BRPI0904083A2 - process of obtaining positively charged amphiphilic vesicles, positively charged amphiphilic vesicles, their pharmaceutical and / or cosmetic compositions and their uses - Google Patents

Abstract

PROCEDURE FOR OBTAINING POSITIVELY LOADED AMPHIFYLIC VESICULES, POSITIVELY LOADED AMPHIFYLIC VESICULES, THEIR PHARMACEUTICAL AND / OR COSMETIC COMPOSITIONS, AND THEIR USES. The present invention provides a method for obtaining positively charged polymeric double-layer amphiphilic and amphiphilic vesicles containing at least two actives with distinct solubilities (water soluble and liposoluble). Additionally, the present application is intended for pharmaceutical and / or cosmetic compositions containing said amphiphilic vesicles and their applications as antineoplastic, antimicrobial and anti-inflammatory agents.

Description

"PROCEDURE FOR OBSERVATIONALLY LOADED AMPHIFYLIC VESTICLES, POSITIVE LOADED AMPHIFYLIC VESicles, PHARMACEUTICAL AND / OR COSMETIC COMPOSITIONS AND THEIR USES"

Field of the Invention

The present invention provides a method of obtaining positively charged polymeric double-layer amphiphilic and amphiphilic vesicles containing at least two actives with distinct solubilities (water soluble and liposoluble). Additionally, the present application is intended for pharmaceutical and / or cosmetic compositions containing said amphiphilic vesicles and their applications as an antineoplastic, antimicrobial and anti-inflammatory agent.

Background of the Invention

Pharmaceutical nanotechnology is the area of pharmaceutical sciences involved in the development, characterization and application of therapeutic systems on a nanometer or micrometer scale. Studies of such systems have been actively carried out worldwide with the purpose of directing and controlling drug release (SAKATA, S.; UCHIDA, K.; KAETSU, I.; KITA, Y. Programming control of intelligent drugreleases in response to single and binaiy environmental stimulationsignals using sensor and electroresponsive hydrogel (Radiat. Phys. Chem., Amsterdam, v.76, p.733-737, 2007).

The application of nanotechnology for the treatment, diagnosis, monitoring and control of biological systems has recently been called "Nanomedicine" by the National Institute of Health in the United States (MOGHIMI, SM; HUNTER, AC; MURRAY, JC Nanomedicine: current status and future prospects. The FASEB J., v.19, p.311-330,2005). This technology emerged in the 1960s with the initial development of microencapsulation, the technique of transforming liquids (polymers and other substances) into micron particle size powders. Microencapsulation is widely used in the food, textile, pharmaceutical and cosmetic industries because it allows the protection of labile and volatile substances, the control of drug release, contributing to the improvement of bioavailability and reduction of dosing and toxicity. Microencapsulation served as a model for more sophisticated techniques, now in the nanometer scale, allowing the development of nanoparticles.

The discovery of liposomes in the 1960s increased the range of tools for the development of pharmaceutical drug technology with lipid drug vectoring systems (LASIC, DD Novel application of liposomes. Trends Biotechnol., Amsterdam, v.16, p.307-321, 1998). SurgicalNeurol., Amsterdam, v.61, p.216-220, 2004 are currently being developed, such as liposomes and nanoparticles, and microsystems such as microparticles, multiple emulsions and microemulsions (SILVA, GA.Introduction to nanotechnology and its applications to medicine. ).

Liposomes are aqueous vesicles surrounded by bicamadalipid and may serve as a vehicle for drugs to be encapsulated in the aqueous vesicle cavity or lipid bilayer (LASIC, DDNovel application of liposomes. Trends Biotechnol, Amsterdam, v. 16, p.307-321, 1998) . Nanoparticles are polymeric particles in the deservatory (capsule) or matrix (polymeric matrix) form in which the drug is encapsulated or adsorbed into the polymeric mesh (BRIGGER, I.; DUBERNET, C.; COUVREUR, P. Nanoparticles in cancer therapy and diagnosis. Adv. DrugDel Rev., Arlington, v.54, p.631-651, 2002).

In the 1990s more sophisticated nanosystems emerged coated with hydrophilic polymers, called stealth systems, which allow a longer circulation time in the body (GREF, R.; MINAMITAKE, Y .; PERACCHIA, Μ.T.; TRUBETSKOY, V .; TORCHILIN, V. LANGER, R. Biodegradable long-circulating polymeric nanospheres (Science, New York, v.263, pp. 1600-1603, 1994). In addition to stealth systems, systems containing so-called lithium-specific surface signaling molecules have been developed for the purpose of targeted drug targeting (BARRAT, GM Therapeuticapplications of colloidal drug carriers. Pharma. Sci. Technol. Today, Cambridge, v.5, pp.163-171, 2000).

Among the advantages that nanosystems can offer are: the protection of the drug in the therapeutic system against possible instabilities in the body, promoting maintenance of plasma levels in constant concentration; increased therapeutic efficacy; the progressive and controlled release of the drug by conditioning it to stimuli of the environment (sensitive pH or temperature malfunction); the significant decrease in toxicity by reducing peak plasma concentrations; the decrease of instability and decomposition of sensitive drugs; the possibility of targeting specific targets (site-specificity); the possibility of incorporating both hydrophilic and lipophilic substances into the devices; decreased therapeutic dose and number of administrations and increased patient acceptance of therapy. Although these advantages are significant, some plausible drawbacks cannot be ignored, such as possible toxicity, lack of biocompatibility of the materials used and the high cost of obtaining nanosystems compared to conventional pharmaceutical formulations (VERMA, RK; GARG, S. Current status ofdrug Technol, v.25, n.2, pp. 1-4, 2001; DUNNE, M.; BIBBY, DC; JONES, JC; CUDMORE, S. Encapsulation of protamine sulphate compacted DNA in polylactide and polyylactide-co-glycolide microparticles J. Control Release, Amsterdam, v.92, p.209-219, 2003; TAO, SL; DESAI, TA Microfabricated drug systems: from partitions to pores. ., Arlington, v.55, p.315-328, 2003).

