WO2013010238A1 - Microparticle pharmaceutical compositions containing antiparasitics for prolonged subcutaneous therapy, use of the pharmaceutical compositions for producing a medicament, and method for treating parasitoses - Google Patents

Microparticle pharmaceutical compositions containing antiparasitics for prolonged subcutaneous therapy, use of the pharmaceutical compositions for producing a medicament, and method for treating parasitoses Download PDF

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WO2013010238A1
WO2013010238A1 PCT/BR2012/000249 BR2012000249W WO2013010238A1 WO 2013010238 A1 WO2013010238 A1 WO 2013010238A1 BR 2012000249 W BR2012000249 W BR 2012000249W WO 2013010238 A1 WO2013010238 A1 WO 2013010238A1
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plga
amphotericin
pharmaceutical compositions
particles
drugs
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French (fr)
Portuguese (pt)
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Bartira ROSSI BERGMANN
Wallace PACIENZA LIMA
Camila ALVES BANDEIRA FALCÃO
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Universidade Federal Do Rio De Janeiro - Ufrj
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A61K31/121—Ketones acyclic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
    • A61K9/1647—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to a pharmaceutical composition containing amphotericin B or antimonial compounds such as meglumine antimoniate, sodium stibogluconate and hydrated antimony (III) potassium tartrate (Sb 111 ), or a nitrated chalcone, in which case it would be 3-nitro-2-hydroxy-4,6-dimethoxycyclones which we have named it CH8.
  • amphotericin B or antimonial compounds such as meglumine antimoniate, sodium stibogluconate and hydrated antimony (III) potassium tartrate (Sb 111 ), or a nitrated chalcone, in which case it would be 3-nitro-2-hydroxy-4,6-dimethoxycyclones which we have named it CH8.
  • CH8 nitrate chalcones
  • This invention also relates to the use of these pharmaceutical compositions, a medicament and the treatment of parasitic diseases.
  • Parasitic diseases including malaria, leishmaniasis, trypanosomes, among others, are considered one of the major public health problems in the world. These diseases, however, are concentrated in countries with low-income populations, such as countries in Africa, Asia, and South America. This is largely due to the lack of public policies on health and sanitation, these countries have the highest number of cases and deaths from these parasites.
  • Leishmaniasis is a complex of diseases caused by various protozoan species of the genus Leishmania, which can cause a spectrum of clinical forms in humans and other vertebrates. These can be classified into Cutaneous form, which includes the simple, diffuse, mucocutaneous, often disfiguring, and often lethal Visceral forms.
  • the parasite exists as a flagellated promastigote in the phlebotomine vector, and as a flush amastigote within mononuclear phagocytes of the vertebrate host.
  • the privileged intracellular location makes it difficult to access active drugs, which makes this disease difficult to treat.
  • Antimonial salts are synthetically obtained from antimonic acid and N-methyl glucamine.
  • the administration Intramuscular or intravenous antimoniate is rapidly absorbed, so that after 48 hours 90% of the initial dose has already been excreted by the kidneys. Repeated high doses are required to maintain the therapeutic level of antimony in the tissues. This results in a variety of side effects such as myalgia, pancreatitis, pancytopenia, hepato and cardiotoxicity. Pancreatitis seems to be the frequent cause of nausea and abdominal pain (GASSER et al, 1994).
  • Pentamidine has similar efficacy to antimonials (TUON et al 2008), but produces other side effects such as hypoglycemia, diabetes, tachycardia, hypotension, nephrotoxicity and local pain on alternate 4mg / kg.day administration for 30 days intramuscularly .
  • Amphotericin B is a polyenic antibiotic isolated from the bacteria Streptomyces sp, initially used as an antifungal drug, especially in systemic infections.
  • a systemic drug should remain in circulation for as long as necessary for its therapeutic effect, within a safe range of high efficacy combined with low toxicity, and a minimum of repetitive doses.
  • encapsulating a drug in appropriate micro and nanoparticles can help direct it to your target tissue or cell.
  • Controlled drug release systems have several advantages over conventional systems, such as: 1) Greater control of the release of the active ingredient, reducing the appearance of toxic and subtherapeutic doses; 2) Use of less quantity of active principle; 3) Resulting in lower cost; 4) Longer administration interval; better treatment acceptance by the patient and the possibility of directing the active ingredient to its specific target.
  • liposomes The first to be developed, and the only ones already approved for intravenous systemic use, are liposomes. From the 1980s onwards various biodegradable polymers were and are being used in the preparation of micro and nanoparticles, including synthetic poly (lactic co-glycolic) polymers (PLGA), in addition to chitosan and polyhydroxyalkanoate (PHA) biopolymers. Of smaller size, in the order of 1 -10 nm, we have the cyclodextrin; and more recently spherical and branched polymers, dendrimers, which are being studied for their pharmaceutical viability.
  • synthetic poly (lactic co-glycolic) polymers PLGA
  • PHA polyhydroxyalkanoate
  • Liposomes were the first particles delineated for antileishmanial drug vectorization.
  • Amphotericin B encapsulated in negatively charged unilamellar liposomes (SUNDAR & RAI, 2002).
  • Recent studies on the toxicity and effectiveness of meglumine antimoniate liposomal formulations in dogs with visceral leishmaniasis have shown that the formulation significantly reduced the parasitic burden of bone marrow in dogs and did not cause significant changes in liver and renal function in the dose (6.5 mg Sb / kg / dose) with four doses (4 days apart) (FRÉZARD, F. & MICHALICK, MS M, 2008).
  • lipid-associated amphotericin B formulations have led to a remarkable improvement in leishmaniasis chemotherapy.
  • These lipid-associated formulations such as AmBisome®, encapsulated in unilamellar liposomes, Abelcet®, incorporated into lipid complex; and Amphocil® as a colloidal suspension in cholesterol were evaluated in clinical tests for visceral and mucocutaneous leishmaniasis.
  • AmBisome® has shown greater efficiency by favoring its approval by the Food and Drug Administration (FDA).
  • Side effects mainly reduced nephrotoxicity by decreasing renal elimination and increasing plasma half-life.
  • Another advantage is the possibility of administering larger doses of the drug in a shorter period of time.
  • the limiting factor of the use of these liposomal formulations is the high phosphatidylcholine, which makes the access to treatment impossible for most of the affected population (GOLENSER et al. 1999).
  • the polymeric micro and nanoparticle polymer systems are drug carrier systems that include capsules and spheres. Its major advantage over liposomes is its greater stability and can be lyophilized for storage. Factors such as type and proportion of polymers in the formulation determine important characteristics such as stability and crystalline structure and biodegradability, which influences the release rate of the drug (SCHAFFAZICK & GUTERRES, 2003).
  • Poly (lactide) polymer, or PLA is one of the most studied for biological application, due to its high biodegradability and biocompatibility, having already approved clinical use in the constitution of sutures and biodegradable orthopedic prostheses.
  • PLA nanospheres have also been successfully tested as carriers of various antileishmaniasis, such as primaquine, pentamidine, bacopasaponin and atovaquone, for the experimental treatment of intravenous visceral leishmaniasis.
  • various antileishmaniasis such as primaquine, pentamidine, bacopasaponin and atovaquone
  • 200 nm PLA nanoparticles were successfully tested by our group on the delivery of nitrated chalcone (CH8) DMC for subcutaneous treatment of L. amazonensis infected mice (TORRES-SANTOS er a /., 1999). .
  • CH8 DMC nitrated chalcone
  • PLGA is also interesting as a controlled drug delivery system. This polymer has also been approved by the FDA as biodegradable structures used in orthopedic screws and studs, sutures, and meniscus and cartilage repair systems (ELIAZ and KOST, 2000). In addition, PLGA is widely used in cosmetic medicine for subcutaneous wrinkle filling, with a high degree of acceptance by patients. Polymer formulations approved for human use are all directed to the treatment of cancer in polymeric microcapsules (> 1000 nm) and have potential application in the case of deposit formation for slow and continuous local release such as in localized vaccines or chronic treatments.
  • the first FDA approved PLGA product was the
  • LupronDepot in 1996 (TAP Pharmaceutical Products Inc), 75:25 lactide / glycolide microparticles containing Leuprolide for the treatment of advanced prostate cancer. Administered locally, LupronDepot forms a depot and releases the drug gradually and can be administered at intervals of up to 4 months, replacing daily injections of free Leuprolide (KADA et al., 1994). PLGA microparticles are already being tested in humans as vaccine delivery systems. This clinical trial is underway in Brazil in patients with advanced neck and neck cancer, developed by SILVA, C. L and collaborators.
  • Poly (lactide) polymer or PLA
  • PLA is one of the most studied for the preparation of microparticles for biological application, due to its high bidegradability and biocompatibility, having already approved clinical use in the constitution of biodegradable suture threads, as well as PLGA
  • PLA nanospheres have also been successfully tested as carriers of various antileishmaniasis, such as primaquine, pentamidine, bacopasaponin and atovaquone, for the experimental treatment of intravenous visceral leishmaniasis.
  • the nitrogenous chaffs referred to herein are 3-nitro-2-hydroxy-4,6-dimethoxychalcona, which we will refer to as CH8 for ease.
  • the first object of this invention relates to pharmaceutical compositions containing a pharmaceutically effective amount of PLGA particle encapsulated antileishmanial drugs or drugs.
  • the second object of this invention relates to the use of pharmaceutical compositions containing a pharmaceutically effective amount of amphotericin B, chalcone nitrated (CH8) or antimony compounds, encapsulated in PLGA particles directed to the manufacture of a medicament for the subcutaneous treatment of inflammatory skin diseases.
  • infectious skin or skin regions such as leishmaniasis, mycoses, leprosy, psoriasis, tropical ulcer, warts, cavernous hemangioma in mammalian animals.
  • the third object of this invention is a pharmaceutical composition containing a therapeutically effective amount of amphotericin B, chalcone nitrite (CH8) or PLGA particle encapsulated antimonial compounds, and pharmaceutically acceptable excipients.
  • the last object of this invention is a method of treating parasitic diseases comprising subcutaneously applying a therapeutically effective amount of amphotericin B, chalcone nitrite (CH8) or antimonial compounds encapsulated in PLGA microparticles to a mammalian animal carrying leishmaniasis.
  • compositions comprising a pharmaceutically effective amount of PLGA particle encapsulated antileishmanial drugs or drugs, having as examples of drugs such as Amphotericin B, chalcone nitrate (CH8) and antimonial compounds.
  • the active agent may constitute about 5 to 85% of the weight of the formulation. Varied concentration of active compounds may enable faster initial release into tissue after local administration. In an exemplary variation as shown below, we use 10% Amphotericin B relative to the polymer (w / w).
  • medicaments or drugs used are amphotericin B, chalcone nitrate (CH8) and antimonial compounds such as meglumine antimoniate, sodium stibogluconate and potassium hydrate (IIIb) tartrate, combined or not.
  • glycolic and lactic acid copolymers are used and the percentage of each poly (lactic-co-glycolic acid) monomer may be about 15-85%, about 25-75%, about 35-65%. %, or the proportion of 50-50% may be employed.
  • the second object of this invention relates to the use of pharmaceutical compositions containing a pharmaceutically effective amount of amphotericin B or antimonial compounds, preferably Glucantime and PLGA particle-encapsulated nitrated chalcone (CH8) for the manufacture of a medicament for the subcutaneous treatment of inflammatory and infectious skin diseases or in skin regions (dermis, epidermis and appendages) such as leishmaniasis, mycosis, leprosy, psoriasis, tropical ulcer, warts, cavernous hemangioma in mammalian animals.
  • a pharmaceutically effective amount of amphotericin B or antimonial compounds preferably Glucantime and PLGA particle-encapsulated nitrated chalcone (CH8) for the manufacture of a medicament for the subcutaneous treatment of inflammatory and infectious skin diseases or in skin regions (dermis, epidermis and appendages) such as leishmaniasis, mycosis, leprosy, psoria
  • the third object of this invention is a pharmaceutical composition containing a therapeutically effective amount of amphotericin B or antimonial compounds such as PLGA particle encapsulated nitrated glucosin and chalcone (CH8) in addition to pharmaceutically acceptable excipients.
  • amphotericin B or antimonial compounds such as PLGA particle encapsulated nitrated glucosin and chalcone (CH8) in addition to pharmaceutically acceptable excipients.
  • the last object of this invention is a method of treating parasitic diseases comprising subcutaneously applying a therapeutically effective amount of Amphotericin B or antimonial compounds such as PLGA microparticle-encapsulated nitrided Glucantime and chalcone (CH8) to a mammalian animal carrying leishmaniasis.
  • Amphotericin B or antimonial compounds such as PLGA microparticle-encapsulated nitrided Glucantime and chalcone (CH8)
  • PLGA particles containing Amphotericin B and antimonial compounds will be produced by the multiple emulsification method (Ai / 0 / A 2 ), followed by the solvent evaporation method.
  • the polymer (PLGA) will be dissolved in dichloromethane (organic phase), to this organic phase will be added 1 ml pH 7.4 buffer containing the drugs (10% relative to the polymer (w / w)), and The mixture of the phases will be promoted under ultra-stirring at 7000rpm for 30 seconds to form a single emulsion (Al 10).
