Veterinary composition for preventing and / or treating leishmaniasis

Abstract

The present invention relates to a veterinary composition comprising artesunate or a salt thereof and a pharmaceutically acceptable excipient, which is used for preventing and / or treating canine leishmaniasis.

Description

Technical Field

[0001] The present invention relates to the field of veterinary medicine and more particularly to the field of parasitology for the treatment of leishmaniasis in dogs. Background Art

[0002] Leishmaniasis (or leishmaniasis), and more specifically, visceral leishmaniasis (VL), is a contagious, severe, chronic systemic disease. It is a major zoonosis with significant clinical and epidemiological control priorities in the world: domestic dogs are the main animal reservoir, while other wild animals such as foxes play a role in the transmission of sylvatic leishmaniasis. Leishmania infantum (also known as Leishmania chagasi) is the causative agent of canine visceral leishmaniasis (CanL) and human visceral leishmaniasis. Over the past few decades, epidemiological changes in VL have been observed, including an increase in morbidity and mortality and its spread to new areas and even urban areas. The number of cases of leishmaniasis worldwide is increasing. Some of the reasons are the lack of vaccines, the inability to control vectors, and the increasing resistance of an increasing number of parasites to chemotherapy.

[0003] CanL is a serious disease that affects millions of dogs in countries on both sides of the Atlantic (mainly in Europe and South America, but also in Africa and Asia) and can be fatal if not treated. In Mediterranean countries, where infection rates are as high as 63% (including at least 2.5 million seropositive dogs), CanL is one of the leading causes of death in dogs. It is an expanding disease and it has become one of the most important canine diseases imported into Central Europe.

[0004] The spectrum of clinical manifestations and immune responses in dogs infected with Leishmania infantum is very wide and variable. Thus, several forms can be represented by their numerous clinical signs, including cutaneous, mucocutaneous, and visceral forms.

[0005] Furthermore, Leishmania infection in dogs can manifest as a subclinical infection, as a self-limited illness, or as a severe, life-threatening illness. Infected dogs may serve as hosts and sources of infection for humans for a long period of time.

[0006] Treatment of CanL is challenging due to the intracellular localization of the parasite. Furthermore, due to the suspected development of resistance in animals with antileishmanial drugs used for human VL, the World Health Organization (WHO) has recommended that such drugs be reserved for human leishmaniasis and not for veterinary use. Nevertheless, the current first-line treatment for CanL is a combination of leishmanicidal agents used as second-line drugs in humans (e.g., pentavalent antimonials, miltefosine) and allopurinol. Some chemotherapeutic compounds used for CanL are included in the 19th edition of the WHO Model List of Essential Medicines (April 2015) for leishmaniasis: pentavalent antimonials, miltefosine, amphotericin B deoxycholate or liposomal amphotericin B, and paromomycin. However, some other drugs are also effective, such as allopurinol, pentamidine, enrofloxacin, marbofloxacin, metronidazole, spiramycin and ketoconazole. The determination of the most preferred drug is based on clinical examination, and the staging of CanL is based on the results of immunodiagnostic tests, clinical symptoms, clinical pathological abnormalities and parasite load. Most of these treatments are expensive and cannot completely cure the disease, and some of them cause important side effects. During the current treatment with pentavalent antimonials and miltefosine, effects such as vomiting, lethargy, diarrhea and nephrotoxicity are common. Long-term administration of allopurinol often induces xanthine urolithiasis. The use of antibiotics to treat leishmaniasis is also problematic because of the risk of resistance in the pathogen population. In addition, because infant Leishmania is easily transmitted from host dogs to humans, not treating infected dogs puts all people living nearby at risk.

[0007] Today, the reference regimen for the treatment of CanL uses meglumine antimoniate and allopurinol (Bourdoiseau and Denerole, Revue Med. Vet., 2000, 151, 5, 395-400; Solano-Gallego et al., Parasites & Vector, 2011, 4: 86). However, in addition to the side effects of meglumine antimoniate and allopurinol, the convenience of the reference regimen is also unsatisfactory because the reference regimen requires the administration of two products and repeated injections for weeks or even months. Such treatment regimens have the risk of compliance failure.

[0008] The antiparasitic activity of artemisinin and its derivatives (ART) has been slightly explored. For example, Loo et al. (Pharmacol Res., 2017, 117, 192-217) have reported the efficacy of ART in treating protozoan infections other than malaria (Loo et al., Pharmacol Res., 2017, 117, 192-217). Chollet et al. (Biomedicine & Pharmacotherapy, 2008, 62, 462-465) have reported the in vitro activity of fluartemisinin against the promastigote form of Leishmania donovani (L. donovani). Cortes et al. (Antimicrobial agents and chemotherapy, 2015, 59 (8), 5032-5035) have shown that the following compounds, i.e. artesunate, deoxyartesunate, dihydroartemisinin, deoxydihydroartemisinin, exhibit in vitro activity against the life stage forms (promastigotes and amastigotes) of Leishmania infantum. However, to date, specific treatment for canine leishmaniasis remains unexplored.

[0009] Therefore, new, highly effective, safe, and affordable drugs are urgently needed to treat canine leishmaniasis. Summary of the invention

[0010] Herein, the inventors have unexpectedly demonstrated the therapeutic significance of artesunate in the treatment of canine leishmaniasis. More specifically, compared to a reference injectable treatment comprising a combination of meglumine antimoniate and allopurinol, the inventors have provided a more efficient and tolerable treatment comprising artesunate.

