HK1158113B - Use of nifurtimox for treating giardiasis - Google Patents

Description

Use of nifurtimox for the treatment of giardiasis

Technical Field

The present invention relates to the use of nifurtimox for the treatment of giardiasis (giardiase), in particular in dogs and cats.

Background

The efficacy of nitroheterocyclic compounds for protozoal diseases is known (1).

Protozoa include mononuclear organisms whose basic structure is a eukaryotic cell. However, more specific taxonomies show great differences in the origin, morphology and biochemical metabolism of the lines, classes, genera and species. This is why, depending on their target and active principle, the chemical substances generally do not act equally well on all protozoa, but only on a certain few groups of protozoa (2, 3, 4).

To date, only nifurtimox has been described as having efficacy against protozoan species of the genus trypanosome, such as trypanosoma brucei and trypanosoma cruzi (5). Trypanosomes have flagella originating from a substrate ("hairy substrate") and attach to the substrate to produce a wavy film. In protozoan systematics, parasites of this basic morphological type belong to the order of the kinetoplastids. Trypanosomes grow primarily in plasma, spreading through blood-sucking arthropods. These pathogens cause chagas disease ("trypanosomiasis") in humans. Nifurtimox is now almost the only compound active against these pathogens. This activity may be based on inhibition of the enzyme trypanothionine reductase, a specific trypanosome enzyme. This enzyme is absent from other protozoan pathogens.

The activity of nifurtimox against trichomonas is described in the parallel patent application documents.

Giardiasis is an infectious disease caused by flagellated, unicellular parasites of the genus giardia. This genus belongs to the order of the Trichomonas. The most important representatives are Giardia lamblia (e.g. Giardia lamblia, Giardia duodenalis). The percentage of positive dog and cat samples, worldwide, is up to 50%, in central europe 2-7%. Infection results in persistent diarrhea and in some cases bloody stool due to inflammation of the duodenum and jejunum, especially in very young animals up to half the year of age. Chronic disease can lead to a delay in current growth (6).

More than 7 genotypes (a-G) have been described in giardia-isolates. Genotype A + B is human-infected and can also occur in cats, dogs, beaver, sheep, calves, horses, pigs and monkeys. This is why this disease is considered to be an "zoonotic disease": humans can become infected with the cysts of dogs and cats through contaminated drinking water. Giardia infections are the most common causative agent worldwide (7) leading to outbreaks of disease in humans by so-called "drinking water infections" (waterborn outbreaks).

Infection is typically by the oral route, by contaminated drinking water or infected faeces, in the form of cysts. In the digestive tract, so-called trophozoites hatch from the cysts. Trophozoites were measured at 11-17 x 7-11 μm, containing 2 nuclei and 8 flagella (flagella). Unlike other types of flagellated parasites, giardia trophozoites do not penetrate intracellularly into epithelial cells. Trophozoites have basal sockets with which they can immobilize themselves outside the digestive tract of humans, monkeys, pigs, dogs and cats, where they divide and reproduce. Extensive attacks can block and alter the absorptive surface of the digestive tract, in some cases resulting in bloody diarrhea. Cysts are regenerated in the cecum and are excreted again through the feces 5-16 days after the first infection. Cysts can be excreted for a period of 4-5 weeks and remain infected for a period of weeks (8, 9).

The important morphological and biochemical differences of the order of the kinetoplast are: the order trichomonas has 8 flagella and two nuclei, but no mitochondria, nor golgi complex, and is known to have no intracellular phase in the life cycle. In contrast to trypanosomes, the surface protein of giardia lacks a GPI anchor (10).

In dogs and cats, metronidazole (Clont) is currently recommended at a dose of 12.5-22 mg/kg body weight^®，Flagyl^®，Elyzol^®) Treatment was given 2 times daily for 5 days (11). Metronidazole and other 5-nitroimidazoles are activated by pyruvate-ferredoxin-oxidoreductase to form free nitro radicals which then act on the metabolism of the parasite DNA. That is why Isonidazole (Iproxan)^®126 mg/l drinking water, over 7 days) and tinidazole (Fasigyn)^® _，44 mg/kg body weight over 3 days) are also suitable as causes of giardiasis in dogs (12, 13).