Liposomes, aqueous vesicles formed by phospholipid concentric bilayers are an excellent form of controlled drug deliberation system due to their structural flexibility (lipid bilayer size, composition and fluidity), as well as their ability to incorporate variety of hydrophilic compounds (ANDRADE, CA; CORREIA , SMTS; RABBIT, LCBB; BIRTH, SC; SANTOS-MAGALHÃES, NS Antitumor activity of Cratylia mollis lectinencapsulated into liposomes. Int. J. Pharm., Amsterdam, v.278, p.435-445,2004) and hydrophobic (BRIDGES) , ACO; CAETANO, MNP; SANTOS-MAGALHÃES, NS Physicochemical characterization and antimicrobialactivity of benzathine penicillin G liposomes. STP Pharma Sci., Paris, v.9, p.419-427, 1999), being the most clinically established nanometric systems for delivery of cytotoxic drugs, genes, and vaccines (DIEBOLD, Y .; JARRÍN, M .; SÁEZ, V .; OAK, ELS; OREA, M.; CALONGE, M .; SEIJO, B .; ALONSO, MJ ivery byliposome-chitosan nanoparticle complexes (LCS-NP). Biomaterials, Amsterdam, v.28, n.8, p. 1553-1564, 2006). Liposomes can be of varying sizes (20 nm to a few micrometres) with lipid layer thickness around 6 nm. Liposomes as controlled drug deliberation systems not only enable drug vectorization and protection, but also allow possible targeting to specific (site-specific) cell or organ sites (BERGSTRAND, N .; ARFVIDSSON, MC; KIM, JM; THOMPSON , DH; EDWARDS, K. Interactions between pH-sensitive liposomes and model membranes Biophys Chem, Amsterdam, v. 104, pp.361-379, 2003; SAPRA, P.; ALLEN,,. Lig Ligant-target liposomal anticancer drugs (Prog.Lipid Res., Amsterdam, v.42, p.439-462, 2003).

Infections and inflammation affecting the eye region cause extreme discomfort and anxiety to patients. Via systemic treatment generally does not allow donor therapeutic concentration to reach the ocular region. Therefore, topical administration is the treatment of choice, but its efficacy may be compromised. Conventional preparations, upon application, are rapidly removed from the eyeball due to the eye's protective mechanisms. In addition, the cornea acts as a barrier contributing to the low concentration of the drug in this organ. Thus, the drug conveyed by such preparations has low availability. This undesirable feature has led researchers to search for innovative preparations with greater therapeutic efficacy.

As for the recent strategies, the uses of nanoparticulate coloidal system as well as the use of bioadhesive property polymers have been evaluated. These strategies aim at deliberate control of the drug and increasing its residence time in the ocular region (AMRITE, AC; KOMPELLA, UB. Size-dependent disposition of nanoparticles and microparticles following subconjunctivaladministration. J. Pharm. Pharmacol v. 57, no. 12, p 1555-1563, 2005; BEJJΑΝΙΑ, R. BE .; BEHAR-COHENA, F.; BENEZRA, D.; GURNY, R.; DELIE, F. Polymeric nanoparticles for drug deletion to the later segment of theeye. 59, no. 6, pp. 344-347, 2005; BUCOLO, C.; MALTESE, A.; PUGLISI, G .; PIGNATELLO, R. Enhanced ocular anti-inflammatory activityof ibuprofen carried by an eudragit RS 100 nanoparticle suspension. Ophthalmic Research, v. 34, no. 5, pp. 319-323, 2002; BUCOLO, C.; MALTESE, A.; MAUGERI, F.; BUSÀ, B .; PUGLISI, G .; PIGNATELLO, R.Eudragit RL 100 nanoparticle system for the ophthalmic deliveiy of chloricromene J. Pharmacy Pharmacology, 56, η 7, ρ 841-846, 2004; BUCOLO, C.; MALTESE,;; PUGLISi, G .; PIGNATELLO, R. Enhancedocular anti-flam matory activity of ibuprofen caried by at Eudragit RS100® nanoparticle suspension. Ophthalmie Research, v. 34, no. 5, p.319-323, 2002; CAVALLI, R .; GASCO, M.R .; CHETONI, P .; BURGALASSI, S.; SETTONE, M.F. Solid lipid nanoparticles (SLN) as ocular deliveiy systemfor tobramycin. Int. J. Pharm., V. 238, v. 1-2, p. 241-245, 2002).

Among the different polymers employed in the preparation of nanoparticulate colloidal systems, chitosan has shown adequate characteristics for its use in ophthalmic preparations. Talsubstance is biodegradable, biocompatible and bioadhesive. This last important feature is due to its ecatonic property, which allows its electrostatic interaction with the negative charges of the mucous layer of the eyeball. In addition, chitosan exhibits antimicrobial activity, healing property, and also has avisolability (DE CAMPOS, AM; SÁNCHEZ, A; ALONSO, MJChitosan nanoparticles: a new vehicle for the improvement of the deliveiyof drugs to the ocular surface. cyclosporine A. Int. J.Pharm., v. 224, No. 1-2, pp. 150-168, 2001; DE CAMPOS, AM; SÁNCHEZ, A.; GRET, R .; CALVO, P .; ALONSO, MJ The effect of PEG versus achitosan coating on the interaction of colloidal drug carriers with the mucous membrane European J. Pharm Sci., V. 20, pp. 73-81, 2003; DECAMPOS, AM; DIEBOLD, Y., OAK , ELS, SÁNCHEZ, A., ALONSO, MJ Chitosan nanoparticles as new ocular drug deliveysystems: in vitro stability, in vivo fate, and cellular toxicity Pharm Res. V.21, n.5, pp 803-810, 2004 ; SALAMANCA, AE, DIEBOLD, Y., CALONGE, M., GARCIA-VAZQUEZ, C., CALLEJO, S., VILA, A., ALONSO, MJ Chitosan nanoparticles as a potential drug delivery system for theocular surface: Toxicit y, uptake mechanism and in vivo tolerance. Investigative Ophthalmology & Visual Sci., ν. 47, no. 4, ρ. 1416-1425, 2006).

