WO2014186623A2 - Methods and compositions for the treatment of a chagas disease - Google Patents

Abstract

The present invention provides, inter alia, methods for treating or ameliorating the effects of a Chagas disease. The methods include administering to a subject in need thereof an effective amount of a first active agent selected from the group consisting of a histamine-H1-receptor antagonist, a selective neutrophil elastase inhibitor, an ergot alkaloid derivative, an inhibitor of 3 β-hydroxysteroid dehydrogenase, a reversible acetylcholinesterase inhibitor, a protease inhibitor, an aldosterone antagonist, a naphthoquinone, and combinations thereof, and a second active agent, which is benznidazole. Also provided are compositions for treating or ameliorating the effects of a Chagas disease. The compositions include azelastine and benznidazole in amounts effective to treat or ameliorate the effect of a Chagas disease and a pharmaceutically acceptable carrier.

Description

METHODS AND COMPOSITIONS FOR THE TREATMENT OF A CHAGAS

DISEASE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present invention claims benefit to U.S. Provisional Application No. 61/824,406 filed May 17, 2013. The entire contents of the above application are incorporated by reference.

FIELD OF INVENTION

[0002] The present invention provides, inter alia, methods and compositions for treating or ameliorating the effects of a Chagas disease.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0003] This application contains references to amino acids and/or nucleic acid sequences that have been filed concurrently herewith as sequence listing text file 0344774.txt, file size of 6 KB, created on May 7, 2013. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. § 1 .52(e)(5).

BACKGROUND OF THE INVENTION

[0004] Chagas disease is caused by the protozoan parasite Trypanosoma cruzi (T. cruzi) and is a primary contributing cause of cardiovascular death and disease in Latin American countries (1, 2). There are currently two primary therapeutics used to treat T. cruzi infections: benznidazole (Radanil®), and nifurtimox (Lampit®). Both therapeutics require protracted courses of treatment, have numerous side effects, and display varying levels of efficacy (3, 4).

[0005] Thus, there is an unmet need for safer, more effective treatments for Chagas disease. The present invention is directed to meeting this and other needs.

SUMMARY OF THE INVENTION

[0006] One embodiment of the present invention is a method for treating or ameliorating the effects of a Chagas disease. This method comprises administering to a subject in need thereof an effective amount of a first active agent selected from the group consisting of a histamine-H1 -receptor antagonist, a selective neutrophil elastase inhibitor, an ergot alkaloid derivative, an inhibitor of 3 β-hydroxysteroid dehydrogenase, a reversible acetylcholinesterase inhibitor, a protease inhibitor, an aldosterone antagonist, a naphthoquinone, and combinations thereof, and a second active agent, which is benznidazole.

[0007] Another embodiment of the present invention is a method for treating or ameliorating the effects of a Chagas disease. This method comprises administering to a subject in need thereof an effective amount of azelastine at 2-200 mg/kg per day and benznidazole at 0.5-2 mg/kg per day.

[0008] An additional embodiment of the present invention is a composition for treating or ameliorating the effects of a Chagas disease. This composition comprises azelastine and benznidazole in amounts effective to treat or ameliorate the effect of a Chagas disease and a pharmaceutically acceptable carrier.

[0009] Another embodiment of the present invention is a method for treating or ameliorating the effects of a Chagas disease. The method comprises administering to a subject in need thereof an effective amount of a first active agent selected from the group consisting of a selective neutrophil elastase inhibitor, an ergot alkaloid derivative, an inhibitor of 3 β-hydroxysteroid dehydrogenase, a reversible acetylcholinesterase inhibitor, a protease inhibitor, an aldosterone antagonist, a naphthoquinone, and combinations thereof, and a second active agent, which is benznidazole.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 shows a Kaplan-Meier survival analysis comparing the effects of saline with various concentrations of benznidazole (BZ).

[0011] Figure 2A shows a Kaplan-Meier survival analysis following sivelestat- BZ combination treatment compared to a saline control. Figure 2B shows a Kaplan- Meier survival analysis following sivelestat-BZ combination treatment compared to BZ treatment at 1 mg/kg.

[0012] Figure 3A shows a Kaplan-Meier survival analysis following donepezil- BZ combination treatment compared to a saline control. Figure 3B shows a Kaplan- Meier survival analysis following donepezil-BZ combination treatment compared to BZ treatment at 1 mg/kg.

[0013] Figure 4A shows a Kaplan-Meier survival analysis following nicergoline-BZ combination treatment compared to a saline control. Figure 4B shows a Kaplan-Meier survival analysis following nicergoline-BZ combination treatment compared to BZ treatment at 1 mg/kg.

[0014] Figure 5A shows a Kaplan-Meier survival analysis following ritonavir- BZ combination treatment compared to a saline control. Figure 5B shows a Kaplan- Meier survival analysis following ritonavir-BZ combination treatment compared to BZ treatment at 1 mg/kg. [0015] Figure 6A shows a Kaplan-Meier survival analysis following trilostane- BZ combination treatment compared to a saline control. Figure 6B shows a Kaplan- Meier survival analysis following trilostane-BZ combination treatment compared to BZ treatment at 1 mg/kg.

[0016] Figure 7A shows a schematic depicting the infection, treatment, and euthanization schedule for all results in which blood was drawn for qPCR analysis.

[0017] Figure 7B shows representative survival curves for combination studies.

[0018] Figure 7C shows parasite enumeration results using a qPCR assay of satellite DNA repeats for two independent experiments.

[0019] Figure 8A shows survival curves for BZ at 1 mg/kg alone compared to BZ at 1 mg/kg dosed in combination with azelastine at 10 mg/kg in two separate experiments.

[0020] Figure 8B shows survival curves for BZ at 1 mg/kg alone compared to BZ at 1 mg/kg dosed in combination with azelastine at 10 mg/kg, 30 mg/kg, and 100 mg/kg.

[0021] Figure 9A shows results from a parasitemia qPCR assay. Blood samples were collected 10 days after parasite inoculation and drug treatment. DNA was purified from blood and assayed by qPCR to detect the relative abundance of T. craz/^'-specific satellite DNA sequences. Parasite burden is expressed normalized relative to levels in vehicle treated subjects.

[0022] Figure 9B shows in vitro parasite growth in cultures treated with varying concentrations of BZ or azelastine. DETAILED DESCRIPTION OF THE INVENTION

[0023] One embodiment of the present invention is a method for treating or ameliorating the effects of a Chagas disease. This method comprises administering to a subject in need thereof an effective amount of a first active agent selected from the group consisting of a histamine-H1 -receptor antagonist, a selective neutrophil elastase inhibitor, an ergot alkaloid derivative, an inhibitor of 3 β-hydroxysteroid dehydrogenase, a reversible acetylcholinesterase inhibitor, a protease inhibitor, an aldosterone antagonist, a naphthoquinone, and combinations thereof, and a second active agent, which is benznidazole.

[0024] As used herein, a Chagas disease means a disease caused by the protozoan parasite Trypanosoma cruzi.

[0025] As used herein, the terms "treat," "treating," "treatment" and grammatical variations thereof mean subjecting an individual subject to a protocol, regimen, process or remedy, in which it is desired to obtain a physiologic response or outcome in that subject, e.g., a patient. In particular, the methods and compositions of the present invention may be used to slow the development of disease symptoms or delay the onset of the disease or condition, or halt the progression of disease development. However, because every treated subject may not respond to a particular treatment protocol, regimen, process or remedy, treating does not require that the desired physiologic response or outcome be achieved in each and every subject or subject, e.g., patient, population. Accordingly, a given subject, e.g., patient, population may fail to respond or respond inadequately to treatment. [0026] As used herein, the terms "ameliorate", "ameliorating" and grammatical variations thereof mean to decrease the severity of the symptoms of a disease in a subject.

[0027] As used herein, a "subject" is a mammal, preferably, a human. In addition to humans, categories of mammals within the scope of the present invention include, for example, agricultural animals, domestic animals, laboratory animals, etc. Some examples of agricultural animals include cows, pigs, horses, goats, etc. Some examples of domestic animals include dogs, cats, etc. Some examples of laboratory animals include rats, mice, rabbits, guinea pigs, etc.

