WO2024196989A1 - Anti-malarial therapy - Google Patents

Abstract

Anti-malarial compounds are disclosed for administration to subjects infected or at risk of being infected with a Plasmodium sp.

Description

ANTI-MALARIAL THERAPY

GOVERNMENT SUPPORT CLAUSE

The invention was made with government support under grant numbers GR107045 and RO1 AI172066 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0001] Malaria still afflicts 247 million people worldwide causing 619,000 deaths in 2021 (WHO Malaria Report 2022). Options for malaria therapy are increasingly becoming limited because of widespread drug resistance. Even artemisinin-based combination therapies (ACTs), the front-line therapeutic choices for uncomplicated falciparum malaria, are gradually becoming ineffective in much of the Southeast Asia. Reports of failure of dihydroartemisinin-piperaquine combination therapy in Cambodia leaves us with very few therapeutic choices. This dire situation underscores the urgent need for new antimalarials that act on novel targets. Furthermore, most of the antimalarials act only on the erythrocytic stages, the drugs for prophylaxis and relapse of malaria are suboptimal. Therefore, there is a need for pharmacophores acting on multiple developmental stages.

SUMMARY

[0002] Dysregulation of the kinase activity is linked to many diseases. Since malaria parasite Plasmodium falciparum kinases are now accepted as druggable targets (Arendse et al, ACS Infect. Dis.7, 518-534), repurposing of human kinase inhibitors for malaria therapy was pursued. In this invention disclosure reports that an aminopyridine compound YLIU-06-026-1 has potent prophylactic and therapeutic antimalarial activities in the rodent malaria model. This compound was identified from screening the Nathanael Gray library developed for cancer therapy. The compound YLIU-6-026-1 has been shown to be a Janus Kinase (JAK) inhibitor (WO 2020/007396).

BRIEF DESCRIPTION OF THE DRAWINGS

[0003] Figure 1 shows that YLIU-06-026-1 eliminates malaria infection. Mice (Swiss webster) infected with P. berghei luc (/W-Luc) parasites (200 pL of iRBC with 10% parasitemia) expressing luciferase were treated with 4 doses of 50 mg/kg YLIU-06-026-1 or the positive

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SUBSTITUTE SHEET (RULE 26) control GNF179 intravenously at 24h, 48h, 72h, and 96h after infection, following the four-day

Peters test. Imaging was done 12 days after infection by IVIS Bioluminescence Imaging.

[0004] Figure 2 shows that YLIU-06-026-1 exhibits prophylactic activity. Infection was initiated using 150,000 F7?-Luc sporozoites administered by IP injection. The drugs were administered IV at 50 mg/kg, single dose 6h before infection. Imaging performed by IVIS Bioluminescence Imaging 12 days after infection.

[0005] Figure 3 shows the test comparing YLIU-06-026-1 and known antimalarial GNF179 against Pb-Luc parasites. Therapeutic (4 doses of 50 mg/kg each time was administered at 24, 48, 72, and 96hr post-infection) activity of YLIU-06-026-1 was evaluated. Groups of 5 mice were injected intravenously with 200pL blood with 10% Pb-Luc parasitemia. The parasitemia was measured every other day for 14 days by flow cytometric analysis of infected RBCs stained with SYBR Green I.

[0006] Figure 4 shows the test comparing YLIU-06-026-1 and GNF179 against Pb-Luc sporozoites. Prophylactic (single dose of 50 mg/kg through IV injection, 6h before infection) activity of YLIU-06-026-1 was evaluated. Groups of 5 mice were infected with 150,000 Pb-Luc sporozoites by IP injection. The parasitemia was quantified by flow cytometric analysis of infected RBCs stained with SYBR Green I every other day for 14 days.

[0007] Figure 5 shows the stage specific activity of and YLIU-06-026-1. Synchronized cultures were exposed to the compound (5 x ECso) at 6h post invasion for 12h and fixed samples were analyzed by flow cytometry of YOYO- 1 labeled cells and by microscopy using Giemsa- stained thin smears. Control cell Giemsa staining is shown in the top panel. Bottom panel shows YLIU- 06-026-1 treated microscopic images and merged flow cytometric profiles. Blue represents control profiles, and red indicates treated profiles. The results are representative of 3 biological replicates.

