WO2014081922A1

WO2014081922A1 - Compositions and methods useful in preventing and treating apicomplexa infections

Info

Publication number: WO2014081922A1
Application number: PCT/US2013/071205
Authority: WO
Inventors: Doran GREENBAUM
Original assignee: The Trustees Of The University Of Pennsylvania
Priority date: 2012-11-21
Filing date: 2013-11-21
Publication date: 2014-05-30

Abstract

The present invention provides compounds that are useful in treating or preventing infection of a mammal by an apicomplexan parasite. The present invention also provides a method of treating infections by an apicomplexan parasite in a mammal, preferably a human. The Apicomplexa are a large group of obligate intracellular protist pathogens, most of which possess a unique organelle called apicoplast and an apical complex structure involved in penetrating a host cell.

Description

TITLE OF THE INVENTION

Compositions and Methods Useful in Preventing and Treating Apicomplexa

Infections

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority from U.S. Provisional Application Ser. No. 61/728,894, filed on November 21, 2012, the entire disclosure of which is incorporated by reference herein as if set forth herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

This invention was made with U.S. government support under grant number 1R01 AI097273-01A1 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The Apicomplexa are a large group of obligate intracellular protist pathogens, most of which possess a unique organelle called apicoplast and an apical complex structure involved in penetrating a host cell. They are unicellular, spore- forming, and are exclusively parasites of animals, including humans. This diverse group includes organisms such as coccidia, gregarines, piroplasms, haemogregarines, and plasmodia. Some diseases caused by apicomplexan organisms include:

babesiosis (Babesia); malaria (Plasmodium); and Coccidian diseases, including cryptosporidiosis (Cryptosporidium parvum), cyclosporiasis (Cyclospora

cayetanensis), isosporiasis (Isospora belli) and toxoplasmosis (Toxoplasma gondii).

Apicomplexan parasites exhibit complex life cycles involving distinct sexual and asexual stages of growth. The asexual phase is comprised of a lytic cycle in which parasites establish an intracellular niche within the host. Plasmodium species infect erythrocytes, while Toxoplasma gondii infects nucleated animal cells. The process of schizogony in Plasmodium (endodyogeny in Toxoplasma) involves replication within a specialized "paras itophorous vacuole" to yield multiple daughter parasites (Nishi et al, 2008, J. Cell Sci. 121 : 1559; Hu et al, 2002, Mol. Biol. Cell 13 :593). The resulting merozoites (tachzyoites in Toxoplasma) must escape from this vacuole and the host cell in order to invade uninfected cells and continue the infection. Egress from the infected cell is a rapid event, requiring only seconds at the end of the -36- to 48-hour intracellular life cycle (Glushakova et al, 2005, Curr. Biol. 15: 1645; Salmon et al, 2001, Proc. Natl. Acad. Sci.. USA. 98:271). Both calcium (Black et al, 2000, Mol. Cell Biol. 20:9399; Nagamune et al, 2008, Nature 451 :207) and proteases (Salmon et al, 2001, Proc. Natl. Acad. Sci. USA. 98:271 ; Hadley et al, 1983, Exp. Parasitol. 55:306; Wickham et al, 2003, J. Biol. Chem. 278:37658;

Akastu-Kapur et al, 2008, Nat. Chem. Biol. 4:203; Yeoh et al, 2007, Cell 131 : 1072) have been implicated in escape from the parasitophorous vacuole and/or host cell membranes. Unlike parasite invasion, the molecular details governing parasite- mediated cytolysis are relatively poorly understood.

Parasite exit has been shown to require the interplay of parasite derived proteins and changes in the host actin cytoskeleton; however, the host protein contribution underlying these changes is not well explored. Parasite-induced host cell cytolysis has been suggested to be a two-step, Ca²⁺-dependent process (Wickham et al, 2003, J. Biol. Chem. 278:37658-37663; Salmon et al, 2001, Proc. Natl. Acad. Sci. USA 98:271-276), requiring both host cell ion loss (Moudy et al, 2001, J. Biol. Chem. 276:41492-41501) and increased membrane poration (Glushakova et al, 2010, Curr. Biol. 20: 1 117-1121 ; Abkarian et al , 2011, Blood 1 17:4118-4124). Recent reports implicate the activity of parasite-derived proteins including a perforin-like protein (Kafsack et al, 2009, Science 323:530-533), a Ca²⁺-dependent kinase (Dvorin et al, 2010, Science 328:910-912), and proteases (Yeoh et al, 2007, Cell 131 : 1072- 1083; Arastu-Kapur et al, 2008, Nat. Chem. Biol. 4:203-213), though reports of host protein contribution to this cytolytic process are limited. The host-derived protease calpain is required for exit of both P. falciparum and T. gondii and proteolyzes the actin cytoskeleton just prior to cytolysis (Chandramohanadas et al, 2009, Science 324:794-797; Arastu-Kapur et al, 2009, Nat. Chem. Biol. 4:203-213). Furthermore, calpain-independent loss of adducin from the host erythrocyte actin cytoskeleton prior to P. falciparum exit has been demonstrated (Millholland et al, 201 1, Mol. Cell Proteomics 10:M1 11 010678).

Protein kinase C (PKC) is a family of protein kinase enzymes that are involved in controlling the function of other proteins through the phosphorylation of hydroxyl groups of serine and threonine amino acid residues on these proteins. PKC enzymes in turn are activated by signals such as increases in the concentration of diacylglycerol (DAG) or calcium ions (Ca²⁺). Hence PKC enzymes play important roles in several signal transduction cascades. The PKC family consists of fifteen isozymes in humans, divided into three subfamilies, based on their second messenger requirements: conventional (or classical), novel, and atypical. Conventional PKCs contain the isoforms α, βΐ, βΙΙ, and γ. These require Ca²⁺, DAG, and a phospholipid such as phosphatidylserine for activation. Novel PKCs include the δ, ε, η, and θ isoforms, and require DAG, but do not require Ca²⁺ for activation. Thus, conventional and novel PKCs are activated through the same signal transduction pathway as phospholipase C. On the other hand, atypical PKCs (including protein kinase Μζ and 1 1 λ isoforms) require neither Ca²⁺ nor diacylglycerol for activation. The term "protein kinase C" usually refers to the entire family of isoforms.

There is a great interest in identifying novel therapeutic interventions that may overcome infestation by these parasites in mammals without causing prohibitively toxic effects on the infected mammal. There is a need to identify small- molecule compounds that disturb the life cycle of apicomplexan parasites and stop infection of mammalian cells by these parasites. The present invention fulfills these needs.

BRIEF SUMMARY OF THE INVENTION

The present invention includes a method of treating or preventing infection by an apicomplexan parasite in a subject in need thereof. The method includes the step of administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising at least one PKC inhibitor or a salt thereof.

In one embodiment, the PKC inhibitor is a PKC-selective inhibitor. In another embodiment, the PKC inhibitor is selected from the group consisting of G56976 (5,6,7, 13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4- c]carbazole-12-propanenitrile); sotrastaurin (3-(lH-Indol-3-yl)-4-[2-(4- methylpiperazin-l-yl)quinazolin-4-yl]pyrrole-2,5-dione), ruboxistaurin ((9S)-9- [(dimethylamino)methyl]-6,7, 10, 11 -tetrahydro-9H, 18H-5,21 : 12, 17- di(metheno)dibenzo [e,k]pyrrolo [3 ,4-h] [ 1 ,4, 13 ] oxadiazacyclohexadecine- 18,20- dione); bisindolylmaleimide-1 (3-[l-[3-(dimethylamino)propyl]-lH-indol-3-yl]-4- (lH-indol-3-yl)-lH-pyrrole-2,5-dione), any salts thereof, and any combinations thereof. In yet another embodiment, the parasite is Plasmodium falciparum. In yet another embodiment, the parasite is Toxoplasma. In yet another embodiment, the subject is a mammal. In yet another embodiment, the subject is human. In yet another embodiment, the administering step is performed through a topical, oral, nasal, buccal, sublingual, rectal, pleural, peritoneal, vaginal, intramuscular, subcutaneous, transdermal, epidural, intrathecal or intravenous route. In yet another embodiment, the subject is further administered at least one additional agent useful for treating infection by an apicomplexan parasite. In yet another embodiment, the at least one additional agent is selected from the group consisting of chloroquine, quinine, amodiaquine, propranolol, artemether-lumefantrine, artesunate-amodiaquine, artesunate-mefloquine, artesunate-sulfadoxine/pyrimethamine, atovaquone-proguanil, cotrifazid, doxycycline, mefloquine, primaquine, proguanil, sulfadoxine- pyrimethamine, hydroxychloroquine, artemisin, semi-synthetic derivatives of artemisin, extracts of the plant Artemisia annua, pyrimethamine, sulfadiazine, clindamycin, spiramycin, minocycline, and atovaquone. In yet another embodiment, the inhibitor and the agent are separately administered to the subject. In yet another embodiment, the inhibitor and the agent are co-administered to the subject. In yet another embodiment, the inhibitor and the agent are co-formulated and coadministered to the subject.

The present invention also includes a pharmaceutical composition comprising a PKC inhibitor and at least one additional agent useful for treating infection by an apicomplexan parasite.

In one embodiment, the at least one additional agent is selected from the group consisting of chloroquine, quinine, amodiaquine, propranolol, artemether- lumefantrine, artesunate-amodiaquine, artesunate-mefloquine, artesunate- sulfadoxine/pyrimethamine, atovaquone-proguanil, cotrifazid, doxycycline, mefloquine, primaquine, proguanil, sulfadoxine-pyrimethamine, hydroxychloroquine, artemisin, semi-synthetic derivatives of artemisin, extracts of the plant Artemisia annua, pyrimethamine, sulfadiazine, clindamycin, spiramycin, minocycline, and atovaquone. In another embodiment, the composition further comprises a pharmaceutically acceptable carrier. In yet another embodiment, the PKC inhibitor is a PKC-selective inhibitor. In yet another embodiment, the PKC inhibitor is selected from the group consisting of G56976 (5,6,7, 13-tetrahydro-13-methyl-5-oxo-12H- indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile); sotrastaurin (3-(lH-Indol-3- yl)-4-[2-(4-methylpiperazin-l-yl)quinazolin-4-yl]pyrrole-2,5-dione), ruboxistaurin ((9S)-9-[(dimethylamino)methyl]-6,7, 10, 1 l-tetrahydro-9H, 18H-5.21 : 12, 17- di(metheno)dibenzo [e,k]pyrrolo [3 ,4-h] [ 1 ,4, 13 ] oxadiazacyclohexadecine- 18,20- dione); bisindolylmaleimide-1 (3-[l-[3-(dimethylamino)propyl]-lH-indol-3-yl]-4- (lH-indol-3-yl)-lH-pyrrole-2,5-dione), any salts thereof, and any combinations thereof. In yet another embodiment, the parasite is Plasmodium falciparum. In yet another embodiment, the parasite is Toxoplasma.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.