[0028] In the present invention, an "effective amount" or a "therapeutically effective amount" of a compound or composition disclosed herein is an amount of such compound or composition that is sufficient to effect beneficial or desired results as described herein when administered to a subject. Effective dosage forms, modes of administration, and dosage amounts may be determined empirically, and making such determinations is within the skill of the art. It is understood by those skilled in the art that the dosage amount will vary with the route of administration, the rate of excretion, the duration of the treatment, the identity of any other drugs being administered, the age, size, and species of mammal, e.g., human patient, and like factors well known in the arts of medicine and veterinary medicine. In general, a suitable dose of a composition according to the invention will be that amount of the composition, which is the lowest dose effective to produce the desired effect. The effective dose of a compound or composition of the present invention may be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day. Exemplary effective dosages of various compounds and compositions are disclosed herein. [0029] In the present invention, the first active agent and the second active agent may be administered together in the same composition, simultaneously in separate compositions, or as separate compositions administered at different times, as deemed most appropriate by a physician.

[0030] In one aspect of this embodiment, the first active agent is a histamine- H1 -receptor antagonist.

[0031] As used herein, an "antagonist" means a substance that does not trigger a response itself upon binding to a receptor, but blocks or dampens agonist- mediated responses. As used herein, an "agonist" means a substance that binds to a receptor of, e.g., a cell, and triggers a response in the cell.

[0032] Preferably, the histamine-H1 -receptor antagonist is selected from the group consisting of 4-methyldiphenhydramine, aceprometazine, acrivastine, alcaftadine, alimemazine, antazoline, aptazapine, astemizole, azatadine, azelastine, bamipine, bepotastine, bilastine, bisulepine, bromazine, brompheniramine, carbinoxamine, cetirizine, chlorcyclizine, chloropyramine, chlorothen, chlorphenamine, chlorphenoxamine, cinnarizine, clemastine, clemizole, dobenzepam, dobenztropine, docinizine, cyanodothiepin, cydizine, cyproheptadine, dacemazine, deptropine, desloratadine, dexbrompheniramine, dexchlorpheniramine, dimenhydrinate, dimetindene, diphenhydramine, diphenylpyraline, doxylamine, ebastine, embramine, emedastine, epinastine, esmirtazapine, etymemazine, fexofenadine, histapyrrodine, homochlorcyclizine, hydroxyethylpromethazine, hydroxyzine, isopromethazine, isothipendyl, ketotifen, latrepirdine, levocabastine, levocetirizine, loratadine, mebhydrolin, meclizine, mepyramine, methafurylene, methapyrilene, methdilazine, mianserin, mirtazapine, mizolastine, moxastine, olopatadine, orphenadrine, oxatomide, pemirolast, phenindamine, pheniramine, phenyltoloxamine, pirolate, promethazine, propiomazine, pyrrobutamine, quifenadine, repirinast, rupatadine, setastine, setiptiline, sominex, talastine, terfenadine, thenalidine, thenyldiamine, thiazinamium metilsulfate, thonzylamine, tolpropamine, tripelennamine, triprolidine, pharmaceutically acceptable salts thereof, and combinations thereof.

[0033] More preferably, the histamine-H1 -receptor antagonist is azelastine available commercially from, e.g., MEDA Pharmaceuticals (Somerset, NJ).

[0034] A suitable, non-limiting example of a dosage of a histamine-H1 - receptor antagonist disclosed herein, such as azelastine, is from about 0.5 mg/kg to about 500 mg/kg per day, such as from about 2 mg/kg to about 200 mg/kg per day, from about 5 mg/kg to about 100 mg/kg per day, including from about 10 mg/kg to about 50 mg/kg per day. Other representative dosages of such a histamine-H1 - receptor antagonist include about 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, 100 mg/kg, 1 10 mg/kg, 120 mg/kg, 130 mg/kg, 140 mg/kg, 150 mg/kg, 160 mg/kg, 170 mg/kg, 180 mg/kg, 190 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg, 400 mg/kg, 400 mg/kg, and 500 mg/kg per day.

[0035] Preferably, azelastine is administered to the subject in an amount from about 2-200 mg/kg per day, and more preferably, in an amount from about 2-50 mg/kg per day.

[0036] A suitable, non-limiting example of a dosage of the second agent, benznidazole, is from about 0.1 mg/kg to 5 mg/kg per day, such as from about 0.5 to 2 mg/kg per day, and more preferably about 1 mg/kg per day. Other representative dosages of such benznidazole include about 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1 .5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, and 5 mg/kg per day.

[0037] In another aspect of this embodiment, the first active agent is a selective neutrophil elastase inhibitor. As used herein, an "inhibitor" decreases or reduces the activity or expression of a protein or a nucleic acid encoding such a protein.

[0038] Preferably, the selective neutrophil elastase inhibitor according to the present invention is selected from the group consisting of serpins, members of the chelonianin family of inhibitors, alpha-1 -macroglobulin, transition state inhibitors, substituted benzoxazinones, substituted azetidine-2,4-diones, N-benzoylpyrazoles, carbamylating agents, heterocyclic inhibitors, peptidyl trifluoromethylketone ZD- 8321 , peptidyl alpha-ketoheterocycle ONO-6818, sivelestat, tripeptide alpha- ketoheterocycles, tripeptide alpha-keto-2-benzoxazoles, pyrimidinone alpha-keto- 1 ,3,4-oxadiazoles, a compound of formula I:

(i)

wherein X and Y are independently selected from the group consisting of oxygen and nitrogen, Cbz is carboxybenzyl and represents an optional double bond, a compound of formula II: (Π)

pharmaceutically acceptable salts thereof, and combinations thereof.

[0039] Non-limiting examples of serpins according to the present invention include alpha-1 -proteinase inhibitor, monocyte/neutrophil elastase inhibitor (Serpin B1 ), pharmaceutically acceptable salts thereof, and combinations thereof.

[0040] Non-limiting examples of members of the chelonianin family of inhibitors according to the present invention include secretory leukocyte proteinase inhibitor (SLPI), elafin, pharmaceutically acceptable salts thereof, and combinations thereof.

[0041] Non-limiting examples of transition state inhibitors according to the present invention include a compound of formula III:

(III)

wherein SP-B1 -25 is a 25 amino acid fragment of human surfactant peptide B (SEQ ID NO: 9), a compound of formula IV: (IV)

pharmaceutically acceptable salts thereof, and combinations thereof. One non- limiting example of a 25 amino acid fragment of formula III include FPIPLPYCWLCRALIKRIQAMIPKG (SEQ ID NO:10).

[0042] Non-limiting examples of substituted benzoxazinones according to the present invention include:

(VI)

(VII)

pharmaceutically acceptable salts thereof, and combinations thereof.

[0043] Non-limiting examples of substituted azetidine-2,4-diones according to the present invention include a compound of formula VIII:

(VIII)

pharmaceutically acceptable salts thereof, and combinations thereof.

[0044] Non-limiting examples of N-benzoylpyrazoles according to the present invention include a compound of formula IX: (IX)

pharmaceutically acceptable salts thereof, and combinations thereof.

[0045] Non-limiting examples of carbamylating agents according to the present invention include a compound of formula X, and pharmaceutically acceptable salts thereof:

(X)

wherein R¹ is isobutyl, R² is selected from the group consisting of 3-methoxybenzyl, 4-methoxybenzyl, 3-fluorobenzyl, phenyl, 2-phenethyl, 3-phenoxyphenyl, and 1 - napthylmethyl, and R³ is 2-phenethyl.

[0046] Non-limiting examples of heterocyclic inhibitors according to the present invention include:

(XVIII)

pharnnaceutically acceptable salts thereof, and combinations thereof.

[0047] Non-limiting examples of tripeptide alpha-ketoheterocycles according to the present invention include carboxybenzyl-Val-Pro-Val-Het or pharmaceutically acceptable salts thereof, wherein Het is selected from the group consisting of 2- benzoxazole, 2-benzofuran, 2-benzothiazole, 2-thiazole, 2-oxazole, 2-oxazoline, 2- (1 -Me-imidazole), 2-(1 -Me-benzimidazole), 2-benzimidazole, and 2-pyridine. [0048] Non-limiting examples of tripeptide alpha-keto-2-benzoxazoles according to the present invention include a compound of formula XX or pharmaceutically acceptable salts thereof,

(XX)

wherein X is selected from the group consisting of hydrogen, CO₂Me, OMe, OH, and CONH₂, and Y is selected from the group consisting of CO₂Me, CO₂-£-Bu, CO₂H, SO₂NH₂, 4-chlorophenylsulfonylaminocarbonylamino, and 4- chlorophenylsulfonylaminocarbonyl.