The recommended substances in the benzimidazole class are mebendazole, albendazole or fenbendazole (orally for 3 days). Benzimidazoles interfere with tubulin polymerization by binding to beta-tubulin subunits. Tubulin is an important cytoskeletal component of the parasite and stabilizes the basal socket of trophozoites in a particular manner.

Substances for use in the human segment include antibiotics (e.g., paromomycin, 25-35 mg/kg/day, 3 times daily, 7-10 days), mepacrine and furazolidone (100 mg t.i.d., 7 days long) or nitazoxanide (500 mg b.i.d., up to 3 days) (14).

Many of these types of active substances have been used for a long time in the treatment of giardiasis, and for the most part the development of resistance can be detected and has been shown to lead to therapeutic failures. Therefore, the development of new active substances and therapeutic concepts is essential (15).

Disclosure of Invention

Surprisingly, we have now found that nifurtimox has activity against giardia species. This activity has not been described to date. This activity acts directly on the pathogenic stages of the digestive tract and prevents cyst formation.

Accordingly, the present invention relates to:

use of nifurtimox in the manufacture of a medicament for the treatment of a disease caused by giardia.

Nifurtimox is a compound of formula (I):

optionally, it is also appropriate to use the compound in the form of a pharmaceutically acceptable salt which is commonly used. It is likewise suitable to also optionally use hydrates or other solvates of the active substances, or optionally, of their salts.

The use may be prophylactic or therapeutic. This activity acts directly on the different stages of the pathogen; in particular, nifurtimox acts on the pathogenic stages of the digestive tract and prevents cyst formation.

Particularly important Giardia species are lamblia (syn. Giardia intestinalis), Giardia duodenalis (Giardia duodenalis), Giardia bovis (Giardia bovis) and Giardia capricorn (Giardia caprae). The species listed here are often used synonymously with each other, they show little host specificity.

Treated according to the invention are animals (carnivores and wildlife), preferably mammals, such as, for example, horses, pigs, rabbits, in particular dogs or cats.

According to one embodiment, the mammal to be treated is preferably a wild animal, in particular a carnivorous animal (dog, cat).

Humans are also amenable to treatment because humans can infect the pathogenic giardia species through contaminated drinking water.

Giardiasis is found primarily in young animals, preferably 3-10 weeks old, and results in severe diarrhea and reduced weight gain.

The active substance is used directly or in the form of a suitable preparation by enteral, parenteral or transdermal route.

By enteral active substance is meant, for example, oral administration in the form of powders, suppositories, tablets, capsules, lozenges, drinks, granules, drenches, boluses, medicated feed or drinking water. Transdermal administration means, for example, working in the form of immersion (dip-dip), spraying (spray), bathing, washing, coating (pouring onto and spotting onto), and brushing. Parenteral administration means, for example, acting in the form of injections (intramuscular, subcutaneous, intravenous, intraperitoneal) or by implants.

Suitable formulations are:

solutions, such as solutions for injection, oral solutions, concentrates for oral administration after dilution, solutions for application on the skin or in body cavities, drench preparations, gels;

emulsions and suspensions for oral or transdermal administration and injection; a semi-solid formulation;

formulations wherein the active substance is incorporated in an ointment base or an oil-in-water or water-in-oil emulsion base;

solid preparations, such as powders, pre-mixes or concentrates, granules, pills, tablets, boluses, capsules; aerosols and inhalants, shaped articles containing an active substance.

Solutions for injection are for example administered intravenously, intramuscularly and subcutaneously.

Oral solutions are administered directly. The concentrate is administered orally after having been previously diluted to the use concentration.

The solution for application to the skin is applied to the skin by dripping, spreading, rubbing, splashing, spraying or by immersion (dip-dip), bathing or washing.

The gel is applied or spread on the skin or introduced into a body cavity.

The coating formulation is poured or spotted onto a defined area of the skin, wherein the active substance permeates through the skin and acts systemically or is distributed over the body surface.

The emulsion is water-in-oil type or oil-in-water type, and can be administered orally, transdermally or as injection.

The suspension can be used orally, transdermally or as an injection.

The semi-solid formulations may be administered orally or transdermally. They differ from the above-mentioned suspensions and emulsions only by their higher viscosity.