  • Such a single emulsion will be dispersed in the continuous phase (A 2 ) under 7000rpm ultrahomogenization for 60 seconds, a 3% aqueous polyvinyl acid (PVA) solution will be added to the system and solvent evaporation will be under stirring at 400 rpm for 4 hours.
  • PVA polyvinyl acid
  • the microparticle suspension will be centrifuged at 4000 rpm for 5 minutes, washed with distilled water 3 times and frozen in -20 ° C freezer for subsequent freeze drying for 16 hours.
  • the active agent may constitute about 5 to 85% of the weight of the formulation.
  • Determination of drug content in the particles is important to ensure the correct dose to be used in the trials.
  • Amphotericin B contents in PLGA particles will be determined by HPLC.
  • Antimony (Sb) levels will be determined by atomic absorption.
  • PLGA particles will have the drug release rate determined in physiological buffer pH 7.2 at 37 ° C. For this, 10 mg of particle will be incubated in 1 ml of PBS, plus 0.1% azide, under constant agitation at 80rpm and 37 ° C. Samples will be collected by centrifugation, taken from 500 ⁇ at times ranging from 1 hour to 270 days. Samples will be analyzed by UV-HPLC to determine the content of the released amphotericin B, the antimony content will be analyzed by atomic absorption. The curve will be constructed:% release x time.
  • Particle size and distribution analysis of b obtained by centrifugation and lyophilization will be performed using the distribution analyzer.
  • particle size Coulter LS230 Small amounts of particles will be dispersed in PA absolute ethanol with the aid of ultrasound until they reach the obscuration index required by the device.
  • the average particle size will be expressed as the average volume diameter (D4,3).
  • the particle diameter will be calculated at D 0 , 9, D 0 , ie D 0 , 5, which means the particle diameter corresponding to 90, 50 and 10% of the cumulative distribution and the polydispersion will be given by the span index, which will be calculated by (OD, 9 - OD, 1 / OD, 5).
  • the particles analyzed in C by SEM will also have their average size evaluated by the Coulter LS230 analyzer.
  • PLGA particles will be dispersed in water with pH corrected to 7.0 and zeta potential measurements will be determined on a Zetamaster apparatus (Malvern Instruments). Microparticle dispersion for potential zeta measurements will be prepared in duplicate and each sample will be measured 0 times.
  • Macrophages will be isolated from the peritoneal of BALB / c mice by adherence to 24-well culture plates infected with L amazonensis GFP promastigotes and treated with the formulations for 72h. After the incubation time, the cells will be scraped off and the parasitic load will be assessed by fluorimeter on a plate fluorimeter (485 nm excitation, 528 nm emission) (ROSSI-BERGMANN, et al., 1999) (FLx800, Bio-Tek Instruments, Inc) for L amazonensis or microscopic counting.
  • Macrophages will be isolated from the BALB / c mouse peritoneum by adherence to 24-well culture plates, empty or drug-containing PLGA particles will be added and after 72 hours the Crop supernatant will be evaluated for NO (Nitric Oxide) production by Griss methodology and for microbicidal activity evaluation and the presence of lactate dehydrogenase (LDH) enzyme for evaluation of necrosis toxicity by lactate dosage (Kit Dolles) .
  • NO Nitric Oxide
  • LDH lactate dehydrogenase
  • the ear swelling test (MEST) test It is recommended by the OECD and ANVISA and will be performed for skin immunotoxicity assessment (allergenicity),
  • MEST evaluates the increase in ear thickness as a sign of skin sensitization.
  • Animals will receive 3 topical applications of 100 ⁇ l of the formulations every 2 days (days 0, 2 and 4) in the dorsal region (induction phase). After 5 days, 10ul of the substances will be topically applied to one ear and to the other 10ul of the formulation will be applied intradermally (challenge phase). This is the time required for the body to produce the immune response. Ear swelling measurements will be evaluated at 6, 18, 36, 48, and 72 hours after challenge. Skin sensitization is positive when there is an increase in ear thickness above 10% compared to the control group.
  • Oxazolone (0.3%) is the most used substance as a positive control in these skin sensitization tests and, according to the literature, promotes a 30% increase in ear thickness in relation to the measurement before the challenge.
  • the animals will be treated intralesionally, blood will be collected and plasma obtained by centrifugation will be pretreated following the methodology proposed by (WANG & MORRIS, 2005; PAGANGA & RICE-EVANS, 1997 and-MANACH et al., 1996). lowering the pH to 4 using H 3 PO 4 ; acidifying with acetonitrile and centrifuging. Acidification of samples is important to break possible binding to plasma proteins. After this procedure plasma samples will be analyzed using UV-HPLC for amphotericin B and atomic absorption for antimonials.
  • Pharmacokinetic parameters (Cmax; ASC 0- -; t1 ⁇ 2; Vd; and Cl) will be obtained using software (ADAPT: Pharmacokinetic / p armacodynamic systems analysis) produced by the University of Southern California, available for free download.
  • ADAPT Pharmacokinetic / p armacodynamic systems analysis
  • the treated ears will be removed after different times and macerated in acetonitrile.
  • the suspensions will be clarified by centrifugation and processed as described for plasma.
  • Figure 1 shows the result of particle size analysis of PLGA particles containing Glucantime (meglumine antimoniate) or Amphotericin B, where Figure 1A represents the empty microparticles, 1 B the microparticles containing Glucantime and 1 C the microparticles containing Amphotericin B.
  • Figure 2 shows the analysis of PLGA particle morphology assessed by SEM and the images obtained by scanning electron microscopy (SEM) of empty PLGA particles (A), Glucantime (B), with Amphotericin B (C). Increases by 1500X, 3500X and 5000X, respectively.
  • the theoretical incorporation of amphotericin B into microparticles is 10% (w / w). Samples were made in triplicate.
  • Figure 4 shows the erosion analysis of amphotericin B-containing PLGA microparticles by scanning electron microscopy (SEM) images. Images A and B at time zero, images C and D with time equal to 30 days in PBS buffer and images E and F with time equal to 120 days in PBS buffer.
  • SEM scanning electron microscopy
  • Figure 5 shows the efficacy of amphotericin B encapsulation in single dose PLGA microparticles in the treatment of intralesional murine cutaneous leishmaniasis, where BALB / c mice were infected with 2x10 6 L.amazonensis GFP promastigotes in the ear. After 10 days of infection, the animals were randomly separated into groups of 4. The free amphotericin B treated group received intralesional injections twice a week with 50 ⁇ g doses of the drug for 4 weeks (total of 8 doses). The PLGA / Amphotericin B treated group received a single dose of the formulation intralesionally with 50 ⁇ g Amphotericin B dose (1 dose).
  • A The lesion growth (infected ear thickness minus thickness before infection) was accompanied during the experiment with the aid of caliper.
  • Figure 6 shows intralesional murine cutaneous leishmaniasis treatment, where BALB / c mice were infected with 2x0 6 L.amazonensis GFP promastigotes in the ear. After 7 days of infection, the animals were randomly separated into groups of 4.
  • treatment 1 the PLGA / amphotericin B treated group received a dose of the formulation intralesionally with a dose of 25 ⁇ g amphotericin B.
  • the free amphotericin B treated group and Liposomal amphotericin B received a dose of 1.25 ⁇ g of the drug.
  • Controls received a single administration of PLGA of 12.5 ⁇ g or 10ul of PBS.
  • the PLGA / amphotericin B treated group received a dose of the formulation intralesionally with 50 ⁇ g amphotericin B dose.
  • the free amphotericin B and liposomal amphotericin B treated group received a 50 ⁇ g dose of the drug.
  • Controls received a single administration of 500ug or 10ul of PBS PLGA.
  • the lesion growth was followed during the experiment with the aid of caliper.
  • Figure 08 shows the assessment of cardiac, hepatic and renal toxicity.
  • Animal blood was drawn 67 days after infection for dosages of: (a) serum TGO levels; (b) serum TGP levels; (c) serum creatinine serum levels.
  • Negative Control PBS.
  • Positive control ICC 4 .
  • Figure 9 shows the determination of the best drug ratio in the formulation.
  • PLGA microparticles containing Amphotericin B added in different proportions to the polymer (5%, 10% and 20% as indicated) were prepared. The particles were then washed and dried. To determine the best process yield, the dried particles were fully dissolved in Acetonitrile after stirring for 24 hours at 100 rpm. The samples were then centrifuged at 10,000 rpm for 5 minutes and the supernatant was removed for quantification of the HPLC incorporated drug content.
  • Table 1 The average particle size expressed as volume average diameter (D 4.3).
  • Table 2 The surface charge of the particles was evaluated by the Zeta Potential. Zeta SD Potential Specification
  • the particle morphology was also evaluated by SEM and the images are represented in Figure 2.
  • SEM morphological analysis of the In particles, we can observe the formation of clusters that may explain the observed heterogeneity in population size distribution when Glucantime or Amphotericin B are encapsulated.
  • EXAMPLE 2 EVALUATION OF EFFECTIVENESS OF IN VITRO PARTICULATION, RELEASE AND DEGRADATION
  • Amphotericin B uptake rate in PLGA microparticles can be seen in Figure 3A.
  • the Average incorporation rate was 89.2%, with this yield the particle amphotericin B content is 8.92% and not the 10% (w / w) Amphotericin B in theoretical PLGA microparticles .
  • EXAMPLE 3 EVALUATION OF THE EFFICACY OF COMPOSITIONS IN THE TREATMENT OF SKIN LEISHMANIASIS
  • amphotericin B-containing PLGA microparticles reduced the lesion growth at a single dose of 50 pg / kg as shown in Figure 5 (A), the Amphotericin-treated group. B free, received intralesional injections twice a week with doses of 50 ⁇ g of the drug for 4 weeks (total of 8 doses). With the evaluation of the parasitic load (B) we can conclude that amphotericin B / PLGA promoted a significant decrease in fluorescence compared to the placebo group, although the size of the lesion decreased there was no statistical difference between the group treated with empty microparticles. and the placebo group when we compared the parasitic load.
  • the group treated with the conventional liposome CH8 formulation was more effective than the group treated with a much higher dose of 200 ⁇ g of free cream lanete CH8.
  • the other groups that were treated with the empty vehicles presented similar profile to the untreated group, indicating specific activity of chalrada nitrada (CH8).

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Abstract

The proposed innovation relates to a pharmaceutical composition containing amphotericin B, nitrochalcone (CH8) or glucantime encapsulated in biodegradable, slow-release polymer microparticles, to the method of encapsulating the pharmaceutical drug inside the particles, to the use of these pharmaceutical compositions, to a medicament and to the treatment of parasitoses.

Description

COMPOSIÇÕES FARMACÊUTICAS MICROPARTICULADAS CONTENDO ANTIPARASITÁRIOS PARA TERAPIA SUBCUTÂNEA PROLONGADA, USO DAS DITAS COMPOSIÇÕES FARMACÊUTICAS PARA A PRODUÇÃO DE UM MEDICAMENTO E MÉTODO DE TRATAMENTO DE PARASITOSES CAMPO DA INVENÇÃO  MICROPARTICULATED PHARMACEUTICAL COMPOSITIONS CONTAINING ANTIPASITARY FOR PROLONGED SUBCUTANEAL THERAPY, USE OF THESE PHARMACEUTICAL COMPOSITIONS FOR THE PRODUCTION OF A MEDICINAL PRODUCT AND METHOD OF TREATMENT OF PARASITOSES FIELD OF THE INVENTION
A invenção se refere a uma composição farmacêutica contendo anfotericina B ou compostos antimoniais tais como, antimoniato de meglumina, estibogluconato de sódio e tartarato de potássio e antimonio (III) hidratado (Sb111), ou ainda uma chalcona nitrada, que nesse caso seria a 3-nitro-2-hidroxi- 4,6-dimetoxichalconas que resolvemos denominá-la de CH8. Essas chalconas nitradas(CH8) encapsulados em partículas de polímeros biodegradáveis de liberação lenta. Esta invenção também se refere ao uso destas composições farmacêuticas, a um medicamento e ao tratamento de parasitoses. The invention relates to a pharmaceutical composition containing amphotericin B or antimonial compounds such as meglumine antimoniate, sodium stibogluconate and hydrated antimony (III) potassium tartrate (Sb 111 ), or a nitrated chalcone, in which case it would be 3-nitro-2-hydroxy-4,6-dimethoxycyclones which we have named it CH8. These nitrate chalcones (CH8) encapsulated in slow release biodegradable polymer particles. This invention also relates to the use of these pharmaceutical compositions, a medicament and the treatment of parasitic diseases.
ANTECEDENTES DA INVENÇÃO  BACKGROUND OF THE INVENTION
As doenças parasitárias, entre elas a malária, as leishmanioses, as tripanossomes, além de outras, são consideradas um dos grandes problemas de saúde pública no mundo. Estas doenças, porém, se concentram nos países que apresentam populações de baixa renda, como por exemplo, países da África, Ásia e América do Sul. Isso ocorre em grande parte devido à falta de políticas públicas na área de saúde e saneamento básico, esses países possuem os maiores números de casos e mortes por essas parasitoses.  Parasitic diseases, including malaria, leishmaniasis, trypanosomes, among others, are considered one of the major public health problems in the world. These diseases, however, are concentrated in countries with low-income populations, such as countries in Africa, Asia, and South America. This is largely due to the lack of public policies on health and sanitation, these countries have the highest number of cases and deaths from these parasites.