[0049] Non-limiting examples of pyrimidinone alpha-keto-1 ,3,4-oxadiazoles according to the present invention include a compound of formula XXI or pharmaceutically acceptable salts thereof,

(XXI)

wherein X is selected from the group consisting of fluorine and hydrogen and R is selected from the group consisting of CH₂(3-Me-C₆H₄), CMe₂(3-Me-C₆H₄), CMe₂Ph, and f-Bu. [0050] More preferably, the selective neutrophil elastase inhibitor is sivelestat:

or pharmaceutically acceptable salts thereof (available commercially from, e.g., Tocris Bioscience (Bristol, U.K.)).

[0051] A suitable, non-limiting example of a dosage of a selective neutrophil elastase inhibitor disclosed herein, such as sivelestat, is from about 2 mg/kg to about 200 mg/kg, such as from about 10 mg/kg to about 100 mg/kg per day, including from about 20 mg/kg to about 80 mg/kg per day, from about 30 mg/kg to about 70 mg/kg per day. Other representative dosages of such agents include about 2 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, 100 mg/kg, 150 mg/kg, or 200 mg/kg per day.

[0052] Preferably, sivelestat is administered to the subject in an amount from about 10-100 mg/kg per day.

[0053] Suitable and preferred dosages of benznidazole are as set forth above.

[0054] In an additional aspect of this embodiment, the first active agent is an ergot alkaloid derivative. As used herein, an "ergot alkaloid derivative" means a compound that shares structural similarities with 6,8-dimethylergoline or lysergic acid. Preferably, the ergot alkaloid derivative according to the present invention is selected from the group consisting of 2-bromo-a-ergokryptine, a-ergokryptine, bromocriptine, cabergoline, D-(+)-lysergic acid, dihydroergotamine, dihydroergotoxine, ergobasine, ergocornine, ergocristine, ergokryptine, ergoline, ergoloid, ergometrine, ergonovine, ergopeptine, ergosecaline, ergotaman, ergotamine, lysergic acid 2-hydroxyethylamide, lysergic acid amide (ergine), lysergic acid diethylamide, methylergonovine, methysergide, nicergoline, paspalic acid, pergolide, pergolide mesylate, pharmaceutically acceptable salts thereof, and combinations thereof. More preferably, the ergot alkaloid derivative is nicergoline:

r

and pharmaceutically acceptable salts thereof (commercially available at, e.g., Tocris Bioscience (Bristol, U.K.)).

[0055] A suitable, non-limiting example of a dosage of an ergot alkaloid derivative disclosed herein, such as nicergoline, is from about 0.5 mg/kg to about 40 mg/kg per day, such as from about 1 mg/kg to about 20 mg/kg per day, including 2 mg/kg to about 15 mg/kg per day, including from about 5 mg/kg to about 10 mg/kg per day. Other representative dosages of such agents include about 0.5 mg/kg, 1 mg/kg, 1 .5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 7.5 mg/kg, 8 mg/kg, 8.5 mg/kg, 9 mg/kg, 9.5 mg/kg, 10 mg/kg, 1 1 mg/kg, 12 mg/kg, 13, mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, and 40 mg/kg per day.

[0056] Preferably, nicergoline is administered to the subject in an amount from about 1 -20 mg/kg per day.

[0057] Suitable and preferred dosages of benznidazole are as set forth above.

[0058] In another aspect of this embodiment, the first active agent is an inhibitor of 3 β-hydroxysteroid dehydrogenase. Preferably, the inhibitor of 3 β- hydroxysteroid dehydrogenase according to the present invention is selected from the group consisting of 17 beta-N,N-diethylcarbamoyl-4-aza-5 alpha-androstan-3- one (4MA), 3-keto-desogestrel, Bis(2-butoxyethyl) phthalate, cyproterone acetate, danazol, desogestrel, dibutyl phthalate, dicyclohexyl phthalate, dipentyl phthalate, dipropyl phthalate, epostane, gestrinone, levonorgestrel, monooctyltin, norethisterone, progesterone, testosterone, tributyltin, trilostane, troglitazone, pharmaceutically acceptable salts thereof, and combinations thereof. More preferably, the inhibitor of 3 β-hydroxysteroid dehydrogenase is trilostane:

or pharmaceutically acceptable salts thereof (available commercially at, e.g., US Biological (Swampscott, MA)).

[0059] A suitable, non-limiting example of a dosage of an inhibitor of 3 β- hydroxysteroid dehydrogenase disclosed herein, such as trilostane, is from about 1 mg/kg to about 100 mg/kg per day, such as from about 3 mg/kg to about 50 mg/kg per day, including from about 5 mg/kg to about 30 mg/kg per day, and from about 10 mg/kg to about 20 mg/kg per day. Other representative dosages of such agents include about 1 mg/kg, 1 .5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 7.5 mg/kg, 8 mg/kg, 8.5 mg/kg, 9 mg/kg, 9.5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, and 100 mg/kg per day.

[0060] Preferably, trilostane is administered to the subject in an amount from about 3-50 mg/kg per day.

[0061] Suitable and preferred dosages of benznidazole are as set forth above.

[0062] In an additional aspect of this embodiment, the first active agent is a reversible acetylcholinesterase inhibitor. Preferably, the reversible acetylcholinesterase inhibitor according to the present invention is selected from the group consisting of 1 ,4-methylenetacrine, ambenonium, amiridine, caffeine, caproctamine, carbamates, delta9-tetrahydrocannabinol (THC), demarcarium, donepezil, dyflos, edrophonium, galanthamine, huperzine A, huprine lactucopicrin, ladostigil, minaprine, neostigmine, neostigmine, phenanthrene derivatives, physostigmine, piperidines, pyridostigmine, pyridostigmine, rivastigmine, tacrine, ungeremine, velnacrine, pharmaceutically acceptable salts thereof, and combinations thereof. More preferably, the reversible acetylcholinesterase inhibitor is donepezil:

or pharnnaceutically acceptable salts thereof (commercially available from, e.g., Eisai Inc. (Woodcliff Lake, NJ)).

[0063] A suitable, non-limiting example of a dosage of a reversible acetylcholinesterase inhibitor disclosed herein, such as donepezil, is from about 0.1 mg/kg to about 50 mg/kg per day, such as 0.5 mg/kg to about 15 mg/kg per day, including from about 1 mg/kg to about 10 mg/kg per day, and from about 2 mg/kg to about 8 mg/kg per day. Other representative dosages of such agents include about 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 1 .5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 7.5 mg/kg, 8 mg/kg, 8.5 mg/kg, 9 mg/kg, 9.5 mg/kg, 10 mg/kg, 10.5 mg/kg, 1 1 mg/kg, 1 1 .5 mg/kg, 12 mg/kg, 12.5 mg/kg, 13 mg/kg, 13.5 mg/kg, 14 mg/kg, 14.5 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, and 50 mg/kg per day.

[0064] Preferably, donepezil is administered to the subject in an amount from about 0.5-15 mg/kg per day.

[0065] Suitable and preferred dosages of benznidazole are as set forth above.

[0066] In another aspect of this embodiment, the first active agent is a protease inhibitor. Preferably, the protease inhibitor according to the present invention is selected from the group consisting of amprenavir, atazanavir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, tipranavir, pharmaceutically acceptable salts thereof, and combinations thereof. More preferably, the protease inhibitor is ritonavir:

or pharmaceutically acceptable salts thereof (commercially available from, e.g., Abbott Laboratories (Chicago, IL)).