To prepare solid preparations, the active substance is mixed with a suitable carrier, optionally with the addition of adjuvants, and in the desired form.

Oral administration is particularly preferred according to the invention, tablets being particularly preferred in the usual oral use forms.

All the abovementioned pharmaceutical forms, the additives and adjuvants used and the preparation of these pharmaceutical forms are basically known to the skilled worker.

The active substance may be present in combination with a synergist or with other active substances. The other active substances are:

anticoccidial agents such as robenidine or apracloniam, in some cases in combination with folic acid antagonists (e.g., pyrimethamine, epiprilin, trimethoprim); antibiotics such as, for example, clindamycin, paromomycin, or spiramycin; sulfonamides such as, for example, sulfadoxine, sulfadimidine, sulfadiazine; anthelmintics such as, for example, cyclodepsipeptides (e.g. Emodepsid, PF1022A), amidine derivatives (tripheniramidine, amirtal, Bay d 9216), praziquantel or benzyl benzoate.

Regular disinfection is recommended as part of an animal care program for long-term therapeutic effect.

Anthelmintics, in particular cyclooctadepsipeptides such as PF1022A or Emodepsid, are suitable for controlling nematode infections in humans and animals (16). In this way, all economically important nematodes in the gastrointestinal tract (also including dogs) are destroyed. Meanwhile, it is known that immune response (GALT = gut associated lymphoid tissue) is used against helminths in the case of non-treated animals (17). This is called the Th2 response of the immune system. In the non-infectious case, the immune response is usually balanced, i.e. an antagonistic way of the Th2 response against protozoa, viruses and bacteria, called Th1 response, is as high as the Th2 response.

If a nematode infection such as a duodenal, roundworm or whipworm infection is present in the animal, e.g., dog, the balance is adversely affected, with the result that there is a significant protozoan infection in the gut, e.g., caused by giardia species. If nematode infestation is now overcome by suitable anthelmintics, the defense against giardia species can be enhanced indirectly by acting on these protozoa via a Th1 response. In the combination of nifurtimox and anthelmintics, nifurtimox now shows a better control of giardia infections, since the latter part is comprised in the results of a Th1 response, wherein said Th1 response is indirectly enhanced as a result of nematode control, i.e. the number of parasites in the digestive tract has been reduced.

According to a preferred embodiment, nifurtimox is used in combination with an anthelmintic.

Preferred anthelmintics used are 24-membered cyclic depsipeptides (cyclooctadepsipeptides). As these, the following may be mentioned:

a compound of formula (IIa)

Wherein

Z represents hydrogen, N-morpholinyl, NH₂Mono-or dimethylamino.

Furthermore, mention may be made of compounds of formula (IIb):

wherein

R¹，R²，R³，R⁴Independently of one another, represents hydrogen, C₁-C₁₀Alkyl or aryl, especially phenyl, optionally substituted by hydroxy, C₁-C₁₀-alkoxy or halogen substitution.

The compounds of the formulｃA (IIb) are known and can be obtained by the processes described in EP-A-382173, DE-A4317432, DE-A4317457, DE-A4317458, EP-A-634408, EP-A-718293, EP-A-872481, EP-A-685469, EP-A-626375, EP-A-664297, EP-A-669343, EP-A-787141, EP-A-865498, EP-A-903347.

Cyclic depsipeptides having 24 ring atoms also include compounds of the general formula (IIc),

wherein

R^1a，R^2a，R^11aAnd R^12aIndependently of one another represent C_1-8Alkyl radical, C_1-8-haloalkyl group, C_3-6-cycloalkyl, aralkyl, aryl,

R^3a，R^5a，R^7a，R^9aindependently of one another, represents hydrogen or straight-chain or branched C_1-8Alkyl, each radical optionally being substituted by hydroxy, C_1-4-alkoxy, carboxyl,Carboxamides, carboxamides,Imidazole, imidazoleRadicals, indolyl, guanidino, -SH or C_1-4Alkylthio-substituted radicals, which in addition represent aryl or aralkyl radicals, each of which may be substituted by halogen, hydroxy, C_1-4-alkyl radical, C_1-4-an alkoxy group substitution,

R^4a，R^6a，R^8a，R^10aindependently of one another, hydrogen, straight chain C_1-5Alkyl radical, C_2-6-alkenyl, C_3-7Cycloalkyl, each of which may optionally be substituted by hydroxy, C_1-4Alkoxy, carboxyl, carboxamide, imidazolyl, indolyl, guanidino, SH or C_1-4Alkylthio-substituted radicals, which in addition represent aryl or aralkyl radicals, each of which may be substituted by halogen, hydroxy, C_1-4-alkyl radical, C_1-4Alkoxy substitution, as well as their optical isomers and racemates.