In general, the topical use of ophthalmic preparations containing steroidal antiinflammatory and antibiotic combination is indicated in superficial infections such as conjunctivitis, keratoconjunctivitis and bacterial blepharitis since such actives do not penetrate the innermost structures of the eye.

In the case of intraocular infections (endophthalmites), these should be treated with the combination of antimicrobials and given as a drug injection into the vitreous humor. Such procedure should be performed by a specialist, and partial vitrectomy (removal of vitreous humor) is also indicated depending on the severity of the infection (LAVINSKY, J.; FIOR, O .; GOLDHARDT. R; DEI RICARDI, LMComplications of Iens displacement into the vitreous Cavity., Ar., Bras.Oftalmol., São Paulo, v. 65, no. 4, 2002; SERRACARBASSA, PD; SERRACARBASSA, LL; RODRIGUES, LD Intravitreous antibiotics. 4, 2003).

Thus, the development of ocular topical product that allows adequate bioavailability of drugs in the external and internal structures of the eye is a therapeutic trend regarding the treatment of eye infections.

Conventional ophthalmic preparations, after application, are rapidly removed from the eyeball due to the eye's protective mechanisms. In addition, the cornea acts as a barrier contributing to the low concentration of the drug in this organ. Thus, the actives conveyed by such preparations have low availability. This undesirable feature has led researchers to search for innovative preparations with greater therapeutic efficacy.

Patent application PI 8802279 relates to the process of preparing polymer particles with amphiphilic molecules implanted on their surface carrying ionogenic or reactive particle groups or aqueous dispersions of particles.

European patent EP 860,166 deals with nanoparticles (with a nanometric dimension and a hydrophilic character), also called nanospheres or latex. Such colloidal systems are composed of a combination of hydrophilic polymers and an active ingredient with a high molecular weight (active macromolecule, molecular weight higher than 1,000 daltons).

US 6,916,488 claims copolymers which are macromolecules composed of two or more distinct types of repeating unit (monomer). The copolymers (diblock and triblock) have hydrophobic and hydrophilic characteristics.

US 6,616,946 deals with polymeric particles for the provision of assets in different permeability states through triblock amphiphilic copolymers (ABA or BAB), (A) being the hydrophilic portion and (B) the hydrophobic portion.

Throughout the foregoing, the Depositor has unexpectedly developed a process of obtaining positively charged polymeric double-layer amphiphilic vesicles for simultaneous administration of various active agents.

Description of the Figures

Figure 1 shows the graphical visualization of (a) diameter (nm) and (b) values of zeta potential values (mV), with the values relative to the data presented in Table 4.

Figure 2 represents the transmission electron microscopy related to dexamethasone acetate-containing nanocapsules (0.1% w / v), being (a) 50K and (b) 120K.

Figure 3 depicts the normal probability, adjusted values, histogram and residual values of the potential zeta relative to the chitosan dispersion optimization assay used in the dexamethasone acetate-containing nanocapsule coating.

Figure 4 proposes the normal probability, adjusted values, histogram, and residual diameter values for the chitosan dispersion optimization assay used in the dexamethasone acetate-containing nanocapsule coating.

Figure 5 shows the contour plot for the assay diameter for optimizing chitosan dispersion in the dexamethasone acetate-containing capsule coating, with valid polymyxin values of 1.5 and A <300; B from 300 to 325; C 325 to 350; D 350 to 375; E from 375 to 400; F from 400 to 425; G from 425 to 450 and H> 450.

Figure 6 shows the contour plot of the potentialzeta in the chitosan dispersion optimization test and coating of dexamethasone acetate-containing nanocapsules, with a fixed polymyxin value of 1.5 and A <3; B from 3 to 6; C from 6 to 9; D from 9 to 12; And from 12 to 15 and F> 15.

Figure 7 represents the diameter-related response surface in the chitosan dispersion optimization test and coating of dexamethasone acetate-containing nanocapsules with valid polymyxin sulfate values of 1.5.

Figure 8 illustrates the potentialzeta response surface in the chitosan dispersion optimization assay and coating of dexamethasone acetate-containing nanocapsules with fixed polymyxin sulfate value of 1.5.

Figure 9 depicts the optimal values of the dequitosan dispersion and final concentration of that dispersion in the dexamethasone acetate-containing capsule suspension for the diameter and potential zeta responses.

Figure 10 shows the graphical visualization of the diameter values (nm) relative to the data presented in Table 11.

Figure 11 shows the graphical visualization of the zeta (mV) (b) potential values for the data presented in Table 11.

Figure 12 shows the electron microscopy of nanocapsules containing chitosan coated dexamethasone acetate, being (a) 50K and (b) 120K.

Figure 13 shows the electron microscopy of nanocapsules containing dexamethasone acetate coated with chitosan and with polymyxin B sulfate, being (a) 50K and (b) 120K.

Figure 14 depicts the standard curve for the assay using high performance liquid chromatography (HPLC) to determine the association rate of dexamethasone acetate to nanocapsule, with y = 35073x + 5313.6 and R2 = 0.9991.

Figure 15 shows the representative illustration of the possible nanostructure obtained containing dexamethasone acetate and depolymyxin B sulfate.

Description of the Invention

The present invention provides a method for obtaining positively charged polymeric double-layer amphiphilic and amphiphilic vesicles containing at least two actives with distinct solubilities (water-soluble and fat-soluble). Additionally, the present application is intended for pharmaceutical and / or cosmetic compositions containing said amphiphilic vesicles and their applications as an antineoplastic, antimicrobial and anti-inflammatory agent.