[0011] Therefore, the present invention relates to a veterinary composition comprising artesunate or a salt thereof and a pharmaceutically acceptable excipient for preventing and / or treating leishmaniasis in dogs. More specifically, the dogs are dogs naturally infected with Leishmania infantum.

[0012] In a specific embodiment, the veterinary composition for use according to the invention comprises a therapeutically effective dose of between 0.1 and 100 mg / kg BW (BW: body weight) per day, preferably between 1 and 50 mg / kg BW per day, more preferably between 20 and 30 mg / kg BW per day. In a preferred embodiment, the veterinary composition comprises a therapeutically effective dose of 25 mg / kg BW per day.

[0013] In another specific embodiment, the veterinary composition for use according to the present invention is administered by oral route. Preferably, the veterinary composition is a tablet.

[0014] In another specific embodiment, the veterinary composition for use according to the invention is administered in a dose once daily.In a preferred embodiment, the veterinary composition is administered for at least 6 days, preferably 6 to 10 days, more preferably 6 days.

[0015] Another object of the invention is a veterinary composition as defined herein for use for controlling clinical symptoms of leishmaniasis and / or Leishmania infantum blood load and / or Leishmania infantum antibodies.

[0016] A preferred object of the present invention is a veterinary composition comprising artesunate or a salt thereof and a pharmaceutically acceptable excipient for use in the prevention and / or treatment of leishmaniasis in dogs naturally infected with Leishmania infantum, wherein the veterinary composition is administered to the infected dog by the oral route at a once daily dose of 25 mg / kg BW per day for 6 days.

[0017] In another embodiment, the veterinary composition for use according to the present invention further comprises at least one antiparasitic agent.

[0018] Another object of the invention is a kit comprising a first compartment comprising artesunate or a salt thereof and a second compartment comprising at least one antiparasitic agent, for simultaneous, separate or sequential use as a combined preparation, in particular for the prevention and / or treatment of leishmaniasis in dogs. In a specific embodiment, the kit comprises precautions or instructions for use. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : CONSORT flow diagram showing the treatment regimen and dogs with leishmaniasis during each phase of the study.

[0020] Figure 2 : Time course of total clinical score (Kruskal-Wallis Test). The clinical score of dogs (n=42) was assessed by a veterinarian at scheduled visits on D0, D+30, D+90 and D+180 (mean ± SD). A. All groups. B. / allopurinol group. C. Artesunate treatment group.

[0021] Figure 3 : / Comparison of mean percentage reduction in clinical scores between allopurinol and artesunate treatment groups (Mann-Whitney Test): A. 30th day after treatment; B. 90th day after treatment; C. 180th day after treatment.

[0022] Figure 4: Monitoring sera. Antibody titers (mean ± SD) were assessed in dogs (n = 42) by quantitative IFAT at scheduled visits on D0, D+30, D+90 and D+180 (Kruskal-Wallis Test / Bilateral). A. All dogs. B. / allopurinol group. C. artesunate group.

[0023] Figure 5 : Comparison of mean percentage reduction in IFAT antibody titers in dogs with leishmaniasis treated with artesunate or Glucantime / allopurinol. Data are reported as mean ± SD for artesunate vs. Glucantime / allopurinol at 30, 90 and 180 days after treatment (Mann-Whitney test). DETAILED DESCRIPTION

[0024] As shown in the Examples, the inventors have demonstrated that artesunate is safer and more effective than meglumine antimoniate / allopurinol in controlling and reducing clinical symptoms, parasite loads and antibodies to leishmaniasis in dogs.

[0025] Therefore, the present invention relates to artesunate or a salt thereof or a veterinary composition comprising artesunate or a salt thereof and a pharmaceutically acceptable excipient for preventing and / or treating leishmaniasis in dogs. The present invention further relates to a veterinary composition comprising artesunate or a salt thereof and a pharmaceutically acceptable excipient for preventing and / or treating canine leishmaniasis (CanL). The present invention also relates to a veterinary composition comprising artesunate or a salt thereof and a pharmaceutically acceptable excipient for preventing and / or treating canine leishmaniasis in dogs naturally infected with Leishmania infantum. In a specific embodiment, the leishmaniasis is visceral leishmaniasis. In another specific embodiment, canine leishmaniasis (CanL) is visceral canine leishmaniasis.

[0026] The present invention further relates to a method for treating canine leishmaniasis, comprising administering an effective amount of artesunate or a salt thereof or a veterinary composition comprising artesunate or a salt thereof to a dog, preferably a dog naturally infected with Leishmania infantum. The present invention also relates to the use of artesunate or a salt thereof for the manufacture of a pharmaceutical composition for preventing and / or treating canine leishmaniasis, preferably a dog naturally infected with Leishmania infantum.

[0027] Artesunate is a sesquiterpene-type organic peroxide, and more specifically, a semisynthetic derivative of the artemisinin group having the following formula:

[0028]

[0029] Currently, artesunate is administered intramuscularly, intravenously or orally (by mouth) as a first-line treatment for severe malaria. For example, it is used in combination products commercialized by Denk pharma with other antimalarial agents, namely pyrimethamine and sulfamethoxypyrazine or amodiaquine.

