MX2008003034A - Method of combating infection - Google Patents

Abstract

A method of combating a parasitic protozoal infection of a host organism, the method comprising administering tretazicar to the host organism. Tretazicar is the compound 5-(aziridin-l-yl)-2,4-dinitrobenzamide (CB 1954), and the parasitic infection is preferably an infection ofTrypanosoma cruzi, T. b. brucei, Leishmaniaspp.particularly L. infantum, Cryptosporidium or Giardiaspp.

Description

METHOD TO COMBAT AN INFECTION DESCRIPTIVE MEMORY This invention relates to a method for combating infection and in particular to the treatment of parasitic protozoan infections. The listing or discussion of a document previously published in this specification should not necessarily be taken as an acknowledgment that the document is part of the state of the art or is common general knowledge. The incidence of drug-resistant parasitic protozoan diseases has grown significantly in recent years, resulting in a greater number of deaths in developing countries than in the western world. The strategies being developed to address this problem include the development of new drugs, the adoption of strict treatment regimens and a broad education program for the public. A possible means to deliver highly active pharmacological agents to their site of action with minimal unwanted side effects in other cells and tissues is the use of prodrugs. Prodrugs have been known for many years and are used in various medical indications. They can be defined as chemical entities that are they modify by means of metabolic or non-metabolic systems, which result in the formation or release of a species with desired pharmacological activity. In many cases, the prodrug forms are used to modify the pharmacokinetics of the drugs by altering a physicochemical property of the drug (such as its lipophilic degree). In these cases, the modification of the prodrug is usually not specific and occurs through non-enzymatic degradation processes or through the metabolic action of ubiquitous enzymes. However, the prodrug forms can also be used to target the active pharmacological agent to specific sites only, usually by exploiting the differential distribution of enzymes capable of catalyzing the prodrug modification reaction. In the field of cancer chemotherapy, the activation of prodrugs by goal-specific enzymes has long been a goal and many potential prodrugs have been synthesized and tested in the hope that a specific enzyme for a tumor would be able to convert them specifically in a powerful antitumor agent. Many researchers still work in this area using prodrugs designed to be activated by a variety of enzymes including β-glucuronidase (Woessner et al (2000) Anticancer Research 20, 2289-2296), DT diaphorase (Loadman et al (2000) Biochemical Pharmacology 59 , 831-837) and thymidylate synthase (Collins et al (1999) Clinical Cancer Research 5, 1976-1981).

