa parasite or facilitates ameliorating the symptoms of infection therewith.

[0016[ The present disclosure also teaches griseofulvin or N-methyl protoporphyrin or a salt, homolog, analog, isoform or enantiomer thereof for use in the treatment or prophylaxis of infection by a member of Phylum Apicomplexa. In an embodiment, griseofulvin or N-methyl protoporphyrin or a salt, homolog, analog, isoform or enantiomer thereof is used in the treatment or prophylaxis of malaria caused by Plasmodium sp.

[0017) The present disclosure enables the use of griseofulvin or N-methyl protoporphyrin or a salt, homolog, analog, isoform or enantiomer thereof in. or in the manufacture of, a medicament for the treatment or prevention of infection by a member of Phylum Apicomplexa. This aspect includes the treatment of malaria.

[0018] In accordance with these embodiments, the composition is generally administered for a time and under conditions sufficient to treat the infection, or reduce the risk of onset of severe infection or to reduce the level of parasite in the subject or population for at least minimal residual disease levels. This aspect includes the treatment, prevention or amelioration of symptoms of malaria. In an embodiment, griseofulvin or N- methyl protoporphyrin or a salt, homolog, analog, isoform or enantiomer thereof inhibits a host and/or parasite ferrochelatase.

[0019| In a further embodiment, administration is in combination or conjunction with one or more further anti-Apicomplexa agents including antimalaria agents.

[0020] Taught herein is a kit comprising griseofulvin or N-methyl protoporphyrin or a salt, homolog. analog, isoform or enantiomer thereof for use in the treatment or prophylaxis of infection by a member of phylum Apicomplexa such as infection by Plasmodium sp.

[0021] In an embodiment, the kit includes instructions for use in the treatment or prophylaxis of malaria and may also include one or more other active agents such as another anti-Apicomplexa agent or an anti-malaria agent. By "anti-malaria agent" includes an agent that targets the parasite such as the intraerythrocytic life-cycle stages of the parasite as well as an agent which ameliorates the symptoms of malaria.

[0022] A drug-screening method is enabled herein comprising (i) testing or selecting or designing a drug for its ability to inhibit ferrochelatase or another enzyme in the heme biosynthetic pathway or a precursor thereof and (ii) if a ferrochelatase inhibitor or another inhibitor of another enzyme in the heme biosynthetic pathway or a precursor thereof is identified, testing the drug, for anti-Apicomplexa activity in vitro or in vivo. In a related embodiment, a screening assay for an anti-malaria drug is provided herein comprising screening for a compound which inhibits ferrochelatase and then testing the drug for its ability to inhibit growth or development of a malarial parasite. An isolated enzyme in the heme biosynthetic pathway for use in screening for a medicament for the treatment of malaria is also contemplated.

{0023] In an embodiment, the Apicomplexa is Plasmodium

|0024) In an embodiment, the enzyme is host ferrochelatase. In an embodiment, the enzyme is a parasite ferrochelatase. By "parasite" is meant the Apicomplexa including Plasmodium sp.

[0025] In an embodiment, the drug is sub-licensed for use in drug testing.

[0026] The above summary is not and should not be seen in any way as an exhaustive recitation of all embodiments herein described.

BRIEF DESCRIPTION OF THE FIGURES

[0027] If figures contain color representations or entities, colored versions of the figures are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.

[0028| Figure 1 is a photographic representation of data showing that host ferrochelatase (Fech) is present in uninfected RBCs (uiRBC) and compartmentalised inside the parasite. (A and B ) Western blot analyses of protein samples prepared from uiRBC and purified P. falciparum parasites (free of red blood cell membrane and cytosol ). Proteins were separated on denaturing SDS polyacrylamidc gels, transferred to nitrocellulose membranes and immuno-blotted with anti-human Fech antibody (A) and anti-human hexokinase antibody (B). A band corresponding to the predicted size of human Fech (43 kD) is present in both the red cell and parasite samples. A band corresponding to the predicted size of human hexokinase ( 1 30 kD) is present only in the red cell sample, indicating that the parasite sample is free from red cell contamination. Note that while the parasite Fech homolog is predicted to be a similar size to human Fech. there is only 26% identity between the proteins and cross reaction of the anti-human Fech antibody to the parasite counterpart is therefore unlikely. (C-G) Images showing P. falciparum infected red blood cells immuno-stained with anti-human FECH and anti-EXP2 antibodies, and stained with the DNA-specific dye DAPI (to detect parasite nuclei). Anti-EXP2 was used to detect the parasitophorous vacuole membrane. The images overlaid (merge) to sho co-localization (anti-FECH in green, anti-EXP2 in red and DAPl in blue). Relatively low intensity anti-FECH staining is evident in uninfected red cells (uiRBC) (C), this staining was distinct as compared to background levels of signal detected by isotype control antibodies (D); Different developmental stages of the parasite are depicted in (E) early ring-stage, (F) mature trophozoite stage, and (G) schizont stage. The extent and intensity of the anti-FECH staining is greatest at the later stages of parasite development (trophozoite and schizont), particularly adjacent to or surrounding the parasite nuclei and parasitophorous vacuole. [0029] Figure 2 is a graphical representation of data showing parasitological kinetics and survival curves of mice infected with P. chabaudi. The mouse strains (on a C57BL/6 genetic background) used were Fech+/+ (WT), Fech+/- (Het) and Fech-/- (Mut). (A and C) Parasitemia (% of iRBC) in female and male mice, respectively. Numbers of mice used are indicated in the figure key. There was a significant difference between Fech+/+ and Fech-/- mice for both sexes at Day 6 of the infection. Fech+/+ n=22, Fcch+/- n= l 7, Fech-/- n=T 2 and 16). (B and D) Survival curves for female and male mice, respectively. Fech+/+. Fech+/- and Fech-/- mice following infection and monitored for survival. The Fech-/- mice showed a significant increase in survival compared to Fech+/+ and Fech+/- for both sexes (p < 0.05 by Mantel-Cox_. log-rank test for survival).

[0030] Figure 3 is a graphical representation of data showing that P. falciparum growth is impaired when cultured in blood from individuals with Fech deficiencies (erythropoietic protoporphyria. EPP). Proportions of cells infected with parasites (% parasitemia) were determined a different time points after in vitro infection of red blood cells purified from blood samples collected from three different individuals w ith EPP (A- C) or a normal individual (D). Data represent the mean of triplicate cultures at each time point; at least 500 cells were counted for each replicate. Error bars indicate SEM.

