FIELD OF THE INVENTION

The present invention relates to the use of a Perhexiline com ound of formula (I):

for the treatment of a pathology caused by a trematodes or flukes, in particular schistosomiasis or bilharzia. Today, Praziquantel is the only widely available drug to treat schistosomiasis, the second most common parasitic disease in the world. The aggressive and repeated treatment campaigns to combat schistosomiasis raise increasing concern about the possible emergence of resistance. Moreover, the insensitivity of immature parasites represents the most serious problem in the clinical use of Praziquantel.

BACKGROUND OF THE INVENTION

Trematodes are commonly referred to as flukes. This term refers to the flattened, rhomboidal shape of the worms. The fiukes can be classified into two groups, on the basis of the system which they infect in the vertebrate host. Tissue flukes infect the bile ducts, lungs, or other biological tissues. This group includes the lung fluke, Paragonimus westermani, and the liver flukes, Clonorchis sinensis and Fasciola hepatica. Blood flukes inhabit the blood in some stages of their life cycle. Blood flukes include species of the genus Schistosoma. They may also be classified according to the environment in which they are found. For instance, pond flukes infect fish in ponds.

Human infections are most common in Asia, Africa, South America, or the Middle East. However, trematodes can be found anywhere where human waste is used as fertilizer. Schistosomiasis (also known as bilharzia, bilharziosis or snail fever) is an example of a parasitic disease caused by one of the species of trematodes (platyhelminth infection, or "flukes"), a parasitic worm of the genus Schistosoma. Other diseases caused by trematodes include Clonorchiasis, Paragonimiasis,

Schistosomiasis, one of the world's greatest human neglected tropical disease, is caused by infection due mainly to Schistosoma mansoni, Schistosoma haematobium, or Schistosoma japonicum. Humans can become infected when their skin comes in contact with freshwater contaminated with the infectious larval stage of the parasite, known as cercariae. Among human parasitic diseases, schistosomiasis ranks second behind malaria in terms of socio-economic, public health importance and prevalence in the developing world, with more than 200 million people currently infected every year in 77 countries worldwide (85% in sub-Saharian Africa). The number of people treated for schistosomiasis rose from 12.4 million in 2006 to 40 million in 2013 (13.1% of people requiring treatment were reached), in spite of the large-scale treatment campaigns at-risk population groups organized by the World Health Organization (WHO). It is estimated that 600 million people are at risk of infection and at least 280.000 deaths per year are associated with the severe consequences of infection, including fibrosis and calcification of the urinary tract, renal failure or bladder cancer (S. hematobium) and acute hepatitis, liver and intestine fibrosis, and portal hypertension (S. mansoni). To date no vaccine is available against schistosomiasis. After intensive research for the development of new chemotherapies in the middle of the last century, limited subsequent research efforts have reduced the therapeutic arsenal against this parasitic disease to one single drug: Praziquantel (PZQ). Since 1980s PZQ is the drug of choice for the treatment of schistosomiasis, because it is orally effective against all species of schistosomes with a single dose treatment. However, the in vivo efficacy of PZQ is dependent on the age of the infection, on the sex of the worms and on their paired o unpaired status. During the earliest stages (from cercariae to the first few days after infection) the parasites are susceptible, followed by progressive insensitivity down to a minimum at around three to four weeks after infection (depending on the schistosome species). Schistosomes then gradually regain susceptibility until they are fully affected by the drug, around weeks 6-7 after infection. The ED50 of PZQ against juvenile S. mansoni worms in mice (4 weeks after infection) was at least 30 times higher than that observed for adult worms (6 or 7 weeks) (Pica-Mattoccia et al., 2004). This can partially explain the low cure rates and rapid re-infection rates in endemic areas where patients are likely to be infected with juvenile and adult parasites concurrently (Dabo et al., 2000; N'Goran et al., 2003). The striking drug insensitivity of immature worms (between 1-5 weeks after infection) is actually the most serious problem in the clinical use of PZQ. For this reason re -treatments are often necessary. Multiple PZQ treatments and repeated rounds of mass treatments raise concerns about the development of resistance to PZQ. Remarkably, it is possible to induce resistance of S. mansoni and S. japonicum to PZQ in mice under laboratory conditions and resistance, reduced susceptibility or low cure rates to PZQ in the field isolates of S. mansoni has been sporadically reported (Fallon et al, 1996; Ismail et al, 1994; Gryseels, 2001; Cioli D, 2004; Melman SD et al, 2009). For the above reasons, the WHO has classified schistosomiasis as an illness for which new therapies are urgently needed (Gray DJ, 2010).

Perhexiline (2-(2,2-dicyclohexylethyl) piperidine) (PHX) is a modulator of myocardial metabolism that is effective in the treatment of patients with refractory angina unsuitable for revascularization (Cole et al, 1990). More recently, it has also been shown to improve myocardial energetics and function in chronic cardiac failure (Lee et al, 2005) and symptomatic hypertrophic cardiomyopathy (Abozguia, et al. 2010). Historically, there have been difficulties in balancing the clinical effectiveness of PHX with significant toxicity, due to marked inter-individual variation in its pharmacokinetics, principally differences in elimination resulting from genetic polymorphisms of CYP2D6. These polymorphisms give rise to approximately 100-fold inter-individual differences in apparent oral clearance and plasma half-lives that range from 1-2 days in most subjects and up to 40 days in poor metabolizers. Severe adverse events, including hepatotoxicity and peripheral neuropathy, can be avoided by therapeutic drug monitoring to maintain plasma PHX concentration within a defined therapeutic range (0.15-0.6 mg Γ ¹ ) (Horowitz, et al 1986). PHX is still believed to hold a critically important place in Australia and New Zealand for the treatment of patients with refractory angina or those who have contraindications to other standard anti-anginal therapies (Ashrafian et al. 2007; Lee et al. 2005). Remarkably, it has been also recently demonstrated in WO2014/036603 that the adverse effects observed upon administration of racemic Perhexiline are unexpectedly associated with the (+) enantiomer and not the (-) enantiomer. PHX's main mode of action is ascribed to its inhibition of long-chain fatty acid oxidation by targeting of carnitine palmitoyltransferase (CPT) 1 and 2 (Kennedy JA et al., 2000). Biochemical assays showed also that PHX compound stimulates autophagy and inhibits mTORCl signaling in mammalian cells maintained in nutrient-rich conditions (Balgi AD et al., 2009).

In a study described by Redman et al., Perhexiline was used to investigate the metabolism of sphingomyelin in schistosomes. In synthesis, treatment of adult parasites with the lysosomotrophic agents NH4CI, Perhexiline and desipramine resulted in no decrease in the rate of BODIPY FL C5- sphyngomyelin breakdown, suggesting that sphyngomyelin breakdown does not occur in the lysosome.

WO2014/052836 discloses a large list of compounds of different structures for use in the treatment of an infection, such list comprises Perhexiline. The compounds are claimed for treating or preventing abacterial infection selected from the group oiEnterobacterium faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinebacter baumannii, Pseudomonas aeruginosa and Enterobacter sp. There is no indication that Perhexiline is effective as schistomicidal agent.

WO2013/182519 relates to pharmaceutical compositions comprising a lysosomotropic agent or agent modulating autophagy and a GSK-3 (glycogen synthase kinase 3) inhibitor, useful in the treatment of cancer, proliferative inflammatory diseases, degenerative diseases and infectious diseases including malaria, hepatitis A to C, African trypanosomiasis, cryptosporidiosis, dengue fever, leishmaniasis, tuberculosis and schistosomiasis. The large list of lysosomotropic agents includes Perhexiline. However there is no indication that Perhexiline alone is effective to treat pathologies caused by a trematode, in particular as schistosomicidal agent, preferably effective against juvenile and adult parasites.

