WO2010020276A1

WO2010020276A1 - Treatment of coccidian parasites

Info

Publication number: WO2010020276A1
Application number: PCT/EP2008/060849
Authority: WO
Inventors: Andreas Obwaller; Oliver Szolar
Original assignee: Orphanidis Pharma Research Gmbh
Priority date: 2008-08-19
Filing date: 2008-08-19
Publication date: 2010-02-25

Abstract

The invention provides a medicament for treatment of coccidiosis of human or vertebrate animals comprising a therapeutically effective amount of miltefosine either alone or in combination with immunomodulatory and/or anti-inflammatory substances.

Description

Treatment of coccidian parasites

The present invention relates to compositions for the treatment and prevention of protozoal infections, specifically infections with apicomplexan parasites, specifically with coccidian parasites as Cryptosporidium sp., or Eimeria sp. or Toxoplasma sp. or Neospora sp. comprising miltefosine as antimicrobial substance. Therapeutic efficacy or tolerability of miltefosine can be enhanced by additional administration of. immunomodulatory and/or antiinflammatory substances as glycyrrhizine or gycyrrhetinic acid or derivatives thereof.

Coccidian parasites of the genera Cryptosporidium or Eimeria infect the mucosal epithelium of a variety of vertebrate hosts, including humans. Infection results from the ingestion of the resistant and infective oocyst stages, which release sporozoites that invade the epithelial cells. The infection may spread throughout the gut, which includes the gastric mucosa and the small and the large intestines, or it may remain localized in segments of the small and/or large intestine. The extent of spread and the sites involved determine whether the infection is clinical or subclinical as well as the overall intensity of the disease. Toxoplasma gondii, and Neospora caninum are related coccidians that can cause systemic illness in many species of animals and in humans.

Toxoplasmosis

Infection is mainly acquired by ingestion of food or water that is contaminated with oocysts shed by cats or by eating undercooked or raw meat containing tissue cysts. Primary infection is usually subclinical but in some patients cervical lymphadenopathy or ocular disease can be present. Infection acquired during pregnancy may cause severe damage to the fetus. In immunocompromised patients, reactivation of latent disease can cause life-threatening encephalitis. The most commonly used therapeutic regimen, and probably the most effective, is the combination of pyrimethamine with sulfadiazine and folinic acid. The treatment of pregnant women who acquire infection during gestation, fetuses or infants who are congenitally infected or latent infections in immunocompromized patients still bear a number of unresolved problems.

Neosporosis

Neospora caninum is an apicomplexan parasite causing neosporosis that affects mainly cows and dogs, although this protozoan has been detected in many other species worldwide. Infection with N. caninum is associated with abortions in both dairy and beef cattle throughout the world and can have a major economic impact on affected herds. The percentage of cattle exposed to N. caninum varies based on geography, with some herds exhibiting seroprevalence rates approaching 100%.

Treatment of neosporosis is rather problematic. Recent studies have shown the in vitro efficacy of thiazolides, in particular nitazoxanide (NTZ), against N. caninum tachyzoites NTZ has been FDA-approved for the treatment of equine myeloencephalitis caused by another member of the apicomplexans, Sarcocystis. However, there can be major side effects in horses treated with NTZ, such as severe enterocolitis.

Eimeriosis

Eimeriosis, often designated as coccidiosis, is the disease caused by Eimeria parasites resulting in severe mucosal damage, weight loss and sometimes even death. The disease is widespread and many species are found in poultry, livestock and small animals such as rabbits.

Infections with Eimeria sp. confined to the distal ileum and/or the large bowel can often result in intermittent diarrhoea or even be asymptomatic. Infections may often involve the pyloric region of the gastric mucosa. Parasite forms displace the microvillus border and eventually lead to the loss of the mature surface epithelium. The rapid loss of surface epithelium causes marked shortening and fusion of the villi and lengthening of the crypts due to acceleration of cell division to compensate for the loss of cells. The combined loss of microvillus border and villus height diminishes the absorptive intestinal surface and reduces uptake of fluids, electrolytes and nutrients from the gut lumen.