As an alternative to this monotherapeutic strategy with prodrug, some researchers have tried to supply the desired enzyme to the tumor, prior to administering a prodrug. Such an approach, generally referred to as antibody directed enzyme prodrug therapy (ADEPT), was described in WO88 / 07378. In this example the enzyme binds to a monoclonal antibody that is capable of binding to an antigen associated with a tumor. In this way the enzyme is delivered to the tumor site where it can act on an appropriate prodrug. A similar strategy was described, for example in WO96 / 03151, in which the gene encoding an enzyme is delivered to the tumor and, once present, expresses the desired enzyme. This strategy is generally referred to as gene directed enzyme prodrug therapy (GDEPT). It is an important principle of many of these targeted strategies that the enzyme delivered to the tumor site is not endogenous to the host (Aghi et al (2000) J Gene Med 2, 148-164). The presence of endogenous enzymes would lead to a non-specific activation of the prodrugs and to the toxicity of normal tissues. Consequently, most of these enzyme-prodrug therapy studies have been carried out using bacterial enzymes such as carboxypeptidase G2, β-lactamase and nitroreductase. Although much of the work to date in the field of activating site-specific prodrugs has focused on anticancer therapy, recent efforts have been made to adapt this technology for use in antibacterial applications. For example, WO 99/321 13 describes the use of prodrugs consisting of a cytotoxic portion and a β-lactam portion. In Gram-negative bacteria that contain β-lactamase enzymes within the periplasmic space, the prodrug is cleaved to release the cytotoxic portion which in turn seeks to interrupt vital cellular functions. These prodrugs, however, have a limited range of application in the sense that not all bacteria express β-lactamase and that Gram positive bacteria tend to excrete ß-lactamase such that many of the beneficial effects of toxin location of the prodrug are they will lose. In Smyth et al ((1998) J. Org Chem 63, 7600-7618), S-aminosulfeniminopenicillins are presented. These compounds are both β-lactamase inhibitors and templates of potential prodrugs for delivery of a variety of agents in β-lactamase positive bacteria. However, like the β-lactamase derivatives discussed above, these compounds are likely to have limited utility in Gram-positive bacteria and, because of their slow permeation through porosity of the outer membrane, may need to be present in concentrations extra unwanted cell phones to achieve significant effects in Gram negative bacteria. A branch of the potential deficiencies of the ß-lactamase-dependent prodrugs was presented by Wei and Pei ((2000) Bioorg, Med. Chem. Lett 10, 1073-1076). These scientists demonstrated conceptually the use of 5'-dipeptidyl derivatives of cytotoxic antibacterial agents such as prodrugs that are activated by the bacterial peptide deformylase. Preliminary results with the use of these prodrugs, however, suggested a weak antibacterial activity and it was assumed that this could be due to poor uptake of the compounds towards the cells. Human parasitic diseases are endemic in many parts of the world. For example, leishmaniasis (a parasitic disease caused by an obligate intracellular protozoan that is transmitted by the bite of some species of gnats) is found in approximately 90 tropical and subtropical countries around the world and in southern Europe. More than 90% of the worldwide cases of cutaneous leishmaniasis are found in Afghanistan, Algeria, Brazil, Iran, Iraq, Peru, Saudi Arabia and Syria. However, approximately 75% of the cases evaluated in the United States occurred in Latin America, where leishmaniasis occurs from northern Mexico (occasionally in rural southern Texas) to northern Argentina. More than 90% of the worldwide cases of visceral leishmaniasis occur in Brazil, Bangladesh, India, Nepal and Sudan. Similarly, the geographic distribution of Chagas disease (caused by a flagellated protozoan parasite, Trypanosoma cruzi, which is transmitted to humans by triatomine insects) extends from Mexico to southern Argentina. The disease affects 16-18 million people and around 100 million, that is, around 25% of the population of Latin America, is at risk of acquiring Chagas disease. Even people who remain for a short time in areas endemic to the parasite can become infected and parasitic diseases are becoming a problem in the developed world as a result of the global increase in travel. Resistance to currently used drugs has been reported. The problems of resistance require the use of more toxic drugs and as the drugs become less and less effective the discovery of new drugs is needed. The inventors have just discovered that, surprisingly, the nitroreductase enzymes also appear to be expressed in certain protozoan parasitic organisms and the inventors suggest that tetrazicar can be highly effective against the infestation of parasites in animals (such as humans)., since the animal host (for example human) is insensitive to this agent while the parasite will be affected toxicly. The inventors have now shown that tetrazicar is highly effective against certain protozoan parasites and it is also an object of the invention to provide tetrazicar as a highly effective antiparasitic agent. A first aspect of the invention provides a method for combating a protozoan parasitic infection of a host organism, the method comprising administering tetrazicar to the host organism.

The host organism preferably is an animal, more preferably a mammal and more preferably a human. Non-human mammals for their treatment by the method of the invention include horses, cows, pigs, goats, sheep, dogs, cats and the like. By "fighting" the given infection is meant that the infection is substantially eradicated or that the infection is substantially inhibited. It will be appreciated that the elimination of all parasites in the host organism may not be necessary to effectively treat the host organism. A second aspect of the invention provides the use of tetrazicar in the manufacture of a medicament for combating a parasitic protozoan infection of an animal. The tetrazicar compound is (5- (aziridin-1-yl) -2,4-dinitrobenzamide (CB1954)), the structure of which is shown below. Tetrazicar has previously been used as an anticancer agent.

