New Approaches to the Development of Anti-Protozoan Drug Candidates : a Review of Patents

As doenças causadas por protozoários afetam hoje em dia uma grande parcela da população mundial, provocando muitas mortes e exercendo grande influência na qualidade de vida e no desenvolvimento de muitos países. Essas doenças afetam principalmente países pobres e por isso, a pesquisa e o desenvolvimento de novos fármacos são negligenciados. De fato, a maioria das drogas usadas no tratamento dessas doenças data de décadas passadas e apresentam muitas limitações, incluindo o aparecimento da resistência às drogas. Este artigo tem como foco os mais recentes desenvolvimentos publicados no campo de patentes, entre 2001-2008, com especial atenção a promissores compostos atuando contra tripanossomíase, leishmaniose, malária, toxoplasmose, amebíase, giardíase, balantidíase e pneumocistose.


Introduction
Neglected parasitic diseases are a group of tropical infections which are especially endemic in low-income populations in developing regions of many countries.Despite affecting millions of people around the world, causing many deaths and having a great and limiting influence on the quality of life, the selection of new molecular targets and the development of more efficient drugs against neglected diseases are scarce (Table 1).
Nowadays, among them, zoonotic diseases attract much attention.Those are defined as diseases shared by animals and humans.In fact, wildlife serves as a reservoir for many diseases common to domestic animals and humans.Generally, disease is more easily prevented than treated.This discussion reviews common zoonotic diseases, including those ailments that are often erroneously cited as being closely linked to wildlife.
Several species of protozoans infect humans and inhabit the body as commensals or parasites.Protozoa have traditionally been divided based on their means of locomotion, although this character is no longer believed to represent genuine relationships: 2,3 (i) Flagellates (Zoomastigophora): Flagellates are characterized by having one or more flagella.Parasitic species generally have more flagella than those that are free living.Pathogens: Giardia intestinalis, Trichomonas vaginalis, Trypanosoma cruzi, Leishmania donovani; (ii) Amoebae (actinopods, rhizopoda): Amoebae may be divided into several morphological categories based on the form and structure of the pseudopods.It can live in a number of places within the human body, but most are found in the intestine.Those in which the pseudopods are supported by regular arrays of microtubules are called actinopods, and forms in which they are not, are called rhizopods, further divided into lobose, filose, and reticulose amoebae.Pathogens: Entamoeba histolytica; (iii) Sporozoans (Apicomplexa, myxozoa, Microsporidia): The members of this group share an "apical complex" of microtubules at one end of the cell that many prefer to the old name of sporozoans.All the members of the phylum are parasites.They do not have a set body plan like the other parasitic protozoans, although they are characterized by having complex life-cycles with an alternation of sexual and asexual generations.The most well known of all the sporozoans are the organisms which cause the malaria disease -Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale-and pneumocystosis; (iv) Ciliates (ciliophora): These are larger protozoans, growing to over 100 µm.They have hundreds of tiny cilia which beat in unison to propel it through the water.Often cilia are fused together in rows or tufts (called cirri) and are used for special functions such as food gathering.In addition to locomotion, the Paramecium and other ciliates like the Stentor use cilia to sweep food down into their central channel or gullet.There is only one species of pathogenic ciliate known to parasitise humans: Balantidium coli; (v) Microsporidia: They are so difficult to diagnose, very little work has been done into their importance in human disease, although they are known to be a major cause of productivity loss in aquaculture facilities such as prawn farms.Microsporidia have also been implicated in causing disease in immunocompromised hosts.
In this paper, we focused on some diseases, such as trypanosomiasis, leishmaniasis, toxoplasmosis, giardiasis, amoebiasis, balantidiasis and malaria.In this context, we believe that a review of patents on the highlighted inventions and innovative ideas in this field could be of importance for scientists, managers and decision-makers in the pharmaceutical industry.

Treatment of African trypanosomiasis
African trypanosomiasis is a parasitic disease of people and animals, caused by protozoa of the species Trypanosoma brucei gambiense or T. brucei rhodesiense and transmitted by the tsetse fly bite.The fly is infected when it bites an infected host and the trypanosome develops in the vector, culminating in infection transmission by its saliva.
The chemotherapy of Human African trypanosomiasis (HAT) is restricted to the clinically approved drugs: suramin (1), pentamidine (2), melarsoprol (3) and eflornithine (4).Suramin and pentamidine are restricted to the treatment of early-stage HAT prior to central nervous system (CNS) infection, while melarsoprol is used once the parasites have penetrated the CNS.Eflornitine is not effective against rhondesiense infections.These drug regimens can cause toxicity and poor efficacy.Thus, the development of novel compounds to provide novel treatment againt HAT is necessary.
Based on the 2 structure, Xavier, University of Louisiana, disclosed bisbenzamidines and bisbenzamidoximes analogs as compounds effective against infection by parasitic hemoflagellates, in particular Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. 21It has been postulated that aromatic bisbenzamidines are selectively taken up by transporters into trypanosomes, where they bind strongly to DNA in the nucleus and mitochondria, inhibiting the topoisomerases II, thus the synthesis of DNA, RNA, and proteins by the parasites will Scheme 1.
be inhibited. 22In mice infected with T. brucei, treatment with the compound 15 at a dose of 5 mg kg -1 per day resulted in a mean survival in days, of 18 (excluding cured animals) and 5 of the 6 treated animals were cured.