Negligenciadas pelo setor privado, o controle dessas endemias depende, quase que exclusivamente, de iniciativas governamentais (universidades, institutos de pesquisa e laboratórios oficiais), havendo uma grande carência de alternativas para o tratamento quimioterápico. A abordagem terapêutica para estas parasitoses é complicada pelo aparecimento de resistência, tal qual mencionado para os agentes anticancerígenos, havendo também nestes casos a necessidade um esforço contínuo visando o desenvolvimento de novos fármacos. A leishmaniose é um complexo de doenças causadas por várias espécies de protozoários do género Leishmania, que podem causar no homem e em outros vertebrados um espectro de formas clínicas. Essas podem ser classificadas em forma Tegumentar, que inclui as formas cutânea simples, difusa e mucocutânea, frequentemente desfigurantes, e forma Visceral muitas vezes letal. Estima-se que haja no mundo 12 milhões de pessoas infectadas, e uma incidência anual de 0,5 milhão de casos da forma visceral e 1 ,5 a 2 milhões de casos da forma tegumentar (WHO, 2004). A Leishmaniose Visceral foi recentemente priorizada pela Organização Mundial de Saúde, junto com a dengue e a tripanossomíase africana, como doenças tropicais de categoria 1 (reemergentes e/ou fora de controle) (REMME et a/. , 2002). O Brasil ocupa posição ímpar, pois é endémico para ambas as formas Tegumentar (simples, mucocutânea e difusa) e Visceral, com uma média anual de 30 mil e 4mil casos notificados, respectivamente, e grau de letalidade para esta última de 8,5%. Aqui, a maioria dos casos ocorre nas regiões Norte e Nordeste, com crescimento recente de casos nas regiões Centro- Oeste, Sudeste e DF (MINISTÉRIO DA SAÚDE, 2006 e 2007a e b). A Leishmaniose Tegumentar Americana (LTA), como são coletivamente chamadas as diferentes formas de leishmanioses tegumentares da América Latina, são causadas por espécies do parasito diferentes das que ocorrem no Velho Mundo. Mais de 80% dos casos são causados pela Leishmania (V) braziliensis, seguida da Leishmania (V.) guyanensis e Leishmania (L.) amazonensis (MINISTÉRIO DA SAÚDE 2007a). O parasito existe como promastigota flagelado no vetor flebotomíneo, e como amastigota aflagelado dentro de fagócitos mononucleares do hospedeiro vertebrado. A localização intracelular privilegiada dificulta o acesso de fármacos ativos, o que torna esta doença de difícil tratamento. Neglected by the private sector, control of these endemics depends almost exclusively on government initiatives (universities, research institutes and official laboratories), and there is a great lack of alternatives for chemotherapy treatment. The therapeutic approach to these parasites is complicated by the emergence of resistance as mentioned for anticancer agents, and in these cases there is also a need for continued efforts to develop new drugs. Leishmaniasis is a complex of diseases caused by various protozoan species of the genus Leishmania, which can cause a spectrum of clinical forms in humans and other vertebrates. These can be classified into Cutaneous form, which includes the simple, diffuse, mucocutaneous, often disfiguring, and often lethal Visceral forms. There are an estimated 12 million infected people worldwide, with an annual incidence of 0.5 million visceral cases and 1, 5 to 2 million integumentary cases (WHO, 2004). Visceral Leishmaniasis has recently been prioritized by the World Health Organization, along with dengue and African trypanosomiasis, as Category 1 tropical diseases (reemerging and / or out of control) (REMME et al., 2002). Brazil occupies a unique position, as it is endemic to both Tegumentary (simple, mucocutaneous and diffuse) and Visceral forms, with an annual average of 30,000 and 4,000 reported cases, respectively, and 8.5% lethality for the latter. . Here, most cases occur in the North and Northeast regions, with recent cases growing in the Midwest, Southeast and DF regions (MINISTRY OF HEALTH, 2006 and 2007a and b). American cutaneous leishmaniasis (ATL), as the different forms of cutaneous leishmaniasis in Latin America are collectively called, are caused by different parasitic species from those in the Old World. More than 80% of cases are caused by Leishmania (V) braziliensis, followed by Leishmania (V.) guyanensis and Leishmania (L.) amazonensis (MINISTRY OF HEALTH 2007a). The parasite exists as a flagellated promastigote in the phlebotomine vector, and as a flush amastigote within mononuclear phagocytes of the vertebrate host. The privileged intracellular location makes it difficult to access active drugs, which makes this disease difficult to treat.
A terapia de pacientes com leishmaniose ainda se baseia no uso de antimoniais pentavalentes. Os sais antimoniais são obtidos sinteticamente a partir do ácido antimônico e da N-metil-glucamina. A administração intramuscular ou endovenosa do antimoniato é rapidamente absorvida, de forma que após 48 horas 90% da dose inicial já foi excretada pelos rins. Faz-se necessário, altas doses repetidas para manter o nível terapêutico do antimônio nos tecidos. Com isso, surge uma diversidade de efeitos colaterais como mialgia, pancreatite, pancitopenia, hepato e cardiotoxicidade. A pancreatite parece ser a causa frequente de náuseas e dores abdominais (GASSER et al, 1994). The therapy of leishmaniasis patients is still based on the use of pentavalent antimonials. Antimonial salts are synthetically obtained from antimonic acid and N-methyl glucamine. The administration Intramuscular or intravenous antimoniate is rapidly absorbed, so that after 48 hours 90% of the initial dose has already been excreted by the kidneys. Repeated high doses are required to maintain the therapeutic level of antimony in the tissues. This results in a variety of side effects such as myalgia, pancreatitis, pancytopenia, hepato and cardiotoxicity. Pancreatitis seems to be the frequent cause of nausea and abdominal pain (GASSER et al, 1994).
A intolerância ou refratariedade do paciente ao tratamento com antimoniais levam ao uso da pentamidina ou da anfotericina B como drogas de segunda escolha, ambas também administradas por via intramuscular ou endovenosa. A pentamidina tem eficácia semelhante aos antimoniais (TUON et al 2008), mas produz outros efeitos colaterais como hipoglicemia, diabetes, taquicardia, hipotensão, nefrotoxidade e dores locais na administração na dose de 4mg/kg.dia alternados, durante 30 dias pela via intramuscular. A anfotericina B é um antibiótico poliênico isolado da bactéria Streptomyces sp, usado inicialmente como fármaco antifúngico, especialmente em infecções sistémicas. É indicada como tratamento alternativo para a leishmaniose visceral e mucocutânea, é considerada a droga que apresenta falha terapêutica mais baixa, sendo que, em regiões refratárias aos antimoniais, como na índia, já é tida como medicamento de primeira linha. A dose recomendada é de 0,75- 1 mg/kg. dia por 20 dias, administrados através de infusões endovenosas lentas em geral com internação do paciente (BERN et al., 2006). A Anfotericina B, também é indicada para o tratamento de pacientes imunocomprometidos, como pacientes HIV positivos, que embora seja mais ativa que os antimoniais, produz graves efeitos colaterais. Esses efeitos são explicados por sua afinidade também pelo colesterol na membrana da célula humana, mesmo que em menor extensão que ao ergosterol e por sua insolubilidade. Os principais efeitos relatados são hipocalemia, cardio e nefrotoxidade (CROFT & COOMBS, 2003). Algumas alternativas de tratamento estão sendo estudadas, como modificações na dose e duração. Uma alternativa com grande potencial de aplicação seria usar, os sistemas de carreamento e liberação de fármacos, destacando-se como uma das áreas da indústria farmacêutica mais promissora, devido à possibilidade de resgatar drogas ativas que foram descartadas por sua toxidez ou baixa biodisponibilidade e o alto custo no descobrimento e desenvolvimento de novas moléculas ativas. Patient intolerance or refractoriness to treatment with antimonials leads to the use of pentamidine or amphotericin B as second-choice drugs, both also administered intramuscularly or intravenously. Pentamidine has similar efficacy to antimonials (TUON et al 2008), but produces other side effects such as hypoglycemia, diabetes, tachycardia, hypotension, nephrotoxicity and local pain on alternate 4mg / kg.day administration for 30 days intramuscularly . Amphotericin B is a polyenic antibiotic isolated from the bacteria Streptomyces sp, initially used as an antifungal drug, especially in systemic infections. It is indicated as an alternative treatment for visceral and mucocutaneous leishmaniasis, and is considered to be the drug with the lowest therapeutic failure, and in regions refractory to antimonials, such as India, it is already considered a first-line drug. The recommended dose is 0.75-1 mg / kg. day for 20 days, administered through slow intravenous infusions usually with patient admission (BERN et al., 2006). Amphotericin B is also indicated for the treatment of immunocompromised patients, such as HIV-positive patients, which although more active than antimonials, produces serious side effects. These effects are explained by their affinity also for cholesterol in the human cell membrane, even to a lesser extent than ergosterol and for its insolubility. The main effects reported are hypokalemia, cardio and nephrotoxicity (CROFT & COOMBS, 2003). Some treatment alternatives are being studied, such as changes in dose and duration. An alternative with great application potential would be to use drug delivery and delivery systems, standing out as one of the most promising areas of the pharmaceutical industry due to the possibility of rescuing active drugs that have been discarded for their toxicity or low bioavailability and high cost in the discovery and development of new active molecules.
Os problemas mais comuns que costumam impedir a aprovação de fármacos potencialmente eficazes são sua rápida metabolização no organismo e os efeitos tóxicos decorrentes de sua baixa solubilidade plasmática e/ou sua ação indiscriminada sobre células sadias. Idealmente, um fármaco de efeito sistémico deve permanecer na circulação o tempo necessário para seu efeito terapêutico, dentro de uma faixa segura de alta eficácia combinada à baixa toxidez, e um mínimo de doses repetitivas. Além de protegê-lo contra a degradação prematura e promover sua solubilização, o encapsulamento de um fármaco em micro e nanopartículas apropriadas pode ajudar a direcioná-lo para o seu tecido ou célula alvo. Os sistemas de liberação controlada de fármacos apresentam várias vantagens em relação aos sistemas convencionais, tais como: 1 ) Maior controle da liberação do principio ativo, diminuindo o aparecimento de doses tóxicas e subterapêuticas; 2) Utilização de menor quantidade do principio ativo; 3) Resultando em menor custo; 4) Maior intervalo de administração; melhor aceitação do tratamento pelo paciente e a possibilidade de direcionamento do principio ativo para seu alvo específico.  The most common problems that often impede the approval of potentially effective drugs are their rapid metabolism in the body and the toxic effects of their low plasma solubility and / or their indiscriminate action on healthy cells. Ideally, a systemic drug should remain in circulation for as long as necessary for its therapeutic effect, within a safe range of high efficacy combined with low toxicity, and a minimum of repetitive doses. In addition to protecting it against premature degradation and promoting its solubilization, encapsulating a drug in appropriate micro and nanoparticles can help direct it to your target tissue or cell. Controlled drug release systems have several advantages over conventional systems, such as: 1) Greater control of the release of the active ingredient, reducing the appearance of toxic and subtherapeutic doses; 2) Use of less quantity of active principle; 3) Resulting in lower cost; 4) Longer administration interval; better treatment acceptance by the patient and the possibility of directing the active ingredient to its specific target.
Os primeiros a serem desenvolvidos, e únicos já aprovados para uso sistémico endovenoso, são os lipossomas. A partir da década de 1980 vários polímeros biodegradáveis foram e estão sendo utilizados no preparo de micro e nanopartículas, incluindo os polímeros sintéticos poli-(lático-co- glicólico) (PLGA), além dos biopolímeros quitosana e polihidroxialcanoatos (PHA). De tamanho mais reduzido, na ordem de 1 -10 nm, temos a ciclodextrina; e mais recentemente os polímeros esféricos e ramificados, os dendrímeros, que estão sendo estudados quanto à sua viabilidade farmacêutica. Alguns destes sistemas já foram estudados para melhorar a solubilidade e/ou diminuir a toxidez dos antimoniais e da anfotericina B na leishmaniose (ROSSI-BERGMANN e FREZARD, 2007). Devido ao fato da Leishmania infectar quase que exclusivamente macrófagos, o uso de sistemas particulados constituiria uma excelente ferramenta para levar fármacos para a célula infectada, em contato íntimo com os parasitos, reduzindo sua toxidez para outras células e o aparecimento de efeitos colaterais. Além disso, limitações farmacocinéticas podem ser contornadas pela liberação lenta dos lipídeos ou polímeros biodegradáveis que constituem as partículas, reduzindo assim sua dosagem. The first to be developed, and the only ones already approved for intravenous systemic use, are liposomes. From the 1980s onwards various biodegradable polymers were and are being used in the preparation of micro and nanoparticles, including synthetic poly (lactic co-glycolic) polymers (PLGA), in addition to chitosan and polyhydroxyalkanoate (PHA) biopolymers. Of smaller size, in the order of 1 -10 nm, we have the cyclodextrin; and more recently spherical and branched polymers, dendrimers, which are being studied for their pharmaceutical viability. Some of these systems have been studied to improve solubility and / or decrease antimonial and amphotericin B toxicity in leishmaniasis (ROSSI-BERGMANN & FREZARD, 2007). Because Leishmania infects almost exclusively macrophages, the use of particulate systems would be an excellent tool for bringing drugs into the infected cell in close contact with the parasites, reducing their toxicity to other cells and the appearance of side effects. In addition, pharmacokinetic limitations can be circumvented by the slow release of the lipid or biodegradable polymers that make up the particles, thereby reducing their dosage.
