US 2011 0118176A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/011817.6 A1 Harder et al. (43) Pub. Date: May 19, 2011

(54) NOVEL POSSIBILITY OF CONTROLLING (30) Foreign Application Priority Data DISEASES CAUSED BY Jul. 2, 2008 (DE) ...... 10 2008 O31 283.5 (75) Inventors: Achim Harder, Koeln (DE); Gisela Greif, Remagen (DE); Robrecht Publication Classification Froyman, Monheim (DE) (51) Int. Cl. (73) Assignee: Bayer Animal Health GMBH A638/00 (2006.01) A6IP33/10 (2006.01) 21) Appl. No.: 13AOOO,871 (21) Appl. No 9 (52) U.S. Cl...... 514/46 (22) PCT Filed: Jun. 20, 2009 (57) ABSTRACT (86). PCT No.: PCT/EP09/04474 The present invention relates to the use of nifurtimox for the S371 (c)(1), treatment of diseases caused by trichomonads, Such as, for (2), (4) Date: Dec. 22, 2010 example, histomoniasis, in particular in turkeys. US 2011/01 1817.6 A1 May 19, 2011

NOVEL POSSIBILITY OF CONTROLLING the most frequent disease in squabs and causes severe dam DISEASES CAUSED BY TRICHOMONADDA age, in particular in breeding flocks. Besides the high mortal ity, symptoms which are observed are digestive disorders, inappetence, reduced consumption of drinking water and 0001. The present invention relates to the use of nifur feed, and limited ability to fly. gallinarum para timox for the treatment of diseases caused by Trichomona sitizes in the appendix of chickens and turkeys. The disease dida and Diplomonadida Such as, for example, histomoniasis, causes delayed growth, severe diarrhoea and necrotic inflam in particular in turkeys. mation of the liver. 0002 The efficacy of nitro-heterocyclic compounds 0007. Histomoniasis is an infectious disease which is against protozoan diseases is known in principle (1). caused by . Histomonads are gut 0003. The include single-nuclear organisms parasites. Histomoniasis is especially important in turkeys, whose basic structure is a eukaryotic cell. The more precise where it is also referred to as blackhead disease. In turkeys, systematics, however, reveal large differences inhabit, mor the disease is caused in particular by the pathogen Histomo phology and the biochemical metabolism of the individual nas meleagridis. Besides turkeys, others which may become strains, classes, genera and species. This is why chemicals, infected with the pathogen include chickens, guinea fowl, depending on their target and active principle, usually do not peacocks, pheasants, partridges and quails, which are also act equally well againstall Protozoa, but only against specific reservoir hosts. groups of Protozoa (2, 3, 4). 0008 Infection with Histomonas meleagridis results in 0004 To date, the efficacy of nifurtimox has only been severe inflammation of the appendiX and liver, since the described against protozoan species of the genus Trypano pathogen damages the gut tissue and, via the blood, reaches Soma, e.g. bruceii and Trypanosoma cruci (5. the liver, where it causes the development of necrosis. An DE-AS-1 170957). Trypanosoma have a which accompanying symptom of the disease is frequently circula originates on the basal body (“kinetosome, hence order ) and, in conjunction with the basal body, tory failure, which can be identified by the blackish-blue develops an undulating membrane. Trypanosoma grow pre heads of diseased animals, which gives the disease its name. dominantly in the blood plasma and are transmitted by blood 0009. In infected flocks, for example in poultry production Sucking arthropods. These pathogens cause units, the disease very rapidly spreads to the entire flock and (“) of humans. Nifurtimox is currently leads to severe economic losses as the result of very high almost the only compound which acts against these patho mortality rates (which may be as high as 100%). gens. This activity is probably based on inhibiting the enzyme 0010. Owing to its structural flagella, Histomonas melea reductase, a Trypanosoma-specific