In general, therapeutic treatment of eimeriosis is considered to be ineffective because the mucosal lesions are already present in the intestine, so tissue damage due to coccidiosis will inevitably result in diarrhoea and clinical disease.

The clinical manifestations of cryptosporidiosis vary according to the host's immunological status. In otherwise healthy individuals, Cryptosporidium spp. typically cause watery or mucoid diarrhoea with abdominal pain that can last several days and occasionally several weeks. The patient in general presents with low fever, colic abdominal pain, headache, loss of appetite, nausea, vomiting and diarrhoea, which is secretory and disabsorptive leading to the loss of various liters of fluid per day. Clinical cure does not coincide with parasito logical cure, since oocyst elimination can last for several weeks. In immunosuppressed patients, including patients with AIDS, the clinical presentation of this protozoosis is severe and consists of intense diarrhoea, with several daily bowel movements, accompanied by voluminous loss of fluid, dehydration and marked weight loss. These manifestations last for several weeks, and cryptosporidiosis can be considered incurable in HIV-infected patients in the advanced phase of the disease.

In immunocompetent patients the therapy of choice is nitazoxanide (Alinia®, Romark), which is approved since 2002 in the USA and in several countries in Latin America. Nitazoxamide is a synthetic agent that has demonstrated activity against a broad range of parasites as well as some bacteria, however no beneficial effect could be found in immunocompromized patients for treatment of cryptosporidiosis. The use of the highly active substance monensin is not approved .

A number of substances for the treatment of cryptosporidiosis have been tested, some of the studies showed encouraging results in pilot studies, in controlled clinical trials none of these substances showed beneficial effect. To date the treatment of cryptosporidiosis in immunocompromised patients is based on the use of antimicrobial agents, passive immunotherapy and immune reconstitution.

There is no antimicrobial chemotherapeutic agent that will reliably eradicate the organism. Mycin, and (most recently) nitazoxanide are commonly used. However, these antiparasitic drugs are at best moderately effective, no benefit could have been demonstrated in AIDS patients. There have been a number of clinical trials over the last few years, but in most cases results were mixed, contrasting or difficult to reproduce, and very few were double-blind clinical trials, which enrolled a very limited number of participants. Spiramycin, a macro lide antibiotic, produced encouraging results in pilot studies, but its efficacy in HIV-positive patients has not been demonstrated in controlled clinical trials. Azithromycin treatment was found to be effective in some studies (children and patients with chronic cryptosporidiosis), whereas other studies reported no significant response. Clarithromycin and rifabutin, alone or in combination, demonstrated some efficacy as prophylactic agents, and a decrease in the risk of cryptosporidiosis was observed. Paromomycin, anaminoglycoside antibiotic that is poorly absorbed from the gastrointestinal tract, has been one of the most widely used agents to treat cryptosporidiosis in AIDS patients however, results of different trials were inconsistent. Only the advent of highly active antiretro viral therapy (HAART) with immune restoration has had a remarkable impact in cryptosporidiosis. The relative hazard of death for cryptosporidiosis decreased after introduction of HAART, for patients failing or without access to HAART however, no effective drug for treatment of crytosporidiosis exists. Several intracellular parasites as coccidia have evolved strategies to inhibit apoptosis by modulation of the expression of signal proteins of apoptosis. Apoptosis, a form of physiological cell death that is used to remove unwanted cells, is a normal part of development and tissue homeostasis. In addition, apoptosis has been recognized as an important defence mechanism to eliminate damaged or infected cells by intracellular pathogens.

The detailed molecular mechanisms by which intracellular parasites cause disease are mostly unknown. An enterotoxinlike activity has been detected in fecal extracts, which may cause abnormal absorption and secretion and impaired epithelial permeability, but no enterotoxin has been purified.

Specific attachment to the apical surface of epithelial cells by the C. parvum sporozoite, as well as molecules inserted into host cells after its attachment, appears to activate secondary signal pathways in the host cell, thereby altering cell function. C. parvum, Eimeria, Neospora and Toxoplasma species also activate the nuclear factor k B (NF£ B) and COX2 system in directly infected biliary epithelial cells. Release of NF£ B-associated cytokines and chemokines has a critical role in the pathogenesis of inflammation associated with cryptosporidiosis or coccidosis.

