WO2005107470A2 - Use of carbonic anhydrase inhibitors for insect control and malaria treatment - Google Patents

Abstract

Disclosed herein are methods and compositions for controlling mosquitoes. Also disclosed herein are methods and compositions for controlling the spread of malaria. Further disclosed herein are methods of treating malaria and Chaga's disease.

Description

TITLE OF THE INVENTION

USE OF CARBONIC ANHYDRASE INHIBITORS FOR INSECT CONTROL AND MALARIA TREATMENT

CROSS-REFERENCE TO RELATED APPLICATIONS [001] This application is related to U.S. Provisional Application No. 60/566,552 to which priority is claimed under 35 USC § 119(e).

BACKGROUND

[002] Malaria is a disease that continues to have an impact in much of the developing world. This disease, which afflicts 200-300 million people, results in considerable morbidity (eg. fever and chills, malaise, anorexia, kidney disease and brain disease) and kills over one million children each year. The intracellular protozoa, Plasmodium falciparum, is the most virulent of human malarias and accounts for greater than 95% of malarial deaths. High levels of parasites in the bloodstream, seen especially in the P. falciparum infection, causes serious complications including severe hemolytic anemia, renal failure, and coma. Cost-effective and easily implementable strategies for controlling the spread of malaria or for treatment of malaria is desired.

BRIEF DESCRIPTION OF THE DRAWINGS

[003] Figure 1. Hansson's histochemistry of larval midguts. Twenty larvae per species were used and representative specimens are shown in this figure. A. Anopheles quadrimaculatus, B. Culex quinquefasciatus larvae, C. Aedes albopictus, D. Ochlerotatus taeniorγnchus (midgut on the right is the same as that on the left after removal of the peritrophic membrane containing the food bolus), E. Culex nigripalpus F. Culex nigripalpus larvae treated with 10^" M methazolamide. The observed darkening in this particular preparation is due to lighting of the tissue and not to CA activity. Arrow heads observed in C. and D. indicate darkening observed in bands around the midgut.

[004] Figure 2. CA content in mosquito larval midgut tissue homogenate. Results shown are the average of duplicate measurements using 45-55 larvae from each species. The presence of CA in the anterior and posterior midgut is species dependent. All species exhibited CA activity in the gastric caeca. Values presented for Ae. aegypti were obtained previously (Corena et al., 2002). [005] Figure 3. An. albimanus anterior midgut CA was inhibited by methazolamide (10 μM). Similar results were obtained for other species. This figure represents enrichment with ¹⁸O in C0₂ over time. Addition of anterior midgut homogenate (AM) and methazolamide (MZ) are indicated by arrows.

[006] Figure 4. Effect of methazolamide and acetazolamide on the pH of the rearing medium of different species of larvae. Lanes corresponding to unavailable instars were left empty. Template shown on the top (Each successive horizontal row in each plate corresponds to first, second, third and fourth instar respectively. Top plate for each species corresponds to larvae in distilled water, bottom plate corresponds to larvae in 2 % seawater. Each plate was treated from left to right with 10^"δ, 10^"5 and 10^"4 M methazolamide followed by acetazolamide at the same concentrations), A. Ochlerotatus taeniorhynchus, B. Aedes albopictus, C. Culex nigripalpus, D. Culex quinquefasciatus, E. Anopheles quadrimaculatus, F. control Aedes aegypti untreated.

[007] Figure 5. Dissected midguts of larvae fed 7?ι-cresol purple. Specimens shown are representative of 20 larvae from each species. The same alkalization pattern with purple anterior midgut and orange/yellow gastric caeca and posterior midgut was observed for all species. A. Anopheles quadrimaculatus, B. Culex quinquefasciatus, C. Aedes albopictus, D. Culex nigripalpus, E. Ochlerotatus taeniorhynchus.

[008] Figure 6. Isolated midgut of Aedes albopictus larvae fed m-cresol purple. Each specimen shown is representative of a group of 20 larvae. A. 24 hours post-treatment with 10^"4 M methazolamide, B. Untreated larvae. Notice decrease in alkalization in posterior midgut.

[009] Figure 7. Semi-intact preparation of mosquito larvae fed m-cresol purple 24 h after treatment with 10^"4 M methazolamide. Specimens shown are representative of groups of 20 larvae each. A. Culex nigripalpus, B. Culex quinquefasciatus, C. Ochlerotatus taeniorhynchus.

DETAILED DESCRIPTION A. Definitions and Terms

[0010] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All patents, patent applications and publications referred to herein are incorporated by reference in their entirety. [0011] As used herein, malaria refers to an acute and sometimes chronic infectious disease caused by or associated with parasitic infection, particularly infection with the protozoan parasites Plasmodium vivax, Plasmodium falciparum, Plasmodium malariae or Plasmodium ovale. The disease is characterized by the presence of the protozoan parasites within red blood cells. Of particular interest herein is malaria caused by or associated with P. falciparum infection.

[0012] As used herein, falcipain refers to a P. falciparum cysteine protease of the papain family.

Falcipain is implicated in hemoglobin degradation in the parasitic food vacuole and in parasite development.

[0013] As used herein, Chagas' disease refers to a parasitic disease associated with or caused by infection with the protozoan parasite Trypanosoma cruzi.

[0014] As used herein, the IC50 refers to a concentration of a particular test compound that achieves a 50% inhibition of a maximal response.

[0015] As used herein, EC50 refers to a concentration of a particular test compound that elicits a dose-dependent response at 50% of maximal expression of a particular response that is induced, provoked or potentiated by the particular test compound.

[0016] As used herein, pharmaceutically acceptable derivatives of a compound include salts, esters, enol ethers, enol esters, acids, bases, solvates, hydrates or prodrugs thereof that may be readily prepared by those of skill in this art using known methods for such derivatization and that produce compounds that may be administered to animals or humans without substantial toxic effects and that either are pharmaceutically active or are prodrugs. For example, acidic groups can be esterified or neutralized.

[0017] As used herein, treatment means any manner in which one or more of the symptoms of a condition, disorder or disease are ameliorated or otherwise beneficially altered. Treatment also encompasses any pharmaceutical use of the compositions herein, such as use as contraceptive agents.

[0018] As used herein, amelioration of the symptoms of a particular disorder by administration of a particular pharmaceutical composition refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of the composition.

[0019] As used herein, biological activity refers to the in vivo activities of a compound or physiological responses that result upon in vivo administration of a compound, composition or other mixture. Biological activity, thus, encompasses therapeutic effects and pharmaceutical activity of such compounds, compositions and mixtures.

[0020] As used herein, a prodrug is a compound that, upon in vivo administration, is metabolized or otherwise converted to the biologically, pharmaceutically or therapeutically active form of the compound. To produce a prodrug, the pharmaceutically active compound is modified such that the active compound will be regenerated by metabolic processes. The prodrug may be designed to alter the metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug. By virtue of knowledge of pharmacodynamic processes and drug metabolism in vivo, those of skill in this art, once a pharmaceutically active compound is known, can design prodrugs of the compound (see, e.g., Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388- 392).

[0021] It is to be understood that the compounds provided herein may contain chiral centers.

Such cl iral centers may be of either the (R) or (S) configuration, or may be a mixture thereof. Thus, the compounds provided herein may be enantiomerically pure, or be stereoisomeric or diastereomeric mixtures. In the case of amino acid residues, such residues may be of either the L- or D-form. The preferred configuration for naturally occurring amino acid residues is L. It is to be understood that the chiral centers of the compounds provided herein may undergo epimerization in vivo. As such, one of skill in the art will recognize that administration of a compound in its (R) form is equivalent, for compounds that undergo epimerization in vivo, to administration of the compound in its (S) form. [0022] As used herein, substantially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis, high performance liquid chromatography (HPLC) and mass spectrometry (MS), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound may, however, be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound.

[0023] As used herein, the term "amino acid" refers to α-amino acids which are racemic, or of either the D- or L-configuration. The designation "d" preceding an amino acid designation (e.g., dAla, dSer, dVal, etc.) refers to the D-isomer of the amino acid. The designation "dl" preceding an amino acid designation (e.g., dlPip) refers to a mixture of the L- and D-isomers of the amino acid. [0024] As used herein the symbols and conventions used in these processes, schemes and examples are consistent with those used in the contemporary scientific literature, for example, the Journal of the American Chemical Society or the Journal of Biological Chemistry. Standard three-letter abbreviations are generally used to designate amino acid residues, which are assumed to be in the L- configuration unless otherwise noted. Unless otherwise noted, all starting materials were obtained from commercial suppliers and used without further purification. Specifically, the following abbreviations may be used in the examples and throughout the specification: g (grams); mg (milligrams); L (liters); mL (milliliters); μL (microliters); psi (pounds per square inch); M (molar); mmol (millimolar); i.v. (intravenous); Hz (Hertz); MHz (megahertz); mol (moles); mmol (millimoles); RT (room temperature); min (minutes); h (hours); mp (melting point); TLC (thin layer chromatography); HPLC (high pressure liquid chromatography); Rt(retention time); RP (reverse phase); MeOH (methanol); i-PrOH (isopropanol); Et3N (triethylamine); TFA (trifluoroacetic acid); THF (tetrahydrofuran); DMSO (dimethylsulfoxide); EtOAc (ethyl acetate); DCM (dichloromethane); 4-NMM (N-methylmorpholine); LAH (lithium aluminum hydride); Dibal-H (diisobutylaluminum hydride); DCE (dichloroethane); DMF (N,N-dimethylformamide); AcOH (acetic acid); HO At (l-hydroxy-7-azabenzotriazole); EDC (ethylcarbodiimide hydrochloride); Boc (tert-butyloxycarbonyl); FMOC (9-fluorenylmethoxycarbonyl); Z (benzyloxycarbonyl); Ac (acetyl); and atm (atmosphere). All references to ether are to diethyl ether; brine refers to a saturated aqueous solution of NaCl; and Rochelle salt refers to sodium potassium tartrate. Unless otherwise indicated, all temperatures are expressed in ° C. (degrees Centigrade). All reactions conducted under an inert atmosphere at room temperature unless otherwise noted.

[0025] 1H NMR spectra were recorded on a Varian Unity Inova-400 instrument. Chemical shifts are expressed in parts per million (ppm, δ units). Coupling constants are in units of hertz (Hz). Splitting patterns describe apparent multiplicities and are designated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad).

[0026] Low-resolution mass spectra (MS) were recorded on a Perkin Elmer SCTE API1 spectrometer. All reactions were monitored by thin-layer chromatography on 0.25 mm E. Merck silica gel plates (60F-254), visualized with UV light, 5% ethanolic phosphomolybdic acid. Flash column chromatography was performed on silica gel (230-400 mesh, Merck).

[0027] In scheme(s) described below, it is well understood that protecting groups for sensitive or reactive groups are employed where necessary in accordance with general principles of chemistry. Protecting groups are manipulated according to standard methods of organic synthesis (T. W. Green and P. G. M. Wuts (1991) Protecting Groups in Organic Synthesis, John Wiley & Sons). These groups are removed at a convenient stage of the compound synthesis using methods that are readily apparent to those skilled in the art. B. Compounds Useful as Carbonic Anhydrase Inhibitors [0028] Compounds and compositions useful as Carbonic Anhydrase inhibitors are provided. The compositions contain compounds that are active in assays that measure Carbonic Anhydrase activity. The compounds and compositions provided herein are thus useful in treatment, prevention, or amelioration of one or more symptoms of malaria and Chagas' disease. The term "carbonic anhydrase inhibitor" as used herein means an agent which blocks or impedes the carbonic anhydrase pathway by inhibiting the enzyme, carbonic anhydrase. Carbonic anhydrase inhibitors (CAIs) as a class are well-known; these compounds have found widespread acceptance in the treatment of elevated intraocular pressure, especially glaucoma. Some of these compounds have also been used as diuretics, for example, in the treatment of congestive heart failure, or in the treatment of allergies. See, for example, the following patents relating to compounds of this type, which are incorporated by reference herein in their entireties and relied upon: U.K. Patent Specification No. 769,757; Clapp et al. U.S. Pat. No. 2,554,816, Young et al. U.S. Pat. No. 2,783,241; Schultz U.S. Pat. No. 2,835,702; Korman U.S. Pat. No. 2,868,800; Yale et al. U.S. Pat. No. 3,040,042; Sircar et al. U.S. Pat. No. 4,092,325; Woltersdorf, Jr. et al. U.S. Pat. No. 4,386,098; Woltersdorf, Jr. et al. U.S. Pat. No. 4,416,890; Woltersdorf, Jr. U.S. Pat. No. 4,426,388; Shepard U.S. Pat. No. 4,542,152; EP 0182691; Shepard et al. U.S. Pat. No. 4,668,697; Baldwin et al. U.S. Pat. No. 4,677,115; EP 0228237; Baldwin et al U.S. Pat. No. 4,797,413; and Marin U.S. Pat. No. 4,619,939.