enzyme. gridis belongs to the Subtribe (Mastigophora) and This enzyme is absent in other protozoan pathogens, in par to the order Trichomonadida. The stages multiply in ticular trichomonads and histomonads. the appendix by division. Starting from the infected appendix, 0005 Within the order Trichomonadida and Diplomona amoeboid-like stages penetrate, via the bloodstream, the dida, the efficacy of nifurtimox has not been described to date. liver, which they destroy via large necroses (7). Trichomonadida are all parasitic Protozoa, for which a plu 0011 Transmission of histomonads via the direct route, rality of, as a rule 4 to 6, flagella are typical. A pronounced for example the oral uptake of histomonad-containing fresh morphological feature is a contractile rod (costa) within the faeces, is rare since outside a host the pathogens are only organisms, which is involved in their movement. In contrast viable for a short time, and since upon passing through the to the Kinetoplastida. Trichomonadida do not have mitochon digestive tract, most of them are killed. Tests carried out by dria, which are important organelles of energy metabolism. American researchers have revealed that in animal experi Instead, the energy metabolism takes place in what are known ments infection in turkeys is much more likely to take place as . In these organelles, the oxidative decar via the cloaca than via the oral route. Since the cloaca gener boxylation of pyruvate is coupled with ATP synthesis and a ates a slight pull after faeces have been deposited, infection ferredoxin-controlled electron transport (6). These unicellu via this route is likely to occur under practice conditions, for lar parasites multiply by division. No sexual stages or cysts example via Soiled litter. The transmission of pathogens via are found. The order Trichomonadida includes many genera intermediate hosts has been proven scientifically beyond (in particular the genera Trichomonas and Histomonas) and doubt. It is in particular the caecal worm Heterakis galli furthermore many species, but most of these are rather harm narum (eggs or larvae) which is known as vector (in particular less and nonpathogenic. However, there are representatives as transport vector of Histomonas meleagridis). Histomonads which trigger severe diseases and cause important economi may remain infectious up to 4 years in embryonated Heter cal damage in animal keeping. These include the genus Tri akis eggs. Further intermediate hosts may be earthworms and chomonas (in particular T. gallinae and T. gallinarum), the arthropods which are contaminated with Heterakis eggs. genus Tritrichomonas (in particular T. foetus and Tsuis) and Another potential risk are chickens and other poultry species. the genus Histomonas (in particular H. meleagridis). They are less sensitive than turkeys and frequently carry the 0006 Trichomonosis of pigeons and of domestic fowl is pathogen without being clinically susceptible; in this way, an infectious disease caused by and T. they contribute to the spreading of the pathogen. gallinarum. T. gallinae parasitizes primarily in the pharynx, 0012 Turkeys can become infected at any age; however, in the oesophagus and in the crop. During the course of the the disease occurs most frequently between age 3 and 12 disease, however, other organs, mainly liver, heart and lung, weeks. The period between infection and appearance of the are also infested. The infection of young pigeons takes place disease is in most cases 7-12 days. Mortality can be as high as as early as during the first feeding with pigeon milk from 100% and reaches its maximum on day 17 post-infection. latently infected older animals. Further sources of infection From day 8, infections can be found in the appendix, from day are infested drinking water or feed. The disease is considered 10 in the liver. US 2011/01 1817.6 A1 May 19, 2011