Taylor CM et al. describe the efficacy of Perhexiline in two nematode species, Haemonchus contortus and Onchocerca lienalis. There is no indication or evidence that the compound is also active on trematodes.

Nematodes and trematodes are very different parasites. Nematodes have a simple body form, often referred to as a "tube within a tube," with a simple digestive system that extends from the mouth at one end to the anus at the other. Trematodes have flat, unsegmented bodies usually shaped like a leaf or an oval. Nematodes have two sexes and reproduce sexually. Except for members of the Schistosoma genus, trematodes are hermaphroditic, meaning they possess reproductive organs of both sexes. Their attachment mechanisms are also different. Nematodes attach to their hosts via liplike or toothlike plates that surround their mouth openings. Food is sucked into the body cavity by the working of muscles that surround the opening. In some species that prey on plants, the mouth cavity has been modified into a hollow spear that can penetrate the plant tissue and withdraw food. Trematodes attach to their hosts with two suckers, one anterior and one posterior. Nematodes can cause a number of serious diseases in humans including ascariasis, hookworm diseases, whipworm disease, trichinosis, pinworm infection and strongyloidiasis. These infections primarily affect the intestines of hosts and are most common in impoverished areas where sanitation standards are low. Trematodes can infect the skin, intestines, liver, blood, brain, lungs and other tissues of hosts, and symptoms can be severe and potentially life -threatening. Further, unlike trematodes, nematodes are major agricultural pests.

Despite the common terminology, the only shared biological features of many "helminthes" are their metazoan origins and the ability to infect mammals. Schistosomes are part of the platyhelminths that include the cestodes (tapeworms) and other trematodes (fiukes or flatworms). The phyla Nematoda (roundworms) include hookworms, whipworms, and filarial parasites. The split that led to Platyhelminthes and ematoda occurred over 1 billion years ago, long predating the split between vertebrates and invertebrates (Hausdorf B., 2000). Therefore results obtained with nematodes cannot be extrapolated to trematodes.

As indicated above, there is still the need for therapy for diseases caused by trematodes' infection. In particular, such treatment needs to overcome drawbacks of current therapy: ineffectiveness against all stage of development of the trematode and risk of resistance.

SUMMARY OF THE INVENTION

In order to search for clinically applicable drugs for trematodes, in particular schistosomiasis, the inventors screened a compound collection also including FDA and/or approved drugs with a luminescent assay based on quantitation of the ATP, which signals the presence of metabolically active cells and organisms. Several hit compounds have been identified and surprisingly among those Perhexiline (2-(2,2-dicyclohexylethyl) piperidine).

Then, the present invention relates to the treatment of parasitic diseases caused by trematodes. Examples of such parasitic diseases include malaria, toxoplasmosis, trypanosomiasis, leishmaniasis, schistosomiasis, clonorchiasis, paragonimiasis, cercarial dermatitis. In particular, the present invention relates to the treatment of schistosomiasis. As a matter of fact, within the present invention it has been surprisingly found that Perhexiline is effective against trematodes, in particular of the Schistosoma genus, more particularly Schistosoma mansoni, Schistosoma haematobium and Schistosoma japonicum.

Perhexiline (2-(2,2-dicyclohexylethyl)piperidine) (PHX) is then a highly promising treatment for pathologies caused by trematodes, in particular as anti-schistosomal compound to be used as an alternative or supplement to PZQ. Remarkably, PHX is active against larvae and both immature (1- 5 weeks old) and adult S. mansoni worms in vitro. The efficacy of PHX was also demonstrated in a murine model infected by S. mansoni. The use of PHX in the treatment of disease caused by trematodes, in particular schistosomiasis can offer a solution to the major limitation that PZQ is not effective against juvenile parasites (4 weeks old). The use of PHX alone or in combination with PZQ can solve the problem of re -treatments. Moreover it can represent a valid alternative in case of more serious cases of emerging resistance to PZQ. Importantly, PHX is currently used in chronic heart failure and refractory angina. Facing substantial obstacles to developing new therapies for neglected diseases, 'repurposing' drugs already approved for other conditions could speed the delivery of new therapies to people in need thereof.

In one aspect, the present invention provides a Perhexiline compound of formula (I):

wherein Ri, R ₂ , R ₃ , R4 are each independently -H or halogen, preferably -H or -F, most preferably -H, pharmaceutically acceptable salts or stereoisomers thereof for use in the treatment of a pathology caused by trematodes.

Preferably the compound is the (-)-enantiomer.

Preferably the pathology caused by trematodes is selected from the group consisting of:

schistosomiasis, clonorchiasis, paragonimiasis and cercarial dermatitis.

Still preferably the pathology caused by trematodes is schistosomiasis. The term "schistosomiasis" includes also neuroschistosomiasis, a severe manifestation of schistosomiasis.

In an embodiment the schistosomiasis is caused by at least one of Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum or a combination thereof.

In a preferred embodiment the trematodes are larvae, immature or juvenile trematodes or adult trematodes.

In a still preferred embodiment the pathology caused by trematodes is resistant to Praziquantel or oxamniquine or other anti-parasitic drug. Other anti-parasitic drug may include antimalarial agents (atovaquone-proguanil, chloroquine, hydroxychloroquine, amodiaquine), metronidazole and tinidazole, nitazoxanide or paromomycin, ivermectin, pyrantel pamoate, albendazole, mebendazole (Joel Thome et al:, 2012).

In one aspect the present invention provides a pharmaceutical composition comprising the Perhexiline compound of formula (I) as defined above and at least one pharmaceutically acceptable excipient for use in the treatment of a pathology caused by trematodes.

Preferably the pharmaceutical composition further comprises at least another active compound. Preferably the other active compound is Praziquantel or Oxamniquine or other anti-parasitic drug as defined above and known in the art. The combination may also comprise an anti-inflammatory agent such as glucocorticoids.

Preferably the other active compound is not a GSK-3 (glycogen synthase kinase 3) inhibitor.

In a preferred embodiment the dosage of Perhexiline ranges between 0.01 mg kg/day to 100 mg/kg/day.

A further aspect of the invention provides a method for the treatment of a pathology caused by trematodes comprising administering to a subject in need thereof a therapeutically effective amount of Perhexiline compound of formula (I :

wherein Ri, R ₂ , R ₃ , R4 are each independently -H or halogen, preferably -H or -F, most preferably -H or pharmaceutically acceptable salts or stereoisomers thereof.

As used herein, the term "Perhexiline" refers to the chemical compound 2-(2,2- dicyclohexylethyl)piperidine: and derivatives thereof. Such derivatives maintain the activity against trematodes of Perhexiline. The activity against trematodes may be tested by known techniques in the art, as well as by methods reported herein.

In particular the derivatives of Perhexiline have the formula I):

wherein Ri, R ₂ , R ₃ , R4 are each independently -H or halogen, preferably -H or -F, most preferably -H or pharmaceutically acceptable salts or stereoisomers thereof. As used herein, the term 'halogen' refers to fluorine, chlorine, bromine and iodine, of which fluorine, chlorine and bromine are preferred.

The synthesis of Perhexiline has been described previously (see US 4, 191 ,828 and US 4,069,222). Substituted derivatives have also been described (see WO 2007/096251). Further derivatives, in which the piperidine has been replaced by other amine -bearing groups have also been described (LeClerc et al., 1982). Derivatives in which the carbon separating the two cyclohexyl groups has been substituted with a hydro xyl group have been reported (see, e.g., Tilford and van Campen, 1954). The synthesis of a deuterated derivative has also been described (see, e.g., Schou, 2010). "hydro xyperhexiline" (i.e., 4-[l-(cyclohexyl)-2-(2- piperidinyl)ethyl]cyclohexanol; CAS Registry No 89787-89-3); "frans-hydro xyperhexiline" (i.e., frans-4-[l-(cyclohexyl)-2-(2- piperidinyl)ethyl]cyclohexanol; CAS Registry No 917877-74-8); and "c/s-hydroxyperhexiline" (i.e., c/s-4-[l-(cyclohexyl)-2-(2- piperidinyl)ethyl]cyclohexanol; CAS Registry No 917877-73-7) are commercially available. 4-monohydroxy metabolites have been identified and isolated by liquid chromatography (see, e.g., Davies et al., 2006).