[0029] Typically, carbonic anhydrase inhibitors have a sulfonamide structure which is attached to a ring system; typically, CAIs are heterocyclic or aryl sulfonamides. At the present time, preferred CAIs for use in the present invention include dorzolamide, acetazolamide, brinzolamide, methazolamide, ethoxzolamide (ethoxyzolamide), butazolamide, dichlorphenamide and flumethiazide. The chemical names for these preferred agents are as follows: dorzolamide: (4S-trans)-4-(ethylamino)-5,6-dihydro-6-methyl-4H-thieno[2,3-b]thiopyran-2- sulfonamide dioxide acetazolamide: N-[5-(aminosulfonyl)-l,3,4-thiadiazol-2-yl]acetamide brinzolamide: (R)-(+)-4-ethylamino-2-(3-methoxypropyl)-3,4-dihydro-2H-thieno [3,2-e]-l ,2-thiazine- 6-sulfonamide- 1 , 1 -dioxide methazolamide: N-[5-(aminosulfonyl)-3 -methyl- 1 ,3,4 thiadiazol-2(3H)-ylidene]acetamide ethoxzolamide: 6-ethoxy-2-benzothiazolesulfonamide butazolamide: N-[5-(aminosulfonyl)-l,3,4-thiadiazol-2-yl)butanamide dichlo henamide: 4,5-dichloro-l ,3-benzenedisulfonamide flumethiazide: 6-(trifluoromethyl)-2H-l ,2,4-benzothiadiazine-7 -sulfonamide 1 , 1 -dioxide See also The Merck Index, 12th edition, ed. Susan Budavari et al. Merck & Co., Inc., Whitehouse Station, N.J., 1996, pp. 10, 250, 520, 579, 641, 701, 1020, incorporated by reference herein; and Physicians' Desk Reference, 54th edition, Medical Economics Company, Inc., Montvale, N.J., 2000, pp. 486-487, 1767-1769, 1778-1779, 1897-1898, also incorporated by reference herein. Other suitable CAIs for use in the present invention will be apparent to those of ordinary skill in the art. C. Formulation of Pharmaceutical Compositions [0030] The pharmaceutical compositions provided herein contain therapeutically effective amounts of one or more CAI's that are useful in the prevention, treatment, or amelioration of one or more of the symptoms of parasitic infections, particularly malaria or Chagas' disease. Preferred compounds for use in the compositions are those that inhibit Carbonic Anhydrase with an IC50 of less than about 100 nM, preferably less that 50 nM, more preferably less than 10 nM.

[0031] The compounds are preferably formulated into suitable pharmaceutical preparations such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs, for oral administration or in sterile solutions or suspensions for parenteral administration, as well as transdermal patch preparation and dry powder inhalers. Typically the compounds described above are formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel Introduction to Pharmaceutical Dosage Forms, Fourth Edition 1985, 126). [0032] In the compositions, effective concentrations of one or more compounds or pharmaceutically acceptable derivatives is (are) mixed with a suitable pharmaceutical carrier or vehicle. The compounds may be derivatized as the corresponding salts, esters, enol ethers or esters, acids, bases, solvates, hydrates or prodrugs prior to formulation, as described above. The concentrations of the compounds in the compositions are effective for delivery of an amount, upon administration, that ameliorates one or more of the symptoms of parasitic infection, particularly malaria or Chagas' disease. Typically, the compositions are formulated for single dosage administration. To formulate a composition, the weight fraction of compound is dissolved, suspended, dispersed or otherwise mixed in a selected vehicle at an effective concentration such that the treated condition is relieved or ameliorated. Pharmaceutical carriers or vehicles suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration. [0033] In addition, the compounds may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients. Liposomal suspensions, including tissue-targeted liposomes, particularly tumor-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art. For example, liposome formulations may be prepared as described in U.S. Pat. No. 4,522,811. [0034] The active compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated. The therapeutically effective concentration may be determined empirically by testing the compounds in known in vitro and in vivo systems (see, e.g., Rosenthal et al. (1996) Antimicrob. Agents Chemother. 40(7):1600-1603; Dominguez et al. (1997) J. Med. Chem. 40:2726-2732; Clark et al. (1994) Molec. Biochem. Parasitol. 17:129; Ring et al. (1993) Proc. Natl. Acad. Sci. USA 90:3583-3587; Engel et al. (1998) J. Exp. Med. 188(4):725-734; Li et al. (1995) J. Med. Chem. 38:5031) and then extrapolated therefrom for dosages for humans.

[0035] The concentration of active compound in the pharmaceutical composition will depend on absoφtion, inactivation and excretion rates of the active compound, the physicochemical characteristics of the compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art. For example, the amount that is delivered is sufficient to ameliorate one or more of the symptoms of parasitic infections, particularly malaria or Chagas' disease.

[0036] Typically a therapeutically effective dosage should produce a serum concentration of active ingredient of from about 0.1 ng/ml to about 50-100 μg/ml. The pharmaceutical compositions typically should provide a dosage of from about 0.001 mg to about 2000 mg of compound per kilo-gram of body weight per day. Pharmaceutical dosage unit forms are prepared to provide from about 1 mg to about 1000 mg and preferably from about 10 to about 500 mg of the essential active ingredient or a combination of essential ingredients per dosage unit form. [0037] The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person admimstering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions. [0038] Preferred pharmaceutically acceptable derivatives include acids, bases, enol ethers and esters, salts, esters, hydrates, solvates and prodrug forms. The derivative is selected such that its pharmacokinetic properties are superior to the corresponding neutral compound. [0039] Thus, effective concentrations or amounts of one or more of the compounds described herein or pharmaceutically acceptable derivatives thereof are mixed with a suitable pharmaceutical carrier or vehicle for systemic, topical or local administration to form pharmaceutical compositions. Compounds are included in an amount effective for ameliorating one or more symptoms of, or for treating or preventing parasitic infections, particularly malaria or Chagas' disease. The concentration of active compound in the composition will depend on absoφtion, inactivation, excretion rates of the active compound, the dosage schedule, amount administered, particular formulation as well as other factors known to those of skill in the art.

[0040] The compositions are intended to be administered by a suitable route, including orally, parenterally, rectally, topically and locally. For oral administration, capsules and tablets are presently preferred. The compositions are in liquid, semi-liquid or solid form and are formulated in a manner suitable for each route of administration. Preferred modes of administration include parenteral and oral modes of administration. Oral administration is presently most preferred.

[0041] Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent; antimicrobial agents, such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates and phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. Parenteral preparations can be enclosed in ampules, disposable syringes or single or multiple dose vials made of glass, plastic or other suitable material.

[0042] In instances in which the compounds exhibit insufficient solubility, methods for solubilizing compounds may be used. Such methods are known to those of skill in this art, and include, but are not limited to, using cosolvents, such as dimethylsulfoxide (DMSO), using surfactants, such as TWEEN®, or dissolution in aqueous sodium bicarbonate. Derivatives of the compounds, such as prodrugs of the compounds may also be used in formulating effective pharmaceutical compositions. [0043] Upon mixing or addition of the compound(s), the resulting mixture may be a solution, suspension, emulsion or the like. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient for ameliorating the symptoms of the disease, disorder or condition treated and may be empirically determined.

[0044] The pharmaceutical compositions are provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil-water emulsions containing suitable quantities of the compounds or pharmaceutically acceptable derivatives thereof. The pharmaceutically therapeutically active compounds and derivatives thereof are typically formulated and administered in unit-dosage forms or multiple-dosage forms. Unit-dose forms as used herein refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of the therapeutically active compound sufficient to produce the desired therapeutic effect, in association with the required phaπnaceutical carrier, vehicle or diluent. Examples of unit-dose forms include ampoules and syringes and individually packaged tablets or capsules. Unit-dose forms may be administered in fractions or multiples thereof. A multiple-dose form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dose form. Examples of multiple-dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons. Hence, multiple dose form is a multiple of unit-doses which are not segregated in packaging. [0045] The composition can contain along with the active ingredient: a diluent such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; a lubricant, such as magnesium stearate, calcium stearate and talc; and a binder such as starch, natural gums, such as gum acaciagelatin, glucose, molasses, polvinylpyrrolidine, celluloses and derivatives thereof, povidone, crospovidones and other such binders known to those of skill in the art. Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, or solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975. The composition or formulation to be administered will, in any event, contain a quantity of the active compound in an amount sufficient to alleviate the symptoms of the treated subject. [0046] Dosage forms or compositions containing active ingredient in the range of 0.005% to

100% with the balance made up from non-toxic carrier may be prepared. For oral admimstration, a pharmaceutically acceptable non-toxic composition is formed by the incoφoration of any of the normally employed excipients, such as, for example pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, talcum, cellulose derivatives, sodium crosscarmellose, glucose, sucrose, magnesium carbonate or sodium saccharin. Such compositions include solutions, suspensions, tablets, capsules, powders and sustained release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others. Methods for preparation of these compositions are known to those skilled in the art. The contemplated compositions may contain 0.001%-100% active ingredient, preferably 0.1-85%, typically 75-95%.

[0047] The active compounds or pharmaceutically acceptable derivatives may be prepared with carriers that protect the compound against rapid elimination from the body, such as time release formulations or coatings.

[0048] The compositions may include other active compounds to obtain desired combinations of properties. The CAIs, or pharmaceutically acceptable derivatives thereof as described herein, may also be advantageously administered for therapeutic or prophylactic puφoses together with another pharmacological agent known in the general art to be of value in treating one or more of the diseases or medical conditions referred to hereinabove, such as malaria or Chagas' disease. It is to be understood that such combination therapy constitutes a further aspect of the compositions and methods of treatment provided herein. 1. Compositions for Oral Administration [0049] Oral pharmaceutical dosage forms are either solid, gel or liquid. The solid dosage forms are tablets, capsules, granules, and bulk powders. Types of oral tablets include compressed, chewable lozenges and tablets which may be enteric-coated, sugar-coated or film-coated. Capsules may be hard or soft gelatin capsules, while granules and powders may be provided in non-effervescent or effervescent form with the combination of other ingredients known to those skilled in the art.

[0050] In certain embodiments, the formulations are solid dosage forms, preferably capsules or tablets. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder; a diluent; a disintegrating agent; a lubricant; a glidant; a sweetening agent; and a flavoring agent.

[0051] Examples of binders include microcrystalline cellulose, gum tragacanth, glucose solution, acacia mucilage, gelatin solution, sucrose and starch paste. Lubricants include talc, starch, magnesium or calcium stearate, lycopodium and stearic acid. Diluents include, for example, lactose, sucrose, starch, kaolin, salt, mannitol and dicalcium phosphate. Glidants include, but are not limited to, colloidal silicon dioxide. Disintegrating agents include crosscarmellose sodium, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar and carboxymethylcellulose. Coloring agents include, for example, any of the approved certified water soluble FD and C dyes, mixtures thereof; and water insoluble FD and C dyes suspended on alumina hydrate. Sweetening agents include sucrose, lactose, mannitol and artificial sweetening agents such as saccharin, and any number of spray dried flavors. Flavoring agents include natural flavors extracted from plants such as fruits and synthetic blends of compounds which produce a pleasant sensation, such as, but not limited to peppermint and methyl salicylate. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene laural ether. Emetic-coatings include fatty acids, fats, waxes, shellac, ammoniated shellac and cellulose acetate phthalates. Film coatings include hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000 and cellulose acetate phthalate. [0052] If oral administration is desired, the compound could be provided in a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition may also be formulated in combination with an antacid or other such ingredient.

[0053] When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents. The compounds can also be administered as a component of an elixir, suspension, syrup, wafer, sprinkle, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors. [0054] The active materials can also be mixed with other active materials which do not impair the desired action, or with materials that supplement the desired action, such as antacids, H2 blockers, and diuretics. The active ingredient is a compound or pharmaceutically acceptable derivative thereof as described herein. Higher concentrations, up to about 98% by weight of the active ingredient may be included.

[0055] Pharmaceutically acceptable carriers included in tablets are binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, and wetting agents. Enteric-coated tablets, because of the enteric-coating, resist the action of stomach acid and dissolve or disintegrate in the neutral or alkaline intestines. Sugar-coated tablets are compressed tablets to which different layers of pharmaceutically acceptable substances are applied. Film-coated tablets are compressed tablets which have been coated with a polymer or other suitable coating. Multiple compressed tablets are compressed tablets made by more than one compression cycle utilizing the pharmaceutically acceptable substances previously mentioned. Coloring agents may also be used in the above dosage forms. Flavoring and sweetening agents are used in compressed tablets, sugar-coated, multiple compressed and chewable tablets. Flavoring and sweetening agents are especially useful in the formation of chewable tablets and lozenges.

[0056] Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Aqueous solutions include, for example, elixirs and syrups. Emulsions are either oil-in-water or water-in-oil.

[0057] Elixirs are clear, sweetened, hydroalcoholic preparations. Pharmaceutically acceptable carriers used in elixirs include solvents. Syrups are concentrated aqueous solutions of a sugar, for example, sucrose, and may contain a preservative. An emulsion is a two-phase system in which one liquid is dispersed in the form of small globules throughout another liquid. Pharmaceutically acceptable carriers used in emulsions are non-aqueous liquids, emulsifying agents and preservatives. Suspensions use pharmaceutically acceptable suspending agents and preservatives. Pharmaceutically acceptable substances used in non-effervescent granules, to be reconstituted into a liquid oral dosage form, include diluents, sweeteners and wetting agents. Pharmaceutically acceptable substances used in effervescent granules, to be reconstituted into a liquid oral dosage form, include organic acids and a source of carbon dioxide. Coloring and flavoring agents are used in all of the above dosage forms. [0058] Solvents include glycerin, sorbitol, ethyl alcohol and syrup. Examples of preservatives include glycerin, methyl and propylparaben, benzoic add, sodium benzoate and alcohol. Examples of non- aqueous liquids utilized in emulsions include mineral oil and cottonseed oil. Examples of emulsifying agents include gelatin, acacia, tragacanfh, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate. Suspending agents include sodium carboxymethylcellulose, pectin, tragacanth, Veegum and acacia. Diluents include lactose and sucrose. Sweetening agents include sucrose, syrups, glycerin and artificial sweetening agents such as saccharin. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether. Organic adds include citric and tartaric acid. Sources of carbon dioxide include sodium bicarbonate and sodium carbonate. Coloring agents include any of the approved certified water soluble FD and C dyes, and mixtures thereof. Flavoring agents include natural flavors extracted from plants such fruits, and synthetic blends of compounds which produce a pleasant taste sensation.