[0030] In the context of the present invention, the salt of artesunate corresponds to its pharmaceutically acceptable salt. Pharmaceutically acceptable salts include inorganic and organic acid salts. Representative examples of suitable inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, etc. Representative examples of suitable organic acids include formic acid, acetic acid, trichloroacetic acid, propionic acid, benzoic acid, cinnamic acid, fumaric acid, maleic acid, methanesulfonic acid, etc.

[0031] As used herein, the expression "effective dose" or "effective amount" or "therapeutically effective dose" means a dose or amount sufficient to prevent and / or treat leishmaniasis. A skilled person can adopt an "effective dose" or "effective amount" or "therapeutically effective dose" according to the severity of leishmaniasis and the overall health of the dog.

[0032] In a specific embodiment, the veterinary composition for use according to the present invention comprises a therapeutically effective dose of between 0.1 and 100 mg / kg BW per day, preferably between 1 and 50 mg / kg BW per day, between 10 and 40 mg / kg BW per day, more preferably between 20 and 30 mg / kg BW per day. "BW" means "body weight". In a preferred embodiment, the therapeutically effective dose comprises between 21 and 29 mg / kg BW per day, between 22 and 28 mg / kg BW per day, between 23 and 27 mg / kg BW per day, and between 24 and 26 mg / kg BW per day. In a more preferred embodiment, the therapeutically effective dose is 25 mg / kg BW per day.

[0033] The veterinary composition can be administered once or several times a day, preferably once or twice a day, more preferably once a day. Therefore, a preferred object of the present invention is a veterinary composition for use, wherein the veterinary composition is administered in a once-daily dose or once a day.

[0034] Treatment as defined herein can be applied daily for a defined period, for example a month (or 30 days), and preferably for a short period of 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 day. In a specific embodiment, the veterinary composition for use according to the invention is applied for at least 6 days, preferably from 6 to 10 days, from 6 to 9 days, from 6 to 8 days, more preferably 6 days. Preferably, a period defined by a number of days from X to Y means that the treatment is applied for a minimum of X days and a maximum of Y days. In other words, the treatment is applied for at least X days and at most Y days.

[0035] As used herein, a "pharmaceutically acceptable excipient" or "veterinarily acceptable excipient" is an ingredient or compound routinely used in the pharmaceutical or veterinary field for pharmaceutical or veterinary compositions and formulations prepared according to the route of administration. Preferably, such acceptable excipients do not have a therapeutic effect. Without limitation, pharmaceutically or veterinarily acceptable excipients include buffers, preservatives, antioxidants, diluents, stabilizers, tonicity agents, solubilizers, suspending agents, surfactants, lubricants, and solvents.

[0036] Without limitation, examples of solvents are propylene glycol, ethanol, sorbitol syrup, water, vegetable oils or combinations thereof. Examples of buffers are calcium phosphate, sodium bicarbonate, sodium phosphate, sodium lactate, sodium citrate or combinations thereof. Examples of suspending agents include methylcellulose, hydroxypropyl methylcellulose, polysorbate 80, hydroxyethyl cellulose, hydroxycellulose, xanthan gum, sodium carboxymethyl cellulose and polyethoxylated sorbitan monolaurate or combinations thereof. Examples of stabilizers include sodium sulfite, sodium metalsulfite and ether. Examples of tonicity agents are mannitol, sodium chloride, sodium citrate, glycerol, lactose, mannitol, sodium chloride, dextrose, sodium sulfate, sorbitol or combinations thereof. Examples of preservatives are ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, methyl p-hydroxybenzoate, sorbic acid, phenol, cresol, chlorocresol, benzoic acid or its salts, sodium metabisulfite, sodium benzoate, benzethonium chloride, cetylpyridinium chloride, benzalkonium chloride, sodium acetate, p-hydroxybenzoates and their salts, sucrose or a combination thereof, or a combination thereof, etc. Examples of solubilizers include polyoxyethylene-solidified castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, polyethylene glycol and castor oil fatty acid ethyl ester or a combination thereof, or a combination thereof, etc. Examples of diluents are calcium carbonate, calcium phosphate, kaolin, sodium carbonate, lactose, sodium phosphate, lactose monohydrate or a combination thereof, etc. Examples of antioxidants include ascorbic acid (vitamin C) or salts thereof, citric acid or salts thereof, malic acid, thioglycerol, fumaric acid, phosphoric acid, sodium metabisulfite, potassium metabisulfite, propionic acid, sodium bisulfite, sodium metabisulfite, potassium metabisulfite, vitamin E (tocopherol), acetone, cysteine, gentisyl ethanolamine, sodium glutamate, sodium formaldehyde sulfoxylate or a combination thereof. Examples of lubricants include oils, waxes, stearic acid, stearates (magnesium stearate, calcium stearate ...), talc, long-chain fatty acids, polyethylene glycol, palmitic acid, hydrogenated vegetable oils, sodium stearyl fumarate, sodium benzoate and mixtures thereof. Examples of surfactants include poloxamer, polysorbate, polyoxyethylene hydrogenated castor oil ...) and mixtures thereof.

[0037] The veterinary composition for use according to the present invention can be administered to dogs by any route, such as oral route, parenteral route including intramuscular and subcutaneous injection, and topical route. In a preferred embodiment, the veterinary composition is administered by oral route. Such oral administration is more convenient for the user (dog or veterinary subject) than the injectable administration of meglumine antimoniate used in the reference treatment with allopurinol.