[0008] Figure d show the killing profile of YLIU-06-026-1, the fast-acting positive control, dihdroartemisinin (DHA), and slow-acting positive control, atovaquone. Parasites were treated with 10 X ECso for 12 h prior to compound wash off. Flow cytometry with SYBR Green I (nuclear dye) and Mitotracker Deep Red FM (mitochondria staining) was performed after

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SUBSTITUTE SHEET (RULE 26) compound wash-off every 24 h after for 6 days (100,000 events/sample) to track parasitemia (data from 3 biological replicates ± SEM).

Figure 7 presents a graph that shows the effects of compounds on parasite proliferation. The graph evaluates number of viable parasites over incubation time.

Figure 8 presents a graph that shows the EC50 of YEIU-06-026-1 ex vivo, in P. falciparum parasites from Ugandan patients.

DETAIEED DESCRIPTION

Definitions

[0009] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods and materials are now described. However, the skilled artisan understands that the methods and materials used and described are examples and may not be the only ones suitable for use in the invention.

Moreover, as measurements are subject to inherent variability, any temperature, weight, volume, time interval, pH, salinity, molarity or molality, range, concentration and any other measurements, quantities or numerical expressions given herein are intended to be approximate and not exact or critical figures unless expressly stated to the contrary.

[0010] Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein, and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed through the present specification unless otherwise indicated. All publications mentioned herein are incorporated herein by reference.

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SUBSTITUTE SHEET (RULE 26) [0011] In the foregoing specification, the invention has been described with reference to specific embodiments thereof. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense. Throughout this specification and the claims, unless the context requires otherwise, the word “comprise” and its variations, such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated item, element or step or group of items, elements or steps but not the exclusion of any other item, element or step or group of items, elements or steps. Furthermore, the indefinite article “a” or “an” is meant to indicate one or more of the item, element or step modified by the article.

[0012] As used herein, the term “about” generally means plus or minus 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the recited value, so that, for example, “about 0.125” means 0.125 ± 0.0125, and “about 1.0” means 1.0 ± 0.1, using 10%. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in specific non-limiting examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements at the time of this writing. Furthermore, unless otherwise clear from the context, a numerical value presented herein has an implied precision given by the least significant digit. Moreover, all ranges disclosed herein are to be understood to encompass any and all sub-ranges subsumed therein. For example, a range of "less than 10" can include any and all sub-ranges between (and including) the minimum value of zero and the maximum value of 10, that is, any and all sub-ranges having a minimum value of equal to or greater than zero and a maximum value of equal to or less than 10, e.g., 1 to 4.

[0013] As used herein, the terms “administering” or "administration" of an agent, compound, drug, or peptide to a subject refers to any route of introducing or delivering to a subject a compound to perform its intended function. The administering or administration can be carried out by any suitable route as deemed appropriate by the practitioner, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, or topically. Administering or administration includes self-administration and the administration by another.

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SUBSTITUTE SHEET (RULE 26) [0014] As used herein, the terms “composition” or “pharmaceutical composition” comprise one or more of the compounds described herein as active ingredient(s), or a pharmaceutically acceptable salt(s) thereof, and preferably also contain a pharmaceutically acceptable carrier and optionally also contain other therapeutic ingredients. The compositions include compositions suitable for oral, rectal, ophthalmic, pulmonary, nasal, dermal, topical, parenteral (including subcutaneous, intramuscular and intravenous) or inhalation administration, or any method of administration deemed appropriate by the practitioner for a particular subject. The most suitable route in any particular case will depend on the nature and severity of the conditions being treated and the nature of the active ingredient(s). The compositions may be presented in unit dosage form and prepared by any of the methods well-known in the art of pharmacy. Dosage regimes may be adjusted for the purpose to improving the therapeutic response. For example, several divided dosages may be administered daily or the dose may be proportionally reduced over time. A person skilled in the art normally is able to determine the effective dosage amount and the appropriate regime.