0013 Infected animals are dull, exhausted, their heads and 0024 Nifurtimox is the compound of the formula (I): wings droop and they, refuse to eat. The passage of Sulphur coloured droppings, diarrhoea, and later even blood in the stools is typical. The circulatory disorders which are associ (I) ated with the disease cause a pronounced blackish-blue col ON O N-N SO oration of the head, which gives the disease its name. 0014. The course of the disease is determined mainly by the age and the intestinal flora of the turkeys. Additional H3C bacterial infections with E. coli, Clostridium perfringens or coccidian may aggravate the course (8). 0025 If appropriate, the use in the form of customary 0015 The diagnosis of histomoniasis can be made using pharmaceutically acceptable salts is also suitable. If appro priate, the use of hydrates or other solvates of the active native specimens from appendiX and liver with the aid of a Substances or, if appropriate, of their salts is furthermore also saline Solution. Amoeboid motile stages are discernible under suitable. the phase-contrast microscope. PAS staining is used for his 0026. The use can be both prophylactic and therapeutic. tological studies. The Trichomonadida include the genera Histomonas, Tri 0016 Up to 1950, arsenic compounds (for example nitar chomonas, Tritrichomonas. From the genus Trichomonas there may be mentioned in particular T. gallinae and T. galli son, carbarson, roXarson) were the only effective compounds narum. From the genus Tritrichomonas there may be men against histomoniasis. However, it is known that arsenic com tioned in particular T. foetus, T suis and T. equi. From the pounds are generally not potent enough to treat established genus Histomonas there may be mentioned, in particular, H. infections. A further disadvantage is that their safety index is meleagridis. extremely low: even twice the dosage of roXarson leads to 0027. The Dipomonadida include the genus . disturbed motor functions in turkeys. From the genus Hexamita, there may be mentioned in par ticular H. Columbae, H. meleagridis and H. Salmonis. 0017. Since 1960, other nitroimidazoles or nitrofurans 0028. It is preferred to control histomoniasis. It is caused have been employed, for example in the feed or drinking by Histomonas spp. It is very especially preferred to control water, but they have been increasingly banned by the EU and in accordance with the invention histomoniasis caused by the USA for use in livestock and us feed additives since the Histomonas meleagridis. The activity of nifurtimox in the mid-90s: dimetridazole was withdrawn from the US market control of histomoniasis is not only directed against the gut pathogenic stages, but also against the liver stages of the in 1997, and banned for use as feed additive in the EU in 2001. pathogens. Since Mar. 31, 2003, even nifursol, the only product still 0029 Organisms which are treated in accordance with the licensed in the EU, may no longer be employed owing to invention are animals. Examples which may be mentioned are safety concerns. Thus, neither pharmaceuticals for therapy mammals such as, for example, cattle, horses, pigs, dogs, nor preparations for the prophylactic control of histomoniasis cats. It is preferred to treat poultry Such as, for example, are available currently and in the future. chickens, guinea fowl, partridges, quails, ducks, geese, pea cocks, pheasants, pigeons and in particular turkeys (synony 0018. The only currently available strategies of avoiding mously used for turkey-cocks and turkey-hens). contracting the disease consist in hygiene measures, optimi Examples of Diseases which May be Emphasized are the Zation of the stocking density and of the nutrient Supply, and Following: the avoidance of spreading the pathogen. These measures are 0030 Trichomonosis of pigeons, turkeys or of domestic insufficient and, on their own, cannot prevent infection and fowl, caused by T. gallinarum and/or T. gallinae. disease. 0031 Histomoniasis in chickens, guinea fowl, peacocks, 0019 Vaccines against histomoniasis are not available. pheasants, partridges, quails and in particular in turkeys. His For example, a vaccination against Histomonas meleagridis tomoniasis in turkeys (blackhead disease) is caused in par is biologically impossible since even natural immunity can ticular by H. meleagridis. not be acquired after infection. Once infected, animals can 0032. The active substances are applied directly or in the fall ill again. Attempts to immunize via attenuated live vac form of Suitable preparations via the enteral, parenteral or cines failed. dermal route. 0033. The enteral administration of the active substances 0020. There is therefore a need for active substances for is effected for example orally in the form of powders, Sup the treatment of diseases caused by Trichomonadida, such as, positories, tablets, capsules, pastes, drinks, granules, for example, histomoniasis, which active Substances have drenches, boluses, medicated feed or drinking water. Dermal good activity and good toxicological properties. administration is effected for example in the form of dipping, 0021 Surprisingly, we have now found that nifurtimox is spraying, bathing, Washing, pouring on and spotting on and active against Trichomonadida, nifurtimoX also having good dusting. Parenteral administration is effected for example in toxicological properties. This activity has not been described the form of an injection (intramuscular, Subcutaneous, intra to date, and the good toxicological properties have not been venous, intraperitoneal) or by implants. expected either. Suitable Preparations are: 0022 The Invention Relates to: 0034 solutions such as solutions for injection, oral solu 0023 The use of nifurtimox for the preparation of phar tions, concentrates for oral administration after dilution, Solu maceuticals for the treatment of diseases caused by Tri tions for use on the skin or in body cavities, pour-on and chomonadida. spot-on formulations, gels; US 2011/01 1817.6 A1 May 19, 2011