Treatment of American trypanosomiasis
American Trypanosomiasis is a tropical parasitic disease caused by the flagellate protozoan Trypanosoma cruzi.The disease may also be spread through an insect vector, blood transfusion and organ transplantation, parasite contaminated food ingestion, and from a mother to her fetus.Nifurtimox (16) or benznidazole (17) is used in the acute Chagas disease phase and in reactivation in immunosupressed patients.Another irrefutable sign refers to the treatment of laboratory accident victims, which demands a very early start for the administration.The action mechanism of 17 may depend on the formation of nitro anion radicals, but precise details are not known.Little is also known regarding mode of action of benznidazole.Because of the adverse events associated with these medications, patients being treated require careful monitoring.In a patent application, Reed and co-workers, 23,24 at the Infectious Disease Research Institute, claimed a fusion protein useful for the diagnosis of Trypanosoma cruzi infection within a biological sample.More specifically, the invention is related to the use of T. cruzi antigenic polypeptides and fusion polypeptides (TcF and ITC-6) in methods for screening individual and blood supplies for T. cruzi infection.Also included are details of a kit which uses the invention to detect T. cruzi involving the fusion protein.It is a protein created through the joining of two or more genes which originally coded for separate proteins.The reactivity of sera from T. cruziinfected individuals and control sera from non-infected individuals against the fusion polypeptides TcF and ITC-6 was determined by ELISA.The data show that the invention is capable of recognizing sera that are negative or low with TcF. 23,24ibell and Watts, at Amura Therapeutics Limited, described novel tetrahydrofuro [3,2-b]pyrrol-3-one compounds (18), their salts, hydrates, complexes or prodrugs.[6][7][8][9][10] Compound 18 exhibited good primary DMPK (dystrophia myotonica-protein kinase) properties along with promising activity in an in vitro cell-based human osteoclast assay of bone resorption.Compound 19 exhibited >80% inhibition at a concentration of 1 µmol L -1 .[6][7][8][9][10] In vitro activity was observed against a range of CAC1 cysteinyl proteinases, however tetrahydrofuro[3,2-b]pyrrol analogs showed a better in vitro activity value. 25 a further patent application with cathepsin K inhibitors against Chagas disease or American trypanosomiasis, SmithKline Beecham Corporation reported new promising trioxo- [1,2]thiazepanylamide derivatives. 26These compounds are substituted asymmetrical imidodicarbonimide diamides derived from hydroxylamines.They include the structure 20 as one of several compounds specifically claimed, but biological data are not presented in this patent.

Treatment of giardiasis
Giardiasis is a disease caused by Giardia lamblia and only the cyst form is infectious by the oral route.People catch Giardia by eating food or drinking water which has been contaminated by the organism -usually from feces.When there is a lot of Giardia present, this generates inflammation, which causes nausea, stomachache and diarrhea.Vol.21, No. 10, 2010   Giardiasis has been recently included in the WHO Neglected Diseases Initiative. 27Giardia lamblia infection in humans is frequently misdiagnosed.Multiple stool examinations are recommended, since the cysts and trophozoites are not consistently shed.Given the difficult nature of testing to find the infection, including many false negatives, some patients should be treated on the basis of empirical evidence; treating based on symptoms.Human infection is conventionally treated with metronidazole (21), tinidazole (22) or nitazoxanide (23). 28Nitroimidazoles are the most effective drugs available and no drug was reported to be unsafe, causing only mild to moderate and transient side effects.Suk et al. 29 reported the design, synthesis, and activity of a potent and non-toxic second generation anti-giardial agent that was designed as an inhibitor of cyst wall synthase.In the formation process of the Giardia cyst wall, the UDP-GalNAc is polymerized by cyst wall synthase into a polysaccharide, which, in conjunction with polypeptides, forms the filamentous outer cyst wall of Giardia. 30The compound 24, a phosphonoxin, was the more potent inhibitor of Giardia cyst formation and should have clinical potential as a new anti-giardia drug.
Currently (2001-2008), there are no patents of novel anti-giardiasis componds.However, oligonucleotide molecules for detection of G. lamblia were disclosed by Macquarie Research Limited. 31Preferably, the oligonucleotide molecule hybridises specifically unique to 18S rDNA/rRNA sequences of G. lamblia under medium to high stringency conditions.The sample can be environmental, from water sources, from waste materials, from medical and body fluids.

Treatment of balantidiasis
Balantidiasis is a disease caused by the large ciliated protozoan, Balantidium coli and tetracycline or iodoquinol is usually used in the treatment.Human infection occurs when human contact with pigs is particularly close.Ingestion of cysts or trophozoites from the infected animal or human feces is the infectious form.
Balantidiasis disease is treated with metronidazole (21)  or tetraciclin (25) for 5-10 days.It is usually effective, but without antibiotic mortality levels.In the treatment of acute balantidiasis, tetracycline is the drug of choice to eliminate B. coli trophozoites in humans.The disease is especially dangerous in immunocompromised patients as pulmonary parenchyma involvement is possible.B. coli is rare in Western Europe and North America and alternative drugs for the treatment of balantidiasis may be 5-nitroimidazole drugs such as tinidazole (22) and ornidazole (26). 32As well as in the case of G. lamblia, currently (2001-2008), there are no new patents for anti-balantidiasis drugs.

Treatment of ameobiasis
Amoebiasis is an infection of the intestine (gut) caused by an amoeba called Entamoeba histolytica, that are found in contaminated food or drink.Virulent strains such as Entamoeba coli and Entamoeba hartmanni, can produce mild diarrhea and dysentery.Only a few cysts are needed to cause infection.Amoebic cysts resist iodine and chlorine if concentration of these chemicals is too low.
Thiosemicarbazones and their metal complexes have been extensively studied during recent years due to their wide variety of biological activities. 34Certain drugs even show enhanced activity when administered as their metal chelates.Copper(II) is a biologically active essential metal ion; its chelating ability and positive redox potential allow participation in biological transport reactions.Sharma et al. 34 reported the synthesis of 5-nitrofuran 2-carboxaldehyde thiosemicarbazones (31) and their subsequent bidentate Cu II complexes (32).These compounds were screened for their anti-amoebic activities against HK-9 strain of Entamoeba histolytica in in vitro experiments and found that the chelation induced significant changes in the biological activity of the ligands and some copper showed better IC 50 value than metronidazole in vitro.