[0059] For a solid dosage form, the solution or suspension, in for example propylene carbonate, vegetable oils or triglycerides, is preferably encapsulated in a gelatin capsule. Such solutions, and the preparation and encapsulation thereof, are disclosed in U.S. Pat. Nos 4,328,245; 4,409,239; and 4,410,545. For a liquid dosage form, the solution, e.g., for example, in a polyethylene glycol, may be diluted with a sufficient quantity of a pharmaceutically acceptable liquid carrier, e.g., water, to be easily measured for administration. [0060] Alternatively, liquid or semi-solid oral formulations may be prepared by dissolving or dispersing the active compound or salt in vegetable oils, glycols, triglycerides, propylene glycol esters (e.g., propylene carbonate) and other such carriers, and encapsulating these solutions or suspensions in hard or soft gelatin capsule shells. Other useful formulations include those set forth in U.S. Pat. Nos. Re 28,819 and 4,358,603.

[0061] In all embodiments, tablets and capsules formulations may be coated as known by those of skill in the art in order to modify or sustain dissolution of the active ingredient. Thus, for example, they may be coated with a conventional enterically digestible coating, such as phenylsalicylate, waxes and cellulose acetate phthalate. 2. Injectables, Solutions and Emulsions [0062] Parenteral administration, generally characterized by injection, either subcutaneously, intramuscularly or intravenously is also contemplated herein. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. In addition, if desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins. Implantation of a slow-release or sustained-release system, such that a constant level of dosage is maintained (see, e.g., U.S. Pat. No. 3,710,795) is also contemplated herein. The percentage of active compound contained in such parenteral compositions is highly dependent on the specific nature thereof, as well as the activity of the compound and the needs of the subject.

[0063] Parenteral administration of the compositions includes intravenous, subcutaneous and intramuscular administrations. Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyopbilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use and sterile emulsions. The solutions may be either aqueous or nonaqueous.

[0064] If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.

[0065] Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable substances. [0066] Examples of aqueous vehicles include Sodium Chloride Injection, Ringers Injection,

Isotonic Dextrose Injection, Sterile Water Injection, Dextrose and Lactated Ringers Injection. Nonaqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, com oil, sesame oil and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations must be added to parenteral preparations packaged in multiple-dose containers which include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium cliloride and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcelluose, hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80 (TWEEN® 80). A sequestering or chelating agent of metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment. [0067] The concentration of the pharmaceutically active compound is adjusted so that an injection provides an effective amount to produce the desired pharmacological effect. The exact dose depends on the age, weight and condition of the patient or animal as is known in the art. [0068] The unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration must be sterile, as is known and practiced in the art.

[0069] Illustratively, intravenous or intraarterial infusion of a sterile aqueous solution containing an active compound is an effective mode of administration. Another embodiment is a sterile aqueous or oily solution or suspension containing an active material injected as necessary to produce the desired pharmacological effect.

[0070] Injectables are designed for local and systemic administration. Typically a therapeutically effective dosage is formulated to contain a concentration of at least about 0.1 %> w/w up to about 90%> w/w or more, preferably more than 1% w/w of the active compound to the treated tissue(s). The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the tissue being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the age of the individual treated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the formulations, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed formulations. [0071] The compound may be suspended in micronized or other suitable form or may be derivatized to produce a more soluble active product or to produce a prodrug. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient for ameliorating the symptoms of the condition and may be empirically determined. 3. Lyophilized Powders

[0072] Of interest herein are also lyophilized powders, which can be reconstituted for administration as solutions, emulsions and other mixtures. They may also be reconstituted and formulated as solids or gels.

[0073] The sterile, lyophilized powder is prepared by dissolving a CAI in a suitable solvent. The solvent may contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbital, fructose, com syrup, xylitol, glycerin, glucose, sucrose or other suitable agent. The solvent may also contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, typically, about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation. Generally, the resulting solution will be apportioned into vials for lyophilization. Each vial will contain a single dosage (10-1000 mg, preferably 100-500 mg) or multiple dosages of the compound. The lyophilized powder can be stored under appropriate conditions, such as at about 4° C. to room temperature.

[0074] Reconstitution of this lyophilized powder with water for inj ection provides a formulation for use in parenteral administration. For reconstitution, about 1-50 mg, preferably 5-35 mg, more preferably about 9-30 mg of lyophilized powder, is added per mL of sterile water or other suitable carrier. The precise amount depends upon the selected compound. Such amount can be empirically determined. 4. Topical Administration

[0075] Topical mixtures are prepared as described for the local and systemic administration. The resulting mixture may be a solution, suspension, emulsions or the like and are formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays, suppositories, bandages, dermal patches or any other formulations suitable for topical administration.

[0076] The compounds or pharmaceutically acceptable derivatives thereof may be formulated as aerosols for topical application, such as by inhalation (see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivery of a steroid useful for treatment inflammatory diseases, particularly asthma). These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such a case, the particles of the formulation will typically have diameters of less than 50 microns, preferably less than 10 microns.

[0077] The compounds may be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application. Topical administration is contemplated for transdermal delivery and also for administration to the eyes or mucosa, or for inhalation therapies. Nasal solutions of the active compound alone or in combination with other pharmaceutically acceptable excipients can also be administered.

[0078] These solutions, particularly those intended for ophthalmic use, may be formulated as

0.01%- 10% isotonic solutions, pH about 5-7, with appropriate salts. 5. Compositions for Other Routes of Administration

[0079] Other routes of administration, such as transdermal patches and rectal administration are also contemplated herein.

[0080] For example, pharmaceutical dosage forms for rectal administration are rectal suppositories, capsules and tablets for systemic effect. Rectal suppositories are used herein mean solid bodies for insertion into the rectum which melt or soften at body temperature releasing one or more pharmacologically or therapeutically active ingredients. Pharmaceutically acceptable substances utilized in rectal suppositories are bases or vehicles and agents to raise the melting point. Examples of bases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax (polyoxyethylene glycol) and appropriate mixtures of mono-, di- and triglycerides of fatty acids. Combinations of the various bases may be used. Agents to raise the melting point of suppositories include spermaceti and wax. Rectal suppositories may be prepared either by the compressed method or by molding. The typical weight of a rectal suppository is about 2 to 3 gm.

[0081] Tablets and capsules for rectal administration are manufactured using the same pharmaceutically acceptable substance and by the same methods as for formulations for oral administration. 6. Combination Therapy

[0082] Also contemplated herein are compositions for use in the methods containing (i) a compound provided herein, or a pharmaceutically acceptable derivative thereof, and (ii) a known antiparasitic compound or composition. The antiparasitic compound or composition may be any known to those of skill in the art, including marketed and experimental therapeutics. Many such compounds are well known to those of skill in the art (see, e.g., Rosenthal (1998) Emerging Infectious Diseases 4(1):49- 57; Rosenthal et al. (1996) Antimicrob. Agents Chemother. 40(7):1600-1603; Dominguez et al. (1997) J. Med. Chem. 40:2726-2732; Li et al. (1996) Bioorg. Med. Chem. 4(9):1421-1427; Ring et al. (1993) Proc. Natl. Acad. Sci. USA 90:3583-3587; and International Patent Application Publication Nos. WO 97/30072, WO 96/40647 and WO 96/40737; see also, Engel et al. (1998) J. Exp. Med. 188(4):725-734). The compositions described above may be more efficacious due to a synergistic effect between the compound provided herein and the known antiparasitic compound or composition. In such cases, the compositions described above may be particularly useful in the treatment of resistant strains of parasitic infection.

[0083] Among the known antiparasitic agents for use in this embodiment are chloroquine, quinine, quinidine, amodiaquine, mefloquine, sulfadoxine, pyrimethamine, a tetracyline antibiotic, clindamycin, a sulfa antibiotic, doxycyline, proguanil, dapsone, primaquine, artemisinin, artesunate, artelinate, artemether, arteether, dihydroartemisinin, halofantrine, atovaquione, pyronaridine, desferrioxamine, azithromycin, SC-50083, Ro 40-4388, "compound 7", ((benzyloxycarbonyl)phenylalanyl)arginyl fluoromethyl ketone, ((moφholinocarbonyl)phenylalanyl)homophenylalanyl fluoromethyl ketone, (((moφholinocarbonyl)leucyl)homophenylalanyl)vinyl phenyl sulfone, oxalic bis((2-hydroxy-l- naphthylmethylene)hydrazide), l-(2,5-dichlorophenyl)-3-(4-qmnolinyl)-2-propen-l-one, and 7-chloro- l,2-dihydro-2-(2,3-dimethoxyphenyl)-5,5-dioxide-4-(lH, 10H)-phenothiazinone. [0084] Other known antiparasitic agents for use in this embodiment include nifurtimox, benznidazole, (((moφholinocarbonyl)phenylalanyl)-homophenylalanyl)vinyl phenyl sulfone, (((moφholinocarbonyl)phenyl-alanyl)lysyl)vinyl phenyl sulfone, (((moφholinocarbonyl)phenylalanyl)- valyl)vinyl phenyl sulfone, (((moφholinocarbonyl)phenylalanyl)-0-benzylseryl)vinyl phenyl sulfone, (((moφholinocarbonyl)leucyl)-homophenylalanyl)vinyl phenyl sulfone, (((moφholinocarbonyl)tyrosyl)- homophenylalanyl)vinyl phenyl sulfone, (((tert-butoxycarbonyι)-2- tetrahydroisoquinolylcarbonyl)homophenylalanyl) phenyl vinyl sulfone, (((moφholinocarbonyl)tyrosyl)homophenylalanyl)vinylphenyl sulfone, (((moφholinocarbonyl)phenylalanyl)homophenylalanyl fluromethylketone and (((moφholinocarbonyl)phenylalanyl)homophenylalanyl)valine benzylamide. 7. Articles of Manufacture [0085] The compounds or pharmaceutically acceptable derivatives may be packaged as articles of manufacture containing packaging material, a compound or pharmaceutically acceptable derivative thereof provided herein, which is effective for inhibiting falcipain or cruzain, or for treatment, prevention or amelioration of one or more symptoms of parasitic infections, particularly malaria or Chagas' disease, and a label that indicates that the compound or pharmaceutically acceptable derivative thereof is used for inhibiting falcipain or cruzain, or for treatment, prevention or amelioration of one or more symptoms of parasitic infections, particularly malaria or Chagas' disease.

[0086] The articles of manufacture provided herein contain packaging materials. Packaging materials for use in packaging pharmaceutical products are well known to those of skill in the art. See, e.g., U.S. Pat. Nos. 5,323,907, 5,052,558 and 5,033,352. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of adininistration and treatment. A wide array of formulations of the compounds and compositions provided herein are contemplated as are a variety treatments for any disorder in which falcipain is implicated as a mediator or contributor to the symptoms or cause. D. Evaluation of the Activity of the Compounds

[0087] Standard physiological, pharmacological and biochemical procedures are available for testing the compounds to identify those that possess biological activities that interfere with, antagonize, inhibit, or otherwise modulate the activity of carbonic anyhydrase. For example, the properties of a potential inhibitor may be assessed as a function of its ability to inhibit carbonic anhydrase including the ability in vitro to antagonize the activity of carbonic anhydrase.

[0088] Using such assays, the relative abilities of the compounds provided herein to inhibit or otherwise modulate the activity of carbonic anhydrase have been and can be assessed. Those that possess the desired in vitro properties, such as specific inhibition of carbonic anhydrase, are selected. The selected compounds that exhibit desirable activities may be therapeutically useful in the methods described herein and are tested for such uses employing the above-described assays from which the in vivo effectiveness may be evaluated. Compounds that exhibit the in vitro activities that correlate with the in vivo effectiveness will then be formulated in suitable pharmaceutical compositions and used as therapeutics. E. Methods of use of Carbonic Anhydrase Inhibitors

[0089] Methods using therapeutically effective concentrations one or more of the CAI compounds, or pharmaceutically acceptable derivatives thereof, for treating, preventing or ameliorating one or more symptoms of parasitic infections, particularly malaria or Chagas' disease, are provided herein.