0035 emulsions and suspensions for oral or dermal tically active in vivo when the treatment is carried out from administration and for injection; semi-solid preparations; the time of infection. The usual anthelmintic treatment is for 0.036 formulations in which the active substance is incor 14 days from the point of time of infection. By a combined porated in an ointment base or in an oil-in-water or water-in treatment together with nifurtimoX and anthelmintics, an oil emulsion base; improved treatment of diseases caused by trichomonads can 0037 solid preparations such as powders, premixes or therefore beachieved. concentrates, granules, pellets, tablets, boluses, capsules; 0056 Anthelmintics which may be mentioned are benz aerosols and inhalers, active-substance-containing shaped imidazoles Such as albendazole, fenbendazole or probenzimi articles. dazoles such as febantel. These substances are active for 0038 Solutions for injection are administered for example example against Heterakis spp., in particulars against Heter intravenously, intramuscularly and Subcutaneously. akis gallinarum, which is known to act as transport vector of 0039 Oral solutions are administered directly. Concen trates are administered orally after previously having been Histomonas meleagridis (10). diluted to the use concentration. 0057. Others which may be mentioned are imidazolethia 0040 Solutions for use for application to the skin are Zoles (levamisol, tetramisol), tetrahydropyrimidines (pyran trickled on, painted on, rubbed on, Splashed on, sprayed on or tel, morantel, oxantel), amidine derivatives, for example ami applied by dipping, bathing or washing. dantel, tribendimidine, and the deacylated amidantel 0041 Gels are applied to or painted onto the skin or intro derivative Bay d9216, and aminoacetonitrile derivatives (see, duced into body cavities. for example, Kaminsky et al., Nature 452, 176-180 (13 Mar. 0042 Pour-on and spot-on formulations are poured onto 2008)), such as, for example, AAD 1470. or spotted onto limited areas of the skin, the active Substance 0.058 Preferred substances which may be mentioned either penetrating the skin and acting systemically or distrib among the anthelmintics are depsipeptide anthelmintics. uting on the body Surface. Depsipeptide anthelmintics such as PF1022A and emodep 0043 Emulsions are either of the water-in-oil type or of side have a broad anthelmintic activity against nematodes in the oil-in-water type and can be applied orally, dermally or as various animal species such as gallinaceous birds, rodents, injections. reptiles, dogs, cats, sheep, cattle, goats, horses (11, 12, 13. 0044 Suspensions can be applied orally, dermally or as an 14). Here, it has been demonstrated that PF1022A and injection. emodepside are active against nematodes of the Superfamily 0045 Semi-solid preparations can be administered orally Heterakoidea. These include for example from the family or dermally. They differ from the above-described suspen Heterakidae, besides Heterakis gallinarum in chickens, the sions and emulsions merely by the fact that they are more murine nematode Heterakis spumosa. PF1022A is active Viscous. against the latter for example at an oral dose of 50 mg/kg, and 0046. To prepare solid preparations, the active substances emodepside for example in a dose range of 1-10 mg/kg (13. are mixed with suitable excipients, if appropriate with addi 15). Furthermore, for example PF1022A has been demon tion of additives, and formulated as desired. strated to be active against a further representative of the 0047. Especially preferred in accordance with the inven Superfamily Heterakoidea, Ascaridia galli in chickens, which tion is the use in poultry. This is preferably done by oral belongs to the family Ascaridiidae. Here, the compound acts administration, in particular via medicated feed or the drink at a dose of 2 mg/kg (16). These substances from the class of ing water. the cyclic depsipeptide compounds are therefore suitable for 0048 All the abovementioned pharmaceutical forms, the the prophylactic control of, in particular, histomoniasis. additives and adjuvants to be used and the preparation of these 0059. Depsipeptide-anthelmintics which are preferably pharmaceutical forms are known in principle to the skilled employed are 24-membered cyclodepsipeptides. The follow worker. ing may be mentioned: 0049. The active substances can exist in combination with 0060 Compounds of the formula (IIa) synergists or with further active substances. Further active Substances which may be mentioned are: 0050 Coccidiostats such as robenidine or amprolium, in (IIa) Some cases in combination with folic acid antagonists (for O example ethopabate, pyrimethamine, epiroprim); 0051 polyether antibiotics such as monensin, salinomy cin, lasalocid, narasin, semduramicin or maduramicin; -, -- 0052 triazinones, such as toltraZuril, ponazuril or dicla Zuril; 0053 sulfonamides (sulfaquinoxalin, sulfadimidin, sulfa diazin). 0054 For a long-time treatment effect, it is recommended to disinfect regularly before housing the animals or after finishing the fattening period. 0055 Helminths, in particular Heterakis spp. (larvae) (9) or.O act as transport vectors in the transfer of histomonads. In the treatment of histomoniasis, it may therefore make sense to carry out a combined treatment together with anthelmintics. 0061 in which Thus, it is known that anthelmintics such as, for example, the 0062 Z represents hydrogen, N-morpholinyl, NH, benzimidazoles albendazole or fenbendazole are prophylac mono- or dimethylamino. US 2011/01 1817.6 A1 May 19, 2011