Figure 1, comprising Figures la-lg, illustrates the finding that siRNA screen identifies novel signaling components required for T. gowi&^'-mediated cyto lysis. Figure la: Model of host siRNA screen for mediators of parasite-induced cyto lysis. Defects in T. go«ii¾^'-mediated cytolysis are scored at 60 hpi: parasites unable to exit persist as vacuoles with >64 parasites, while parasites able to exit host cells reinvade new cells. Figure lb: Primary siRNA screen in U20S cells:

unruptured vacuoles (intact vacuoles with >64 parasites) are compared at 44 and 60 hpi to identify host gene involvement in parasite release (*p<0.05; **p<0.01). Figure lc: Secondary screen utilizing multiple oligos validates individual host genes and gene pairs whose knockdown resulted in accumulation of unruptured vacuoles by 60 hpi. Figure Id: Pearson correlation graph indicates strong relation between oligos (r=0.91). Figure le: Parasite doublings measurements upon siRNA knockdown of hits. Figure If: Western blot analysis confirms siRNA-mediated knockdown of target proteins, as compared to Scr oligo. Figure lg: Representative images of P30- GFP vacuoles in knockdown cell lines at 44 and 60 hpi.

Figure 2, comprising Figures 2a-2d, illustrates the finding of conserved function of host proteins in P. falciparum-mediated cytolysis. Figure 2a:

Immunodepletion studies of soluble erythrocyte protein hits confirmed by western blot (M: Mock; ID: immunodepletion; IP: immunoprecipitation - target protein elution from sepharose beads). Figure 2b: Representative Giemsa images following immunodepletion of host Gaq, PLC l, PKCa/β, and CaM-1 results in persistence of schizont stage parasites by 60 hpi versus mock or PKAa/ cat depletion, which resulted in exit and reinvasion by 60 hpi. Figure 2c: Flow cytometric quantitation of schizont-stage (left) and ring-stage (right) parasites in depleted erythrocytes. Figure 2d: Ring parasitemia assessed 12 hours following needle shearing of infected, depleted erythrocytes at the times indicated (ND: Not determined). Data shown are means of at least three experiments +/-SEM.

Figure 3, comprising Figures 3A-3L, illustrates the finding that host

PKC is activated downstream of host Gaq late in the parasite life cycle and functions in adducin loss from the host cell cytoskeleton. Figures 3A-3C: Representative images of 3D7-CKAR parasites (Figure 3A: control images at 35 hpi; Figure 3B: life cycle time course) and CFP/YFP FRET fluorimetry (Figure 3C) to assess PKC activity. 100 nM PMA is used as a positive control, while PKCa/β depletion and 500 nM G56976 are used as negative controls for CKAR activity. Figures 3D-3F:

Representative images of T. go«ii¾^'-infected U20S cells expressing cytoplasmic CKAR (Figure 3D: control images at 5 parasite divisions; Figure 3E: life cycle time course) and CFP/YFP FRET fluorimetry (Figure 3F). Positive control: PMA;

negative controls: PKCa/β knockdown, 500 nM G56976. Data shown are mean fluorescence +/- SEM. Figure 3G: Western blot analysis of host cytoskeletal fractions confirmed adducin disappearance from P. falciparum-m^' fected erythrocytes (top) and also demonstrated PKCa/β immunodepletion from erythrocytes (bottom). . Figure 3H: Western blot analysis confirmed adducin disappearance from T. gondii- infected U20S cells (top) and also demonstrated shRNA knockdown in U20S cells (bottom).. Figures 3I-3L: A549 cells expressing S716A/S726A phospho-mutant adducin or overexpressing adducin- 1 assessed for T. gondii life cycle progression. Figure 31: Adducin and phospho-adducin western blot from A549 cytoskeletal fractions following T. gondii infection at 44 hpi. Loading control: actin. Figure 3J: Representative 60 hpi images and quantitation (Figure 3K) of unruptured vacuoles at 60 versus 44 hpi. Data shown are mean +/- SEM. Figure 3L: Parasite doublings throughout life cycle following host cell manipulation.

Figure 4, comprising Figures 4a-4d, illustrates that host signaling cascade enhances calpain-mediated cytoskeletal proteolysis just prior to parasite exit. Figure 4a: DCG04-labeling of active calpain in P. falciparum-iniQctQd erythrocyte fractions at 50 hpi in cells depleted of Gaq, PLC l, PKCa/β, and CaM- 1 by immunodepletion. Biotin blot confirms calpain activity upon labeling. Negative control: Calpain-1 depletion; positive control: ΡΚΑα/β depletion; loading control: actin. Figure 4b: a-Spectrin cleavage by western blot at 50 hpi in erythrocyte cytoskeletal fractions following depletion of signaling components. Figure 4c:

a- Spectrin cleavage by western blot following in vitro incubation of U20S cell cytoskeletal fractions with activated CaMKII prior to incubation with recombinant human calpain- 1. Figure 4d: Host CAMKII/calmodulin-1 coimmunoprecipitation through the T. gondii life cycle and presence of p-CAMKII (the activated form) by western blot.

Figure 5, comprising Figures 5A-5E, illustrates the finding that host [Ca²⁺] increases throughout the last third of the intracellular cycle, in a TRPC6- dependent manner. Figure 5A: Representative images of erythrocytes loaded with Fura-2-dextran prior to challenge with synchronous P. falciparum parasites (left). [Ca²⁺] as a function of 340nm/380nm emission ratio (right). Figure 5B:

Representative images of T. go«ii¾^'-infected U20S cells expressing YC3.6 (left). [Ca²⁺] as a function of CFP/YFP FRET (right). Data shown are mean Ca²⁺ measurements +/- SEM. Figure 5C: Representative images and quantitation (Figure 5D) at 44 and 60 hpi of siRNA-mediated knockdown of cation channels prior to T. gondii infection. Positive control: Capns 1 ; negative control: scr oligo. Figure 5E: Representative images (left) and [Ca²⁺] measurements as a function of CFP/YFP FRET (right) at 5 T. gondii divisions (-35 hpi) following siRNA-mediated knockdown of cation channels in U20S cells expressing YC3.6.

Figure 6, comprising Figures 6a-6d, illustrates the finding that mammalian PKC inhibitors show antiparasitic activity in vivo. Figure 6a: T. gondii parasite burden in cells from the spleen (left) and peritoneal cavity (PECS; right) upon treatment with 10 mg/kg G56976, quantified by flow cytometry. Plots shown are gated on single, live cells by FSC and SSC (n=15 for all conditions; ***p<0.0001). Figure 6b: P. yoelli parasitemia quantified via Giemsa tail vein blood smear upon 10 mg/kg G56976 treatment once daily for 4 days. Figure 6c: P. berghei ANKA parasitemia quantified via Giemsa tail vein blood smear upon 50 mg/kg Sotrastaurin treatment via gastric gavage once daily for 4 days. (**p<0.001). Figure 6d: Survival curves following sotrastaurin treatment using the standard Mantel-Cox log rank test (*p<0.05).

Figure 7 illustrates a model of host GPCR-mediated cytolysis. Parasite ligands, perhaps upregulation of parasite TCA cycle intermediates during periods of high parasite replication, activate GPCRs on the PV membrane and initiate a signaling cascade through host Gaq. PKC-mediated phosphorylation liberates adducin from the host cell cytoskeleton, activating the mechanosensitive plasma membrane cation channel TRPC6. CaMKII-activation following Ca²⁺ influx mediates phosphorylation of cytoskeletal substrates, which, upon rapid influx of Ca²⁺ from the extracellular media, are proteolyzed by calpain to allow for cytoskeletal dissolution and parasite release.

Figure 8 illustrates the design of siRNA screen to identify host genes required for T. gowi&^'-mediated cytolysis.

Figure 9, comprising Figures 9A-9B, illustrates the finding that depletion of ΡΙ βΙ and calmodulin- 1 from erythrocytes is sufficient to block P. falciparum-mediated cytolysis. Figure 9A: Erythrocytes depleted of either PLCpi, PLCy, or both PLCpi and PLCy were assayed for schizont persistence (left panel) or reinvasion of new rings (right panel) at 60 hpi. Figure 9B: Erythrocytes depleted of either calmodulin- 1 , calmodulin-2, or both calmodulin- 1 and calmodulin-2 were assayed for schizont persistence (left panel) or reinvasion of new rings (right panel) at 60 hpi. Data shown are means of at least three experiments ± SEM; n=3.