Treatment of Plasmodium falciparum infection
Malaria is a mosquito-borne disease caused by small, one-celled parasites called Plasmodium that infect and destroy red blood cells.Four different plasmodia can cause disease in humans: Plasmodium.falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax.People get malaria from the bite of an infected mosquito.A mosquito can become infected when it bites a person who has malaria organisms in the blood.
The effectiveness of antimalarial drugs differs with different species of the parasite and with different stages of the parasite's life cycle.Drugs include chloroquine, mefloquine, primaquine, quinine, pyrimethaminesulfadoxine (Fansidar), and doxycycline.Two aspects have stimulated new efforts regarding the development of chemotherapy, vaccines and sanitary studies on malaria: the resistance to currently available antimalarial drugs and the inefficacy of antimalarial vaccines. 35Clark et al, 36 at the SmithKline Beecham Corporation, described a process able to prepare commercial quantities of benzofuran-2-carboxylic acid {(S)-3-methyl-1-[(4S,7R)-7-methyl-3-oxo-1-(pyridine-2-sulfonyl)-azepan-4-yl-carbamoyl]-butyl}-amide, 33, and analogs.The method starting from the cycloaddition of 3-chloro-1-butene with potassium phthalimide in the presence of an alkali metal carbonate to form compound 36 N-(alfa-methylallyl) phthalimide.The compound 36 reacts with alkyldiamine followed by azeotropic distillation with ethanol and gaseous HCl treatment to give (R)-3-amino-1-butene hydrochloride, 37. 2-Chlorosulfonyl pyridine is coupled with 37 in the presence of a thialkylamine base to form the pyridine sulfonamide 38.Mitsunobu reactions of (2S,3R)-1,2-epoxy-4-penten-3-ol lead to the nitrogenprotected epoxide fragment 40.The reaction of sulfonamide fragment 39 and epoxide fragment 40 provides 33.There are twelve oxidation reaction steps to yield the final product (Scheme 2).The compound 33 is the Relacatib used for treatment of malaria.
SmithKline Beecham Corporation has also patented new promising dihydrotriazine derivatives. 37These compounds derived from oxo-amino-sulfonyl-azapanes 41 also are cathepsin K inhibitors and useful for the treatment of 5 is optionally substituted C 3-7 cycloalkyl, C 3-7 cycloalkenyl, aryl, heterocycloalkyl.8][39][40][41][42][43][44] For instance, the scheme for preparation of the compound 42 with substituent CH 2 -cyclohexyl is shown in Scheme 3.Those compounds were tested for treating diseases characterized by bone loss such as osteoporosis, periodontitis and gingivitis, and diseases characterized by parasitic infections particularly malaria, trypanosomiasis and leishmaniasis.In the patent, 45 the Corporation related a method of treatment of parasitic diseases, especially malaria.The compound 44 exhibited a Ki value of 1.9 nmol L -1 against the falcipain cysteine protease.

