BR102020018304A2 - Pharmaceutical formulation, process of producing a pharmaceutical formulation, drug, treatment method, and, use of a pharmaceutical formulation - Google Patents

Abstract

The present invention generally relates to pharmaceutical formulations comprising, as an active ingredient, the compound 17-Dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) and at least one pharmaceutically acceptable excipient. The invention further provides a process for producing said formulation, medicine, method of treating leishmaniasis and use of the formulation. The formulations of the invention constitute an innovative therapeutic alternative in the control of leishmaniasis.

Description

PHARMACEUTICAL FORMULATION, PROCESS OF PRODUCTION OF A PHARMACEUTICAL FORMULATION, DRUG, TREATMENT METHOD, AND, USE OF A PHARMACEUTICAL FORMULATION FIELD OF THE INVENTION

[001] The present invention relates to pharmaceutical formulations comprising the compound 17-Dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG). More specifically, the present invention relates to topical or systemic pharmaceutical formulations comprising 17-DMAG and the use thereof as an antiparasitic agent, preferably for the treatment of leishmaniasis. The present invention further provides a process for producing said formulation, medicine, method of treating leishmaniasis and use of the formulation.

FUNDAMENTALS OF THE INVENTION

[002] Leishmaniasis forms a complex of diseases transmitted by vectors called sandflies of the genus Lutzomyia in the New World and by the genus Phlebotomus in the Old World (MITROPOULOS, 2010).

[003] This complex of diseases affects 12 million people on the planet (AKHOUNDI, 2017). According to the WHO (2019), in 2017, 94% of new cases of leishmaniasis reported to the World Health Organization (WHO) occurred in seven countries: Brazil, Ethiopia, India, Kenya, South Sudan and Sudan.

[004] Leishmaniasis manifests in two main clinical forms: visceral leishmaniasis (VL), which can be fatal if left untreated, and cutaneous leishmaniasis, whose mucocutaneous (CML) and cutaneous (LC) forms cause lesions that usually heal spontaneously. , but they can leave scars or deformities, which leads this infectious disease to be classified as one of the six most important on the planet given its severity and prevalence (GOTO, 2010, AKHOUNDI, 2017).

[005] The treatment of choice for visceral leishmaniasis is pentavalent antimonials. In more severe cases, or in patients who do not respond adequately to treatment with antimonials, Amphotericin B is the second choice. Ultimately, the drug of choice is Pentamidine (MCGWIRE, 2014).

[006] The US Food and Drug Administration (FDA) approved in 2014 miltefosine (Impavido, Paladin Therapeutics), an oral alkylphosphocholine analogue, for the treatment of VL, CL and CML. It is the first oral drug to be approved for the treatment of VL (SUNYOTO, 2018).

[007] Current treatments for leishmaniasis have several limitations, such as high toxicity, low efficacy, high cost, development of parasite resistance and poor patient adherence, which can lead to therapeutic failure. Thus, the most recent interest in the class of antiparasitic drugs is focused on increasing efficacy, decreasing side effects and low costs (MONGE-MAILLO, 2013; COPELAND, 2015).

Pentavalent Antimonials

[008] The recommended first-line treatment for most forms of leishmaniasis is the systemic use of pentavalent antimonials (Sb+5) such as N-methylglucamine antimoniate or meglumine antimoniate (Glucantime®) and sodium stibogluconate (Pentostam). ®). For systemic use, these compounds can be administered intravenously or intramuscularly, and are also used intralesionally for CL.

[009] Sodium stibogluconate and N-methylglucantime exhibit similar pharmacodynamics and pharmacokinetics. Pharmacokinetics indicate that more than 80% of the administered dose is excreted unchanged within 6 hours in urine. Different dosage regimens have been proposed and tested over the years in an attempt to find the minimum effective therapeutic dose with the least toxic side effects (WHO, 1995; MITROPOULOS, 2010).

[0010] The World Health Organization recommends that doses of antimony should not exceed 20 mg/kg/day, not exceeding the limit of 850 mg of antimony, due to its high toxicity. (WHO, 1995)

[0011] Despite being widely used, pentavalent antimonials cause severe side effects and chemotherapy with these drugs is necessary for a prolonged period. Side effects such as cardiac arrhythmias, nephrotoxicity, pancreotoxity and hepatotoxicity have already been described in several studies (MCGWIRE, 2014; MONGE-MAILLO, 2013; HALDAR, 2011, GOTO, 2010).

[0012] Another challenge for the clinic is the need to progressively increase the daily dose of antimony and the duration of therapy to combat the lack of response and resistance of organisms to therapy. In India, 65% of patients not previously treated with antimonials do not respond promptly or relapse after therapy. In Brazil, cure rates after Sb+5 therapy are becoming increasingly low and range from 50% to 90%. (HALDAR, 2011, MACHADO, 2010)

[0013] The development of parasite resistance to a drug used for decades and the irregular adherence of patients to treatment, due to the parenteral daily dose for 20 days, may be the main factors in determining the increasing failure rate of antimonials. Other alternative drugs, such as pentamidine and amphotericin B, are also used parenterally (MITROPOULOS, 2010).

Amphotericin B (AmB)

[0014] Amphotericin preparations have typically been used in the treatment of VL and mucosal leishmaniasis (LM). In some regions of the world (eg United States of America and Europe) it is the drug of first choice (MITROPOULOS, 2010, GOTO, 2010).

[0015] AmB is available in four drug formulations: amphotericin deoxycholate (d-AmB), liposomal amphotericin (L-AmB; Ambisome®), lipid complex amphotericin B (ABLC; Abelcet®); amphotericin B colloidal dispersion (ABCD; Amphotec® or Amphocil®) (GOTO, 2010).

[0016] In general, these formulations share similar efficacy, but have differences regarding side effects. Common side effects of amphotericin B are renal failure and electrolyte abnormalities, which are most commonly seen in association with d-AmB. (MCGWIRE, 2014; MITROPOULOS, 2010; COPELAND, 2015).