Figure 10, comprising Figures 1 OA- 101, illustrates the finding that host Gaq and PLCp/y are required upstream for host PKCa/β function in adducin loss prior to parasite-mediated cytolysis. Figure 10A: PKC inhibitor IC₅₀ values and percent parasite reinvasion upon 500 nM treatment on P. falciparum and T. gondii. Data shown are means of at least three experiments ± SEM, n=3. Figure 10B: T. gondii viability following block of host cell cytolysis in host cells upon G56976 treatment, PKCa/β knockdown, or S716A/S726A adducing expression. Data shown are mean T. gondii viability upon mechanical release at the timepoints shown following arrest at egress, of at least 3 experiments ± SEM. Figure IOC: 3D7-CKAR parasites removed from their host cells via saponin treatment were assessed for CKAR fluorescence to account for digestive vacuole uptake. Representative 30-hour image (left) and population-based fluorometry throughout the life cycle (right) show digestive vacuole fluorescence, which is removed as background from all 3D7-CKAR measurements in Figure 3. Data shown are means of at least three experiments ±

SEM. Figure 10D: 3D7-CKAR fluorescence at 35 hpi following immunodepletion of key host signaling components. Representative images show that Gaq and PLCpi immunodepletion diminished CKAR signal in the host cell compartment, which was rescued by 100 nM PMA. Data shown are means of at least three experiments ± SEM. Figure 10E: Erythrocyte CKAR FRET-based fluorescence of 3D7-CKAR parasites and life cycle progression as measured by quantitation of ring stage parasites at 60 hpi following depletion of Gaq or PLCpi . Data shown are means of at least three experiments ± SEM. Figure 1 OF: Adducin immunofluorescence and western blot of erythrocyte cytoskeletal fractions at 35 hpi of P. falciparum following knockdown of key signaling components. PMA rescues adducing loss following depletion of Gaq or PLCP by immunofluorescence (left) or Western blot (right). PMA could not induce adducin loss following depletion of PKCa/β. Figure 10G: Adducin immunofluorescence and western blot of U20S cell cytoskeletal fractions at five parasite divisions following knockdown of key signaling components. PMA rescues adducin loss following knockdown oignaq or plcbl/plcgl by

immunofluorescence or Western blot. PMA could not induce adducin loss following prkca/prkcb knockdown. Figure 10H: CKAR FRET-based fluorescence at five T. gondii divisions following stable knockdown of key host signaling components.

Representative images show that gnaq or plcbl/plcgl knockdown diminished CKAR signal in the host cell compartment, which was rescued by 100 nM PMA. Data shown are means of at least three experiments ± SEM. Figure 101: CKAR FRET- based fluorometry within T. gondii-m^' fected U20S cells at five parasite divisions. PMA addition rescues PKC activity following depletion of gnaq or plcbl/plcgl . Data shown are means of at least three experiments ± SEM.

Figure 11, comprising Figures 1 lA-1 ID, illustrates the finding that PKC activity, as mediated by Gaq and PLC, is required for Ca²⁺ influx at the end of the parasite life cycle. Figure 1 1A: Giemsa images at specified timepoints following treatment beginning at 30 hpi with 500 nM cation channel inhibitors GsTMx-4, 2- APB, SKF-63563, or Pyr3. Figure 1 IB: Population level measurement of schizont persistence (left) and new ring formation (right) following inhibition of cation channel inhibitors at 30 hpi. Data shown are means of at least three experiments ± SEM. Figure 1 1C: Representative FRET images and population level fluorometry at five T. gondii parasite divisions of U20S cells expressing YC3.6 following knockdown of gnaq, plcbl/plcgl, or prkca/prkcb show that [Ca²⁺] is limited upon knockdown of these signaling members. 100 nM PMA treatment rescues Ca²⁺ influx following gnaq or plcbl/plcgl knockdown, though it cannot be rescued following prkca/prkcb knockdown. Data shown are means of at least three experiments ± SEM. Figure 1 ID: Representative 45 hpi images and population-level Ca²⁺ measurements of P. falciparum-mfected erythrocytes loaded with Fura-2-dextran, and immunodepleted of Gaq, PLCpi, or PKCa/β. Ca²⁺ influx was limited in cells depleted of these effectors, but could be rescued upon PMA treatment following Gaq or ΡΙΧβΙ depletion. The mechanosensitive channel inhibitor GsTMx-4 abrogated PMA-induced Ca²⁺ influx, indicating that is influx was cation channel-dependent. Data shown are means of at least three experiments ±- SEM.

Figure 12, comprising Figures 12A-12D, illustrates the finding that parasite TCA cycle intermediates initiate the host cytolytic network. Figure 12A:

Representative images of P30-GFP-infected U20S knockdown cells at 44 and 60 hpi. Confirmation of siRNA-mediated knockdown of host GPCRs and the positive control CAPNSl are shown by Western blot. Figure 12B: Quantitation of enlarged vacuoles through 60 hpi. Data shown are means of at least three experiments ± SEM. Figure 12C: Time until T. gondii parasite exit upon treatment with 50 μΜ exogenous aKG/succinate or 50 μΜ lactate, versus 10 μΜ A23187 (positive control for egress induction). Data shown are mean time of at least 3 experiments ± SEM. Figure 12D: Representative Giemsa images following a 12-hour treatment with 50 μΜ exogenous aKG/succinate, lactate, or DMSO control.

Figure 13, comprising Figures 13A-13B, is a table illustrating mediators of parasite- induiced cytolysis as identified by siRNA screen in U20S cells.

Figure 14 is a table illustrating multi-isoform families and components in initial U20S cell siRNA screen.

Figure 15 is a table illustrating isoform pair siRNA-mediated knockdowns.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the discovery of a host-signaling network common to both T. gondii and P. falciparum that results in host PKC- induced weakening of the host cell cytoskeleton. In one embodiment, the host PKC activity plays a role in parasitic disease progression. As described herein, the general mammalian PKC inhibitor G56976 demonstrated antiparasitic activity in murine models of toxoplasmosis (in vitro IC₅₀ = 404 ± 93.2 nM) and malaria (in vitro IC₅₀ = 253 ± 43.2 nM), showing parasite clearance following 4 days of treatment at 10 mg/kg dosage. Furthermore, experiments in mice indicate that treatment with the human classical PKC isoform-specific inhibitor Sotrastaurin (AEB071; a drug being tested in the treatment of psoriasis and renal transplant rejection) is protective in murine models of severe cerebral malaria and in vitro against P. falciparum (IC₅₀ = 89.4 ± 28.1 nM).

In one aspect, the invention provides a method of treating, inhibiting, suppressing or preventing infection by an apicomplexan parasite in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising at least one compound useful within the methods of the invention. In one embodiment, the at least one compound is a PKC inhibitor or a salt thereof. In another embodiment, the at least one compound is a PKC-specific inhibitor or a salt thereof. In yet another embodiment, the parasite is Plasmodium falciparum. In yet another embodiment, the parasite is Toxoplasma. Preferably the subject is a mammal, more preferably the subject is a human.

Host cell cytolysis was thought to be largely parasite-mediated, mainly due to growing pressure on the host cell membrane and cytoskeleton by the growing parasite body (Glushakova et al, 2010, Curr. Biol. 20: 1 117-1 121; Glushakova et al, 2005, Curr. Biol. 15: 1645-1650). The data described herein indicates the existence of a complex host-derived signaling pathway, suggesting that host cell cytolysis is a highly regulated process requiring a complex interplay of host-derived components.

As Gaq, PLC, and PKC converge in established GPCR signaling networks, GPCRs play a role in this host cytolysis pathway. Considering that parasites do not encode GPCRs, a parasite-derived small molecule ligand may be used to signal to GPCRs of host origin through direct interaction with the host cell membrane to mediate signaling through Gaq. Several parasite metabolites peak during schizogony, especially the TCA cycle intermediate alphaketoglutarate (aKG) (Olszewski et al, 2009, Cell. Host Microbe 5: 191-199). As aKG and other TCA cycle intermediates including succinate have recently been shown to signal through Gaq-coupled GPCRs, such as oxoglutarate receptor 1 (oxgrl) and succinate receptor 1 (sucnrl) (He et al, 2004, Nature 429: 188-193; Qi et al, 2004, Purinergic Signal 1 :67-74), knockdown studies were performed to assess their contribution to host cell cytolysis. A significant delay in T. gondii exit (p<0.05) from cells depleted of both GPCRs was observed (Figures 12A-12B). A mixture of exogenous aKG/succinate initiated a hastening of T. gondii exit (Figure 12C) and induced premature lysis of P. falciparum parasites leading to parasite death (Figure 12D). As parasites rely primarily on glucose fermentation for their energy needs, the requirement for oxidative phosphorylation in the parasite mitochondrion has been widely disputed, though the majority of TCA enzymes are expressed throughout the life cycle. aKG as well as other TCA cycle intermediates have been shown to diffuse out as putative waste products (Olszewski et ah, 2010, Nature 466:774-778) due to a branched TCA pathway. As suggested herein, the diffusion of TCA metabolites into the host cell space serves to induce a host cascade to facilitate parasite release.

In the model presented herein (Figure 7), heightened parasite metabolism causes an increase in TCA metabolic intermediates outside the parasite (in the PV space and host cell), resulting in overstimulation of host metabolite-sensing GPCRs. These GPCRs engage a Gaq-mediated signaling pathway converging on PKC activation that results in the complete loss of a key cytoskeletal component, and leads to pathological Ca2+ influx through mechanosensitive plasma membrane TRP channels. High cytoplasmic [Ca+] induces global calpain activation, which results in the proteolysis of various substrates, and in turn causes the loss of host cell plasma membrane integrity to allow for parasite release. PKC and calpain play key executioner functions within this cascade. Though it is unclear whether the sequential activation of these enzymes necessarily mediates cell death, PKC activity may be an essential upstream component of pathological calpain activation in the context of parasite infection. Given the antiparasitic activity of PKC inhibitors in mouse models of both toxoplasmosis and malaria, it is likely that inhibiting multiple host proteins upstream of PKC activity limits parasite growth, and represent a multitude of potential antiparasitic targets.

Definitions

As used herein, each of the following terms has the meaning associated with it in this section.

Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well-known and commonly employed in the art.

As used herein, the articles "a" and "an" refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

As used herein, the term "about" will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term "about" is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1 %, and still more preferably ±0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods.

As used herein, the term "hpi" refers to hours post-invasion.

As used herein, the term "polypeptide" refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds. Synthetic polypeptides may be synthesized, for example, using an automated polypeptide synthesizer. As used herein, the term "protein" typically refers to large polypeptides. As used herein, the term "peptide" typically refers to short polypeptides.

Conventional notation is used herein to represent polypeptide sequences: the left- hand end of a polypeptide sequence is the amino-terminus, and the right-hand end of a polypeptide sequence is the carboxyl-terminus.