Tetrazicar has a number of records C.A.S. No 21919-05-1 and its synthesis is described in Khan & Ross (1969/70) "Tumor growth inhibitor nitrophenylazridines and related compounds: structure-activíty relationships" Chem-Biol ¡nteractions 1, 27-47 and in Cobb et al (1969) Biochem. Pharmacol. 18, 1519-1527. This compound is capable of eradicating a specific rat tumor ("Walter tumor") although it has little or no effect on a variety of other tumors (Cobb et al (1969) Biochem Pharmacol 18, 519-1527) and shows no benefit Therapeutic in clinical studies of oncology (Knox et al (1993) Cancer and Metastasis REv 12, 195-212). It has been shown that a nitroreductase enzyme present in the Walter tumor is capable of activating CB1924 by reducing its 4-nitro group to form the compound 5-azridino-4-hydroxylamino-2-nitrobenzamine, a potent DNA crosslinking agent. electrophilic in the presence of intracellular thioesters (Knox et al (1988) Biochem Pharmacol 37, 4661-4669; Knox et al (1991) Biochem Pharmacol 42, 1691-1697). The corresponding enzyme in human cells has a relatively slow kinetics of reduction, thus rendering human cells insensitive to the effects of CB1954 (Boland et al (1991) Biochem Pharmacol 41, 867-875). In mechanistic studies in bacteria, it has been shown that CB1954 attenuated the toxicity in negative nitroreductase strains (Venitt and Crofton-Sleigh (1987) Mutagenesis 2, 375-381). The tetrazicar is activated to form an antiparasitic agent by enzymes associated with protozoan parasites. The term "enzyme associated with protozoan parasite" means an enzyme or isoform thereof that is either specific to the protozoan parasite that constitutes the infection or which is expressed functionally to such a low degree by the host organism as to make any activation of tetrazicar by the latter insufficient to cause unacceptable toxicity in the host, but whose enzyme is expressed by the protozoan parasite. In general, the enzyme associated with the parasite is at least 10 times or 20 times or 50 times or 100 times or 500 times or 1000 times more active to activate the compound than an enzyme present in the host organism. It will be noted that tetrazicar remains substantially unchanged by enzymes endogenous to the host organism and that it is substantially activated by one or more enzymes in the protozoan parasite. "Activated to form an antiparasitic agent" includes the meaning that tetrazicar is converted to a form that is cytotoxic, in particular to the protozoan parasite. Similarly, it will be noted that the methods and medicaments of the invention are particularly suitable for combating infection by protozoan parasites wherein the protozoan parasite is one that contains an enzyme system that is capable of activating the tetrazicar in a substantially cytotoxic form. A method to determine if a protozoan parasite responds to tetrazicar is described in the example. Such protozoan parasites can be destroyed by tetrazicar. Ideally, protozoan parasitic infections that can be treated with tetrazicar are those for which the tetrazicar has an IC5o of less than 10 micromolar, preferably less than 5 micromolar and more preferably less than 1 micromolar. Other methods for determining whether a protozoan parasite contains an enzyme capable of activating the tetrazicar to a substantially cytotoxic form will be known to those skilled in the art. These include, without restriction, the use of suitable computer programs, for example the GAP program of the Genetic Computation Group of the University of Wisconsin, to compare the protozoan gene sequence with that of the known species containing nitroreductase or the use of classical techniques with which the relevant enzyme is detected, is isolated and purified prior to tetrazicar tests. It is believed that tetrazicar is able to covalently crosslink the nucleic acid of the protozoan parasite once it has been activated by an enzyme system present in the parasite to the cytotoxic form. Since tetrazicar only becomes capable in such a manner with activation by an enzyme associated with a parasite, the presence of tetrazicar in host cells is thought to pose no danger since it has no activity compatible with the enzymes. The binding of tetrazicar to host cell components by the aziridine (or mustard) group will present only a minor risk of cell disruption since it is believed that any monofunctionally linked compound is cleavable by enzymatic processes of host repair (Knox et al (2003) Current Pharmaceutical Design 9, 2091-2104). It is believed that the enzyme associated with the parasite responsible for activating the compound has nitroreductase activity, for example under oxic and hypoxic conditions. In one embodiment, tetrazicar can be used to selectively inhibit those parasites that have developed resistance to one or more antibiotics currently used. In one embodiment of the invention, the animal is also administered one or more compounds known to be useful for combating a parasitic infection. The tetrazicar and one or more compounds as manifested may be administered together or in sequence. Compounds known to be useful for combating a parasitic infection include misonidazole, nitroheterocyclics such as Nifurtimox and RSU 1069, benzinidazole antimonials such as stibogluconate, and acetylcholine derivatives such as miltefosine. Thus, further aspects of the invention offer the use of a combination of tetrazicar and one or more compounds known to be useful for combating a parasitic infection of an animal in the manufacture of a medicament for combating a parasitic infection of an animal; as well as the use of tetrazicar in the manufacture of a drug to combat a parasitic infection of an animal, where the animal is administered one or more compounds known to be useful for combating a parasitic infection of an animal; as well as the use of one or more compounds known to be useful for combating a parasitic infection of an animal in the manufacture of a medicament for combating a parasitic infection of an animal, wherein the animal receives tetrazicar. The methods and medicaments of the invention find particular use to combat infections with one or more of Trypanasoma cruzi, T. brucei, Leishmania spp. in particular L. infaritum, Cryptosporidium spp. and Giardia spp. A further aspect of the invention provides the combination of tetrazicar with one or more compounds known to be useful for combating a protozoan parasitic infection of a host organism. The combination can be packaged and presented for use in medicine. In a further embodiment, the combination can be further mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition. A pharmaceutical composition may include, for example, tetrazicar and sterile, pyrogen-free water. In general, the pharmaceutical composition is in liquid form in polyethylene glycol / A / -methylpyrrolidone (PEG / NMP) diluted with saline (Cheng-Faye et al (2001) Clinical Cancer Research 7, 2662-2668). A preferred embodiment is a pharmaceutical composition for oral administration. Conveniently, the pharmaceutical composition is a gelatin capsule that contains the tetrazicar. Typically, the pharmaceutical composition is a capsule or tablet that allows for enteric release, for example by virtue of a coating that dissolves in the intestine. Methods for making such capsules and tablets are well known in the art. The one or more compounds as defined may be administered to the host organism in any suitable form and in any effective amount to combat the infection. Ideally, the treating veterinarian (in the case of non-human animals) or treating physician (in the case of humans) can analyze the appropriate administration route and the appropriate dose or dosage regimen. An amount of tetrazicar is administered, either as a single or multiple doses, in an amount effective to combat the parasitic infection. For administration to a mammal (including humans), appropriate routes of administration include no intravenous, transdermal, and inhalation restrictions. Generally, for administration to a human by infusion, tetrazicar could be administered in a dose of up to 30 mg / m2. Oral administration is also adequate, such as using a gelatin capsule as discussed above. Tetrazicar can be given by a variety of routes depending on the nature and location of the infectious agent. Accordingly, a variety of compositions of different pharmaceutical form is provided, as would be understood by one skilled in the art.