Fluoro -perhexiline (FPER) compounds have been described (WO 2014/184561 , incorporated by reference herein) and they have been protected against the major route of metabolism that acts upon Perhexiline (specifically, CYP2D6-mediated oxidation of the 4-position of one or both cyclohexyl groups, to give an alcohol) by the replacement of the hydrogen with a fluorine. In addition to preventing CYP2D6-mediated metabolism (which is regarded as one of the major drawbacks of Perhexiline, and which prevents wider clinical use), these modifications have also increased metabolic stability as a whole and have not led to the introduction of other CYP-mediated routes of oxidative metabolism. Therefore compounds that have one or two fluoro groups at the para-position of one or both of the cyclohexyl groups of Perhexiline are part of the present invention.

Substantially Purified Forms

One aspect of the present invention pertains to a Perhexiline compound of formula I, as described herein, in substantially purified form and/or in a form substantially free from contaminants. In one embodiment, the substantially purified form is at least 50% by weight, e.g., at least 60% by weight, e.g., at least 70% by weight, e.g., at least 80% by weight, e.g., at least 90% by weight, e.g., at least 95% by weight, e.g., at least 97% by weight, e.g., at least 98% by weight, e.g., at least 99% by weight.

Unless specified, the substantially purified form refers to the compound in any stereoisomeric or enantiomeric form. For example, in one embodiment, the substantially purified form refers to a mixture of stereoisomers, i.e., purified with respect to other compounds. In one embodiment, the substantially purified form refers to one stereoisomer, e.g., optically pure stereoisomer. In one embodiment, the substantially purified form refers to a mixture of enantiomers. In one embodiment, the substantially purified form refers to an equimolar mixture of enantiomers (i.e., a racemic mixture, a racemate). In one embodiment, the substantially purified form refers to one enantiomer, e.g., optically pure enantiomer.

In one embodiment, the contaminants represent no more than 50% by weight, e.g., no more than 40% by weight, e.g., no more than 30% by weight, e.g., no more than 20% by weight, e.g., no more than 10% by weight, e.g., no more than 5% by weight, e.g., no more than 3% by weight, e.g., no more than 2% by weight, e.g., no more than 1 % by weight.

Unless specified, the contaminants refer to other compounds, that is, other than stereoisomers or enantiomers. In one embodiment, the contaminants refer to other compounds and other stereoisomers. In one embodiment, the contaminants refer to other compounds and the other enantiomer.

In one embodiment, the substantially purified form is at least 60% optically pure (i.e., 60% of the compound, on a molar basis, is the desired stereoisomer or enantiomer, and 40% is the undesired stereoisomer(s) or enantiomer), e.g., at least 70% optically pure, e.g., at least 80% optically pure, e.g., at least 90% optically pure, e.g., at least 95% optically pure, e.g., at least 97% optically pure, e.g., at least 98% optically pure, e.g., at least 99% optically pure.

Kits

One aspect of the invention pertains to a kit comprising (a) a Perhexiline compound of formula I as described herein, or a composition comprising a Perhexiline compound of formula I as described herein, e.g., preferably provided in a suitable container and/or with suitable packaging; and (b) instructions for use, e.g., written instructions on how to administer the compound or composition. In one embodiment, the kit further comprises one or more (e.g., 1 , 2, 3, 4) additional therapeutic agents, as described herein.

The written instructions may also include a list of indications for which the active ingredient is a suitable treatment.

Where the compound is a salt, an ester, an amide, a prodrug, or the like, the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately.

It may be noted that, currently, perhexiline is administered as racemic perhexiline maleate, at an initial dosage of 100 mg/day, and increased or decreased if required.

For "poor metabolizers" (PM), the dosage is typically 10-25 mg/day. For "extensive metabolizers" (EM), the dosage is typically 100-250 mg/day. For "ultra metabolizers" (UM), the dosage is typically 300-500 mg/day. The optimal steady state plasma concentration of perhexiline is about 0.15-0.6 mg/L.

Included in the instant invention is the free base of Perhexiline as well as the pharmaceutically acceptable salts. The encompassed pharmaceutically acceptable salts include all the typical non-toxic pharmaceutically acceptable salts of the free form of the compound of formula (I). The free form of the specific salt compounds described may be isolated using techniques known in the art. For example, the free form may be regenerated by treating the salt with a suitable dilute aqueous base solution such as dilute aqueous NaOH, potassium carbonate, ammonia and sodium bicarbonate. The free form may differ from their respective salt forms somewhat in certain physical properties, such as solubility in polar solvents, but the salts are otherwise pharmaceutically equivalent to their respective free forms for purposes of the invention. Conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like, as well as salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic and the like. Preferred salts are the maleate salt, the hydrochloride salt or the lactate salt.

Perhexiline exists in two enantiomeric forms, and occurs as racemate and single enantiomers, the (+)-enantiomer and the (-)-enantiomer, all such stereoisomers being included in the present invention. As used herein, the term Perhexiline encompasses the racemate and the single enantiomers.

Perhexiline may be administered to a subject in a suitable form. In this regard the term "administering" includes administering Perhexiline as a racemic mixture or single enantiomer, and/or administering a salt, prodrug or derivative of Perhexiline, that will form an effective amount of the active agent within the body of the subject. The term includes routes of administration that are systemic (e.g. via injection such as intravenous injection, orally in a tablet, pill, capsule, or other dosage form useful for systemic administration of pharmaceuticals). Methods of drug administration are generally known in the art.

The invention also provides pharmaceutical compositions comprising Perhexiline, alone or in combination with other anti-parasitic agents, and a pharmaceutically acceptable carrier.

When Perhexiline is administered into a human subject, the daily dosage regimen will normally be determined by the prescribing physician with the dosage generally varying according to the age, weight, sex and response of the individual patient, as well as the severity of the patient's symptoms. In one exemplary application, oral dosages of the present invention will range between about 0.01 mg per kg of body weight per day (mg/kg/day) to about 100 mg kg/day, preferably 0.01 to 10 mg/kg/day, and most preferably 0.1 to 5.0 mg/kg/day.

Included within the scope of the present invention is Perhexiline in combination with known therapeutic schistosomiasis agents for simultaneous, separate or sequential administration and for treatment of polyparasitism that appears to be the rule, rather than the exception, both at the population level and among individuals residing in developing countries.

In an embodiment, Perhexiline may be used in combination with known agents useful for treating or preventing parasitic diseases, including malaria, toxoplasmosis, trypanosomiasis, chagas disease, leishmaniasis, schistosomiasis, amebiasis, giardiasis, clonorchiasis, fasciolopsiasis, lymphatic filariasis, onchocerciasis, thricomoniasis and cestodiasis. Combinations of Perhexiline with other agents useful for treating or preventing parasitic disease are within the scope of the invention. A person of ordinary skill in the art would be able to discern which combinations of agents would be useful based on the particular characteristics of the drugs and the disease involved.

In particular, the present invention refers to a combination comprising Perhexiline and/or Praziquantel and/or Oxamniquine. The combination or pharmaceutical composition of the invention does not include a glycogen synthase 3-kinase (GSK-3) inhibitor.