As used herein, the polypeptides include natural peptides, recombinant peptides, synthetic peptides or a combination thereof. A peptide that is not cyclic will have an N-terminus and a C-terminus. The N-terminus will have an amino group, which may be free (i.e., as a H₂ group) or appropriately protected (for example, with a BOC or a Fmoc group). The C-terminus will have a carboxylic group, which may be free (i.e., as a COOH group) or appropriately protected (for example, as a benzyl or a methyl ester). A cyclic peptide does not necessarily have free N- or C-termini, since they are covalently bonded through an amide bond to form the cyclic structure. The term "peptide bond" means a covalent amide linkage formed by loss of a molecule of water between the carboxyl group of one amino acid and the amino group of a second amino acid. As used herein, amino acids are represented by the full name thereof, by the three letter code corresponding thereto, or by the one-letter code corresponding thereto, as indicated in the following table:

Full Name Three-Letter Code One-Letter Code

Aspartic Acid Asp D

Glutamic Acid Glu E

Lysine Lys K

Arginine Arg R

Histidine His H

Tyrosine Tyr Y

Cysteine Cys C

Asparagine Asn N

Glutamine Gin Q

Serine Ser s

Threonine Thr T

Glycine Gly G

Alanine Ala A

Valine Val V

Leucine Leu L

Isoleucine He I

Methionine Met M

Proline Pro P

Phenylalanine Phe F

Tryptophan Trp W

As used herein, a "disease" is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subject's health continues to deteriorate.

As used herein, a "disorder" in a subject is a state of health in which the subjecl is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject's state of health. As used herein, the term "prevent" or "prevention" means no disorder or disease development if none had occurred, or no further disorder or disease development if there had already been development of the disorder or disease. Also considered is the ability of one to prevent some or all of the symptoms associated with the disorder or disease.

As used herein, the terms "patient" and "subject" refer to a human or a non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. Preferably, the subject is human.

As used herein, the terms "effective amount," "pharmaceutically effective amount" and "therapeutically effective amount" refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result can be reduction and/or alleviation of the frequency and/or severity of signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

As used herein, the term "pharmaceutical composition" refers to a mixture of at least one compound of the invention with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients. The pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to: intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.

As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the subject such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the invention, and not injurious to the subject. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;

powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the invention, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions. The "pharmaceutically acceptable carrier" may further include a pharmaceutically acceptable salt of the compound useful within the invention. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

As used herein, the language "pharmaceutically acceptable salt" refers to a salt of the administered compounds prepared from pharmaceutically acceptable non-toxic acids including inorganic acids, organic acids, solvates, hydrates, or clathrates thereof. Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include sulfate, hydrogen sulfate, hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids (including hydrogen phosphate and dihydrogen phosphate). Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric, salicylic, galactaric and galacturonic acid. Suitable pharmaceutically acceptable base addition salts of compounds of the invention include, for example, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, Ν,Ν'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine ( -methylglucamine) and procaine. All of these salts may be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound.

As used herein, the "instructional material" includes a publication, a recording, a diagram, or any other medium of expression that may be used to communicate the usefulness of the compounds of the invention. In some instances, the instructional material may be part of a kit useful for effecting alleviating or treating the various diseases or disorders recited herein. Optionally, or alternately, the instructional material may describe one or more methods of alleviating the diseases or disorders in a cell or a tissue of a mammal. The instructional material of the kit may, for example, be affixed to a container that contains the compounds of the invention or be shipped together with a container that contains the compounds. Alternatively, the instructional material may be shipped separately from the container with the intention that the recipient uses the instructional material and the compound cooperatively. For example, the instructional material is for use of a kit; instructions for use of the compound; or instructions for use of a formulation of the compound.

Compounds

The compounds useful within the invention may be synthesized using techniques well-known in the art of organic synthesis. The compounds useful within the invention may be derived from a naturally occurring compound, or synthesized from a non-natural starting material. The compounds useful within the invention may also be chemically synthesized, using, for example, solid phase synthesis methods.

In one embodiment, any PKC inhibitor or a salt thereof is useful within the methods of the invention. In another embodiment, any PKC-specific inhibitor or a salt thereof, which is selective over other kinases such as PKA and/or PKB, is useful within the methods of the invention. In another embodiment, any PKC-specific inhibitor, specific to classical (c)PKCa/p/y isoforms over novel (n)PKC isoforms or atypical (a)PKC isoforms, or a salt thereof is useful within the methods of the invention.

In one embodiment, the compound useful within the invention is G56976, also known as 5,6,7, 13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3- a]pyrrolo[3,4-c]carbazole-12-propanenitrile, or a salt thereof:

In one embodiment, the compound useful within the invention sotrastaurin, also known as 3-(lH-Indol-3-yl)-4-[2-(4-methylpiperazin-l- yl)quinazolin-4-yl]pyrrole-2,5-

In one embodiment, the compound useful within the invention is ruboxistaurin, also known as (9S)-9-[(dimethylamino)methyl]-6,7, 10, l l-tetrahydi 9H, 18H-5,21 : 12,17-di(metheno)dibenzo[e,k]pyrrolo[3,4-h] [ 1 ,4, 13 ]

oxadiazacyclohexadecine- 18,20-dione, or a salt thereof:

In one embodiment, the compound useful within the invention is bisindolylmaleimide-1, also known as 3-[l-[3-(dimethylamino)propyl]-lH-indol-3- yl]-4-(lH-indol-3-yl)-lH-pyrro -2,5-dione, or a salt thereof:

Methods of the Invention

The invention includes a method of treating or preventing infection by an apicomplexan parasite in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising at least one PKC inhibitor or a salt thereof.

In one embodiment, the PKC inhibitor is a PKC-selective inhibitor. In another embodiment, the PKC inhibitor is selected from the group consisting of:

G56976 (5,6,7, 13-tetrahydro-l 3-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4- c]carbazole- 12-propanenitrile);

sotrastaurin (3-(lH-Indol-3-yl)-4-[2-(4-methylpiperazin-l-yl)quinazolin-4- yl]pyrrole-2 , 5 -dione) ;

ruboxistaurin ((9S)-9-[(dimethylamino)methyl]-6,7,10, l l-tetrahydro-9H, 18H- 5,21 : 12, 17-di(metheno)dibenzo[e,k]pyrrolo[3,4-h] [1,4,13] oxadiazacyclohexadecine- 18,20-dione);

bisindolylmaleimide-1 (3-[l-[3-(dimethylamino)propyl]-lH-indol-3-yl]-4- (lH-indol-3-yl)-lH-pyrrole-2,5-dione); any salts thereof, and any combinations thereof.

In one embodiment, the parasite is Plasmodium falciparum. In another embodiment, the parasite is Toxoplasma. In yet another embodiment, the subject is a mammal. In yet another embodiment, the subject is human. In yet another embodiment, the administering step is performed through a topical, oral, nasal, buccal, sublingual, rectal, pleural, peritoneal, vaginal, intramuscular, subcutaneous, transdermal, epidural, intrathecal or intravenous route. In yet another embodiment, the subject is further administered at least one additional agent useful for treating infection by an apicomplexan parasite. In yet another embodiment, the at least one additional agent is selected from the group consisting of chloroquine, quinine, amodiaquine, propranolol, artemether-lumefantrine, artesunate-amodiaquine, artesunate-mefloquine, artesunate-sulfadoxine/pyrimethamine, atovaquone-proguanil, cotrifazid, doxycycline, mefloquine, primaquine, proguanil, sulfadoxine- pyrimethamine, hydroxychloroquine, artemisin, semi-synthetic derivatives of artemisin, extracts of the plant Artemisia annua, pyrimethamine, sulfadiazine, clindamycin, spiramycin, minocycline, and atovaquone. In yet another embodiment, the inhibitor and the agent are separately administered to the subject. In yet another embodiment, the inhibitor and the agent are co-administered to the subject. In yet another embodiment, the inhibitor and the agent are co-formulated and co- administered to the subject.

Combination Therapies

The invention includes a pharmaceutical composition comprising a PKC inhibitor and at least one additional agent useful for treating infection by an apicomplexan parasite. In one embodiment, the parasite is Plasmodium falciparum. In another embodiment, the parasite is Toxoplasma. In one embodiment, the at least one additional agent is selected from the group consisting of chloroquine, quinine, amodiaquine, propranolol, artemether-lumefantrine, artesunate-amodiaquine, artesunate-mefloquine, artesunate-sulfadoxine/pyrimethamine, atovaquone-proguanil, cotrifazid, doxycycline, mefloquine, primaquine, proguanil, sulfadoxine- pyrimethamine, hydroxychloroquine, artemisin, semi-synthetic derivatives of artemisin, extracts of the plant Artemisia annua, pyrimethamine, sulfadiazine, clindamycin, spiramycin, minocycline, and atovaquone. In another embodiment, the composition further comprises a pharmaceutically acceptable carrier. In one embodiment, the PKC inhibitor is a PKC-selective inhibitor. In another embodiment, the PKC inhibitor is selected from the group consisting of:

G56976 (5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4- c]carbazole-12-propanenitrile);

bisindolylmaleimide-1 (3-[l-[3-(dimethylamino)propyl]-lH-indol-3-yl]-4-

(lH-indol-3-yl)-lH-pyrrole-2,5-dione);

any salts thereof, and any combinations thereof.

The compounds useful within the invention are intended to be useful, e.g., in the methods of present invention, in combination with one or more additional compounds useful for treating infection by an apicomplexan parasite. These additional compounds may comprise compounds of the present invention or compounds, e.g., commercially available compounds, known to treat, prevent, or reduce the symptoms of infection by an apicomplexan parasite.

In non-limiting examples, the compounds of the invention may be used in combination with one or more of the following anti- infectives against

apicomplexan parasites:

(a) malaria: chloroquine, quinine, amodiaquine, propranolol, artemether- lumefantrine (Coartem® and Riamet®), artesunate-amodiaquine, artesunate- mefloquine, artesunate-sulfadoxine/pyrimethamine, atovaquone-proguanil

(Malarone®), cotrifazid, doxycycline, mefloquine (Lariam®), primaquine, proguanil, sulfadoxine-pyrimethamine, hydroxychloroquine (Plaquenil®), artemisin, semisynthetic derivatives of artemisin, and extracts of the plant Artemisia annua.

(b) toxoplasmosis: pyrimethamine, sulfadiazine, clindamycin, spiramycin, minocycline, and atovaquone.