[0067] A suitable, non-limiting example of a dosage of a protease inhibitor disclosed herein, such as ritonavir, is from about 0.1 mg/kg to about 100 mg/kg per day, such as 0.2 mg/kg to about 40 mg/kg per day, including from about 5 mg/kg to about 30 mg/kg per day, and from about 10 mg/kg to about 20 mg/kg per day. Other representative dosages of such agents include about 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 1 1 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 21 mg/kg, 22 mg/kg, 23 mg/kg, 24 mg/kg, 25 mg/kg, 26 mg/kg, 27 mg/kg, 28 mg/kg, 29 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, and 100 mg/kg per day. [0068] Preferably, ritonavir is administered to the subject in an amount from about 0.2-40 mg/kg per day.

[0069] Suitable and preferred dosages of benznidazole are as set forth above.

[0070] In an additional aspect of this embodiment, the first active agent is an aldosterone antagonist. Preferably, the aldosterone antagonist according to the present invention is selected from the group consisting of spironolactone, epierenone, canrenone, prorenone, mexrenone, pharmaceutically acceptable salts thereof, and combinations thereof. More preferably, the aldosterone antagonist is epierenone:

or pharmaceutically acceptable salts thereof (commercially available from, e.g., G.D. Searle (New York, NY)).

[0071] A suitable, non-limiting example of a dosage of aldosterone antagonist disclosed herein, such as epierenone, is from about 1 mg/kg to about 1000 mg/kg per day, such as from about 10-400 mg/kg per day, including from about 50 mg/kg to about 300 mg/kg per day, and about 100 mg/kg to about 200 mg/kg per day. Other representative dosages of such agents include about 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, 100 mg/kg, 1 10 mg/kg, 120 mg/kg, 130 mg/kg, 140 mg/kg, 150 mg/kg, 160 mg/kg, 170 mg/kg, 180 mg/kg, 190 mg/kg, 200 mg/kg, 220 mg/kg, 240 mg/kg, 260 mg/kg, 280 mg/kg, 300 mg/kg, 350 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg and 1000 mg/kg per day.

[0072] Preferably, eplerenone is administered to the subject in an amount from about 10-400 mg/kg.

[0073] Suitable and preferred dosages of benznidazole are as set forth above.

[0074] In another aspect of this embodiment, the first active agent is a naphthoquinone. Preferably, the naphthoquinone according to the present invention is selected from the group consisting of 1 ,2-naphthoquinone, 1 ,4-naphthoquinone, 2,6-naphthoquinone, and atovaquone. More preferably, the naphthoquinone is atovaquone:

or pharmaceutically acceptable salts thereof (commercially available from, e.g., GlaxoSmithKline (Research Triangle Park, NC)).

[0075] A suitable, non-limiting example of a dosage of a naphthoquinone disclosed herein, such as atovaquone, is from about 1 mg/kg to about 1000 mg/kg per day, such as from about 10 mg/kg to about 400 mg/kg per day, including about 50 mg/kg to about 300 mg/kg per day, and about 100 mg/kg to about 200 mg/kg per day. Other representative dosages of such agents include about 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, 100 mg/kg, 1 10 mg/kg, 120 mg/kg, 130 mg/kg, 140 mg/kg, 150 mg/kg, 160 mg/kg, 170 mg/kg, 180 mg/kg, 190 mg/kg, 200 mg/kg, 220 mg/kg, 240 mg/kg, 260 mg/kg, 280 mg/kg, 300 mg/kg, 350 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg and 1000 mg/kg per day.

[0076] Preferably, atovaquone is administered to the subject in an amount from about 10-400 mg/kg per day.

[0077] Suitable and preferred dosages of benznidazole are as set forth above.

[0078] Another embodiment of the present invention is a method for treating or ameliorating the effects of a Chagas disease. This method comprises administering to a subject in need thereof an effective amount of azelastine at 2-200 mg/kg per day and benznidazole at 0.5-2 mg/kg per day. Preferably, azelastine is administered to the subject in an amount from about 2-50 mg/kg per day, and benznidazole is administered to the subject in an amount from about 0.5-1 mg/kg per day. Other suitable and preferred dosages of azelastine and benznidazole are as set forth above.

[0079] An additional embodiment of the present invention is a composition for treating or ameliorating the effects of a Chagas disease. The composition comprises azelastine and benznidazole in amounts effective to treat or ameliorate the effect of a Chagas disease and a pharmaceutically acceptable carrier. [0080] In one aspect of this embodiment, azelastine is administered to the subject in an amount from about 2-200 mg/kg per day, and benznidazole is administered to the subject in an amount from about 0.5-2 mg/kg per day. In another aspect of this embodiment, azelastine is administered to the subject in an amount from about 2-50 mg/kg per day, and benznidazole is administered to the subject in an amount from about 0.5-1 mg/kg per day. Other suitable and preferred dosages of azelastine and benznidazole are as set forth above.

[0081] Another embodiment of the present invention is a method for treating or ameliorating the effects of a Chagas disease. This method comprises administering to a subject in need thereof an effective amount of a first active agent selected from the group consisting of a selective neutrophil elastase inhibitor, an ergot alkaloid derivative, an inhibitor of 3 β-hydroxysteroid dehydrogenase, a reversible acetylcholinesterase inhibitor, a protease inhibitor, an aldosterone antagonist, a naphthoquinone, and combinations thereof, and a second active agent, which is benznidazole. Suitable and preferred selective neutrophil elastase inhibitors, ergot alkaloid derivatives, inhibitors of 3 β-hydroxysteroid dehydrogenase, reversible acetylcholinesterase inhibitors, protease inhibitors, aldosterone antagonists, and naphthoquinones, including dosages of these agents, are as disclosed herein. Suitable and preferred dosages of benznidazole are also disclosed herein.

[0082] The compositions of the present invention may be administered to a subject in any desired and effective manner: for oral ingestion, or as an ointment or drop for local administration to the eyes, or for parenteral or other administration in any appropriate manner such as intraperitoneal, subcutaneous, topical, intradermal, spray/inhalation, intrapulmonary, rectal, vaginal, sublingual, intramuscular, intravenous, intraarterial, intrathecal, or intralymphatic. Further, a composition of the present invention may be administered to a subject in conjunction with other treatments. A composition of the present invention may be encapsulated or otherwise protected against gastric or other secretions, if desired.

[0083] The compositions of the invention comprise one or more active ingredients in admixture with one or more pharmaceutically-acceptable carriers and, optionally, one or more other compounds, drugs, ingredients and/or materials. Regardless of the route of administration selected, the agents/compounds of the present invention are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. See, e.g., Remington, The Science and Practice of Pharmacy (21 ^st Edition, Lippincott Williams and Wilkins, Philadelphia, PA.).

[0084] Pharmaceutically acceptable carriers are well known in the art (see, e.g., Remington, The Science and Practice of Pharmacy (21 ^st Edition, Lippincott Williams and Wilkins, Philadelphia, PA.) and The National Formulary (American Pharmaceutical Association, Washington, D.C.)) and include sugars {e.g., lactose, sucrose, mannitol, and sorbitol), starches, cellulose preparations, calcium phosphates (e.g., dicalcium phosphate, tricalcium phosphate and calcium hydrogen phosphate), sodium citrate, water, aqueous solutions (e.g., saline, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection), alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol), polyols (e.g., glycerol, propylene glycol, and polyethylene glycol), organic esters (e.g., ethyl oleate and tryglycerides), biodegradable polymers (e.g., polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides)), elastomeric matrices, liposomes, microspheres, oils (e.g., corn, germ, olive, castor, sesame, cottonseed, and groundnut), cocoa butter, waxes (e.g., suppository waxes), paraffins, silicones, talc, silicylate, etc. Each pharmaceutically acceptable carrier used in a pharmaceutical composition of the invention must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Carriers suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable carriers for a chosen dosage form and method of administration can be determined using ordinary skill in the art.