[0031 ] Figure 4 is a graphical representation of data described in Example 3 showing the anti-plasmodial effect of NMPP (a specific Fech inhibitor) and griseofulvin. (A) Growth inhibition of P. falciparum cultured in the presence of different concentrations of NMPP (relative to an imtreated culture). (B) Growth inhibition of P. falciparum in cultured in red blood cells that were treated prior to infection with various concentrations of griseofulvin. Red blood cells were treated with griseofulvin for 3 days, with the medium replaced each day; griseofulvin was also included in the parasite cultures. Three different strains of P. falciparum were tested: 3D7 (chloroquine susceptible), l (chloroquine resistant) and W2mef (chloroquine and metloquine resistant). In A and B. each data point represents the mean of two independent experiments performed in triplicate (at least 500 cells counted in each replicate). Error bars indicate SEM. Further results are provided in Figure 7 described in Example 5. [0032] Figure 5 is a graphical representation of data described in Example 4 showing that administration of griseofulvin to human subjects results in red cells unable to support parasite growth ex vivo. In all experiments isolated red cells were used. They were prepared by centrifugation and washing (in phosphate-buffered saline) the blood sample to remove contaminating white blood cells and platelets. (A) Percentage of parasite-infected cells (% parasitemia) in blood collected before (untreated) and follow ing a 7 day course of griseofulvin (treated; 500mg/day taken orally). Purified red blood cells (untreated and treated) were prepared and infected with P. falciparum strain 3D7 at the same day. The mean (SD) initial percentage of infected cells was 1 .01 % (0.34). Parasite cultures were smeared after 24 hours, stained with Giemsa stain and numbers of infected cells enumerated using light microscopy. Seven different individuals were tested. For all the individuals tested, parasite growth was significantly impaired in blood after 7 days of griseofulvin treatment (p < 0.01 ). Data points represent the mean of 3 replicate cultures. At least 1000 cells were counted per replicate. Error bars indicate SEM. (B) Parasite growth in blood collected each day from one individual taking a daily 500mg/day course of griseofulvin for five days. Growth (determined as described in A) was compared as a proportion with cultures using untreated blood. The proportion of growth in the treated blood is significantly less that untreated after four and five days of griseofulvin (pO.01 ). Data points represent the mean of 3 replicate cultures. At least 1000 cells were counted per replicate. Error bars indicate SEM. (C) Levels of griseofulvin measured in blood during a five day course of griseofulvin (500mg/day taken orally ; same individual as in B). Levels were determined by HPLC-coupled MS-MS analysis in conjunction with a griseofulvin standard concentration curve. (D) Parasite growth in blood collected from one individual at indicated time points after taking one single dose (2 g) of griseofulvin. Growth is compared as a proportion with cultures using untreated blood. The proportion of growth in the treated blood is significantly less that untreated 8, 24 and 48 hours after taking the drug (pO.O l ). Data points represent the mean of 3 replicate cultures. At least 1000 cells were counted per replicate. Error bars indicate SEM. (E) Levels of griseofulvin measured in blood after a single dose of griseofulvin (2000mg taken orally; same individual as in D). Levels were determined by HPLC analysis using a griseofulvin standard concentration curve. Further results are described in Example 6 and illustrated in Figures 8 or 9. [0033) Figure 6 is a graphical illustration of results, wherein P. falciparum is grown in red cells from individuals with erythropoietic protoporphyria (EPF) and X-linked dominant protoporphyria (XLDPP). Results are shown as a percentage of parasitemia of test blood relative to normal red cells. In all experiments, infection was initiated by the addition of purified trophozoite-stage infected cells (grown in normal cells) to the test blood (EPP. XLDPP or normal) to a final concentration of 1 %. Relative parasitemias were determined at the times indicated. Average percentages (± SD) of infected cells measured in the normal blood were, 0 hr, 1 .1 (±0.9); 24 hr. 2.0 (±1. 1 ); 48 hr. 3.2 (± 1.3); 72 hr. 6. 1 (± 1 .7). The data (± SEM) represent the mean percentage of parasite growth in cells from multiple patients (n=4 for EPP, n=3 for XLDPP). Duplicate independent assays (each in triplicate) were performed for each cell sample.

(0034] Figure 7 provides a graphical illustration of results described in Example 5 extending the results provided in Example 3 and Figure 4 showing pharmacological inhibition of FECH using griseofulvin and NMPP and growth inhibition of P. falciparum infected human red cells. Parasite growth inhibition (relative to untreated control) with increasing concentrations of NMPP (0. 1 to 100000 iiM NMPP) (A) and griseofulvin (0.08 to 250 μΜ griseofulvin) (B). Assays were conducted using P. falciparum 3D7. l and W2mef strain parasites and growth was determined after 48 h incubation. Data (± SEM) represent the mean of two independent assays (each concentration assayed in triplicate). Titration of parasite growth inhibition using NMPP (50μΜ) or griseofulvin (250μΜ) and increasing concentrations of PPIX. Assays were conducted using P. falciparum 3D7 parasites and growth was determined relative to an untreated control after 48 hours incubation. Data (± SEM) represents mean of two independent assays (each concentration assayed in triplicate). * P < 0.05. * * p < 0.01 , * * * p < 0.001 compared to untreated cells.

[0035] Figure 8 illustrates the results described in Example 6 wherein griseofulvin treated human red blood cells are unable to support parasite growth ex vivo Panel A shows the percentage of parasite growth in red cells collected from seven individuals taking griseofulvin (500 mg/day) after seven days: (panel B) or each day for five days; (panel C) or at eight hours and (panel D) at indicated time points after a single dose of 2000 mg-. All assays were conducted by inoculating red cells (with purified trophozoite-stage infected cells (mean parasitaemia, 1 ±0.5%). Growth was determined relative to an untreated control after 24 hours incubation Data (± SEM) represent the mean of 3 replicate cultures. * * p< 0.01 and * * * p < 0.001 compared to untreated cells.

[0036] Figure 9 provides an illustration of the results of ultra performance liquid chromatography-mass spectrometry (UPLC-MS) analysis of griseofulvin in red cells from individuals taking griseofulvin for five days (500 mg/day) (A) or once (2000 mg) (B), respectively. Blood samples were collected and analysed at the indicated times, and quantified by comparison to a stEindard curve of known griseofulvin amounts. Data (± SEM) represent the mean of three independent assays.

[0037] Figure 10 provides an illustration of the results of ultra performance liquid chromatography-mass spectrometry (UPLC-MS) analysis of griseofulvin in red cells treated with 10 μΜ griseofulvin for up to five - days, and sampled every 24 hours. Griseofulvin-containing medium was replaced every 24 hours. Griseofulvin levels were quantified by comparison to a standard curve of known griseofulvin amounts. Data (± SEM) represent the mean of three independent assays.

DETAILED DESCRIPTION

[0038] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or element or method step or group of integers or elements or method steps but not the exclusion of any other integer or element or method step or group of integers or elements or method steps.

[0039] As used herein the singular forms "a^", "an^"' and "'the'- include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a Plasmodium sp." Includes one Plasmodium sp., as well as two or more species of Plasmodium; reference to "an Apicomplexa member" includes one member, as well as two or more members; reference to "the disclosure" includes a single and multiple aspects taught by the disclosure; and so forth. Aspects taught, described and/or claimed herein are encompassed by the term "invention" and are within the width of the disclosure.

[0040] The present disclosure teaches the use of griseofulvin or N-methyl protoporphyrin or a salt, homOlog, analog, isoform or enantiomer thereof to specifically inhibit growth and development of a member of Phylum Apicomplexa thereby being useful in the treatment or prophylaxis of infection in a subject by the member. Further taught herein is the amelioration of symptoms of disease caused by the infection.

[0041 j Griseofulvin has the chemical formula C 1 7 H 17 CI06 (The Merek Index ( 1983 ) 10th Edition, pp. 4433-34)) and its structure is set forth in Formula (I).

Formula (I)

[0042| The term "griseofulvin" includes its salts, homolog, analog, isoforms and enantiomers. An "enantiomer" including steriosomers. Functional equivalent forms of griseofulvin are also contemplated herein. A functional equivalent includes a molecule sterically similar to griseofulvin and having the same ability to inhibit a host cell ferrochelatase and/or inhibit development of a stage of the parasite life-cycle. An example of a functional equivalent includes the change or modification of a substituent on the griseofulvin molecule, selecting a particular isoform, natural ly occurring or prepared by physical or chemical manipulation and selecting a molecule with the same physical or chemical properties as griseofulvin. [0043] Griseofulvin administrations results in pharmacological inhibition of Fech leading to reduced parasite growth and/or death of intraerythrocytic li fe cycle stages. N- methy protoporphyrin is produced from griseofulvin in the host via a cytochrome- dependent mechanism and shares the chemical property of griseofulvin as a FECH inhibitor by competing with PPIX for ferrochelatase.