A synergistic effect may be calculated, for example, using suitable methods such as, for example, the Sigmoid-E_max equation (Holford & Scheiner, 19981, Clin. Pharmacokinet. 6:429-453), the equation of Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul. 22:27-55). Each equation referred to above may be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively.

Administration/Dosage/Formulations

Routes of administration of any of the compositions of the invention include topical, oral, nasal, buccal, sublingual, rectal, pleural, peritoneal, vaginal, intramuscular, subcutaneous, transdermal, epidural, intrathecal or intravenous route.

The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the subject either prior to or after the onset of an infection by an apicomplexan parasite. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

Administration of the compositions of the present invention to a subject, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to treat an infection by an apicomplexan parasite in the subject. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the subject; the age, sex, and weight of the subject; and the ability of the therapeutic compound to treat infection by an apicomplexan parasite in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound of the invention is from about 1 and 5,000 mg/kg of body weight/per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.

In particular, the selected dosage level will depend upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well, known in the medical arts.

A medical doctor, e.g. , physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the

pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of an infection by an apicomplexan parasite in a subject.

In one embodiment, the compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In one embodiment, the pharmaceutical compositions of the invention comprise a therapeutically effective amount of a compound of the invention and a

pharmaceutically acceptable carrier.

The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

In one embodiment, the compositions of the invention are administered to the subject in dosages that range from one to five times per day or more. In another embodiment, the compositions of the invention are administered to the subject in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It will be readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the invention will vary from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the invention should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any subject will be determined by the attending physical taking all other factors about the subject into account.

Compounds of the invention for administration may be in the range of from about 1 μg to about 10,000 mg, about 20 μg to about 9,500 mg, about 40 μg to about 9,000 mg, about 75 μg to about 8,500 mg, about 150 μg to about 7,500 mg, about 200 μg to about 7,000 mg, about 3050 μg to about 6,000 mg, about 500 μg to about 5,000 mg, about 750 μg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 50 mg to about 1,000 mg, about 75 mg to about 900 mg, about 100 mg to about 800 mg, about 250 mg to about 750 mg, about 300 mg to about 600 mg, about 400 mg to about 500 mg, and any and all whole or partial increments therebetween.

In some embodiments, the dose of a compound of the invention is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound of the invention used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound (i.e., an apicomplexan parasite anti- infective) as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments therebetween.

In one embodiment, the present invention is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the invention, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of an infection with an apicomplexan parasite in a subject.

Granulating techniques are well known in the pharmaceutical art for modifying starting powders or other particulate materials of an active ingredient. The powders are typically mixed with a binder material into larger permanent free-flowing agglomerates or granules referred to as a "granulation." For example, solvent-using "wet" granulation processes are generally characterized in that the powders are combined with a binder material and moistened with water or an organic solvent under conditions resulting in the formation of a wet granulated mass from which the solvent must then be evaporated.

Melt granulation generally consists in the use of materials that are solid or semi-solid at room temperature (i.e. having a relatively low softening or melting point range) to promote granulation of powdered or other materials, essentially in the absence of added water or other liquid solvents. The low melting solids, when heated to a temperature in the melting point range, liquefy to act as a binder or granulating medium. The liquefied solid spreads itself over the surface of powdered materials with which it is contacted, and on cooling, forms a solid granulated mass in which the initial materials are bound together. The resulting melt granulation may then be provided to a tablet press or be encapsulated for preparing the oral dosage form. Melt granulation improves the dissolution rate and bioavailability of an active (i.e. drug) by forming a solid dispersion or solid solution.

U.S. Patent No. 5, 169,645 discloses directly compressible wax- containing granules having improved flow properties. The granules are obtained when waxes are admixed in the melt with certain flow improving additives, followed by cooling and granulation of the admixture. In certain embodiments, only the wax itself melts in the melt combination of the wax(es) and additives(s), and in other cases both the wax(es) and the additives(s) will melt.

In a further embodiment, the present invention relates to a method for manufacturing a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds of the invention, and a further layer providing for the immediate release of a medication for infection with an apicomplexan parasite. Using a wax/pH-sensitive polymer mix, a gastric insoluble composition may be obtained in which the active ingredient is entrapped, ensuring its delayed release.

Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelate, carbohydrates such as lactose, amylose or starch, magnesium stearate talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxymethylcellulose, and polyvinylpyrrolidone. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents. For oral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gelcaps. The compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients which are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate. The tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients. Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.

The term "container" includes any receptacle for holding the pharmaceutical composition. For example, in one embodiment, the container is the packaging that contains the pharmaceutical composition. In other embodiments, the container is not the packaging that contains the pharmaceutical composition, i.e., the container is a receptacle, such as a box or vial that contains the packaged

pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition. Moreover, packaging techniques are well known in the art. It should be understood that the instructions for use of the pharmaceutical composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product. However, it should be understood that the instructions may contain information pertaining to the compound's ability to perform its intended function, e.g., treating, preventing, or reducing infection by an apicomplexan parasite in a subject.

The compounds for use in the invention may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans )buccal, (trans )urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous,

intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein. Oral Administration

For oral administration, the compounds of the invention may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., polyvinylpyrrolidone,

hydroxypropylcellulose or hydroxypropylmethylcellulose); fillers (e.g., cornstarch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrates (e.g., sodium starch glycollate); or wetting agents (e.g., sodium lauryl sulphate). If desired, the tablets may be coated using suitable methods and coating materials such as OPADRY™ film coating systems available from Colorcon, West Point, Pa. (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY™ White, 32K18400). Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions. The liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxy benzoates or sorbic acid).

Parenteral Administration

For parenteral administration, the compounds of the invention may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and/or continuous infusion. Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and/or dispersing agents may be used.

Additional Administration Forms

Additional dosage forms of this invention include dosage forms as described in U.S. Patents Nos. 6,340,475, 6,488,962, 6,451,808, 5,972,389, 5,582,837, and 5,007,790. Additional dosage forms of this invention also include dosage forms as described in U.S. Patent Applications Nos. 20030147952,

20030104062, 20030104053, 20030044466, 20030039688, and 20020051820. Additional dosage forms of this invention also include dosage forms as described in PCT Applications Nos. WO 03/35041, WO 03/35040, WO 03/35029, WO 03/35177, WO 03/35039, WO 02/96404, WO 02/32416, WO 01/97783, WO 01/56544, WO 01/32217, WO 98/55107, WO 98/1 1879, WO 97/47285, WO 93/18755, and WO 90/11757.

Controlled Release Formulations and Drug Delivery Systems

In certain embodiments, the formulations of the present invention may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.

The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.

For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds. As such, the compounds for use the method of the invention may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.

In a preferred embodiment of the invention, the compounds of the invention are administered to a subject, alone or in combination with another pharmaceutical agent, using a sustained release formulation.

The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.

The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.

The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration. As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments therebetween after drug administration after drug administration.

As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments therebetween after drug administration.

Dosing

The therapeutically effective amount or dose of a compound of the present invention will depend on the age, sex and weight of the subject, the current medical condition of the subject and the nature of the infection by an apicomplexan parasite being treated. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.

A suitable dose of a compound of the present invention may be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day. The dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses.

It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.

The compounds for use in the method of the invention may be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosage for subjects undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of this invention and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction times, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing/oxidizing agents, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present application.

It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present invention. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application.

The following examples further illustrate aspects of the present invention. However, they are in no way a limitation of the teachings or disclosure of the present invention as set forth herein.

EXAMPLES

The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the invention is not limited to these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein.

Materials and Methods

Reagents and antibodies:

Unless otherwise noted, all starting materials and resins were obtained from commercial suppliers and used without purification. DCG04 was synthesized as reported previously (Greenbaum et al, 2000, Chem. Biol. 7:569-581). Streptavidin-HRP was purchased from Vector Labs (Burlingame, CA), and Fura-2-Dextran and SYTOX Green from Invitrogen (Grand Island, NY). Purified human calpain-1 was purchased from Sigma-Aldrich (St. Louis, MO), and calpastatin domain I from EMD Chemicals (Darmstadt, Germany).

Commercially available antibodies were obtained from the following sources: anti- OXGR1, anti-GPR91, and anti-TRPC6 from Abeam (Cambridge, MA); anti-ankyrin- 1, anti-GAPDH, anti-calmodulin 1, anti-calmodulin 2, anti-CAMKI, anti-CAMKII, anti-PKAcat, anti-PLCp, anti-PLCy from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-PKCa and anti-PKC from Cell Signaling (Danvers, MA). Anti-a- and β- spectrin antibodies were kindly provided by D. Speicher (Wistar Institute, USA).

Growth and maintenance of P. falciparum cultures:

Strain 3D7 Plasmodium parasites were cultured in human red blood cells (4% hematocrit) under standard conditions (37°C, 5% O₂/5%CO₂/90%N₂) in RPMI buffer supplemented with HEPES and hypoxanthine. Replication was tightly synchronized via serial treatments with D-sorbitol to lyse trophozoite- and schizontinfected erythrocytes. The remaining ring-stage parasites were cultivated to yield schizonts (-42 hr post-infection), which were selectively purified using a Miltenyi Biotec magnetic separator. Cultures were staged by microscopy of Giemsa- stained blood smears and flow cytometry using SYTOX Green (Invitrogen) as a marker of DNA content. Cultures were fixed with 4% paraformaldehyde/0.0016% gluteraldehyde for 1 hour and permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature prior to incubation with 5 μΜ SYTOX Green for 10 minutes and analysis on an Accuri Flow Cytometer.