[0090] In certain cases, the medicament containing the compound is injected into the circulatory system of a subject in order to deliver a dose to the targeted cells. Targeting may be effected by linking the compound to a targeting agent specific for the desired cells, such as, but not limited to, cells associated with the malaria parasite. See, e.g., U.S. Pat. Nos. 5,456,663, 4,764,359, 5,543,391, 5,820,879, 5,026,558. Dosages may be determined empirically, but will typically be in the range of about 0.01 mg to about 100 mg of the compound per kilogram of body weight as a daily dosage. [0091] Methods of inhibiting the development or growth of parasites, particularly malarial parasites or parasites that are the causative agent of Chagas' disease, more particularly Plasmodium falciparum, Trypanosoma cruzi or Tiypanosoma brucei, are also provided. F. Control of Hosts [0092] Control of parasites can be accomplished by a variety of methods known to those skilled in the art. In the case of malaria, the parasite may be controlled by affecting the carriers of the parasite. The inventors have shown that CAIs are lethal to parasite hosts. 1. Basic Pesticidal Formulations and Application Thereof [0093] Amounts and locations for application of CAIs are generally determined by the habits of the insect pest, the lifecycle stage at which the pest is to be attacked, the site where the application is to be made and the physical and functional characteristics of the polypeptide.

[0094] CAIs are generally administered to the insect pest by oral ingestion, but may also be administered by means which permit penetration through the cuticle or penetration of the insect respiratory system. CAIs may also be administered with other insect control chemicals, for example, the CAI compositions may employ various chemicals designed to affect insect behavior, such as attractants and or repellents. The pesticidal polypeptides may also be administered with other insect control agents, such as chemosterilants.

[0095] Where the CAI compositions are formulated to be orally administered to the insect pests, they can be administered alone or in association with an insect food. The CAI composition are preferably so associated with the food that it is not possible for the insect to feed on the food without ingesting the CAI composition. Preferred foods for mosquito larvae are algae (particularly green, unicellular) and yeast. The food may comprise live organisms or killed organisms. The CAI composition may also be mixed with an attractant to form a bait that will be sought out by the pest. Further, the CAI composition may be applied as a systemic poison that is absorbed and distributed through the tissues of a plant or animal host, such that an insect feeding thereon will obtain an insecticidally effective dose of the pesticidal polypeptide.

[0096] The CAIs may also be formulated as contact pesticides which penetrate the insect cuticle or enter through the spiracles of the respiratory system. In one aspect, the CAIs are formulated and applied as sprays, preferably using water as the principal carrier, although volatile oils may also be used. [0097] CAIs may be encapsulated, included in a granular form, solubilized in water or other appropriate solvent, powdered, and included in any appropriate formulation for direct application to the pest.

[0098] CAIs may be used either alone or in combination with other active or inactive substances and may be applied by any method known in the art including, for example, spraying, pouring, dipping, in the form of concentrated liquids, solutions, suspensions, sprays, powders, pellets, briquettes, bricks and the like, formulated to deliver a pesticidally effective concentration of a CAI composition. The pesticidal formulations may be applied in a pesticidally effective amount to an area of pest infestation or an area susceptible to infestation, a body of water or container, a bam, a caφet, pet bedding, an animal, clothing, skin, and the like.

[0099] For some applications the CAIs are bound to a solid support for application in powder form or in a "trap". As an example, for applications where the composition is to be used in a trap or as bait for a particular pest, the compositions of the present invention can be bound to a solid support or encapsulated in a time-release material. Examples of delivery systems include starch-dextran, and the like. See Yuan et al., Fundamental and Applied Toxicology (1993) 20: 83-87, for examples of delivery systems.

[00100] In all formulations described herein, materials which can lead to reduction in the pesticidal effectiveness of the CAI composition should be avoided but may be employed in appropriate circumstances where such materials do not entirely eliminate the pesticidal properties of the CAI composition.

[00101] The pesticidal compositions may also include various pesticidally acceptable adjuvants known in the art. The term "adjuvant" is used herein to mean a substance added to a composition to aid the operation of the main ingredient. The adjuvants are pesticidally acceptable in that they do not completely diminish the pesticidal properties of the CAI composition. Spray adjuvants are commonly employed in the application of agricultural chemicals. An effective spray adjuvant may be formulated to contain one or more surfactants, solvents or co-solvents.

[00102] Formulated CAIs can also be applied as a seed-coating or root treatment or total plant treatment at later stages of the crop cycle. Plant and soil treatments may be employed as wettable powders, granules or dusts, by mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the like). The formulations may include spreader-sticker adjuvants, stabilizing agents, other pesticidal additives, or surfactants. Liquid formulations may be aqueous-based or non-aqueous and employed as foams, gels, suspensions, emulsifiable concentrates, or the like. The ingredients may include rheological agents, surfactants, emulsifiers, dispersants, or polymers. [00103] CAIs according to the instant invention can be utilized, in the form of the usual compositions or compositions with conventional inert (e.g., plant and/or animal compatible or herbicidally mammacidally inert) pesticide diluents or extenders, i.e. diluents, carriers or extenders of the type usable in conventional pesticide compositions or compositions, e.g. conventional pesticide dispersible carrier vehicles such as gases, solutions, emulsions, suspensions, emulsifiable concentrates, spray powders, pastes, gels, soluble powders, dusting agents, granules, etc. These are prepared, for example, by extending the pesticidal polypeptides with conventional pesticide dispersible liquid diluent carriers and/or dispersible solid carriers optionally with the use of carrier vehicle assistants, e.g. conventional pesticide surface-active agents, including emulsifying agents and/or dispersing agents, whereby, for example, in the case where water is used as diluent, organic solvents may be added as auxiliary solvents. The following may be chiefly considered for use as conventional carrier vehicles for this puφose: aerosol propellants which are gaseous at normal temperatures and pressures, such as freon; inert dispersible liquid diluent carriers, including inert organic solvents, such as aromatic hydrocarbons (e.g. benzene, toluene, xylene, alkyl naphthalenes, etc.); halogenated, especially chlorinated, aromatic hydrocarbons (e.g. chlorobenzenes, etc.); cycloalkanes (e.g. cyclohexane, etc.); paraffins (e.g. petroleum or mineral oil fractions); chlorinated aliphatic hydrocarbons (e.g. methylene cliloride, chloroethylenes, etc.); alcohols (e.g. methanol, ethanol, propanol, butanol, glycol, etc.), as well as ethers and esters thereof (e.g. glycol monomethyl ether, etc.), amines (e.g. ethanolamine, etc.), amides (e.g. dimethyl formamide, etc.), sulfoxides (e.g. dimethyl sulfoxide, etc.), acetonitrile, ketones (e.g. acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, etc.), and/or water; as well as inert dispersible finely divided solid carriers, such as ground natural minerals (e.g. kaolins, clays, alumina, silica, chalk, i.e. calcium carbonate, talc, attapulgite, montmorillonite, kieselguhr, etc.) and ground synthetic minerals (e.g. highly dispersed silicic acid, silicates, e.g. alkali silicates, etc.); whereas the following may be chiefly considered for use as conventional carrier vehicle assistants, e.g. surface-active agents, for this puφose: emulsifying agents, such as non-ionic and or anionic emulsifying agents (e.g. polyethylene oxide esters of fatty acids, polyethylene oxide ethers of fatty alcohols, alkyl sulfates, alkyl sulfonates, aryl sulfonates, etc., and especially alkyl arylpolyglycol ethers, magnesium stearate, sodium oleate, etc.); and/or dispersing agents, such as lignin, sulfite waste liquors, methyl cellulose, etc. The pesticidal polypeptides may also be encapsulated in a liposomal composition (Belles et al in Pesticide Biochem. Physiol. 32, 1-10 (1988)). Esters, such as succinate ester or citrate esters, can be employed to control the buoyancy of the composition.

[00104] CAI compositions of the present invention can be delivered to the environment using a variety of devices known in the art of pesticide administration; particularly preferred devices are those which permit continuous extended or pulsed extended delivery of the pesticidal composition. For example, U.S. Pat. No. 5,417,682 discloses a fluid-imbibing dispensing device for the immediate, or almost immediate, and extended delivery of an active agent over a prolonged period of time together with the initially delayed pulse delivery of an active agent to a fluid environment of use. [00105] Other dispensing means useful for dispensing the pesticidal compositions of the present invention include, for example, osmotic dispensing devices which employ an expansion means to deliver an agent to an environment of use over a period of hours, weeks, days or months. The expansion means absorbs liquid, expands, and acts to drive out beneficial agent composition from the interior of the device in a controlled, usually constant manner. An osmotic expansion device can be used to controllably, usually relatively slowly and over a period of time, deliver the pesticidal compositions of the present invention. In one aspect, the invention provides a method for using such a device to deliver the pesticidal compositions of the present invention. In one aspect, the osmotic expansion device floats on water and delivers CAI to the surface of the water.

[00106] The compositions of the present invention may also be employed as time-release compositions, particularly for applications to animals, or areas that are subject to reinfestation, such as mosquito-infested ponds or animal quarters. Various time-release formulations are known in the art. Common analytical chemical techniques are used to determine and optimize the rate of release to ensure the delivery of a pesticidally effective concentration of the CAIs. The amount of the time-release composition necessary to achieve a pesticidally effective concentration of pesticide in the environment where the pesticide is applied, e.g., a body of water, is based on the rate of release of the time-release formulation. In one aspect, the time-release formulations may be formulated to float on top of the water. In another aspect, the formulation may be formulated to rest on the bottom, or below the surface of the body of water, and to gradually release small particles which themselves float to the surface, thereby delivering the pesticidal composition to the niche of the pest, e.g., mosquito larvae. [00107] Delayed or continuous release can also be accomplished by coating the CAI composition with a dissolvable or bioerodable coating layer, such as gelatin, which coating dissolves or erodes in the environment of use, such as in a pond, to then make the pesticidal polypeptide available, or by dispersing the peptides in a dissolvable or erodable matrix.

[00108] Such continuous release and/or dispensing means devices may be advantageously employed in a method of the present invention to consistently maintain a pesticidally effective concentration of CAIs in a specific pest habitat, such as a pond or other mosquito-producing body of water. In a preferred mode, the continuous release compositions are formulated by means known in the art, such that they can float on a body of water, thereby delivering the CAI composition to the surface layer of the water inhabited by insect larvae.

[00109] As would be appreciated by a person skilled in the art, the pesticidal concentration will vary widely depending upon the nature of the particular foπnulation, particularly whether it is a concentrate or to be used directly. The pesticide will be present in at least about 0.0001% by weight and may be 100% by weight. The dry formulations will have from about 1-95% by weight of the pesticide while the liquid foπnulations will generally be from about 0.0001-60% by weight of the solids in the liquid phase.

2. Methods for Controlling Insect Populations in a Geographical Locus [00110] The CAI compositions of the present invention may be used advantageously to control an insect population of a specific geographical area. The specific geographical area can be as large as a state or a county and is preferably 1/2 to 10 square miles, more preferably one square mile, and more preferably 1/2 to one square miles, and may also be much smaller, such as 100-200 square yards, or may simply include the environment surrounding and/or inside an ordinary building, such as a barn or house. [00111] In general, the CAIs or compositions containing one or more of the CAIs are introduced to an area of infestation. For example, the composition can be sprayed on as a wet or dry composition on the surface of organic material infested with a target pest, or organic material or habitat susceptible to infestation with a target pest. Alternately, the composition can be applied wet or dry to an area of infestation where it can come into contact with the target pest. The CAI composition also be applied to an area of larvae development, for example, an agricultural area or a body of water such as a pond, rice paddy, watering hole or even a small puddle. [00112] The methods of the present invention are carried out by exposing a target pest populatiori to a pesticidally effective amount of a CAI and thereby decrease or eliminate the population of that pest in an area. The method of introduction of the CAI into the target pest can be by direct ingestion by the target pest from a trap, or by feeding of a target pest on nutrient-providing organic matter treated with the CAI, (e.g., killed yeast or algae in the case of mosquito larvae). In some instances, the pesticide may be absorbed by the pest, particularly where the composition provides for uptake by the outer tissues of the pest, particularly a larval or other pre-adult form of the pest, such as a detergent composition. [00113] The method of use of the CAIs and compositions will depend at least in part upon the pest to be treated and its feeding habits, as well as breeding and nesting habits.