[0038] The veterinary composition for use according to the present invention can be formulated in any form, such as a liquid solution, suspension, solid or semisolid, powder, pellet, capsule, granule, sugar-coated pellet, capsule, spray, pill, tablet, paste, implant or gel. For oral administration, a liquid solution, suspension, solid or semisolid, powder, chewable, pellet, capsule, granule, sugar-coated pellet, or tablet is preferred. In a more preferred embodiment, the veterinary composition for use according to the present invention is a tablet. Tablets suitable for practicing the present invention are sold under the trademark Asunate Commercial artesunate tablets.

[0039] For formulation in tablets or pills suitable for oral administration, lactose monohydrate, magnesium stearate, microcrystalline cellulose, polyvinyl pyrrolidone, crospovidone, flavoring agents and compressible sugars may be used as excipients in the solvent.

[0040] A preferred object of the present invention is a veterinary composition comprising artesunate or a salt thereof and a pharmaceutically acceptable excipient for the prevention and / or treatment of leishmaniasis in dogs naturally infected with Leishmania infantum, wherein the veterinary composition is administered to the infected dog by the oral route at a once daily dose of 25 mg / kg BW per day for 6 days. Another preferred object of the present invention is a method for the treatment of leishmaniasis in dogs naturally infected with Leishmania infantum, comprising orally administering to the dog a veterinary composition comprising artesunate or a salt thereof and a pharmaceutically acceptable excipient at a once daily dose of 25 mg / kg BW per day for 6 days.

[0041] As used herein, the term "treatment" or "treating" refers to any action intended to improve the health status of the dog, such as to treat, prevent, control, delay and limit the development of leishmaniasis as disclosed herein. In certain embodiments, the term refers to the improvement or eradication of leishmaniasis or symptoms associated with the disease caused by parasitic infestation such as Leishmania infantum. In other embodiments, the term refers to minimizing the spread or exacerbation of leishmaniasis caused by parasitic infestation by administering a composition as disclosed herein to an infected dog.

[0042] The term "treatment" or "treating" also includes the prophylactic treatment of dogs for leishmaniasis caused by parasitic infestation, which means treatment before the dog is exposed to or comes into contact with the pathogen of the disease, such as a parasite, or after the exposure / contact but before the onset of symptoms or in the early stages of the disease due to parasitic infestation. In a specific embodiment, the term "treatment" or "treating" means protecting a dog from leishmaniasis, for example, from the effects of exposure to a pathogen, or from the development of leishmaniasis in an exposed dog. The present invention is particularly suitable for protecting dogs from leishmaniasis caused by parasitic infection or infestation. Therefore, an object of the present invention is a method for protecting a dog from leishmaniasis, which comprises administering to the dog an effective amount or an effective dose of a veterinary composition as disclosed herein.

[0043] The terms "treatment" or "treating" also include alleviation of symptoms, such as reduction of clinical symptoms or clinical manifestations, such as general conditions (particularly emaciation, anorexia, apathy, anemia and hyperthermia), skin symptoms (particularly localized alopecia, pruritus, scaling, hyperkeratosis, onychomycosis and ulcers), visceral lesions (particularly lymphadenopathy, splenomegaly, hepatomegaly and renal disease), ophthalmic conditions (particularly hair loss around the "glassed" eyes, mucopurulent conjunctivitis, keratitis and / or uveitis), oral conditions (particularly oral erosions), bone conditions (particularly arthritis, synovitis and diffuse pain after training), and other conditions such as epistaxis, chronic diarrhea and neurological symptoms. Other symptoms are parasite blood load and antibodies, more specifically, Leishmania infantum blood load and Leishmania infantum antibodies.

[0044] Another object of the invention is a veterinary composition for use as defined herein for controlling clinical symptoms of leishmaniasis.Another object is a method for controlling clinical symptoms of leishmaniasis in dogs comprising administering a veterinary composition as defined herein.

[0045] Another object of the invention is a veterinary composition for use for controlling the blood load of Leishmania infantum as defined herein.Another object is a method for controlling the blood load of Leishmania infantum in dogs comprising administering a veterinary composition as defined herein.

[0046] Another object of the invention is a veterinary composition for use as defined herein for controlling antibodies to Leishmania infantum. Another object is a method for controlling antibodies to Leishmania infantum in dogs comprising administering a veterinary composition as defined herein.

[0047] As used herein, the terms "control" and "controlling" mean, but are not limited to, reducing, limiting development, eradicating, lowering, and eliminating. In a specific embodiment, these terms refer to the prophylactic treatment of infected dogs, thereby preventing human infection caused by Leishmania infantum.

[0048] As used herein, the terms "comprise(s)" or "comprising" (and other similar terms, e.g., "containing" and "including") are "open ended" and can be generally interpreted as including all of the specifically mentioned features as well as any optional, additional and unspecified features. Depending on the specific embodiment, it can also be interpreted as the phrase "consisting essentially of", which includes the specified features as well as any optional, additional and unspecified features that do not materially affect the basic and novel characteristics of the claimed invention, or the phrase "consisting of", which includes only the specified features unless otherwise specified.