Preferred compounds of the formula (IIc) are those in which

R^1a，R^2a，R^11aAnd R^12aIndependently of one another, represents methyl, ethyl, propyl, isopropyl, n-, sec-, tert-butyl or phenyl, each of which is optionally substituted by halogen, C_1-4Alkyl, OH, C_1-4-alkoxy-substituted, in addition to representing benzyl or phenylethyl, each of which may be optionally substituted by the residue indicated for phenyl;

R^3a-R^10ahave the meaning as described above.

Particularly preferred compounds of the formula (IIc) are those in which

R^1a，R^2a，R^11aAnd R^12aIndependently of one another, represents methyl, ethyl, propyl, isopropyl or n-, sec-, tert-butyl,

R^3a，R^5a，R^7a，R^9arepresents hydrogen, straight or branched chain C_1-8Alkyl, in particular methyl, ethyl, propyl, isopropyl, n-, sec-, tert-butyl, each of which may optionally be substituted by C_1-4-alkoxy radicalRadicals being, in particular, methoxy, ethoxy, imidazolyl, indolyl or C_1-4Alkylthio is in particular methylthio, ethylthio substituted, furthermore represents phenyl, benzyl or phenethyl, each of which may optionally be substituted by halogen, in particular chlorine.

R^4a，R^6a，R^8a，R^10aIndependently of one another, represent hydrogen, methyl, ethyl, n-propyl, n-butyl, vinyl, cyclohexyl, each of which may optionally be substituted by methoxy, ethoxy, imidazolyl, indolyl, methylthio, ethylthio, furthermore isopropyl, sec-butyl, furthermore phenyl, benzyl or phenylethyl, which are optionally substituted by halogen.

The compounds of the formulｃA (IIc) can also be obtained by the processes described in EP-A-382173, DE-A4317432, DE-A4317457, DE-A4317458, EP-A-634408, EP-A-718293, EP-A-872481, EP-A-685469, EP-A-626375, EP-A-664297, EP-A-669343, EP-A-787141, EP-A-865498, EP-A-903347.

The very particularly preferred depsipeptides are the compound PF1022 known from EP-OS 382173; it is a compound of formula (IIa) in which both substituents Z represent hydrogen. PF1022 therefore has the following formula (IId):

other preferred depsipeptides are the compounds known from PCT application WO 93/19053, which are compounds of the formula (IIa),

wherein

Z represents N-morpholinyl, NH₂Mono-or dimethylamino.

Very particularly preferred among these compounds is the depsipeptide Emodepside (PF 1022-. This is a compound of formula (IIa) wherein both residues Z represent morpholinyl residues. INN Emodepside represents a compound with the following system name: cyclo [ (R) -lactyl-N-methyl-L-leucinyl- (R) -3- (p-morpholinophenyl) lactyl-N-methyl-L-leucinyl- (R) -3- (p-morpholinophenyl) lactyl-N-methyl-L-leucinyl. Emodepside is described in WO 93/19053 and has the formula:

depending on their structure, the active substances mentioned above in suitable combinations can be present in stereoisomeric form or as a mixture of stereoisomers, for example as enantiomers or racemates. Mixtures of stereoisomers as well as pure stereoisomers may be used according to the invention.

Furthermore, the following are optionally used: salts of the active substance with pharmaceutically acceptable acids or bases, in particular solvates, in particular hydrates, of the active substance or of their salts.

When nifurtimox and a second active substance, in particular a cyclodepsipeptide, are used in combination, they can be used separately or staggered. In this case, nifurtimox and the second active substance are each formulated as separate medicaments.

Simultaneous use is also possible. The active substances of the composition are formulated in a medicament according to the use form suitable for the case.