US Army Medical Research Institute of Infectious
Diseases patented novel tricyclic compounds that reduced side effects and restore the clinical efficacy of antimalarial drugs, including mefloquine (45) and chloroquine (46). 46heir salts and prodrugs, and their use as chemosensitizing agents and for modulating resistance to a drug, particularly an antimalarial drug is also claimed.IC 50 values were determined for each candidate compound alone and in combination with chloroquine.The fractional inhibitory concentration indices (FIC, actual IC 50 of one compound in the presence of a second compound but is expressed as a fraction of its IC 50 when used alone) for 1:1 combinations were determined using a chloroquine-resistant P. falciparum W2 clone (Table 2).The combination of the compounds 47b and 47e and chloroquine showed the best MDR (multidrug resistance)-reversing activity.Chloroquine with compounds containing saturared (47i and 47m) and unsaturated seven-membered central rings (47q and 47r) possessed similar activity.The compounds 47s and 47t were not as potent as their tricyclic ring counterparts.A FIC index of less than 1.0 represents synergy or potentiation and a FIC index greater than 1.0 represents antagonism (Table 2).
Mepha Pharma AG studied the administration of the combination of artesunate (48) and mefloquine (30)  on humans with acute, uncomplicated P. falciparum malaria.Artemisinin and its derivatives are the most rapidly acting antimalarial drugs, however the duration of antimalarial activity is short. 47Thus, in those studies, patients were randomized to receive accordingly in: Group Aartesunate 200 mg per day and mefloquine 250 mg per day simultaneously once daily for 3 days; Group B -artesunate 200 mg per day and mefloquine 750 mg (no dose on the 1 st day, 250 mg on the 2 nd day and 500 mg on the 3 rd day) once daily for 3 days.The cure rate after 14 days and 28 days was monitored.The results show that group A had a 100% cure rate and group B had a cure rate of 98% after 14 days.Also in group A, the low incidence rate of early vomiting and overall vomiting was observed.
Three companies, Universidade de Lisboa, Cenix BioScience GmbH and Instituto De Medicina Molecular, reported the use of an inhibitor of a scavenger receptor class protein (ScarB1) for the therapy and/or prophylaxis of a malaria infection. 48ScarB1 appears to mediate HDLtransfer and uptake of cholesterol.It was observed that inhibitors of ScarB1 function inhibit the growth of protozoa in liver cells, thus, inhibitors of ScarB1 can be used to treat infectious diseases involving liver cells and because the ScarB1 is expressed in erythrocytes, in hematopoietic cells.Human hepatoma cells were treated and the influence of the compounds on proliferation and infection with Plasmodium sporozoites was calculated as percentage of the plate mean for all samples, with the mean set to 100%.In each compound a score was assigned between 0 and 4 for inhibition of infection.The compounds 49, 50 and 51 showed score 4 (corresponding to an IC 50 of 1 mmol L -1 or lower), 1 (IC 50 between 3 and 4 mmol L -1 ) and 0 (IC 50 between 4 mmol L -1 or large), respectively.
Fansidar is a combination of sulfadoxin (52) and pyrimethamine (53) used for the treatment of chloroquine (47) resistent P. falciparum. 49Council of Scientific and Industrial Research patented a kit of α,ß-arteether (an artemisinin derivative), for increasing the sites of action on the parasites and thus will be more effective in controlling them.The result shows that intramuscular α,ß -arteether (7.5 mg kg -1 ) and the combination of sulfadoxin and pyrimethamine (5 mg kg -1 ) produced 100% curative efficacy compared to the individual compounds alone. 49e Holding Company For Biological Products And Vaccines developed a scorpion venom (Pandinus imperator) drug that has the ability to stop the development of asexual life cycle from ring to schizont stage. 50Doses of the venom stimulated the immune system producing specific immune response against malaria parasite.The venom is free of side effects and does not cause histamine release.Curiously, the scorpion venom, is used as a carrier to deliver radioactive iodine into tumour cells left behind after surgery, has removed the bulk of the tumour.The P. falciparum erythrocytic stage causes several million deaths yearly, primarily in Africa.Thus, National Institutes of Health developed a novel vaccine formulation comprising a P. falciparum erythrocyte binding protein, BAEBL. 51BAEBL polynucleotide is used to induce an immune response to a Plasmodium parasite, because it has homology to EBA-175, a P. falciparum receptor that binds specifically to glycophorin A on erythrocytes.Like EBA-175, the erythrocyte receptor for BAEBL is destroyed by neuraminidase and trypsin, however BAEBL can bind to erythocytes that lack glycophorin A and Gerbich erythrocytes (predent glycophorin C and absent glycophorin D) bind BAEBL much more weakly than normal erythrocytes.The compound of this invention can be employed in admixture with conventional excipients and can be combined with other active agents, e.g., vitamins.In addition, Jensen et al. 52 isolated VAR4, VAR5 or VAR6 polypeptide (from var genes) and its nucleic acid for use as vaccines for the diagnosis and treatment of malaria.The proteins are responsible for inducing the immunoglobulin G (IgG) antibody with specificity to P. falciparum erythocyte membrase protein 1 (PfEMP1) on infected red blood cells.Thus, severe malaria is caused by parasites expressing a subset of PfEMP1 molecules and antibodies directed against these will be responsible for the protection against severe disease acquired early in life.
Yet in vaccine preparation, Claudianos et al., 53 at the Imperial College Innovations Limited, claimed novel secreted scavenger receptor (PfSR) proteins from the P. falciparum genome database, and their use in the preparation of vaccines against malaria and in diagnosis.PfSR protein was shown to have 64% homology to proteins found in the mouse malarial parasites P. berghei (PbSR) and P. yoelii (PySR), whilst nucleic acid comparisons showed 74% homology.It contains Limulus clotting factor C, lipid binding, cysteine rich scavenger receptor and pentraxin domains.The protein causes disruption of the normal expression of the proteins, so the malaria parasite dies at an early stage in its life cycle.Parasite disruption via drugs or vaccines is therefore more likely to be effective when parasite numbers are low, i.e., at the early infective stages: sporozoites in human and ookinete in mosquito.
Walter and Eliza Hall Institute of Medical Research developed a vaccine for inducing an immune response to P. falciparum using a glycosylphosphatidylinositol (GPI) inositolglycan domain or its derivative or equivalent. 54GPI has been identified as a candidate toxin of parasite origin.
Experiments using GPI in mice with cerebral malaria showed relatively low parasitemia levels between days 5 to 12 post-infection.