[0015] As used herein, the term “analog” refers to a compound having a structure similar to that of another compound but differing from the other compound with respect to a certain component or substituent. The compound may differ in one or more atoms, functional groups, or substructures, which may be replaced with other atoms, groups, or substructures. In one aspect, such structures possess at least the same or a similar therapeutic efficacy.

[0016] As used herein, "derivative" refers to a compound derived or obtained from another and containing essential elements of the parent compound. In one aspect, such a derivative possesses at least the same or similar therapeutic efficacy as the parent compound.

[0017] The term enumerated compounds refers to the compounds set forth in Table 2.

[0018] As used herein, the term “pharmaceutically acceptable salt” is intended to include nontoxic base addition salts. Suitable salts include those derived from organic and inorganic acids such as, without limitation, hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, methanesulfonic acid, acetic acid, tartaric acid, lactic acid, sulfinic acid, citric acid, maleic acid, fumaric acid, sorbic acid, aconitic acid, salicylic acid, phthalic acid, and the like. The term “pharmaceutically acceptable salt” as used herein is also intended to include salts of acidic groups, such as a carboxylate, with such counterions as ammonium, alkali metal salts,

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SUBSTITUTE SHEET (RULE 26) particularly sodium or potassium, alkaline earth metal salts, particularly calcium or magnesium, and salts with suitable organic bases such as lower alkylamines (methylamine, ethylamine, cyclohexylamine, and the like) or with substituted lower alkylamines (e.g. hydroxyl-substituted alkylamines such as diethanolamine, triethanolamine or tris(hydroxymethyl)-aminomethane), or with bases such as piperidine or morpholine.

[0019] As used herein, the term “prodrug” refers to a compound that is converted to a therapeutically active compound after administration, and the term should be interpreted as broadly herein as is generally understood in the art. Generally, but not necessarily, a prodrug is inactive or less active than the therapeutically active compound to which it is converted. For example, a methyl ester prodrug can be converted to a free carboxylic acid drug in vivo via the action of non-specific serum esterases.

[0020] As used herein, As used herein, the term "stereoisomer" refers to a compound which has the identical chemical constitution but differs with regard to the arrangement of the atoms or groups in space.

[0021] As used herein, As used herein, the terms “treating” or “treatment” or “alleviation” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the objective is to prevent or slow down (lessen) the targeted pathologic condition or disorder. The term “treatment regimen” refers to a course of treatment or a series of doses administered to a subject over time. Thus, treatment includes administration of a compound to cure the disease, to reduce the length of infection, to reduce the severity of the disease, to inhibit the disease or a symptom thereof, to reduce the parasitemia in a subject suffering from the disease, and the like.

[0022] As used herein, the term “preventing” means causing the clinical symptoms of the disease state not to worsen or develop, e.g., inhibiting the onset of disease, in a subject that may be exposed to or predisposed to the disease state, but does not yet experience or display symptoms of the full disease state, e.g., malaria. Thus, prevention and/or prophylaxis includes administration of a compound to completely prevent the occurrence of the disease, to reduce the occurrence of the disease in a population, to reduce the likelihood of occurrence of the disease in a subject, to reduce the severity of the disease when it does occur, to inhibit the disease or a symptom thereof, to reduce parasitemia in a subject, and the like.

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SUBSTITUTE SHEET (RULE 26) [0023] According to certain embodiments, provided are methods of preventing or treating malaria in a subject or preventing or treating a subject exhibiting a symptom of malaria by administering effective amounts of one or more compounds described herein. Malaria in humans typically produces a string of recurrent attacks, or paroxysms, each of which has three stages — chills, followed by fever, and then sweating. Along with chills, the person is likely to have headache, malaise, fatigue, muscular pains, occasional nausea, vomiting, and diarrhea. Within an hour or two, the body temperature rises, and the skin feels hot and dry. Then, as the body temperature falls, a drenching sweat begins. The person, feeling tired and weak, is likely to fall asleep. Thus, a subject exhibiting one, two or more of the foregoing symptoms is considered a subject in need.