[0049] Thus, the veterinary composition may further comprise at least one antiparasitic agent. These antiparasitic agents include, in particular, insect growth regulators, antibiotic pesticides, botanical pesticides, organophosphorus compounds, carbamates, amidines, organochlorinated compounds, phenylpyrazole compounds, pyrethroids, benzylureas, pyrethroid acids, formamidines, semicarbazones, neonicotinoids, diamides, spinosaccharins, isoxazolines, copper-containing pesticides, anthelmintics, benzimidazoles, probenzimidazole precursors, salicylanilides, imidazothiazoles, aminoacetonitrile derivatives (AAD), spiroindoles, pyrimidines, substituted phenols and anticoccidial agents.

[0050] For example, an IGR may be a chitin synthesis inhibitor or a synthetic juvenile growth hormone. hormones, for example azadirachtin, bistrifluron, diofenolan, fenoxycarb, hydroprene, kinoprene, methoprene, pyriproxyfen, tetrahydroazadirachtin, chlorfluazuron, cyromazine, diflubenzuron, fluazuron, flucycloxuron, flufenoxuron, hexaflumuron, lufenuron, novaluron, noviflumuron, tebufenozide, teflubenzuron or triflumuron. The antimicrobial pesticide may be a toxin of Bacillus thuringiensis subsp. israelensis. The botanical pesticide may be d-limonene, thymol, geraniol, linalol, carvacrol, nicotine, ryania or pyrethrins. The organophosphorus compound may be selected from dicrotophos, terbufos, dimethoate, diazinon, disulfoton, trichlorfon, azinphos-methyl, chlorpyrifos, malathion, oxydemeton-methyl, methamidophos, acephate, ethyl-parathion, parathion-methyl, mevinphos, phorate, carbofenthion, phosalone, naphthalophos or pyraclofos.Carbamates can be selected from carbaryl, carbofuran, aldicarb or carbofuran. Chlorinated organic compounds can be selected from methoxychlor, dicofol or cyclopentadienes such as endosulfan. Pyrethroid acids can be selected from allethrin, resmethrin, permethrin, flumethrin, deltamethrin and cypermethrin. An example of formamidine is amitraz. An example of semicarbazone is metaflumizone. Neonicotinoids can be selected from imidacloprid, nitenpyram or dinotefuran. The copper-containing pesticide may be copper (II), or basic copper chloride or copper sulfate, i.e. (Cu2Cl(OH)3) mixed with (Cu4(OH)6(SO4). The anthelmintic may be selected from macrolides, such as avermectins (e.g. ivermectin and moxidectin) or milbemycins (e.g. milbemycin oxime). oxime)). The benzimidazole may be albendazole or thiabendazole. The salicylanilide may be closantel or oxyclozanide. Levamisole may be one of the imidazothiazoles. Pyrantel may be one of the pyrimidines. Nitroxynil may be one of the substituted phenols. Fipronil may be one of the phenylpyrazoles. The isoxazoline may be 4-{5-[3-chloro-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)phenyl]-

[0063] In some embodiments, the present invention relates to 4-[(5-(3,5-dichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl}-N-{2-oxo-2-[(2,2,2-trifluoroethyl)amino]ethyl}naphthalene-1-carboxamide (afoxolaner), 4-[5-(3,5-dichlorophenyl)-5(trifluoromethyl)-4,5-dihydro-1,2-oxazol-3-yl]-3-yl]-2-methyl-N-{2-oxo-2-[(2,2,2-trifluoroethyl)amino]ethyl}benzamide (fluranaler) or N-{2-chloro-{5-(3,5-dichlorophenyl)-5-trifluoromethyl-4,5-dihydroisoxazol-3-yeenzyl}cyclopropanecarboxylic acid.

[0051] Various categories of parasites and categories of antiparasitic agents and the active agents for sale can be found on the parasitipedia website at the following address http: / / parasitipedia.net / and on the European Scientific Advisor Companion Animal Parasites (ESCCA) website at the following address http: / / www.esccap.org / . Another object of the present invention is a kit comprising a first compartment and a second compartment, which are used simultaneously, separately or sequentially as a combined preparation, in particular for the prevention and / or treatment of leishmaniasis in dogs, wherein the first compartment comprises artesunate or a salt thereof and the second compartment comprises at least one antiparasitic agent as defined herein. In a specific embodiment, the kit comprises precautions or instructions for use. As used herein, the term "separate" or "sequential" means the sequential administration of artesunate or a salt thereof and at least one antiparasitic agent. For example, artesunate or a salt thereof is first administered to a dog, and the at least one antiparasitic agent is secondly administered to the same dog, or vice versa. In this case, artesunate or a salt thereof and the at least one antiparasitic agent are sufficiently physically distinct when administered separately or sequentially.

[0052] On the other hand, the veterinary composition for use according to the invention does not contain an additional antiparasitic agent. According to this embodiment, artesunate is the only active ingredient in the veterinary composition against leishmaniasis. Therefore, another object of the present invention is a veterinary composition consisting essentially of artesunate or a salt thereof and a pharmaceutically acceptable excipient for the prevention and / or treatment of leishmaniasis in dogs. Another object of the present invention is a veterinary composition consisting of artesunate or a salt thereof as the only active ingredient and a pharmaceutically acceptable excipient for the prevention and / or treatment of leishmaniasis in dogs.