In a first embodiment the present application is for the method of obtaining amphiphilic vesicles by precipitation of the preformed polymer consisting of the steps of (a) adding the organic phase containing caprolactone polymers and cationic polysaccharides over the aqueous phase containing at least one surfactant preferably under stirring at about 250 rpm, temperature between 37 and 40 ° C for 60 minutes, followed by the elimination of solvent and part of the water, (b) coating of the combiopolymer nanometer vesicle, with concentrations of the chitosan colloidal dispersion about 1.87% (w / v) and the final concentration of about 0.497% (v / v), under 50 rpm agitation for about 60 minutes, in the suspension containing asnanocapsules, (c) incorporation of polypeptides, preferably sodium sulfate. polymyxin B.

The organic phase of the present invention may contain lactones, such as caprolactone polymers, preferably poly (s-caprolactone) (PCL), lactic and glycolic acid, such as lactic and glycolic acid polymers, preferably poly (lactic acid). (PLGA) and poly (lactic acid) (PLLA), acetone and other organic solvents such as alcohols alone and / or in combination with acetone, preferably acetone, capric and caprylic acids such as capric acid triglycerides / caprylic, preferably a mixture of caprylic and caprylic acids, triglycerides, oleic, myristic, linoleic, linolenic, stearic, palmitic, eicosanoic, eicosenoic, lauric, butyric acids. Vegetable oils such as Brazil nuts, acai, buriti, pequi, tucumã, annatto, passion fruit, babassu coconut, olive, sunflower, soybean, rice, sesame, palm, sweet almonds may also be used in the organic phase. , cocoa butters, cupuassu butter, demurumuru butter and ucuuba butter. In addition to the components described, the organic phase may contain lipophilic surfactants such as sorbital monostearate and monooleate, ethoxylated stearyl alcohol with 2 and 20 moles of ethylene oxide, cetostearyl alcohol with 20 moles of ethoxide, fatty ester mixture and ethoxylated fatty acid. and a mixture of ethoxylated fatty alcohol, phospholipids and / or soy lecithin.

Among the different polymers employed in the preparation of nanoparticulate colloidal systems, chitosan (biopolymer) demonstrates adequate characteristics, as it is a biodegradable, biocompatible substance and presents bioadhesiveness. The bioadhesiveness is due to its cationic property, which allows its electrostatic interaction with negative charges of the layer to be applied. Optionally, alginate, hyaluronic acid, polysaccharides of microbial or non-microbial origin and / or chitin may also be used in the coating.

The aqueous phase of the present application may contain ethoxylated hydrophilic surfactants such as ethoxylated desorbitan monolaurate, ethoxylated sorbitan monostearate and ethoxylated desorbitan monooleate, preferably sorbitanethoxylated monostearate.

The active ingredients of the organic phase can be chosen from dexamethasone (as anti-inflammatory agent), antiglaucomas, antihypertensives, antimalarials and other antiprotozoans, immunosuppressants, vitamins, antivirals, analgesic-antipyretics, hormones and antagonists of hormones, hypnotics and sedatives, antidepressants, anesthetics, anxiolytics. local and general, antiarrhythmic, diuretics, antiemetics, anticholinesterase agents, sympathomimetics and adrenergic receptor antagonists, serotonin receptor agonists and antagonists, cardiotonics, antiulcer and anti-secretory acid, antihypercholesterolemic, antidiarrheal, peptides, gene and peptides.

In the coating phase, the actives can be chosen from interferon-derived vaccines, antivirals and antiparasitics, antidiabetic, antimicrobials such as silver derivatives, antitumor agents, for example platinum and amino acid derivatives, antirheumatic agents such as gold derivatives and / or other noble, antineoplastic metals including amino acid-based, debradicinin, antimicrobial and / or anti-inflammatory peptide-based antagonists of synthetic and / or natural origin.

The solvent consisting of acetone, ethanol and / or mixture of acetone ketone and ethanol and part of the water (between 20 and 150 ml) is removed by rotary evaporator and the suspension volume adjusted in a volumetric flask. After preparation, the diameter and polydispersion of the nanocapsules obtained were determined as well as the zeta potential. Additionally, a morphological analysis of the nanoparticles using transmission electron microscopy (MET) is performed.

The zeta potential of the nanostructures is +22.80 mV and a diameter of approximately 390 nm.

In particular, the present invention addresses the combination of polymyxin B sulfate (polypeptide) in the double polymeric layer (chitosan + poly-s-caprolactone) using preformed chitosan-polymyxin B complex. , to capsules containing dexamethasone acetate. The rate for performing the process of obtaining said gallbladder ranges from 30 to 250 rpm, preferably 50 rpm and the stirring time from about 10 to 120 minutes, preferably 60 minutes for the effective association of polypeptide to the nanostructure.

The peptides used in the present invention are selected from peptides of natural and / or synthetic origin, such as T-2022, a synthetic amino acid peptide composed of 36 amino acids, gomesin, exogenous porcine natural pulmonary surfactant, antimicrobial activity decapeptides, highly biologically and pharmaceutically active peptides, for example for the treatment of sarcoidosis-related disease; beta-amyloid peptide, filosseptin-1 (PS-1), antimicrobial belonging to the family of cationic peptides called phyllseptins, found in the cutaneous secretion of Phyllomedusahypochondnalis tree frog among other peptides with biological and pharmacological activity derived from biological or synthetic sources. Amounts of these peptides may range from 300,000 IU / mL to 6,000UI / mL.

In a second embodiment the present application specifically relates to the positively charged amphiphilic vesicles obtained by the process described above having a diameter of 100nm to 750nm, in particular between 300nm and 600nm, preferably between 450nm and 550nm. Thus, such vesicles are termed as positively charged amphiphilicanovesicles.

Yet the nanovesicle of the present invention has an association rate and / or encapsulation efficiency of dedexamethasone acetate ranging from 90% (w / v) to 100% (w / v) in its oily core, which is comprised of capric acid triglycerides / caprylic.