[0024] As the term is used herein, the term “subject” includes any animal, preferably a mammal, including mammals such as a human, a laboratory animal, a companion animal, and a livestock animal. Preferably, the subject is human, however the invention contemplates treatment of animals such as rats, mice and rabbits, livestock such as cattle, sheep, pigs and the like, and companion animals such as dogs, cats and other pets. A "subject in need” refers to any mammal, particularly a human, who suffers from malaria or a symptom of malaria, has been diagnosed with malaria, or is likely to become infected by the malarial parasite, for example by being in an area endemic to the disease.

[0025] As used herein, the terms “pharmaceutically acceptable carrier, excipient, vehicle, or diluent” refer to a medium which does not interfere with the effectiveness or activity of an active ingredient and which is not toxic to the hosts to which it is administered. A carrier, excipient, vehicle, or diluent includes but is not limited to binders, adhesives, lubricants, disintegrants, bulking agents, buffers, and miscellaneous materials such as absorbents, taste-masking agents, pH modifiers, and the like, that may be needed in order to prepare a particular composition.

[0026] As used herein, “anti-malarial” or “anti-malarial activity” includes any activity that decreases the infectivity, the reproduction, or inhibits the progress of the lifecycle of a malaria parasite. “Anti-malarial activity” also includes inhibition of the growth of malaria infection by all of the means observed with current anti-malarial drugs.

[0027] As used herein, the term “anti-malarial agent” refers to any compound according to the various embodiments, compounds referred to in the Tables below and the accompanying figures,

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SUBSTITUTE SHEET (RULE 26) and any combinations, prodrugs, pharmaceutically acceptable salts, analogs, and derivatives thereof. The compositions and methods described herein are useful for the treatment and/or prevention of malaria. The determination of a therapeutically effective dose is well within the capability of those skilled in the art.

[0028] The term "conjunctive anti-malarial agent" as used herein refers to an anti-malarial agent other than YLIU-06-026-1. Conjunctive anti-malarial agents include but are not limited to one or more agents selected from quinine, quinidine, cinchonine, cinchonidine, chloroquine, hydroxychloroquine, amodiaquine, pyrimethamine, proguanil, a sulfonamide, mefloquine, atovaquone, primaquine, artemisinin and derivatives thereof, halofantrine, lumefantrine, doxycycline, and clindamycin.

[0029] As used herein, a “therapeutically effective dose” refers to that amount of active ingredient which causes reduction or elimination of malaria in a subject and includes a single dose as well as a course of doses or a regimen of doses administered to a subject.

[0030] As used herein, the term “effective amount,” “amount effective,” “therapeutically effective amount,” or the like, means an amount or number of effective dosages administered for periods of time necessary to achieve the desired result. For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans, as is understood by persons of skill in the art.

[0031] Therapeutic efficacy and toxicity, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. These data are commonly used by the person of ordinary skill to determine an appropriate dose or range of doses.

[0032] The half maximal inhibitory concentration (IC50) is a measure of the potency of a substance in inhibiting a specific biological or biochemical function. IC50 is a quantitative

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SUBSTITUTE SHEET (RULE 26) measure that indicates how much of a particular inhibitory substance is needed to inhibit, in vitro, a given biological process or biological component by 50%.

[0033] As used herein, The half maximal effective concentration (ECso) is a statistical estimate of the concentration of a toxicant in the ambient medium necessary to produce a particular effect in 50% of a very large population under specified conditions.

Pharmaceutical Formulations and Administration

[0034] Various embodiments of the invention relate to pharmaceutical compositions comprising an effective amount of one or more of the enumerated compounds or to various analogs of those compounds or combinations, derivatives, prodrugs, stereoisomers, or pharmaceutically acceptable salts thereof. The pharmaceutical compositions exhibit antiplasmodium potency against P. berghei, P. falciparum, P. vivax, P. ovale, P. malariae, or P. knowlesi. According to various embodiments, the pharmaceutical compositions may further comprise a pharmaceutically acceptable carrier and/or a conjunctive anti-malarial agent.