Ready-to-use preparations generally contain the respective active substance in a concentration of from 10 ppm to 20% by weight, preferably from 0.1 to 10% by weight.

The formulations diluted before use comprise the respective active substances in a concentration of from 0.5 to 90% by weight, preferably from 5 to 50% by weight. In concentrated solutions for metered addition to drinking water, the respective active substance is present in a concentration of, for example, 0.5 to 20% by weight, preferably 1 to 15% by weight, particularly preferably 2 to 10% by weight.

In general, for effective results, it has proven advantageous to administer an amount of active substance of from about 0.05 to about 400 mg, preferably from 0.1 to 200 mg, per kg of body weight per day.

In mixtures with other anticoccidial, antibiotic or anthelmintic agents, the active substances according to the invention may be present in a ratio of 1: 0.01-50 up to 1: 1-50.

The active substance can also be administered with the feed or drinking water of the animal.

The feed and food contain 0.005-1000 ppm, preferably 0.05-500 ppm of active substance, in combination with a suitable edible substance.

Such feeds and foods may be used for therapeutic and prophylactic purposes.

The disinfectant may be used to supplement treatment with nifurtimox. The disinfectant is used to disinfect the site where the animal (or human) is located during treatment. The disinfectant preferably eliminates the resistant phase of the parasite by destroying the excreted cyst form, thus preventing reinfection after the end of the treatment. Thus, the disinfectant may be used prior to treatment with nifurtimox; however, in general, it is preferred to use it simultaneously with or at least prior to completion of nifurtimox treatment.

Disinfectants are, for example, those based on biocidal phenols and/or phenol derivatives. Biocidal phenols are understood to mean phenolic compounds which have free OH groups and have biocidal activity. These phenols may also have ring substituents such as, for example, halogen (especially chlorine), C_1-6Alkyl radical, C_3-6-cycloalkyl, phenyl, chlorophenyl, benzyl and/or chlorobenzyl.

Non-chlorinated biocidal phenols are for example: 2-methylphenol, 3-methylphenol, 4-ethylphenol, 2, 4-dimethylphenol, 2, 5-dimethylphenol, 3, 4-dimethylphenol, 2, 6-dimethylphenol, 4-n-propylphenol, 4-n-butylphenol, 4-n-pentylphenol, 4-n-hexylphenol, thymol (5-methyl-2-isopropylphenol), 2-phenylphenol, 4-phenylphenol, 2-benzylphenol. The preferred non-chlorinated biocidal phenol used is 2-phenylphenol.

Chlorinated biocidal phenols are for example: 4-chloro-3-methylphenol (PCMC, p-chloro-m-cresol), 4-chloro-3-ethylphenol, 2-n-pentyl-4-chlorophenol, 2-n-hexyl-4-chlorophenol, 2-cyclohexyl-4-chlorophenol, 4-chloro-3, 5-xylenol (PCMX, p-chloro-m-xylenol), 2, 4-dichloro-3, 5-xylenol (DCMX, dichloro-p-xylenol), 4-chloro-2-phenylphenol, 2-benzyl-4-chlorophenol, benzyl-4-chloro-m-cresol, 4-chlorobenzyl dichloro-m-cresol. Preferred chlorinated biocidal phenols are 2-benzyl-4-chlorophenol, 4-chloro-3, 5-xylenol, 2, 4-dichloro-3, 5-xylenol, especially 4-chloro-3-methylphenol.

Throughout the present invention, phenol derivatives are understood to be phenol-derived compounds in which the OH groups are derivatized, and therefore they do not contain free OH groups. They are preferably phenol ethers, in particular those of aliphatic alcohols having 1 to 6 carbon atoms. As a preferred example, phenoxyethanol may be mentioned.

Preferably, the disinfectant described in WO 2007/009606 is used, comprising a biocide and a keratolytic agent. Suitable biocides or combinations of biocides with suitable keratolytic agents are detailed in WO 2007/009606, which is hereby expressly incorporated by reference.