Thus, it appears that Cryptosporidium or Eimeria species possesses a complex virulence capacity to invade epithelial cells and induce survival signals (e.g., NF£ B) in the infected cells, so that the organism can propagate, while simultaneously triggering alterations (e.g., apoptosis) in uninfected neighboring cells to impair the absorptive and secretive functions of epithelial cells, thus causing disease. This may moreover suggest that COX2 and NF-kB mediate the inflammatory response owing to infection with coccidian parasites. This indicates that pharmacological interventions that can inhibit COX2 and NF-kB may prove beneficial in the treatment of infections with coccidian parasites. However, suppression of inflammatory reactions of the host might also abrogate the natural immune response against the parasite and may also have diametral therapeutic effects.

Miltefosine was referred as anti-protozoal agent for the treatment of visceral and cutaneous leishmaniosis (WO9937289). In WO2007/071658 a huge number of phospholipid analogues including miltefosine was listed amongst many other substances for the treatment of infectious diseases including gastrointestinal coccidian infections like cryptosporidiosis or eimeriosis, yet no information was disclosed showing any efficacy of miltefosine against one of the said infectious agents.

Although nitazoxamide has been approved for the treatment of immunocompetent patients suffering from cryptosporidiosis, no benefit could be objected in immunocompromized patients after treatment with this substance. Thus, there is still a high need on effective therapeutic drugs.

It has been surprisingly found that miltefosine is highly effective against diseases induced by apicomplexan parasites, specifically by the coccidian parasites Cryptosporidium sp., Eimeria sp, Toxoplasma sp. or Neospora sp., specifically by Cryptosporidium parvum, Toxoplasma gondii or Neospora caninum.

The term "sp." in connection with the microorganisms is used to comprise all members of a given genus, including species, subspecies and others.

The present invention therefore provides a medicament comprising miltefosine for the treatment of coccidiosis, more specifically of cryptosporidiosis, eimeriosis, neosporosis or toxoplasmosis.

Figures:

Figure 1 shows inhibitory effects of miltefosine on replication of C. parvum, compared to monensin and buffer control.

Figure 2 shows inhibitory effects of miltefosine on replication of T. gondii compared to Atovaquone (Wellvone) und the combination of Pyrimethamin/Sulfadiazine.

The present invention provides a medicament comprising miltefosine for the treatment of infections with coccidian parasites, specifically for the treatment of cryptosporidiosis, eimeriosis toxoplasmosis or neosporosis. Miltefosine can therefore be used for the treatment of coccidiosis diseases in animals and specifically in humans.

Alkylphosphocho lines (APCs), such as miltefosine, are a group of compounds consisting of phosphocholine esterified to various long-chain aliphatic alcohols and exhibit in vitro and in vivo antineoplastic activity with different biologic effects, including induction of cell differentiation and apoptosis. Whereas the majority of the conventional anticancer drugs may cause severe side effects due to bone marrow suppression, miltefosine is known to exert minimal hematologic toxicity. Also normal resting vascular endothelial cells are not affected by miltefosine.

Although the precise mechanism responsible for miltefosine induced cytotoxicity is unknown, the plasma membrane has been identified as the primary site of action of these compounds. In vitro studies demonstrate that miltefosine stimulates T cells and macrophages to secrete activating cytokines, including interferon (IFN)-gamma and enhances macrophage production of microcidal reactive nitrogen and oxygen intermediates. Intracellular killing was retained in T cell deficient mice suggesting that miltefosine induced visceral leishmanicidal effect does not require host T cell-dependent or activated macrophage- mediated mechanisms.

According the invention miltefosine can be administered in any form suitable for the treatment of coccidiosis, preferably it is formulated as a tablet, a capsule or a sachet.

Miltefosine can be applied in a solid or fluid form.