Os lipossomos foram as primeiras partículas delineadas para vetorização de drogas antileishmaniais. Hoje em dia, a única formulação comercialmente disponível e aprovada para o uso humano para o tratamento da leishmaniose é a Anfotericina B encapsulada em lipossomos unilamelares carregados negativamente (Ambisome®) (SUNDAR & RAI, 2002). Recentes estudos relacionados com a toxicidade e efetividade de formulações lipossomais de antimoniato de meglumina em cães com leishmaniose visceral demonstraram que a formulação reduziu significantemente a carga parasitária da medula óssea dos cães e não provocou mudanças significantes nas funções hepáticas e renais na dose (6.5 mg Sb/kg/dose) com quatro doses (4 dias de intervalos) (FRÉZARD, F. & MICHALICK, M. S. M, 2008). Liposomes were the first particles delineated for antileishmanial drug vectorization. Today, the only commercially available formulation approved for human use for the treatment of leishmaniasis is Amphotericin B encapsulated in negatively charged unilamellar liposomes (Ambisome®) (SUNDAR & RAI, 2002). Recent studies on the toxicity and effectiveness of meglumine antimoniate liposomal formulations in dogs with visceral leishmaniasis have shown that the formulation significantly reduced the parasitic burden of bone marrow in dogs and did not cause significant changes in liver and renal function in the dose (6.5 mg Sb / kg / dose) with four doses (4 days apart) (FRÉZARD, F. & MICHALICK, MS M, 2008).
A aplicação clínica de formulações de anfotericina B associada a lipídeos levou a uma melhora notável na quimioterapia de leishmaniose. Essas formulações associadas a lipídeos como o AmBisome®, encapsulada em lipossomos unilamelares, o Abelcet®, incorporada em complexo lipídico; e o Amphocil®, como uma suspensão coloidal em colesterol foram avaliadas em testes clínicos para leishmaniose visceral e mucocutânea. Das três formulações, o AmBisome® demonstrou maior eficiência favorecendo sua aprovação pelo FDA (Food and Drug Administration). Os efeitos colaterais diminuíram principalmente a nefrotoxidade, por diminuir sua eliminação renal, além de aumentar sua meia vida plasmática. Outra vantagem é a possibilidade de se administrar doses maiores do fármaco em menor espaço de tempo. Porém, o fator limitante do uso dessas formulações lipossomais é o alto da fosfatidilcolina, o que inviabiliza o acesso ao tratamento por grande parte da população afetada (GOLENSER et al. 1999). Clinical application of lipid-associated amphotericin B formulations has led to a remarkable improvement in leishmaniasis chemotherapy. These lipid-associated formulations such as AmBisome®, encapsulated in unilamellar liposomes, Abelcet®, incorporated into lipid complex; and Amphocil® as a colloidal suspension in cholesterol were evaluated in clinical tests for visceral and mucocutaneous leishmaniasis. Of the three formulations, AmBisome® has shown greater efficiency by favoring its approval by the Food and Drug Administration (FDA). Side effects mainly reduced nephrotoxicity by decreasing renal elimination and increasing plasma half-life. Another advantage is the possibility of administering larger doses of the drug in a shorter period of time. However, the limiting factor of the use of these liposomal formulations is the high phosphatidylcholine, which makes the access to treatment impossible for most of the affected population (GOLENSER et al. 1999).
Os sistemas poliméricos micro e nanopartículas poliméricas são sistemas carreadores de fármacos que incluem cápsulas e esferas. Sua maior vantagem em relação aos lipossomos é a maior estabilidade, podendo ser liofilizados para armazenagem. Fatores como tipo e proporção dos polímeros na formulação determinam características importantes como estabilidade e estrutura cristalina e biodegradabilidade, que influencia na velocidade de liberação do fármaco (SCHAFFAZICK & GUTERRES, 2003). O polímero de poli (lactídeo), ou PLA, é um dos mais estudados para aplicação biológica, por sua alta biodegradabilidade e biocompatibilidade, já tendo inclusive uso clínico aprovado na constituição de fios de suturas e próteses ortopédicas biodegradáveis. As nanoesferas de PLA já foram também testadas com certo sucesso como carreadoras de vários antileishmaniasis, como a primaquina, pentamidina, bacopasaponina e atovaquona, para o tratamento experimental da leishmaniose visceral por via endovenosa. Na leishmaniose cutânea, As nanopartículas de PLA de 200 nm foram testadas com sucesso pelo nosso grupo, na veiculação da chalcona nitrada(CH8) DMC para o tratamento subcutâneo de camundongos infectados com L. amazonensis (TORRES- SANTOS er a/., 1999).  The polymeric micro and nanoparticle polymer systems are drug carrier systems that include capsules and spheres. Its major advantage over liposomes is its greater stability and can be lyophilized for storage. Factors such as type and proportion of polymers in the formulation determine important characteristics such as stability and crystalline structure and biodegradability, which influences the release rate of the drug (SCHAFFAZICK & GUTERRES, 2003). Poly (lactide) polymer, or PLA, is one of the most studied for biological application, due to its high biodegradability and biocompatibility, having already approved clinical use in the constitution of sutures and biodegradable orthopedic prostheses. PLA nanospheres have also been successfully tested as carriers of various antileishmaniasis, such as primaquine, pentamidine, bacopasaponin and atovaquone, for the experimental treatment of intravenous visceral leishmaniasis. In cutaneous leishmaniasis, 200 nm PLA nanoparticles were successfully tested by our group on the delivery of nitrated chalcone (CH8) DMC for subcutaneous treatment of L. amazonensis infected mice (TORRES-SANTOS er a /., 1999). .
Os primeiros registros do uso de partículas poliméricas para veiculação de antileishmaniais datam de 1987, quando micropartículas de poliacrilamida com diâmetro superior a 1 pm foram utilizadas para complexação do antimonial pentavalente estibogluconato de sódio (DHANARAJUA, M. D. et al., 2006; ESPUELAS, M. S. et ai, 2002). Como mencionado acima, por seu tamanho relativamente grande, as micropartículas não são indicadas para uso parenteral pelo risco de formação de êmbolos e trombos nos capilares sanguíneos, sendo seu uso terapêutico geralmente restrito às vias subcutânea, intramuscular, oral ou intranasal. Até hoje nenhuma formulação polimérica foi disponibilizada comercialmente para tratamento da leishmaniose humana. Além do PLA, o PLGA também é interessante como sistema de liberação controlada de drogas. Este polímero também já foi aprovado pelo FDA, como estruturas biodegradáveis usadas em parafusos e tachas ortopédicos, fios de sutura e sistemas de reparo de menisco e cartilagem (ELIAZ e KOST, 2000). Além disso, o PLGA é amplamente utilizado na medicina cosmética no preenchimento subcutâneo de rugas, com alto grau de aceitação pelos pacientes. Os medicamentos formulados em polímeros e aprovados para uso humano são todos dirigidos para o tratamento do câncer em microcápsulas poliméricas (> 1000 nm) e têm aplicação potencial nos casos em que interessa a formação de depósitos para liberação local lenta e contínua, como por exemplo, em vacinas ou tratamentos crónicos localizados. The earliest records of the use of polymeric particles for antileishmanial delivery date from 1987, when microparticles of Polyacrylamides larger than 1 µm in diameter were used for complexation of the pentavalent sodium stibogluconate antimonial (DHANARAJUA, MD et al., 2006; ESPUELAS, MS et al, 2002). As mentioned above, because of their relatively large size, microparticles are not indicated for parenteral use because of the risk of embolism and thrombus formation in the blood capillaries, and their therapeutic use is generally restricted to subcutaneous, intramuscular, oral or intranasal routes. To date no polymeric formulation has been commercially available for treatment of human leishmaniasis. In addition to PLA, PLGA is also interesting as a controlled drug delivery system. This polymer has also been approved by the FDA as biodegradable structures used in orthopedic screws and studs, sutures, and meniscus and cartilage repair systems (ELIAZ and KOST, 2000). In addition, PLGA is widely used in cosmetic medicine for subcutaneous wrinkle filling, with a high degree of acceptance by patients. Polymer formulations approved for human use are all directed to the treatment of cancer in polymeric microcapsules (> 1000 nm) and have potential application in the case of deposit formation for slow and continuous local release such as in localized vaccines or chronic treatments.
O primeiro produto de PLGA aprovado pelo FDA foi o The first FDA approved PLGA product was the
LupronDepot em 1996 (TAP Pharmaceutical Products Inc), micropartículas de 75:25 lactideo/glicolídeo contendo Leuprolide para o tratamento de câncer avançado de próstata . Administrado localmente, o LupronDepot forma um depósito e libera a droga gradualmente, podendo ser administrado em intervalos de até 4 meses, substituindo as injeções diárias de Leuprolide livre (KADA et al., 1994). Micropartículas de PLGA já estão sendo testadas, em humanos, como sistemas de liberação de vacina. Trata-se de teste clínico está em andamento no Brasil em pacientes com câncer avançado de colo e pescoço, desenvolvida por SILVA, C. L e colaboladores. Os primeiros registros do uso de partículas poliméricas para veiculação de antileishmaniais datam de 1987, quando micropartículas de poliacrilamida com diâmetro superior a 1 ιη foram utilizadas para complexação do antimonial pentavalente estibogluconato de sódio. Os trabalhos que se seguiram utilizaram primaquina e IFN-gama como ativos, também utilizando a via endovenosa em camundongos, e mostraram a sua potencialização e acúmulo principalmente no fígado (FORTINA, et ai, 2005; ITO, et al., 2005). LupronDepot in 1996 (TAP Pharmaceutical Products Inc), 75:25 lactide / glycolide microparticles containing Leuprolide for the treatment of advanced prostate cancer. Administered locally, LupronDepot forms a depot and releases the drug gradually and can be administered at intervals of up to 4 months, replacing daily injections of free Leuprolide (KADA et al., 1994). PLGA microparticles are already being tested in humans as vaccine delivery systems. This clinical trial is underway in Brazil in patients with advanced neck and neck cancer, developed by SILVA, C. L and collaborators. The earliest records of the use of antileishmanial polymeric particles date back to 1987, when polyacrylamide microparticles larger than 1 µm in diameter were used to complex the pentavalent sodium stibogluconate antimonial. The following studies used primaquine and IFN-gamma as active, also using the intravenous route in mice, and showed its potentiation and accumulation mainly in the liver (FORTINA, et al, 2005; ITO, et al., 2005).
O polímero de poli (lactídeo), ou PLA, é um dos mais estudados para o preparo de micropartículas para aplicação biológica, por sua alta bidegradabilidade e biocompatibilidade, já tendo inclusive uso clínico aprovado na constituição de fios de suturas biodegradáveis, assim como o PLGA, as nanoesferas de PLA já foram também testadas com certo sucesso como carreadoras de vários antileishmaniasis, como a primaquina, pentamidina, bacopasaponina e atovaquona, para o tratamento experimental da leishmaniose visceral por via endovenosa.  Poly (lactide) polymer, or PLA, is one of the most studied for the preparation of microparticles for biological application, due to its high bidegradability and biocompatibility, having already approved clinical use in the constitution of biodegradable suture threads, as well as PLGA , PLA nanospheres have also been successfully tested as carriers of various antileishmaniasis, such as primaquine, pentamidine, bacopasaponin and atovaquone, for the experimental treatment of intravenous visceral leishmaniasis.
Pinero J. e colaboradores 2006, mostraram que implantes de filmes de PLA e PLGA veiculando uma trans-chalcona foram testadas em camundongos BALB/c infectados com L. amazonensis na pata. Nesta avaliação cada indivíduo recebeu um implante de 3 mm na orelha 5 semanas após a infecção. Os resultados mostraram que os camundongos implantados com a droga em PLA e PLGA induziram uma redução significativa no tamanho das lesões. Já os camundongos que receberam implantes de PLA vazias também apresentaram diminuição no tamanho da lesão devido aparentemente a uma atividade pro-inflamatória intrínseca do PLA que poderia estar ativando mecanismos antimicrobicidas dos macrófagos.  Pinero J. et al., 2006, showed that PLA and PLGA film implants carrying a trans-chalcona were tested in L. amazonensis infected BALB / c mice in the paw. In this evaluation each individual received a 3 mm ear implant 5 weeks after infection. Results showed that mice implanted with the drug in PLA and PLGA induced a significant reduction in lesion size. Mice that received empty PLA implants also had decreased lesion size apparently due to an intrinsic PLA proinflammatory activity that could be activating antimicrobicidal mechanisms of macrophages.