The chitosan-coated nanocapsules containing dexamethasone acetate have concentrations of 1.0 to 3.0% w / v, particularly 1.87% (w / v) for the concentration of the dequitosan colloidal dispersion and 0.2 to 0.5 % v / v, particularly 0.497% (v / v) for final concentration, in the suspension containing the nanocapsules.

The nanostructure of the present application further offers a special, double-polymeric, simultaneous carrier activity with hydrophilic (polypeptide) and hydrophobic (corticoid) characteristics being positively charged.

The association rate of polymyxin B sulfate reached an index greater than 50%.

Chitosan-coated nanostructures containing dexamethasone acetate and polymyxin B sulfate showed zetapositive potential from approximately + 10.00 to +25.00 mV, particularly + 17.00 mV.

Also the nanostructure of the present invention is close to neutrality between 5.5 to 7.5, preferably 6.8. As an optional formulation the nanostructure has a residence time property in its application which generates the possibility of controlled, prolonged and extended release. / or modified from the active ingredients used in the nanovesicle.

In a third embodiment the present invention claims pharmaceutical and / or cosmetic compositions containing said nanostructures. Such compositions may be powders, capsules, tablets, coated tablets and tablets; compressed powder (eye shadow; facial) and uncompressed powder (talc, starch, powder and bath salts) ointments, creams, suppositories and ova, lipstick, deodorant and antiperspirant, sunscreen and eye shadow, emulsions and suspensions, mask, sunscreen, shaving and aftershave, enamel, antiseptic, erroneous, otological, oral drops and syrups, shampoo, hair tonic, tonicofacial, makeup remover, nail polish remover, eye drops, small and large injections, preferably in suspensions and ophthalmic ointments, but not limited to them.

Nanostructures, in particular, were employed in ophthalmic suspensions for the treatment of eye infections caused by gram-negative bacteria accompanied by an inflammatory reaction.

Finally, the present application indicates the use of said nanostructures as antineoplastic, antimicrobial and antiinflammatory agents in pharmaceutical and / or cosmetic compositions.

Examples

Nanocapsule Preparations Containing Chitosan Coated Dexamethasone Acetate εPolymyxin B Sulphate

The process of obtaining the positively charged amphiphilic nanovesicles takes place through the steps of (a) preformed polymer precipitation, (b) preparation of the coating dispersion of the biopolymer and hydrophilic active nanovesicles and (c) coating of the nanovesicles.

In step (a), the organic phase was added to the phase after complete dissolution of the polymer (1 hour under agitation at 250 rpm and 37-40 ° C). Step (b) preparation of the coating dispersion of the nanopolymer containing nanopolymer (chitosan) and the hydrophilic active (polymyxin B sulfate) was prepared by employing low molecular weight chitosan with a minimum degree of 85% acetylation. 1.87 g of the biopolymer was dispersed in 100 ml of purified water under stirring using a magnetic stirrer (200 rpm) for 15 minutes. The pH was adjusted to approximately 5.0 using 0.1 N hydrochloric acid.

In 25 mL of the obtained dispersion (1.87% w / v) was added aliquot of 0.1 mL of aqueous solution containing 600,000 IU of polymyxin sulfate resulting in a final concentration of 24,000 IU / mL of polymyxin sulfate in the dispersion. The system (chitosan + polymyxin B) was subjected to magnetic stirring at a speed of 50 rpm for a period of 60 minutes.

As for the coating of nanovesicles, using the previously obtained dispersion, a 2.5 mL aliquot of the complex formed in the previous step (chitosan + polymyxin B) was transferred to 7.5 mL of nanocapsule suspension. The mixture was stirred for 1 hour at 50 rpm. The final concentration of chitosan and polymyxin B desulfate in the nanocapsule suspension was 0.5% (v / v), respectively (2.5 mL of the 2% w / v dispersion contains 0.05 g of chitosan) and 6,000 IU / mL (2.5 mL Chitosan + Polymyxin B complex Contains 60,000 IU / mL). Thus, the final product volume obtained (10mL) contains 0.5% (v / v) chitosan and 6,000 IU / mL depolymyxin B sulfate.

In general, to obtain nanocapsules containing hexamethasone, the preformed polymer precipitation method was used, which consists of the stirring addition of the organic phase containing poly (ε-caprolactone) (PCL), acetone, dexamethasone and triglyceride mixture of capric / caprylic acids over an aqueous phase containing polysorbate 80 (Table 1).

Solvent and part of the water were removed using a rotary evaporator and the suspension volume was adjusted to balloon knitting volume. After preparation, the diameter and polydispersion of the obtained capsules were determined as well as the zeta potential.

Additionally, the morphological analysis of nanoparticles using transmission electron microscopy (MEiT) was performed.

Physical Characterization of Nanocapsule Suspensions containing

Dexamethasone AcetateThe physical characterization of suspensions was performed as described by SCHAFFAZICK and his collaborators (SCHAFFAZICK, SR; GUTERRRES, SS; FREITAS, LL; POHLMANN, AR. Characterization and physical-chemical stability of nanoparticulate polymer systems for drug administration. See Quim. Nova , v.26, no.5, 2003).

Transmission electronic microscopy

The morphological analysis (surface and shape) of the microparticles was performed using scanning electron microscopy. The nanoparticles were previously metallized with gold.

Particle Size Distribution

The diameter of the suspended particles was determined by dynamic light scattering by diluting the samples and observing scattered light at an angle of 90 ° C. The results corresponded to the average of 3 (three) determinations. Particle size was determined using Zetasizer 3000 equipment. Samples were measured in suspension after preparation. Zeta Potential

Zeta potential was measured using Zetasizer 3000 equipment. Samples were diluted in sterile distilled water of the measurement to optimize signal strength. For each sample 10 (ten) measurements were made, and the resulting mean was used in the additional calculations.

Table 1 below shows the nanocapsule formula obtained by the precipitation method containing dexamethasone acetate for final volume of 10, 50 and 100 mL.