[0017] In addition to the high toxicity, d-AmB must be administered by slow intravenous infusion that lasts for hours, which suggests the need for medical care during the application of the treatment in a hospital environment (MCGWIRE, 2014).

[0018] Amphotericin B lipid formulations were developed as a strategy to prolong the half-life of the drug and reduce toxicity. L-AmB has been showing good results for (VL) but the high cost of this drug is still a limiting factor for access to treatment. Furthermore, the use of this drug for (CL) in humans still lacks more conclusive clinical trials (COPELAND, 2015).

pentamidine

[0019] Pentamidine, a synthetic derivative of amidine, is mainly used as an option to pentavalent antimonials in individuals intolerant or resistant to antimony treatment. This drug also has activity against protozoa (certain strains of Trypanosoma and Babesia) and fungi (Candida albicans). The precise mode of its antiprotozoal action is still not fully understood (MITROPOULOS, 2010; MCGWIRE, 2014).

[0020] Pentamidine therapy has the advantage of having a short duration. Despite this, frequent adverse reactions with moderate morbidity have been associated with its use, including an unusually high rate (50%) of hyperglycemia, likely as a result of pancreatic damage and hypotension, tachycardia, and cardiologic changes. Patients using this medication require careful observation (MITROPOULOS, 2010; MCGWIRE, 2014).

miltefosine

[0021] Miltefosine was the first oral drug approved for the treatment of leishmaniasis. The drug has shown very promising results in VL in the Indian population (95% cure rates) and in CL in Pakistan (cure rates comparable to pentavalent antimony) and is undergoing clinical trials for use in several other countries. The main adverse effects reported associated with treatment are non-specific nausea and vomiting. The drug is teratogenic and contraindicated in pregnancy. (MITROPOULOS, 2010).

[0022] However, a study done of the use of miltefosine in CL therapy in Colombia and Guatemala revealed therapeutic results that varied according to the causative species. In regions of Colombia where L. panamensis is common, researchers reported cure rates of 91% for the oral miltefosine group versus 38% for the placebo group. The cure rate of miltefosine was comparable to treatment with standard pentavalent antimony. On the other hand, in the regions of Guatemala where L. braziliensis and L. mexicana are predominant, the cure rate for the miltefosine-treated group was 53% versus 21% in the placebo group (MITROPOULOS, 2010).

[0023] Another study evaluated the efficacy and safety of miltefosine versus pentavalent antimony in the treatment of CL caused by L. braziliensis and L. guyanensis. The result showed a cure rate above 75% with miltefosine and 60% with Sb+5. However, the used dose of 150 mg/kg/day for 28 days presented high cost and side effects such as nausea and vomiting in half of the patients (MACHADO, 2010).

Heat Shock Protein 90 (HSP90) Inhibitors

[0024] The drugs currently used in the treatment of leishmaniasis were discovered decades ago and/or have serious adverse effects and limitations of access to the drug due to the high cost. Several research centers around the world have been supporting basic science in understanding the biology of parasites, composing important knowledge that facilitates the choice of therapeutic targets. Among the molecular targets with potential to be explored pharmacologically, heat shock protein 90 (HSP90) may be an alternative for new, more effective antiparasitic drugs, with fewer side effects and low cost.

[0025] HSP90 acts as a molecular chaperone helping the correct folding of nascent proteins, thus preventing the formation of proteins with incorrect tertiary structure and formation of protein aggregates. HSP90 assists the structural maturation of newly synthesized proteins under conditions of cellular homeostasis and stabilizes proteins when cells are under stress conditions, such as those caused by temperature increases, pH changes and nutritional deprivation (PRATT, 2003).

[0026] Many HSP90 client proteins are involved in different oncogenic pathways. Thus, HSP90 came to be recognized as a potential molecular target against different types of cancer.

[0027] Geldanamycin (GA) was one of the first natural compounds identified as being able to inhibit the activity of HSP90. GA is a natural antibiotic derived from the fungus Streptomyces hygroscopicus, which has antiproliferative activity against cancer cells and belongs to the benzoquinone ansamycin family. GA has low solubility, limited in vivo stability and is hepatotoxic, making it a compound with low chances of becoming marketable (SCHULTE, 1998; ISAACS, 2003).

[0028] Semi-synthetic compounds were produced from GA. The first HSP90 inhibitor to enter clinical trials for cancer was a GA derivative, 17-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin). 17-AAG showed a greater number of positive results against breast cancer and multiple myeloma compared to GA, but also problems with solubility, low pharmaceutical properties and the toxic profile mainly related to the benzoquinone moiety (NECKERS, 2012; HOLLINGSHEAD, 2005). .

[0029] 17–Dimethylaminoethylamino–17-demethoxygeldanamycin (17-DMAG, alvespimycin), another derivative of geldanamycin, has improved formulation and pharmacokinetics over 17-AAG. However, preclinical studies have indicated that, in the treatment of cancers, 17-DMAG can cause hepatotoxicity, with a lower maximum tolerated dose than that tolerated by 17-AAG (HOLLINGSHEAD, 2005).

[0030] The research for HSP90 inhibitor molecules still encompasses some questions and challenges, such as, for example, difficulty in defining the maximum tolerated dose of drugs and better anticancer results with approaches that explore other targets such as HSP40, HSP70 or CDC37 for certain types of cancer. tumors (DEN, 2012).

HSP90 from protozoan parasites

[0031] In protozoan parasites, HSP90 is involved in processes related to the development of specific stages and the pathogenesis of the disease (SHONHAI, 2011).

[0032] The parasite undergoes stress due to sudden changes in temperature, pH, reactive oxygen species and the attack of hydrolytic enzymes when it infects host cells. The increased expression of chaperones by the parasite in response to stress is related to adaptation to new environmental conditions, which activates the process of cell differentiation. Evidence indicates that chaperones directly influence the virulence of the parasite and its survival within macrophages of the vertebrate host (SHONHAI, 2011).