[0035] The present disclosure specifically includes pharmaceutical compositions and formulations of the enumerated compounds described here which contain an active agent as described herein and a pharmaceutically acceptable carrier or excipient. In typical embodiments, pharmaceutical compositions comprise therapeutically effective amounts of the enumerated compounds, which amounts treat or prevent Plasmodium infection in a subject as a single dose or as part of a series of doses or a treatment regimen. Pharmaceutical compositions for use in the present methods include therapeutically effective amounts of one or more of the enumerated compounds, in an amount sufficient to prevent or treat the diseases described herein in a subject, formulated for systemic administration. In an optional embodiment, one or more of the enumerated compounds may be co-administered with at least one other active agent.

[0036] Thus, pharmaceutical compositions comprising one or more enumerated compounds as described herein also include one or pharmaceutically acceptable carriers. Such compositions may contain any excipient known in the art of pharmaceutics to function in making the composition suitable for administration to a subject. For example, excipients can function as a filler, a carrier, a binder, a disintegrant, a glidant, a preservative, a stabilizing agent, a wetting

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SUBSTITUTE SHEET (RULE 26) agent or surfactant, a pH modifier, an emulsifier, a solvent, an antimicrobial, an osmotic agent, and the like as known in the pharmaceutical arts, or may serve several of such functions.

[0037] The formulations can be prepared for any suitable route of administration, including liquid, solid, or gas formulations. Formulations such as solutions, suspensions, lipid emulsions, and the like are suitable for injection into the subject, including intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, and the like, in a bolus dose or an infusion. Solid formulations can be prepared for administration orally, or as a suppository, implant, transdermal patch, a powder inhalant, and the like. Preferred administration is by intravenous or intraperitoneal injection.

[0038] The pharmaceutical compositions of this disclosure may be in a variety of forms, which may be selected according to the preferred modes of administration. These include, for example, solid, semi-solid and liquid dosage forms such as tablets, lozenge, pills, powders, liquid solutions or suspensions, suppositories, and injectable and infusible solutions. The preferred form depends on the intended mode of administration and therapeutic application.

[0039] For oral administration, one or more enumerated compounds can be formulated as dispersible tablet, orally disintegrating tablet, effervescent tablet, chewable tablet, sprinkle granules, oral solution, dry suspension or dry syrup for reconstitution, quick melt wafers, lozenge, or chewing gum. Examples of suitable excipients for solid dosage forms for oral administration include, water, saline, dextrose, glycerol, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions typically contain l%-95% of active ingredient, preferably 2%-70% active ingredient.

[0040] Aqueous solution compositions can be packaged for use as is or for dilution before use, filtered under aseptic conditions and lyophilized to be reconstituted with a sterile aqueous solution, such as water or saline solution, prior to administration. The compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. Additionally, lipidic suspension compositions can include lipid-protective agents which protect lipids against free -radical and lipid-peroxidative damages on storage. Lipophilic free-

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SUBSTITUTE SHEET (RULE 26) radical quenchers, such as a-tocopherol and water-soluble iron-specific chelators, such as ferrioxamine, are suitable for inclusion as well. The pharmaceutical preparations optionally are sterilized by conventional, well known sterilization techniques.

[0041] Formulations for parenteral administration by injection include aqueous and non-aqueous sterile injection solutions, which optionally contain anti-oxidants, buffers, bacteriostats, and components that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions for injection which optionally include suspending agents and thickening agents. The formulations can be presented in unit-dose or multi-dose containers, for example prepared syringes or injector pens, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example saline or water-for-injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

[0042] The disclosure has references to specific embodiments. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the disclosure. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense. The disclosure is illustrated herein by the experiments described above and by the following examples, which should not be constmed as limiting. The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference.