Table 1

<table> table see original document page 19 </column> </row> <table>

Chitosan Dispersion Optimization for the coating of nanocapsules containing dexamethasone acetate

For the coating was used chitosan with low molecular weight and minimum acetylation degree of 85%. Amount equal to 2.0, 3.0 and 4.0 g of the biopolymer was dispersed in 100 mL of purified water under stirring using magnetic stirrer (200 rpm) for 15 minutes. The pH was adjusted to approximately 5 , Employing 0.1 N hydrochloric acid.

The factorial design used for optimization consisted of continuous and categorical variables with different levels. Three and four levels were respectively used for the concentration of chitosan dispersion (2.0, 3.0 and 4.0% w / v) and for the final concentration of chitosan in the formula (0.2, 0.3, 0.4 and 0.5% v / v). For the categorical variable only two levels were included: presence or absence of polymyxin B desulphate at 6,000 IU / mL, as shown in Table 2. Statistical analysis was performed using Minitab® version 15 software.

In addition, preliminary tests were performed in order to obtain the best conditions for the coating. Thus, the addition of chitosan (concentrations indicated in Table 2) to nanocapsule suspension was performed as follows: from each obtained dispersion (2.0, 3.0 and 4.0% w / v) was transferred aliquot vis-à-vis final concentration in the suspension. 0.2, 0.3, 0.4 and 0.5% v / v. After addition, the system was subjected to magnetic stirring for 1 hour at 50 rpm stirring speed. For assays predicting the addition of depolymyxin B sulfate, concentrated antimicrobial solution (600,000 IU / 0.1 ml) was prepared. A 0.1 mL aliquot of this solution was transferred to 100 mL of chitosan dispersion (2.0 3.0 and 4.0% w / v). The system remained under magnetic stirring at a speed of 50 rpm for a period of 60 minutes. After this period, the final aliquot was transferred with a final concentration of 0.2, 0.3, 0.4 and 0.5% v / v. After addition, the system was subjected to magnetic stirring for 1 hour at 50 rpm stirring speed.

Table 2 refers to the assay matrix for chitosan dispersion optimization in the coating of dexamethasone acetate containing nanocapsules. Where PDI: polydispersion; N: Polymyxin eSulfate assay number: (1) absent and (2): present at 6,000 IU / mL

Table 2

<table> table see original document page 21 </column> </row> <table> <table> table see original document page 22 </column> </row> <table>

Association of Polymyxin B Sulphate to the Ouitosan Layer of the Nanocapsule Containing Dexamethasone Acetate

Regarding the addition of polymyxin B sulfate to be associated with the chitosan layer, the following procedure was performed: an adequate volume of 2.0% (w / v) chitosan dispersion was transferred to reach a final concentration in the preparation equal to 0.5% (v / v). At this volume, polymyxin B sulfate was added with final concentration of 6,000 IU / ml. The dispersion was homogenized using a speed of 50 rpm for 1 hour. After this period, an adequate volume of nanocapsule suspension containing dexamethasone acetate was added and homogenized for 1 hour at 50 rpm. After the addition of polymyxin B sulfate in nanocapsule suspension, the diameter, dapolidispersion and zeta potential of the obtained preparation were determined. Additionally, the morphological analysis of nanoparticles using electron microscopic transmission (MET) was performed.

Comparison of Nanocapsule Physical Characteristics

Three different suspensions were prepared: nanocapsules containing dexamethasone acetate 0.1% (w / v) (1); nanocapsules containing chitosan coated 0.1% (w / v) dexamethasone acetate (2); enanocapsules containing dexamethasone acetate 0.1% w / v and depolymyxin B sulfate (6,000 IU / mL) associated with chitosan (3). The suspensions were prepared as described above using optimal concentration for chitosan coating.

Determination of Dexamethasone Acetate Association Rate using high performance liquid chromatography

The concentration of dexamethasone acetate bound to the particulate system was assessed by centrifugation of the suspended particles at 14,000 rpm for 2 hours. Supernatant sample (non-particle bound drug content) was analyzed using traditional method. For dexamethasone a high performance liquid chromatographic method (HPLC) was used.

Determination of Polymyxin Sulfate Association Rate Using the microbiological method

The assay for the determination of the polymyxin B sulfate association rate was performed according to the official compendium (USPXXVI).

Comparative assays were performed covering prepared samples as follows: (1) nanocapsules containing chitosan coated dedexamethasone acetate (0.10% w / v). The addition of polymyxin sulfate (6,000 IU / mL) to the nanocapsule suspension was performed immediately prior to the assay using manual stirring until complete dissolution of the powder; (2) nanocapsules containing dexamethasone acetate (0.10% w / v) coated with polymyxin B sulfate-associated chitosan (6,000 IU / mL) prepared as described above; (3) supernatant from the suspension of chitosan-coated dexamethasone acetate nanocapsules associated with polymyxin B sulfate (6,000 IU / mL) after centrifugation at 14,000 rpm for a period of 2 hours; (4) nanocapsules containing dexamethasone acetate (0.10% w / v) coated with optimized chitosan concentration without the addition of polymyxin B sulfate (control).

The association rate of polymyxin B sulfate was calculated by the difference between the results obtained from the suspension containing 100% of the unassociated and therefore free antimicrobial (1) and from the suspension containing the associated antimicrobial (3).

Preparation of Ophthalmic Suspension containing Chitosan-Coated Nanocapsules containing Dexamethasone Acetate Sulfate

Polymyxin B

The suspension preparation containing the nanocapsules was aseptically analyzed. The components below (Table 3), except for the preservative and polymer, were homogenized using magnetic stirring (300 rpm) for 20 minutes. For this solution 45 ml of distilled water was used. The solution was sterilized by filtration using 0.22 Mm pore size membrane and 45 mm diameter. 45 ml volume of sterile 0.50% (w / v) hydroxypropyl methylcellulose (hypromellose) dispersion (steam sterilization 121 ° C for 20 minutes) was added to the solution.