0063 Moreover, compounds of the following formula (IIb) may be mentioned:

(-COH), (IIb) carboxamide,

(-O-C-NH),

imidazolyl, indolyl, guanidino, -SH or C-alkylthio, and which furthermore represent aryl or aralkyl, each of which can be substituted by halogen, hydroxyl, C-alkyl, Ca alkoxy, 0070. R. R. R. R' independently of one another represent hydrogen, straight-chain C-s-alkyl, C-alk in which enyl, C-7-cycloalkyl, each of which can optionally be Substituted by hydroxyl, C-alkoxy, carboxyl, carboxa 0064) R', R. R. R. independently of one another repre mide, imidazolyl, indolyl, guanidino, SH or C-alkylthio. sent hydrogen, C-Co-alkyl or aryl, in particular phenyl, and representaryl or aralkyl, each of which can be substi each of which is optionally substituted by hydroxyl. tuted by halogen, hydroxyl, C-alkyl, C-alkoxy, C-Co-alkoxy or halogen. and their optical isomers and racemates. 0065. The compounds of the general formula (IIb) are 0071 Preferred compounds of the formula (IIe) are those known and can be obtained by the processes described in in which R', R", R'' and R' independently of one EP-A-382173, DE-A 4317 432, DE-A 4 317 457, DE-A 4 another represent methyl, ethyl, propyl, isopropyl. n-, s-, t-bu 317458, EP-A-634 408, EP-A-718293, EP-A-872481, EP tyl or phenyl, each of which is optionally substituted by A-685 469, EP-A-626 375, EP-A-664. 297, EP-A-669 343, halogen, C-alkyl, OH, C-alkoxy, and represent benzylor EP-A-787 141, EP-A-865498, EP-A-903 347. phenylethyl, each of which can optionally be substituted by 0066. The cyclic depsipeptides with 24 ring atoms also the radicals mentioned for phenyl: include compounds of the general formula (Ic) 0072 R to R' have the abovementioned meanings. 0073 Especially preferred compounds of the formula (IIc) are those in which R', R. R'' and R' independently of one another represent methyl, ethyl, propyl, isopropyl or n-, (IIc) S-, t-butyl. R5a RI la O 0074 R. R. R', R represent hydrogen, straight chain or branched Cis-alkyl, in particular methyl, ethyl, N propyl, i-propyl. n-, S-, t-butyl, each of which can option O O ally be substituted by C-alkoxy, in particular methoxy, O - O R6a R7a ethoxy, imidazolyl, indolyl or C-alkylthio, in particular R4a O methylthio, ethylthio, furthermore represent phenyl, ben Zyl or phenethyl, each of which can optionally be substi tuted by halogen, in particular chlorine, 0075 R', R, R, R' independently of one another represent hydrogen, methyl, ethyl, n-propyl. n-butyl, vinyl, cyclohexyl, each of which can optionally be substituted by methoxy, ethoxy, imidazolyl, indolyl, methylthio, eth ylthio, and represent isopropyl. S-butyl, and furthermore represent optionally halogen-substituted phenyl, benzyl or phenylethyl. 0076. The compounds of the formula (IIc) can also be 0067 in which obtained by the processes described in EP-A-382173, DE-A 0068 R'', R, R'' and R' independently of one 4317 432, DE-A4317457, DE-A4317 458, EP-A-634 408, another represent Cis-alkyl, Cs-haloalkyl, C-cy EP-A-718 293, EP-A-872 481, EP-A-685 469, EP-A-626 cloalky, aralkyl, aryl, 375, EP-A-664. 297, EP-A-669 343, EP-A-787 141, EP-A- 0069. R. R. R', R' independently of one another 865498, EP-A-903 347. represent hydrogen or a straight-chain or branched C 0077. A very especially preferred depsipeptide which may alkyl which can optionally be substituted by hydroxyl, be mentioned is the compound PF 1022, which is known from C-alkoxy, carboxyl, EP-OS 382 173; it is the compound of the formula (IIa) in US 2011/01 1817.6 A1 May 19, 2011 which both substituents Z represent hydrogen. PF 1022 there I0082. The following may furthermore optionally be used: fore has the following formula (IId): salts of the active Substances with pharmaceutically accept able acids or bases, and also solvates, in particular hydrates, of the active substances or of their salts. 0083 Use in combination means either that nifurtimox (IId) and the second active Substance, in particular a cyclodep sipeptide, can be employed separately or staggered. In this case, nifurtimoX and the second active Substance are formu lated respectively as a separate pharmaceutical. I0084. The simultaneous use is also feasible. According to a use form which is suitable for this case, the active sub stances of the combination are formulated together in one composition. I0085 Ready-to-use preparations usually contain the active Substance in question in concentrations of 10 ppm to 20% by weight, preferably from 0.1 to 10% by weight. I0086 Preparations which are diluted prior to use contain the active Substance in question in concentrations of from 0.5 to 90% by weight, preferably from 5 to 50% by weight. In concentrated Solutions for metering into the drinking water, the active Substance in question is present for example in concentrations of from 0.5 to 20% by weight, preferably 1 to 15% by weight, especially preferably 2 to 10% by weight. 0078. Further preferred depsipeptides are compounds I0087. In general, it has proved advantageous to administer which are disclosed in the PCT application WO 93/19053, amounts of from approximately 0.05 to approximately 200 which are compounds of the formula (IIa) in which mg, preferably from 0.1 to 100 mg. ofactive Substance per kg 0079 represents N-morpholinyl, NH, mono- or dimethy body weight per clay in order to achieve effective results. lamino. I0088. In the mixture with other coccidiostats or polyether 0080 Very especially preferred among these compounds antibiotics, the active substances according to the invention is the depsipeptide emodepside (PE 1022-221). This is the are generally present in the weight ratio 1 to 0.01-50 up to 1 compound of the formula (IIa) in which both radicals Z rep to 1-50. resent the morpholinyl radical. The INN emodepside repre 0089. The active substances can also be administered sents the compound with the systematic name: cyclo(R)- together with the animals' feed or drinking water. lactoyl-N-methyl-L-leucyl-(R)-3-(p-morpholinophenyl) (0090 Feed and foodstuffs contain 0.005 to 1000 ppm, lactoyl-N-methyl-L-leucyl-(R)-lactoyl-N-methyl-L-leucyl preferably 0.05 to 500 ppm, of the active substance in com (R)-3-(p-morpholinophenyl)lactoyl-N-methyl-L-leucyl. bination with a suitable edible material. Emodepside is described in WO 93/19053 and has the fol 0091 Such a feed and foodstuff can be used both for lowing formula: therapeutic and for prophylactic purposes.