Methods of Use

[0043] Methods of using the enumerated compound(s) described herein are contemplated as part of this invention. The compounds, or pharmaceutical compositions containing the compounds, are administered to a subject. For the prevention or treatment of disease, the appropriate dosage will depend on the severity of the disease, whether the drug is administered for protective or therapeutic purposes, previous therapy, the patient's clinical history and response to the drugs and the discretion of the attending physician. The formulations also can contain more than one of the inventive active compounds and/or contain additional active agents useful in the treatment or prevention of malaria. Such additional agents suitably are present in combination in amounts that are effective for the purpose intended. It is understood that the methods and uses of the

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SUBSTITUTE SHEET (RULE 26) embodiments of the invention disclosed here can be employed for prophylaxis/prevention as well as (more suitably) in the treatment of subjects suffering from Plasmodium infection. Thus, the enumerated compounds can be administered with additional compounds (1) simultaneously in a single combination dosage form, (2) simultaneously in separate dosage forms, or (3) sequentially.

[0044] The duration of treatment with the described compounds can extend over any suitable period of time as determined by the clinician, depending on the condition of the subject, with the treatment preferably continuing until the Plasmodium infection is sufficiently reduced or eliminated. The progress of therapy is easily monitored by conventional techniques and assays that may be used to adjust dosage and the dosage regimen to achieve a desired therapeutic effect.

[0045] The subject is preferably a human but can be non-human as well. A suitable subject for treatment can be any individual who is suspected of having, has been diagnosed as having, or is at risk of developing a Plasmodium infection. A suitable subject for prophylaxis can be any individual who resides in a region endemic for malaria or is visiting such an area.

[0046] These and other features, aspects, and advantages of various embodiments will become better understood with reference to the following description, figures, examples and claims.

EXAMPLES

Example 1. Screening of kinase inhibitor libraries identifies asexual and liver stage antiplasmodial hits

[0047] To establish the utility of kinase inhibitor compounds synthesized in the Gray laboratory as a source of novel antimalarial activities, the libraries containing 1,100 compounds were screened using the well-established SYBR Green I-based fluorescence assay. This screen yielded 34 compounds with EC50 <500 nM in the CQ^R Dd2 strain. These hits were subjected to counterscreening against human HepG2 cells using CellTiter 96® AQueous (Promega) MTS-based cell proliferation assay. After down- selecting compounds based on novelty, potency, and selectivity, the compound YLIU-06-026-1 was identified as a hit compound.

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SUBSTITUTE SHEET (RULE 26)

Table 1. Antiplasmodial hits from the Gray libraries. P. falaciparum Dd2 ECso was determined using SYBR Green I assay. For selectivity index (Dd2 EC5o/HepG2 human liver cell EC50) cytotoxicity was determined using CellTiter 96® AQueous MTS. The liver stage assay was performed using P. berghei luciferase expressing parasites. All assays were performed with technical triplicates and 3 biological replicates. Assays have a Z’ factor >0.7.

Example 2. YLIU-06-026-1 exhibits therapeutic and prophylactic activities

[0048] As shown in Fig 1, YLIU-06-026-1 eliminates established P. berghei infection at 50 mg/kg administered intravenously once daily for 4 days. Experiment shows that YLIU-06-026-1 has prophylactic activity (Fig 2) when administered as a single dose (50 mg/kg, IV) 6 h prior to infection of mice with sporozoites. These results establish the utility of YLIU-06-026-1 as a starting point for optimization of prophylactic and therapeutic compounds. It is clearly evident from Fig 3 and 4 that YLIU-06-026-1 has both prophylactic and therapeutic activities, a desired property of current malaria target product profile.

Example 3. YLIU-06-026-1 inhibits asexual stage maturation early, exhibit fast killing rate, and exhibit metabolic stability

[0049] The stage specific action of the prioritized hits was determined. As shown in Fig 5, the compound YLIU-06-026-1 blocks ring to trophozoite maturation when treated at early ring. This is significant, as other than artemisinin none of the current antimalarials act early at the ring stage. The compound also blocks maturation of asexual parasites when added at the trophozoite stage (data not shown). As shown in Fig 6, YLIU-06-026-1 exhibited pronounced irreversible killing effect following 12 h exposure similar to that with dihydroartemisinin (DHA). YLIU-06-026-1 also exhibit desirable microsomal stability in mouse liver microsomes (ti/2= 2.42 hr zv, 6.72 hr po) and an excellent oral bioavailability (F% = 129.2).