The invention will now be described in more detail by reference to the following figures and non-restrictive example. Figure 1 shows the activity of tetrazicar against HU3 of L. donovani in BALB / c mice (daily IP administration). Figure 2 shows the activity of tetrazicar against HU3 of L. donovani in BALLB / c mice (daily IV administration). Figure 3 shows the activity of tetrazicar against HU3 of L. donovani in SCID mice (daily IV administration).

EXAMPLE 1 Antiparasitic activity of tetrazicar In vitro activity The tetrazicar was analyzed against a series of parasitic organisms using an integrated in vitro analysis system and compared against the treatment of choice against that organism (Table 1). As shown in Table 1, tetrazicar is extremely active against Leishmania infantum and Trypanasoma cruzi and is much more active than established agents. Cytotoxicity assays against a range of human cell lines always gives an IC50 value>. 50μ? and the therapeutic relationships against these two organisms are dramatic (> 16,000 for T. cruzi and> 625 for L. infantum). Although not as potent as suramin, tetrazicar was active against T. brucei with a therapeutic relationship > 10. No activity was observed against the tested strains of T. colubriformis or Plasmodium falciparum in theory because it does not express an enzyme systems that is capable of activating tetrazicar in a cytotoxic form. Given the proven clinical acceptability of tetrazicar, this agent represents a novel prodrug approach to the treatment of parasitic infestation, in particular leishmaniasis and Chagas disease.