In the present invention, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). In many embodiments, a subject is a human being. A human includes pre and postnatal forms. In certain embodiments of the present invention the subject is an adult, an adolescent or an infant. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term "subject" is used herein interchangeably with "individual" or "patient." A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder. Also contemplated by the present invention are the administration of the pharmaceutical compositions and/or performance of the methods of treatment in-utero.

It is preferable that the therapeutic agent (PHX or the compound (s) used in association thereof) is administered to the patient in the form of a pharmaceutical composition that includes a pharmaceutically acceptable carrier.

The pharmaceutical compositions of the present invention are preferably in the form of a single unit dosage form that contains an amount of the therapeutic agent that is effective to treat and/or prevent a pathology caused by trematodes of the type described herein. The pharmaceutical composition can also include suitable excipients, or stabilizers, and can be in solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, or emulsions. Typically, the composition will contain from about 0.01 to 99 percent, preferably from about 5 to 95 percent of active compound(s), together with the carrier. The therapeutic agent, when combined with a suitable carrier and any excipients or stabilizers, and whether administered alone or in the form of a composition, can be administered orally, parenterally, subcutaneously, transdermally, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by implantation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, by application to mucous membranes, such as, that of the nose, throat, and bronchial tubes (i.e., inhalation), or by intracerebral administration. The administration may be buccal, sublingual, rectal, topical, transdermal, intravesical, or using any other route of administration.

For most therapeutic purposes, the therapeutic can be administered orally as a solid or as a solution or suspension in liquid form, via injection as a solution or suspension in liquid form, or via inhalation of a nebulized solution or suspension.

The solid unit dosage forms containing the therapeutic agent can be of a conventional type. The solid form can be a capsule, such as an ordinary gelatin type containing the therapeutic agent and a carrier, for example, lubricants and inert fillers such as, lactose, sucrose, or cornstarch. In another embodiment, the therapeutic agent is tableted with conventional tablet bases such as lactose, sucrose, or cornstarch in combination with binders like acacia or gelatin, disintegrating agents such as cornstarch, potato starch, or alginic acid, and a lubricant such as stearic acid or magnesium stearate. For injectable dosages, solutions or suspensions of the therapeutic agent can be prepared in a physiologically and pharmaceutically acceptable diluent as the carrier. Such carriers include sterile liquids, such as water and oils, with orwithout the addition of a surfactant and other pharmaceutically and physiologically acceptable components, including adjuvants, excipients or stabilizers. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions.

For use as aerosols, the therapeutic agent in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. The therapeutic agent also may be administered in a non-pressurized form such as in a nebulizer or atomizer.

In addition to the above-described formulations which are intended to immediately deliver the therapeutic agents to the patient, sustained release formulations are also contemplated. Preferably, the sustained release formulation is an implantable device that includes a matrix in which the therapeutic agent is captured. Release of the agents can be controlled via selection of materials and the amount of drug loaded into the vehicle. A number of suitable implantable delivery systems are known in the art, such as U.S. Pat. No. 6,464,687 to Ishikawa et al, U.S. Pat. No. 6,074,673 to Guillen, each of which is hereby incorporated by reference in its entirety.

Implantable, sustained release drug delivery systems can be formulated using any suitable biocompatible matrix into which an agent can be loaded for sustained-release delivery. These include, without limitation, microspheres, hydrogels, polymeric reservoirs, cholesterol matrixes, polymeric systems and non-polymeric systems, etc. Exemplary polymeric matrixes include, without limitation, poly(ethylene-co-vinyl acetate), poly-L-lactide, poly-D-lactide, polyglycolide, poly(lactide-co-glycolide), polyanhydride, polyorthoester, polycaprolactone, polyphospagene, proteinaceous polymer, polyether, silicone, and combinations thereof.

Use of perhexiline in combination with one or more other therapeutic agents is also contemplated. Thus, the present invention also relates to formulations and therapeutic systems comprising two or more active agents, one of which is a Perhexiline compound of formula I.

"Pharmaceutically acceptable salts" comprise conventional non-toxic salts obtained by salification with organic or inorganic bases. The inorganic salts are, for example, metal salts, particularly alkali metal salts, alkaline-earth metal salts and transition metal salts (such as sodium, potassium, calcium, magnesium, aluminum). Salts may be also obtained with bases, such as ammonia or secondary or tertiary amines (such as diethylamine, triethylamine, piperidine, piperazine, morpholine), or with basic amino-acids, or with osamines (such as meglumine), or with aminoalcohols (such as 3- aminobutanol and 2- aminoethanol). In addition, the compound of the present invention can exist in unsolvated as well as in solvated forms with pharmaceutically acceptable solvents such as water, ethanol and the like.

The compounds of this invention can be administered via any of the accepted modes of administration or agents for serving similar utilities.

The compounds can be pharmaceutically formulated according to known methods. The pharmaceutical compositions can be chosen on the basis of the treatment requirements. Such compositions are prepared by blending and are suitably adapted to oral or parenteral administration, and as such can be administered in the form of tablets, capsules, oral preparations, powders, granules, pills, injectable or infusible liquid solutions, suspensions or suppositories.

Tablets and capsules for oral administration are normally presented in unit dose form and contain conventional excipients such as binders, fillers, diluents, tableting agents, lubricants, detergents, disintegrants, coloring agents, flavoring agents and wetting agents. The tablets can be coated using methods well known in the art.

Suitable fillers include cellulose, mannitol, lactose and other similar agents. Suitable disintegrants include polyvinylpyrrolidone and starch derivatives such as sodium glycolate starch. Suitable lubricants include, for example, magnesium stearate. Suitable wetting agents include sodium lauryl sulfate.

The oral solid compositions can be prepared by conventional methods of blending, filling or tableting. The blending operation can be repeated to distribute the active principle throughout compositions containing large quantities of fillers. Such operations are conventional.

Oral liquid preparations can be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or can be presented as a dry product for reconstitution with water or with a suitable vehicle before use. Such liquid preparations can contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel, or hydrogenated edible fats; emulsifying agents, such as lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which can include edible oils), such as almond oil, fractionated coconut oil, oily esters such as esters of glycerine, propylene glycol, or ethyl alcohol; preservatives, such as methyl or propyl /?-hydroxybenzoate or sorbic acid, and if desired, conventional flavoring or coloring agents.

Oral formulations also include conventional slow-release formulations such as enterically coated tablets or granules. For parenteral administration (e.g. bolus injection or continuous infusion), fluid unit dosages (e.g. in ampoules or in multi-dose containers) can be prepared, containing the compound and a sterile vehicle. The compound can be either suspended or dissolved, depending on the vehicle and concentration. The parenteral solutions are normally prepared by dissolving the compound in a vehicle, sterilising by filtration, filling suitable vials and sealing. Advantageously, adjuvants such as local anaesthetics, preservatives and buffering agents can also be dissolved in the vehicle. To increase stability, the composition can be frozen after having filled the vials and removed the water under vacuum. Parenteral suspensions are prepared substantially in the same manner, except that the compound can be suspended in the vehicle instead of being dissolved, and sterilized by exposure to ethylene oxide before suspending in the sterile vehicle . Advantageously, a surfactant or wetting agent can be included in the composition to facilitate uniform distribution of the compound of the invention.

For buccal or sublingual administration the compositions may be tablets, lozenges, pastilles, or gel. The compounds can be pharmaceutically formulated as suppositories or retention enemas, e.g. containing conventional suppositories bases such as cocoa butter, polyethylene glycol, or other glycerides, for a rectal administration.