Treatment of Leishmaniasis
Cutaneous leishmaniasis is caused by Leishmania mexicana and L. brasiliensis in the Americas, and L. tropica in the Old World; and by visceral leishmaniasis L. donovani, L. infantum and L. chagasi.Sandfly vectors transmit cutaneous leishmaniasis.Treatment remains inadequate because of drug toxicity (sodium antimony gluconate), long courses required, and frequent need for hospitalization.
For the treatment of Leishmaniasis the currently used drugs are limited to four.The first line compounds are the two pentavalent antimonials, sodium stibogluconate and meglumine antimoniate.They were used for the first time in 1947 and 1950, respectively.Failures and relapses occur in all forms of leishmaniasis and constitute approximately 10-25% of cases.If these drugs are not effective, the second line compounds of pentamidine (2) and amphotericin B (54) are used, which were introduced in 1940 and 1959, respectively.
Fuertes and Jimenez 55 at the Mologen Holding AG Dana-Farber Cancer Institute, claimed to have discovered a new DNA-expression construct for treatment of infections with leishmaniasis and a corresponding vaccine.The DNA expression constructs may code for one or more Leishmania antigens, including the p36 LACK antigen.p36 Antibodies were determined in mice following immunization.
Duran et al. 56 (Universidad Complutense de Madrid) described a novel method of obtaining water-dispersible albumin microspheres containing amphotericin B. The invention is based on the atomisation of the albumin with the drug that is microencapsulated for treatment of leishmaniasis and not requiring the pre-formation of an emulsion, avoiding the use of oils and organic solvents.This composition is less toxic, has fewer side effects than current amphotericin B formulations, such as Fungizone®, and is easy to produce on an industrial scale.The product is suitable for parenteral administration or intravenously.
8][59] The inclusion of procaine or lidocaine in the formulation reduces the pain of injection.A representative tablet composition comprises the following compounds: 350 mg of diminazene diaceturate (55), 250 mg of chloroquine (47)  and 400 mg of antipyrine (57).
The Institut de Recherche pour le Developpement reported the use of niacin (vitamin B 3 , 58a, R = OH) or nicotinamide (58b, R = NH 2 ) for treating parasitic diseases.Nicotinamide is currently in trials as therapy to prevent cancer recurrence and insulin-dependent (type I) diabetes.In particular, they can be used to treat protozoan parasitic diseases such as leishmaniasis in immunodepressed patients. 60It has been recently demonstrated that nicotinamide is a substrate of SIR2-like enzymes in vitro. 61he data presented that nicotinamide strongly inhibited the proliferation of both promastigotes and amastigotes with promastigote forms showing less sensitivity to nicotinamide than amstigotes.Also the patent reported the composition and administration in combination with another active agent selected from, e.g., amphotericin B, paromomycin, melarsopol and antimonials, using oral, intravenous, topical or intralesional routes.In addition, the Institut described in a patent, the nucleic acid constructs, which comprise nucleic acid encoding an immunogenic protein from a promastigote or amastigote of Leishmania. 62,63ine-borane compounds exhibit high similarity to organic compounds mainly due to the atomic radii and the characteristics of the B-N bond, which resemble those of carbon-carbon bonds.Thus, novel families of amineborane compounds, including fluorinated aminoboranes, novel bis-aminoboranes and aminoboranes having saturated and unsaturated alkyl chains are provided by Soreq Nuclear Research Center Israel Atomic Energy Commission and Yissum Research Development Co. 64 Processes for preparing, pharmaceutical compositions and methods utilizing these novel compounds are also provided.Radiolabeled aminoboranes and uses thereof in radioimaging (e.g., PET) and radiotherapy are further provided.They are used for the treatment of leishmaniasis and demonstrated excellent antimicrobial activity with reduced adverse side effects and improved pharmacokinetic profile.The in vitro anti-leishmanial effect of compound 59 (dimethyl-undecyl-amine cyanoborane) was determined on promastigotes and amastigotes.According to Table 3, the measured radioactivity, which is indicative of the parasite, diminishes rapidly with the increased dosage of amineborane, indicating a strong anti-leishmanial effect.
In a further patent, Soreq Nuclear Research Center Israel Atomic Energy Commission and Yissum Research Development Co. disclose modified polymer conjugates.The conjugate of a polymer (e.g., polysaccharide) and a drug (e.g.amphotericin B, 54) reduced the toxicity relative to the unmodified parent compound, increased the solubility and retained substantially the same degree of therapeutic activity of the unmodified parent compound. 65It is an antiparasitic composition against L. donovani and formulated in the form of a nanoparticle, micellar dispersion and a liposome.The test was performed in dextran-AmB imine (60) conjugate with or without ethanolamine (Table 4).The result shows that the conjugate had an IC 50 value of 0.25 µg mL -1 compared to the Dextran-AmB imine alone (1.2 µg mL -1 ).
Valtion Teknillinen Tutkimuskeskus reported the use of betulin (62) derivatives or their salts against leishmaniasis Dextran-AmB-ethanolamine imine 0.25 400 >500 a IC 50 values were derived from the activity test of AmB and different dextran-AmB conjugates against L. donovani.b IC 50 values were derived from cytoxicity test AmB and different dextran-AmB canjugates against the murine RAW 264.7 cell line.c Hemolysis was evaluated visually after 1 h incubation at 37 ºC with sheep erythrocytes. 65 pharmaceutical industry applications.66 Betulin is the most abundant triterpenoid of the lupane series and is found in the bark of some tree species, particularly in birch bark (Betula). Itis a component of various food additives and cosmetic products.The activity of the betulin derivatives was tested for both L. donovani and L. tropica species (Table 5).Kemin Pharma Europe patented the use of bicyclic carbohydrates for the treatment of leishmaniasis.67 Compound 63 and 64, bicyclic carbohydrates with  68 National Institutes of Health reported a novel dinitroaniline sulfonamide based on the herbicide oryzalin 66 69 preferably less cytotoxic to normal cells than oryzalin.70 Tubulin, a critical eukaryotic protein responsible for formation of the mitotic spindle has been implicated as the target of dinitroaniline analogs.Compounds 67 and 68 are significantly more potent than compound 66 in blocking leishmanial tubulin assembly (Table 6).The compound 68 had K d (dissociation constant) values of 57 µmol L -1 leishmanial tubulin, and the corresponding value for oryzalin were 170 µmol L -1 .These data are consistent with the hypothesis that tubulin is the target of the dinitroanilines in Leishmania.
In 2006, De Souza and Chequer Bou-Habib 71 related a prophylaxis vaccine for blocking transmission of Leishmania infections.It is based on L. donovani saponin and promastigotes or amastigotes fractions called Fucose Mannose ligand (FML, an isolated glycoprotein complex).Leishmune® vaccine (FML vaccine) developed 92-95% protection against canine visceral leishmaniasis and showed immunotherapy potential.In 2008, due to high production cost of Leishmune®, Gazzinelli and co-workers 72 developed a vaccine formulation comprising amastigote A2 antigen.
The antibodies produced by the dogs in the vaccination process with this antigen are non-reactive to serologic diagnostic infection tests.The formulation is composed of 50 to 200.00 mg mL -1 of recombinant A2-HIS (rA2) protein of Leishmania, produced in E. coli, 0.125 to 0.500 mg mL -1 of Saponin, 1.00 mL of q.s.p buffered saline solution and 0.01 mL of thimerosal.Therefore, the main innovation of this vaccine formulation is the production of antibodies specific against the vaccine antigen.The main innovation of this formulation is that the animals vaccinated present serologic reaction against the antigen, but remain seronegative in the reaction with the total parasite extract, e.g., the antibodies do not react with the extract of the promastigote forms of Leishmania in the ELISA tests, for example.
Rosalind Franklin University of Medicine & Science disclosed the use of a live mutant Leishmania as a suicidal vaccine. 73The mutant can be selected from natural Leishmania species or constructed by genetic engineering.The invention also exploits the virtual absence of heme biosynthesis pathway in trypanosomes to identify or construct the mutant porphyric Leishmania as suicidal vaccines.The vaccinated hamsters showed ca.10 fold reduction of parasite loads than the control group.