[00114] The CAIs may be employed alone or in mixtures with one another and/or with such solid and/or liquid dispersible carrier vehicles as described herein or as otherwise known in the art, and/or with other known compatible active agents, including, for example, insecticides, acaricides, rodenticides, fungicides, bactericides, nematocides, herbicides, fertilizers, growth-regulating agents, etc., if desired, in the form of particular dosage preparations for specific application made therefrom, such as solutions, emulsions, suspensions, powders, pastes, and granules as described herein or as otherwise known in the art which are thus ready for use. For example, a dosage form for a pond environment may be provided in the form of time releasable bricks, briquettes, pellets, powders, liquids, and the like, comprising at least one pesticidal polypeptide according to the present invention and at least one other active ingredient selected from the group consisting of insecticides, acaricides, rodenticides, fungicides, bactericides, nematocides, herbicides, fertilizers, and growth-regulating agents, for administration to the pond. [00115] Various polypeptide fungicides useful in the methods and compositions of the present invention are disclosed in U.S. Pat. No. 5,872,152, entitled "Use of MMP inhibitors," issued to Brown, et al.; U.S. Pat. No. 5,861,478 , entitled "Lytic peptides," issued to Jaynes, et al.; U.S. Pat. No. 5,804,588, entitled "Quinoline Carboxanides and their Therapeutic Use" issued to Dyke, et al.; U.S. Pat. No. 5,773,467, entitled "Benzofuran Sulphonamides," issued to Dyke, et al.; U.S. Pat. No. 5,773,413, entitled "Method of Combating Mammalian Neoplasias, and Lytic Peptides Therefor," issued to Jaynes, et al.; U.S. Pat. No. 5,744,445, entitled "Method of Treating Pulmonary Disease States With Non-Naturally Occuring Amphipathic Peptides," issued to Jaynes, et al.; U.S. Pat. No. 5,717,064, entitled "Methylated Lysine-Rich Lytic Peptides and Method Of Making Same by Reductive Alkylation," issued to Jaynes, et al; U.S. Pat. No. 5,597,946, entitled "Method for Introduction of Disease and Pest Resistance Into Plants and Novel Genes Incoφorated Into Plants Which Code Therefor," issued to Jaynes, et al.; and U.S. Pat. No. 5,597,945, entitled "Plants Genetically Enhanced for Disease Resistance," issued to Jaynes, et al. G. Control of Parasites through Host Administration

[00116] For some applications it will be advantageous to deliver the CAI composition to the location of the pest colony. In other applications, it will be preferable to apply the CAI composition to a prey or host of the pest, such as a human or other animal. In a preferred embodiment CAIs are administered to humans known to be infected with plasmodium. When pests, such as mosquitoes, have a blood meal of an infected but treated individual, the mosquito ingests the CAI. Preferably, a medicament containing the compound is administered orally, although administration by other methods, such as, but not limited to, topical, parenteral, intravenous (IV) and local administration may be tolerated in some instances. In agricultural areas, non-toxic amounts of CAIs may be fed to livestock. Carbonic anhydrase is localized in the midgut of adult mosquitoes. The highest amount of enzymatic activity is localized in the posterior midgut of the female adult mosquitoes. Carbonic anhydrase catalyzes the hydration of carbon dioxide to produce bicarbonate. It also catalyzes the reverse reaction, the dehydration of bicarbonate to produce carbon dioxide. Malaria parasites such as Plasmodium falciparum and Plasmodium cynomolgi require bicarbonate for their development inside the female mosquito midgut. The parasites develop preferentially in the posterior midgut which also contains the highest amount of carbonic anhydrase.

[00117] Malaria parasites undergo several stages throughout their development in the mosquito midgut. The parasites enter the midgut after the female ingests a blood meal from an infected host. The blood meal contains Plasmodium male and female gametocytes. The male gametocytes undergo a process known as exflagellation and after this process, the male gametocytes fertilize the female gametocytes and the resulting ookinetes migrate towards the midgut epithelium. After crossing this epithelium, the ookinetes develop into oocysts that remain attached to the midgut wall facing the hemolymph on the basal side. After rupture of the oocyst, sporozoites are relased into the hemolymph where they migrate to the salivary glands. When the female takes a second blood meal, it injects the sporozoites into the new host, starting the cycle again.

[00118] Exflagellation of the male gametocytes is one of the first steps inside the mosquito midgut. It has been discovered that in order for this process to occur

Introduction

[00119] The larval mosquito midgut is divided into three main regions: gastric caeca, anterior and posterior stomach (also called anterior and posterior midgut). The pH inside the midgut varies depending on the species but it is maintained within the 10.5-11.0 range in the anterior midgut (Clements, 1992). In Ae. aegypti maintenance of this pH has been attributed to a plasma membrane H* V-ATPase (Zhuang et al., 1999; Boudko et al, 2001a) and a high concentration of bicarbonate/carbonate ions (58.1 mM) present in the lumen of the anterior midgut. In contrast, in the posterior midgut lumen and the hemolymph bicarbonate/carbonate levels are close to 4.0 mM (Boudko et al., 2001a). Additional evidence of the involvement of bicarbonate and more specifically of the role of CA in this mechanism was provided by time-lapse video assays of pH profiles in vivo. The assays revealed that ingestion of acetazolamide (a CA specific inhibitor) at 10^"4M eliminates lumen alkalization (Boudko et al., 2001a). It has been previously shown, using Hansson's histochemical staining and in situ hybridization, that CA activity and mRNA are localized preferentially in the posterior midgut and gastric caeca of Ae. aegypti larvae and that inhibition of the pH gradient within the midgut can be accomplished with acetazolamide and another CA inhibitor, methazolamide (Corena et al., 2002). Furthermore, the enzyme has been cloned from the midgut of Ae. aegypti (Corena et al., 2002). These results combined suggest that the presence of CA is crucial to maintaining alkalization in the midgut of mosquito larvae. In the present study, the inventors have localized CA in the midgut of different species of mosquito larvae from salt marsh inhabitants to container breeders. Additionally, the inventors have quantitated CA activity in the gastric caeca, anterior and posterior midgut using ¹⁸0-isotope exchange coupled to mass spectrometry. This method is based on the original method developed by Mills and Urey where the velocity constants of the hydration of C0₂ and the dehydration of carbonic acid were determined by observing the redistribution of ^lsO from ¹⁸0- labeled bicarbonate between bicarbonate, C0₂ and water (Mills and Urey, 1940). The ¹⁸0-exchange reaction reaches equilibrium nearly two orders of magnitude more slowly than the reaction between water, C0₂, bicarbonate and protons. Therefore, it is possible to monitor the slow time course of the approach to isotopic equilibrium of ¹⁸0 between bicarbonate, C0₂ and water. Due to the large amount of water in comparison to the Cθ₂~bicarbonate system, almost all of the original ¹⁸0 introduced in the form of labeled bicarbonate gets incoφorated in the water. The loss of ¹⁸0 in the water causes a decay of ¹⁸0- labeled C0₂ (producing a gas peak (m = 46)) detectable by mass spectrometry. Such decay is slow in the absence of CA.

[00120] The technique developed by Mills and Urey was adapted later by Itada and Forster to measure intracellular CA activity in a cell suspension (Itada and Forster, 1977). The result was a theoretical expression that allowed calculation of CA activity in intact red blood cells. They also demonstrated that addition of intact cells containing CA to the reaction mixture produced a double- exponential disappearance curve for the m 46 peak ¹⁸0-labeled C0₂. The inventors have used this method with its adaptations to determine CA content in the midgut of mosquito larvae.

[00121] In addition, the inventors used CA specific inhibitors (methazolamide, acetazolamide and dorzolamide) to further study the role of CA in the physiology of the larval midgut. The inhibitors were added to the larval rearing media and the effects on the alkalization of the midgut were recorded. Additionally, the inventors determined LC₅₀ and LC₉₀ values for each compound and each species. [00122] Methazolamide (N-(4-Methyl-2-sulfamoyl-Δ²-l,3,4-tlιiadiazolin-5-ylidene) acetamide), acetazolamide (N-(5-[Aminosulfonyl]-l,3,4-thiadiazol-2-yl) and dorzolamide hydrochloride ((4S-^ra«^)- 4-(ethylanuno)-5,6-dihydro-6-methyl-4H-thieno[2,3-έ]thiopyran-2-sulfonamide 7,7-dioxide monohydrochlori.de) are sulfonamides primarily used to control intraocular pressure in the treatment of glaucoma in humans. These drugs reduce the transport of ions and decrease aqueous secretion through a local osmotic effect most likely due to their interaction with CA (Parasrampuria and Gupta, 1989). Although there are multiple reports on the clinical use of methazolamide and acetazolamide in vertebrates and invertebrates, including their use to study ion transport and acid-base regulation, this is the first comparative report on the use of CA inhibitors to study their effect in the physiology of the midgut in different mosquito larvae species.

[00123] Sulfonamides such as benzenesulfonamide and /j-amino benzenesulfonamide

(sulfanilamide) have been used in the past to determine larvicidal activity (Beesley, 1971; Beesley, 1973). CA inhibitors such as methazolamide and acetazolamide are sulfonamides which have been chemically modified but still possess the sulfonamide group. The inventors are presenting here the effect of these CA inhibitors on mosquito larval physiology with the prospect to contribute in the future, to the development of novel safe larvicides that would target specifically mosquito larvae.

Materials and methods

Mosquito species

[00124] Mosquito larvae from Ae. aegypti , C. quinquefasciatus, Cx. nigripalpus, O. taeniorhynchus, Ae. albopictus and An. quadrimaculatus were raised either from eggs obtained from colonies maintained either at the United States Department of Agriculture (USD A, Gainesville, FL) or at

Florida Medical Entomology Laboratory (FMEL, Vero Beach, Florida).

[00125] Larvae were reared in distilled water except Oc. taeniorhynchus which were reared in either 50% or 2% artificial seawater. Species were reared at 25 ±1°C under a 12 h light/dark cycle.

Different instars were separated by size and by following the number of hours post-hatching.

Solutions and test formulations

Artificial sea water (100%) was prepared fresh every time . Final concentrations were 411.04 mM NaCl, 9.94 mM KC1, 10.25 mM CaCl₂, 53.6 mM MgCl₂, 28.24 mM Na₂S0₄ ; the pH was adjusted to 8.1-8.3 with NaOH. This stock solution was diluted to prepare 50% and 2% artificial seawater culture medium. [00126] Hemolymph substitute solution (HSS) was prepared according to Clark et al. (1999). The solution consisted of 42.5 mM NaCl, 3 mM KC1, 0.6 mM MgS0₄, 5 mM CaCl₂, 5 mM NaHC0₃, 5 mM L-succinic acid, 5 mM L-malic acid, 5 mM L-proline, 9.1 mM L-glutamine, 8.7 mM L-histidine, 3.3 mM L-arginine, 10 mM dextrose, and 25 mM Hepes pH 7.0 adjusted with NaOH.

[00127] Methazolamide, acetazolamide, -cresol puφle, bromothymol blue, dimethylsulfoxide

(DMSO) and protease inhibitor cocktail (4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), pepstatin A, E-64, bestatin, leupeptin and aprotinin) were obtained from Sigma-Aldrich Coip., St. Louis, MO, USA. Dorzolamide was obtained from Merck & Co. Inc., Rahway, NJ, USA. Stock solutions of methazolamide, acetazolamide and dorzolamide were prepared in DMSO (10^"2 M or 10^"3 M) were diluted in distilled water. Bromothymol blue and m-cresol puφle stock solutions (0.2%) were prepared in DMSO or water. Aliquots of 100 μL of these stocks were added per mL of rearing medium to achieve 0.02%. CA histochemistry

[00128] Histochemistry was performed using a modified version of the method described by

Hansson (1967). Ten to fifteen larval midguts were dissected in HSS and incubated overnight at 4°C in 3% glutaraldehyde in 0.1M sodium phosphate buffer pH 7.3 in Sylgard coated dishes. On the following day, the midguts were rinsed three times with 0.1M sodium phosphate buffer (pH 7.3) followed by a five minute incubation in a solution made by combining 17 mL of solution A (1 mL 0.1M solution of C0SO₄ mixed with 6 mL 0.5M H₂S0₄ and 10 mL 0.066M KH₂P0₄) with 40 mL of solution B (0.75 g of NaHC0₃ in 40 mL distilled water). After incubation, the guts were rinsed again in sodium phosphate buffer and incubated in 0.5% (NH₄)₂S for two min followed by a rinse with distilled water. The midguts were placed on depression slides and digitally imaged using a Zeiss Axiovert 135 TV inverted microscope (Carl Zeiss Inc., Themwood, NY, USA) equipped with a CCD camera. Darkening was inhibited by pre-incubating living larvae in 10^"4M methazolamide for two hours prior to dissecting the midguts.

¹⁸O isotope exchange

[00129] Forty five to 55 mosquito larvae from each species were cold anesthetized in HSS. The larvae were pinned by the head to Sylgard coated dishes and the midguts were removed in ice-cold HSS. The isolated midguts were rinsed with HSS. For each preparation, the Malpighian tubules and hind gut were removed. The midguts were cut into three different sections: gastric caeca, anterior midgut and posterior midgut. Each one of the gut sections was placed in either 100 or 150 μL of 0.1 M ice-cold Hepes (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer (pH= 7.6) containing protease inhibitors (1:1 ,000). The tissue was then sonicated in this solution using a Heat Systems-Ultrasonics, Inc. sonicator (W-220) equipped with a microtip. The resulting homogenate was centrifuged at 12,000 g at 4 °C for 1 min. The supernatant was used to measure CA activity using the ¹⁸0 exchange method. Individual measurements of CA activity were performed in duplicates with tissue homogenates corresponding to gastric caeca, anterior midgut and posterior midgut. The reaction medium was 10 mM ¹⁸0-labeled NaHC0₃ in 0.1M Hepes buffer, pH 7.6, at 9.5°C. Each experiment was initiated by placing 950 μL or 925 μL of this solution in a membrane inlet vessel and allowing it to reach chemical equilibrium during 1-2 min. At this time, the uncatalyzed ¹⁸0-exchange rate was measured. The disappearance of ¹⁸0 isotopes from C0₂ and or bicarbonate upon addition of the enzyme preparations was monitored by mass spectrometry using a gas permeable probe. Enzyme preparations contained 50 or 75 μL of mosquito midgut homogenate added sequentially while the temperature and pH were maintained constant. Mosquito homogenate activity was calculated by comparison with the activity of a mutant form of human CA-II and expressed as equivalents of human CA-II (Silverman and Tu, 1986). Samples containing CA activity were inhibited by addition of 10 μM methazolamide to confirm that the activity measured was due to CA (Corena, et al., 2002). [00130] Since the peritrophic membrane containing the food bolus was not removed for these measurements, a control containing only food was tested for CA activity. The inventors did not detect any CA activity in the food.