0081. Depending on their structure, the abovementioned 0092 Such a feed or foodstuff is prepared by mixing, with active substances which are suitable for the combination may customary feeds, a concentrate or a premix which comprises be present in Stereoisomeric forms or as stereoisomer mix 0.5 to 30% by weight, preferably 1 to 20% by weight, of an tures, for example as enantiomers or racemates. Both the active Substance in admixture with an edible organic or inor Stereoisomer mixtures and the pure stereoisomers can be used ganic carrier. Edible carriers are, for example, maize meal or in accordance with the invention. maize and soya meal or mineral salts which preferably com US 2011/01 1817.6 A1 May 19, 2011

prise a small amount of an edible anti-dust oil, for example 0100 Such a feed contains 18% crude protein, 5% crude maize oil or soya oil. The premix thus obtained can then be fibre, 1% Ca. 0.7% Pand, per kg, 1200 IU vitaminA, 1200 IU added to the complete feed before the latter is fed to the vitamin D3, 10 mg vitamin E, 20 mg zinc bacitracin. animals. 0093. The Use in Histomoniasis May be Described by REFERENCES Way of Example: 0101 1. Raether W., Hänel H. (2003): Nitroheterocyclic 0094 For the curative treatment and prophylaxis of histo drugs with broad spectrum activity Parasitol Res. 90:19 moniasis in poultry, in particular in chickens, ducks, geese or 39. turkeys, 0.005 to 1000 ppm, preferably 0.05 to 500 ppm, of an 0102 2. Harder A., Greif G., Haberkorn A. (2001a): Che active substance are mixed with a suitable edible material, for motherapeutic approaches to protozoa: Haemosporina— example a nutritious feed. If desired, these amounts can be current level of knowledge and outlook. increased, in particular when the active Substance is well (0103) 3. Harder A. Greif G. Haberkorn A. (2001b): Che tolerated by the recipient. The administration via the drinking motherapeutic approaches to protozoa: , Tri water can be carried out analogously. chomonas and Entamoeba-current level of knowledge 0095 Nevertheless, it may occasionally be necessary to and outlook. deviate from the abovementioned quantities, in particular as a 0104. 4. Greif G. Harder A., Haberkorn A. (2001): Che function of the body weight of the experimental animal or of motherapeutic approaches to protozoa: Coccidia—current the type of route of administration, but also depending on the level of knowledge and outlook. animal species and its individual reaction to the active Sub 0105. 5. Harder A., Greif G., Haberkorn A. (2001 c): Che stance, or on the type of formulation and the time or interval motherapeutic approaches to protozoa. Kinetoplastida— at which it is administered. Thus, it may suffice in some cases current level of knowledge and outlook. Parasitol Res to use less than the abovementioned minimum amount, while 87.778-780. the mentioned upper limit has to be exceeded in other cases. 0106 6. Kulda J. (1999): Trichomonas, hydrogenosomes When larger amounts are administered, it may be expedient to and drug resistance. International Journal for Parasitology divide them into several individual doses over the course of 29: 199-212. the day. 0107 7. McDougald L. R. (2005): Blackhead Disease 0096. The activity of the compounds according to the (Histomoniasis) in Poultry: A critical Review. Avian Dis invention can be demonstrated for example in cage experi eases 49 (4):462-476. ments with the following experimental design, where the 01.08 8. McDougald, Hu. J. (2001): Blackhead Disease animals are treated with the respective active Substance. (Histomonas meleagridis) aggravated in broiler chickens 0097. An active-substance-containing feed is prepared in by concurrent infection with cecal coccidiosis (Eimeria Such a way that the required amount of active Substance is tenella). Avian Diseases 45:307-312. mixed thoroughly with a nutritionally balanced animal feed, 0109) 9. Hegngi F. N. Doerr J., Cummings T. S., Schwartz for example with the chick feed specified hereinbelow. R. D., Saunders G. Zajac A. Larsen C. T., Pierson F. W. 0098. If it is intended to prepare a concentrate or a premix (1999): The effectiveness of benzimidazole derivatives for which is eventually to be diluted in the feed to the values the treatment and prevention of histomonosis (blackhead) mentioned in the experiment, a general procedure is to mix in turkeys. Veterinary Parasitology 81:29-37. approximately 1 to 30%, preferably approximately 10 to 20% 0110 10. Hegngi, F. N. Doeerr, J., Cummings, T. S., by weight, of active Substance with an edible organic or Schwartz, R. D., Saunders. G. Zajac, A., Larsen, C. T., inorganic carrier, for example maize and soya meal or mineral Pierson. F. W. (1999): The effectiveness of benzimidazole salts, which contain a small amount of an edible dc-dusting derivatives for the treatment and prevention of histomono oil, for example maize oil or Soya oil. The premix thus sis (blackhead) in turkeys. Veterinary Parasitology 81:29 obtained can then be added to the complete poultry feed 37. before being administered. 0111 11. Von Samson-Himmelstjerna G. Harder A., 0099. A suitable example for the use of the substances Schnieder T. Kalbe.J. Mencke N. (2000): In vivo activities according to the invention in poultry feed is the following of the new anthelmintic depsipeptide PF1022A. Parasitol. composition: ReS. 86:194-199. 0112 12. Mehlhorn H. Nicolay F., Harder A. von Sam son-Himmelstjerna A. (2000): Synergistic action of Bay S2.00% crushed feed grain: 40% maize, 12% wheat 44-4400 and piperazine on nematodes of the mouse invitro 17.00% extracted Soybean meal and in Vivo: a light and transmission electron microscopic S.00% maize gluten feed study. Parasitol. Res. 86:982-992. S.00% wheat feed meal 3.00% fish meal 0113 13. Harder A. Schmitt-Wrede H.-P. Kricken J., 3.00% mineral blend Marinovski P., Wunderlich F., Willson J., Amliwala K., 3.00% lucerne-grass meal Holden-Dye L. Walker R. (2003): Cyclooctadepsipep 2.50% vitamin blend tides—an anthelmintically active class of compounds 2.00% comminuted wheat germs 2.00% Soya oil exhibiting a novel mode of action. Int. J. Antimicrobial 2.00% meat-bone meal Agents 22:318-331. 1.50% powdered whey 0114 14. Mehlhorn H. Schmahl G., Frese M., Mevissen 1.00% molasses I., Harder A. Krieger K. (2005): Effects of a combination 1.00% brewer's yeast mixed with spent grain emodepside and praziquantel on parasites of reptiles and 100.00% rodents. Parasitol. Res. 97 Suppl 1:65-69. 0115 15. Bernt U., Junkersdorf. B., Londershausen M., Harder A., Schierenberg E. (1998): Effects of anthelm US 2011/01 1817.6 A1 May 19, 2011