[0033] HSP90 is an abundant protein in Leishmania, representing about 2.8% of the total cytoplasmic proteins of this parasite. Some Leishmania spp. HSP90 client proteins. have a role in the cell cycle of the parasite (SHONHAI, 2011).

[0034] The study by SANTOS (2014) evaluated the ability of the HSP90 inhibitor, 17-AAG, to reduce in vitro and in vivo Leishmania braziliensis infection. Treatment with 17-AAG of infected mice resulted in a reduction in the size of the skin lesion and in the parasite load at the site of infection (from 107 in the control animals to 106 in the treated animals), however, it was not able to reduce the parasite load. in the draining lymph node of the treated animals, showing that the results observed were not as robust as those described for other protozoan parasites. It is possible that the persistence of L. braziliensis parasites in the initial site of infection and in the lymph node of these animals shows that there is a difficulty for 17-AAG to reach peripheral tissues, such as the dermis and epidermis. The use of dermal dressings or ointments containing the drug may be a strategy to increase the efficiency of the treatment of CL. Thus, the drug could reach higher concentrations at the site of infection, however, there is a difficulty in the technique in identifying the tolerated dose.

[0035] The study by Palma et al. (2019) demonstrates the different affinity of Hsp90 inhibitors - geldaminicin and its analogues 17-AAG and 17-DMAG - in a leishmaniasis parasite species. From an in silico study it was possible to identify that the inhibitors have a greater tendency to bind to the N-terminal domain of the Leishmania amazonensis protein Hsp83 compared to the human protein Hsp90. It is disclosed that the existing intermolecular differences allow the use in anti-Leishmania therapies (PALMA, 2019)

[0036] The present invention is built with the use of the compound 17-DMAG that proved to be effective against Leishmania amazonensis and Leishmania braziliensis in LC models in vitro and in vivo. In addition to the technical contributions, combining the 17-DMAG molecule with other inputs and under specific technical parameters in a pharmaceutical formulation active against leishmaniasis.

[0037] In short, the invention described herein aims to provide a pharmaceutical formulation comprising 17-DMAG that: (1) enables the administration of the drug that is released more adequately and, therefore, resulting in a more effective formulation than the free compound; (2) defines the in vivo active concentration range of 17-DMAG in the formulation; (3) shows a significant reduction in lesion size compared to in vitro and in vivo assays in cutaneous leishmaniasis models on the species L. amazonensis and L. braziliensis.

[0038] The invention is therefore to obtain alternatives for the treatment of parasitic diseases. In this sense, the present investigation is based on formulations comprising 17-DMAG and its leishmanicidal activity superior to the state of the art with reduction of side and adverse effects.

SUMMARY OF THE INVENTION

[0039] In a more general aspect, the invention provides a pharmaceutical formulation which comprises, as an active ingredient, a therapeutically effective amount of the 17-DMAG compound and at least one pharmaceutically acceptable excipient.

• (a) solubilização do princípio ativo em excipientes adequados;
• (b) formação das nanopartículas a partir da diluição em meio previamente selecionado e processamento por emulsificação;
• (c) recuperação das nanopartículas pela eliminação dos solventes empregados no processo e extração física.

[0040] In another aspect, the production process of a pharmaceutical formulation is provided, comprising the steps of:

• (a) solubilization of the active ingredient in suitable excipients;
• (b) formation of the nanoparticles from the dilution in a previously selected medium and processing by emulsification;
• (c) recovery of nanoparticles through the elimination of solvents used in the process and physical extraction.

[0041] In another aspect, a medicament is provided, which comprises said pharmaceutical formulation

[0042] In yet another aspect, the invention provides a method of treating leishmaniasis using the formulation described above.

[0043] In a further aspect, the invention provides the use of the formulation described above for the manufacture of a medicament for treating leishmaniasis.

BRIEF DESCRIPTION OF THE FIGURES

[0044] Figure 1 refers to the chemical structure of the 17-DMAG molecule.

[0045] Figure 2 refers to the in vitro cell viability assay with axenic promastigotes of L. amazonensis and differentiated THP-1 cells treated with 17-DMAG in a 12-step serial dilution assay (initial concentration: 2 µM and 50 µM, respectively). The graph represents IC50 (L. amazonensis) and CC50 (THP-1 cells) values for 17-DMAG. Mann Whitney test *** p = 0.001.

[0046] Figure 3 refers to the cytotoxicity assay in human lung fibroblasts, MRC-5 cells, treated with 17-DMAG (initial concentration: 50 µM) in a 12-step serial dilution assay, performed in triplicate. The graph represents values of cell viability percentages.

[0047] Figure 4 refers to the intracellular viability assay in THP-1 cells infected by L. amazonensis and treated with 17-DMAG at different concentrations. The graph represents the number of viable cells.

[0048] Figure 5 refers to the effect of different treatment regimens with 17-DMAG on the lesion size of BALB/c mice infected by L. braziliensis. The graphs express the difference in the thickness of the lesion in the infected ear (left ear - LE) in relation to the contralateral ear (right ear - OD) of the treated and untreated animals. Where, (A) all groups of animals, treated and untreated; (B) area under the curve (AUC) of lesion thickness shown in “A”. ANOVA, (*** p < 0.0001; * p < 0.05).

[0049] Figure 6 refers to images of the effect of different treatment regimens with 17-DMAG on the lesion size of BALB/c mice infected with L. braziliensis. (A – D) lesions in the control group, applied intraperitoneally with a 5% glucose solution; (E – H) lesions in the group treated with 20 mg/kg daily; (I – L) lesions in the group treated with 30 mg/kg every other day; (M – P) lesions in the group treated with 50 mg/kg every five days.

[0050] Figure 7 refers to the kinetics of 17-DMAG treatment (20 mg/kg concentration, daily dose for two, four or seven weeks) on the lesion size of BALB/c mice infected with L. braziliensis. Where, (A) lesion development in treated or control groups, monitored weekly (B) Area under the curve (AUC) of lesion development shown in “A”. Student's t test, ***p< 0.0001.