Treatment of pneumocystosis
Pneumocystosis is a pneumonia that is characterized by the accumulation of very large numbers of a eucaryotic single-celled organism called Pneumocystis carinii, which has not been cultured.Pneumocystosis also occurs in many other mammalian species.It is not yet established whether Pneumocystis carinii is a fungus or a protozoan.
Pneuocystis carinii causes P. carinii pneumonia (PCP) in people with depressed immune systems such as AIDS patients, patients undergoing chemotherapy, or transplant patients being treated with immunosuppressants.The infection is treated with pentamidine (2) and the combination of trimethoprim (77) and sulfamethoxazole, however there are side effects and the mortality rate remains high.An alternative treatment is the preparation containing methioninase in combination with an antibiotic, either pentamidine (2) or combination of trimethoprim (77)  and sulfamethoxazole related by AntiCancer Inc. 74 Infection by P. carinii can be treated by administering methioninase optionally in combination with additional therapeutic agents, such as antibiotics, either pentamidine or combination of trimethoprim and sulfamethoxazole.The use of methioninase depletes plasma levels of methionine and deprives the parasites of S-adenosylmethionine.
In a further patent application and development, the preparation of ten carbamate and carbonate prodrugs of bisamidinophenyl furans was patented by The University of North Carolina at Chapel Hill. 75Compounds were tested against P. carinii infected immunosuppressed rats.The more potent compound, 69, inhibited cyst formation in the lung by 100 and 97.9% inhibition at doses of 22 and 33 µmol kg -1 per day, respectively.
Dihydrofolate reductase (DHFR) is a key enzyme in the treatment of Pneumocystosis.Its role in thymidine biosynthesis (Figure 1) is the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF) using the cofactor nicotinamide adenine phosphate (NADPH).After reduction, serine hydroxymethyltransferase (SHMT) catalyses the regeneration of 5,10-methylenetetrahydrofolate; and deoxyuridine monophosphate (dUMP) is methylated to give deoxythymidylate (dTMP) in a reaction catalysed by thymidylate synthase (TS).This reaction converts methylene-tetrahydrofolate back to dihydrofolate, completing the cycle.Therefore, the inhibition of DHFR prevents the biosynthesis of thymidine and, as a consequence, DNA biosynthesis. 77Novel substituted 2,4-diamino-5-benzylpyrimidine tested as inhibitors of P. carinii DHFR, was claimed by Forsch et al. 78 In summary (Table 7), the results indicated that, 5´-(5-carboxy-1-pentynyl) substitution is more favorable when the benzyl ring is 3′,4′,5′-trisubstituted than when it is 2′,5′-disubstituted.Compound 73 has the highest species selectivity for DHFR P. carinii.For this reason this novel analogue may be viewed as a novel lead for further structure-activity optimization of DHFR binding.Rosowsky et al. 79 reported a concise new route allowing easy access to five 2,4-diaminopyrido [2,3-d]pyrimidine analogs containing a substituted phenyl or other aromatic ring attached to C6 via by a short bridge.The derivatives were tested against P. carinii DHFR, however none of the quinazolines or pyridopyrimidines tested (IC 50 0.087-72 µmol L -1 ) were more potent against the P. carinii enzyme than the structurally related reference compound piritrexim (77, IC 50 = 0.013 mmol L -1 ).In the patent application, Forsch and Rosowsky, 80 at the Dana-Farber Cancer Institute Inc., synthesized several lipophilic DHFR inhibitors having an aromatic group and a heteroaromatic group linked by a methylene group in the treatment of P. carinii.Trimethoprim (77) and piritrexim (76) are lipid-soluble antifolates that have been used clinically for the prophylaxis and treatment of P. carinii infections in patients with AIDS.In addition, Rosowsky and Forsch, 81 synthesized novel 77 derivatives (Table 8), compositions comprising them and their use as DHFR inhibitors.The most potent of the O-alkyl derivatives against P. carinni DHFR was compound 78c (n = 6).Moreover there was an increase in potency as the length of the 5′-o-alkyl group increased from n = 4 to n = 6, followed by a decrease in potency as this length increased from n = 6 to n = 8.The most potent of the 5´-o-(w-carboxyalkyl) analogs was 60g (n = 4), 245 times more potent than 77.
The activity of four triazolyl analogues (79a-d) was evaluated as inhibitors of P. carinii DHFR by Chan et al. 82 The two most potent compounds are 80a and 80b, against the P. carinii DHFR.Four compounds with a nitrogen bridge (benzanilides and benzylamines) were also tested for activity.Compounds in the benzanilide series (80a,b) are less potent than the benzylamines (81a,b; Table 9).With an IC 50 value of 0.12 mmol L -1 , compound (81a) is the most potent member of the group as an inhibitor of P. carinii DHFR.Interestingly, methylation of the amino bridge (81b) showed a 10-fold reduction of activity towards the P. carinii enzyme and nearly a 7-fold gain of potency towards the rat liver DHFR.
Hallberg et al. 83 reported a novel series of DHFR inhibitors, where the methylenamino-bridge of nonclassical inhibitors was replaced with an ester function.The most potent of the new ester-based DHFR inhibitors, the 1-naphthyl derivative (82), exhibits an IC 50 -value of 110 nmol L -1 , and is less active than trimetrexate (83) (IC 50 = 42 nmol L -1 ).
Cushion and co-workers 84 synthesized a series of pentamidine (2) congeners and screened for their in vitro activity against P. carinii.The ATP assay was used to evaluate the effects of this pentamidine on the viability of A549 epithelial lung cell monolayers derived from a human carcinoma.All diamide derivatives (84a-e, Table 10), including the most potent agents 84c and 84e, showed no cytotoxicity at 100 times the IC 50 concentration against P. carinii.This study identified the simple small molecules 84c and 84e as two very promising therapeutic leads, which deserve further pre-clinical and clinical testing.
In a patent application, Walzer et al., 85 University of Cincinnati, disclosed a method of combating infectious agents, such as Pneumocystis pneumonia, and a method of treating a subject in need of such treatment.The method comprises administering a bis-benzamidoxime to the subject.The most active compound of the series, compound 85, had an IC 50 value of 0.00087 µg mL -1 against P. carinii.In comparison, pentamidine (2) had an IC 50 value of 0.300 µg mL -1 .