Another means of administering the compounds of the invention regards topical treatment. Topical formulations can contain for example ointments, creams, lotions, gels, solutions, pastes and/or can contain liposomes, micelles and/or microspheres. Examples of ointments include oleaginous ointments such as vegetable oils, animal fats, semisolid hydrocarbons, emulsifiable ointments such as hydroxystearin sulfate, anhydrous lanolin, hydrophilic petrolatum, cetyl alcohol, glycerol monostearate, stearic acid, water soluble ointments containing polyethylene glycols of various molecular weights. Creams, as known to formulation experts, are viscous liquids or semisolid emulsions, and contain an oil phase, an emulsifier and an aqueous phase. The oil phase generally contains petrolatum and an alcohol such as cetyl or stearic alcohol. The emulsifier in a cream formulation is chosen from non-ionic, anionic, cationic or amphoteric surface-active agents. Dispersing agents such as alcohol or glycerin can be added for gel preparation. The gelling agent can be dispersed by finely chopping and/or mixing.

A further method of administering the compounds of the invention regards transdermal delivery. Typical transdermal formulations comprise conventional aqueous and non-aqueous vectors, such as creams, oils, lotions or pastes or can be in the form of membranes or medicated patches. A reference for the formulations is the book by Remington ("Remington: The Science and Practice of Pharmacy", Lippincott Williams & Wilkins, 2000).

Administration through synthetic nanoparticles engineered to target specific sites is also comprised within the present invention (Davis et al, 2010, Nature 464, 1067-1071; Dashi et al, 2012 Adv Mater., 24,3864-3869). Perhexiline may be encapsulated in such nanoparticles.

The above mentioned uses and methods also include the possibility of co-administration of additional therapeutic agents, simultaneously or delayed with respect to the administration of Perhexiline. The dosage of the administered compounds can vary depending upon a variety of factors including the patient type and condition, the degree of disease severity, mode and time of administration, diet and drug combinations. As an indication, they can be administered within a dose range of between 0.001 and 1000 mg/kg/day. The determination of optimum dosages for a particular patient is well known to one skilled in the art. Preferred dose range is between 0.01 and 100 mg/kg/day, preferably between 0.1 to 50 mg/kg/day, preferably between 1 and 10 mg/kg/day, most preferred range is between 10 and 100 mg/kg/day. Still preferred dose range is between 100 and 200 mg/kg/day. Yet preferred dose range is between 200 and 500 mg/kg/day. Still preferred dose range is between 500 and 1000 mg/kg/day. Preferably the compound of formula I of the invention is administered orally. The compound of the invention is suitably administered to the patient at one time or over a series of treatments.

Depending on the type and severity of the disease, the candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion may be determined. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs.

However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

The "therapeutically effective amount" of the compound to be administered will be governed by such considerations, and is the minimum amount necessary to prevent, ameliorate, or treat a disease or disorder.

As is common practice, the compositions are normally accompanied by written or printed instructions, such as package insert for use in the treatment in question. The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products. As used herein, "treatment" (and grammatical variations thereof such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing relapse, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, compound of the invention is used to delay development of a disease or to slow the progression of a disease.

In the present invention, the treatment of any pathology caused by trematodes is contemplated. Trematodes may be classified according to their habitat.

Blood flukes include Schistosoma haematobium, Schistosoma mansoni, Schistosoma japonicum, Schistosoma mekongi, and Schistosoma intercalatum.

Liver flukes include F hepatica, Fasciola gigantica, C sinensis, Opisthorchis felineus, O viverrini, Dicrocoelium dendriticum, and Dicrocoelium hospes.

Pancreatic flukes include Eurytrema pancreaticum, Eurytrema coelomaticum, and Eurytrema ovis. Lung fiukes include Pwestermani, Paragonimusheterotremus, Paragonimus kellicoti, Paragonimusmexicana, Paragonimus skrjabin, Paragonimus miyazakii, Paragonimus compactus, and Paragonimushueit 'ungensis.

Intestinal flukes include F buski, M yokogawai, Echinostoma ilocanum, Watsonius watsoni, H heterophyes, and Gastrodiscoides hominis.

Concerning lung flukes, the genus Paragonimus contains more than 30 species that have been reported to cause infections in animals and humans. Among these, approximately 10 species have been reported to cause infection in humans, of which Pwestermani is the most important.

Pwestermani, also known as the Oriental lung fluke, is the most widespread species in Africa, South America, and parts of Asia. Among other species of Paragonimus that have been reported to cause human disease from around the world is Paragonimus heterotremus, which has been reported from northeastern parts of the Indian subcontinent.

P westermani is a thick, fleshy, reddish brown, egg-shaped worm (7.5-12 mm in length, 4-6 mm in breadth, and 3.5-5 mm in thickness). It inhabits parenchyma of the lung close to bronchioles in humans, foxes, wolves, and various feline hosts (eg, lions, leopards, tigers, cats).

The infection is typically transmitted via ingestion of metacercariae contained in raw freshwater crabs or crayfish. Additionally, consumption of the raw meat of paratenic hosts (eg, omnivorous mammals) may also contribute to human infection. Freshwater snails and crabs are first and second intermediate hosts of Paragonimus species, respectively. In the duodenum, the cyst wall is dissolved, and the metacercariae are released. The metacercariae migrate by penetrating through the intestinal wall, peritoneal cavity, and, finally, through the abdominal wall and diaphragm into the lungs. There, the immature worms finally settle close to the bronchi, grow, and develop to become sexually mature hermaphrodite worms.

Adult worms begin to lay the eggs, which are unembryonated and are passed out in the sputum. However, if they are swallowed, they are excreted in the feces. The eggs develop further in the water. In each egg, a ciliated miracidium develops during a period of 2-3 weeks. The miracidium escapes from the egg and penetrates a suitable species of snail (first intermediate host), in which it goes through a generation of sporocysts and 2 generations of rediae to form the cercariae. The cercariae come out of the snail, invade a freshwater crustacean (crayfish or crab), and encyst to form metacercariae. When ingested, these cause the infection, and the cycle is repeated.

Liver flukes comprise C sinensis and F hepatica. C sinensis is a widespread parasite found in Southeast Asia that infects the biliary passage in humans. The fluke is oblong, flat, transparent, and relatively small (10-25 mm long by 3-5 mm wide). It has a pointed anterior and rounded posterior end. Humans are infected by eating raw or partially cooked freshwater fish or dried, salted, or pickled fish infected with the metacercariae. In the duodenum, the cyst is digested and an immature larva released. The larva enters the biliary duct, where it develops and matures into an adult worm. The adult worm feeds on the mucosal secretions and begins to lay fully embryonated operculated eggs, which are excreted in the feces.

Upon reaching fresh water and upon ingestion by a suitable species of operculate snails (first intermediate host), the eggs hatch to produce a miracidium. Inside the snail, the miracidia multiply asexually through a single generation of sporocysts and generations of rediae to fork-tailed cercariae. The cercariae escape from the snail to the water and penetrate under scales of freshwater cyprinid fish (second intermediate host). In the fish, the cercariae lose their tails and encyst in the scale or muscle of the fish to the metacercariae, which are infectious to humans. When ingested, the infected fish cause infection in humans.

Fascioliasis, a zoonotic disease caused by infection with F hepatica (a digenetic trematode), is a major disease of livestock that is associated with important economic losses due to mortality; liver condemnation; reduced production of meat, milk, and wool; and expenditures for anthelmintics. The disease has a cosmopolitan distribution, with cases reported from Scandinavia to New Zealand and southern Argentina to Mexico.

F hepatica, also known as the sheep liver fluke, is a large liver fluke. This fluke primarily causes zoonotic disease in sheep and other domestic animals. Humans are infected by eating watercress and other aquatic plants contaminated by the metacercariae, which enter the duodenum and excyst. They then penetrate the intestinal wall, peritoneal cavity, and liver capsule (Glisson capsule) to reach the bile duct of the liver, where they develop and mature into adult worms.