[0053] Further aspects and advantages of the invention will be disclosed in the following experimental section.The following examples illustrate the invention and are provided as non-limiting illustrations.

[0054] Examples

[0055] Materials and methods

[0056] Ethics statement

[0057] The study protocol was also approved by the Faculty of Science of the Institute of Veterinary Sciences of the University of Constantine I, Algeria (Procès-Verbal du CSI N°6, 2018). To facilitate the field work, a collaboration was established with veterinary doctors and their assistants working in these institutions. All dog owners gave their verbal informed consent and samples were collected by veterinary specialists. The risk assessment was also submitted to and approved by the Ethics Committee and the Decision Committee of Veterinary Practitioners of the Provinces of Northern Algeria. These institutions are affiliated to the Ministry of Agriculture and Rural Development of Algeria (Directions des Services Vétérinaires).

[0058] Research

[0059] This was an open-label, randomized, comparative clinical field trial conducted in northern Algeria. The prevalence observed in the area was 36%. Dogs of any age, breed, or sex owned by the client were enrolled and monitored by 11 participating veterinary staff. All dogs had previously been naturally infected with Leishmania infantum and presented clinical signs. The dogs were fully monitored and treatment could be changed to meglumine antimoniate / allopurinol at the owner's request if clinical lesions were noted. A total of 187 dogs were enrolled for CanL. Among them, 42 adult dogs (30 males and 12 females) with an average age of 3.8 years (minimum = 1 year, maximum = 11 years), weights between 3 and 37 kg, and different breeds (8 German shepherds, 1 Belgian shepherd malinois, 2 sloughys, 5 pointers, 6 braques, 1 bagnole, 1 American staffordshire terrier, 1 beagle and 17 mongrel dogs) were selected for this study.

[0060] Inclusion, Exclusion, and Efficacy Criteria

[0061] The main inclusion criteria were: i) dogs with at least two clinical manifestations associated with CanL as detailed in Table 1) below, and ii) dogs with a positive test result based on a qualitative rapid immunochromatographic test. Dogs with positive serology for Leishmania (Zoetis, France) and / or quantitative indirect immunofluorescence antibody test (IFAT ≥ 100) or positive PCR results obtained in blood.

[0062] Dogs with significant changes in renal and / or hepatic function or with other infectious diseases were excluded. Dogs were excluded if they had complications that could interfere with the assessment of the response to treatment, if they had been vaccinated or treated with leishmanicidal agents for 3 months or with leishmanistatics for 1 month before pre-inclusion. Finally, no prior treatment with systemic antibiotics or antifungals within 7 days before pre-inclusion or with systemic long-acting corticosteroids within 1 month before pre-inclusion was allowed. Pregnant and lactating female dogs were excluded. Dogs could be withdrawn from the study at any time if they showed intolerance to the treatment or if the owner requested.

[0063] The efficacy criteria were determined by a reduction in clinical scores (i.e., the sum of clinical scores was lower than before treatment) with a reduction in parasite DNA and Leishmania-specific antibody titers. Scoring was defined according to the severity of each clinical symptom and the final value was the sum of all values ​​obtained (Table 1). Quantitative parasite load was estimated by qPCR and antibody titers were estimated by quantitative IFAT.

[0064] Table 1: Clinical signs associated with CanL defined to assess the clinical score of dogs

[0065]

[0066] Each manifestation was assigned a severity score ranging from 0 to 3, where 0 = none, 1 = mild, 2 = moderate, and 3 = severe (maximum total clinical score = 21). Mortality associated with CanL was assessed by the maximum score (22).

[0067] Experimental design

[0068] The selected dogs were randomized into one of two treatment arms. Dogs in Group 1 (N=26): (positive control) received daily subcutaneous injections of 100 mg / kg meglumine antimoniate ( Merial, France) for 28 days, and 30 mg / kg allopurinol orally daily 300 mg, France) for one month. Dogs in Group 2 (N=16): (chemotherapy of the present invention) received oral artesunate 25 mg / kg (Asu- Denk Pharma, Germany) for 6 days. After each administration, the dogs were observed for 30 minutes to monitor for responsiveness, vomiting and / or regurgitation to ensure complete absorption of the drug. The dogs were followed up for 180 days ( Figure 1 ).

[0069] Visit schedule and sample collection

[0070] Animals were examined on D0, D+30, D+90 and D+180 days after the start of treatment and blood samples were collected at each time point. Clinical signs and lesions associated with leishmaniasis as well as infection status (parasite load - qPCR and serology) were assessed and monitored at these points.

[0071] Clinical evaluation

[0072] The efficacy of the treatment was assessed at each time point based on the clinical response to treatment (evolution of clinical scores over time and percentage reduction in total clinical scores). For each dog, the same veterinarian performed all clinical assessments to maintain consistency. The clinical scores were categorized according to the severity of clinical signs listed in Table 1. Mortality due to CanL constitutes a higher score (22). The percentage reduction was calculated according to the following formula:

[0073] CS% reduction percentage = (CS at D+XX - CS at D+0) / CS at D+0) x 100

[0074] Parasite load-qPCR

[0075] Blood samples collected at each time point were analyzed for quantification of parasite load by qPCR using primers and probes for detection and quantification of Leishmania infantum DNA and targeting conserved regions of kinetoplastid minicircle DNA (kDNA) (several 1000-fold repeated sequences). Prior to DNA extraction, 200 μL of blood was digested with proteinase K and incubated overnight at +56°C. Extraction was performed using a commercially available DNA extraction kit (QIAamp DNA Mini [Qiagen, Courtaboeuf, France]) and were performed on a BIOROBOT EZ1 (Qiagen, Qiagen, Courtaboeuf, France) according to the manufacturer's instructions. DNA was eluted in 200 μl of distilled water and stored at -20°C until analysis. qPCR was prepared and performed as follows: a 20 μL final volume containing 10 μL of Eurogentec Master Mix Roche (Eurogentec, Liège, Belgium), 0.5 mM of each primer and probe, 0.5 μL of UDG, 3 μL of DNAse- and RNAse-free distilled water, and 5 μL of DNA sample was amplified in a CFX96 Real-Time System (BioRad Laboratories, Foster City, CA, USA) using the following temperature control technique: an incubation step at 50°C for two minutes and an initial denaturation step at 95°C for three minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds and annealing extension at 60°C for 30 seconds. Quantification was based on a standard curve of 10 μL of each kDNA region from the PCR product. 8 8-fold serial dilutions of a plasmid containing 10000 DNA copies, equivalent to 10,000 parasites per mL; 5 μL of the serial dilutions ranging from 10,000 to 0.001 parasites per mL were poured into a reaction tube. The results are expressed as the number of Leishmania parasites present in 1 ml of blood, taking into account the volume introduced during the extraction process (200 μL of blood) and the eluent (200 μL).

[0076] Serological monitoring

[0077] On day 0, a qualitative rapid immunochromatographic test was performed using one drop (10 μL) of whole blood from each dog immediately after sampling according to the manufacturer's instructions. Leishmania (Zoetis, France). Quantitative IFAT of titration of anti-infant Leishmania specific immunoglobulin G (IgG) was performed on serum for monitoring as previously described. In brief, the plate wells were sensitized via 20 μL of infant Leishmania commercial antigen (Zoetis, France). After dilution to 1:50, 20 μL of each serum dilution was added to each well and the plate was incubated at 37 ° C for 30 minutes. The plate was washed twice with PBS for 5 minutes each time and then washed once with distilled water. 20 μL of anti-canine IgG conjugated with fluorescein isothiocyanate (FITC) (Sigma-Aldrich, St. Louis, Missouri, USA) was added to each well. The plate was incubated at 37 ° C for 30 minutes in the dark. After another washing step, a few drops of mounting agent were added to the coverslip and read. To avoid observation errors, all samples were checked by two different researchers, and positive and negative controls were added to each plate. All samples that were negative at 1:50 were considered negative. Positive results were further investigated using a five-fold serial dilution IFAT ranging from 1:100 to 1:1600. At this point, the sample was classified as highly positive for Leishmania infantum (>1:1600).

[0078] Data analysis

[0079] XLSTAT Addinsoft version 2018.7 was used for statistical analysis. Data were analyzed descriptively according to the nature of the variables for each follow-up visit and assigned treatment. The non-parametric Kruskal-Wallis / bilateral Test was used to compare the means of the two groups, group 1 and group 2 alone, obtained at the assessment time points. Multiple pairwise comparisons after the Dunn / Bilateral Test procedure were used for CS, parasitemia, and antibody titers. The treatment effects were compared between the two groups by analyzing clinical symptoms, parasite load, and the percentage reduction of Leishmania antibodies. The Mann-Whitney test was used to compare the efficacy of the treatment using the differences between D0 and D+30 days, between D0 and D+90 days, and between D0 and D+180 days. The significance level used for the test was 5% (Bonferroni-corrected significance level: 0.0083).

[0080] result

[0081] Dogs included and monitored

[0082] There were no statistically significant differences between the two groups in age, sex, breed (mongrel or breeder), clinical condition of the dogs, mean values ​​of parasitemia and Leishmania antibody titers on the day of inclusion (P values>0.05).

[0083] Clinical examination of the selected dogs showed that 92.85% (39 / 42) had general symptoms, 83.33% (35 / 42) had skin symptoms, 38.1% (16 / 42) had visceral symptoms, 23.8% (10 / 42) had ophthalmological symptoms, and 4.76% (2 / 42) had musculoskeletal symptoms. One dog (2.30%) showed oral erosions, another dog showed epistaxis, and two dogs (4.76%) had neurological diseases.

[0084] Table 2: Characteristics of dogs assigned to each treatment arm and homogeneity analysis data expressed as mean ± SD and frequency (%)

[0085]

[0086] %*: (number of positives by PCR / total number of positives)×100.

[0087] Statistical analysis was performed on 42 dogs, although one dog in Group 2 was lost to follow-up before the D+30 time point. During the 6-month follow-up, four dogs in Group 1 were lost to follow-up two months after the start of treatment, two of which could not be returned for the final revisit at D+90 and D+180 because the client failed to return them. The other two were due to deaths not related to CanL. In addition, six dogs died between D+60 after the start of treatment and before visit D+90, as clinical signs related to CanL increased. Four of these dogs were in Group 1 and two were in Group 2 ( Figure 1 ).