Assim, tendo em vista a biocompatibilidade comprovada do polímero de PLGA, inclusive na forma de micropartículas para injeção subcutânea em preenchimento cosméticos, neste estudo propomos avaliar o potencial das micropartículas de PLGA para melhoramento da eficácia do tratamento da leishmaniose cutânea através da administração intralesional de compostos antimoniais, chalcona nitrada(CH8) e Anfotericina B. A terapêutica convencional baseada em séries de injeções intramusculares contribui de certa forma para o insucesso do tratamento, porque além da elevada toxicidade destes fármacos o regime terapêutico longo com múltiplas doses diminui a adesão de muitos pacientes. A administração intralesional de antimoniais vem sendo usada visando minimizar os efeitos colaterais sistémicos e apresentam resultados satisfatórios, mas provoca dor no local de administração devido à necessidade de múltiplas injeções locais. Thus, in view of the proven biocompatibility of PLGA polymer, including in the form of microparticles for subcutaneous injection in cosmetic filler, in this study we propose to evaluate the potential of PLGA microparticles for improving the efficacy of treatment of cutaneous leishmaniasis through intralesional administration of antimonial compounds, chalcone nitrada (CH8) and amphotericin B. Conventional therapy based on series of intramuscular injections contributes somewhat to the treatment failure, because besides the high toxicity of these drugs the therapeutic regimen Long dosing with multiple doses decreases adherence of many patients. Intralesional administration of antimonials has been used to minimize systemic side effects and yield satisfactory results, but causes pain at the site of administration due to the need for multiple local injections.
SUMÁRIO DA INVENÇÃO  SUMMARY OF THE INVENTION
Para melhor esclarecimento desta invenção, as chalconhas nitrogenadas que aqui nos referimos tratam-se da 3-nitro-2-hidroxi-4,6-dimetoxichalcona, que para facilitar iremos denomina-la de CH8.  For the sake of clarity of this invention, the nitrogenous chaffs referred to herein are 3-nitro-2-hydroxy-4,6-dimethoxychalcona, which we will refer to as CH8 for ease.
O primeiro objetivo desta invenção trata de composições farmacêuticas contendo uma quantidade farmaceuticamente efetiva de medicamentos ou fármacos antileishmaniais encapsulados em partículas de PLGA.  The first object of this invention relates to pharmaceutical compositions containing a pharmaceutically effective amount of PLGA particle encapsulated antileishmanial drugs or drugs.
O segundo objeto desta invenção trata do uso das composições farmacêuticas contendo uma quantidade farmaceuticamente efetiva de Anfotericina B, chalcona nitrada (CH8) ou compostos antimoniais, encapsulados em partículas de PLGA voltada para a produção de um medicamento para o tratamento subcutâneo de doenças cutâneas inflamatórias e infecciosas da pele ou em regiões da pele (derme, epiderme e anexos), tais como, leishmaniose, micoses, hanseníase, psoríases, úlcera tropical, verrugas, hemangioma cavernoso em animais mamíferos. The second object of this invention relates to the use of pharmaceutical compositions containing a pharmaceutically effective amount of amphotericin B, chalcone nitrated (CH8) or antimony compounds, encapsulated in PLGA particles directed to the manufacture of a medicament for the subcutaneous treatment of inflammatory skin diseases. infectious skin or skin regions (dermis, epidermis and appendages) such as leishmaniasis, mycoses, leprosy, psoriasis, tropical ulcer, warts, cavernous hemangioma in mammalian animals.
O terceiro objeto desta invenção é uma composição farmacêutica contendo uma quantidade terapeuticamente eficaz de Anfotericina B, chalcona nitrada (CH8) ou de compostos antimoniais encapsulados em partículas de PLGA, além de excipientes farmaceuticamente aceitáveis. O último objeto desta invenção trata de método de tratamento de parasitoses que compreende a aplicação subcutânea de uma quantidade terapeuticamente efetiva de Anfotericina B, chalcona nitrada (CH8) ou compostos antimoniais, encapsulados em micropartículas de PLGA a um animal mamífero portador da leishmaniose. The third object of this invention is a pharmaceutical composition containing a therapeutically effective amount of amphotericin B, chalcone nitrite (CH8) or PLGA particle encapsulated antimonial compounds, and pharmaceutically acceptable excipients. The last object of this invention is a method of treating parasitic diseases comprising subcutaneously applying a therapeutically effective amount of amphotericin B, chalcone nitrite (CH8) or antimonial compounds encapsulated in PLGA microparticles to a mammalian animal carrying leishmaniasis.
DESCRIÇÃO DETALHADA DA INVENÇÃO DETAILED DESCRIPTION OF THE INVENTION
Esta invenção trata de composições farmacêuticas compreendendo uma quantidade farmaceuticamente efetiva de medicamentos ou fármacos antileishmaniais encapsulados em partículas de PLGA, tendo como exemplos de fármacos como a Anfotericina B, chalcona nitrada(CH8) e compostos antimoniais. O agente ativo pode constituir cerca de 5 a 85% do peso da formulação. Concentração variada de compostos ativos pode possibilitar uma liberação inicial mais rápida no tecido após a administração local. Em uma variação exemplificativa como será mostrado a seguir, utilizamos 10% de Anfotericina B em relação ao polímero (p/p).  This invention relates to pharmaceutical compositions comprising a pharmaceutically effective amount of PLGA particle encapsulated antileishmanial drugs or drugs, having as examples of drugs such as Amphotericin B, chalcone nitrate (CH8) and antimonial compounds. The active agent may constitute about 5 to 85% of the weight of the formulation. Varied concentration of active compounds may enable faster initial release into tissue after local administration. In an exemplary variation as shown below, we use 10% Amphotericin B relative to the polymer (w / w).
Exemplos de medicamentos ou fármacos utilizados são a Anfotericina B, chalcona nitrada(CH8) e compostos antimoniais tais como, antimoniato de meglumina, estibogluconato de sódio e tartarato de potássio e antimonio (III) hidratado (Sblll), combinados ou não.  Examples of medicaments or drugs used are amphotericin B, chalcone nitrate (CH8) and antimonial compounds such as meglumine antimoniate, sodium stibogluconate and potassium hydrate (IIIb) tartrate, combined or not.
Um processo de encapsulamento desses fármacos, Anfotericina B, chalcona nitrada(CH8) ou compostos antimoniais em partículas de PLGA de tamanhos variando entre 0,5 e 500pm. Em uma variação, copolímeros de ácido glicólico e lático são usados e o percentual de cada monômero de ácido poli (lático-co-glicólico) pode ser, cerca de 15-85%, cerca de 25-75%, cerca de 35- 65%, ou a proporção de 50-50% podem ser empregadas.  A process of encapsulating these drugs, amphotericin B, chalcone nitrada (CH8) or antimonial compounds in PLGA particles of sizes ranging from 0.5 to 500pm. In one variation, glycolic and lactic acid copolymers are used and the percentage of each poly (lactic-co-glycolic acid) monomer may be about 15-85%, about 25-75%, about 35-65%. %, or the proportion of 50-50% may be employed.
O segundo objeto desta invenção trata do uso das composições farmacêuticas contendo uma quantidade farmaceuticamente efetiva de Anfotericina B ou compostos antimoniais, preferencialmente, Glucantime e chalcona nitrada(CH8) encapsulados em partículas de PLGA voltada para a produção de um medicamento para o tratamento subcutâneo de doenças cutâneas inflamatórias e infecciosas da pele ou em regiões da pele (derme, epiderme e anexos), tais como leishmaniose, micoses, hanseníase, psoríases, ulcera tropical, verrugas, hemangioma cavernoso em animais mamíferos. The second object of this invention relates to the use of pharmaceutical compositions containing a pharmaceutically effective amount of amphotericin B or antimonial compounds, preferably Glucantime and PLGA particle-encapsulated nitrated chalcone (CH8) for the manufacture of a medicament for the subcutaneous treatment of inflammatory and infectious skin diseases or in skin regions (dermis, epidermis and appendages) such as leishmaniasis, mycosis, leprosy, psoriasis, tropical ulcer, warts, cavernous hemangioma in mammalian animals.
O terceiro objeto desta invenção é uma composição farmacêutica contendo uma quantidade terapeuticamente eficaz de Anfotericina B ou de compostos antimoniais como Glucantime e chalcona nitradas(CH8) encapsulados em partículas de PLGA, além de excipientes farmaceuticamente aceitáveis.  The third object of this invention is a pharmaceutical composition containing a therapeutically effective amount of amphotericin B or antimonial compounds such as PLGA particle encapsulated nitrated glucosin and chalcone (CH8) in addition to pharmaceutically acceptable excipients.
O último objeto desta invenção trata de método de tratamento de parasitoses que compreende a aplicação subcutânea de uma quantidade terapeuticamente efetiva de Anfotericina B ou compostos antimoniais como Glucantime e chalcona nitradas(CH8) encapsulados em micropartículas de PLGA a um animal mamífero portador da leishmaniose.  The last object of this invention is a method of treating parasitic diseases comprising subcutaneously applying a therapeutically effective amount of Amphotericin B or antimonial compounds such as PLGA microparticle-encapsulated nitrided Glucantime and chalcone (CH8) to a mammalian animal carrying leishmaniasis.
PROCESSO DE ENCAPSULAMENTO PRODUÇÃO DAS MICROPARTÍCULAS DE PLGA: Encapsulation Process Production of Plga Microparticles:
As partículas PLGA contendo a Anfotericina B e os compostos antimoniais serão produzidos pelo método de emulsificação múltipla (Ai / 0 / A2), seguida pelo método de evaporação de solvente. Inicialmente, o polímero (PLGA) será dissolvido em diclorometano (fase orgânica), a esta fase orgânica, será adicionado 1 ml de solução tampão pH 7,4 contendo os fármacos (10% em relação ao polímero (p/p)), e a mistura das fases será promovida sob ultra agitação de 7000rpm durante 30 segundos, para a formação de uma emulsão simples (Ai I 0). Essa emulsão simples será dispersa na fase contínua (A2) sob ultra-homogeneização de 7000rpm por 60 segundos, uma solução aquosa de 3% de ácido polivinílico (PVA) será adicionada ao sistema e a evaporação do solvente será sob agitação de 400 rpm durante 4horas. Na etapa final, a suspensão de micropartículas será centrifugada a 4000 rpm durante 5 minutos, lavada com água destilada 3 vezes e congelada em freezer -20°C para posterior liofilização por 16 horas. O agente ativo pode constituir cerca de 5 a 85% do peso da formulação. PLGA particles containing Amphotericin B and antimonial compounds will be produced by the multiple emulsification method (Ai / 0 / A 2 ), followed by the solvent evaporation method. Initially, the polymer (PLGA) will be dissolved in dichloromethane (organic phase), to this organic phase will be added 1 ml pH 7.4 buffer containing the drugs (10% relative to the polymer (w / w)), and The mixture of the phases will be promoted under ultra-stirring at 7000rpm for 30 seconds to form a single emulsion (Al 10). Such a single emulsion will be dispersed in the continuous phase (A 2 ) under 7000rpm ultrahomogenization for 60 seconds, a 3% aqueous polyvinyl acid (PVA) solution will be added to the system and solvent evaporation will be under stirring at 400 rpm for 4 hours. In the final step, the microparticle suspension will be centrifuged at 4000 rpm for 5 minutes, washed with distilled water 3 times and frozen in -20 ° C freezer for subsequent freeze drying for 16 hours. The active agent may constitute about 5 to 85% of the weight of the formulation.
DETERMINAÇÃO DE CARACTERÍSTICAS QUÍMICAS DAS PARTÍCULAS DETERMINATION OF CHEMICAL CHARACTERISTICS OF PARTICULARS
A) TAXA DE INCORPORAÇÃO DOS FÁRMACOS  A) INCORPORATION RATE OF PHARMACEUTICALS
A determinação do teor dos fármacos nas partículas é importante para a garantia da dose correta a ser utilizada nos ensaios. Os teores de Anfotericina B nas partículas de PLGA serão determinados por CLAE. Os teores de antimônio (Sb) serão determinados por absorção atómica.  Determination of drug content in the particles is important to ensure the correct dose to be used in the trials. Amphotericin B contents in PLGA particles will be determined by HPLC. Antimony (Sb) levels will be determined by atomic absorption.
B) TAXA DE LIBERAÇÃO DOS FÁRMACOS  B) DRUG RELEASE RATE
As partículas de PLGA terão a velocidade de liberação dos fármacos determinada em tampão fisiológico pH 7,2 a 37°C. Para tal, 10 mg de partícula serão incubados em 1 ml de PBS, acrescido de 0, 1 % de azida, sob agitação constante de 80rpm e temperatura de 37°C. A coleta das amostras será feita através de centrifugação, retirada de 500 μΙ em tempos variando de 1 hora a 270 dias. As amostras serão analisadas por UV-CLAE para determinar o teor da anfotericina B liberada, o teor de antimônio será analisado por absorção atómica. Será construída a curva: % de liberação x tempo.  PLGA particles will have the drug release rate determined in physiological buffer pH 7.2 at 37 ° C. For this, 10 mg of particle will be incubated in 1 ml of PBS, plus 0.1% azide, under constant agitation at 80rpm and 37 ° C. Samples will be collected by centrifugation, taken from 500 μΙ at times ranging from 1 hour to 270 days. Samples will be analyzed by UV-HPLC to determine the content of the released amphotericin B, the antimony content will be analyzed by atomic absorption. The curve will be constructed:% release x time.
c) DEGRADAÇÃO POLIMÉRICA POR MICROSCOPIA ELETRÔNICA DE VARREDURA c) POLYMERIC DEGRADATION BY ELECTRONIC SCAN MICROSCOPY
A degradação e morfologia das micropartículas de PLGA em tampão PBS por microscopia eletrônica de varredura (MEV). As partículas de PLGA obtidas em tempos variados em b acima serão liofilizadas e tratadas para visualização por MEV.  The degradation and morphology of PLGA microparticles in PBS buffer by scanning electron microscopy (SEM). PLGA particles obtained at varying times in b above will be lyophilized and treated for SEM viewing.