[0044] N-methyl protoporphyrin has the chemical formula C35H36N404 and its structure is set forth in Formula (II)

Formula (II)

[0045] The term "N-methyl protoporphyrin" (also referred to as NMPP) includes its salts, homolog, analog, isomer, ester, isoforms and enantiomers. An "enantiomer" including steriosomcrs. Functional equivalent forms of N-methyl protoporphyrin arc also contemplated herein. A functional equivalent includes a molecule sterically similar to N- methyl protoporphyrin and having at least the same ability to inhibit ferrochelatase and/or inhibit development of a stage of the parasite life-cycle. An example of a functional equivalent includes the change or modification of a substituent on the N- methylprotoporphyrin molecule, selecting a particular active form, natural ly occurring or prepared by physical or chemical manipulation and selecting a molecule with the same desirable physical or chemical properties as N-methyl protoporphyrin. [0046J Hence, taught herein is a method for the treatment or prophylaxis of a subject infection with or who has or who may have exposure to a member of Phylum Apicomplexa, the method comprising administering to the subject an effective amount of griseofulvin or N-methyl protoporphyrin or its salt, homolog, analog, isoform or enantionmer.

[0047] The griseofulvin or N-methyl protoporphyrin may also be given in a composition. Hence, taught here is a method for the treatment or prophylaxis of a subject infected with or who has or who may have or who had exposure to a member of Phylum Apicomplexa. the method comprising administering to the subject an effective amount of a composition comprising griseofulvin or N-methyl protoporphyrin or its salt, homolog. analog, isoform or enantiomer. The composition may further comprise one or more pharmaceutically acceptable carriers, di luents or excipients. The composition may also contain another active agent such as another anti-Apicomplexa agent or anti-malarial agent or an agent which ameliorates symptoms of an infection by a member of Phylum Apicomplexa. The agent may also specifically target a ferrochelatase produced by a host cell or by the parasite or both.

(0048) As taught herein, an example of a Apicomplexa member is a species of Plasmodium (Plasmodium sp.). Examples of Plasmodium sp. include Plasmodium falciparum, Plasmodium virax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi.

[0049] Hence, the present specification is instructional for the treatment or prophylaxis of a subject for infection with or who has or who may have exposure to Plasmodium sp.. the method comprising administering to the subject an effective amount of griseofulvin or N-methyl protoporphyrin or its salt, homolog, analog, isoform or enantiomer.

[0050] A related embodiment taught herein is a method for the treatment or prophylaxis of a subject for infection with or who has or who may have exposure to Plasmodium sp., the method comprising administering to the subject an effective amount of a composition comprising griseofulvin or N-methyl protoporphyrin or its salt, homolog, analog, isoform or enantiomer. [0051 ] As above, the composition may further comprise one or more pharmaceutically acceptable carriers, diluents or excipients. The composition may also contain another active agent such as another anti-Apicomplexa agent or an agent which ameliorates symptoms of an infection by Plasmodium sp.

[0052] In an embodiment, the Plasmodium sp. is selected from the list consisting of P. falciparum, P. virax, P. ovale, P. malariae and P. knowlesi.

[0053] Hence, the present specification is instructional for the treatment or prophylaxis of a subject for infection with or who has or who may have exposure to Plasmodium sp. selected from the list consisting of P. falciparum, P. virax, P. ovale, P. malariae and P. knowlesi, the method comprising administering to the subject an effective amount of griseofulvin or N-mcthyl protoporphyrin or its salt, homolog, analog, isoform or enantiomer.

[0054] A related embodiment taught herein is a method for the treatment or prophylaxis of a subject for infection with or who has or who may have exposure to Plasmodium sp. selected from the list consisting of P. falciparum, P. virax, P. ovale, P. malariae and P. knowlesi, the method comprising administering to the subject an effective amount of a composition comprising griseofulvin or N-methyl protoporphyrin or its salt, homolog, analog, isoform or enantiomer.

[0055] As above, the composition may further comprise one or more pharmaceutically acceptable carriers, diluents or excipients. The composition may also contain another active agent such as another anti-Apicomplexa agent or an agent which ameliorates symptoms of an infection by Plasmodium sp.

[0056] In an embodiment, the Plasmodium sp. is P. falciparum.

[0057| Hence, the present specification is instructional for the treatment or prophylaxis of a subject for infection with or who has or who may have exposure to P. falciparum, the method comprising administering to the subject an effective amount of griseofulvin or N- methyl protoporphyrin or its salt, homolog, analog, isoform or enantiomer. [0058] A related embodiment taught herein is a method for the treatment or prophylaxis of a subject for infection with or who has or who may have exposure to P. falciparum, the method comprising administering to the subject an effective amount of a composition comprising griseofulvin or N-methyl protoporphyrin or its salt, homolog, analog, isoform or enantiomer.

[0059] As above, the composition may further comprise one or more pharmaceutically acceptable carriers, diluents or excipients. The composition may also contain another active agent such as another anti-Apicomplexa agent or an agent which ameliorates symptoms of an infection by Plasmodium sp.

[0060] In an embodiment, the subject is in need of treatment or prophylaxis for malaria. In this case, the subject may be infected with Plasmodium sp. or is at risk of becoming infected.

[0061 ] In an embodiment, administration is prior exposure to Plasmodium sp.

[0062] In an embodiment, a subject or population of subjects is (i) tested for infection with a member of Phylum Apicomplexa and (ii ) administered a composition comprising griseofulvin or N-methyl protoporphyrin or a salt, homolog. analog, isoform or enantiomer thereof if the subject or population of subjects tests positive for infection by a member of Apicomplexa.

[0063] In an embodiment, the subject is (i) tested for infection with Plasmodium sp. and (ii) administered a composition comprising griseofulvin or N-methyl protoporphyrin if the subject tests positive for Plasmodium sp.

[0064] In an embodiment, the subject is tested for infection or infection levels with Plasmodium sp. after administration of griseofulvin.

[0065] In an embodiment, the subject is treated after exposure to Plasmodium sp.

[0066) Pharmaceutical compositions are conveniently prepared according to conventional pharmaceutical compounding techniques. See, for example. Remington's Pharmaceutical Sciences. 18th Ed., ( 1990) Mack Publishing, Company. Faston, PA. U.S.A. These compositions may comprise, in addition to griseofulvin or N-methyl protoporphyrin or a salt, homolog. analog, isoi rm or enantiomer thereof, a pharmaceutically acceptable excipient, carrier, diluent or which includes a buffer, stabilizer or other material well known in the art. Such materials are generally non-toxic and do not interfere with the efficacy of the active ingredient. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g. intravenous, oral, parenteral or topical.

[0067] The present disclosure is also directed griseofulvin or N-methyl protoporphyrin or a salt, homolog, analog, isoform or enantiomer thereof for use in the treatment or prophylaxis of infection by a member of Phylum Apicomplexa. This aspect includes the use of griseofulvin or N-methyl protoporphyrin or a salt, homolog. analog, isoform or enantiomer thereof in. or in the manufacture of a medicament for, the treatment or prevention of infection of a member of Phylum Apicomplexa.

[0068] The present disclosure is also directed to griseofulvin or N-methyl protoporphyrin or a salt, homolog, analog, isoform or enantiomer thereof for use in the treatment or prophylaxis of infection by Plasmodium sp. This aspect includes the use of griseofulvin or N-methyl protoporphyrin or a salt, homolog, analog, isoform or enantiomer thereof in, or in the manufacture of a medicament for, the treatment or prevention of infection Plasmodium sp.