Treatment of toxoplasmosis
Toxoplasmosis is a disease caused by protozoan Toxoplasma gondii.Transmission to man occurs via ingestion of meat containing cysts or tachyzoites, ingestion of oocysts become infective after sporulation or transplacentally.
Toxoplasma gondii (T.gondii) is an ubiquitous parasitic protozoan that infects up to one-third of the US population and up to 90% of certain European populations. 86Risk factors for infection include exposure to infected cats, consumption of rare meats and contact with contaminated soil.Medications that are prescribed for toxoplasmosis are pyrimethamine (53) (an antimalarial medication) and sulfadiazine (86) (an antibiotic).Thus, the need for new chemotherapeutics active against T. gondii is therefore acute.Sanofi-Aventis developed herbimycin (87) derivatives, a benzoquinone ansamycin antibiotic, as heat shock protein 90 (Hsp90) inhibitors, useful for the treatment of toxoplasmosis. 87Hsp90 is a molecular chaperone and is one of the most abundant proteins expressed in cells.It is highly conserved and expressed in a variety of different organisms from bacteria to mammals.In an in vitro assay, 88 had an IC 50 of 0.928 µmol L -1 for the inhibition of the ATPase activity of Hsp82.
One feature that distinguishes T. gondii from its human host is its inability to synthesize purine "salvage". 88ifferently from its human host, the T. gondii recovers purine precursors from the adenosine kinase, enzyme which converts adenosine in adenosine monophosphate (AMP) (Figure 2).Through it, all other purine nucleotides can be synthesized.Then, the inhibition of the adenosine kinase activity interrupts the purine recovery route ("salvage"), offering a number of potential targets for the antiparasite chemotherapy.It is known in the literature that benzylthioinosine analogues are substrates for the parasite adenosine kinase, but not for human  89 Rais et al. 90,91 described a series of 6-benzyladenosine analogues that act as potent and selective substrates for T. gondii adenosine kinase.6-Benzylthioinosine analogs (Table 11) were identified as excellent subversive substrates of T. gondii adenosine kinase.
Currently, there are no tests which can differentiate between oocyst ingestion versus tissue cyst ingestion as the infection route. 93Recombinant proteins have been developed by US Department of Agriculture for the detection of T. gondii oocyst proteins for example in biological fluids.Isolates and recombinant T. gondii-specific proteins (rDGP5p) can be adsorbed to the surface of microtiter plates or to immunoblotting membranes and used in an ELISA format for detection of antibodies.Recombinant antigens also can be used to prepare monoclonal antibodies which selectively identify T. gondii oocyst proteins.In addition, primers directed to T. gondii-specific regions of the DNA sequences can be produced for sensitive detection of the parasite by polymerase chain reaction.Using and MAT, (microscopic agglutination test) assays were performed on 127 human sera known to have resulted from oocyst exposure, five sera from infections resulting from congenital infection, and 76 sera from MAT negative individuals.From the 76 uninfected sera, all were negative using the recombinant ELISA and from the 127 oocyst induced infections, the recombinant antigen ELISA detected 119, and did not detect 8. Sera from 40 oocyst infected pigs and 45 tissue cyst infected pigs also were tested using ELISA and MAT.From the 40 pigs infected with oocysts, 39 were detected using ELISA assay.
In the development of vaccine patents, GlaxoSmithKline B i o l o g i c a l s p a t e n t e d a va c c i n e c o m p o s i t i o n comprising the toxoplasma protein, SAG3, the major glycosylphosphatidylinositol (GPI)-anchored surface protein of T. gondii. 94SAG1 mediates the host-cell invasion and mono/polyclonal antibodies directed to SAG1 inhibited invasion of cells by tachyzoites, probably by interfering at the parasite attachment level. 95The binding of SAG1 and SAG3 to CHO K1 cells (Chinese hamster ovary cells) was examined.Just SAG3 displays affinity for sulfated proteoglycans.The presence of alternative cellular receptors used by T. gondii was already suggested, as inhibition of parasite attachment by soluble GAGs or by heparinase treatments of host cells was not complete.The studies show that soluble heparin (glycosaminoglycan) inhibited SAG3 binding by up to 90%.Dermatan sulfate, a glycosaminoglycan that is a structural component of certain body tissues also inhibited the binding of SAG3, but chondroitin sulfate A, a chondrin derivative, did not.In a further patent application on vaccines, Kyushu TLO Co Ltd [Kyushu University] developed a novel fused DNA vaccine produced by constructing a fused gene comprising a gene from an intracellular protozoal parasite, such as toxoplasma, with a ubiquitin gene. 96This vaccine is said to form an ubiquitinated pathogen antigen which is processed with a proteasome which strongly induces the production of CD8 + T cells.