The adult worms begin to lay the unembryonated eggs, which are excreted in the stool. They develop further in the fresh water. A miracidium hatches out of the egg and invades the appropriate snail host. Inside the snail host, the larva multiplies asexually through a single generation of sporocysts and 2 generations of rediae to finally develop into cercariae. Upon exiting the snail, the cercariae encyst on aquatic plants to form metacercariae. When humans and sheep eat these plants, they become infected, repeating the life cycle.

Dicrocoeliasis is a parasitic disease caused by the small liver flukes D.dendriticum and D.hospes. The disease represents a worldwide and widespread problem in grazing livestock, and it is sometimes

(although rarely) found in humans. However, because of its unusual method of transmission by ingestion of infected ants, human dicrocoeliasis remains a relatively rare occurrence in humans.

Cases of human dicrocoeliasis have been reported throughout Eastern Europe, Western Europe,

Africa, Australia, India, and Saudi Arabia.

Pancreatic flukes comprise E pancreaticum , E coelomaticum , E ovis. These flukes have a thick body and are 8-16 mm long and 6 mm wide. They parasitize the pancreatic ducts and occasionally the bile ducts of sheep, pigs, and cattle in Brazil and Asia. Three species, E pancreaticum, E coelomaticum, and E ovis, are recognized.

The first intermediate hosts are terrestrial snails (Bradybaena species), and the cercariae encyst in grasshoppers (Conocephalus species), which are the second intermediate host. After a suitable animal hosts ingests a grasshopper with cercariae, the immature flukes are released and migrate to the pancreatic duct, where they mature and produce eggs within approximately 11-14 weeks.

There are no obvious clinical signs of infection with these parasites. Dicrocoelium -like eggs can be demonstrated in feces. Light infections cause proliferative inflammation of the pancreatic duct, which may become enlarged and occluded. In heavy infections, fibrotic, necrotic, and degenerative lesions develop. Losses are reported due to condemned pancreas, but the pathogenesis suggests an additional loss of production. Intestinal flukes comprises F buski, H heterophyes, Myokogawai , G hominis. F buski is the most common intestinal nematode that causes infections in humans. The trematodes H heterophyes, Myokogawai, and G hominis are less-common causes of human infection.

F buski, known as the giant intestinal fluke, is found in the duodenum and jejunum of pigs and humans and is the largest intestinal fluke to parasitize humans. Humans are infected by eating freshwater aquatic plants such as water caltrops, water chestnuts, and water bamboo, which can harbor the metacercariae. In the intestine, the metacercariae excyst, attach to the duodenum or jejunum, develop, and grow into adult worms. They lay unembryonated eggs, which are excreted in the feces.

In water, inside the egg, a ciliated miracidium develops, comes out, and penetrates a suitable snail host. Inside the snail, after several stages of asexual multiplication, large numbers of cercariae are produced. The latter emerge from the snail and encyst on the surface of aquatic plants to metacercariae. Ingestion of these plants causes infection in humans, and the cycle is repeated. Clinical presentation of schistosomiasis includes acute and chronic manifestations:

Acute manifestations

Cercarial dermatitis, also known as swimmer's itch, is an allergic reaction caused by the penetration of cercariae in persons who have been exposed to cercariae in fresh water. Cercarial dermatitis manifests as petechial hemorrhages with edema and pruritus, followed by maculopapular rash, which may become vesicular. The process is usually related to avian schistosomal species of the genera Trichobilharzia,Gigantobilharzia, and Orientobilharzia, which do not develop further in humans. Katayama syndrome corresponds to maturation of the fluke and the beginning of oviposition. This syndrome is caused by high worm load and egg antigen stimuli that result from immune complex formation and leads to a serum sickness-like illness. This is the most severe form and is most common in persons with S mansoni and S japonicum infections. Symptoms include high fever, chills, headache, hepatosplenomegaly, lymphadenopathy, eosinophilia, and dysentery. A history of travel in an endemic area provides a clue to the diagnosis.

Chronic manifestations

Symptoms depend on the Schistosoma species that causes the infection, the duration and severity of the infection, and the immune response of the host to the egg antigens.

Terminal hematuria, dysuria, and frequent urination are the main clinical symptoms of urinary schistosomiasis. The earliest bladder sign is pseudotubercle, but, in longstanding infection, radiography reveals nests of calcified ova (sandy patches) surrounded by fibrous tissue in the submucosa.

Dysentery, diarrhea, weakness, and abdominal pain are the major symptoms of intestinal schistosomiasis.

A reaction to schistosomal eggs in the liver causes granuloma formation and neoangiogenesis with periportal fibrotic reaction termed Symmers clay pipestem fibrosis manifested as portal hypertension, splenomegaly and esophageal varices.

Hemoptysis, palpitation, and dyspnea upon exertion are the symptoms of schistosomal cor pulmonale that develops as a complication of hepatic schistosomiasis.

Headache, seizures (both generalized and focal), myeloradiculopathy with lower limb and back pain, paresthesia, and urinary bladder dysfunction are the noted symptoms of CNS schistosomiasis due to S japonicum infection.

Neuroschistosomiasis is a severe manifestation of schistosomal infection. The neurological symptoms result from the inflammatory response of the host to the deposition of eggs in the brain and spinal cord. Myelopathy is the most common neurological complication of S mansoni infection. Clinical presentation of Paragonimiasis includes acute manifestations such as acute pulmonary infection that is characterized by low-grade fever, cough, night sweats, chest pain, and blood-stained rusty-brown sputum. Chronic manifestations can include lung abscessor pleural effusion. Fever, hemoptysis, pleurisy pain, dyspnea, and recurrent attacks of bacterial pneumonia are the common symptoms. The condition mimics pulmonary tuberculosis.

Fever, headache, nausea, vomiting, visual disturbances, motor weakness, and localized or generalized paralysis are the symptoms of cerebral paragonimiasis.

Pulmonary paragonimiasis has been found to mimic metastatic pulmonary tumors on evaluation with imaging methods such as computed tomography (CT) and positron emission tomography (PET) scanning.

Paragonimiasis can affect all parts of the human body, and reports have described cerebral paragonimiasis in the last few years. The rate of cerebral paragonimiasis has been found to be about 0.8% of all active cases of paragonimiasis.

Liver fluke infections comprise acute and chronic manifestations:

Acute manifestations Fascioliasis is mostly subclinical. Acute manifestations are due to migration of larva through lung parenchyma. Malaise, intermittent fever, night sweats, and pain in the right costal area are early symptoms of acute infection.

Clonorchiasis is frequently asymptomatic. A serum sickness-like illness with symptoms of high fever, eosinophilia, and rash occurs in individuals with acute infection.

Chronic manifestations

Chronic fascioliasis is frequently asymptomatic. In symptomatic patients, irregular fever, anemia, hepatobiliary manifestations (colicky pain, jaundice), and secondary bacterial infections are present. In its end stage, chronic clonorchiasis may be complicated by recurrent pyogenic cholangitis and jaundice associated with cholangiocarcinoma.

Intestinal fluke infections are frequently asymptomatic. Diarrhea and abdominal pain are common symptoms in individuals with acute infection.

Generalized abdominal pain; ascites; and edema of the face, abdomen wall, and lower limbs are the main symptoms.

Acute schistosomiasis infections manifest hepatosplenomegaly, lymphadenopathy, and rashes.

Chronic schistosomiasis manifests anemia, pedal edema, ascites, and abdominal distension with distended abdominal veins. Patients may also have intestinal polyposis and signs of malnutrition. Abdominal mass, pain in the abdomen, and mucosanguineous diarrhea characterize abdominal paragonimiasis.

Patients with chronic liver fluke infection such as clonorchiasis may have tender hepatomegaly, progressive ascites, catarrhal cholecystitis, progressive edema, and jaundice.