D) ANÁLISE DA DISTRIBUIÇÃO E DEGRADAÇÃO POLIMÉRICA DAS PARTÍCULAS  D) ANALYSIS OF DISTRIBUTION AND POLYMERIC DEGRADATION OF PARTICULES
A análise de tamanho e distribuição das partículas obtidas em b por centrifugação e liofilização será feita usando o analisador de distribuição granulométrica Coulter LS230. Pequenas quantidades de partículas serão dispersas em etanol absoluto P.A. com auxílio de ultrassom até atingirem o índice de obscuração requisitado pelo aparelho. O tamanho médio de partícula será expresso com o diâmetro médio em volume (D4,3). O diâmetro das partículas será calculado em D0,9, D0, i e D0,5 , que significam o diâmetro de partícula correspondente a 90, 50 e 10 % da distribuição acumulada e a polidispersão será dada pelo índice span, o qual será calculado por (DO, 9 - DO, 1 /DO, 5). As partículas analisadas em C por MEV terão também seu tamanho médio avaliado pelo analisador Coulter LS230. Particle size and distribution analysis of b obtained by centrifugation and lyophilization will be performed using the distribution analyzer. particle size Coulter LS230. Small amounts of particles will be dispersed in PA absolute ethanol with the aid of ultrasound until they reach the obscuration index required by the device. The average particle size will be expressed as the average volume diameter (D4,3). The particle diameter will be calculated at D 0 , 9, D 0 , ie D 0 , 5, which means the particle diameter corresponding to 90, 50 and 10% of the cumulative distribution and the polydispersion will be given by the span index, which will be calculated by (OD, 9 - OD, 1 / OD, 5). The particles analyzed in C by SEM will also have their average size evaluated by the Coulter LS230 analyzer.
E) ANÁLISE DE CARGA SUPERFICIAL (POTENCIAL ZETA) E) SURFACE LOAD ANALYSIS (POTENTIAL ZETA)
As partículas de PLGA serão dispersas em água com pH corrigido para 7,0 e as medidas de potencial zeta serão determinadas em um aparelho Zetamaster (Malvern Instruments). A dispersão das micropartículas para as medidas de potencial zeta será preparada em duplicata e cada amostra será medida 0 vezes.  PLGA particles will be dispersed in water with pH corrected to 7.0 and zeta potential measurements will be determined on a Zetamaster apparatus (Malvern Instruments). Microparticle dispersion for potential zeta measurements will be prepared in duplicate and each sample will be measured 0 times.
ESTUDOS IN VITRO IN VITRO STUDIES
A) AVALIAÇÃO DA ATIVIDADE ANTIAMASTIGOTA INTRACELULAR  A) EVALUATION OF INTRACELLULAR ANTI-MASTASTIC ACTIVITY
Macrófagos serão isolados do peritôneo de camundongos BALB/c, por aderência em placas de cultura de 24 poços, infectados com promastigotas de L amazonensis GFP e tratados com as formulações por 72h. Após o tempo de incubação, as células serão raspadas e a carga parasitária será avaliada por fluorimetria em fluorímetro de placa (excitação 485 nm, emissão 528 nm) (ROSSI-BERGMANN, et al., 1999) (FLx800, Bio- Tek Instruments, Inc) para L amazonensis ou contagem em microscópica.  Macrophages will be isolated from the peritoneal of BALB / c mice by adherence to 24-well culture plates infected with L amazonensis GFP promastigotes and treated with the formulations for 72h. After the incubation time, the cells will be scraped off and the parasitic load will be assessed by fluorimeter on a plate fluorimeter (485 nm excitation, 528 nm emission) (ROSSI-BERGMANN, et al., 1999) (FLx800, Bio-Tek Instruments, Inc) for L amazonensis or microscopic counting.
B) EFEITO CITOTÓXICO OU ADJUVANTE SOBRE OS ACRÓFOGOS  B) CYTOTOXIC OR ADJUVANT EFFECT ON ACROPHOGES
Macrófagos serão isolados do peritôneo de camundongos BALB/c, por aderência em placas de cultura de 24 poços, partículas de PLGA vazias ou contendo os fármacos serão adicionadas e após 72 horas o sobrenadante das culturas será avaliado quanto à produção de NO (Oxido Nítrico) pela metodologia de Griss e para avaliação da atividade microbicida e a presença da enzima lactato desidrogenase (LDH), para avaliação de toxicidade por necrose, pela dosagem do lactato (Kit Dolles). Macrophages will be isolated from the BALB / c mouse peritoneum by adherence to 24-well culture plates, empty or drug-containing PLGA particles will be added and after 72 hours the Crop supernatant will be evaluated for NO (Nitric Oxide) production by Griss methodology and for microbicidal activity evaluation and the presence of lactate dehydrogenase (LDH) enzyme for evaluation of necrosis toxicity by lactate dosage (Kit Dolles) .
C) HlSTOPATOLOGIA DA PELE  C) SKIN HATTOPATHOLOGY
Para avaliar o efeito inflamatório intralesional provocado pelas formulações e ao mesmo tempo avaliar a degradação das partículas de PLGA in situ, os animais serão sacrificados em diferentes tempos após a administração. As orelhas serão fixadas e serão feitos cortes histológicos das patas infectadas e tratadas para análise histopatológica por microscopia ótica. D) AVALIAÇÃO DE IMUNOTOXICIDADE CUTÂNEA DAS MICROFORMULAÇÕES DE EM CAMUNDONGOS HAIRLESS  To evaluate the intralesional inflammatory effect caused by the formulations and at the same time to evaluate the degradation of PLGA particles in situ, the animals will be sacrificed at different times after administration. The ears will be fixed and histological sections of the infected paws will be made and treated for histopathological analysis by light microscopy. D) CUTANEOUS IMMUNOTOXICITY ASSESSMENT OF HAIRLESS MICE MICROFORMULATIONS
O teste de medida de edema de orelha (mouse ear swelling test - MEST). É recomendado pela OECD e ANVISA e será realizado para avaliação de imunotoxicidade cutânea (alergenicidade),  The ear swelling test (MEST) test. It is recommended by the OECD and ANVISA and will be performed for skin immunotoxicity assessment (allergenicity),
- Teste de medida de edema de orelha (mouse ear swelling test - MEST) - Ear swelling test (MEST)
O MEST avalia o aumento da espessura da orelha como sinal de sensibilização cutânea. Os animais receberão 3 aplicações tópicas de 100ul das formulações a cada 2 dias (dias 0, 2 e 4) na região dorsal (fase de indução). Após 5 dias, será feita a aplicação tópica de 10ul das substancias em uma orelha e na outra 10ul da formulação será aplicada intradermicamente (fase de desafio). Esse é o tempo necessário para que o organismo produza a resposta imunológica. As medidas do inchaço das orelhas serão avaliadas nos tempos 6, 18, 36, 48 e 72 horas depois do desafio. A sensibilização cutânea é positiva quando há um aumento da espessura da orelha acima de 10% em relação ao grupo controle. O oxazolone (0,3%) é a substância mais utilizada como controle positivo nestes testes de sensibilização cutânea e, segundo a literatura, promove um aumento de 30% da espessura da orelha em relação à medida antes do desafio. E) AVALIAÇÃO DE PARÂMETROS FARMACOCIN ÉTICOS MEST evaluates the increase in ear thickness as a sign of skin sensitization. Animals will receive 3 topical applications of 100 µl of the formulations every 2 days (days 0, 2 and 4) in the dorsal region (induction phase). After 5 days, 10ul of the substances will be topically applied to one ear and to the other 10ul of the formulation will be applied intradermally (challenge phase). This is the time required for the body to produce the immune response. Ear swelling measurements will be evaluated at 6, 18, 36, 48, and 72 hours after challenge. Skin sensitization is positive when there is an increase in ear thickness above 10% compared to the control group. Oxazolone (0.3%) is the most used substance as a positive control in these skin sensitization tests and, according to the literature, promotes a 30% increase in ear thickness in relation to the measurement before the challenge. E) EVALUATION OF PHARMACOCIN ETHICAL PARAMETERS
Os animais serão tratados pela via intralesional, o sangue será coletado e o plasma obtido por centrifugação será pré-tratado seguindo a metodologia proposta por (WANG & MORRIS, 2005; PAGANGA & RICE- EVANS, 1997 e-MANACH et al., 1996) baixando o pH para 4 utilizando H3PO4; acidificando com Acetonitrila e centrifugando. A acidificação das amostras é importante para quebrar possíveis ligações com proteínas plasmáticas. Após este procedimento as amostras de plasma serão analisadas utilizando UV- CLAE para a anfotericina B e absorção atómica para os antimoniais. Os parâmetros farmacocinéticos (Cmax; ASC0--; t½ ; Vd; e Cl) serão obtidos com auxilio de um software (ADAPT: Pharmacokinetic/p armacodynamic systems analysis) produzido pela University of Southern Califórnia, disponível gratuitamente para download. Alternativamente, para determinação da quantidade de droga liberada localmente, as orelhas tratadas serão removidas após diferentes tempos, e maceradas em acetonitrila. As suspensões serão clarificadas por centrifugação e processadas como descrito para o plasma. The animals will be treated intralesionally, blood will be collected and plasma obtained by centrifugation will be pretreated following the methodology proposed by (WANG & MORRIS, 2005; PAGANGA & RICE-EVANS, 1997 and-MANACH et al., 1996). lowering the pH to 4 using H 3 PO 4 ; acidifying with acetonitrile and centrifuging. Acidification of samples is important to break possible binding to plasma proteins. After this procedure plasma samples will be analyzed using UV-HPLC for amphotericin B and atomic absorption for antimonials. Pharmacokinetic parameters (Cmax; ASC 0- -; t½; Vd; and Cl) will be obtained using software (ADAPT: Pharmacokinetic / p armacodynamic systems analysis) produced by the University of Southern California, available for free download. Alternatively, to determine the amount of locally released drug, the treated ears will be removed after different times and macerated in acetonitrile. The suspensions will be clarified by centrifugation and processed as described for plasma.
DESCRIÇÃO DAS FIGURAS A Figura 1 mostra o resultado da análise de tamanho das partículas de PLGA contendo o Glucantime (antimoniato de meglumina) ou Anfotericina B, onde a figura 1A representa as micropartículas vazias, 1 B as micropartículas contendo Glucantime e 1 C as micropartículas contendo Anfotericina B.  DESCRIPTION OF THE DRAWINGS Figure 1 shows the result of particle size analysis of PLGA particles containing Glucantime (meglumine antimoniate) or Amphotericin B, where Figure 1A represents the empty microparticles, 1 B the microparticles containing Glucantime and 1 C the microparticles containing Amphotericin B.
A figura 2 mostra a análise da morfologia das partículas de PLGA avaliada por MEV e as imagens obtidas por microscopia eletrônica de varredura (MEV) das partículas de PLGA vazias (A), Glucantime (B), com Anfotericina B (C). Aumentos de 1500X, 3500X e 5000X, respectivamente.  Figure 2 shows the analysis of PLGA particle morphology assessed by SEM and the images obtained by scanning electron microscopy (SEM) of empty PLGA particles (A), Glucantime (B), with Amphotericin B (C). Increases by 1500X, 3500X and 5000X, respectively.
A figura 3A mostra o ensaio in vitro da incorporação de Anfotericina B em micropartículas de PLGA, onde as micropartículas de PLGA/Anfotericina B (1 mg/ml) foram dissolvidas previamente em DMSO, em seguida, a amostra foi diluída em uma solução aquosa de Ácido Fosfórico (0,001 %) e Acetonitrila (60/40) nas concentrações A=50ug/ml; B=100ug/ml e C=200ug/ml. A Incorporação teórica de Anfotericina B nas micropartículas é de 10% (p/p). As amostras foram feitas em triplicatas. Figure 3A shows the in vitro assay of incorporation of Amphotericin B into PLGA microparticles, where the microparticles of PLGA / Amphotericin B (1 mg / ml) were previously dissolved in DMSO, then the sample was diluted with an aqueous solution of Phosphoric Acid (0.001%) and Acetonitrile (60/40) at concentrations A = 50ug / ml; B = 100ug / ml and C = 200ug / ml. The theoretical incorporation of amphotericin B into microparticles is 10% (w / w). Samples were made in triplicate.