Total protein

[00131] Total protein was determined using Coomassie Plus Protein Assay Reagent Kit (Pierce

Biotechnology, Rockford, IL, USA) following a modification of Bradford's method (Bradford, 1976).

Bovine Serum Albumin was used as reference protein. Tissue homogenates of gastric caeca, anterior and posterior midgut (50 μL) were diluted appropriately in distilled water according to the manufacturer's instructions.

Effect of CA inhibitors on the pH of the rearing medium

[00132] Rearing medium pH was monitored using bromothymol blue as described (Corena et al.,

2002). Culture plate wells were used to rear five mosquito larvae from each species. Each plate contained 24 wells arranged in 4 rows and 6 columns. Each well was filled with ImL of distilled water. Rows from top to bottom contained larvae from first, second, third and fourth instars respectively. A replicate plate was also set up for each species by replacing the distilled water in the wells with 2% artificial seawater. Plates containing Oc. taeniorhynchus larvae were filled with 2% seawater only. 100 μL of 0.2% bromothymol blue in DMSO were added to each well and larvae were allowed to acclimate for 3 h at room temperature. Following acclimation, each plate was treated with CA inhibitors. The first three columns on each plate from left to right were treated with acetazolamide (in DMSO) to achieve 10^"6, 10^"5 and 10^"4M final concentration. The three columns on the right were treated with methazolamide at the same concentrations. After 24 h, the plates were scanned using an Epson Perfection 1640SU scanner. In addition, the pH of the medium was recorded before addition of the inhibitors and 24 h post-treatment. Controls included 5 larvae (for each instar) placed in ImL of distilled or sea water containing 0.02% of bromothymol blue. Treatments and controls were adjusted to 0.01% DMSO. The controls did not contain inhibitors. Measurements of pH reported in this paper are the mean average of three pH readings.

Effect of CA inhibitors on midgut pH

[00133] Groups of 20 early fourth instar larvae were placed in distilled water or 2% seawater containing 0.02%> m-cresol puφle in DMSO. The larvae were allowed to acclimate for 30 min to one h and then dissected in HSS on depression slides and digitally imaged using a Zeiss Axiovert 135 TV inverted microscope (Carl Zeiss Inc., Thernwood, NY, USA) equipped with a CCD camera. To test the effect of CA inhibitors in the midgut, methazolamide at 10^"4 M final concentration was added to the medium. Twenty early fourth instar larvae from each species were used as controls and left untreated. Larvae were dissected 2 h after addition of the inhibitor and digitally imaged. Effect of CA inhibitors on the vitality of different species of mosquito larvae [00134] The procedure for the bioassays was adapted from Mulla et al. (1966) and Mulla and

Khasawinah (1969). Twenty early first, second, third and fourth instar larvae were placed in a 120 mL disposable cup containing 100 mL of distilled water, 50% or 2% artificial sea water depending on the species. Five concentrations (10^"3, 10^"4, 10^"5, 10^"6 and 10^"7M) of dorzolamide, methazolamide and acetazolamide were tested. Stock solutions were prepared in DMSO. Controls contained DMSO only. Treatments and controls were replicated two or three times. Larval mortality was recorded 24 and 48 h post-treatment. LC₅₀ and LC₉₀ values were determined by log-dose probit regression analysis (U.S. Environmental Protection Agency, 1994). Control mortality, if any, was corrected for using Abbott's formula (WHO, 1963).

Results

CA histochemistry of different species of mosquito larvae

[00135] Using Hansson' s histochemical method, a black precipitate is produced at the site of CA enzymatic activity. Results varied depending on the species, therefore they are presented separately. An. quadrimaculatus and Cx. quinquefasciatus larvae exhibited intense darkening of the anterior midgut and the gastric caeca (Fig. 1 panels A and B). In le. albopictus larvae, darkening was observed as two rings around the middle and the posterior portions of the gut with little darkening of the gastric caeca (Fig. 1 panel C). Oc. taeniorhynchus larvae exhibited intense darkening in the middle of the midgut. The gastric caeca in this species did not exhibit intense darkening (Fig. 1 panel D). In Cx. nigripalpus larvae, darkening of the posterior midgut and gastric caeca was observed in a pattern similar to that observed in Ae. aegypti (Fig. 1 panel E). Results of methazolamide inhibition for Cx. nigripalpus larvae are shown in figure 1 panel F. Similar results were obtained for all species (results not shown).

Total protein

[00136] Total soluble protein for tissue homogenates from gastric caeca, anterior and posterior midgut was calculated for each species after homogenization and centrifugation of the tissue to remove food and insoluble material. These values were corrected for the amount of protease inhibitors added to each individual sample (Table 1). Sincere, aegypti is one of the models used to study mosquito physiology, the inventors have included data obtained previously fromAe. aegypti larvae (Corena et al., 2002) for comparison pvuposes. Although the inventors did not calculate the amount of total protein per larva, the majority of the species tested exhibited the highest soluble protein content in gastric caeca homogenates. Since the larvae vary in size depending on the species, a comparison of protein content from one portion of the gut in one species to the same portion in another species is imprecise. [00137] The highest amount of total soluble protein was found in gastric caeca, anterior and posterior midgut tissue homogenates of O. taeniorynchus larvae. The gastric caeca exhibited a value close to 4 μg of total soluble protein per μL of homogenate. The inventors also observed high values in tissue homogenates of An. quadrimaculatus and Cx. quinquefasciatus (Table 1).

¹⁸O isotope exchange

[00138] The inventors define CA content as the percentage obtained by calculating μg of CA in tissue homogenate (expressed as equivalents of the recombinant human CA-II) per μg of total soluble protein present in the homogenate (Fig. 2). The inventors found that all species exhibited CA activity in the gastric caeca. The highest enzymatic activity in gastric caeca homogenates appeared to be associated with three of the species: C. nigripalpus, An. quadrimaculatus and Ae. albopictus. All other species displayed lower levels.

[00139] The inventors observed species differences in the distribution of CA in the posterior and anterior midgut: three species exhibited significant activity in the posterior midgut. The highest value was observed in Ae. albopictus followed by C. quinquefasciatus and Cx. nigripalpus. The inventors have detected previously low levels of CA activity in^e. aegypti posterior midgut homogenates (Corena et al., 2002) (included in Fig. 2). The inventors were unable to detect CA activity in posterior midgut homogenates from An. quadrimaculatus and An. αlbimαnus. Both species exhibited the highest CA percentage in the anterior midgut when compared to other species. Additionally, Cx. quinquefasciatus and Ae. albopictus larvae also exhibited CA activity in the anterior midgut. The inventors did not detect CA activity in the anterior midgut of Cx. nigripalpus. The inventors were also unable to detect enzymatic activity in the anterior midgut of Ae. aegypti in their previous studies (Corena et al., 2002).

[00140] In summary, the highest values for CA content in larval midgut tissue homogenates were found in the anterior midgut of An. quadrimaculatus (0.27%) followed by the gastric caeca of Cx. nigripalpus (0.18%) and the gastric caeca of An. quadrimaculatus (0.15%). The inventors were unable to detect CA activity in the posterior midgut of both An. albimanus and An. quadrimaculatus and the anterior midgut of Cx. nigripalpus and _^4e. aegypti using this method.

[00141] The inventors observed inhibition of activity when 10 μM methazolamide was added.

Fig. 3 illustrates the results the inventors obtained when An. albimanus anterior midgut CA was inhibited by methazolamide. As seen in this figure, the baseline was left to stabilize for approximately 150 sec to allow the solution to reach chemical equilibrium. The straight line in this portion of the graph represents no enzymatic activity. At 150 sec, tissue homogenate from the anterior midgut of An. albimanus was added. The decrease in the curve represents the depletion of ¹⁸0 in the C0₂ that reaches the enzyme's active site indicating enzymatic activity. The reaction was allowed to proceed and at approximately 400 sec, addition of methazolamide caused inhibition of the reaction which is reflected by the return of the curve to a straight line (slower uncatalyzed rate). CA activity in all tissue samples was inhibited by methazolamide in a similar way to that shown in Fig. 3.

Effect of C A inhibitors on the pH of the rearing medium

[00142] Mosquito larvae excrete bicarbonate, which in turn, leads to alkalization of their surrounding aqueous medium (Stobbart, 1971). It has been reported previously that the pH of the rearing medium decreased over time when CA inhibitors were added to the medium containing fourth instar Ae. aegypti larvae (Corena et al., 2002). To determine if these inhibitors had a similar effect on the alkalization of the rearing medium for other species of mosquito larvae, the inventors added acetazolamide or methazolamide to larval cultures of the species mentioned above. Bromothymol blue was added to visually monitor changes in pH. The color of this indicator changes gradually from yellow (around pH 6.0) to yellow-green followed by green-blue as the pH increases, to blue at pH values around 7.6 or higher. Although inaccurate, the indicator offers a simple and convenient method to monitor changes of pH in the rearing medium. The inventors used microelectrodes to obtain more accurate pH measurements. In the absence of inhibitor the color of the rearing medium gradually changed from yellow to blue in as little as 3 h for most species. After 24 h, all wells containing larvae in the absence of inhibitor appeared blue suggesting a pH around 7.6 or above. Microelectrodes showed that the pH was in the range of 7.4 (for second instar larvae) to 7.6 (for third and fourth instar larvae). [00143] In the presence of inhibitor, first and second instar larvae showed a considerable decrease in the pH of the medium 24 h after addition of methazolamide or acetazolamide (Fig. 4). DMSO at the concentration used had no significant effect on the alkalization of the media. The strongest effect on pH was observed when 10^"3 M methazolamide was used. However, at this high concentration, precipitation of methazolamide was observed as well as rapid larval mortality for some species; therefore, the inventors set the upper limit in concentration at 10^"4M.

[00144] After 24 h, the pH of the medium containing 10^"4M methazolamide was lower than in the controls. In the majority of the species, the difference was as much as 0.5 pH units for first instar larvae. Some species were more susceptible to treatment with these inhibitors than others (Figure 4). When Oc. taeniorhynchus and An. quadrimaculatus larvae were treated, the pH decreased by as much as 0.4 units in the rearing medium containing first and second instars (Fig. 4 panels B and F). This effect was less pronounced in first and second instar larvae of Ae. albopictus, Cx. nigripalpus and Cx. quinquefasciatus (Fig. 4 panels C,D, and E).

[00145] Figure 4 panel G shows a control plate containing untreated Ae. aegypti larvae, and after

24 h all wells exhibited a pH in the vicinity of 7.6. At concentrations below 10^"4M methazolamide, the pH of the rearing medium for most species remained the same or was not lower over 24 h for third and fourth instars. However, unlike the other species, the pH of the medium containing Cx. nigripalpus third and fourth instar larvae increased 0.4 and 0.3 pH units in the presence of 10^"5 and 10^"4 M inhibitor, respectively. This change in pH was detected with microelectrodes and it was not apparent when using bromothymol blue, since this indicator remains blue above pH 7.6 . It has not been deteixnined if the change in pH observed for third and fourth instars of Cx. nigripalpus is in response to a change during development.

Effect of inhibitors on midgut pH

[00146] Midguts were dissected from early fourth instar larvae acclimated in distilled water containing m-cresol puφle and then photographed immediately after dissection. All species revealed a similar pattern: a dark puφle anterior midgut indicative of pH 9.0 or higher and an orange/yellow posterior midgut and gastric caeca indicating a pH close to 7.4 (Fig. 5).

[00147] To determine the effect of CA inhibitors in the alkalization of the midgut, the inventors treated early fourth instar larvae with 10^"4 M methazolamide. It has been shown that the alkalization of the anterior midgut in early fourth instar larvae of Ae. aegypti decreases after treatment with 10^" M methazolamide, as indicated by the change of color of the indicator from puφle to yellow (Boudko et al, 2001b). The inventors were able to reproduce the results observed for Ae. aegypti (results not shown). Interestingly, the only species where midgut pH seemed unaffected by the inhibitors was Ae. albopictus. Nevertheless, a closer look at the isolated midguts revealed that the region of lower pH in the posterior midgut was extended further towards the anterior midgut in the larvae treated with methazolamide (Fig. 6 panel A). Although this was a minor effect, it was consistently detected in all twenty larvae dissected for this species. When larvae from other species were treated with 10^" M methazolamide in the presence of m-cresol puφle, the pattern of alkalization of the midgut varied depending on the species (Fig. 7). Cx. nigripalpus larvae were unable to maintain alkalization in the midgut as indicated by the yellow color observed in this region. Interestingly, the posterior midgut appeared more alkaline than the anterior as Fig 7 (panel A) shows a darker-orange color of the posterior compared to a yellow anterior midgut. [00148] Larvae of Cx. quinquefasciatus (Fig. 7 panel B) and Oc. taeniorhynchus (Fig. 7 panel C) were able to maintain some alkalization even after 24 h in the presence of 10^"4M methazolamide. The pattern of alkalization of the midgut An. quadrimaculatus larvae treated with methazolamide did not differ from that of untreated larvae, but these results were unable to be reproduced due to larval availability issues.

[00149] To summarize: of the six species studied, two species (Ae. albopictus and An. quadrimaculatus) were able to maintain alkalization of the anterior midgut even after treatment with methazolamide. In Oc. taeniorhynchus and Cx. quinquefasciatus larvae, the effect of methazolamide in the alkalization of the midgut was more pronounced. In two species (Ae. aegypti and Cx. nigripalpus), the larvae failed to maintain alkalization after treatment with the inhibitor.