Growth and maintenance of T. gondii cultures:

Wild type RH strain Toxoplasma gondii tachyzoites, and parasites constitutively expressing P30-GFP or P30-RFP were cultivated in confluent monolayers of human foreskin fibroblasts, human U20S cells, or human A549 cells. Cells were grown to confluence in Dulbecco's modified Eagle's medium containing 10% newborn bovine serum (D10), replaced upon infection with minimal essential medium containing 10% dialyzed fetal bovine serum (ED I). Immunodepletion and loading of erythrocytes:

Erythrocyte ghosts were prepared by hypotonic lysis as previously described utilizing 5 mM K₂HPO4 at <4°C for 15 minutes (Chandramohanadas et ah, 2009, Science 324:794-797). To immunodeplete target proteins, target antibodies were pre-conjugated to Protein G-Sepharose (Upstate/Millipore) following a titration to achieve maximal immunodepletion (1-10 μg antibody per 10⁶ erythrocytes). These sepharose beads were incubated with ghosts for 3 hours on ice with gentle mixing and repeated with fresh antibody-sepharose conjugates up to 3 times. The slurry was separated by centrifugation and followed by another round of immunodepletion for cells immunodepleted of multiple proteins. Ghosts were resealed by gradual addition of 5X resealing buffer (475 mM KOAc, 25 mM Na₂HP0₄, 25 mM MgCl₂, 237.5 mM KC1, pH 7.5) over 1 hr at 37°C. Parallel studies were carried out using anti-PKAcat, and mock treated erythrocytes were incubated with Sepharose G beads without antibodies.

Analysis of host protein function during parasite exit from immunodepleted erythrocytes:

To assay for host protein function during egress, resealed erythrocytes were prepared as described above. Schizont stages were isolated from 50 ml parasite culture by magnet purification ~40 hr after sorbitol synchronization, and added to either mocktreated, PKCa/p-depleted, ΡΙΧβΙ -depleted, calmodulin- 1 -depleted, Gaq- depleted, or calpain-1 -depleted erythrocytes to a final hematocrit of 4%. Parasite progress through the intraerythrocytic cycle was monitored by Giemsa smear.

Schizont-stage parasites were followed to assess egress and the establishment of rings in newly-infected erythrocytes from -46-60 hr post-infection. Flow cytometry was exploited for quantitative evaluation of P. falciparum development in resealed erythrocytes. Beginning at 45 hpi, culture aliquots were harvested at 3 hr intervals, fixed in 4% formaldehyde/ 0.0075% glutaraldehyde in PBS, permeabilized 10 min in 0.25% Triton X-100, and stained 5 min with 5 μΜ SYTOX-Green at RT. 10⁶ events were collected per sample using an Accuri flow cytometer and analyzed using Accuri software, gating to exclude debris defined by scatter characteristics, and uninfected erythrocytes based on low fluorescence; rings and trophozoites were distinguished from schizonts based on DNA content. Immunofluorescence studies:

Synchronous P. falciparum-m^' fected erythrocytes or P30-GFP/P30- RFP T. gondii infected U20S cells were fixed for 1 hour at RT with 4%

paraformaldehyde/ 0.008% gluteraldehyde, washed three times with PBS, permeabilized using 0.1% Triton X-100 for 10 minutes at 37°C, and blocked using 5% milk in PBS for 1 hour at RT. Cells were incubated with primary antibodies to adducin-1 (Santa Cruz) in 5% milk in PBS overnight at 4°C, washed three times with PBS, and incubated with secondary antibodies preconjugated to Alexa-568, Alexa- Cy3, or Alexa-488 (Life Technologies) for two hours at RT. Cells were washed three times prior to incubation with Hoechst DNA stain and imaging on a Leica DMI6000B epifluorescent scope.

Western blotting:

U20S cells or erythrocytes were fractionated using Triton X-100 prior to separation by SDS-PAGE, transfer to PVDF membrane, and probing for target proteins of interest using primary antibodies at a concentration of 1 : 1000 (Santa Cruz, Abeam) and secondary antibodies conjugated to HRP (Sigma) at a concentration of 1 : 10000. Monitoring caipain activation and membrane binding:

Immunodepleted cells were prepared as above and challenged with synchronous, magnet-purified schizont stage parasites. Following 48-hour incubation, infected cells were incubated with DCG04 (5 μΜ) and membrane fractions were prepared from each sample by ultracentrifugation (2 hr at 200,000 x g). Equal amounts of solubilized protein from each sample was separated by SDS-PAGE, transferred to PVDF membrane, and probed for biotin using streptavidin-HRP, revealing DCG04 labeling of active caipain. As a loading control, the same immunoblots were probed with anti-calpain. T. gondii invasion, replication, and egress in knockdown cultures:

Intracellular parasite growth was measured by counting the number of parasites per parasitophorous vacuole at 6, 24 and 44 hr post-infection (prior to egress from the initial host cell). Confluent monolayers of ~5 x 10⁵ host cells in 60 mm dishes were infected with 10⁶ T. gondii parasites. At least 100 vacuoles were counted per time point, and doubling rates were determined as the average log₂ number of parasites per vacuole. siRNA Screen and shRNA stable knockdown generation:

Primary siRNA oligos were purchased from Santa Cruz, oligos for follow up screens were purchased from Ambion. siR As were added at a final concentration of 20 nM in 96-well plates and titrated to achieve maximal knockdown by 72 hours post-transfection, along with 10⁶ U20S cells in culture medium lacking phenol red. Reverse transfection was carried out using Lipofectamine 2000

(Invitrogen) according to the manufacturer's instructions. Transfected host cells were inoculated with 10⁵ P30-GFP-expressing T. gondii tachyzoites 24 hr post-transfection, and followed throughout the intracellular cycle to assess invasion, replication, and egress rates at 6, 24, 44, and 60 hpi. siRNA knockdown was confirmed via western blot of mirrored cultures utilizing validated antibodies from Santa Cruz. Stable knockdown cell lines were generated following transfection with huSH shRNA plasmids (Origene) encoding target shRNAs conjugated to soluble cytoplasmic RFP to affirm knockdown and selection with puromycin (1 μg/ml) for 2 passages, prior to isolation of clonal populations. Double knockdown cells were generated utilizing pGFP-B-RS expression vectors (Origene) and selection with blasticidin. Following confirmation of knockdown via western blot, cells were cultured in in the absence of selection pressure.

Calcium studies:

Parasites infecting fura-2-dextran-loaded erythrocytes were followed by fluorescent microscopy and plate-based fluorometry throughout the life cycle to assess host erythrocyte cytoplasmic [Ca²⁺] via the equation:

[Ca²⁺] = K_d x Q x(R-R_mi„y R_max-R),

where R represents the fluorescence intensity ratio at 340 nm/380 nm excitation and emission at 510 nm. U20S cells were transduced using the Premo Cameleon Calcium Sensor (Invitrogen) according to manufacturer's instructions to facilitate cytoplasmic expression of YC3.6 prior to synchronous T. gondii infection. Host [Ca²⁺] was measured throughout the parasite life cycle via FRET microscopy on a Leica

DMI6000B and FRET-based fluorimetry to assess [Ca²⁺] as a function of YFP/CFP emission ratio. CKAR transfection and PKC activity measurement:

CKAR plasmid (Addgene) and transfected into U20S cells using Lipofectamine 2000 (Life Technologies) according to manufacturer's instructions. 3D7-CKAR construct was generated via addition of a minimal export PEXEL motif N-terminal to CKAR (following PCR amplification from the CKAR plasmid). This was achieved via amplification of the N-terminus of the exported protein KAHRP (PF3D7_0202000) from cDNA of asynchronous 3D7 parasite cultures and recombination with the CKAR motif into the P. falciparum expression vector p- HHVPatt using MultiSite Gateway (Life Technologies; Marti et al, 2004, Science 306: 1930-1933). Constructs were transfected into the P. falciparum 3D7 line and selected using 10 nM WR99210. 3D7-CKAR transgenic parasites and T. gondii- infected U20S cells expressing CKAR were cultured under standard conditions and assessed for PKC activity via FRET -based fluorimetry as a function of YFP/CFP emission ratio and FRET imaging on a Leica DMI6000B.

CaMKII phosphorylation studies

U20S cells were fractionated using Triton X-100 and the cytoskeletal fraction was incubated with 100 nM recombinant rat CAMKII (New England Biolabs) in IX NEBuffer for Protein Kinases supplemented with 200 μΜ ATP, 1.2 μΜ calmodulin and 2 mM CaCl₂ and incubated for 1 hour at 37°C. 100 nM recombinant human calpain (Biovision) was added to the mixture and incubated for 30 minutes at 37 °C prior to separation by SDS-PAGE, transfer to PVDF membrane, and probing for alphaspectrin (primary antibody gift from David Speicher) at a concentration of 1 :5000 and anti-rabbit secondary antibodies conjugated to HRP (Sigma) at a concentration of 1 : 10000.

The results of this example are now described. Identification of a Pathway Necessary for T. go«<i»-Mediated Cytolysis

To identify host genes essential for parasite-mediated cytolysis, an RNAi screen was performed in U20S cells (T. gondii host cells) focused on canonical Ca²⁺-signaling components, given earlier studies that implicated host calpain in T. gondii and P. falciparum exit (Chandramohanadas et al, 2009, Science 324:794-797; Millholland et al, 2011, Mol. Cell Proteomics 10: Ml 1 1 010678). To circumvent the potential for functional redundancy between host gene isoforms, pooled siRNAs were tested for simultaneous knockdown of gene families, as well as individual genes present in both U20S cells and erythrocytes. This primary screen included 45 individual gene knockdowns and 1 1 multi-isoform knockdowns (Figures 13 and 14). Pooled siRNAs (three siRNAs/gene) were arrayed in quadruplicate, reverse- transfected into U20S cells, and infected 24 hours post-transfection with transgenic T. gondii tachyzoites (MOI of 0.1) that constitutively secrete GFP into the PV, allowing for facile identification of PV diameter as a function of life cycle progression (P30- GFP; Figure la, Figure 8) (Striepen et al, 1998, Mol. Biochem. Parasitol. 92:325- 338). siRNAs against calpain small subunit (capnsl) and a scrambled oligo (Scr) were included as positive and negative controls, respectively. siRNA knockdown efficiency was maximal at 72 hours post-transfection, which corresponded with parasite-mediated cytolysis in this cell type (-50 hpi). Following synchronous T. gondii infection (Kafsack et al, 2004, Mol. Biochem. Parasitol. 136:309-31 1), plates were fixed and imaged at 6, 24, 44, or 60 hpi and intracellular parasite life cycle progression through cytolysis was assessed. Genes involved in parasite-mediated cytolysis showed an accumulation of PVs containing >64 parasites at 60 hpi upon knockdown (Z score +1.5, p < 0.05), -10 hours after host cell rupture typically occurs in this cell type.