intics with different modes of action on the behaviour and development of Caenorhabditis elegans. Fundam. Appl. TABLE 1 Nematol. 21:251-263. 011 6 16. Sasaki T. Takagi M. Yaguchi T. Miyadoh S, Okada T. Koyama S (1992): A new anthelmintic cyclodep Study design 210 animals sipeptide, PF1022A. Journal of Antibiotics 45:692-697. Treatment EXAMPLES Medicated Feed Group Code Compound Concentration Day Infection 0117 Medicated feeds can be prepared by admixing pull Verulent nifurtimox in concentrations of 50, 100, 200 and 400 Uninf. D untreated ppm to the feed mix detailed hereinbelow. Inf. contr. F untreated -- 30 ppm E nifurtimox 30 ppm -4 to +14 -- 60 ppm B inifurtimox 60 ppm -4 to +14 -- S2.00% crushed feed grain: 40% maize, 12% wheat 120 ppm C inifurtimox 120 ppm -4 to +14 -- 17.00% extracted Soybean meal 200 ppm G nifurtimox 200 ppm -4 to +14 -- S.00% maize gluten feed S.00% wheat feed meal Nita. A nitarSone 187.5 ppm -4 to +14 -- 3.00% fish meal 3.00% mineral blend 3.00% lucerne-grass meal 2.50% vitamin blend 2.00% comminuted wheat germs TABLE 2 2.00% Soya oil 2.00% meat-bone meal Study design 210 animals 1.50% powdered whey 1.00% molasses Treatment 1.00% brewer's yeast mixed with spent grain

100.00% Group Code Compound Concentration Day Infection

Uninf. G untreated Inf. contr. A untreated -- Tablets 100 ppm D nifurtimox 100 ppm -4 to +14 -- 200 ppm F nifurtimox 200 ppm -4 to +14 -- 0118 Nifurtimox tablets are known and available as a 300 ppm B inifurtimox 300 ppm -4 to +14 -- pharmaceutical for example under the trade name Lampit R. 400 ppm C inifurtimox 400 ppm -4 to +14 -- A. Biological Example Nita. E nitarsone 187.5 ppm -4 to +14 -- Cage Experiments; Histomonad-Activity in Turkeys 0119 Histomonad-free male turkeys aged 10 days TABLE 3 received nifurtimox or the comparative compound nitarson in the concentration given in “ppm together with the feed from Mortality caused by histomoniasis day -4 (-4 days prior to infection) to day 14. Infection is Treatment Mortality Treatment Mortality carried out on day 0, 10 animals are kept per group cage. 1 to 3 Such groups are employed per dose. inf. contr. 7 inf. contr. 11 30 ppm 5 100 ppm 13 0120. The infection is carried out with a histomonad field 60 ppm 6 200 ppm 4 strain which is passaged in the laboratory and stored in liquid 120 ppm 5 300 ppm 2 nitrogen. On day 0, in each case 5 animals of a cage (with the 200 ppm 4 400 ppm O exception of the noninfected control) are infected intracloa Nita. 1 Nita. 2 cally with 250 000 histomonads in each case 1 ml of Dwyer's medium (-direct infection). After a few days, these infected animals excrete fresh histomonads and thus transmit the TABLE 4 pathogen to the remaining 5 animals of the cage (indirect infection). Mean weight gain (g) between day O and the time of death 0121 To assess the activity, the criteria of McDougald and Hu 2001 (7) are taken into consideration: Direct infection Direct infection 0.122 the infection-caused mortality Treatment O yes Treatment O yes I0123 the weight gain from the beginning to the end of Uninf. 791 uninf. 819 the experiment Inf. contr. 741 197 inf. contr. 666 259 0.124 feed consumption 30 ppm 730 235 100 ppm 581 135 0.125 feed conversion 60 ppm 838 304 200 ppm 525 270 120 ppm 744 182 300 ppm 750 450 0.126 macroscopic assessment of the infection-related 200 ppm 744 264 400 ppm 832 691 lesions in the appendices (caecum) and in the liver. In Nita. 782 541 Nita. 725 426 this assessment, score 0 no lesions and score 4-severe lesions. US 2011/01 1817.6 A1 May 19, 2011