Current and Future Developments
Neglected tropical diseases are widely related to poverty and disadvantage.The poorest populations often living in remote, rural areas, urban slums or in conflict zones are the most affected.Actually, the neglected tropical diseases show a clear link between health and development.Adding HIV, 97 tuberculosis, 98-100 dengue, [101][102][103] Chagas disease, [104][105][106] leishmaniasis 107 and malaria [108][109][110][111] to the mix, the diseases kill several million each year, shorten lives and reduce productivity.
Biologists have identified more than 50,000 species of protozoa, of which a fifth are parasitic and some can reach humans by food and water.The majority of food and waterborne infections of parasitic origin are related to poverty, low sanitation, and old food habits.Parasitic protozoa do not multiply in foods, but they may survive in or on moist foods for months in damp environments.
Diseases caused by intestinal protozoa are increasing and new treatments are very important because of their association with acute and chronic diarrhea in immunocompromised patients as well as those with a normal immune system.
However, as in the case of G. lamblia, currently (2001-2008), there are no new patents for anti-balantidiasis drugs.Balantidiasis disease is especially dangerous in immunocompromised patients as pulmonary parenchyma involvement is possible.Alternative drugs for the treatment of balantidiasis may be 5-nitroimidazole drugs, such as tinidazole (22) and ornidazole (26). 32n this scenario, 5-nitrofuran 2-carboxaldehyde thiosemicarbazones (31) and their subsequent bidentate Cu II complexes (32) showed promising results (IC50 value in vitro) when compared to the metronidazole (conventional compound used for the Treatment of Amebiasis). 34egarding malaria, two points call special attention: the resistance to currently available antimalarial drugs and the inefficacy of antimalarial vaccines. 35Thus, new promising cathepsin K inhibitors (dihydrotriazine derivatives) and inhibitors of a scavenger receptor class protein (ScarB1) were proposed. 37,48[53][54] It is interesting to mention that Dihydrofolate reductase (DHFR) is key in the treatment of Pneumocystosis.Novel series of DHFR inhibitors may be viewed as a novel lead for further structure-activity optimization of DHFR binding.In this line, Hallberg and co-workers 83 reported a serie of DHFR inhibitors, where the methylenamino-bridge of non-classical inhibitors was replaced with an ester function leading to interesting results.
The need for new chemotherapeutics active against T. gondii is also acute.On this point, herbimycin (87) derivatives, protein 90 (Hsp90) inhibitors, and 6-benzylthioinosine analogs, which are subversive substrates of T. gondii adenosine kinase, were identified as promising compounds in the treatment of toxoplasmosis.
Nowadays, ultraviolet (UV) irradiation is regarded as being widely effective against all pathogens, bacteria, protozoa and viruses that can be transmitted through drinking water. 94However, more research is needed to understand the extent to which particles in water may hinder the UV treatment efficacy by interacting with microbial pathogens.UV irradiation is a primary disinfection technology for use in water and wastewater effluents.It has been demonstrated that UV radiation is very effective against (oo)cysts of giardia, a pathogenic microorganism of major importance for the safety of drinking water. 112n Europe, UV has been widely applied for drinking water disinfection since the 1980s, for the control of incidental contamination of vulnerable groundwater and for reduction of Heterotrophic Plate Counts.Protozoan infections are especially endemic and affect millions of people around the word.In spite of their importance, the development of more efficient drugs against those diseases is still scarce.Furthermore, surprisingly few patents for new drug candidates acting on these diseases have appeared.][115][116][117] Therefore, we strongly feel that molecular modeling studies, combining docking and molecular dynamic simulations, can improve our understanding of the inhibitor-protein interactions.][120][121] However, it should be kept in mind that computers are an essential tool in modern medicinal chemistry.Currently, computational approaches and 3D visualization are not used simply to depict pretty pictures of molecules in biological systems; these powerful computational tools allow one to obtain insights on the interaction between enzyme-substrate, mechanism reaction, statistical behavior of molecules and much more, at the molecular level, contributing significantly to the problem solving in biological systems.

Figure 1 .Table 7 .New
Figure 1.Role of DHFR in the synthesis of thymine.

Table 2 .
FIC (fractional inhibitory concentration -1:1 combination of drug and chloroquine) indices of new modulators in P. falciparum W2 clone

Table 3 .
Concentration of the amine-borane is given in µg mL -1 versus the counts per minute (cpm) recorded for the parasite sample

Table 4 .
In vitro activity against Leishmania donovani, cytotoxicily and hemolysis of conjugates

Table 5 .
Activity in vivo of the compounds tested for L. donovani.Initially, the concentration of the compounds was 50 mmol L -1 .Amphotericin B (1 mmol L -1 ) was included as positive control.Broth containing DMSO was used as negative control -1for miltefosine 65.The use of 65 in leishmaniasis therapy should be carefully considered.

Table 6 .
Activity in vivo of Oryzalin and new dinitroniline compounds against L. donovani and leishimanial tubulin in vitro

Table 9 .
Inhibition of P. carinii DHFR by benzanilides and benzylamines derivatives

Table 8 .
Inhibition of P. carinii DHFR by trimethoprim derivatives

Table 11 .
Inhibition of T. gondii AK by 6-benzyladenosine derivatives Reaction catalyzed by the adenosine kinase enzyme.