Detailed Description

Examples

Preparation examples

Examples 1 ( Liquid preparation ) ：

A suspension of glycerol formaldehyde/glycerol polyethylene glycol ricinoleate (Cremophor EL)/water in a mixing ratio of 100 ml of 1:10 having the following drugs:

● 500 mg nifurtimox

● 1000 mg nifurtimox

Examples 2 ( Liquid preparation ) ：

The mixing ratio in 100 ml is 1: 5 Cremophor EL/Water suspensions of the following drugs

● 500 mg nifurtimox

● 1000 mg nifurtimox

Examples 3 ( Solid preparation )

The following amounts of active substance were filled in the form of powder into gelatin capsules:

● 250 mg nifurtimox

Examples 4 ( Tablet formulation )

Nifurtimox tablets are known and are commercially available as pharmaceuticals, for example under the trade name Lampit @.

Biological Experimental example

Examples 1

Puppy, 10-11 weeks old, which had been carried into the center of the animal 11 days prior to infection, was orally infected with 50000 giardia duodenalis cysts. Cysts for infection are evacuated with the aid of a sucrose gradientObtained from faeces of giardia cathartica cyst dogs, stored in Bacto-Casitone medium at 4 ℃ for no more than 2 weeks. From day 10 post-infection, puppies were kept in separate cages to collect total daily feces from each individual. Quantitative determination of cyst excretion was performed 4 days before treatment (days-3 to 0), and puppies were randomly divided into two groups for examination of cyst excretion. On day 0, an oral dose of 50 mg/kg nifurtimox (Lampit)^®) The treatment group of 7 puppies was given 1 time, while the control group of 6 dogs remained untreated. Quantification of cyst excretion was continued on days 1 to 8 after treatment.

As a result: the activity was calculated using the formula:

the activity calculated after a single administration of nifurtimox was 90.4% (see table 1).

The method comprises the following steps: a modified version of Hewlett (18) was used for quantitative determination of cyst excretion: 4 g of faeces were dissolved in 100 ml of water, sieved and allowed to settle. The sediment was applied to a 1M sucrose gradient (specific gravity 1.13), the cysts were concentrated after centrifugation in a sucrose-water-separating layer and aspirated with a pipette. After a washing step following centrifugation, the cysts in the pellet were counted. Then, the precipitated aliquots (Aliquot) were counted under a microscope and the number of cysts per g of stool was calculated.

Table 1: giardia cyst excretion before and after treatment with nifurtimox

T-3 to T-1: each day before treatment

T0: day of treatment

T1 to T8: each day after treatment

Examples 2

Puppy of 11-15 weeks old, which had been carried into the center of the animal for at least 2 weeks prior to infection, was orally infected with 50000 giardia duodenalis cysts prior to study initiation. Cysts for infection were obtained from faeces of dogs excreting giardia cysts with the aid of a sucrose gradient and stored in Bacto-Casitone medium at 4 ℃ for no more than 2 weeks. In this study 18 puppies were included who excreted giardia cysts, and they were kept in separate cages to collect total daily feces from each individual. Quantitative determination of cyst excretion was performed as in experiment 145.717, the improvement consisting in counting precipitated aliquots under a microscope with the aid of a Fuchs-Rosenthal cytometer. Quantitative determination of cyst excretion was performed 4 days before treatment (days-3 to 0), and puppies were randomly divided into three groups for examination of cyst excretion. Three consecutive days (days 0, 1 and 2), in the morning, with an oral dose of 50 mg/kg of nifurtimox (Lampit)^®) The 6 puppies of treatment group 1 were treated, while the 6 dogs of treatment group 2 were treated with an oral dose of 50 mg/kg of fenbendazole (fenbendazole tablet) in the morning of the three days. The 6 dogs in the control group remained untreated. Quantification of cyst excretion was continued on days 1 to 8 after treatment.

As a result: the calculated activity of nifurtimox was 98.6% whereas the activity of fenbendazole was 48.3% (see table 2).

Table 2: pre-and post-treatment giardia cyst excretion with nifurtimox compared to fenbendazole treatment

T-3 to T-1: each day before treatment

T0, T1, T2: day of treatment

T3-T8: each day after treatment

Reference to the literature

Claims (2)

1. Use of nifurtimox in the manufacture of a medicament for the treatment of a disease caused by giardia.

2. Use according to claim 1 for the treatment of a disease caused by giardia lamblia.