[0051] Figure 8 refers to the parasite load of the ear and lymph node of mice infected with L. braziliensis treated with 17-DMAG intraperitoneally (concentration of 20 mg/kg, daily dose for two, four or seven weeks). Where, (A) lymph nodes after two weeks, (B) ear after two weeks, (C) lymph node after four weeks, (D) ear after four weeks; (E) lymph node after seven weeks; (F) ear after seven weeks. Student's t test, (*** p< 0.0001; ** p< 0.01; * p< 0.05).

[0052] Figure 9 refers to images of lesions in the ear and lymph nodes of BALB/c mice infected with L. braziliensis and treated with 17-DMAG injected intraperitoneally (concentration of 20 mg/kg, daily dose for two, four or seven weeks). Where, (A – D) ear and lymph node of the animals in the control group, which received a 5% glucose solution daily for two weeks intraperitoneally; (E – H) ear and lymph node of infected animals treated with 20 mg/kg daily for two weeks; (I – L) ear and lymph node of the animals in the control group, which received a 5% glucose solution daily for four weeks intraperitoneally; (M – P) ear and lymph node of infected animals treated with 20 mg/kg daily for four weeks

[0053] Figure 10 refers to the toxicity image of topical application of free 17-DMAG, incorporated into a hydrogel in BALB/c mice. Healthy BALB/c mice were daily exposed to 17-DMAG incorporated in hydrogel, at concentrations of 0.15 mg/g; 0.20 mg/g; 0.25mg/g or 0.30mg/g for four weeks. As a control, animals were exposed to the white hydrogel without 17-DMAG. The results are expressed as the difference in the thickness of the exposed ear compared to the contralateral ear. (* p < 0.05, Kruskal-Wallis test).

[0054] Figure 11 refers to the electron microscopy image of the polymeric nanoparticles of 17-DMAG (A and B), of the polymeric nanoparticles contrasted with osmium (A' and B'), with the measurements of the nanoparticles.

DETAILED DESCRIPTION OF THE INVENTION

[0055] It is believed that one skilled in the art may, based on the description contained in the present invention, use the same to its fullest extent. The specific embodiments below are to be interpreted as merely illustrative, and not limiting in any respect to the remainder of the description.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one skilled in the art to which the present invention pertains. The specification also provides definitions of terms to aid in the interpretation of what is described herein and the claims. The terminology used in describing the invention is for the purpose of describing preferred embodiments only, and is not intended to limit the scope of its teachings.

[0057] Unless otherwise indicated, all numbers expressing amounts, percentages and proportions, and other numerical values used in the specification and claims, shall be understood to be modified, in all cases, by the term " about". Thus, unless otherwise indicated, the numerical parameters shown in the specification and claims are approximations that may vary depending on the properties to be obtained.

[0058] All patent or non-patent bibliographic references cited in this application are hereby incorporated by reference in their entirety.

[0059] The present invention, in its most general aspect, provides a pharmaceutical formulation that comprises, as an active ingredient, a therapeutically effective amount of the 17-DMGA compound and at least one pharmaceutically acceptable excipient, such that this formulation improves the effectiveness of the drug. treatment of leishmaniasis, while reducing its toxicity.

[0060] The term "active" is used herein to refer to a material that induces a desired effect and includes derivatives and analogues of the specifically mentioned material that also induces the desired effect.

[0061] As used herein, the term "therapeutically effective amount" means an amount sufficient to induce the desired effect, but low enough to avoid serious side effects. For any compound, the therapeutically effective dose can be estimated initially, either in cell culture assays or in animal models, usually mice, rabbits, dogs or pigs. The animal model can also be used to determine the appropriate concentration range and route of administration. Information of this type can then be used to determine useful doses and routes of administration in humans.

[0062] In one embodiment, the drug 17-DMAG is comprised in said formulation in the range of 0.50 mg/g to 0.05 mg/g in relation to the total weight of the formulation. According to the present invention, the administration of said pharmaceutical formulations can be carried out by the routes of administration: oral, sublingual, nasal, intravenous, intramuscular, intraperitoneal, intra-articular, subcutaneous, cutaneous, transdermal, but not limited to these.

[0063] Pharmaceutically acceptable excipients are selected depending on the final presentation of the formulation of the present invention, which can be in the form of capsules, tablets or suspension for oral administration, suspension for nasal administration, injectable suspension for intramuscular, intravenous, cutaneous or subcutaneous, semi-solid formulations for topical administration. Pharmaceutically acceptable excipients do not show toxicity to the recipient organism at the dosages and concentrations employed and include buffers such as phosphates, citrates and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkyl parabens such as methyl and propyl paraben, catechol, resorcinol and m-cresol; polymeric excipients such as polyvinylpyrrolidones and polyethylene glycols; sweeteners and flavoring agents; stabilizing agents such as EDTA or EGTA; non-ionic surfactants such as polysorbates 20 and 80; diluents such as lactose, starch, mannitol or microcrystalline cellulose; lubricants such as stearic acid, stearates (magnesium, zinc), sodium stearyl fumarate; flow promoters such as talc and colloidal silicon dioxide; granulating agents such as cellulose derivatives (HPC, HPMC), pregelatinized starch, PVP k-30; disintegrants such as sodium starch glycollate, croscarmellose sodium or crospovidone; wetting agents such as sodium lauryl sulfate, sodium docusate, polysorbates 20, 60, 80 (Tween®); coating agents such as cellulose derivatives (methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, cellulose acetate). References known to the skilled artisan on pharmaceutically acceptable excipients are the publication "Remington's Pharmaceutical Sciences", 18th edition, (1990), Mack Publishing Co., USA, and the publication "Handbook of Pharmaceutical Additives" by Michael and Irene Ash, 1995, Gower (England).

[0064] For purposes of this invention, the term "pharmaceutically acceptable" means that the excipient is suitable for use in contact with tissues without toxicity, incompatibility, instability, irritation or undue allergic reaction, or the like.