A figura 3B mostra o ensaio in vitro da liberação de Anfotericina B, onde as micropartículas de PLGA/Anfotericina B, (6mg/ml contendo 535 g de Anfotericina B /ml), foram incubadas em solução de PBS 0, 1 % de Azida, pH=7.2 e mantido a 37°C em agitação constante de 80rpm. Em diferentes tempos as amostras foram centrifugadas a 1500rpm por 10 minutos e aiíquotas foram retiradas do sobrenadante para dosagem em HPLC da Anfotericina B liberada. O teor de Anfotericina B nas partículas é de 8,92% (Gráfico 6.2.3).  Figure 3B shows the in vitro Amphotericin B release assay where PLGA / Amphotericin B microparticles (6mg / ml containing 535g Amphotericin B / ml) were incubated in 0.1% PBS Azide solution, pH = 7.2 and kept at 37 ° C under constant agitation of 80rpm. At different times the samples were centrifuged at 1500rpm for 10 minutes and aliquots were taken from the released amphotericin B HPLC supernatant. Amphotericin B content in the particles is 8.92% (Graph 6.2.3).
A figura 4 mostra a análise da erosão das micropartículas de PLGA contendo anfotericina B, através das imagens obtidas por microscopia eletrônica de varredura (MEV). As imagens A e B no tempo zero, as imagens C e D com tempo igual a 30 dias em tampão PBS e, as imagens E e F com tempo igual a 120 dias em tampão PBS.  Figure 4 shows the erosion analysis of amphotericin B-containing PLGA microparticles by scanning electron microscopy (SEM) images. Images A and B at time zero, images C and D with time equal to 30 days in PBS buffer and images E and F with time equal to 120 days in PBS buffer.
A figura 5 mostra a eficácia do encapsulamento de Anfotericina B em micropartículas de PLGA administrado em dose única no tratamento de leishmaniose cutânea murina por via intralesional, onde camundongos BALB/c foram infectados com 2x106 promastigotas de L.amazonensis GFP na orelha. Após 10 dias de infecção, os animais foram aleatoriamente separados em grupos de 4. O grupo tratado com Anfotericina B livre, recebeu injeções intralesionais duas vezes por semana com doses de 50 ug do fármaco por 4 semanas (total de 8 doses). O grupo tratado com PLGA/Anfotericina B recebeu uma única dose da formulação intralesionalmente com dose 50 ug de Anfotericina B (1 dose). Os controles receberam uma única administração de PLGA de 500ug (1 doses), ou 10ul de PBS. (A) O crescimento lesão (espessura da orelha infectada menos espessura antes da infecção) foi acompanhado durante o experimento com o auxílio de paquímetro. (B) Carga parasitária medida por unidades de fluorescência específica, obtida 42 dias após a infecção. Fluorescência basal = 76728,5 ±1472. (C) Fotografia das orelhas infectadas, o halo vermelho indica o local de infecção. Média ±SD (n=4), ** p< 0,01 e *** p< 0,001 . Figure 5 shows the efficacy of amphotericin B encapsulation in single dose PLGA microparticles in the treatment of intralesional murine cutaneous leishmaniasis, where BALB / c mice were infected with 2x10 6 L.amazonensis GFP promastigotes in the ear. After 10 days of infection, the animals were randomly separated into groups of 4. The free amphotericin B treated group received intralesional injections twice a week with 50 µg doses of the drug for 4 weeks (total of 8 doses). The PLGA / Amphotericin B treated group received a single dose of the formulation intralesionally with 50 µg Amphotericin B dose (1 dose). Controls received a single administration of 500ug (1 dose) PLGA, or 10ul of PBS. (A) The lesion growth (infected ear thickness minus thickness before infection) was accompanied during the experiment with the aid of caliper. (B) Parasitic loading measured by specific fluorescence units obtained 42 days after infection. Basal fluorescence = 76728.5 ± 1472. (C) Photograph of the infected ears, the red halo indicates the site of infection. Mean ± SD (n = 4), ** p <0.01 and *** p <0.001.
A figura 6 mostra o tratamento de leishmaniose cutânea murina por via intralesional, onde camundongos BALB/c foram infectados com 2x 06 promastigotas de L.amazonensis GFP na orelha. Após 7 dias de infecção, os animais foram aleatoriamente separados em grupos de 4. No tratamento 1 o grupo tratado com PLGA/Anfotericina B recebeu uma dose da formulação intralesionalmente com dose 1 ,25 ug de Anfotericina B. O grupo tratado com Anfotericina B livre e Anfotericina B Lipossomal, recebeu uma dose 1 ,25 ug do fármaco. Os controles receberam uma única administração de PLGA de 12,5 ug ou 10ul de PBS. No tratamento 2 o grupo tratado com PLGA/Anfotericina B recebeu uma dose da formulação intralesionalmente com dose 50 ug de Anfotericina B. O grupo tratado com Anfotericina B livre e Anfotericina B Lipossomal, recebeu uma dose 50 ug do fármaco. Os controles receberam uma única administração de PLGA de 500ug ou 10ul de PBS. O crescimento lesão (espessura da orelha infectada menos espessura antes da infecção) foi acompanhado durante o experimento com o auxílio de paquímetro. Figure 6 shows intralesional murine cutaneous leishmaniasis treatment, where BALB / c mice were infected with 2x0 6 L.amazonensis GFP promastigotes in the ear. After 7 days of infection, the animals were randomly separated into groups of 4. In treatment 1 the PLGA / amphotericin B treated group received a dose of the formulation intralesionally with a dose of 25 µg amphotericin B. The free amphotericin B treated group and Liposomal amphotericin B, received a dose of 1.25 µg of the drug. Controls received a single administration of PLGA of 12.5 µg or 10ul of PBS. In treatment 2 the PLGA / amphotericin B treated group received a dose of the formulation intralesionally with 50 µg amphotericin B dose. The free amphotericin B and liposomal amphotericin B treated group received a 50 µg dose of the drug. Controls received a single administration of 500ug or 10ul of PBS PLGA. The lesion growth (thickness of infected ear minus thickness before infection) was followed during the experiment with the aid of caliper.
A figura 07 mostra a avaliação da carga parasitária na lesão após o tratamento intralesional com as micropartículas de PLGA contendo Anfotericina B: O tratamento de leishmaniose cutânea murina por via intralesional com micropartículas de PLGA com Anfotericina B diminui a carga parasitária na lesão de animais infectados com L. amazonensis. As cargas de parasita no lisado da lesão foram medidas no dia 67 de infecção. O número de parasitas na lesão (A) e no linfonodo auricular (B) foi determinado pelo método de diluição limitante (LDA), a carga parasitária em (C) foi medida em unidades de fluorescência específica (FU). « p< 0,01 em relação ao grupo PBS. T p< 0,001 em relação ao grupo PLGA. · p< 0,05 em relação ao grupo PLGA. Média ± DP (n = 4). Figure 07 shows the evaluation of the parasitic burden on the lesion after intralesional treatment with amphotericin B-containing PLGA microparticles: Treatment of intralesional murine cutaneous leishmaniasis with amphotericin B-PLGA microparticles decreases the parasitic burden on lesion of animals infected with amphotericin B. L. amazonensis. Parasite loads in the lesion lysate were measured on day 67 of infection. The number of parasites in the lesion (A) and atrial lymph node (B) was determined by the limiting dilution method (LDA), the parasitic load in (C) was measured in specific fluorescence units (FU). 'P <0.01 relative to the PBS group. T p 0.001 in relation to the PLGA group. · P <0.05 in relation to the PLGA group. Mean ± SD (n = 4).
A figura 08 mostra a avaliação de toxidez cardíaca, hepática e renal. O sangue dos animais foi retirado 67 dias após a infecção para dosagens de: (a) níveis séricos de TGO; (b) níveis séricos de TGP; (c) níveis séricos de creatinina no soro. Controle negativo: PBS. Controle positivo: CCI4. Figure 08 shows the assessment of cardiac, hepatic and renal toxicity. Animal blood was drawn 67 days after infection for dosages of: (a) serum TGO levels; (b) serum TGP levels; (c) serum creatinine serum levels. Negative Control: PBS. Positive control: ICC 4 .
A figura 9 mostra a determinação da melhor proporção de droga na formulação. Foram preparadas micropartículas de PLGA contendo Anfotericina B adicionada em diferentes proporções em relação ao polímero (5%, 10% e 20%, como indicado). As partículas foram então lavadas e secadas. Para determinação do melhor rendimento do processo, as partículas secas foram totalmente dissolvidas em Acetonitrila após agitação por 24 horas a 100 rpm. As amostras foram então centrifugadas a 10.000 rpm por 5 minutos e o sobrenadante foi retirado para quantificação do teor da droga incorporada por CLAE.  Figure 9 shows the determination of the best drug ratio in the formulation. PLGA microparticles containing Amphotericin B added in different proportions to the polymer (5%, 10% and 20% as indicated) were prepared. The particles were then washed and dried. To determine the best process yield, the dried particles were fully dissolved in Acetonitrile after stirring for 24 hours at 100 rpm. The samples were then centrifuged at 10,000 rpm for 5 minutes and the supernatant was removed for quantification of the HPLC incorporated drug content.
Tabela 1 : O tamanho médio de partícula expresso como diâmetro médio em volume (D 4,3).  Table 1: The average particle size expressed as volume average diameter (D 4.3).
Distribuição do diâmetro das partículas (μηι) Particle Diameter Distribution (μηι)
Figure imgf000019_0001
Figure imgf000019_0001
Tabela 2: A carga superficial das partículas foi avaliada pelo Potencial Zeta. Especificação Potencial Zeta SD Table 2: The surface charge of the particles was evaluated by the Zeta Potential. Zeta SD Potential Specification
PLGA vazia -1 1 ,0 2,6 Empty PLGA -1 1, 0 2.6
PLGA / GLUCANTIME -13,2 4,5PLGA / GLUCANTIME -13.2 4.5
PLGA / ANFOTERICINA B -10,6 2,3 PLGA / ANFOTERICIN B -10.6 2.3
EXEMPLOS EXAMPLES
EXEMPLO 1 : CARACTERIZAÇÃO FÍSICA DAS PARTÍCULAS DE PLGA CONTENDOEXAMPLE 1: PHYSICAL CHARACTERIZATION OF PLGA PARTICLES CONTAINING
GLUCANTIME OU ANFOTERICINA B ANÁLISE DE TAMANHO E DISTRIBUIÇÃO GLUCANTIME OR ANFOTERYCIN B SIZE AND DISTRIBUTION ANALYSIS
A análise da distribuição granulométrica (polidispersão) nas partículas vazias e nas partículas com Glucantime encapsulada, gerando as distribuições conforme figura B. O tamanho médio de partícula foi expresso com o diâmetro médio em volume (D 4,3). As diferentes % de distribuição acumulada estão indicadas na tabela 1. Ao analisarmos a distribuição granulométrica das partículas com Glucantime, podemos observar uma população mais heterogénea, com span de 1 ,69um para partículas de Glucantime em PLGA. A carga superficial das partículas foi caracterizada, avaliadas pelo Potencial Zeta. Os valores são mostrados na tabela 2.  Analysis of particle size distribution (polydispersion) in the empty particles and in the particles with encapsulated Glucantime, generating the distributions as shown in Figure B. The average particle size was expressed as the volume average diameter (D 4.3). The different% of cumulative distribution are shown in Table 1. When analyzing the particle size distribution with Glucantime, we can see a more heterogeneous population with a span of 1.69um for Glucantime particles in PLGA. The surface charge of the particles was characterized, evaluated by the Zeta Potential. The values are shown in table 2.
A análise da distribuição granulométrica (polidispersão) nas partículas vazias e nas partículas com Anfotericina B encapsulada, gerando as distribuições conforme figura 1C. O tamanho médio de partícula foi expresso com o diâmetro médio em volume (D 4,3). As diferentes % de distribuição acumulada estão indicadas na tabela 1. Ao analisarmos a distribuição granulométrica das partículas com Anfotericina B, podemos observar uma população mais heterogénea, com span de 1 ,75 um para partículas de Anfotericina B em PLGA. A carga superficial das partículas foi caracterizada, avaliadas pelo Potencial Zeta. Os valores são mostrados na tabela 2.  The analysis of particle size distribution (polydispersion) in the empty particles and in the particles with encapsulated amphotericin B, generating the distributions according to figure 1C. The average particle size was expressed as the mean volume diameter (D 4.3). The different% accumulated distribution are shown in Table 1. When analyzing the particle size distribution of amphotericin B particles, we can see a more heterogeneous population with a span of 1.75 µm for amphotericin B particles in PLGA. The surface charge of the particles was characterized, evaluated by the Zeta Potential. The values are shown in table 2.
ANÁLISE MORFOLÓGICA DAS PARTÍCULAS MORPHOLOGICAL ANALYSIS OF PARTICULES
A morfologia das partículas também foi avaliada por MEV e as imagens estão representadas na Figura 2. Pela análise morfológica das partículas, podemos observar a formação de aglomerados que podem explicar a heterogeneicidade observada na distribuição de tamanho da população, quando o Glucantime ou a Anfotericina B estão encapsulados. The particle morphology was also evaluated by SEM and the images are represented in Figure 2. By the morphological analysis of the In particles, we can observe the formation of clusters that may explain the observed heterogeneity in population size distribution when Glucantime or Amphotericin B are encapsulated.