[0069] The present disclosure is also directed griseofulvin or N-methyl protoporphyrin or a salt, homolog, analog, isoform or enantiomer thereof for use in the treatment or prophylaxis of malaria. This aspect includes the use of griseofulvin or N-methyl protoporphyrin or a salt, homolog, analog, isoform or enantiomer thereof in, or in the manufacture of a medicament for, the treatment or prevention of malaria.

[0070] Griseofulvin or N-methyl protoporphyrin or a salt, homolog. analog, isoform or enantiomer thereof or a composition comprising same is generally administered for a time and under conditions sufficient to inhibit infection or attenuate or reduce the ability for parasite or life-cycle stages thereof to cause an adverse infection. In an embodiment, griseofulvin or N-methyl protoporphyrin or a salt, homolog, analog, isoform or enantiomer thereof or a composition comprising same is administered for a time and under conditions sufficient to ameliorate by symptoms of malaria or reduce the risk of onset of a severe from of malaria. In an embodiment, the griseofulvin or N-methyl protoporphyrin or a salt, homolog, analog, isoform or enantiomer thereof is administered for a time and under conditions sufficient to inhibit a host and/or parasite, ferrochelatase.

[0071] The terms "effective amount" includes "therapeutically effective amount" and "prophylactically effective amount" and means a sufficient amount of a composition comprising griseofulvin or N-methyl protoporphyrin or a salt, homolog. analog, isoform or enantiomer thereof either in a single dose or as part of a series or slow release system which provides the desired therapeutic, preventative, epidemiological or physiological effect in some subjects. Undesirable effects, e.g. side effects, may sometimes manifest along with the desired therapeutic effect: hence, a practitioner balances the potential benefits against the potential risks in determining an appropriate "effective amount". The exact amount of composition required will vary from subject to subject, depending on the species, age, and sex general condition of the subject, mode of administration and the like. Thus, it may not be possible to specify an exact "effective amount". However, an appropriate "effective amount" in any individual case may be determined by one of ordinary skill in the art using routine skills or experimentation. One of ordinary skill in the art would be able to determine the required amounts based on such factors as prior administration of the compositions or other agents, the subject's size and age, the severity of a subject's symptoms or the severity of symptoms in an infected population, member of Phylum Apicomplexa, species of Plasmodium, and the particular composition or route of administration selected. In an example, from about l mg to about 50g of griseofulvin or N- methyl protoporphyrin is administered to a subject. Examples of this dosage range include from about 20mg to about 20g such as 30, 50. 80, 100, 200, 300, 500, 700. 800. l OOOmg, 1.5g, 3g, 5g and l Og as well as amounts in between. Administration may also be measured in terms of plasma concentration. Hence, an effective amount includes plasma concentration levels of griseofulvin from about 0^g/ml blood to about 20μg/ml blood including about 1 , 2, 3. 4, 5, 6, 7, 8, 9, 10, I I , 12, 13, 14, 15, 16, 17. 18, 19 and 20μg/ml blood. A single or multiple doses may be administered such as hourly, daily, weekly, monthly or yearly. For example, a dose of from 100-2000mg (e.g.. 500mg or 2000mg) may be given daily lor 7 days. The effective amount may also be determined on the minimal amount to inhibit or reduce activity of host and/or parasite ferrochelatasc. The effective amount may also be determined on the minimal amount to inhibit or reduce inraerythrocytic malarial parasite growth or development. The IC50 of griseofulvin was found to be about 10 μπτ to 50 μηι. Effective amounts of NMPP sufficient to reduce parasite growth in red cells are in the 25nM range (See Figure 4A) or at least in the l OnM to Ι ΟΟηΜ range (see Figure 7A). l OOOOOnM NMPP was effective in completely inhibiting parasite growth ( 100% inhibition) in three different P. faciparum strains.

[0072] The term "treatment" refers to any measurable or statistically significant amelioration in at least some subjects in one or more symptoms of infection or a disease caused thereby such as malaria or in the risk of developing severe symptoms of or the risk of transmitting the disease. The term treatment also includes a measurable decrease in host and/or parasite ferrochelatase.

[0073] The terms "prevention", "attenuation" and "prophylaxis" and the like are used interchangeably and include administration of griseofulvin or N-methyl protoporphyrin or a salt, homolog, analog, isoform or enantiomer thereof or a composition comprising same to a subject not known to be infected with a member of Apicomplexa for the purpose of prevention or reducing the risk of becoming infected or reducing the severity or onset of a disease caused or exacerbated by infection by Apicomplexa sp. In an example, the member is Plasmodium sp. and the disease is malaria.

[0074] In one embodiment, a composition comprising griseofulvin or N-methyl protoporphyrin or a salt, homolog, analog, isoform or enantiomer thereof is provided to a subject either before the onset of infection (so as to prevent or attenuate an anticipated infection) or after the initiation of an infection to treat the infection or a disease caused thereby, such as malaria. In an embodiment, a composition is provided to a subject before or after onset of infection, to reduce transmission of infectious agents between subjects.

[0075] In a further embodiment, administration is in combination or conjunction with one or more further anti-Apicomplexa agents, including anXi-Plasmociium sp. agents as well as anti-malaria agents. [0076] Compositions taught herein can be administered as the sole active pharmaceutical agent, or used in combination with one or more agents to treat or prevent Apicomplexa infection or symptoms associated with Apicomplexa infection. These agents include, but are not limited to, host immune modulators (for example, interferon-alpha, pegylated interferon-alpha, consensus interferon, interferon-beta, interferon-gamma. CpG oligonucleotides and the like); in the case of Plasmodium sp. infection anti-malarial compounds/agents such as artemether, artemisinin, artemotil and artesunate. quinine, alone or in further combination with one or more of lumefantrine, malarone, chloraquine. mefloquine, · pyronaridine, amodiaquine, piperaquine, sulphadoxine-pyrimethamine. clindamycin, doxycycline, dihydroartemisinin. primaquine, and the like: antibodies or antigen-binding portions that directly or indirectly inhibit or reduce infection; compounds that enhance the development of an effective antibody and/or T cell response; interfering RNA; anti-sense RNA; vaccines comprising malaria antigens or antigen adjuvant combinations directed against Apicomplexa member such as Plasmodium sp.

[0077] Administration is generally for a time and under conditions sufficient to elicit the desired response. Administration may be parenteral or non-parenteral, systemic or local. Other contemplated routes of administration are by patch, cellular transfer, implant, sublingually, intraocularly, topically, orally, rectally, vaginally, nasally or transdermal ly. Griseofulvin or N-methyl protoporphyrin may be administered orally, as known in the art.

[0078] Taught here is a kit comprising griseofulvin or N-methyl protoporphyrin or a salt, homolog, analog, isoform or enantiomer thereof and optionally one or more further agents.

[0079] In an embodiment, the kit includes instructions for use in the treatment or prophylaxis of infection by a member of Phylum Apicomplexa such as Plasmodium sp. including those which cause malaria. Hence, the kit may contain an anti-malaria agent. By "anti-malaria agent includes an agent which targets the parasite or a life-cycle form of the parasite as well as an agent which ameliorates the symptoms of malaria or which targets host or parasite ferrochelatase activity or expression. [0080] A drug-screening method is provided herein comprising (i) testing or selecting or designing a drug for its ability to inhibit ferrochelatase or another enzyme in the heme biosynthetic pathway or a precursor thereof and (ii) if an inhibitor is identified, testing the drug, for anti-Apicomplexa activity in vitro or in vivo. In a related embodiment, a screening assay for an anti-malaria drug is provided herein comprising screening for a compound which inhibits ferrochelatase and then testing the drug for its ability to inhibit growth or development of a malarial parasite.