[0085] The compositions of the invention may, optionally, contain additional ingredients and/or materials commonly used in pharmaceutical compositions. These ingredients and materials are well known in the art and include (1 ) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium starch glycolate, cross-linked sodium carboxymethyl cellulose and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, and sodium lauryl sulfate; (10) suspending agents, such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth; (1 1 ) buffering agents; (12) excipients, such as lactose, milk sugars, polyethylene glycols, animal and vegetable fats, oils, waxes, paraffins, cocoa butter, starches, tragacanth, cellulose derivatives, polyethylene glycol, silicones, bentonites, silicic acid, talc, salicylate, zinc oxide, aluminum hydroxide, calcium silicates, and polyamide powder; (13) inert diluents, such as water or other solvents; (14) preservatives; (15) surface-active agents; (16) dispersing agents; (17) control-release or absorption-delaying agents, such as hydroxypropylmethyl cellulose, other polymer matrices, biodegradable polymers, liposomes, microspheres, aluminum monostearate, gelatin, and waxes; (18) opacifying agents; (19) adjuvants; (20) wetting agents; (21 ) emulsifying and suspending agents; (22), solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan; (23) propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane; (24) antioxidants; (25) agents which render the formulation isotonic with the blood of the intended recipient, such as sugars and sodium chloride; (26) thickening agents; (27) coating materials, such as lecithin; and (28) sweetening, flavoring, coloring, perfuming and preservative agents. Each such ingredient or material must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Ingredients and materials suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable ingredients and materials for a chosen dosage form and method of administration may be determined using ordinary skill in the art. [0086] Compositions of the present invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, powders, granules, a solution or a suspension in an aqueous or non-aqueous liquid, an oil-in-water or water-in-oil liquid emulsion, an elixir or syrup, a pastille, a bolus, an electuary or a paste. These formulations may be prepared by methods known in the art, e.g., by means of conventional pan-coating, mixing, granulation or lyophilization processes.

[0087] Solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like) may be prepared, e.g., by mixing the active ingredient(s) with one or more pharmaceutically-acceptable carriers and, optionally, one or more fillers, extenders, binders, humectants, disintegrating agents, solution retarding agents, absorption accelerators, wetting agents, absorbents, lubricants, and/or coloring agents. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using a suitable excipient. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using a suitable binder, lubricant, inert diluent, preservative, disintegrant, surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine. The tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein. They may be sterilized by, for example, filtration through a bacteria-retaining filter. These compositions may also optionally contain opacifying agents and may be of a composition such that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. The active ingredient can also be in microencapsulated form.

[0088] Liquid dosage forms for oral administration include pharmaceutically- acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. The liquid dosage forms may contain suitable inert diluents commonly used in the art. Besides inert diluents, the oral compositions may also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions may contain suspending agents.

[0089] Compositions of the present invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more active ingredient(s) with one or more suitable nonirritating carriers which are solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound. Compositions of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such pharmaceutically-acceptable carriers as are known in the art to be appropriate.

[0090] Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops and inhalants. The active agent(s)/compound(s) may be mixed under sterile conditions with a suitable pharmaceutically-acceptable carrier. The ointments, pastes, creams and gels may contain excipients. Powders and sprays may contain excipients and propellants.

[0091] Compositions of the present invention suitable for parenteral administration comprise one or more agent(s)/compound(s) in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Proper fluidity can be maintained, for example, by the use of coating materials, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain suitable adjuvants, such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption.

[0092] In some cases, in order to prolong the effect of a drug (e.g., pharmaceutical formulation), it is desirable to slow its absorption from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility.

[0093] The rate of absorption of the active agent/drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered agent/drug may be accomplished by dissolving or suspending the active agent/drug in an oil vehicle. Injectable depot forms may be made by forming microencapsule matrices of the active ingredient in biodegradable polymers. Depending on the ratio of the active ingredient to polymer, and the nature of the particular polymer employed, the rate of active ingredient release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.

[0094] The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.

[0095] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0096] For recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6,9, and 7.0 are explicitly contemplated.

[0097] The following examples are provided to further illustrate the methods of the present invention. These examples are illustrative only and are not intended to limit the scope of the invention in any way. EXAMPLES

Example 1

Materials and Methods for the In Vivo Primary Screen

Animals and Husbandry

[0098] Female, Balb/c mice ordered from Harlan weighing 19-21 grams were acclimated to housing conditions and handled in accordance with Animal Use Protocol number TP-17-09. They were 6-8 weeks old on Day 1 of the experiment. The animals were fed irradiated Teklad Global Rodent Diet (Harlan) and water ad libitum. Mice were housed in static cages with Bed-O'Cobs bedding inside Biobubble clean rooms that provide H.E.P.A. filtered air into the bubble environment at 100 complete air changes per hour. All treatments and infectious challenges were carried out in the Biobubble environment. The environment was controlled to a temperature range of 74° ± 4°F and a humidity range of 30-70%. Mice were fully immunocompetent. All animals were observed for clinical signs twice daily.

Parasite Cultures

[0099] A virulent Trypanosoma cruzi, Tulahuen strain, was procured from the laboratory of Frederick Buckner, MD (Department of Medicine, Division of Allergy and Infectious Diseases, University of Washington, Seattle, WA). The parasites were seeded onto sub-confluent cultures of 3T3 cells (ATCC CCL-92) in the presence of Dulbecco's Modified Eagle Medium (DMEM) with high glucose, and supplemented with 10% fetal bovine serum (FBS). The parasites were seeded onto the 3T3 cells at an approximate multiplicity of infection (MOI) of 4 parasites per cell. At 24 hours following exposure to the parasites, the cultures were washed to remove non-internalized parasites, and the growth media was replenished. Cultures were maintained at 37°C. At approximately 4 days following parasite exposure, new trypomastigotes began to emerge from the culture. The media was changed one day prior to use as a challenge inoculum to ensure that newly emerged trypomastigotes were used in the study.

Challenge

[0100] Cultures were removed from the incubator and the growth media was pipetted up and down, on the culture, to remove all free parasites. The media was transferred to a sterile conical tube and a sample was transferred to a hemacytometer to enumerate live parasites. The culture was diluted to provide a challenge concentration of 2.0x10³ parasites/ml, yielding a 1 .0x10³ challenge in 0.5 cc. Cages were prepared with five mice per cage and animals were divided into test groups. Each group had 5 animals. Some of the test groups are detailed in Table 1 . Each animal in the study was administered an intraperitoneal challenge of the diluted T. cruzi culture with a total volume of 0.5 cc. Instillation of the parasitic challenge constituted time Oh for the study.

Table 1 . Animal Challenge and Dosing Schedule

Sivelestat IP 50 1 saline

saline containing 0.4% tween 80

Trilostane IP 30 1

Formulation and Dosing

[0101] The commercial comparator (benznidazole, BZ) was formulated for oral gavage using a formulation vehicle of 5% PEG200 in 0.5% methylcellulose (aqueous) ("PEG/MC"). The test compounds that, in combination with BZ, were found to be effective against T. cruzi, were formulated as set forth above in Table 1 . Once formulated, all treatments were stored at 4°C and light protected until time of use. Formulations were allowed to warm to room temperature prior to dosage and visually assessed for signs of precipitation or turbidity. Dosing began 4 days following parasitic challenge. Treatments were freshly formulated on days 4, 7, and 10 following challenge.

Measurements and Endpoints

[0102] The primary endpoint used to assess progress of the infection and drug activity was mortality over a 21 day period following challenge. Percent survivorship per time was calculated through use of Microsoft Excel. Graphic interpretation of the survival results (Kaplan-Meier) were performed using GraphPad Prism v. 4.0. Survival curve comparisons were performed by Logrank analysis (Mantel-Cox Test) in GraphPad Prism v. 4.0. Curve comparisons returning a p value less than or equal to 0.05 were considered significantly different. Curve comparisons returning a calculated p value less than or equal to 0.005 were considered highly significantly different. Example 2

Results of the In Vivo Primary Screen

Infection Progression/General Observations

[0103] All challenge, dosing, and data tracking procedures were performed as detailed above. The 21 day survival tracking results were used for the Kaplan-Meier analyses used to generate the data in Table 2.

[0104] One test group, the combination of Bz with Tegaserod, demonstrated no signs of acute toxicity at the doses delivered; however, this cohort of mice all died prior to the final day of dosing, with a median day of survival of 10 days. None of the remaining study subjects displayed any overt signs of toxicity, adverse events or early disease onset associated with the treatments. The survival data values are listed in Table 2 and are also shown in Figures 2-6. The untreated control mice began to exhibit clinical signs of parasitemia starting at day 10 following challenge and died on days 14 and 15 post challenge, with a median survival day at day 15. These results are consistent with previous mortality rates provided by a 1 .0x10³ parasite challenge with the Tulahuen strain in this model.