After addition of the preservative, the dispersion was homogenized for 15 minutes using magnetic stirrer (300 rpm). A 10 mL aliquot containing the nanocapsules was transferred to this dispersion. The obtained suspension was homogenized for 10 minutes using magnetic stirrer (300 rpm).

Table 3 describes a basic ophthalmic suspension formulation containing nanocapsules containing 0.1% (w / v) dedexamethasone acetate and chitosan coated 6000 IU / mL polymyxin B sulfate.

Table 3

Basic Formulation - Ophthalmic Suspension <table> table see original document page 25 </column> </row> <table>

Isotonicity Assessment

The isotonicity of the suspensions was determined by cryoscopic lowering after 7 days of preparation using the DigiMatic Osmometer 3D model. For this, a volume of 10 mL of previously homogenized sample was used in duplicate.

Determination of Suspension pH

The pH values of the suspensions were obtained using Micronal Model B474 potentiometer, at 25 ° C, immediately after preparation and after 7 days. For this purpose a volume of 10 mL of previously homogenized sample will be used in duplicate.

Results

Table 3 presents the results regarding the physical characterization of dexamethasone acetate-containing nanocapsules immediately after preparation.

Table 4 shows the mean diameter, polydispersion, and dopotential zeta of dexamethasone acetate-containing nanocapsules.Table 4

<table> table see original document page 26 </column> </row> <table>

Table 5 presents the results of the study for optimizing the chitosan concentration for the dexamethasone acetate-containing danocapsule coating. Table 5 also presents the diameter, diameter dispersion and potentialzeta values relative to the test matrix for the optimization of debrisization in the coating of nanocapsules containing dedexamethasone acetate, where PDI: polydispersion; N: Assay number and depolymyxin sulphate: (1) absent and (2) present at 6,000 IU / mL

Table 5

<table> table see original document page 26 </column> </row> <table> <table> table see original document page 27 </column> </row> <table>

Table 6 presents a test for the significance of the regression coefficients and fit indices of the model selected in the chitosan dispersion assay for nanocapsule diameter containing dexamethasone acetate. Where Coef .: coefficients; SE Coef .: standard deviation of coefficients; p: significance level; T: T statistic (T test) - Coef./SE Coef.

Table 6

<table> table see original document page 27 </column> </row> <table> <table> table see original document page 28 </column> </row> <table>

Table 7 presents the analysis of variance (ANOVA) to test the significance of the regression for the data obtained in the chitosan dispersion test for nanocapsule diameter containing dexamethasone acetate, where DF: degree of freedom; Seq SS: sum of squares; Adj SS: sum of the adjusted squares; F: F statistic (F test); Adj. MS: adjusted quadratic mean; P: designation level.

Table 7

<table> table see original document page 28 </column> </row> <table>

Table 8 represents the test for the significance of the regression coefficients and fit indices of the selected model in the chitosan dispersion assay for the zeta potential of dexamethasone acetate-containing nanocapsules, with Coef.:coefficients; SE Coef: standard deviation of coefficients; p: designation level; T: T statistic (T test) - Coef./SE Coef.

Table 8

<table> table see original document page 28 </column> </row> <table> <table> table see original document page 29 </column> </row> <table>

Table 9 shows the analysis of variance (ANOVA) to test the significance of regression for chitosan dispersion assay data for zeta potential of nanocapsules containing dexamethasone acetate, where DF: degree of freedom; Seq SS: sum of squares; Adj SS: sum of the adjusted squares; F: F statistic (F test); Adj. MS: adjusted quadratic mean; P: designation level.

Table 9

<table> table see original document page 29 </column> </row> <table>

Table 10 shows the mean diameter (nm), polydispersion, and zeta potential (mV) of dexamethasone acetate (0.10% w / v) nanocapsules (1), dexamethasone acetate (0.10% w / v) nanocapsules v) coated with chitosan (2) and nanocapsules containing dexamethasone acetate and polymyxin B, coated with chitosan (3), being * average between 3 measurements and ** average between 6 measurements.

Table 10

<table> table see original document page 30 </column> </row> <table>

Table 11 shows the concentrations in μg / mL of dexamethasone acetate and their areas relative to the standard curve of the assay using HPLC to determine the nanocapsule association rate, where A: area.

Table 11

<table> table see original document page 30 </column> </row> <table>

Table 12 shows the efficiency of associating depolymyxin B sulfate in the double-polymeric layer of dexamethasone acetate-containing nanocapsules, (1) Suspension containing unassociated (free) polymyxynan sulfate; (2) Suspension containing associated polymyxin sulfate; (3) Supernatant of depolymyxine sulfate-associated nanocapsules after centrifugation: 14,000 rpm for a period of 2 hours; (4) Control suspension (absence of polymyxin sulfate); E: essay performed in triplicate; and (-) not detected

Table 12

<table> table see original document page 31 </column> </row> <table>

The average pH value for the preparation containing the nanocapsule suspension with the associated actives was 6.8. Determination of osmolarity revealed the isotonicity of the preparation.

Claims (26)