[0050] In summary, YLIU-06-026-1 meets many of the MMV (Medicine for Malaria Venture) Target Product Profiles (TPP) criteria as it shows activity against multiple stages Pf parasite development with mechanism of action different from current antimalarials, act rapidly, and with acceptable PK criteria for parasite clearance.

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SUBSTITUTE SHEET (RULE 26) Table 2 below provides EC50 data for select compounds

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

Example 4

[0051] Further results from two additional assays which underscores the potential of YLIU-06- 026-1 as a promising antimalarial compound. The first is the parasite reduction ratio, an assay which more precisely describes the compound’s rapid killing kinetics. The second assay evaluates the compound’s potency against malaria parasites obtained from Ugandan patients, which reflect the currently circulating strains of P. falciparum in that region.

Parasite Reduction Ratio:

[0052] This assay can describe in detail the killing kinetics of a compound and compare it to two known antiplasmodial compounds: the fast- acting dihydroartemisinin (DHA) and the slow-

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SUBSTITUTE SHEET (RULE 26) killing atovaquone. In this assay synchronized ring stage P. falciparum parasites are treated with lOx EC50 of compound, and compound is washed out and readministered every 24 hours for a total of 96 hours. Parasites are maintained without treatment for 21 days, and parasite proliferation is determined by a SYBR Green I-based assay. The graph below depicts the viable parasites over incubation time with either YLIU-06-026-1 or the two control compounds. As shown, YLIU-06-026-1 behaves in a manner most similar to the rapidly killing antiplasmodial compound, DHA; YLIU-06-026-1 acts rapidly on ring stage parasites, lacks a lag phase, and results in clearance of 99.9% of parasites by 39 hours (Figure 7). This is promising, as an ideal antimalarial compound should be capable of killing the parasite quickly and early in the life cycle.

Evaluation in Clinical Isolates:

[0053] In addition to in vitro assays, we evaluated the potency of YLIU-06-026-1 ex vivo, in P. falciparum parasites obtained from Ugandan patients, using a classic SYBR Green I-based parasite proliferation assay, in which parasites are treated with a serial dilution of compound for 72 hours. As shown in the Figure 8, YLIU-06-026-1 performed very well against the ex vivo isolates, with an average EC50 comparable to the lab-adapted P. falciparum strains Dd2 and 3D7 (Table 3). This suggests that YLIU-06-026-1 is effective against currently circulating P. falciparum strains in Uganda, bolstering the clinical relevance of the compound.

Table 3

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SUBSTITUTE SHEET (RULE 26)

Claims

CLAIMS What is claimed is

1. A method for treatment or prevention of Plasmodium infection in a subject in need thereof, comprising administering an effective amount of one or more compounds selected from

2. The method of claim 1 , wherein the Plasmodium comprises P. berghei, P. falciparum, P. vivax, P. ovale, P. malariae, or P. knowlesi.

3. The method of claim 1, wherein the one or more compounds is

, or a pharmaceutically acceptable salt

thereof.

4. The method of any of claims 1-3, wherein the one or more compounds are administered in a composition comprising a pharmaceutically acceptable carrier.

5. The method of claim 4, wherein the composition is formulated for oral or parenteral administration.

6. The method of any of claims 1-5, further comprising co-administering at least one conjunctive anti- malarial agent.

7. A composition comprising

, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

8. The composition of claim 7, formulated for oral or parenteral administration.

9. The composition of claim 7, wherein the composition is a solution, suspension, or lipid emulsion formulated for injection into a subject, including intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal in a bolus dose or an infusion.

10. The composition of claim 7, wherein the composition is a solid formulation for oral administr ation to the subject, including a table, lozenge, pill, powder, or capsule.

11. The composition of any of claims 7-10, further comprising at least one conjunctive anti-malarial agent.