[0081 1 In an embodiment, the Apicomplexa is Plasmodium sp.

[00821 ^{m an} embodiment, the enzyme is ferrochelatase.

[0083] In an embodiment, the enzyme is a host heme synthetic enzyme.

[0084| In an embodiment, the enzyme is a host ferrochelatase. In an embodiment, the enzyme is a parasite ferrochelatase. By "parasite" is meant the Apicomplexa species or genera, including Plasmodium sp.

[0085] Furthermore, a screening assay is enabled for an anti-malaria drug, the assay comprising screening a compound which inhibits human ferrochelatase and the testing the drug for its ability to inhibit growth or development of Plasmodium sp. such as P. falciparum.

[0086| The terms "treating" and "treatment" as used herein refer to reduction in severity and/or frequency of symptoms of an infection, elimination of the infection and/or improvement or remediation or amelioration of a disease condition following infection by the Apicomplexa member. In general terms, treatment may involve actively treating a disease arising from or exacerbated by the infection treatment or treating the infection.

[0087[ "Treating" a subject, therefore, may involve prevention or reduction in extent of development of infection or a condition arising therefrom as well as treatment of a clinically symptomatic individual by ameliorating the symptoms of the infection or treating as a symptomatic subject exposed to potential infection. [0088] In an embodiment, the infection is by a Plasmodium sp. such as a Plasmodium sp. selected from the list consisting of human infective species, P. falciparum. . virax, . ovale, P. malariae and P. knowlesi.

[0089| Also enabled herein is a method for the treatment or prophylaxis of a subject with, suspected of having or previously exposed to malaria, the method comprising administering to the subject an effective amount of griseofulvin or N-methyl protoporphyrin or its salt, homolog. analog, isoform or enantiomer.

[0090] In an embodiment, taught herein is a method for the treatment or prophylaxis of a subject with, suspected of having or previously exposed to malaria, the method comprising administering to the subject an effective amount of a composition comprising griseofulvin or N-methyl protoporphyrin or its salt, homolog. analog, isoform or enantiomer.

[0091 ] A "subject" as used herein refers to an animal, such as a mammal including a human who can benefit from the pharmaceutical agents and formulations and methods of the present disclosure. Animal models arc encompassed. A subject regardless of w hether a human or non-human animal may also be referred to as an individual, patient, animal, host or recipient.

[0092] Aspects taught and enabled herein are further described by the following non- limiting Examples. In the Examples, the following methods and materials are employed.

[0093J P. falciparum culture. P. falciparum strains 3D7, l and W2mef were maintained at between 0.5 and 10% parasitemia in purified AB+ human erythrocytes in a 1 % 02/5% C02 atmosphere according to the method of Trager and Jensen (Trager et al.. (1976)). The cell culture medium (CCM) comprised of RPMI 1640 supplemented with I X Glutamax, 0.2% Albumax (all from Gibco), 4% pooled AB+ human serum (Invitrogen), 10 mM D-glucose, 25 ng/ml gentamycin, 6 mM HEPES and 0.2 mM hypoxanthine (al l from Sigma). [0094] Collection and preparation of purified red blood cells. Blood from individuals with porphyrias and from individuals treated with griseofulvin was collected by venepuncture into 5 ml sodium citrate tubes. Blood was then centrifuged at 1 70g for 1 3 minutes and the plasma and white cel l fractions removed. Blood was washed two times in RPMI and stored at 4 °C, and then washed further prior to use.

|0095| P. falciparum growth inhibition assay. Synchronized P. falciparum ring-stage parasites were grown in the presence of increasing concentrations of N- methylprotoporphyrin (NMPP, Frontier Scientific) for 48 hours prior to analysis of growth and subsequent comparison with an untreated control. For the growth assays involving in vitro griseofulvin treatment, uninfected RBC were incubated with griseofulvin for 3 days prior to the addition of synchronized ring-stage P. falciparum parasites. The medium and griseofulvin was replaced every 24 h during the pre-incubation and growth assay periods. For PPIX titration experiments, PPIX was added in varying concentrations to RBCS with either 50μΜ NMPP, 250μΜ griseofulvin (as pre-incubated above) or PPIX alone and incubated for 48 h prior to analysis.For growth assays using the porphyric blood and blood from the griseofulvin-treated individuals, parasites grown in normal untreated blood were harvested by Percoll density gradient centrifugation or MACS column (Rivadeneira et al.. ( 1983); Ribaut el al., (2008)) and then added to the test blood at a final percentage parasitemia of approximately 0.5%. Thin-blood smears were obtained after incubation of the parasites for up to 72 hours, stained with 10% Giemsa stain and the percentage of infected parasites (% parasitemia) was determined by counting cells under a light microscope. At least 1000 cells were counted on each slide. In some experiments parasite growth was also quantified by flow cytometry analysis using YOYO- 1 dye (Li el al.. (2007)). Percentage parasite growth and percentage growth inhibition were determined by comparison of growth in cultures treated without drug, or in blood from normal, untreated individuals.

[0096] Experimental P. chabaudi infection. Fech^{m X fAi} mice, originally produced from an ENU-mutagenesis screen at the Pasteur Institute (Paris) were kindly provided by X. Montagutelli. Female and male Fech+/+, Fech+I- and Fech-I- mice (genotyped as per Abitbol el al., (2005)) on a C57BL/6 background were infected with the rodent malarial species Plasmodium chabaudiadami DS at 7- 12 weeks of age. Female mice were routinely infected intravenously with 5 x l O'iRBC/mL and male mice were infected with 2.5 x 105iRBC/mL RBC. Blood stage parasitemia (% infected red blood cells) for each mouse during infection was determined by counting cells on Giemsa-stained thin blood smears taken from mice during the course of infection. Percentage parasitemia was calculated by counting at least 500 cells per slide.

[0097] UPLC-MS analysis of griscofulvin levels in blood. Quantification of griseofulvin in blood from griseofulvin treated volunteers or griscofulvin pre-incubated red cells was analysed by UPLC-MS using a Waters Acquity H-series UPLC coupled to a Waters Xevo triple quadrupole mass spectrometer as described previously (Mensch el al., (2007)).

[0098) lmmunoblotting. Late-stage parasitized cells (trophozoites and schizonts) were harvested from P. falciparum cultures (at approximately - 10% parasitemia) using Percoll density gradient centrifugation (Rivadeneira el al., ( 1983)); 80-90% of the harvested cells were infected. To isolate the parasites from their host red cells, the harvested parasites were treated with 10 volumes of saponin (0.1 5 % w/v) for 1 0 minutes on ice. Follow ing centrifugation, the resulting supernatant (containing the hemoglobin, red cell cylosol components and plasma membranes of uninfected cells) was removed and the parasite pellet was washed with RPM1 and then resuspended in PBS containing protease inhibitors (Complete® protease cocktail from Roche). The parasites were then subjected to sonication (3 x 30 sec) and protein quantified using a Bradford protein assay. Puri fied parasites and equivalent numbers of uninfected red blood cells were lysed in equal volume of 2x SDS loading buffer (containing 0.5 % β-mercaptoefhanol), heated at 95 °C for 5 min. and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 12% gel from Biorad), and then transferred to nitrocellulose membrane. Membranes, blocked in 1 % blocking reagent (Roche) overnight, were then blotted with the fol lowing antibodies (in 0.5% blocking reagent): mouse monoclonal anti-human FECH ( 1/200 dilution; sc-271434), mouse polyclonal anti-human hexokinase ( 1 /200 dilution; sc-46695 ; both from Santa Cruz, CA) and rabbit anti-mouse conjugated peroxidase ( 1 /2000 dilution; Sigma). Blotted membranes were visualized using enhanced chemiluminescence (ECL) reagents (Thermoscientific).