[0065] Non-limiting examples of excipients suitable for the invention include surfactants such as tweens, spans and myrjs; gelling agents such as carboxymethyl, hydroxyethyl, hydroxypropyl celluloses, nonionic emulsifiers such as monoglycerides, fatty acid esters, monooleates, polysorbates; immobilizing agents such as cyclodextrins: alpha, beta, beta hydroxypropyl, alginates and gelatins; film-forming or film-forming agents such as polyvinyl alcohol and cetostearyl alcohol; epithelializing agents such as allantoin and cepalin; dispersing agents such as sorbitol, polyethylene glycols, stearates; and preserving and stabilizing agents, such as parabens and imidasolinidylurea, used alone or in combination with each other.

[0066] In one embodiment, the excipient is selected from the group consisting of diluents, carriers, wetting agents, dispersants, disintegrants, flow promoters, coating agents, stabilizers, gelling agents, emulsifiers, sweeteners, flavors and preservatives.

[0067] In one embodiment, the excipients are in the range of 50% to 95% in relation to the total weight of the formulations. Preferably, the excipients are in the range of 60% to 90%. More preferably, the excipients are in the range of 65% to 80%.

[0068] In addition, the formulations described herein may optionally comprise additives in order to increase ease of administration, storage capacity, resistance to degradation, bioavailability, half-life, provide isotonic preparations, etc. Additives useful for the preparation of pharmaceutical formulations are well known in the art.

[0069] Further, said formulations may comprise modified drug delivery systems. Modified delivery systems are selected from the group comprising liposomes, osmotic pumps, enteric coatings, polymer matrix systems, transdermal systems, implants, micro and nanoemulsions and others well known in the art.

[0070] The pharmaceutical formulations of the present invention can be oral, topical or systemic.

[0071] In one embodiment, said formulations are topical formulations.

[0072] For the purposes of this invention, formulations for topical use are understood as any compositions for application directly to the skin, more specifically, to skin lesions and their surroundings.

[0073] The formulations of the invention can be used to manufacture a wide variety of types of products that include, but are not limited to, ointments, creams, pastes, gels, hydrogels, lotions, solutions, suspensions, sprays, plasters, among others.

[0074] In a preferred embodiment, said formulations are in the form of a cream or hydrogel.

[0075] For the purposes of this invention, the cream formulation obtained from the agitation of the oil phase in the aqueous phase under high speed to obtain a homogeneous cream with smaller particles (droplets) and with better texture and stability.

[0076] In a preferred embodiment, the aqueous phase contains nipagin glycerin and distilled water. In a preferred embodiment, the oil phase contains wax, petroleum jelly, cetyl alcohol or nizapol.

[0077] In a preferred embodiment, the cream formulation containing 17-DMAG in a concentration range of 0.05mg-0.30mg/g, diluted in water.

[0078] For the purposes of this invention, the hydrogel formulation is obtained by solubilizing 17-DMAG in distilled water and adding carboxymethylcellulose (CMC) solution.

[0079] In a preferred embodiment, the hydrogel formulation containing 17-DMAG in a concentration range of 0.05mg-0.30mg/g, diluted in distilled water, added to a 2% solution of CMC.

[0080] In one embodiment, the pharmaceutical formulation is for the treatment of leishmaniasis.

[0081] In a preferred embodiment, the pharmaceutical formulation is for the treatment of Visceral Leishmaniasis and/or Cutaneous Leishmaniasis.

[0082] In a more preferred embodiment, the leishmaniasis is American Cutaneous Leishmaniasis

[0083] Particularly, the American Cutaneous Leishmaniasis is caused by the species L. amazonensis and L. braziliensis, responsible for the disease in the diffuse cutaneous form.

• (a) solubilização do princípio ativo em excipientes adequados;
• (b) formação das nanopartículas a partir da diluição em meio previamente selecionado e processamento por emulsificação;
• (c) recuperação das nanopartículas pela eliminação dos solventes empregados no processo e extração física.

[0084] In another aspect, the production process of a pharmaceutical formulation is provided, comprising the steps of:

• (a) solubilization of the active ingredient in suitable excipients;
• (b) formation of the nanoparticles from the dilution in a previously selected medium and processing by emulsification;
• (c) recovery of nanoparticles through the elimination of solvents used in the process and physical extraction.

[0085] In another aspect, a medicament is provided, which comprises the combination described above.

[0086] As used in the present invention, the term "drug" refers to a pharmaceutical product, technically obtained or elaborated, with prophylactic, curative, palliative or diagnostic purposes.

[0087] In yet another aspect, the invention provides a method of treating leishmaniasis using the formulation described above.

[0088] The precise effective amount for a human subject will depend on the severity of the disease state, the general health of the subject, the age, weight, and sex of the subject, the diet, the time and frequency of administration, the combination /drug combinations, reaction sensitivities, and tolerance/response to therapy. Thus, doses to be delivered depend on a number of factors that cannot be measured before clinical studies are done. The person skilled in the art, however, knows how to arrive at adequate doses for different treatments.

[0089] In a further aspect, the invention provides the use of the combination described above for the manufacture of a medicament for treating leishmaniasis, preferably for the treatment of Visceral Leishmaniasis and/or Cutaneous Leishmaniasis, more preferably for the treatment of American Cutaneous Leishmaniasis.

[0090] While various embodiments embodying the teachings of the present invention have been shown and described in detail herein, those skilled in the art will readily be able to envision many other varied embodiments that may further embody those teachings. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the preceding description. Such modifications are also intended to be within the scope of the appended claims.

[0091] The present invention will now be described by way of examples, which are intended to be merely illustrative of the present invention and are in no way limiting as to the scope and scope of the present invention.

EXAMPLES EXAMPLE 1: In vitro cell viability assay for 17-DMAG

[0092] Initially, the difference in susceptibility of the parasite and the host cell to 17-DMAG was evaluated. For this, cell viability assays were performed to evaluate the leishmanicidal activity and cytotoxicity (in human cell line THP-1) of 17-DMAG.