[0105] Both dose concentrations of BZ performed with efficacies consistent with previously benchmarked results. The 10 mg/kg dose provided 100% survival for the duration of the study. While the 1 mg/kg dose concentration of BZ ended the 21 day observation period with 0% survival, the median day of survival was calculated at 17 days, which is consistent with previously benchmarked standards for this dose concentration (Table 2 and Figure 1 ).

[0106] When survival curves were compared between that generated for the group treated with BZ alone (1 mg/kg) and groups treated with the various combination therapies, two compounds were found to significantly improve the survival outcomes: flutrimazole and sivelestat. Flutrimazole provided complete protection for the infected mice and appeared to be well tolerated. The survival curve for mice treated with flutrimazole was significantly better than that for mice treated with BZ at 1 mg/kg alone (p = 0.003, Table 2). Sivelestat treatment provided 60% survival for the infected mice at the end of the 21 day observation period and extended the median day of survival beyond 21 days (Table 2). The survival curve for mice treated with sivelestat was significantly better than that for mice treated with BZ at 1 mg/kg alone (p = 0.02, Figure 2).

[0107] These data, together, demonstrate that both flutrimazole and sivelestat, in combination with a suboptimal dose of BZ, have in vivo activity against T. cruzi- mediated disease.

[0108] Additional survival curves for some of the other tested compounds are shown in Figures 3-6. These test compounds, in combination with BZ at 1 mg/kg, confirmed benefit to the animals in comparison to saline treated ones. Comparative survival curves of mice treated with BZ at 1 mg/kg and mice treated with BZ at 1 mg/kg combined with a test drug are shown in part B of each of Figures 2-6. Comparative survival curves of mice treated with saline control and mice treated with BZ at 1 mg/kg combined with a test drug are shown in part A of each of Figures 2-6.

Example 3

Confirmation Study and Dose Variation Study

Study Design for the Dose Variation

[0109] 7-8 week old female BALB/c mice were acclimated for 1 1 days before treatment. On day 0, mice were dosed by intraperitoneal site injection with 1000 viable T. cruzi trypomastigotes. Parasites were harvested following serial passage in murine NIH 3T3 cells. Starting daily, animals were dosed with antiparasitic BZ, either alone or in combination with experimental compounds. Combinations were administered orally with a once-daily sub-efficacious dose of 1 mg/kg q.d. BZ plus the experimental compound. A curative dose of 10 mg/kg BZ was used as a positive control.

[0110] Mice were monitored daily. In some studies, blood samples were obtained on Day 10 either by tail vein collection or following animal sacrifice (Figure 7A). Parasitemia was assessed either by quantitative PCR for satellite repeat sequences in the T. cruzi genome or by microscopic enumeration by hemacytometer. Moribund animals were monitored and sacrificed through the study. Cohorts with remaining animals were sacrificed at 24 to 30 days post-infection (Figure 7A).

[0111] Typical survival curves for combination studies are shown in Figure 7B. All animals treated with 10 mg/kg BZ survived until study end. Untreated animals or animals treated with 1 mg/kg BZ were moribund and sacrificed over 20 weeks. [0112] Typical parasite enumeration results using a qPCR assay of satellite DNA repeats are shown for two independent experiments in Figure 7C. 10 mg/kg BZ reduces parasite burden within 10 days, while parasite burden in 1 mg/kg BZ cohorts is equivalent to untreated animals. Groups are N = 5 with SEM shown.

Results of In Vivo Primary Screen and Confirmation Studies

[0113] Mantel-Cox survival analysis was performed (GraphPad), comparing the efficacy of 1 mg/kg BZ alone to combination treatment with the indicated compound and 1 mg/kg BZ (Table 3). For each comparison, a chi-square statistic (log-rank) is reported, along with p value and hazard ratio (Mantel-Haenszel). Selected compounds that showed protective hazard functions in primary screening were tested in confirmatory studies at the same dose level and administration route. Compounds that showed protective hazard functions in both screening and confirmatory studies included eplerenone, atovaquone, and azelastine. These compounds were tested in dose-range response studies. Compounds were administered at doses above and below the screening dose.

Table 3: Primary Screen and Confirmation Results

^* 100% survival in experimental arm.

Azelastine efficacy and survival analysis

[0114] Azelastine consistently exhibited a protective hazard function in screening, confirmatory, and dose-range response studies. Summary statistics are shown in Table 4. Exemplary Kaplan-Meier survival curves are shown in Figure 8A and 8B.

Table 4. Azelastine testing

[0115] Azelastine exhibited a protective hazard ratio in multiple survival studies. Blood samples were collected 10 days after parasite inoculation and drug treatment. DNA was purified from blood, and assayed by qPCR to detect relative abundance of T. cruzi specific satellite DNA sequences. QPCR primers targeted a T. cruzi-specific satellite repeat reference sequence (SEQ ID NOs: 1 and 2) with FAM dye-labeled Roche Universal Probe #66 as the probe (Cat. No. 04688651001 ) (SEQ ID NO. 3), producing the amplicon shown as SEQ ID NO. 4. Other qPCR primers targeted the Mus musculus ApoB gene (SEQ ID NOs.: 5 and 6) with the VIC dye-labeled probe shown as SEQ ID NO. 7. SEQ ID NO. 8 shows the resultant amplicon.

[0116] The qPCR assay was run using an Applied Biosystems 7900HT Fast System. Each qPCR reaction consisted of 10 μΙ test sample DNA, 20 μΙ of 1 X TaqMan Universal PCR Master Mix, T. cruzi specific repeat reference sequence forward and reverse primers (300 nM each), Mus musculus ApoB forward and reverse primers (900 nM each), FAM labeled Roche Universal Probe #66 probe (250 nM), VIC labeled Mus musculus ApoB probe (250 nM), and PCR grade water. Cycling conditions were as follows: Stage 1 - 50°C for 2 minutes, Stage 2 - 95°C for 10 minutes, Stage 3 - 95°C for 15 seconds, 60°C for 1 minute, with 40 cycles for this stage.

[0117] QPCR derived parasite burden is expressed in Figure 9A normalized relative to levels in vehicle treated subjects (N = 3, SEM shown).

Example 4

In Vitro Parasite Growth Assay

[0118] To investigate the effects of azelastine on parasite growth in murine 3T3 cells, T. cruzi was co-cultured by serial passage with murine 3T3 host/feeder cells. In brief, epimastigote stage parasites were used to infect cells at a multiplicity of infection of 4, one day following passage of the cells. Non- internalized parasites were washed from the culture following a 24 hour incubation period. All cells were cultured in the presence of Dulbecco's Modified Eagle Medium with high glucose, and supplemented with 10% fetal bovine serum. Cells were cultured in a humid atmosphere, with 5% CO₂ at 37°C. At 4 days following parasite exposure, the cells began to lyse free epimastigotes. The media was changed on the culture one day prior to infection to ensure that newly emerged parasites were used for the infection. Challenge inoculate provided approximately 1000 viable trypomastigotes in a volume of 0.5 ml.

[0119] Cultures were treated at 16 hours post infection with T. cruzi for 72 hours with varying concentrations of BZ or azelastine as shown in Table 5.