10099] Immunofluorescence staining. Thin blood smears prepared from P. falciparum cultures were airdried, fixed for 30 s in 100% methanol, and then fixed for 20 min in 1 % paraformaldahyde in PBS. Slides were washed in PBS and blocked with 1 % BSA/0. 1 % Triton X 100 in PBS (PBT). Slides were then incubated in the following primary antibodies (overnight at 4 °C, diluted in PBT); mouse monoclonal anti-human FECH and rabbit anti-/⁵ EXP2, and after washing, incubated in donkey anti-mouse Alexa Fluor® 488 ( 1 : 1000) or goat anti-rabbit Alexa Fluor® 594 ( 1 : 1000; both from Invitrogen) for 90 min at room temperature in the dark. Slides were counter-stained with DAPI ( 1 :5000), washed and mounted in Slowiade Gold (Invitrogen). Slides were viewed on a Nikon inverted microscope at 60x magnification with a water immersion lens at room temperature. Images were obtained using an Evolve camera and NIS-Elements software.

|01001 Statistical Analysis. P values were calculated using two-tailed t-tests assuming equal variance.

EXAMPLE 1 : Detection of host ferrochelatase in malarial parasites

[0101 ] Immunohistochemical staining using anti-human ferrochelatase (anti-hFech) antibodies show diffuse and even staining within the cytoplasm of uninfected red cells (Figure 1 C), consistent with presence of hFech (EC 4.99.1 .1 ) in mature cells that is not localized to a particular cell compartment. In infected cells distinctive and more intense staining is observed in close association with the parasite nucleus. This confirmed the findings of others (Varadharajan el al , (2004)) that mature erythrocytes contain FECH and that host FECH is located with Plasmodium parasites. The parasite apparently uses the enzyme to supplement its own synthesis of heme (Varadharajan et al. , (2004)). The staining intensity is greater in more mature forms of the parasite (trophozoite) compared to earlier developmental stages (ring forms) (Figure 1 C-G). The distinctive localization and intensity of staining in infected cells is consistent with the presence of hFech protein within the parasite (Figure 1 A). Probing the same filters with host hexokinase antibody, employed as a cytoplasmic red cell marker, ruled out host red cell contamination in the parasite preparation (Figure I B). |0102] Taken together, the western blot and immunohistochemical analyses indicate that the hFech protein is present within the mature erythrocyte and within the parasite.

EXAMPLE 2: A deficiency in host ferrochelatase is detrimental to parasite growth

[0103| To determine the role ferrochelatase (Fech) plays in sustenance of a malarial infection, deficiencies in human Fech (hFech) are investigated and found to slow malarial growth.

[0104| The contribution of host Fech to malarial infection was investigated using two separate approaches. In the first approach, the growth rates of P. falciparum were examined in blood cells collected from individuals with naturally occurring Fech enzyme deficiency, called erythropoietic protoporphyria (EPP). The growth rates of parasites cultured in EPP blood is markedly delayed (Figures 3A through C). The degree of growth inhibition depended on the type and severity of the Fech deficiency, with more severely affected red cells supporting less parasite growth. These results indicate that a functional red cell Fech enzyme is important for parasite growth in red blood cells.

[0105] This study was extended and the results are represented graphically in Figure 6. Ferrochelatase deficiency manifests in humans as erythropoielicprotoporphyria ( F.PP; MIM 1 77000) (Puy et. al., (2010)). The condition is inherited in a pattern that resembles autosomal dominance with low penetrance due to the co-inheritance of a common hypomorphic E H allele (Gouya et al. , (2002)). Red cells from four human EPP patients, all with laboratory confirmed FECH mutations (see Table 1 ) were infected in vitro with synchronized P. falciparum (late-stage trophozoites and schizonts) and parasite growth kinetics were examined for 72 hours. Erythrocytes from all four FECH deficient individuals showed significantly reduced P. falciparum growth compared to normal controls (Figure 6). EPP erythrocytes contain elevated levels of the FECFI substrate, protoporphyrin IX (PPIX) (Table 1 ). PPIX is known to be toxic at elevated concentrations (Holme et al., (2007)). To exclude the possibility that PPIX was the cause of impaired parasite growth in EPP cells, the growth of parasites in erythrocytes from patients with X- linked dominant protoporphryia was tested (XLDPP; MIM 300752). FECH activity is normal in these cells. But they contain elevated levels of PPIX due to a mutation in the gene encoding aminolevulinate synthase-2. The mutant enzyme upregulates the entry of succinyl-CoA and glycine precursors into the heme synthesis pathway, resulting in substrate buildup at the next rate limiting step (FECH) (Whatley et ai, (2008)). Parasite growth was identical between XLDPP and normal erythrocytes (Figure 6), excluding a growth-inhibitory effect of PPIX. Instead the data indicate, as before, that erythrocytic FECH is required for growth (increasing parasitaemia) of stasis of intraerythrocylic P. falciparum parasites.

(0106) In the second approach, mice ( isogenic to a C57BL/6 background) with a genetically induced deficiency in murine Fech were infected with a rodent malarial parasite {Plasmodium chahaudi) and its resulting parasitaemia and survival was monitored. This model of malaria infection is comparable with human P. falciparum infections, as the P. chahaudi parasites infect mouse red cells and infected mice eventually succumb to hyper-parasitemia and severe anemia. The Fech-deficient strain, called Fe ch^{m 1 Pd}\ contains a single nucleotide mutation in the murine Fech gene. Mice homozygous for the mutation express a mutant Fech enzyme with only 5% of normal Fech activity (Lyoumi er ai . (2007)), while heterozygotes have 45-65% activity compared to wild type mice.

[0107| Comparing males (Figures 2C and 2D) and females (Figures 2 A and 213) . separately (due to well-known sex differences in the response to infection). F h^{m { ?i} homozygotes are significantly more resistant to P. chahaudi infection. A 30% decrease in parasitemia at peak parasitemia compared to non-mutant mice is observed and their survival is significantly greater (Figure 2A to D). compared to heterozygotes and wild type mice. The levels of parasite growth and survival arc significantly improved, irrespective of the sex of the mice. Therefore host FECH is necessary to sustain a normal malaria infection in mice.

[0108] Taken together, these two independent, but complementary approaches demonstrate that the host Fech enzyme is necessary for normal parasite growth and development of life-threatening malaria infection. Whilst the protective effect of Fech deficiency in the Fech^{m ?ai} mouse may be due to any number of host response mechanisms that involve Fech, the P. falciparum culture experiments which are performed with purified Fech-deficient red blood cells indicate the anti-malarial effect of l^;ech deficiency is red cell autonomous.

EXAMPLE 3: Inhibition of Fech greatly diminishes the ability of protozoan parasite to grow in culture

(0109) N-methylprotoporphyrin (NMPP) is a FF,CH substrate analogue and a potent competitive inhibitor of the enzyme (Km - 10 nM) (Shi and Ferreira (2006) Biochem J., 399:21 -28). Increasing concentrations of NMPP are tested for their ability to inhibit the growth of P. falciparum under culture conditions. NMPP is observed to have potent anti- plasmodial activity with an 1C50 of 25nM (Figure 4A).