Effect of CA inhibitors on the vitality of different species of mosquito larvae [00150] It has been previously observed that CA inhibitors have a lethal effect on Ae. aegypti larvae when administered to the rearing medium. Mortality of this species was higher with methazolamide than acetazolamide and dorzolamide (Corena et al., 2001). In order to determine if these compounds have a lethal effect on larvae from other species of mosquitoes, larvae from each of the species mentioned previously were treated with these inhibitors. While some species were susceptible as demonstrated by larval mortality observed 24 h post-treatment, other species were not affected. Results for methazolamide and acetazolamide are presented with their corresponding LC₅₀ (Table 2) and LC₉₀ values (Table 3). Results for dorzolamide are not shown in these tables but are mentioned below for each species. Due to the differences observed in the effect of each inhibitor, the inventors present a summary of the results separately for each species.

Anopheles quadrimaculatus

[00151] Treatment of An. quadrimaculatus larvae with methazolamide at concentrations higher than 10^"4M resulted in the death of 80% of the larvae after 24 h. In contrast, mortality with dorzolamide and acetazolamide at these concentrations reached only 20%). Addition of CA inhibitors at concentrations lower than 10^"4M resulted in the larvae appearing lethargic and moving slowly. All controls including DMSO were unaffected. The inventors observed that third instar were more susceptible than fourth instar larvae. In general, a comparison of the LC₅₀ values obtained for this species with those obtained for other species of larvae indicates that An. quadrimaculatus larvae are very sensitive to the effect of CA inhibitors.

Culex quinquefasciatus

[00152] The three CA inhibitors tested do not have a strong effect on the vitality of third and fourth instar larvae of this species when added to the rearing medium. Only 20% of third instar larvae were dead after 24 h when treated with 10^"3 M methazolamide. Addition of 10^"3 M acetazolamide resulted in 10% mortality. Dorzolamide and DMSO alone had no effect. Table 2 shows that the LC₅o values for methazolamide and acetazolamide were close to 10^"3M and that the LC₉0 values were identical

(10^{"2 2} M) (Table 3).

Culex nigripalpus

[00153] Third instar larvae were sensitive to methazolamide and dorzolamide. Treatment at 10^"4

M resulted in 70% mortality in 24 h. Although this species was not as sensitive to acetazolamide as to methazolamide and dorzolamide, it seemed sensitive to the presence of DMSO as indicated by the high mortality of the controls. Therefore, it was difficult to calculate LC₅₀ values for this species for methazolamide and LC₉₀ values for acetazolamide. The LC₅₀ value obtained for acetazolamide was 10^"1'2 M corroborating that this species is not very sensitive to this inhibitor (Table 2). Ochlerotatus taeniorhynchus

[00154] This particular species inhabits salt marshes in Florida. Therefore, the effect of CA inhibitors in this species was tested by placing the larvae in 2% and 50% artificial sea water instead of distilled water. Treatment of third instar larvae with methazolamide, acetazolamide and dorzolamide resulted in 100%> mortality after 24 h independent of the concentration used. On the other hand, mortality of early fourth instar larvae reached 50% at 10^" M after 24 h with acetazolamide and methazolamide. Mortality observed upon treatment with 10^"4M dorzolamide was not higher than 20%>. Mortality of the DMSO controls was on average 20%. The LC₅₀ values obtained for O. taenyorhynchus were very similar to those obtained for An. quadrimaculatus indicating that these two species were the most sensitive of all the species tested to the effect of CA inhibitors (Table 2). In addition, third instar larvae of O. taenyorhynchus were the most sensitive exhibiting 100%_> mortality after treatment.

Aedes albopictus

[00155] Third and fourth instar larvae were not affected by any of the CA inhibitors used and therefore the LC₅₀ and LC₉₀ values were not calculated. There was no mortality in the DMSO controls. Furthermore, larvae of this species did not slow down and appeared active and alive in the presence of CA inhibitors.

Aedes aegypti

[00156] Third instar larvae of Ae. aegypti are more susceptible than fourth instar larvae when treated with CA inhibitors. Third instar larval mortality was 100% with methazolamide and acetazolamide at 10^"4M. Mortality of third instar larvae with dorzolamide at the same concentration only reached 22%. Treatment of fourth instar larvae with dorzolamide resulted in no mortality. The inventors did not observe larval mortality in the DMSO controls.

[00157] In summary, some species of larvae are more susceptible to the effects of methazolamide and acetazolamide than others (Tables 2 and 3). In general, higher doses of acetazolamide were necessary to obtain the same mortality as that obtained with lower doses of methazolamide. Interestingly, Ae. albopictus larvae were not susceptible to the inhibitors or to DMSO at any of the concentrations tested. On the contrary, An. quadrimaculatus and Oc. taeniorhynchus seemed more susceptible than any of the other species as reflected by the lower LC₅₀ and LC₉₀ values. Larvae of Cx. nigripalpus were very sensitive to DMSO. The LC₅₀ value for Cx. nigripalpus larvae treated with acetazolamide was higher than for any other species. Accordingly, the mortality observed in Cx. nigripalpus larvae might reflect the combined effect of the inhibitor and DMSO. Discussion

[00158] Mosquito larvae have adapted to different environments. Most species live in fresh water but approximately 5% live in brackish or saline water (Clements, 1992). The fresh water species used in this study were Ae. aegypti, Ae. albopictus, Cx. quinquefasciatus, Cx. nigripalpus sad An. quadrimaculatus. The inventors also used Oc. taeniorhynchus, a species that populates salt marshes and has been found in habitats with extreme pH values ranging from 3.3 to 8.1 (Petersen and Chapman, 1970). The fresh water species chosen develop in different habitats. For example, Ae. aegypti and Ae. albopictus are typically considered container breeders. Cx. quinquefasciatus, Cx. nigripalpus and An. quadrimaculatus are capable of laying eggs in containers as well, but seem to prefer to lay eggs and develop mainly in rain pools. These species also vary in their feeding behavior and the kind of food they ingest. Most species of larvae ingest detritus or "particulate organic matter". However, differences have been found even among species that inhabit the same containers. Cx. quinquefasciatus larvae ingest free- swimming bacteria and algae while Ae. aegypti larvae kept in the same container ingested bacterial film, algae, fungal mycelia, protists, rotifers and the eggs of aquatic worms (Riviere, 1985). [00159] Despite the differences in the environments that these species populate, their differences in feeding modes and food ingested, all these species exhibited a similar alkalization pattern in the midgut and gastric caeca when fed a pH indicator dye (m-cresol puφle). The pH in the gastric caeca has been reported to be in the range of 7.0 to 8.5 depending on the species (Ramsay, 1950; Dadd, 1975; Stiles and Paschke, 1980; Charles and de Barjac, 1981). Observations with pH indicators in the gastric caeca indicate a pH close to 7.4. In all the species that the inventors tested, the pH in the posterior midgut was close to 7.4 and it increased towards the anterior portion of the midgut where the pH was close to 9.0 or higher. Similar observations have been reported previously for different aedine species (Ramsay, 1950; Dadd, 1975; Stiles and Pasclike, 1980; Charles and de Barjac, 1981). The high pH in the anterior midgut has subsequently been associated with a high concentration of bicarbonate/carbonate ions and hence presence of CA (Boudko et al., 2001b, Corena et al., 2002). The inventors have previously cloned CA from the midgut of Ae. aegypti. Furthermore, the inventors have shown that CA is present in the gastric caeca and posterior midgut of Ae. aegypti. Additionally, larvae of this species, treated with methazolamide, exhibit a decrease in the alkalization of the anterior midgut (Corena et al., 2002). These results combined suggest that CA plays an important role in the alkalization mechanism in the midgut of Ae. aegypti larvae. Is CA present in the midgut of other species of mosquitoes? Does it follow the same distribution pattern observed in Ae. aegypti? To address these questions, the inventors determined the localization of the enzyme in the midgut of all the species used in this study by means of Hansson's histochemistry. The inventors then compared these results with those obtained for Ae. aegypti. Contrasting the results obtained previously with Ae. aegypti, larvae of Ae. albopictus, another container breeder, exhibited darkening in two portions of the posterior midgut as evidenced by bands of darkened cells around the midgut. Histochemistry on two species of culicine larvae, Cx. nigripalpus and Cx. quinquefasciatus, one species of anopheline larvae, An. quadrimaculatus and a salt-marsh mosquito, Oc. taeniorhynchus revealed that the pattern of localization of CA is species dependent. Only Cx. nigripalpus larvae exhibited darkening in the posterior midgut and gastric caeca as it was observed iaAe. aegypti. Darkening in the anterior midgut was observed in An. quadrimaculatus and Cx. quinquefasciatus larvae. In Oc. taeniorhynchus larvae localization of CA activity seems to be concentrated towards the middle of the midgut as opposed to the posterior end as observed in Ae. aegypti. Darkening in all species was inhibited by addition of methazolamide to the rearing medium.

[00160] CA localization by Hansson's histochemical method revealed that the enzyme is present in different regions of the midgut according to each particular species. Even species closely related such as Ae. aegypti and Ae. albopictus displayed different distribution of CA in the midgut. The inventors have previously observed a correlation between Hansson's histochemical method and quantitation of CA using the ¹⁸0 isotope exchange method in larval Ae. aegypti midgut (Corena et al., 2002). Therefore, the inventors decided to quantitate CA in midgut tissue homogenates from other species and compare the results with those obtained with histochemistry. Analysis of the results presented in this study revealed that there is in fact a correlation between the quantitative results obtained using the ¹⁸0-isotope exchange method and the qualitative results obtained using Hansson's histochemistry. The only noticeable exception was Cx.quinquefasciatus. Histochemistry of isolated larval midguts for this species showed strong darkening of the anterior midgut while the ^lsO-exchange method resulted in the detection of only low levels of activity in this region of the midgut. Since only soluble (cytosolic) has been determined CA using the ¹⁸0-exchange method, it is then possible that any discrepancy in the results is due to the presence of membrane-bound CA.

[00161] Are the levels of CA observed in this study directly related to the amount of protein present in each one of the midgut regions? The inventors' findings in terms of the total soluble protein content and CA content in the midgut of these species suggest that there is not an apparent relationship between the amount of soluble protein present in the tissue and the amount of cytosolic CA detected. For example, larvae of Oc. taeniorhynchus exhibited the highest total soluble protein content in all three regions of the midgut but displayed some of the lowest values for CA content. This is further demonstrated by the absence of CA activity in the posterior midgut of An. quadrimaculatus where the total protein content was among the highest in this region of the midgut. The results presented in this paper do not provide information related to membrane-bound carbonic anhydrase that could have been lost in the pellet after centrifugation and prior to analysis.

[00162] In terms of CA localization in the different regions of the mosquito larval midgut, it is apparent now that all the species tested exhibit CA activity in the midgut although to various degrees. Localization of CA was consistent in the gastric caeca for all species. Localization in the posterior and anterior midgut on the other hand, appears to be species dependent. The inventors detected CA activity in the anterior midgut for most species with two exceptions: Ae. aegypti and Cx. nigripalpus. In the posterior midgut, the inventors were unable to detect CA activity using this method in two anopheline species, An. albimanus and An. quadrimaculatus.