Table 5: Azelastine Dosage Schedule

Treatment Dose T. cruzi Study

Group Treatment n (wells)

Dose (μΜ) Schedule Challenge Endpoint

Parasite

16 hours

1 Untreated Control NA l .OxlO³ 3 enumeration p.i.^a

72 Hours

Parasite

2 Benznidazole 1 16 hours p.i. l .OxlO³ 3 enumeration

72 Hours

Parasite

2 Benznidazole 5 16 hours p.i. l .OxlO³ 3 enumeration

72 Hours

Parasite

2 Benznidazole 35 16 hours p.i. l .OxlO³ 3 enumeration

72 Hours

Parasite

3 Azelastine 0.0256 16 hours p.i. l .OxlO³ 3 enumeration

72 Hours

Parasite

4 Azelastine 0.0767 16 hours p.i. l .OxlO³ 3 enumeration

72 Hours

Parasite

5 Azelastine 0.23 16 hours p.i. l .OxlO³ 3 enumeration

72 Hours

Parasite

6 Azelastine 0.7 16 hours p.i. l .OxlO³ 3 enumeration

72 Hours

Parasite

7 Azelastine 2.1 16 hours p.i. l .OxlO³ 3 enumeration

72 Hours Parasite

8 Azelastine 6.2 16 hours p.i. l .OxlO³ 3 enumeration

72 Hours

Parasite

9 Azelastine 18.5 16 hours p.i. l .OxlO³ 3 enumeration

72 Hours

Parasite

10 Azelastine 55.6 16 hours p.i. l .OxlO³ 3 enumeration

72 Hours

Parasite

11 Azelastine 166.7 16 hours p.i. l .OxlO³ 3 enumeration

72 Hours

Parasite

12 Azelastine 500 16 hours p.i. l .OxlO³ 3 enumeration

72 Hours ^lp.i. = post-infection

[0120] Parasites were then enumerated in culture supernatants by hemacytometer and are expressed on a per ml basis in Figure 9B. Data shown are N = 3 replicates, +/- SEM.

DOCUMENTS

1 . WHO. Control of Chagas disease. World Health Organ Tech Rep Ser, 2002. 905: p. i-vi, 1 -109, back cover.

2. Schofield, C.J., Jannin, J., Salvatella, R. The future of Chagas disease control. Trends Parasitol, 2006. 22: p. 583-588.

3. Rodriques Coura J., de Castro, S.L. A critical review on Chagas disease chemotherapy. Mem Inst Oswaldo Cruz, 2002. 97: p. 3-24.

4. Bern, C, Montgomery, S.P., Herwaldt, B.L., Rassi, A., Jr., Marin-Neto, J.A., et al. Evaluation and treatment of Chagas disease in the United States: a systematic review. JAMA, 2007. 298: p. 2171 -2181 .

[0121] All documents cited in this application are hereby incorporated by reference as if recited in full herein.

[0122] Although illustrative embodiments of the present invention have been described herein, it should be understood that the invention is not limited to those described, and that various other changes or modifications may be made by one skilled in the art without departing from the scope or spirit of the invention.

Claims

WHAT IS CLAIMED IS:

1 . A method for treating or ameliorating the effects of a Chagas disease comprising administering to a subject in need thereof an effective amount of a first active agent selected from the group consisting of a histamine-H1 -receptor antagonist, a selective neutrophil elastase inhibitor, an ergot alkaloid derivative, an inhibitor of 3 β-hydroxysteroid dehydrogenase, a reversible acetylcholinesterase inhibitor, a protease inhibitor, an aldosterone antagonist, a naphthoquinone, and combinations thereof, and a second active agent, which is benznidazole.

2. The method according to claim 1 , wherein the first active agent is a histamine-H1 -receptor antagonist.

3. The method according to claim 2, wherein the histamine-H1 -receptor antagonist is selected from the group consisting of 4-methyldiphenhydramine, aceprometazine, acrivastine, alcaftadine, alimemazine, antazoline, aptazapine, astemizole, azatadine, azelastine, bamipine, bepotastine, bilastine, bisulepine, bromazine, brompheniramine, carbinoxamine, cetirizine, chlorcyclizine, chloropyramine, chlorothen, chlorphenamine, chlorphenoxamine, cinnarizine, clemastine, clemizole, clobenzepam, clobenztropine, clocinizine, cyanodothiepin, cyclizine, cyproheptadine, dacemazine, deptropine, desloratadine, dexbrompheniramine, dexchlorpheniramine, dimenhydrinate, dimetindene, diphenhydramine, diphenylpyraline, doxylamine, ebastine, embramine, emedastine, epinastine, esmirtazapine, etymemazine, fexofenadine, histapyrrodine, homochlorcyclizine, hydroxyethylpromethazine, hydroxyzine, isopromethazine, isothipendyl, ketotifen, latrepirdine, levocabastine, levocetirizine, loratadine, mebhydrolin, meclizine, mepyramine, methafurylene, methapyhlene, methdilazine, mianserin, mirtazapine, mizolastine, moxastine, olopatadine, orphenadrine, oxatomide, pemirolast, phenindamine, pheniramine, phenyltoloxamine, pirolate, promethazine, propiomazine, pyrrobutamine, quifenadine, repirinast, rupatadine, setastine, setiptiline, sominex, talastine, terfenadine, thenalidine, thenyldiamine, thiazinamium metilsulfate, thonzylamine, tolpropamine, tripelennamine, triprolidine, pharmaceutically acceptable salts thereof, and combinations thereof.

4. The method according to claim 3, wherein the histamine-H1 -receptor antagonist is azelastine.

5. The method according to claim 4, wherein azelastine is administered to the subject in an amount from about 2-200 mg/kg per day.

6. The method according to claim 4, wherein azelastine is administered to the subject in an amount from about 2-50 mg/kg per day.

7. The method according to claim 4, wherein benznidazole is administered to the subject in an amount that is about 1 mg/kg per day.

8. The method according to claim 1 , wherein the first active agent is a selective neutrophil elastase inhibitor.

9. The method according to claim 8, wherein the selective neutrophil elastase inhibitor is selected from the group consisting of serpins, members of the chelonianin family of inhibitors, alpha-1 -macroglobulin, transition state inhibitors, substituted benzoxazinones, substituted azetidine-2,4-diones, N- benzoylpyrazoles, carbamylating agents, heterocyclic inhibitors, peptidyl trifluoromethylketone ZD-8321 , peptidyl alpha-ketoheterocycle ONO-6818, sivelestat, tripeptide alpha-ketoheterocycles, tripeptide alpha-keto-2- benzoxazoles, pyrimidinone alpha-keto-1 ,3,4-oxadiazoles, a compound of formula I:

(I)

wherein X and Y are independently selected from the group consisting of oxygen and nitrogen, Cbz is carboxybenzyl, and represents an optional double bond

a compound of formula II: (Π)

Me0₂C-Val-Pro-Val pharmaceutically acceptable salts thereof, and combinations thereof.

10. The method according to claim 9, wherein the serpin is selected from the group consisting of alpha-1 -proteinase inhibitor, monocyte/neutrophil elastase inhibitor (Serpin B1 ), pharmaceutically acceptable salts thereof, and combinations thereof.

1 1 . The method according to claim 9, wherein members of the chelonianin family of inhibitors are selected from the group consisting of secretory leukocyte proteinase inhibitor (SLPI), elafin, pharmaceutically acceptable salts thereof, and combinations thereof.

12. The method according to claim 9, wherein the transition state inhibitor is selected from the group consisting of a compound of formula III:

(III)

wherein SP-B1 -25 is a 25 amino acid fragment of human surfactant peptide B,

a compound of formula IV:

(IV)

pharmaceutically acceptable salts thereof, and combinations thereof.

13. The method according to claim 9, wherein the substituted benzoxazinone is selected from the group consisting of: (VI)

pharmaceutically acceptable salts thereof, and combinations thereof.

14. The method according to claim 9, wherein the substituted azetidine-2,4- dione is a compound of formula VIII: (VIII)

pharmaceutically acceptable salts thereof, and combinations thereof.

15. The method according to claim 9, wherein the N-benzoylpyrazole is a compound of formula IX:

(IX)

pharmaceutically acceptable salts thereof, and combinations thereof.

16. The method according to claim 9, wherein the carbamylating agent is a compound of formula X, and pharmaceutically acceptable salts thereof: (X)

wherein R¹ is isobutyl, R² is selected from the group consisting of 3- methoxybenzyl, 4-methoxybenzyl, 3-fluorobenzyl, phenyl, 2-phenethyl, 3- phenoxyphenyl, and 1 -napthylmethyl, and R³ is 2-phenethyl.

17. The method according to claim 9, wherein the heterocyclic inhibitor is selected from the group consisting of:

56

Ġ ĨXV)

(XVIII)

pharmaceutically acceptable salts thereof, and combinations thereof.