[0110) A search for additional Fech inhibitors revealed that griseofuivin has an anti- Fech activity that is a side-effect to its normal anti-fungal activity. Griseofuivin concentrates in skin cells, binding to fungal microtubules, making keratin precursor cells resistant to fungal infection. It has the reported additional effect of inhibiting FECH. which manifests in susceptible humans and mice as porphyria. It is determined that griseofuivin takes several days to accumulate in erythrocytes ( Figure 5 D). Red blood cells were cultured and were treated with various concentrations of griseofuivin. Red blood cells were treated with griseofuivin for 3 days with medium replaced each day. Three different strains of P. falciparum were tested, 3D7 (chloroquine susceptible) K l (chloroquine resistant) and W2mcf (multidrug resistant). Intra-erythropoietic griseofuivin inhibits parasite growth 48 hours after the addition of parasites to pre-treated uninfected erythrocytes with an IC50 of 10-50 μιη (Figure 4B).

EXAMPLE 4: Blood from subjects treated with griseofuivin resists parasite growth

(01 1 1 ] The anti-plasmodial activity of griseofuivin administered to human individuals is tested. Human subjects took a clinically standard course of griseofuivin (500mg/day for 7 days) and blood is taken to test ex vivo the ability to support P. falciparum parasite growth. Following the blood collection, the red cells are purified from contaminating white blood cells and platelets by washing the blood sample three times with equal volumes of a neutrally-buffered saline solution (phosphate-buffered saline, PBS). Parasites grow normally in purified red cells collected before beginning the course of griseofulvin, but are unable to grow in blood following 7 days of treatment. (Figure 5A). The numbers of parasites measured in cultures using the seven-day griseofulvin-treatment samples are less than the numbers added at the start of the infection. This indicates that the parasites are killed and are not just growth retarded. The anti-plasrhodial effect of the treatment is pronounced as early as 4 days of griseofulvin treatment, but effects are negligible at earlier collection time points (Figure 5B). This observation coincides with an observed time- dependent accumulation of griseofulvin in the red blood cells. Samples of the purified red cells are analyzed for levels of griseofulvin using HPLC-coupled MS-MS analysis. Griseofulvin levels increased steadily during the first few days of treatment and reached a steady-state maximum after four days (Figure 5C). To further examine the relationship between rates of accumulation and dose, subjects are given single oral dose of 2g of griseofulvin and blood samples collected at shorter time intervals (8. 24 and 48 hr). Parasites are unable to grow in red cells taken 8 hours after the treatment whereas some parasite growth occurred in the later two time points, although growth is still less than in the pre-treatment blood (Figure 5D). These observations coincide with the levels of griseofulvin measured in the red cells, which are maximal at 8 hours and declined at 24 and 48 hours. Griseofulvin levels are not detectable in human red cells one-week after the 2g treatment ( Figure 5E). These results indicate that a single high dose of griseofulvin renders host red cells impervious to parasite growth 8 hours later.

|01 12| Hence, a single dose of griseofulvin if given in sufficient amounts is effective in preventing parasite growth in an ex vivo setting and is, therefore, a useful therapeutic agent to treat malaria. Griseofulvin may be used alone or in conjunction with other antimalarial drugs. Its use alone could be valuable in areas where other drugs are compromised by underlying parasite drug resistance. The anti-plasmodial activity of griseofulvin is related to its rates of accumulation in red blood cells. EXAMPLE 5: NMPP and griseofulvin inhibit parasite growth by specifically targeting FECH

[0113] The translation of the genetic interruptions of FECH function to pharmacological targeting was examined. N-methylprotoporphyrin (NMPP) is a FECH substrate analogue and a potent competitive inhibitor of the enzyme (Km - 10 nM) (Shi et ai, (2006)) P. falciparum parasites cultured with NMPP were highly sensitive to the compound with IC50 values of around 25nM (Figure 4A). The action of N MPP was found to be independent of the drug resistance status of the parasite (Figure 7A). The plasmodiacidal activity of NMPP was inhibited with increasing concentrations of PP1X (FECH substrate), proving that NMPP was preventing parasite growth by specifically targeting FECH (Figure 7C). NMPP has nM activity, its IC50 is similar to antimalarial drugs and it acts rapidly. ·

[0114] The antifungal drug, griseofulvin is reported to have an anti-FECH activity (Bellingham et ai , ( 1995); Holley et ai, ( 1991 )). It is commonly used to treat human dermatophyte fungal infections. It accumulates in dermatophytes and binds to fungal microtubules, preventing fungal growth (Develoux (2001 )). Griseofulvin is also metabolized by a cytochrome-dependent mechanism that results in the alkylation of protoporphyrin rings and the formation of NMPP (Bellingham el ai, ( 1995)). Griseofulvin was therefore tested to determine if it could inhibit growth of P. falciparum parasites. Preliminary experiments showed that the drug takes several days to accumulate in erythrocytes (Figure 10). Red cells pretreated with griseofulvin for three days were infected with synchronized P. falciparum (late-stage trophozoites and schizonts) and parasite growlh kinetics were examined. Marked growth inhibition after 48 h incubation was observed, with calculated 1C50 values ranging between 10 and 50 μΜ. depending on the parasite strain (Figure 7B). The specific involvement of FECH inhibition in the drugs' action was demonstrated by titration of the effect with PP1X. ( Figure 7C). The generation of NMPP from griseofulvin may occur within the parasite as the red cell lacks the appropriate systems to catalyze the conversion. EXAMPLE 6: Anti-plasmodial activity of oral griseofulvin wherein a loading dose leads to rapid anti-parasite effects in humans

[0115J Griseofulvin has been used in the clinic for over 50 years and has only minimal side effects (Develoux, (2001 )). The anti-plasmodial activity of oral ly-administered griseofulvin was examined ex vivo. Firstly, seven healthy volunteers each took 500 mg griseofulvin orally, daily for seven days and provided a blood sample before and after the 7 day treatment. Red cells from the samples were infected in vitro with P. falciparum and the effect on parasite growth examined. An almost complete lack of parasite growth was observed in all of the post-treatment blood samples (Figure 8 A). The kinetics of the growth inhibition effect was examined by infecting blood taken each day from a volunteer on the same griseofulvin regimen. Parasite growth was reduced in blood taken after three days, and was completely absent by four days (Figure 8B). The kinetics of inhibition coincided with the rate of accumulation of the drug in the red cells, which increased for the first three days and then remained at a relatively steady state level (Figure 9A).

[01 16] Given the apparent relationship between red cell griseofulvin levels and plasmodiacidal effect, the effect of a griseofulvin loading dose was examined. Volunteers were bled, given a single 2 g dose of griseofulvin and then bled again after eight hours; one volunteer was also bled at additional time points for 3 days. Complete growth inhibition was observed in all the samples taken 8 hours after the 2 g dose (Figure 8C). The effect remained significant after 24 and 48 hours, and then declined at the subsequent time points (Figure 9D), which coincided with a reduction in intracellular concentrations of the compound (Figure B). Therefore, a single 2 g orally administered dose of griseofulvin renders red cells incapable to supporting parasite growth for at least 24 hours.

[0117) As described herein, FECH is essential for intraerythrocytic parasite growth and blood stage malarial infection. Cells and animals in which FECH activity was reduced through genetic changes additionally suggest the parasite has a high reliance for the host version of the enzyme. This supports other observations that host erythrocyte FECH is imported by the parasite and is functionally active (Varadharajan et ai , (2004)). A total absence of FECI I is lethal and it is therefore relevant to test the involvement of partial mutants of the parasite enzyme. EPP individuals retain at least 1 0% FECI I activity (Gouya et ai , (2006)); this is likely to be surpassed by NMPP and griseofulvin mediated inhibition of FECH and may explain the differences between the genetic and pharmacologic effects.