[0093] Logarithmic axenic promastigotes of L. amazonensis were distributed in 96-well plates (4x105 parasites/well) in complete Schneider medium and treated with 17-DMAG (LC Laboratories, Woburn, MA, USA) in serial dilutions of 12 steps (1:2) at an initial concentration of 2 μM. After 72 h of treatment at 24º C, the parasites were incubated with 10% Alamar Blue® (Invitrogen, Carlsbad, CA, USA) for 1 h and 30 min. Then, optical density (OD) was read at wavelengths of 570 and 600 nm in a spectrophotometer (SPECTRA Max 340 PC) to determine cell viability, expressed in percentage terms, used to calculate the IC50 value. This value was determined by applying sigmoidal regression to the concentration-response curves using the GraphPad Prism (version 5.0) of at least three independent experiments performed in triplicates.

[0094] Human THP-1 cells were centrifuged at 300xg for 10 min at 4oC and resuspended (105 cells/100µL) in complete RPMI medium containing phorbol-12-myristate-13-acetate (PMA) at 100 nM and plated. of 96 wells. Cultures were incubated at 37°C for 72 h to induce macrophage differentiation. The wells were then washed twice with saline, the complete RPMI medium (without PMA) was replenished, and the cells were re-incubated at 37°C for an additional 48 h. The cultures were then treated with 17-DMAG in 12-step serial dilutions (1:2) using an initial concentration of 50 μM. After 72 h of treatment at 37°C, the cells were incubated with 10% Alamar Blue® for 22 h. Then, the plates were read at wavelengths of 570 and 600 nm in a spectrophotometer (SPECTRA Max 340 PC). The data obtained were used to calculate the percentage of cell viability and, later, the CC50 value was determined from the sigmoidal regression of the concentration-response curves using GraphPad Prism (version 5.0) of at least three independent experiments performed in triplicate.

[0095] From these assays, IC50 and CC50 values (Leishmania and THP-1 cell, respectively) were calculated, observed in Figure 2. The IC50 value for 17-DMAG was 0.09 μM in L. amazonensis promastigotes , while in THP-1 cells, a CC50 value of 10.6 μM (p = 0.001) was observed, resulting in a selectivity index of 117.8. The results in Figure 2 demonstrate that 17-DMAG has greater toxicity against L. amazonensis compared to the host cell.

EXAMPLE 2: In vitro cytotoxicity assay of MRC-5 cells to 17-DMAG

[0096] In addition to the THP-1 assay and with the aim of evaluating another cell type, the cytotoxicity assay was performed with human lung fibroblasts (MRC-5 cells) treated with 17-DMAG. For this purpose, MRC-5 cells were centrifuged at 300xg for 10 min at 4°C and resuspended in complete RPMI medium and plated at a concentration of 2.5 x 10 4 per well, then incubated at 37°C for 24 h. Subsequently, the cells were treated with 17-DMAG (initial concentration: 50 μM) following the same protocol used for THP-1 cells. After 72 h, Alamar Blue® was added and the plates were read in a spectrophotometer. The CC50 value was determined in the same way as for THP-1 cells.

[0097] The test result is shown in Figure 3. It is observed that 17-DMAG reduced the percentage of cell viability only at concentrations above 6.25 µM. As for the THP-1 cells, the CC50 value was calculated and presented a value of 13.24 μM

EXAMPLE 3: In vitro intracellular viability assay for 17-DMAG

[0098] To assess whether the toxicity observed in promastigotes would also be observed in amastigotes, an intracellular viability assay was performed on macrophages infected by L. amazonensis and treated with 17-DMAG at different concentrations. THP-1 cells (5x105 per well) were plated in complete RPMI medium with PMA (100 nM) to promote differentiation into macrophages. After 72 h, the cells were washed with saline and the medium (without PMA) was replenished. After 48 h in medium without PMA, the cells were washed again and infected by L. amazonensis promastigotes in stationary phase (10:1) for 6 h at 35º C. To remove non-internalized parasites, the cells were washed twice with saline. , the RPMI medium was replaced and concentrations of 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, 150 nM, 200 nM, 300 nM and 400 nM (five replicates for each concentration) of 17-DMAG were added and the cells were incubated for a period of 72 h. Then the cells were washed with saline and complete Schneider medium was added. After four days, viable parasites were counted in a Neubauer chamber and the IC50 value was calculated for 17-DMAG.

[0099] Figure 4 demonstrates that, as in promastigotes, 17-DMAG also showed toxicity against the amastigote forms in a dose-dependent manner. It is observed that at concentrations above 100 nM of 17-DMAG, viable parasites were no longer observed. Interestingly, the IC50 value obtained against the intracellular form of amastigote was 13.4 nM, showing that 17-DMAG is about 7 times more effective against the amastigote than against the flagellated promastigote form of L. amazonensis, whose IC50 was 90 nM.

EXAMPLE 4: In vivo assay with mice infected by L. braziliensis with 17-DMAG intraperitoneally at doses of 20, 30 or 50 mg/kg.

[00100] BALB/c mice were infected in the left ear dermis with 105 L. braziliensis parasites in a total volume of 10 µL of sterile saline and treated with intraperitoneal injections of 17-DMAG at doses of 20 mg/kg daily, 30 mg/kg every other day and 50 mg/kg every five days.

[00101] Treatment started 15 days after L. braziliensis infection. For this, 35 mice were divided into four groups: (i) untreated control animals, which received 5% glucose application daily; (ii) animals treated with 20mg/kg of 17-DMAG intraperitoneal daily; (iii) animals treated with 30mg/kg of 17-DMAG intraperitoneally every other day; (iv) animals treated with 50mg/kg of 17-DMAG intraperitoneally every five days.

[00102] Before each treatment application, the animals were weighed to determine the drug volume. The thickness of the infected and contralateral ears was measured weekly, comparatively.