From this screen, 5 gene families whose knockdown blocked parasite- mediated host cell cytolysis were initially identified: calmodulin (CaM),

Ca²⁺/calmodulin dependent kinases (CaMK), Ga subunits (Ga), protein kinase C (PKC), and phospholipase C (PLC) (Figure lb). To deconvolute the specific genes required for parasite-mediated cytolysis from these gene family hits, two unique siRNAs targeting individual and pairs of genes were tested (Figure lc, Figure 15). Single knockdown oignaq (p<0.002), and knockdown of the gene pairs p^a/prkcb (p<0.002), plcbl/plcgl (p<0.002), calml/calm2 (p<0.09) and camkl /camk2a

(p<0.09), showed a statistically significant and correlated accumulation of unruptured PVs at 60 hpi (r=0.91; Figure Id). Knockdown of these genes had no effect on parasite doublings upon maximal knockdown, reinforcing their function in cytolysis rather than parasite replication (Figure le). Knockdown efficiency was measured by western blot analysis of total protein as compared to scr (Figure If). Representative images are shown for positive hits that display unruptured PVs in comparison to controls (scr) or negatives (prkaca/prkacb) that show newly reinvaded parasites at 60 hpi (Figure lg).

Conserved Host Protein Function in P. falciparum-Mediated Cytolysis as Indicated by Immunodepletion Studies

Antibody-mediated depletion studies were used to investigate whether the protein products of the validated gene hits in T. gondii functioned similarly in P. falciparum-m^' duced erythrocyte cytolysis. Efficiency of target protein

immunodepletion in erythrocytes was determined by western blot and only immunodepletions showing >80% knockdown were further analyzed (Figure 2a)

(Chandramohanadas et al, 2009, Science 324:794-797). Synchronous P. falciparum parasites were assessed for life cycle progression by Giemsa smear (Figure 2b) and flow cytometry of DNA content (Figure 2c) to distinguish blocked multinucleated schizonts from newly invaded, ring-form parasites with a single nucleus.

Immunodepletion of Gaq, ΡΙΧβΙ, CaM-1, or the simultaneous immunodepletion of both PKCa and PKC resulted in an accumulation of schizont stage parasites and a lack of newly invaded rings at 60 hpi, indicating a block in parasite-mediated cytolysis and corroborating the results of the RNAi screen in T. gondii. The relative levels of PLC and CaM isoforms in erythrocytes may differ from that of U20S cells, as there is no requirement to deplete multiple isoforms to block cytolysis (Figure 9). To assess parasite viability, trapped parasites were mechanically released from depleted host cells and assessed for their invasive capability via flow cytometric quantitation of mononuclear ring-stage parasites 12 hours following mechanical release (Figure 2d). Parasites trapped within erythrocytes were able to invade erythrocytes upon needle-shearing up to 54 hpi, indicating that parasite death likely occurs following 6 hours of blocked cytolysis.

Host PKC is Activated during Schizogony to Remove Adducin from the Host Cytoskeleton

As PKC is a major downstream effector of Ga_q-coupled GPCR signaling via PLC-mediated generation of diacylglycerol (DAG; Castagna et al, 1982, J. Biol. Chem. 257:7847-7851 ; Rhee et al, 1989, Science 244:546-550) and PKC inhibitors showed potent antiparasitic activity and the ability to block parasite- mediated cytolysis in vitro (Figure 10A), the role of this critical host enzyme in parasite life cycle progression was further investigated. To measure host PKC activity during the P. falciparum life cycle, a transgenic P. falciparum line that secretes a FRET -based PKC activity indicator was generated (CKAR; Violin et al, 2003, Cell Biol. 161 :899-909 into the infected erythrocyte cytoplasm (3D7-CKAR) via expression of a chimeric construct with an N-terminal signal peptide and PEXEL motif (Gallegos et al, 2006, J. Biol. Chem. 281 :30947-30956; Hiller et al, 2004, Science 306: 1934-1937; Marti et al, 2004, Science 306: 1930-1933). Figure 3A displays the specificity of the CKAR reporter for PKC activity with representative positive control FRET images (maximal FRET signal) of 3D7-CKAR-infected erythrocytes treated with the PKC agonist phorbol myristate acetate (PMA) and negative control images (minimal FRET signal) upon treatment with the PKC inhibitor G56976 or PKC immunodepletion prior to infection. As with any protein present in the erythrocyte cytoplasm, a significant amount of CKAR was taken up into the parasite digestive vacuole, causing an intense CKAR FRET signal within this organelle likely due to intermolecular FRET. In order to account for this artifact in the measurements of host PKC activity, 3D7-CKAR parasites were removed from their host cells and intracellular FRET signal was quantified to assess this background signal within the digestive vacuole (Figure IOC). By FRET microscopy (Figure 3B) and background-corrected fluorometry to remove digestive vacuole signal (Figure 3C), limited host PKC activity throughout the ring and trophozoite stages (0-25 hpi) with a sharp increase during schizogony (30-40 hpi) was observed. Immunodepletion of G(Xq or PLCpi ablated PKC activity during the entire infectious cycle, reinforcing the importance of the host Ga_q signaling pathway upstream of PKC activation (Figure 10D). PMA restored PKC activity in these parasite-infected erythrocytes depleted of signaling components and also rescued the associated exit defect (Figure 10E), further indicating a critical role for host PKC. PKC activity reporter cell lines were also generated via stable expression of CKAR in U20S host cells prior to T. gondii infection. The CKAR signal output throughout the T. gondii life cycle in these reporter U20S cells largely mirrored the results obtained in P. falciparum, with maximal FRET signal occurring in the last third of the intracellular life cycle (Figures 3D-3F). Knockdown of host gnaq or plcbl/plcgl ablates PKC activity but this activity was restored by the PKC agonist PMA (Figures 10H-10I). The host cytoskeletal protein adducin is lost from the erythrocyte actin cytoskeleton at ~35 hpi of P. falciparum in a calpain-independent manner

(Millholland et al, 2011, Mol. Cell Proteomics 10:M1 11 010678). As adducin cytoskeletal association is regulated by the PKC-mediated phosphorylation of residues S716/S726 (Matsuoka et al, 1996, J. Biol. Chem. 271 :25157-25166), PKC depletion on host adducin cytoskeletal association during parasite infection was assessed. Western blot and immunofluorescence analysis of host cytoskeletal fractions confirmed adducin disappearance from both P. falciparum-iniQctQd erythrocytes (Figure 3G, top; Figure 10F) and T. gondii-m^' fected U20S cells late in the intracellular cycle (Figure 3H, top; Figure 10G). However,

PKCa/β immunodepletion from erythrocytes (Figure 3G, bottom) or shRNA knockdown in U20S cells (Figure 3H, bottom) abrogated this loss and maintained adducin cytoskeletal association through the end of both parasite life cycles. The S716/S726 phosphorylated adducin species (p-adducin) accumulated in mock-treated cells in the last third of the life cycle by Western blot while the unphosphorylated form was lost from cytoskeletal fractions, unless PKCa/β was depleted from host cells. G(Xq or ΡΙΧβΙ immunodepletion from erythrocytes (Figure 10F), or knockdown in U20S cells (Figure 10G) also caused adducin persistence within the host cytoskeleton, highlighting the function of this upstream cascade.

In order to examine the effect of adducin persistence on T. gondii life cycle progression, the PKC phospho-mutant adducin (S716A/S726A; Matsuoka et al, 1998, J. Cell Biol. 142:485-497) as well as wild-type adducin was overexpressed in host A549 cells. S716A/S726A adducin expression resulted in minimal

phosphorylation by western blot upon T. gondii infection (Figure 31), while wild-type adducin overexpressors showed an abundance of p-adducin. Similar to

PKCa/β stable knockdown, S716A/S726A adducin expression resulted in the persistence of enlarged vacuoles by 60 hpi (Figures 3J-3K), with no effect on parasite replication (Figure 3L), suggesting that PKC-induced adducin loss directly mediated host cell cytolysis. Tachyzoites trapped within host cells show reduced viability following 18-24 hours of blocked cytolysis within host cells expressing the adducin phospho-mutant S716A/S726A, similar to PKC inhibition or knockdown (Figure 10B), highlighting the feasibility of targeting this phase of the parasite life cycle as an antiparasitic strategy. Requirement of Host Signaling Cascade For Calpain-Mediated Cytoskeletal

Proteolysis

Host calpain activation results in cytoskeletal proteolysis necessary for parasite exit (Chandramohanadas et ah, 2009, Science 324:794-797; Arastu-Kapur et ah, 2008, Nat. Chem. Biol. 4:203-213). Calpain activation following

immunodepletion of signaling components from erythrocytes was assessed using an activity -based probe for cysteine proteases, DCG04 (Greenbaum et ah, 2000, Chem. Biol. 7:569-581) (Figure 4a). As opposed to negative control ΡΚΑα/β

immunodepleted cells which showed a clear active calpain band at the conclusion of the life cycle, PKCa/β, ΡΙΧβΙ, and Gaq immunodepletion disallowed calpain activation at the end of the parasite life cycle, similar to calpain- immunodepleted cells. CaM-1 immunodepletion resulted in diminished calpain activation, indicating that CaM-1 may facilitate this process. As a corollary to calpain activation, spectrin cleavage was examined via western blot, a key calpain substrate. Immunodepletion of pathway components abrogated spectrin proteolysis in P. falciparum-m^' fected erythrocytes at 50 hpi, while ΡΚΑα/β-immunodepleted cells showed multiple spectrin fragments (Figure 4b). Interestingly, in vitro incubation of U20S cell membrane fractions with activated CaMKII prior to incubation with calpain resulted in multiple spectrin cleavage products (Figure 4c), suggesting that CaMKII phosphorylation of cytoskeletal substrates may enhance calpain-mediated proteolysis. CaMKII activation was shown by CaM-1 co-immunoprecipitation and autophosphorylation by p-CaMKII western blot analysis near the end of the T. gondii life cycle (Figure 4d), suggesting that host CaMKII activity may enhance cytoskeletal proteolysis to facilitate parasite release.