[0088] Clinical Results

[0089] Compared with the initial clinical scores, an overall improvement in the total clinical scores was observed in both treatment groups throughout the study. Taking into account the higher scores for mortality outcomes (22), the mean values ​​of the clinical scores decreased over time from 7.16 ± 3.47 at D0 to 4.24 ± 4.37 at visit D+30, and increased slightly again at D+90 (5.75 ± 8.25), but they were still inferior to those at D0 and remained constant after D+180 (5.60 ± 8.35). The same dynamics of clinical scores were observed in both groups ( Figure 2 ).

[0090] The mean percentage of clinical score reduction was 43.3 ± 47.8% with clinical scores (CS) at D0 and D+30 as the starting and end points, respectively. At each time point: D+30, D+90 and D+180, statistically significant differences (p<0.0001) were observed in the mean percentage of clinical score reduction between the two groups. It was noted that group 2 (artesunate) was significantly better than group 1 ( / allopurinol) showed more improvement ( Figure 3 At the level of individual responses, approximately 26.66% (4 / 15) of dogs in Group 2 recovered completely (CS of 0) compared with 18.18% (4 / 22) in Group 1, while approximately half of the dogs in both groups showed partial improvement at 6 months of follow-up, with 54.54% (12 / 22) in the control group and 46.66% (7 / 15) in the artesunate group. On D+180, the rates of clinical exacerbations were very similar in both groups, with 27.27% (6 / 22) in the control group, including 18.18% (4 / 22) mortality, and 26.66% (4 / 15) in the artesunate group, including 13.33% (2 / 15) mortality. Details of the clinical changes in the dogs followed over time are described in Table 3.

[0091] Adverse Events

[0092] No serious adverse effects related to these treatments were reported in any dog. Both study compounds were well tolerated in all dogs except for those given Two dogs treated with allopurinol for 15 days had mild local pain and pruritus.

[0093] Table 3. Individual clinical changes in dogs over time

[0094]

[0095] Including CanL-related mortality

[0096] Quantitative real-time PCR kinetics of parasitemia

[0097] At D0, blood from 12 / 42 (28.5%) dogs was positive by qPCR. Parasite clearance was observed over time after artesunate treatment, and all dogs that were initially positive (5 / 5) became negative for Leishmania. However, 6 / 7 dogs treated with artesunate did not clear parasites. The PCR-positive dogs treated with 1 / 7 allopurinol became negative, and 1 / 7 dogs remained positive. / allopurinol-treated dogs were positive, whereas they were negative previously. Table 4 shows the individual evolution of parasitemia during follow-up.

[0098] Table 4. Parasite load, number of parasites per mL of blood and percent reduction over time

[0099]

[0100] All other dogs were negative and remained negative at all follow-up visits.

[0101] N / A*: Mortality associated with CanL

[0102] N / A: Lost to follow-up

[0103] Serological status

[0104] Initially, all dogs The test and IFAT were positive and no significant differences were observed between the groups (P>0.05). After treatment, the mean values ​​of Leishmania antibody titers decreased over time (mean ± SD, 444.05 ± 467.50 at D0, 181.25 ± 234.71 at D+30, 120.69 ± 298.36 at D+90, and 118.97 ± 297.73 at D+180) ( Figure 4 A significant decrease compared with baseline values ​​was observed on D+30 in both groups (P<0.012, IFAT serology) (mean ± SD, artesunate 128.57 ± 212.78; / allopurinol 209.61±244.96). At D+180, they continued to decrease (mean±SD, artesunate 37.50±60.77; / allopurinol 176.471±379.604)( Figure 3 When the groups were compared, the artesunate group showed a higher percentage reduction in antibody titers at the end of the study (P<0.0001)( Figure 5 ). On an individual level, 78.57% (11 / 14) of dogs treated with artesunate required a reduction in antibody titers on D+30 and 50% (7 / 14) of these dogs were absolutely seronegative. In the 1 / allopurinol group, 57.7% (15 / 26) required serological reduction on D+30 and only 15.38% (4 / 26) were seronegative. At the end of follow-up, 41.37% (12 / 29) of dogs became seronegative at the end of follow-up, including: 29.41% (5 / 17) in Group 1 vs. 58.33% (7 / 12) in Group 2 (P value, Z test / two-sided = 0.227).

[0105] In conclusion, these results suggest that artesunate is an improved and effective treatment for canine visceral leishmaniasis compared to the reference combination meglumine antimoniate / allopurinol. In addition, artesunate is safer, easier to administer with short-term treatment (oral), and is cheaper than meglumine antimoniate and allopurinol.

Claims

1. Use of a veterinary composition comprising artesunate or a salt thereof and a pharmaceutically acceptable excipient in the preparation of a medicament for preventing and / or treating canine visceral leishmaniasis in dogs.

2. The use according to claim 1, wherein the dog is a dog naturally infected with Leishmania infantum.

3. The use according to claim 1, wherein the drug is administered by oral route.

4. The use according to claim 1, wherein the medicament is a tablet.

5. The use according to claim 1, wherein the veterinary composition further comprises at least one antiparasitic agent.

6. Use of a kit comprising a first compartment and a second compartment in the preparation of a therapeutic kit for preventing and / or treating canine visceral leishmaniasis in dogs, wherein the first compartment comprises artesunate or a salt thereof, the second compartment comprises at least one antiparasitic agent, the kit optionally comprising precautions or instructions for use, And wherein the artesunate or its salt and at least one antiparasitic agent are used simultaneously, separately or sequentially as a combined preparation.

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