Patients with mild intestinal fluke infection are usually asymptomatic. Patients with severe infections may have ascites and edema of the face, abdomen wall, and lower limbs.

The compound of formula (i) of the present invention treat and/or prevent at least one of the above indicated manifestations or symptoms.

In the present invention, the genus Schistosoma is composed of over twenty species, infecting mammalian hosts. The genus has been divided into four groups — indicum, japonicum, haematobium and mansoni. Thirteen species are found in Africa. Twelve of these are divided into two groups— those with a lateral spine on the egg (mansoni group) and those with a terminal spine (haematobium group). The four mansoni group species are: S. edwardiense, S. hippotami, S. mansoni and S. rodhaini. The nine haematobium group species are: S. bovis, S. curassoni, S. intercalatum, S. guineensis, S. haematobium, S. kisumuensis, S. leiperi, S. margrebowiei and S. matthei. The indicum group has three species: S. indicum, S. nasale and S. spindale. This group appears to have evolved during the Pleistocene. All use pulmonate snails as hosts. The japonicum group has three species: S. japonicum, S. malayensis and S. mekongi. All the species are part of the invention.

In the present invention the term "larvae" means the free swimming stage infective for the definitive host, the term "immature or juvenile worms" means trematodes, in particular schistosomes, aged up to 6 weeks post infection, the term "adult worms" means trematodes, in particular schistosomes aged from 6 weeks post infection.

The present invention will be described by means of non-limiting examples referring to the following figures:

Figure 1. Adult worms (8 weeks old, S. mansoni) in vitro survival following ovemight treatment with ΙΟμΜ PHX, ΙΟμΜ Gambogic Acid, ΙΟμΜ Praziquantel (PZQ) or DMSO (vehicle). Parasites damage was assessed by optical examination each day using the following criteria: reduction of motility, tegumental damages and darker appearance. Data shown are the means of results of at least three experiments.

Figure 2. Juvenile worms (4 weeks old, S. mansoni) in vitro survival following ovemight treatment with ΙΟμΜ PHX, ΙΟμΜ Gambogic Acid, ΙΟμΜ Praziquantel (PZQ) or DMSO (vehicle). Parasites damage was assessed by optical examination each day using the following criteria: reduction of motility, tegumental damages and darker appearance. Data shown are the means of results of at least three experiments.

Figure 3. Effect of PHX on total worm count (A) and egg burdens (B) in mice infected with S. mansoni. One-way analysis of variance (one-way Anova) was used to compare means of the samples. The p-values are indicated. PZQ (Praziquantel, oral administration, 500 mg/kg), PERHEX LOW (PHX, oral administration, 23 mg kg), PERHEX HIGH (PHX, oral administration, 70 mg/kg). DETAILED DESCRIPTION OF THE INVENTION MATERIAL AND METHODS

Compounds, reagents, medium

Gambogic Acid, Perhexiline maleate salt, Praziquantel, Dimethyl sulphoxide (DMSO), Percoll (starting density 1.13g/ml) and Foetal bovine serum (FBS) were purchased from Sigma-Aldrich. CellTiter-Glo reagent, used in the luminescent viability schistosomula assay, was purchased from Promega. BioWhittaker Dulbecco's Modified Eagle's Medium (DMEM) lacking phenol red but containing 4500 mg/1 glucose (Lonza), supplemented with ImM Hepes (Lonza), 2mM Z-glutamine (Lonza), lx antibiotic-antimycotic reagent (Life Technologies) and 10% FBS was the completed tissue culture media for schistosomula. Juvenile and adult worms (S. mansoni) were cultured in BioWhittaker Dulbecco's Modified Eagle's Medium (DMEM) containing 4500mg/l glucose (Lonza) supplement with 2mM Z-glutamine (Lonza), Penicilline lOOU/ml, Streptomycine 10(^g/ml (Lonza), Amphothericin B 0^g/ml (Cambrex) and 10% heath inactivated FBS.

Ethics statement

All animals were subjected to experimental protocols as reviewed and approved by the Public Veterinary Health Department of the Italian Ministry of Health (Rome, Italy) (Authorization N. 25/2014-PR), according to the ethical and safety rules and guidelines for the use of animals in biomedical research provided by the relevant Italian laws and European Union's directives.

Maintenance of the S. mansoni life cycle

A Puerto-Rican strain of S. mansoni was maintained by passage through albino Biomphalaria glabrata, as the intermediate host, and ICR (CD-I) outbred female mice (Harlan Laboratories). The snails had been individually infected with 8-12 miracidiae per snail. Snails were kept in tanks with dechlorinated tap water in a humid room simulating a 12 hour day and night cycle. First shedding of cercariae occurred from 4 weeks post infection. Approximately 100-200 snails (size of snails: 6 - 1 1mm) were placed twice under a direct 2000 lux lamp for 60 min at 27°C. The cercarial suspension was collected and used for the preparation of schistosomula.

Adult parasites were harvested by reversed perfusion of the hepatic portal system of infected mice previously euthanized with peritoneal (i.p.) injections of Tiletamina/Zolazepam (800 mg/kg) + Xylazina (100 mg kg). Animals and S. mansoni infection

Female ICR (CD-I) outbred and C57BL/6, 4-7 weeks old mice (Harlan Laboratories) were housed under controlled conditions [(22 ± 2) °C; (65 ± 15)% relative humidity; 12/12 h light/dark cycle]. The mice received standard food and water ad libitum. Female ICR (CD-I) outbred mice were infected transcutaneously with approximately 80 (for mixed infection) or 200 (single sex) S. mansoni cercariae for life cycle maintainance and in vitro worm assays. For in vivo experiments, C57BL/6 mice were infected with 140 cercariae and administered with selected compounds. Preparation of schistosomula

Cercariae, shed from infected snails, were subsequently converted to schistosomula by mechanical transformation using an optimised version of the protocol of Brink et al., 1977 previously described (Protasio et al. 2013). Briefly, the cercarial suspension (approximately 50.000 cercariae) was placed in a glass 40 ml tube on ice for 60 minutes in order to reduce parasite motility. Tail detachment was obtained by shaking cercariae vigorously for approximately 30 seconds in a vortex mixer before passing these through a 22G syringe needle approximately 10-12 times. Next, the separation of heads/schistosomula and tails was obtained by placing the heads plus tails suspension on 4-5 ml of ice-cold 70% Percoll in tube and centrifugating (600 xg) for 10 minutes at 4°C. Finally, the schistomula preparations were washed twice (with DMEM complete media lacking FBS) and microscope examination was used to assess the quantity and quality of purified schistosomula (less than 1% tails). Schistosomula were cultured at 37°C in 6 wells tissue culture plates containing 3 ml schistosomula complete media in an atmosphere of 5% CO2 for 24 hr before any further experimental manipulations proceeded. Negligible parasite death occurred in this media during the 24 hr culturing period. Following this, schistosomula were aliquoted into flat-bottom 384-well black-sided for compound assays.

Screening of compounds

A compounds collection including also FDA and/or EMA approved drugs was tested according to the following procedure. a) Compound storage and transfer to assay plates.