1. METHOD OF OBTAINING AMPHIFYLIC VESICULES, characterized in that it comprises the steps (a) addition of the organic phase containing polymers of cationic caprolactones epolysaccharides over the aqueous phase containing at least one ethoxylated hydrophilic surfactant (biopolymer) coating (b) c) incorporation of polypeptides. The method of obtaining amphiphilic vesicles according to claim 1, characterized in that the step (a) preferably occurs under agitation of about 250 rpm at a temperature between 37 and 40 ° C and for at least 60 minutes. The method of obtaining amphiphilic vesicles according to claim 1, characterized in that the step (a) further comprises the elimination of the solvent and part of the water. The method of obtaining amphiphilic vesicles according to claim 1, characterized in that the solvent comprises acetone, ethanol and / or a mixture of acetone, ethanol and / or water. The method of obtaining amphiphilic vesicles according to claim 1, characterized in that the coating step (b) comprises concentrations of the chitosan dispersion of about 1.87% (w / v) and the final concentration of about 0.497%. (v / v). The method of obtaining amphiphilic vesicles according to claim 5, characterized in that the coating step (b) comprises in particular a stirring of 50 rpm for about 60 minutes. The method of obtaining amphiphilic vesicles according to claim 1, characterized in that the peptide incorporation step (c) preferably comprises polymyxin B sulfate. The method of obtaining amphiphilic vesicles according to claim 7, characterized in that the peptide factors further comprise peptides of natural and / or synthetic origin. The method of obtaining amphiphilic vesicles according to claims 7 and 8, characterized in that the amounts of the peptides comprise concentrations of -3,000 IU / mL to 6,000 IU / mL. The method of obtaining amphiphilic vesicles according to claim 1, characterized in that the organic phase comprises lactones such as decaprolactone polymers, preferably poly (s-caprolactone) (PCL), lactic and glycolic acid polymers. lactic and glycolic acids, preferably poly (lactic-co-glycolic acid) (PLGA) and poly (acid-lactic) (PLLA), acetone and other organic solvents such as alcoholsisolated and / or in combination with acetone, preferably acetone, capric acids and caprylic, such as caprylic / caprylic acid triglycerides, preferably mixture of capric and caprylic acid triglycerides, oleic, myristic, linoleic, linolenic, stearic, palmitic, eicosanoic, laicosic, butyric acids. The method of obtaining amphiphilic vesicles according to claim 10, characterized in that the organic phase fat still comprises vegetable oils, such as Brazil nut oils, acai, buriti, pequi, tucumã, passion fruit, babassu coconut, olive. , sunflower, soy, rice, sesame, palm, sweet almonds, cocoa butters, cupuaçú butter, demurumuru butter and ucuuba butter. The method of obtaining amphiphilic vesicles according to claims 10 and 11, characterized in that the organic phase further comprises lipophilic surfactants such as sorbital monostearate and monostearate, ethoxylated stearyl alcohol with 2 and 20 moles of ethylene oxide ethoxide, with 20 moles of ethylene oxide, ethoxylated fatty acid fatty ester mixture and ethoxylated fatty alcohol mixture, phospholipidose / or soy lecithin. The method of obtaining amphiphilic vesicles according to claim 1, characterized in that the biopolymer method of the coating step (b) comprises alginate, hyaluronic acid, polysaccharides of microbial or non-chitin origin, preferably chitosan. The method of obtaining amphiphilic vesicles according to claim 1, characterized in that the aqueous phase comprises ethoxylated hydrophilic surfactants such as ethoxylated sorbitan monolaurate, ethoxylated sorbitan, ethoxanate preferred ethoxanate ethoxylated sorbitan monooleate. Amphiphilic vesicles according to claims 1 to 14, characterized in that the amphiphilic vesicles comprise positively charged structures. Amphiphilic vesicles according to claim 15, characterized in that the vesicles comprise a diameter of 100nm to 750nm, in particular between 300nm and 600nm, preferably between 450nm and 550nm. 17. Amphiphilic vesicles according to claims 15 and 16, characterized in that the vesicles comprise a potential zeta of the nanostructures of +10.00 to + 25.00 mV, particularly +17.00 mV and the diameter of approximately 390 nm. 18. Amphiphilic vesicles according to claims 15 to 17, characterized in that the vesicles comprise a double-polymeric layer simultaneously carrying assets with hydrophilic and hydrophobic characteristics. 19. Amphiphilic vesicles according to claims 15 to 18, characterized in that the active ingredients of the organic phase comprise dexamethasone, antiglaucoma, antihypertensive, antimalarial and antiprotozoal, immunosuppressants, vitamins, antivirals, analgesic-antipyretic, sedative hormones, eantagonist hormones, antidepressants, anxiolytics, local and general anesthetics, antiarrhythmics, diuretics, antiemetics, anticholinesterase agents, sympathomimetics, adrenergic receptor antagonists, agonists and antagonists of serotonin receptors, cardiotonics, antiulcerides, antihypercholesterolemic, anti-hypercholesterolemic, genes, epeptides. Amphiphilic vesicles according to claims 15 to 19, characterized in that the active coatings phase comprises interferon-derived vaccines, antivirals and antiparasitics, antidiabetic agents, silver-derivatives, antitumor agents, for example, platinum derivatives and amino acids, antacid agents such as gold and / or other noble metals, antineoplastic derivatives including those of amino acids, bradykinin antagonists, antimicrobials and / or anti-inflammatory peptides based on synthetic and / or natural origin. 21. Amphiphilic vesicles according to claims 15 to 20, characterized in that the vesicles comprise pH close to neutrality between 5.5 and 7.5, preferably 6.8. PHARMACEUTICAL AND / OUCOSMETIC COMPOSITIONS according to Claims 1 to 21, characterized in that the nanovesicles are comprised in the form of powders, capsules, tablets, coated tablets and dragees; compacted and / or uncompressed powder, ointments, creams, ovarian suppositories, lipstick, deodorant and antiperspirant, sunscreen and eye shadow, mascara, sunscreen, shaving and aftershave, enamel, antiseptic, errino, otological , oral drops and syrups, shampoo, hair tonic, facial tonic, makeup remover, nail polish remover, eye drops, small and large injectables, preferably in suspensions and ophthalmic ointments. Use of amphiphilic vesicles according to claims 1 to 22, characterized in that they are applied to pharmaceutical and / or cosmetic compositions as antineoplastic, antimicrobial and anti-inflammatory agents. Use of amphiphilic vesicles according to claim 23, characterized in that the pharmaceutical and / or cosmetic compositions comprise controlled, prolonged and / or modified release systems of the active ingredients. Use of amphiphilic vesicles according to claims 23 to 24, characterized in that it is preferably applied in ophthalmic suspensions for the treatment of eye infections. Use of amphiphilic vesicles according to claim 23, characterized in that infections are caused by gram-negative bacteria.