B. Biological Example TABLE 5 Anthelmintic Properties of the Cyclic Octadepsipep Mean feed consumption between day 0 and day 14 tides Feed Feed consumption consumption I0127. Male mice (strain Bor CFW, body weight between Treatment (kg) Treatment (kg) 25 and 30 g) are kept in Makrolon cages (3 animals/cage) and fed ad lib with water and SNIFF rat food (10 mm pellets). Uninfected control 12.01 Uninfected control 12.44 Infected control 8.84 Infected control 9.S6 Mice are infected with Heterakis spumosa by oral adminis 30 ppm 9.OO 100 ppm 6.69 tration of 90 embryonated eggs. The eggs had been obtained 60 ppm 9.68 200 ppm 9.14 from female worms which had been isolated from the mices 120 ppm 8.70 300 ppm 10.40 colon 40 days post-infection. The eggs were incubated for a 200 ppm 9.59 400 ppm 11.79 further 3 weeks at 37° C. 35 days post-infection, the mice Nita. 11.64 Nita. 9.82 were treated with the respective dose of PF1022A oremodep side on four consecutive days. PF1022A is suspended in Cremophor EL. On day 7 after the treatment, the mice are TABLE 6 sacrificed, the ileum/caecum/colon region is removed, and the worms are counted with the naked eye. The ratio of the Mean feed conversion between day 0 and day 14 number of expelled worms in percent of the total number of Feed worms in untreated, infected control animals is defined as the Treatment conversion Treatment Feed conversion measure for the anthelmintic activity. Uninfected control 1.52 Uninfected control 1.63 I0128. The experiments on chickens are described in (14) Infected control 1.89 Infected control 2.10 and the literature cited therein. 30 ppm 1.88 100 ppm 2.32 60 ppm 1.70 200 ppm 2.33 TABLE 9 120 ppm 1.88 300 ppm 1.83 200 ppm 1.91 400 ppm 1.55 Anthelmintic activity of PF1022A and emodepside against Nita. 1.77 Nita. 1.79 nematodes from the Superfamily Heterakoidea Nematode Dose with full activity TABLE 7 PF1022A Average lesions in the appendix Heterakis spinosa (mouse) 50 mg/kg Ascaridia gali (chicken) 2 mg/kg Direct infection Direct infection Emodepside

Treatment O yes Treatment O yes Heterakis spinosa (mouse) 1-10 mg/kg Uninfected O.O3 Uninfected O.OO control control Infected control O.29 3.93 Infected control O.93 2.93 30 ppm O.29 3.73 100 ppm 1.08 3.79 60 ppm O.OO 3.53 200 ppm 18O 3.67 1-6. (canceled) 120 ppm O.S3 4.00 300 ppm 0.79 2.53 7. A pharmaceutical formulation comprising nifurtimox 200 ppm O.47 3.80 400 ppm 0.27 1.20 and an anthelmintic. Nita. O.OO 2.26 Nita. O.29 3.07 8. The pharmaceutical formulation of claim 7, wherein the anthelmintic is a depsipeptide. 9. A method of treating a disease caused by Trichomona TABLE 8 dida in an animal comprising administering to the animal in Infection-related lesions in the liver need thereofnifurtimox. 10. A method of treating a disease caused by Histomonads Direct infection Direct infection in an animal comprising administering to the animal in need Treatment O yes Treatment O yes thereofnifurtimox. 11. The method of claim 10, wherein the Histomonads are Uninf. O.OO uninf. O.OO Inf. contr. O.29 3.47 inf. contr. O.14 18O caused by the pathogen Histomonas meleagridis. 30 ppm O.29 2.53 100 ppm O.S8 2.00 12. The method of claim 9, wherein the Trichomonadida is 60 ppm O.OO 2.53 200 ppm 0.73 1.33 of the genus Trichomonas. 120 ppm 0.27 18O 300 ppm O.O7 O.40 200 ppm O.13 1.33 400 ppm O.OO O.OO 13. The method of claim 9, wherein the Trichomonadida is Nita. O.OO O.13 Nita. O.21 0.79 of the genus Tritrichomonas.

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