Compounds are stored as solution of 100% DMSO at - 20 °C under inert atmosphere. Intermediate storage microplates, in the 384 well plate format, are prepared on demand and stored in the same controlled environment as the stock solutions. Intermediate microplate stored compounds are transferred to assay plates by the acoustic droplet ejection technology (ATS- 100, EDC Biosystems USA) which ensures a safe, contactless, pre-dilution free delivery. The test set was transferred to 384 well, black, tissue culture treated destination plate. The initial test was carried out at a single concentration of 10uM. b) Assay protocol. A luminescence-based medium-throughput assay has been used. Briefly, a suspension of schistosomula, in DMEM added with 10 % FBS, was transferred to each well of the compound containing assay plates in order to have 100 schistosomula per well in a final volume of 30 μL·. The plate set was let to incubate with the compounds at 37 °C/5% C0 ₂ for 24 hours. At the end of the incubation period each well was filled by and equal volume (30μί) of CellTiterGlo (Promega Corp. P/N G7570) and let to incubate for 30 minutes at room temperature. The light based signal emitted by the reaction is proportional to the ATP amount in the culture and ultimately reflects the mitochondrial function and thus the schistosomula viablity. The readout, luminescence signal,, was carried out by a CCD based detector (ViewLux, PerkinElmer USA). Each plate contains 16 DMSO treated samples as negative controls and 16 gambogic acid treated samples as positive controls (C. Lalli et al, 2015). c) Active compound selection and confirmation.

In order to select the best cytotoxic compounds, the effect of each molecule was rescaled between 0% cytotoxicity (DMSO treated samples) and 100% cytotoxicity (gambogic acid treated samples). A threshold was set to 70% cytotoxicity that, together with other presumably cytotoxic compounds, surprisingly showed PHX as positive compound.

The latter molecule was purchased from Sigma Aldrich (SML0120) as fresh lot and subjected to the UPLC-MS quality control. After having passed the QC, the new batch of PHX was tested in a dose response manner. To this aim, a serial dilution of the compound was carried out in DMSO in order to cover the concentration range between 50 μΜ and 20 nM. The transfer of the serially diluted compounds and the assay protocol were identical to those described in the previous paragraphs.

In vitro studies with S. mansoni juvenile (4 weeks old) and adult (8 weeks or older) worms

Seven-eight male worms or 4-5 worm pairs were recovered under aseptic conditions from infected mice by perfusion of mesenteric veins at 28 days (juvenile) or 56-days post infection (adult pairs) or 2-4 months post-infection (adult males). After washing, the worms were transferred into a 35 mm plates containing 3 mL of DMEM complete worms media and incubated at 37 °C in a humid atmosphere containing 5% C0 ₂ with drugs overnight. Next, the worms were washed 3 times with drug-free medium and were incubated for 5 days at 37°C/5% C0 ₂ and monitored daily under a stereo microscope for mobility, tegumental damage, viability and egg output (worm pairs). The experiment was repeated at least three times. In vivo studies with S. mansoni infected mice

C57B/L6 inbred female mice (5-6 weeks old) were divided in 4 groups of 7 animals each and treated with DMSO (control), PZQ or two dosages of PHX. All compounds were administered orally (vehicle is 2.5% Cremophor EL) 42 days post infection. The amount of PHX administered (23 mg/kg and 70 mg/kg) was guided by available information of therapeutic dose in use for angina treatment (Ashrafian H., 2007); PZQ was used at the standard dose (500mg/Kg). Toxicity of compounds (e.g., death and behavioral changes) was assessed daily during and after treatment until the time of euthanasia. At 56 days post infection, mice were euthanized with an intraperitoneal injection of Tiletamina/Zolazepam (800mg/kg) + Xylazina (l OOmg/kg) and adult worms perfused as described above.

Compound efficacy in vivo, measured using a number of criteria, was compared to that of the anti- schistosomal drug, PZQ. The criteria include the numbers of male and female worms (total worm count) recovered by perfusion and hepatic egg burden (number of eggs in the liver). To recover eggs trapped in liver, whole livers from individual mice were excised, weighed and incubated in a 4% KOH solution over night at 37°C. Eggs were counted under a dissecting microscope as previously described (Cheever, A. W., 1968).

RESULTS

Efficacy of PHX on schistosomula in vitro

In order to investigate the effect of PHX on S. mansoni larvae viability, newly transformed schistosomula were cultured in vitro for 24h in presence of a range of drug concentrations (0.019- 50μΜ). Parasites survival was assessed by using a CellTiterGlo luminescent assay (C. Lalli et al, , 2015) and the CCso (calculated by fitting the four parameters equation) is shown in Table 1 .

Table 1. Perhexiline and Gambogic Acid CCso of newly transformed schistosomula

CCso values of two independent experiments Gambogic Acid, previously described as a potent killer agent for schistosomes (Peak et al, 2010), was used as positive control and DMSO (drug vehicle) as negative control. Gambogic Acid, was confirmed to be effective in killing the schistosomula within the expected concentration range. Perhexiline was also shown to be effective in this validated model.

Efficacy of PHX on different stages ofS. mansoni in vitro

Male adult worms (7-8 worms/sample) obtained from infected mice were cultured at 37°C, 5% CO2 in DMEM complete medium (10% FBS) in presence of different concentrations of PHX (3-10 μΜ) and for variable time (12 h to 5 days). For all experiments Gambogic Acid (l -ΙΟμΜ) and PZQ (1 - 10μΜ) were used as positive controls while DMSO was used as negative control. Overnight incubation with 10 μΜ PHX resulted in a statistically significant decreased viability of parasites after 5 days as shown in Fig. l . Parasites damage was assessed by optical examination each day using the following criteria: reduction of motility, tegumental damages and darker appearance.

PHX was also tested at the concentrations of 5 μΜ and 3 μΜ leading to a reduced viability (around 50%) in the first case and having no effect in the second one.

Juvenile worms (4 weeks old parasites) were also tested at the same conditions reported above. Following overnight incubation of PHX (10 μΜ), viability of parasites was reduced by 80% after 7 days of observation (Fig. 2). Remarkably, PZQ (at the same concentration) treated worms started to recover after the wash at day 1 and PZQ treatment showed a 50% survival rate at 5 days. This is in accordance with previous report on PZQ showing a survival of approximately 80% of juvenile worms at 8 days (Livia-Pica et al, 2004).

In order to test the effect of PHX on egg production, adult worm pairs were treated overnight using the sub-lethal dose of 5 μΜ. Total number of eggs laid by female parasites was determined 3 and 6 days after the treatment. To do so, the medium containing the eggs was harvested and briefly centrifuged. The pellet containing the eggs was re-suspended in 500 μΐ of saline solution and the egg number was determined by microscopy analysis. Results showed that PHX treatment led to a reduction in egg production of around 60% after both 3 and 6 days compared to the untreated control incubated with DSMO alone.

In vivo efficacy of PHX in mice infected with S. mansoni Based on the strong in vitro effects of PHX against larvae, juvenile (4 weeks old) and adult worms, its in vivo efficacy was tested in mice infected with S. mansoni. Based on previous cardiovascular studies of the drug (Ashrafian H., 2007), two different concentrations of PHX were used: a low dose (23 mg/Kg) and a high dose (70 mg/Kg). The compound was administered orally as single dose in 2.5% Cremphor EL to mice with patent S. mansoni infection (56 days post infection). As positive control, PZQ was administered orally in a single dose at the standard concentration of 500 mg/Kg. Two weeks after the treatment, mice were sacrificed and the total worm count and egg burden (number of eggs in the liver) were determined (Fig. 3).

A significant decrease in total worm counts was observed after treatment with both LOW and HIGH doses of PHX (35 and 63% respectively). For egg burden, a reduction of 50 and 60% was shown after treatment with LOW and HIGH doses of PHX, respectively.

Based on the present data, it is demonstrated that Perhexiline is a potent inhibitor of schistosoma worms with CCso in the low micromolar range. Worthy of specific note is the fact that the compound is active against larvae and both juvenile and adult worms, thus overcoming one of the major limitations of PZQ, the only drug currently in use for the treatment of schistosomiasis.

In addition, repurposing FDA or EMA-approved products has several practical advantages over novel compounds that are as yet unapproved for use in treating human diseases. With the tested dosages and formulation, approved products have not only demonstrated their pharmacological activity but have known toxicity profiles in humans and have well-studied pharmacokinetics and pharmacodynamics.

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