TABLE 1 In vitro data to tetrazicate against certain parasites In vivo activity The in vivo activity of tetrazicar against L. donovani was tested in BALB / c and SCID mice. The infection and treatment of the animals was performed as described by Croft and Yardley and their references (Croft &Yardley (1999) Animal models of viceral leishmaiasis, Handbook of Animal Models of Infection, Zak, O. (ed) pp 783- 787, Academia Press, London). At the end of the treatment the mice were weighed to have an estimate of drug toxicity. The liver of freshly killed animals was removed and weighed. Smears were then prepared from the livers in microscope slides and fixed with methanol and stained with Giemsa stain. The number of parasites per 500 liver cells was determined microscopically for each animal tested. This figure is multiplied by the total weight of the liver (mg) and this figure (the Leishman-Donovan unit (LDU)) is used as a basis to calculate the difference in parasite load between treated and untreated animals (Croft & amp;; Yardley, 1999 supra). As shown in Figures 1 to 3, there is a dramatic log / linear response to the dose of parasite inhibition with tetrazicar concentration. No drug-related toxicity was observed as a function of body weight loss in these experiments and no significant toxicity was observed until a dose of 10 mg / kg x 5 was used. The ED5o values obtained are shown in the table 2. The equivalent activity of tetrazicar in immunodeficient BALB / c and SCID mice shows that the therapy is not immune dependent. This is would forecast from the proposed mechanism of action. The stibogluconate in its MTD (15 mg / kg x 5 SC) was used as the control compound for these experiments. This dose only achieves an inhibition of 52 ± 15.2% in parasite counting in BALB / c mice and little effect in the SCID mouse model. The in vivo efficacy of stibogluconate is known to depend on T lymphocytes (Murray et al (1993) Antimicrob Agents Chemother, 37, 1504-505). The activity of tetrazicar is markedly greater than that of standard anti-leishmanial drugs in mouse models (compare the data in Table 1 with those of (Croft &Yardley, 1999 supra.) Tetrazicar is also effective against T. cruz] In vivo As shown in Table 3, infected but untreated BALB / c mice survived only an average of 13.8 days At a dose of 0.3 mg / kg (IP x 5, daily) all mice survived for 50 days before However, at this dose, parasites could still be detected in the blood at day 13. A dose of 3.0 mg / kg (IP x 5, daily) cleared all parasites from the blood. a dose of 45 mg / kg (po x 5 daily) of benzinidazole to produce an equivalent effect.

TABLE 2 Activity of tetrazicar against HU3 of L. donovani TABLE 3 Activity of tetrazicar against T cruzi Given the approved clinical acceptability of tetrazicar, this agent represents a novel prodrug approach for the treatment of parasitic infestation, in particular leishmaniasis and Chagas disease.

Methods of in vitro analysis Chagas disease: in vitro analysis model of T.cruzi (MHOM / CL / 00 / Tulahuen); T. cruzi (MHOM / BR / 00 / Y) Parasite and cell cultures Trypanosoma cruzi (MHOM / CL / 00 / Tulahuen) transfected with the β-galatosidase gene (Lac Z) (Buckner et al (1996) Antimicrobial Agents and Chemotherapy 40 (11), 2592-2597) was used. The strain was maintained in a cell layer L-6 (rat skeletal myoblasts cell line obtained from the European Animal Cell Culture Deposit (ECACC, Salisbury, United Kingdom)) in RPMI 1640 without phenol red medium added to it. % heat inactivated fetal calf serum All cultures and assays were performed at 37 ° C under a 5% C02 atmosphere in air.

Drug Sensitivity Tests Stock solutions of tetrazicar in 100% DMSO (dimethylsulfoxide) at 20 mg / ml were prepared. The solutions were kept at room temperature in the dark prior to use. For the tests, the compound was further diluted to the appropriate concentration using a complete medium. The assays were performed in sterile 96-well microtiter plates, and each well contained 100 μ? of medium with 2x103 of L cells 6. After 24 hours, 50 μ? of a Trypanosoma suspension containing 5x103 of trypomastigote forms from the culture to the wells. 48 hours later the medium was removed from the wells and replaced with 100 μ? of fresh medium with or without serial drug dilution. After 72 hours of incubation the plates were inspected under an inverted microscope to ensure the growth of the controls and their sterility, as well as to determine the minimum inhibitory concentration (MIC): this is the lowest concentration of drug to which they can not be observed Trypanosomas with normal morphology compared to control wells. Nifurtimox was used as the reference drug. The CPRG / Nonidet substrate (50 μ?) Was added to all wells. A color reaction became visible at 2-6 hours and was read photometrically at 540 nm. The results, expressed as percentage of reduction in parasite loads compared to control wells, were transferred to a graphics program (EXCEL), the sigmoidal inhibition curves were determined and the IC5o values were calculated.

Primary analysis The compounds were tested in triplicate in four concentrations (30-10-3-1 μg / ml). Nifurtimox was included as the reference drug. The compound is classified as inactive when IC50 is greater than 15 μg / ml. When IC50 is between 15 and 5 μg / ml, the compound is consider as moderately active. When IC50 is less than 5 μg / ml, the compound is classified as highly active and is further evaluated in a secondary analysis.

Secondary analysis The same protocol was used and ICsoS was determined using an extended and adjusted dose scale as appropriate.