[01 18] Pharmacologic mimicry of the genetic changes in EPP using NMPP or griseofulvin offers therapeutic promise. Although the parasite's reliance on the erythrocyte enzyme suggests NMPP and griseofulvin are acting in a host-directed manner, Plasmodium also produces its own active FECH (Varadharajan et al., (2004); Sato et al., (2003)). Therefore NMPP and griseofulvin may be targeting both host and Plasmodium FECH. and any such treatments may be vulnerable to drug resistance. However, resistance would require the unlikely simultaneous mutations in the parasite that both reduce the enzyme's affinity for the NMPP substrate analog (without affecting catalysis of PPI X to heme) and up-regulate expression to compensate for the lack of active host FECH.

(0 I 19| Griseofulvin and NMPP are therefore potential antimalarial, agents. Griseofulvin is active in vitro against drug-resistant parasites and ex vivo studies show that it could be used as a treatment. In some embodiments, griseofulvin is used in combination with shorter acting drugs such as artesunate. and as a prophylactic, given its well- established proven safety profile, for long term use.

[0120] Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise. The disclosure of every publication cited herein is hereby incorporated herein by reference in its entirety. Many modifications will be apparent to those skilled in the art without departing from the scope of the present invention. Table 1. Study porphryria subjects

Protoporphyrins

Specific in RBC nmol/L „ _t

Patient U FECH mutation enzyme RBC ^C _r ^0Untry

activity* o I °' origin

(normal:<l 8nmoI °

)

Q104X (C339T),

EPP001 n.a. 37.6 Australia

IVS3-48c/t

Q104X (C339T).

EPP002 n.a. 84.8 Australia

IVS3-48c/t

Q104X (C339T),

EPP003 n.a. 43.9 Australia

IVS3-48c/t

EPP004 ^P-^R,64W <^C-^C490T). _{n £} 17.9 Australia c^Γυυ lVS3-48c/t c.l706-1709delAGTG ALAS2:

XLDPP

(p.E569GfsX24) +280%*. 86.3 France 001

ALAS2 gene FECH: 4.1* c.l706-1709delAGTG ALAS2:

XLDPP 002 (p.E569GfsX24) +220%*. 51.2 France

ALAS2 gene FECH: 3.7*

Af AS-?'

XLDPP 003 ^C 1M20T (PQ548X) France

25.6

ALAS2 gene _FECH: 3.9* n.a.: not available

* : - Alas2 mutants were expressed as % of Wt activity using prokaryotic expression vector; Normal: 100% - Fcch activity was measured in Lymphocytes and expressed as nmol/mg_pr01cillsh ; N: > 3.5 nmol/mg_p,_ol0,_ni.₅.h BIBLIOGRAPHY

Abitbol et al.. (2005), Am. J. Physiol. Gastrointest. Liver Physiol.2 S:G1208

Bellingham et al., (1995). Biochem. J. J07(Pt2):5O5

Develoux (2001). Ann. Dermatol. Venereol.. / 5:1317

Gouya et al., (2002), Nat Genet 30:21

Gouya et al., (2006). Am. J. Hum. Genet.78:2

Holley et al., (1991 ), Biochem J.274 (Pt 3j:S4

Holme et al., (2007), Blood.7/0:4108

Li et al., (2007), Cytometry A 77:297

Lyoumi e/ a/.. (2007) Boot 109:811

Mensch e al.. (2007), ./. Chromatogr. B. Analyi. Technol. Biomed. Life Sci.84

Puy et al., (2010), Lancet 375:924

Ribaut e/ a/., (2008), Malar. J. 7:45

Rivadeneira et al.. (1983), J. Protozoal 30:367

Sato <?/ a/., (2003), Cwr. Gew/.42:292

Shi «?/ al.. (2006). Biochem J.399:21

Trager «?/ a/.. (1976), Science 193:673

Varadharajan et al., (2004), Biochemical Journal 384:429

Whatle el al., (2008), Am J Hum Genet 53:408

Claims

CLAIMS:

1 . A method for the treatment or prophylaxis of a subject infected with or who has or had exposure to a member of Phylum Apicomplexa, said method comprising administering to said subject an effective amount of griseofulvin or N-methyl protoporphyrin (NMPP).

2. The method of Claim 1 wherein the subject is a human

3. The method of Claim 1 or 2 wherein the member of Phylum Apicomplexa is Plasmodium sp.

4. The method of Claim 1 or 2 or 3 wherein the effective amount is an amount of griseofulvin or NMPP is effective to reduce the activity of host cell ferrochelatasc.

5. The method of Claim 3 or 4 wherein the effective amount is the amount of griseofulvin or NMPP effective to reduce the growth or development of Plasmodium sp.

6. The method of any one of Claims 1 to 5 wherein the effective amount of griseofulvin is from about l Omg to about 20g per dose.

7. The method of Claim 6 wherein the effective amount of griseofulvin is from about 200mg to about 5g per dose. .

8. The method of any one of Claims 1 to 7 wherein griseofulvin or NMPP is provided orally, systemically, topically or parentally.

9. The method of any one of Claims 1 to 8 wherein the effective amount of NMPP reduces parasite growth in red blood cells at concentrations of at least about 25nM or at least about l OnM to ! OOnM.

10. The method of any one of Claims 1 to 9 wherein the griseofulvin or N MPP is provided in a composition, the composition comprising a pharmaceutically acceptable carrier, diluent and/or excipient and/or another anti-Apicomplexa agent.

1 1 . The method of Claim 10 wherein the anti-Apicomplexa agent is an anti-malarial agent.

12. The method of Claim 1 1 wherein the anti-malarial agent selected from artemether, artemotil, artesunate, quinine and chloroquine.

1 3. A method for the treatment or prophylaxis of a human subject with, exposed to. or suspected of having malaria or a complication arising therefrom, said method comprising administering to said subject an effective amount of griseofulvin or N- ethyl protoporphyria ( MPP) or a composition comprising same.

14. The method of Claim 1 3 wherein the subject is infected with or exposed to falciparum.

15. The method of Claim 13 or 14 wherein the effective amount' is an amount of griseofulvin or NMPP is effective to reduce the activity of human host cell ferrochelatase.

16. The method of Claim 13 or 14 wherein the effective amount is an amount of griseofulvin or NMPP effective to reduce the growth or development of Plasmodium sp.

17. The method of any one of Claims 13 to 16 wherein the effective amount of griseofulvin is from about l Omg to about 20g per dose or from about 200mg to about 5g per dose.

1 8. The method of any one of Claims 1 3 to 15 wherein the effective amount of NMPP reduces parasite growth in red blood cells at concentrations of at least about 25nM or at least about Ι ΟηΜ to Ι ΟΟηΜ.

19. The method of Claim 13 wherein the composition comprises a pharmaceutically acceptable carrier, diluent and/or excipicnt and/or another antimalarial agent.

20. The method of claim 19 wherein the antimalarial agent is selected from artemether, artemotil, artesunate, quinine and chloroquine.

21 . Use of griseofulvin or NMPP in the manufacture of a medicament for the treatment or prophylaxis of an infection by a member of Phylum Apicoplexa such as Plasmodium sp,

25. Griseofulvin or NMPP thereby for use in the treatment or prophylaxis of an in ection by a member of Phylum Apicomplexa, such as Plasmodium sp.

24. A screening assay for an antimalarial drug, said assay comprising screening for a compound which inhibits ferrochelatase and then testing the drug for its ability to reduce growth or development of Plasmodium sp.

* * *