The term therapeutically effective amount of miltefosine is defined as a daily application of the drug between 4 and 2000 mg, preferably between 20 and 10000 mg, preferably between 10 and 500 mg.

Miltefosine is well absorbed after oral administration and is widely distributed. However, gastrointestinal symptoms such as vomiting (38 %) and diarrhoea (20 %) can occur. Gastrointestinal symptoms could be of more consequence in severely ill patients such as those who are malnourished and dehydrated.

According to a specific embodiment of the invention, the medicament comprising miltefosine, can be combined with immunomodulatory and/or inflammatory components, preferably a substance of the group consisting of glycyrrhicine, glycyrrhetinic acid and derivatives thereof.

Glycyrrhizic acid (GL), the glycoside of glycyrrhetinic acid (GA), is one of the major ingredients in licorice, which is one of the most often used drugs in traditional Chinese medicine (TCM) prescriptions and also used as sweetener. The first report of medicinal use licorice comes from Greeks, who recommended it for the treatment of gastric and peptic ulcers. In Asia and Europe, the extract is used in the treatment of psoriasis. Licorice is used to relieve 'Vata' and 'Kapha' inflammations, eye diseases, throat infections, peptic ulcers, arthritic conditions, and liver diseases in Indian Ayurveda system. GL has been used in the treatment of hepatitis and chronic hepatitis. and is also effective against allergic disorder and gastric. Both GL and GA possess the anti-inflammatory activity.

The term therapeutically effective amount of anti-inflammatory substance is defined as daily application of the drug between 500 mg and 100 g of glycyrrhizine or glycyrrhetinic acid, preferably between 1 mg and 20 g. The drugs or a combination of drugs can be applied in a solid or fluid form.

By combining miltefosine with other immunomodulatory and/or anti-inflammatory compounds the therapeutic efficacy and tolerability can be enhanced. These compounds specifically can be glycyrrhicine, glycyrrhetinic acid and derivatives thereof Specifically also in view of the fact that immunocompromised patients mostly die due to the loss of water and ions and not due to tissue damage induced by the parasites. Additionally, a very recent study showed that miltefosine inhibits replication of the HI-virus (Chugh et al. 2008, Retro virology, 5: 11) which could have also positive effects on treatment of cryptosporidiosis in AIDS patients.

One approach of this invention is the elimination of the parasite by combination treatment with miltefosine and the attenuation of life-threatening diarrhoea by treatment with glycyrrhizine or gycyrrhetinic acid.

According to the invention the medicament can be used for treatment of animals and humans, more specifically for animals of the group consisting of lambs, calves, pigs, rabbits, chicken dogs, cows, sheep, goats and horses.

The medicament according to the invention can also comprise miltefosine and a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means approved by regulatory authorities like FDA or EMEA. The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which the preparation is administered. Saline solutions, dextrose and glycerol solutions as liquid carriers or excipients like glucose, lactose, sucrose or any other excipients as known in the art to be useful for pharmaceutical preparations can be used. Additionally, also stabilizing agents can be included to increase shelf live of the medicament.

Examples The following examples are describing the invention in more detail without limiting the scope of the invention

Example 1 :

The in vitro efficacy of miltefosine on C. parvum multiplication in human ileocaecal adenocarcinoma cells (HTC-8) was tested. For this purpose 5x10⁴ C. parvum oocysts were directly excysted on HCT-8 cell monolayers and then incubated with different concentrations of miltefosine, monensin (positive control) and buffer control. The C. parvum multiplication in infected cells was measured by using real time PCR based on quantitative detection of parasite HSP 70 gene.

Example 2:

We tested the in vitro efficacy of miltefosine, glycyrrhizin and glycyrrhetinic acid on C. parvum multiplication in human ileocaecal adenocarcinoma cells (HTC-8). For this purpose

5x10⁴ C. parvum oocysts were directly excysted on HCT-8 cell monolayers and then incubated with different concentrations of miltefosine, glycyrrhizin and glycyrrhetinic and monensin as control. In previous experiments treatment with 0.5μg miltefosine showed similar effects as buffer control and was determined as was ineffective. The C. parvum multiplication in infected cells was measured by using real time PCR based on quantitative detection of parasite HSP 70 gene.