Leishmaniasis: in vitro analysis Parasite and cell cultures A strain of Leishmania spp. (Leishmania donovani MHOM / ET / 67 / L82, also known as LV9, HU3). The strains are kept in the Syrian hamster (Mesocricetus auratus). Amastigotes were collected from the glass of an infected hamster and the load of parasites in the spleen was evaluated using the Stauber technique. Primary mouse peritoneal macrophages (CDI) were collected 1 or 2 days after stimulation for macrophage production with an intraperitoneal injection of 2 ml of 2% soluble starch. All cultures and tests were carried out at 37 ° C under an atmosphere of 5% C02.

Drug Sensitivity Tests 20 mg / ml of a tetrazicar stock solution in 100% DMSO was prepared and maintained at room temperature in the dark. The solution was prediluted to 60 μg / ml in RPMI 1640 + 10% heat inactivated fetal calf serum. The assays were performed on tissue culture plates of 16 sterile wells, each containing 50 μ? of the dilutions of the compound together with 100 μ? of the macrophage / parasite inoculum (4x105 macrophages / ml and 4x 06 parasites / ml). The inoculum was prepared in RPMI-1640 medium, added with 10% heat inactivated fetal calf serum. The growth of the parasites was compared with the control wells (growth of parasites at 100%). After 5 days of incubation, the growth of the parasites was evaluated under a microscope after staining the cells with a 10% Giemsa solution. The infection / well level was evaluated by counting the number of infected macrophages per 100 macrophages. The results expressed as percent reduction in the parasite load compared to the control wells were transferred to a graphics program (EXCEL), the sigmoidal inhibition curves were determined and the IC5o values were calculated.

Primary analysis The compounds were tested in quadruplicates in four concentrations (30-10-3-1 μg / ml). Pentostam® (sodium stibogluconate) was included as the reference drug.

The compound is classified as inactive when IC50 is greater than 15 μ? /? T. When IC50 is between 15 and 5 μg / ml, the compound is considered as moderately active. When IC50 is less than 5 μg / ml, the compound is classified as highly active and is further evaluated in a second analysis.

Secondary analysis The same protocol was used, and IC50 was determined using an extended dose range and adjusted as appropriate. Pentostam® was included as the reference drug.

Claims (16)

NOVELTY OF THE INVENTION CLAIMS

1. - The use of tretrazlcar in the manufacture of a drug useful for combating a parasitic protozoan infection of an animal.

2. - The use as claimed in claim 1, wherein the animal is a mammal.

3. - The use as claimed in claim 2, wherein the mammal is a human.

4. The use as claimed in any of the preceding claims, wherein the parasite that causes the infection is associated with an enzyme system capable of activating tetrazicar in a cytotoxic form.

5. The use as claimed in claim 4, wherein the enzyme is a nitroreductase.

6. - The use as claimed in any of the preceding claims, wherein the animal is one that does not endogenously contain an enzyme system that activates tetrazicate to a degree that causes unacceptable toxicity to the animal.

7. - The use as claimed in any of the preceding claims, wherein the parasite that causes the infection is resistant to antibiotics.

8. - The use as claimed in claim 2, wherein the parasitic infection of the animal is caused by any one or more of the following parasites: Trypanosoma cruzi, T. brucei, Leishmania spp.l in particular L. infantum, Cryptosporidium and Giardia ssp

9. The use as claimed in claim 1, wherein the medicament is also adapted to be administrable with one or more compounds known to be useful for combating a parasitic infection.

10. The use of a combination of tetrazicar and one or more compounds known to be useful for combating a protozoan parasitic infection of an animal in the manufacture of a medicament useful for combating a parasitic infection of an animal. eleven .

The use of tetrazicar in the manufacture of a medicament useful for combating a protozoan parasitic infection of an animal, wherein the medicament is adapted to be administrable with one or more compounds known to be useful for combating a parasitic infection of an animal.

12. The use of one or more compounds known to be useful for combating a protozoan parasitic infection of an animal in the manufacture of a medicament useful for combating a parasitic infection of an animal, wherein the animal is administered tetrazicar.

13. A combination of tetrazicar and one or more compounds known to be useful for combating a protozoan parasitic infection of a host organism.

14. - The combination according to claim 13, further characterized in that the host organism is an animal.

15. A combination according to claim 14 for use in medicine.

16. A pharmaceutical composition comprising the combination as defined in claim 14 and a pharmaceutically acceptable carrier.