[00103] The effect of the treatment of different therapeutic regimens with 17-DMAG in its injectable formulation against L. braziliensis in BALB/c mice can be seen in Figure 5.

[00104] The group of animals undergoing treatment with 20 mg/kg showed a significant reduction in lesion size compared to the control group (Figure 5A). In relation to the group treated with 30 mg/kg every other day, it was also possible to observe a reduction in the size of the lesion in relation to the control, but less expressive than in the group treated daily, even at a lower concentration (Figure 5B). Mice treated with a concentration of 50 mg/mL every five days showed a less significant reduction in lesion size compared to control (Figure 5C). We assume that the lower efficacy of the 50 mg/kg dose compared to animals treated with 20 mg/kg was due to the time interval between applications being daily in the group treated with 20 mg/kg, compared to the space of five days. in the group treated with 50 mg/kg.

[00105] Figure 5B shows the result of the area under the curve (AUC) of the lesion size of the different experimental groups. Despite the difference between the AUC of the treated animal in relation to that of the untreated animal, being higher at the dose of 20 mg/kg of 17-DMAG applied daily, at all concentrations tested the differences between the AUCs in relation to that of the untreated animals was statistically significant.

[00106] Corroborating the results of lesion thickness, images taken from the ears of infected animals show a clear difference between the control group and the treated groups (Figure 6). Control group mice showed a well-developed lesion (Figure 6A-D). The ears of the treated animals showed a smaller lesion (Figure 6E–P). The dermis of the ears of the untreated animals presented a more reddish appearance, when compared to the skin of the mice treated at any concentration. Animals treated with 20 mg/kg (Figure 6E-H) and 30 mg/kg (Figure 6I-L) showed little or no injury. Mice treated with 50 mg/kg every five days (Figure 5M-P) showed lesions, however, with a less inflamed appearance than the lesions in untreated animals.

[00107] The results obtained with this trial show that the drug 17-DMAG, when applied intraperitoneally at doses of up to 30 mg/kg, significantly reduces the size of the lesion caused by L. braziliensis, regardless of the therapeutic regimen used. However, the findings show that, when applied at long intervals between applications, the therapeutic effect was less effective.

EXAMPLE 5: In vivo assay with mice infected by L. braziliensis with 17-DMAG intraperitoneally for 2, 4 or 7 weeks.

[00108] After demonstrating that mice treated daily with a dose of 20 mg/kg of 17-DMAG did not show symptoms of toxicity and still showed an effective response that promoted a decrease in the thickness of the lesion as well as the appearance of the infected ear, it was decided to to carry out a kinetics study following the lesion and the parasite load at different times after treatment and for longer periods: two, four and seven weeks of treatment.

[00109] BALB/c mice were infected in the left ear dermis with 105 L. braziliensis parasites in a total volume of 10 µL of sterile saline and, after two weeks, treated with intraperitoneal injections of 17-DMAG at a concentration of 20 mg /kg daily for two, four or seven weeks.

[00110] Treatment with 17-DMAG was started 15 days after L. braziliensis infection. For this, 34 mice were divided into six groups: (i) untreated control animals that received 5% glucose application daily for two weeks; (ii) animals treated for two weeks with application of 20 mg/kg of 17-DMAG daily; (iii) control animals untreated for four weeks that received 5% glucose application daily; (iv) animals treated for four weeks with applications of 20 mg/kg of 17-DMAG daily; (v) control animals untreated for seven weeks that received 5% glucose applications daily; (vi) animals treated for seven weeks with applications of 20 mg/kg of 17-DMAG daily.

[00111] Before each application the mice were weighed to determine the drug volume. Ear lesion thickness was measured weekly. After two, four or seven weeks of treatment, the animals were euthanized and the infected ear and draining lymph node were removed to quantify the parasite load by limiting dilution.

[00112] Figure 7 shows the variation in lesion size of mice infected with L. braziliensis and treated with 17-DMAG for two, four or seven weeks of treatment. Mice treated for two weeks showed little lesion development during the first week and decreased thickness in the second week, when compared to the control. The mice treated for four weeks showed a significant decrease in the size of the lesion in relation to the previous weeks, unlike the control group, which showed an increase in the lesion (Figure 7B). In mice treated for seven weeks, the lesion showed a significant decrease in relation to the control, reaching zero between the infected and uninfected ear between the fifth and sixth week of treatment.

[00113] Figure 8 demonstrates evaluation of the parasite load by limiting dilution. After two weeks of treatment, a marked reduction was observed, both in the ear and in the lymph node of the treated mice in relation to the control (Figure 8A – B). With four weeks of treatment, the reduction in the parasite load is even more expressive in the treated group compared to the control. Five animals in the group treated for four weeks did not show ear load and six did not show lymph node load, while all mice in the control group showed load (Figure 8C – D). After seven weeks of treatment, no parasite burden was found either in the ear or in the lymph node of the treated mice, while the control animals had a median of 55,000 parasite burden in the lymph node and 5,500 parasites in the lesion (Figure 8E – F).

[00114] Figure 9 shows images acquired from the ear and draining lymph node of mice that show the difference between the treated and control groups. In the animals followed for two weeks, the control showed a well-developed lesion and an enlarged lymph node (Figure 9A – D), while the animals that received treatment for two weeks had no lesion or a small lesion and the lymph node was reduced 1, 6 times compared to the control (Figure 9E – H). Similar aspects were observed in the lesions and lymph nodes of the mice in the fourth week after treatment, when the control presented a well-developed lesion, with a more inflamed aspect than the two-week-old animals, in addition to the lymph node enlarged by three times (Figure 9I – L), while the treated mice showed no lesions and the lymph node showed a three-fold reduction compared to the control (Figure 9M – P).

EXAMPLE 6: In vivo toxicity assay in uninfected mice treated with 17-DMAG topically in hydrogel formulations.