Analysis of Host Ca²⁺ dynamics Implicates TRPC6 in Ca²⁺ influx

Finally, changes in host [Ca²⁺] that drive this network were determined. To assess erythrocyte [Ca²⁺] during P. falciparum infection, a Ca²⁺ indicator was confined to the erythrocyte space via loading of a fura-2-dextran conjugate. Within the U20S cell cytoplasm during T. gondii infection, Ca²⁺ measurements were achieved via expression of the ratiometric FRET -based Ca²⁺ indicator yellow cameleon 3.6 (YC3.6) in U20S cells (Nagai et ah, 2004, Proc. Natl. Acad. Sci. USA 101 : 10554-10559; Palmer & Tsien, 2006, Nat. Protoc. 1 : 1057-1065). By microscopy and background-corrected fluorometry (Figures 5A-5B), the relative changes in host cell [Ca²⁺] were similar between both parasite systems, with host [Ca²⁺] hovering near resting levels (100 nM) throughout the first two-thirds of the life cycle, followed by a sharp increase in [Ca²⁺] at the point of parasite release.

Given the large [Ca²⁺] increase seen in both T. gondii and P.

falciparum host cells, host plasma membrane cation channels may play a role in this influx. siR A-mediated knockdown studies of mechanosensitive transient receptor potential (TRP) channels (trpcl, trpc3, trpc6) as well as canonical endoplasmic reticulum Ca²⁺ pumps (ryr3, ip3rl) identified TRPC6 as a specific mediator of T. gondii exit, as trpc6 knockdown resulted in an accumulation of unruptured vacuoles by 60 hpi (p<0.05; Figures 5C-5D). Trpc6 knockdown using multiple siRNA oligos abrogated the large increase in cytoplasmic [Ca²⁺] while ip3rl knockdown diminished the initial minor increase in [Ca²⁺] observed earlier in T. gondii infection, indicating that PLC activity and generation of IP3 may be responsible for the minor increase in [Ca²⁺] seen only in T. gowifc^'-infected cells (Figure 5E).

Pharmacological studies in P. falciparum-m^' fected erythrocytes confirmed the importance of mechanosensitive cation channel function in parasite- mediated cytolysis (Figure 1 1A). [Ca²⁺] diminished in cells depleted of Ga_q, ΡΙΧβ, or PKCa/β (Figures 1 lB-1 1C), but was rescued by PKC activation with PMA in cells depleted of Ga_q or ΡΙΧβ, indicating that PKC activity facilitates later Ca²⁺ influx, perhaps via adducing phosphorylation and loss. PMA could not rescue Ca²⁺ influx in cells depleted of PKCa/β, further confirming the necessity of PKC activity for this process. Taken together, these data highlight the importance of TRPC6-mediated cation influx in host cell cytolysis, perhaps as a mechanosensitive response to cytoskeletal rearrangement.

Anti-Parasitic Activity in Vivo

Reinforcing the importance of host PKC activity in parasite life cycle progression, the mammalian classical PKC inhibitor G56976 showed antiparasitic activity in murine models of toxoplasmosis and malaria. Indicating that these new host egress mediators represent an untapped resource of antiparasitic targets.

Intraperitoneal injection of 10 mg/kg G56976 limited parasite burden in both the spleen (pO.0001 ; Figure 6a, left) and peritoneal exudate cells (p<0.0001 ; Figure 6a, right). Similarly, 10 mg/kg G56976 caused a significant decrease in parasitemia in a mouse P. yoelii malaria model (Figure 6b). The consistency in antiparasitic activity in vivo further suggests the importance of host PKC function in both parasitic infections and underscores the conserved function of this host-signaling pathway.

As further corroboration of the importance of host PKC activity in malaria disease progression, the orally-bioavailable specific PKC inhibitor sotrastaurin was studied in a mouse model of experimental cerebral malaria (Figures 6c-6d). 50 mg/kg by gastric gavage in a standard 4-day Peters' suppression test led to a significant decrease in P. berghei ANKA parasitemia (p<0.001 ; Figure 6c) and significantly increased survival versus vehicle-treated controls (p<0.001 ; Figure 6d). Identification of the function of this host pathway within the parasite life cycle directly translates to drug discovery, as this inhibitor has passed Phase I trials and is undergoing Phase II trials for numerous indications, making it a clear antimalarial drug candidate.

Design of siR A Screen to Identify Host Genes Required for T. go«<i»-Mediated Cytolysis

As illustrated in Figure 8, for the primary screen, pooled siR As targeting a select set of 50 genes and 8 multiple gene families (three siRNAs/gene) were arrayed in collagen-coated 96-well plates (in quadruplicate), reverse-transfected into U20S cells. Following synchronous T. gondii infection at 24 hours post- transfection, plates were fixed and imaged by fluorescence microscopy at 6, 24, 44 or 60 hpi. Image analysis was used to calculate parasite doublings as a measure of parasite replication and parasites per PV to assess progression of the entire intracellular parasite life cycle including cytolysis. Hits were selected based on persistence of large vacuoles. The secondary screen included 19 individual genes and degenerate pairs of the multiple gene family hits, for a total of 17 multiple gene knockdowns using multiple oligos for validation. Hits from the secondary screen (individual genes or gene pairs) were tested in the final screen in 24-well plates. Images/Western blots of stable shRNA knockdown cells were utilized in Figures If-

Ig- Depletion of PLC i and Calmodulin- 1 from Erythrocytes is Sufficient to Block P.q/czpan<m-mediated Cytolysis

As illustrated in Figure 9A, erythrocytes depleted of either ΡΙΧβΙ, PLCy, or both ΡΙΧβΙ and PLCy were assayed for schizont persistence (left panel) or reinvasion of new rings (right panel) at 60 hpi. Though PLCpi depletion caused a significant persistence of schizont stage parasites and a reciprocal decrease in newly reinvaded rings (*p<0.05), indicating a block in cytolysis, PLCy depletion did not alter normal life cycle progression. Dual depletion of both PLCpi and PLCy did not significantly exaggerate the block in cytolysis from PLCP 1 alone, indicating that PLCy does not have a significant role in this process.

As illustrated in Figure 9B, erythrocytes depleted of either calmodulin- 1, calmodulin-2, or both calmodulin- 1 and calmodulin-2 were assayed for schizont persistence (left panel) or reinvasion of new rings (right panel) at 60 hpi. Though calmodulin- 1 depletion caused a significant persistence of schizont stage parasites and a reciprocal decrease in newly reinvaded rings, indicating a block in cytolysis

(*p<0.05), calmodulin-2 depletion did not alter normal life cycle progression. Dual depletion of both calmodulin- 1 and calmodulin-2 did not significantly exaggerate the block in cytolysis from calmodulin- 1 alone, indicating that calmodulin-2 does not have a significant role in this process. Data shown are means of at least three experiments ± SEM; n=3.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.

While the invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed:

1. A method of treating or preventing infection by an apicomplexan parasite in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising at least one PKC inhibitor or a salt thereof.

2. The method of claim 1, wherein the PKC inhibitor is a PKC- selective inhibitor.

3. The method of claim 1, wherein the PKC inhibitor is selected from the group consisting of:

sotrastaurin (3-(lH-Indol-3-yl)-4-[2-(4-methylpiperazin-l-yl)quinazolin-4- yl]pyrrole-2,5-dione),

bisindolylmaleimide-1 (3-[l-[3-(dimethylamino)propyl]-lH-indol-3-yl]-4- (lH-indol-3-yl)-lH-pyrrole-2,5-dione),

any salts thereof, and any combinations thereof.

4. The method of claim 1, wherein the parasite is Plasmodium falciparum.

5. The method of claim 1, wherein the parasite is Toxoplasma.

6. The method of claim 1, wherein the subject is a mammal.

7. The method of claim 6, wherein the subject is human.

8. The method of claim 1, wherein the administering step is performed through a topical, oral, nasal, buccal, sublingual, rectal, pleural, peritoneal, vaginal, intramuscular, subcutaneous, transdermal, epidural, intrathecal or intravenous route.

9. The method of claim 1, wherein the subject is further administered at least one additional agent useful for treating infection by an apicomplexan parasite.

10. The method of claim 9, wherein the at least one additional agent is selected from the group consisting of chloroquine, quinine, amodiaquine, propranolol, artemether-lumefantrine, artesunate-amodiaquine, artesunate- mefloquine, artesunate-sulfadoxine/pyrimethamine, atovaquone-proguanil, cotrifazid, doxycycline, mefloquine, primaquine, proguanil, sulfadoxine-pyrimethamine, hydroxychloroquine, artemisin, semi-synthetic derivatives of artemisin, extracts of the plant Artemisia annua, pyrimethamine, sulfadiazine, clindamycin, spiramycin, minocycline, and atovaquone.

1 1. The method of claim 9, wherein the inhibitor and the agent are separately administered to the subject.

12. The method of claim 9, wherein the inhibitor and the agent are co-administered to the subject.

13. The method of claim 12, wherein the inhibitor and the agent are co-formulated and co-administered to the subject.

14. A pharmaceutical composition comprising a PKC inhibitor and at least one additional agent useful for treating infection by an apicomplexan parasite.

15. The composition of claim 14, wherein the at least one additional agent is selected from the group consisting of chloroquine, quinine, amodiaquine, propranolol, artemether-lumefantrine, artesunate-amodiaquine, artesunate-mefloquine, artesunate-sulfadoxine/pyrimethamine, atovaquone-proguanil, cotrifazid, doxycycline, mefloquine, primaquine, proguanil, sulfadoxine- pyrimethamine, hydroxychloroquine, artemisin, semi-synthetic derivatives of artemisin, extracts of the plant Artemisia annua, pyrimethamine, sulfadiazine, clindamycin, spiramycin, minocycline, and atovaquone.

16. The composition of claim 14, further comprising a pharmaceutically acceptable carrier.

17. The composition of claim 14, wherein the PKC inhibitor is a PKC-selective inhibitor.

18. The composition of claim 14, wherein the PKC inhibitor is selected from the group consisting of:

any salts thereof, and any combinations thereof.

19. The composition of claim 14, wherein the parasite is

Plasmodium falciparum.

20. The composition of claim 14, wherein the parasite is

Toxoplasma.