EXEMPLO 2: AVALIAÇÃO DA EFICÁCIA DE ENCAPSULACÃO, LIBERAÇÃO E DEGRADAÇÃO DAS PARTÍCULAS IN VITRO EXAMPLE 2: EVALUATION OF EFFECTIVENESS OF IN VITRO PARTICULATION, RELEASE AND DEGRADATION
A Taxa de incorporação de Anfotericina B nas micropartículas de PLGA pode ser observada na figura 3A. Pela análise do teor de anfotericina B das partículas quando estas foram previamente dissolvidas em DMSO e analisada em HPLC. Podemos observar que a Média da taxa de incorporação foi de 89,2%, com esse rendimento o teor de anfotericina B na partícula é de 8,92% e não os 10% (p/p) de Anfotericina B em micropartículas de PLGA teórico. Amphotericin B uptake rate in PLGA microparticles can be seen in Figure 3A. By analyzing the amphotericin B content of the particles when they were previously dissolved in DMSO and analyzed on HPLC. We can observe that the Average incorporation rate was 89.2%, with this yield the particle amphotericin B content is 8.92% and not the 10% (w / w) Amphotericin B in theoretical PLGA microparticles .
No ensaio de liberação da anfotericina B das micropartículas de PLGA in vitro, as partículas foram incubadas em solução de PBS 0, 1 % de Azida, pH=7.2 e mantido a 37°C em agitação constante de 80rpm, em diferentes tempos as amostras foram centrifugadas e alíquotas foram retiradas do sobrenadante para dosagem da anfotericina B por cromatografia de alta eficiência (CLAE). Com esta análise podemos observar que aproximadamente 60% da anfotericina B que estava incorporada às micropartículas foi liberada para o sobrenadante nas condições analisadas, como podemos observar na Figura 3B. Análise da erosão das micropartículas.  In the amphotericin B release assay of PLGA microparticles in vitro, the particles were incubated in 0.1% PBS solution of Azide, pH = 7.2 and kept at 37 ° C under constant agitation at 80rpm, at different times the samples were incubated. Centrifugations and aliquots were removed from the supernatant for amphotericin B dosing by high performance chromatography (HPLC). With this analysis we can observe that approximately 60% of the amphotericin B that was incorporated into the microparticles was released to the supernatant under the analyzed conditions, as we can see in Figure 3B. Microparticle erosion analysis.
A análise da degradação e morfologia das micropartículas de PLGA contendo anfotericina B em tampão PBS foi avaliada por microscopia eletrônica de varredura (MEV). As micropartículas de PLGA foram obtidas em tempos variados, liofilizadas e tratadas para microscopia eletrônica. A incorporação da anfotericina B nas micropartículas de PLGA não promove mudanças significativas no tamanho, formato ou carga das partículas como observamos nos resultados anteriores (exemplo 1). No estudo da degradação as partículas vazias e contendo a anfotericina B não apresentaram diferenças morfológicas e nem mudanças na erosão da partícula (dados não mostrados). Como podemos observar na Figura 4 (A e B) as micropartículas contendo anfotericina B no tempo zero apresentavam um formato esférico de tamanhos bastante variados, na Figura 4 (C e D) observamos a morfologia no tempo de 30 dias em solução tampão, onde podemos observar a presença de estruturas quadriculadas em um número bastante significativo, na Figura 4 (E e F) observamos a morfologia no tempo igual a 120 dias em solução tampão, podemos observar uma predominância de estruturas quadriculadas oriundas da degradação das micropartículas de PLGA. Neste ponto, aproximadamente 60% da anfotericina B já foi liberada (exemplo 1). The degradation and morphology analysis of PLGA microparticles containing amphotericin B in PBS buffer was evaluated by scanning electron microscopy (SEM). PLGA microparticles were obtained at various times, lyophilized and treated for electron microscopy. Incorporation of amphotericin B into PLGA microparticles does not promote significant changes in particle size, shape or charge as observed in previous results (Example 1). In the study of degradation the empty particles containing amphotericin B showed no morphological differences or changes in particle erosion (data not shown). As we can see in Figure 4 (A and B) the microparticles containing amphotericin B at time zero had a spherical shape of widely varying sizes, in Figure 4 (C and D) we observed the morphology at 30 days in buffer solution, where we can observe the presence of squared structures in a very significant number, In Figure 4 (E and F) we observe the morphology in time equal to 120 days in buffer solution, we can observe a predominance of squared structures derived from degradation of PLGA microparticles. At this point, approximately 60% of amphotericin B has already been released (example 1).
EXEMPLO 3: AVALIAÇÃO DA EFICÁCIA DAS COMPOSIÇÕES NO TRATAMENTO DA LEISHMANIOSE CUTÂNEA  EXAMPLE 3: EVALUATION OF THE EFFICACY OF COMPOSITIONS IN THE TREATMENT OF SKIN LEISHMANIASIS
Na avaliação da atividade in vivo das micropartículas de PLGA contendo Anfotericina B. As micropartículas de PLGA com anfotericina B promoveram diminuição no crescimento da lesão na dose única de 50 pg/Kg como podemos observar na Figura 5 (A), o grupo tratado com Anfotericina B livre, recebeu injeções intralesionais duas vezes por semana com doses de 50 ug do fármaco por 4 semanas (total de 8 doses). Com a avaliação da carga parasitária (B) podemos concluir que a Anfotericina B/PLGA promoveu uma diminuição significativa da fluorescência comparando com o grupo tratado com placebo, apesar do tamanho da lesão ter diminuído não houve diferença estatística entre o grupo tratado com as micropartículas vazias e o grupo tratado com placebo quando comparamos a carga parasitária. Podemos observar em C, que as lesões das orelhas dos camundongos tratados estão diminuídas e menos inflamadas que as dos não tratados. A Anfotericina B encapsulada administrada em uma única dose promoveu uma diminuição do tamanho da lesão e na carga parasitária bastante significativa. Na avaliação de atividade in vivo com o tratamento pela via tópica, o tratamento tópico com os LCs contendo 6,6 μg CH8 apresentou a mesma atividade que o controle positivo intralesional, que recebeu 3,3 μg da CH8 livre (Figura 4). O mesmo efeito é obtido com a avaliação da carga parasitária ao final do experimento (Figura 5). Este resultado demonstra a capacidade da formulação em lipossomas convencionais (LCs) de penetrar a pele e veicular a chalcona nitrada(CH8) à região infectada, possibilitando assim, o tratamento por uma via não invasiva. O grupo tratado com a formulação de CH8 em lipossomas convencionais apresentou maior eficácia que o grupo tratado com uma dose bem mais alta de 200 ug de CH8 livre em creme lanete. Os demais grupos que foram tratados com os veículos vazios (lipossomas convencionais LCs, lipossomas peguilados LPs e creme lanete) apresentaram perfil semelhante ao grupo não tratado, indicando atividade específica da chalcona nitrada(CH8). In the evaluation of the in vivo activity of amphotericin B-containing PLGA microparticles The amphotericin B-PLGA microparticles reduced the lesion growth at a single dose of 50 pg / kg as shown in Figure 5 (A), the Amphotericin-treated group. B free, received intralesional injections twice a week with doses of 50 µg of the drug for 4 weeks (total of 8 doses). With the evaluation of the parasitic load (B) we can conclude that amphotericin B / PLGA promoted a significant decrease in fluorescence compared to the placebo group, although the size of the lesion decreased there was no statistical difference between the group treated with empty microparticles. and the placebo group when we compared the parasitic load. We can see in C that the damage to the ears of treated mice is diminished and less inflamed than untreated mice. Single-dose encapsulated amphotericin B promoted a significant reduction in lesion size and parasite burden. In the in vivo activity evaluation with topical treatment, topical treatment with LCs containing 6.6 μg CH8 showed the same activity as the intralesional positive control, which received 3.3 μg of free CH8 (Figure 4). The same effect is obtained with load evaluation. parasite at the end of the experiment (Figure 5). This result demonstrates the ability of the conventional liposome formulation (LCs) to penetrate the skin and deliver nitrated chalcone (CH8) to the infected region, thus enabling treatment by a non-invasive route. The group treated with the conventional liposome CH8 formulation was more effective than the group treated with a much higher dose of 200 µg of free cream lanete CH8. The other groups that were treated with the empty vehicles (conventional LCs liposomes, LPS pegylated liposomes and lanete cream) presented similar profile to the untreated group, indicating specific activity of chalrada nitrada (CH8).
O uso da formulação de chalcona nitrada(CH8) encapsulada em lipossomas convencionais é a mais vantajosa, pois apresenta o mesmo desempenho que os lipossomas peguilados, sem necessidade de incluir a etapa adicional de incorporação de PEG, na avaliação de retenção cutânea em células de Franz. E sua eficácia foi maior que o peguilado no tratamento do animal pela via tópica, apresentando o mesmo controle da infecção que o grupo tratado com chalcona nitrada(CH8) intralesional, com a vantagem de ser esta uma via não invasiva. Estes resultados podem ser verificados nas Figuras 3 e 4.  The use of conventional liposome encapsulated nitrated chalcone (CH8) formulation is the most advantageous as it performs the same as pegylated liposomes without the need to include the additional PEG incorporation step in the evaluation of skin retention in Franz cells . And its efficacy was greater than the pegylated treatment of the animal by the topical route, presenting the same infection control as the intralesional chalceda nitrated (CH8) group, with the advantage of being a noninvasive route. These results can be seen in Figures 3 and 4.

Claims

REIVINDICAÇÕES
1 - Composições farmacêuticas caracterizadas por conter uma quantidade farmaceuticamente efetiva de medicamentos ou fármacos antileishmaniais encapsulados em partículas de PLGA. Pharmaceutical compositions characterized in that they contain a pharmaceutically effective amount of PLGA particle-encapsulated antileishmanial drugs or drugs.
2- Composições farmacêuticas de acordo com a reivindicação Pharmaceutical compositions according to claim
1 , caracterizadas por serem os medicamentos ou fármacos a Anfotericina B, compostos antimoniais, tais como antimoniato de meglumina, chalcona nitrada (CH8), estibogluconato de sódio e tartarato de potássio e antimonio (III) hidratado (Sblll), combinados ou não, encapsulados em partículas de PLGA, além de excipientes farmaceuticamente aceitáveis. 1, characterized in that the drugs or drugs are amphotericin B, antimonial compounds such as meglumine antimoniate, chalrada nitrada (CH8), sodium stibogluconate and hydrated potassium antimony (Sblll), combined or not, encapsulated in PLGA particles in addition to pharmaceutically acceptable excipients.
3 - Composições de acordo com as reivindicações 1 e 2, caracterizado pelo fato da quantidade do medicamento ou fármaco constituir-se de cerca de 1 a 70% do peso da formulação.  Compositions according to claims 1 and 2, characterized in that the amount of the medicament or drug is about 1 to 70% of the weight of the formulation.
4 - Composições de acordo com as reivindicações 1 e 2, caracterizado pelo fato da porcentagem ótima do encapsulamento do medicamento ou fármaco ser de 10% em relação ao polímero.  Compositions according to claims 1 and 2, characterized in that the optimum percentage of encapsulation of the medicament or drug is 10% relative to the polymer.
5 - Composições de acordo com as reivindicações 1 e 2, caracterizado pelo fato das partículas de PLGA apresentarem tamanhos variando entre 0,5 e 500μηη.  Compositions according to claims 1 and 2, characterized in that the particles of PLGA have sizes ranging from 0.5 to 500μηη.
6- Composições de acordo com a reivindicação 1 , caracterizado por uma variação copolímeros de ácido glicólico e lático serem usados e o percentual de cada monômero de ácido poli (lático-co-glicólico) ser, cerca de 15-85%, cerca de 25-75%, cerca de 35-65%, ou a proporção de 50- 50%.  Compositions according to claim 1, characterized in that a variation of glycolic and lactic acid copolymers is used and the percentage of each poly (lactic-co-glycolic) monomer is about 15-85%, about 25%. -75%, about 35-65%, or the proportion of 50-50%.
7- USO das composições farmacêuticas descritas nas reivindicações de n° 1 a 4, caracterizado por ser voltado para a produção de um medicamento para o tratamento de doenças cutâneas inflamatórias e infecciosas da pele ou em regiões da pele (derme, epiderme e anexos), tais como, leishmaniose, micoses, hanseníase, psoríases, ulcera tropical, verrugas, hemangioma cavernoso em animais mamíferos. Use of the pharmaceutical compositions described in claims 1 to 4, characterized in that it is directed to the manufacture of a medicament for the treatment of inflammatory and infectious skin diseases or in regions of the skin (dermis, epidermis and appendages). such such as leishmaniasis, mycoses, leprosy, psoriasis, tropical ulcer, warts, cavernous hemangioma in mammalian animals.
8- Método de tratamento de parasitoses, caracterizado por compreender a aplicação subcutânea de uma quantidade terapeuticamente efetiva da composição conforme definida nas reivindicações de n° 1 a 4, a um animal mamífero portador da leishmaniose.  A method of treating parasitic diseases, comprising subcutaneously applying a therapeutically effective amount of the composition as defined in claims 1 to 4 to a mammalian animal carrying leishmaniasis.
PCT/BR2012/000249 2011-07-18 2012-07-17 Microparticle pharmaceutical compositions containing antiparasitics for prolonged subcutaneous therapy, use of the pharmaceutical compositions for producing a medicament, and method for treating parasitoses WO2013010238A1 (en)

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