[00115] To assess the toxicity of 17-DMAG on a semi-solid basis, uninfected BALB/c mice were exposed to 17-DMAG incorporated into a topical hydrogel base at different concentrations. For this, 12.0 mg, 10.0 mg, 8 mg and 6 mg of 17-DMAG were individually diluted in 40 mL of distilled water. For gelation, each solution was heated to 37 °C for the addition of 1% sodium carboxymethylcellulose (CMC-Na), producing, respectively, hydrogel doses of 0.30, 0.25, 0.20 and 0.15 mg/ g.

[00116] The different therapeutic regimens were applied daily to uninfected animals at doses of 0.15, 0.20, 0.25, 0.30 mg/g of hydrogel. For this, 30 mice were divided into five groups, a control group of mice treated with hydrogel without the drug or white hydrogel (i); and another four groups were treated with hydrogel containing 17-DMAG at concentrations of 0.15 mg/g (ii); 0.20 mg/g (iii); 0.25 mg/g (iv); 0.30 mg/g (v) to evaluate the toxicity of 17-DMAG in hydrogel in vivo, exposing the formulation on the skin of the ear of mice for 4 weeks. The 30 mice were treated daily and ear thickness was measured weekly.

[00117] The results obtained are represented in Figure 6, where it is observed that only the hydrogel with the highest concentration, 0.30 mg/g, showed a significant difference in ear thickness when compared to the white. However, although the hydrogel compound did not cause an increase in thickness at a concentration of 0.25 mg/g, an increase in sensitivity, stiffness and slight redness was observed in the treated ear when compared to the control (white). These data show that the dose to be used for efficacy testing should start at a concentration of 0.20 mg/g of hydrogel, a concentration of 300 µM, 15,000 times higher than that of the IC50.

EXAMPLE 7: Production of polymeric nanoparticles containing 17-DMAG.

[00118] For the production of 17-DMAG polymeric nanoparticles. 2-5 mg of the drug are dissolved in 1.5 to 5 ml of an aqueous solution containing 0-5% PEG 300. Then this sample is poured into 2 to 8 ml of dichloromethane containing 100 to 400 mg of PLGA and processed by ultrasonication at 20-40% amplitude in 30 second cycles. This emulsion is poured into 10 mL of an aqueous solution containing 0.5-2% PVA and taken to the ultrasonicator again. The final emulsion formed is poured into 30 ml of an aqueous solution containing 0.5-2% PVA and left for up to 10 min under magnetic stirring. In a preferred embodiment, the following proportions were used: 3mg of 17-DMAG; 2.5% PEG in a volume of 1.5 mL; 100 mg of PLGA in 2 ml of dichloromethane; 1% PVA; 40% sonication amplitude in 30 second cycles.

[00119] Then the solvent is evaporated in a rotary evaporator at 40 °C and 200 mBar for 1 hour and the solution is ultracentrifuged at 166,713.1 m/s² (17,000 x g) three times and resuspended in type II water. After the last ultracentrifugation, the sample is resuspended in 5 mL of water and the samples are frozen at -80°C and lyophilized for 24h at -30°C.

[00120] The nanoparticles achieved an encapsulation efficiency of 40% of 17-DMAG. This measurement was performed by high performance liquid chromatography (HPLC). The size of the nanoparticles, obtained by dynamic light scattering, showed an average value equal to 282 nm and a polydispersion index of 0.1. The Zeta potential was acquired by electrophoretic potential, having a value equal to -33 mV. Figure 11 shows the image obtained by transmission electron microscopy (TEM) where it is observed that the nanoparticles are spherical and with sizes ranging from 70 to 400 nm. Cell viability was evaluated and the IC50 obtained was 14.9 nM.

References

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Claims (11)

Pharmaceutical formulation, characterized in that it comprises, as an active ingredient, a therapeutically effective amount of the compound 17-Dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) and at least one pharmaceutically acceptable excipient Pharmaceutical formulation according to claim 1, characterized in that the amount of 17-DMAG is from 0.50 m/g to 0.05 mg/g in relation to the total weight of the formulation. Pharmaceutical formulation according to claim 2, characterized in that the pharmaceutically acceptable excipient is selected from the group consisting of diluents, carriers, protectors, surfactants, dispersing agents, immobilizing agents, film-forming or film-forming agents, healing agents, tissue regenerators , stabilizing agents, gelling agents, epithelializing agents, nonionic emulsifiers, and preserving agents. Pharmaceutical formulation according to claim 3, characterized in that the sum of the percentage of pharmaceutically acceptable excipients is in the range of 60% to 90%, more preferably, the excipients are in the range of 65% to 80% in relation to weight total of the formulation. Pharmaceutical formulation according to any one of claims 1 to 4, characterized in that it is a topical or systemic formulation. Pharmaceutical formulation according to any one of claims 1 to 5, characterized in that it is in the form of ointments, creams, pastes, gels, hydrogels, lotions, solutions, suspensions, sprays, plasters, among others, preferably in the form of a hydrogel . Pharmaceutical formulation according to any one of claims 1 to 6, characterized in that it is for the treatment of leishmaniasis, preferably for the treatment of Visceral Leishmaniasis and/or Cutaneous Leishmaniasis, more preferably for the treatment of American Cutaneous Leishmaniasis. Production process of a pharmaceutical formulation as defined in any one of claims 1 to 7, characterized in that it comprises the steps of:

• (a) solubilization of the active ingredient in suitable excipients;
• (b) formation of the nanoparticles from the dilution in a previously selected medium and processing by emulsification;
• (c) recovery of nanoparticles through the elimination of solvents used in the process and physical extraction.

Medicine, characterized in that it comprises the pharmaceutical formulation as defined in any one of claims 1 to 7. Leishmaniasis treatment method, characterized by the fact that it uses the pharmaceutical formulation as defined in any one of claims 1 to 7. Use of a pharmaceutical formulation as defined in any one of claims 1 to 7, characterized in that it is for the manufacture of a drug to treat leishmaniasis, preferably.

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