[00163] Regarding the differences observed among the mosquito species the inventors used, the highest enzymatic activity appeared to be associated with the gastric caeca in three of the species: Cx. nigripalpus, An. quadrimaculatus, and Ae. albopictus. Three species exhibited significant activity in the posterior midgut: Ae. albopictus, Cx. quinquefasciatus and Cx. nigripalpus. It is concluded that CA distribution in the midgut of all the species of mosquito larvae tested in this study appears to be species dependent. Is the localization of CA related to the mechanisms of ion regulation in the midgut for each particular species? A detailed analysis of CA distribution in the midgut and other organs such as Malpighian tubules and rectum for each particular species and its relationship with ion transport mechanisms will be necessary in order to answer this question. A correlation between CA localization and ion transport in the midgut could be made afterwards by using Self-referencing Ion Selective (SERIS) microelectrodes to measure ion fluxes in the midgut (Boudko et al., 2001b). [00164] Does CA play a key role in the alkalization mechanism in the midgut of the species tested? Most mosquito larvae have a remarkable capacity for ionic and osmotic regulation when compared to other organisms in the animal kingdom. Their ability to survive under extreme conditions has been attributed to strong transport processes that take place in the anal papillae as well as the midgut epithelium (Clements, 1992). It has been postulated that the activity of the gut cells in the first three abdominal segments is responsible for the high alkalinity of the lumen and that this region extends toward the posterior midgut by the backward movement of the gut contents as well as the anterior flow of the fluid produced by the Malpighian tubules. Evidence of this anterior flow has been linked to the observation that the gastric caeca contents become acidic when larvae are ligated just behind the thorax (Zhuzhikov and Dubrovin, 1969). In the present study, treatment of larvae from different species with methazolamide affected the pH of the midgut. Although the majority of the species were affected by the presence of inhibitor, the extent of the decrease in pH in the anterior midgut was different among the species. These observations suggest that CA is crucial in the maintenance of pH within the midgut in all the species tested. How does methazolamide affect alkalization of the midgut? It is possible that this compound inhibits the production of bicarbonate, therefore interfering with the ion transport processes that occur in the midgut epithelium ultimately altering the alkalization mechanism. The extent of decrease in pH in the anterior midgut was species dependent suggesting that perhaps the mechanism of ion transport in this region of the midgut is more efficient in some species than in others. CA inhibitors have been shown to have an effect on ion transport processes in other organs besides the midgut of mosquito larvae (Patrick et al., 2002). This study focuses on the effect of CA inhibitors in the midgut but it is possible that by treating the larvae with these compounds, inhibiting the enzyme in other regions of the larvae such as the hindgut, the anal papillae or the Malpighian tubules also may occur. [00165] The inventors treated separately, larvae from different instars of different species with two CA inhibitors. Since the inventors did not measure bicarbonate/carbonate content in the rearing medium for any of the species, the inventors can only speculate about the effect of the inhibitors on bicarbonate excretion. A detailed analysis of the excretion products released into the water by the larvae will lead to a better understanding of the effect of CA inlύbitors on excretion. The inventors observed that when larvae of different instars (of the same species) were treated with methazolamide or acetazolamide the pH of the medium decreased over time. Methazolamide appears to have a stronger effect on first and second instar larvae of most species when compared to acetazolamide. Since the contents of the midgut appear to have a pH close to neutrality at the posterior midgut, it would not make sense if only the products of digestion generated in the midgut were secreted into the medium and were responsible for the observed alkalization. Several studies have shown that the fluid secreted by the larvae is a combination of fluid produced by the Malpighian tubules and the rectum which is in turn supplied with fluid from the posterior midgut. Unfortunately, there is very little information available on the excretion products of mosquito larvae. Studies by Strange and collaborators (Strange et al, 1982) have demonstrated that the rectum is an important site of HC0₃ ^" excretion, as originally suggested by Bradley and Phillips (1977). It has been shown in^e. dorsalis larvae, that the anterior compartment of the rectum is the principal site of bicarbonate and carbonate secretion (Strange and Phillips, 1984) and that addition of acetazolamide reduced the bicarbonate secretion of rectal preparations by as much as 80%. This observed reduction in the secretion of bicarbonate might account for the decrease in pH of the rearing medium when the larvae were treated with CA inhibitors. However, these studies do not indicate if the bicarbonate secreted by the larvae is produced entirely by the rectum alone or if it is a combination of secretions from other organs including possibly, the midgut. Furthermore, most of the studies involving excretion of mosquito larvae have been accomplished with species that live in extreme environments such as very alkaline lakes. Therefore, the amount of information on excretion products generated by larvae that inhabit fresh water habitats such as containers is almost non-existent. In addition, very little is known of the nitrogenous excretory products of mosquito larvae and it is possible that the larvae excrete ammonia. [00166] The inventors did not observe significant differences in the alkalization by mosquito larvae reared in distilled water versus 2%_> sea water. This might indicate that the effectiveness of the inhibitor is not dependent on salt concentration.

[00167] With regards to the strong effect observed on first and second instar larvae when compared to third and fourth instar larvae the inventors can only speculate. Perhaps the effect was more pronounced in the early instars due to the differences in biomass between the different larval instars. First and second instar larvae being smaller probably do not excrete the same amount of bicarbonate that third and fourth instar excrete in a given period of time. Actual measurements of bicarbonate would be necessary to determine if in fact, this is the case. [00168] In order to study whether CA inhibitors are lethal to all species and whether mortality is related to inhibition of CA in the midgut, the inventors determined the LC₅₀ and LC₉₀ values for each species using methazolamide or acetazolamide. It is concluded from these experiments that some species of larvae are more susceptible to the effects of methazolamide and acetazolamide than others. Remarkably, Ae. albopictus larvae were not susceptible to treatment with these compounds. It is also concluded that there is no apparent relationship between the localization of CA and the mortality observed when the larvae were treated with the inhibitors. For instance, Oc. taeniorhynchus and An. quadrimaculatus larvae showed different patterns of distribution but were affected in similar ways by the inhibitors. Cx. nigripalpus larvae appear more sensitive to the effects of CA inhibitors tha Ae. aegypti even though CA localization in the midgut of both species is similar. Interestingly, both species show a similar pattern of change in alkalization when treated with methazolamide. In Ae. albopictus, the pattern of alkalization was similar in the rnethazolamide-treated and the untreated larvae with only minor differences indicating that the inhibitors do not have a substantial effect in the alkalization mechanism of the anterior midgut in this species. Since this species showed no mortality when treated with the inhibitors, it is possible then to hypothesize that Ae. albopictus larvae used in these studies survived because the alkalization mechanism in the midgut was not affected. Consequently, larvae of other species that were susceptible to CA inhibitors exhibited also signs of pH imbalance in the midgut. As a result of certain observations, the inventors believe that inhibition of CA in the midgut resulted in a pH imbalance and inhibition of ion transport processes which led to larval mortality. Did treatment with CA inhibitors affect other metabolic processes such as respiration? The inventors' LC₅₀ values indicate that Oc. taeniorhynchus and An. quadrimaculatus larvae were more sensitive to the effects of methazolamide and acetazolamide than the other species. These two species differ, among other things, in the way they breathe. Anopheline larvae lie horizontally in the water and breathe through spiracles that are flush with the dorsal surface of the abdomen, where they have direct contact with air. In Oc. taeniorhynchus larvae, the terminal spiracles are situated within the respiratory siphon that projects dorsally from the abdomen. There is no apparent relationship between the mortality the inventors observed by inhibition of CA and the mode of respiration of larvae since both larvae that breathe in different manners were affected in similar ways by the inhibitors. The possibility that inhibiting CA could affect respiration by interfering with the removal of accumulated C0₂ in the larvae should also be considered. Mosquito larvae require access to atmospheric oxygen to breathe. However, it has been demonstrated that the larvae can resist long periods of time submerged, making use of dissolved oxygen. Ae. aegypti larvae have been shown to live up to 53 days and develop to fourth instar even when denied access to the surface (Macfie, 1917). Furthermore, Ae. aegypti and Cx. quinquefasciatus larvae that had their entire tracheal systems filled with hexadecane were able to survive and develop to adulthood (Micks and Rougeau, 1976). Therefore, it is unlikely that inhibition of respiration is related to the mortality observed over 24 and 48 hours when the larvae were treated with CA inhibitors. [00169] In terms of the potential use of CA inhibitors as mosquito larvicides, it is concluded based on the results that sulfonamides such as acetazolamide and methazolamide are more effective than benzenesulfonamide and/^ammobenzenesulfonamide (sulfanilamide) as reported previously (Beesley, 1973). The LD₅0 values obtained by Beesley were reported as percentages (g/ml) in the range of 0.01- 0.05 % for An. Stephens! and^e. aegypti. The values the inventors calculated for methazolamide and acetazolamide based on the inventors' results are 0.002% for Ae. aegypti and 0.00002% for another anopheline species, An. quadrimaculatus. The values obtained for the other species used in this study are very similar. The LD₅₀ values the inventors obtained indicate that methazolamide and acetazolamide are more lethal to mosquito larvae than those compounds used by Beesley. Since the development of methazolamide and acetazolamide, novel CA inhibitors more potent than these two drugs have been developed (Scozzafava, et al. 2000). Perhaps by taking advantage of the alkaline pH inside the midgut as well as of these novel inhibitors it might be possible to develop formulations containing CA inhibitors that become active only at very high pH. Since different CA isozymes are present in living organisms, an effective mosquito larvicide based solely on CA inhibition would have to selectively target the mosquito CA without affecting other species. In order to develop such larvicides, further studies are necessary to have a better understanding of the structure of the mosquito CA and the environment that surrounds it. [00170] In conclusion, the inventors' results indicate that CA plays a key role in the midgut alkalization mechanism for all the species tested with the possible exception of Ae. albopictus. Furthermore, the inventors believe that inhibition of CA has a profound effect in the mechanism of ion transport in the midgut epithelium with lethal consequences for some species. Strong evidence linking CA with ion transport mechanisms in the midgut and the alkalization mechanism was provided by the inventors' studies with Oc. taeniorhynchus larvae. Mosquito larvae that live in salt water have an extremely high capacity for ionic regulation (Bradley and Phillips, 1977). Since the salt marsh habitat where this species lives is liable to dry out, larvae of Oc. taeniorhynchus must develop quickly and must have a very effective mechanism for ionic regulation to be able to survive the fluctuations in ionic composition of the medium around them. Evidence for effective ion transport mechanisms in this species has been provided by Nayar and Sauerman. In their studies, larvae from this species were able to maintain the chloride concentration and osmotic pressure of the hemolymph close to normal values when placed in dilute as well as in concentrated media (Nayar and Sauerman, 1974). The inventors' studies indicate that treatment of Oc. taeniorhynchus larvae with CA inhibitors leads to a decrease in the alkalization of the anterior midgut, a decrease in the pH of the rearing medium and larval mortality. Since this species was one of the two most sensitive to the effect of the inhibitors, the inventors believe that by treating Oc. taeniorhynchus with CA inhibitors the inventors interfered with their mechanism for ionic regulation inside the midgut with lethal consequences for the larvae. Furthermore, most mosquito larvae depend on their ability to regulate the ionic composition of the hemolymph to survive. Perhaps by treating mosquito larvae with CA inhibitors the inventors disrupted ion transport processes not only in the midgut but in other organs of the larvae, interfering with the mechanisms necessary to maintain a balance in the ionic composition of the hemolymph and ultimately affecting larval survival.

References

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The teachings of the references cited in the specification are incoφorated herein in their entirety by this reference to the extent they are not inconsistent with the teachings herein. While various embodiments of the present invention have been shown and described herein, it will be obvious that such embodiments are provided by way of example only. Numerous variations, changes and substitutions may be made without departing from the invention herein.

Claims

CLAIMS What is claimed is:

1. A method for treating malaria comprising administering to a human in need of such treatment an effective amount of a composition comprising a carbonic anhydrase inhibitor.

2. The method of claim 1 wherein said composition comprises dorzolamide, acetazolamide, brinzolamide, methazolamide, ethoxzolamide (ethoxyzolamide), butazolamide, dichloφhenamide or flumethiazide, or a combination thereof.

3. A method for treating malaria comprising administering to a patient in need of such treatment an effective amount of a carbonic anhydrase inhibitor used in the method of claim 1 , in combination with an effective amount of an additional antiparasitic agent.

4. A method for treating malaria comprising administering to a patient in need of such treatment an effective amount of the composition used in the method of claim 2, in combination with an effective amount of an additional antiparasitic agent.

5. The method of claim 3, wherein said carbonic anhydrase inhibitor is administered prior to, concurrent to or subsequent to the administration of said additional antiparasitic agent and or said agent for reversing antimalarial resistance.

6. The method of claim 3 wherein said additional antiparasitic agent is selected from the group comprising: a) quinolines, b) folic acid antagonists, c) sulfonamides, and d) antibiotics.

7. The method of claim 4, wherein said carbonic anhydrase inhibitor is administered prior to, concurrent to or subsequent to the administration of said additional antiparasitic agent.

8. The method of claim 4 wherein said additional antiparasitic agent is selected from the group comprising: a) quinolines, b) folic acid antagonists, c) sulfonamides, and d) antibiotics.

9. A pharmaceutical composition comprising an effective amount of a carbonic anhydrase inhibitor used in the method of claim 1, an additional antiparasitic agent, and a pharmaceutically acceptable carrier.

10. A method of controlling the spread of malaria comprising administering a composition comprising a carbonic anhydrase inhibitor to a subject known to be infected with plasmodium.

11. A method of controlling an insect population of a geographical location comprising administering a composition comprising a carbonic anhydrase inhibitor to said geographical location.

12. The method of claim 11, wherein said composition is administered in the form of concentrated liquids, solutions, suspensions, sprays, powders, pellets, capsules, briquettes, or bricks formulated to deliver a pesticidally effective concentration of a carbonic anhydrase inhibitor.

13. The method of claim 11 , wherein said composition is administered to bodies of water contained with said geographical location.

14. The method of claim 13, wherein said composition is in a form for delayed or continuous release in water.

15. The method of claim 11 , wherem said composition is in a form for delayed or continuous release of said composition.

16. A method for treating Chaga's disease comprising administering to a human in need of such treatment an effective amount of a composition comprising a carbonic anhydrase inhibitor.

17. The method of claim 16 wherein said composition comprises dorzolamide, acetazolamide, brinzolamide, methazolamide, ethoxzolamide (ethoxyzolamide), butazolamide, dichloφhenamide or flumethiazide, or a combination thereof.

18. A method for treating Chaga' s disease comprising administering to a patient in need of such treatment an effective amount of a carbonic anhydrase inhibitor used in the method of claim 16, in combination with an effective amount of an additional antiparasitic agent.

19. A method for treating Chaga' s disease comprising administering to a patient in need of such treatment an effective amount of the composition used in the method of claim 17, in combination with an effective amount of an additional antiparasitic agent.

20. The method of claim 18, wherein said carbonic anhydrase inhibitor is administered prior to, concurrent to or subsequent to the administration of said additional antiparasitic agent and or said agent for reversing antimalarial resistance.

21. The method of claim 18 wherein said additional antiparasitic agent is selected from the group comprising: a) quinolines, b) folic acid antagonists, c) sulfonamides, and d) antibiotics.