18. The method according to claim 9, wherein the tripeptide alpha- ketoheterocycle is carboxybenzyl-Val-Pro-Val-Het or pharmaceutically acceptable salts thereof, wherein Het is selected from the group consisting of 2- benzoxazole, 2-benzofuran, 2-benzothiazole, 2-thiazole, 2-oxazole, 2-oxazoline, 2-(1 -Me-imidazole), 2-(1 -Me-benzimidazole), 2-benzimidazole, and 2-pyhdine.

19. The method according to claim 9, wherein the tripeptide alpha-keto-2- benzoxazole is a compound of formula XX or pharmaceutically acceptable salts thereof,

(XX)

wherein X is selected from the group consisting of hydrogen, CO2Me, OMe, OH, and CONH_2, and Y is selected from the group consisting of CO₂Me, CO₂-£-Bu, CO2H, SO2NH2, 4-chlorophenylsulfonylaminocarbonylamino, and 4- chlorophenylsulfonylaminocarbonyl.

20. The method according to claim 9, wherein the pyrimidinone alpha-keto- 1 ,3,4-oxadiazole is a compound of formula XXI or pharmaceutically acceptable salts thereof,

(XXI)

wherein X is selected from the group consisting of fluorine and hydrogen and R is selected from the group consisting of CH₂(3-Me-C₆H₄), CMe₂(3-Me-C₆H₄), CMe₂Ph, and f-Bu.

21 . The method according to claim 9, wherein the selective neutrophil elastase inhibitor is sivelestat.

22. The method according to claim 21 , wherein sivelestat is administered to the subject in an amount from about 10-100 mg/kg per day.

23. The method according to claim 21 , wherein benznidazole is administered to the subject in an amount that is about 1 mg/kg per day.

24. The method according to claim 1 , wherein the first active agent is an ergot alkaloid derivative.

25. The method according to claim 24, wherein the ergot alkaloid derivative is selected from the group consisting of 2-bromo-a-ergokryptine, a-ergokryptine, bromocriptine, cabergoline, D-(+)-lysergic acid, dihydroergotamine, dihydroergotoxine, ergobasine, ergocornine, ergocristine, ergokryptine, ergoline, ergoloid, ergometrine, ergonovine, ergopeptine, ergosecaline, ergotaman, ergotamine, lysergic acid 2-hydroxyethylamide, lysergic acid amide (ergine), lysergic acid diethylamide, methylergonovine, methysergide, nicergoline, paspalic acid, pergolide, pergolide mesylate, pharmaceutically acceptable salts thereof, and combinations thereof.

26. The method according to claim 24, wherein the ergot alkaloid derivative is nicergoline.

27. The method according to claim 26, wherein nicergoline is administered to the subject in an amount from about 1 -20 mg/kg per day.

28. The method according to claim 26, wherein benznidazole is administered to the subject in an amount that is about 1 mg/kg per day.

29. The method according to claim 1 , wherein the first active agent is an inhibitor of 3 β-hydroxysteroid dehydrogenase.

30. The method according to claim 29, wherein the inhibitor of 3 β- hydroxysteroid dehydrogenase is selected from the group consisting of 17 beta- N,N-diethylcarbamoyl-4-aza-5 alpha-androstan-3-one (4MA), 3-keto-desogestrel, Bis(2-butoxyethyl) phthalate, cyproterone acetate, danazol, desogestrel, dibutyl phthalate, dicyclohexyl phthalate, dipentyl phthalate, dipropyl phthalate, epostane, gestrinone, levonorgestrel, monooctyltin, norethisterone, progesterone, testosterone, tributyltin, trilostane, troglitazone, pharmaceutically acceptable salts thereof, and combinations thereof.

31 . The method according to claim 29, wherein the inhibitor of 3 β- hydroxysteroid dehydrogenase is trilostane.

32. The method according to claim 31 , wherein trilostane is administered to the subject in an amount from about 3-50 mg/kg per day.

33. The method according to claim 31 , wherein benznidazole is administered to the subject in an amount that is about 1 mg/kg per day.

34. The method according to claim 1 , wherein the first active agent is a reversible acetylcholinesterase inhibitor.

35. The method according to claim 34, wherein the reversible acetylcholinesterase inhibitor is selected from the group consisting of 1 ,4- methylenetacrine, ambenonium, amiridine, caffeine, caproctamine, carbamates, delta9-tetrahydrocannabinol (THC), demarcarium, donepezil, dyflos, edrophonium, galanthamine, huperzine A, huprine lactucopicrin, ladostigil, minaprine, neostigmine, neostigmine, phenanthrene derivatives, physostigmine, piperidines, pyridostigmine, pyridostigmine, rivastigmine, tacrine, ungeremine, velnacrine, pharmaceutically acceptable salts thereof, and combinations thereof.

36. The method according to claim 34, wherein the reversible acetylcholinesterase inhibitor is donepezil.

37. The method according to claim 36, wherein donepezil is administered to the subject in an amount from about 0.5-15 mg/kg per day.

38. The method according to claim 36, wherein benznidazole is administered to the subject in an amount that is about 1 mg/kg per day.

39. The method according to claim 1 , wherein the first active agent is a protease inhibitor.

40. The method according to claim 39, wherein the protease inhibitor is selected from the group consisting of amprenavir, atazanavir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, tipranavir, pharmaceutically acceptable salts thereof, and combinations thereof.

41 . The method according to claim 39, wherein the protease inhibitor is ritonavir.

42. The method according to claim 41 , wherein ritonavir is administered to the subject in an amount from about 0.2-40 mg/kg per day.

43. The method according to claim 41 , wherein benznidazole is administered to the subject in an amount that is about 1 mg/kg per day.

44. The method according to claim 1 , wherein the first active agent is an aldosterone antagonist.

45. The method according to claim 44, wherein the aldosterone antagonist is selected from the group consisting of spironolactone, epierenone, canrenone, prorenone, mexrenone, pharmaceutically acceptable salts thereof, and combinations thereof.

46. The method according to claim 44, wherein the aldosterone antagonist is epierenone.

47. The method according to claim 46, wherein epierenone is administered to the subject in an amount from about 10-400 mg/kg per day.

48. The method according to claim 46, wherein benznidazole is administered to the subject in an amount that is about 1 mg/kg per day.

49. The method according to claim 1 , wherein the first active agent is a naphthoquinone.

50. The method according to claim 49, wherein the naphthoquinone is selected from the group consisting of 1 ,2-naphthoquinone, 1 ,4-naphthoquinone, 2,6-naphthoquinone, and atovaquone.

51 . The method according to claim 49, wherein naphthoquinone is atovaquone.

52. The method according to claim 51 , wherein atovaquone is administered to the subject in an amount from about 10-400 mg/kg per day.

53. The method according to claim 51 , wherein benznidazole is administered to the subject in an amount that is about 1 mg/kg per day.

54. A method for treating or ameliorating the effects of a Chagas disease comprising administering to a subject in need thereof an effective amount of azelastine at 2-200 mg/kg per day and benznidazole at 0.5-2 mg/kg per day.

55. The method according to claim 54, wherein azelastine is administered to the subject in an amount from about 2-50 mg/kg per day, and benznidazole is administered to the subject in an amount from about 0.5-1 mg/kg per day.

56. A composition for treating or ameliorating the effects of a Chagas disease, the composition comprising azelastine and benznidazole in amounts effective to treat or ameliorate the effect of a Chagas disease and a pharmaceutically acceptable carrier.

57. The composition according to claim 56, wherein azelastine is administered to the subject in an amount from about 2-200 mg/kg per day, and benznidazole is administered to the subject in an amount from about 0.5-2 mg/kg per day.

58. The composition according to claim 56, wherein azelastine is administered to the subject in an amount from about 2-50 mg/kg per day, and benznidazole is administered to the subject in an amount from about 0.5-1 mg/kg per day.

59. A method for treating or ameliorating the effects of a Chagas disease comprising administering to a subject in need thereof an effective amount of a first active agent selected from the group consisting of a selective neutrophil elastase inhibitor, an ergot alkaloid derivative, an inhibitor of 3 β-hydroxysteroid dehydrogenase, a reversible acetylcholinesterase inhibitor, a protease inhibitor, an aldosterone antagonist, a naphthoquinone, and combinations thereof, and a second active agent, which is benznidazole.