The multiplication of Cryptosporidium developmental stages was reduced after 48h incubation with miltefosine, miltefosine/GL, miltefosine/GA and monensin. GL and GA alone showed no or moderate reduction.

The control substance monensin showed highest inhibition, GL and GA showed only moderate inhibition. Best inhibition showed the combination miltefosine/GA (Tab 1).

Tab. 1 : Number of DNA-copies of C. parvum detected after incubation with test substances

Monensin 0.1 μg 4.071

Miltefosine 0.5μg/ml 576.600

Miltefosine 2μg/ml 25.100

Miltefosine 5 mg/ml 16.530

Miltefosine 1 Oμg/ml 11.160

GL 84 μg/μl 339.600 GL 420 μg/μl 283.800

GA 2,4 μg/ml 259.500

GA 9,6 μg/ml 236.000

GL 84 μg/μl + Miltefosine 5 μg/ml 16.780

GL 420 μg/μl + Miltefosine 5 μg/ml 13.620

GA 2,4 μg/ml + Milefosine 5 μg/ml 12.620

GA 9,6 μg/ml + Miltefosine 5 μg/ml 10.820

Example 3 :

We tested the efficacy of miltefosine of Toxoplasma gondii replication in the mouse model. Mice were infected with T. gondii oocysts and then treated with miltefosine, and standard therapy (atovaquone and pyrimethamine/sulfadiazine). Miltefosine showed a reduction of the replication similar or superior to standard therapy (Fig 2).

Example 4:

We tested the efficacy of miltefosine against the replication of N. caninum in- vitro and in vivo. Therefore HFF-cells are infected with the coccidian parasite Neospora caninum and treated with miltefosine. Proliferation of parasites are determined by quantitative PCR. In in- vivo experiments, mice are infected with N. caninum and treated with different doses of miltefosine as described by Gottstein et al. (Parasitol Res, 2001. 87(1): p. 43 - 8). Efficacy of treatment was determined by detection of parasite specific DNA, assessing clinical symptoms and histopathological findings.

Claims

1) A medicament comprising miltefosine for the treatment of coccidian parasites.

2) Medicament according to claim 1 wherein the parasites are selected from the group consisting of Cryptosporidium sp., Eimeria sp, Toxoplasma sp. and Neospora sp..

3) Medicament according to claims 1 or 2, wherein miltefosine is formulated as a tablet, a capsule or a sachet.

4) Medicament according to any one of claims 1 to 3, wherein miltefosine is contained in an amount between 4 to 2000 mg, preferably in an amount between 10 and 500 mg.

5) Medicament according to any one of claims 1 to 4 comprising an immunomodulatory and/or inflammatory component, preferably a substance of the group consisting of glycyrrhicine, glycyrrhetinic acid and a derivative thereof.

6) Medicament according to any one of claims 1 to 5, wherein glycyrrhizine or glycyrrhetinic acid or a derivative thereof is contained in an amount between 500 mg and 100 g, preferably between 1 mg and 20 g.

7) Use of miltefosine for manufacturing a medicament for the treatment of coccidian parasites.

8) Use according to claim 7 wherein the parasites are selected from the group consisting of Cryptosporidium sp., Eimeria sp, Toxoplasma sp. and Neospora sp..

9) Use according to claims 7 or 8, wherein miltefosine is formulated as a tablet, a capsule or a sachet.

10) Use according to any one of claims 7 to 9 wherein miltefosine is contained in an amount between 4 to 2000 mg, preferably in a amount between 10 and 500 mg.

11) Use according to any one of claims 7 to 10 comprising an immunomodulatory and/or inflammatory component, preferably a substance of the group consisting of glycyrrhicine, glycyrrhetinic acid and a derivative thereof. 12) Use according to any one of claims 7 tol 1 wherein glycyrrhizine or glycyrrhetinic acid or a derivative thereof is contained in an amount between 500 mg and 100 g of, preferably between 1 mg and 20 g.