WO2018090112A1

WO2018090112A1 - Method for obtaining nanostructured amphiphilic cationic lipid and amphiphilic cationic polymer compounds, thus obtained nanostructured compounds and uses thereof

Info

Publication number: WO2018090112A1
Application number: PCT/BR2017/050297
Authority: WO
Inventors: Nadia ARACI BOU CHACRA; Milton RUIZ ALVES
Original assignee: Universidade De São Paulo - Usp; Tecnan Brasil - Pesquisa, Desenvolvimento E Inovação Ltda
Priority date: 2016-11-18
Filing date: 2017-09-29
Publication date: 2018-05-24
Also published as: BR102016027137A2

Abstract

The present invention relates to a method for obtaining nanostructured amphiphilic cationic lipid and amphiphilic cationic polymer compounds that can contain at least two pharmaceutical drugs, a hydrophobic and a hydrophilic drug, associated with an oil phase and an aqueous phase, and coated with chitosan. The method comprises four steps which allow the nanostructures to be obtained in a controlled manner. Thus, it is possible to produce a cationic nanoparticle in a controlled manner. The present invention also relates to the thus obtained compounds and to the use thereof for developing pharmaceutical or cosmetic products in which the cationic nature is desirable, preferably ophthalmic products.

Description

COMPOUND PROCESSING

NAMESTRUCTURED AMPHYPHILIC CATIHONIC LIPID AND AMPHYPHIC CATIHONIC POLYMERIC, OBTAINED NUTRUCTURAL COMPOUNDS AND THEIR USES

Field of the invention:

[001] The present invention falls within the field of application of medical science, more specifically in the field of

as it refers to a process for obtaining an amphiphilic cationic lipid or polymeric nanoparticulate compound for the development of pharmaceutical or

cosmetics where the cationic characteristic is desirable,

preferably ophthalmic products.

Background of the invention:

Conventional ophthalmic products, especially eye drops, have low bioavailability and therefore low therapeutic efficacy. This undesirable feature is due to eye protection mechanisms (blinking and watering reflex) that expel any foreign material from the surface of the eye.

In addition, the instilled material may be drained via the nasolacrimal duct and may cause an undesirable systemic effect. Another possibility of undesirable systemic effect is the absorption of the material via conjunctiva, highly vascularized structure.

In addition, the cornea and tear film also constitute a natural barrier contributing to the reduction of the therapeutic efficacy of eye drops in combating eye disease in the anterior and posterior segment of the eye. Thus, the patient must instill the eye drops several times a day, making it difficult to adhere to treatment.

However, the tear film also has a particular feature that can be used to improve the residence time of the eye drops and thereby increase the therapeutic efficacy of this product. In its composition, this structure presents mucin, protein glycolyzate containing sialic acid, which is negatively charged at neutral pH.

[006] Thus, the development of cationic (or positive) eye drops aims to increase its therapeutic efficacy.

Thus, as existing ophthalmic products have poor therapeutic efficacy, the present invention proposes an amphiphilic cationic lipid or polymeric nanoparticulate compound.

Thus, these amphiphilic cationic nanostructured compounds aim to obtain innovative ophthalmic products that present greater efficacy and safety to the patient, since the average particle size, in nanometer scale, and the interaction of these compounds with sialic acid (charged

negatively) of highly O-glycosylated glycoproteins (mucins) will allow longer residence of the compound on the surface of the eye, allowing treatment of the anterior and posterior segment of the eye.

In addition, this nanostructure can be used for the development of other pharmaceutical or cosmetic products where the cationic characteristic is desirable.

[010] By way of example, the present compound may be used for the development of medicaments for neglected diseases. In the case of leishmaniasis, the parasite infects macrophages and thus the positively charged nanostructured compound can be internalized into this cell, release the drug and eliminate the parasite.

State of the art:

[01 1] Some prior art documents describe a nanoparticulate compound associated with active substances.

[012] "Evaluation of cationic polymer-coated nanocapsules as ocular drug carriers" by Calvo et al. (1997) describes a nanostructure that presents only the lipophilic drug and the coating employing chitosan. Unlike the present invention, it does not present in the same nanostructure the association of another drug. Furthermore, the simple addition of a cationic hydrophilic drug to the chitosan coated nanostructure does not result in the present invention since the document reports a + 37 mV zeta potential nanostructure after coating. Thus, the addition of a positive cationic hydrophilic drug does not allow its association with the nanostructure reported in this document, since the electrical force between two charges with the same signal is repulsive.

US2015290092 describes nanoparticles containing mono, di or trivalent ions which have a functional core comprising drug containing one or more subsequent polymeric layers of opposite charges for use in dentistry. However, in contrast, the present invention proposes amphiphilic cationic lipid or polymeric nanoparticles which do not contain ions of any nature associated with the nanostructure for the development of cationic ophthalmic products.

[014] Document BRPI0904083 describes a process for obtaining cationic amphiphilic nanovesicles for application in ophthalmic suspensions in the treatment of eye infections. However, unlike the present invention, the polymer and hydrophilic drug are simply mixed and added to the nanostructure. Such procedure results in high variability in the nanostructure zeta potential characteristics revealing low reproducibility of the method.

[015] US2013189368 presents the same technical problem, where the nanostructure is obtained in one step

comprising the simultaneous combination of the oil phase and the aqueous phase.

[016] To solve the presented technical problem, the present invention proposes a process that has additional steps that allow control of the amount of hydrophilic drug associated with

nanoparticle, as well as the amount of chitosan, so it is possible to cationize the nanoparticle in a controlled manner. In addition, through this process, it is possible to associate at least two drugs to the nanostructure, one hydrophilic and one hydrophobic, and other hydrophilic drugs may be added.

[017] The cationic hydrophilic drug class includes a series of cationic peptide substances, cationic peptides with antimicrobial action such as vancomycin.

Therefore, no prior art document describes a process for obtaining an amphiphilic cationic lipid or polymeric nanoparticulate compound for the development of pharmaceutical or cosmetic products where the cationic characteristic is desirable as proposed by the present invention.

Brief Description of the Invention:

[019] The present invention relates to a process for obtaining amphiphilic cationic lipid and amphiphilic cationic polymeric nanostructured compounds capable of comprising at least two drugs, one hydrophobic and one hydrophilic, associated with an oily and aqueous phase and coated with chitosan . This process comprises four steps, which allow the control of obtaining the nanostructures. Thus, it is possible to cationize the nanoparticle in a controlled manner: control of the zeta potential, control of the amount of cationic substance (SC), and control of the amount of drug or active substance. Accordingly, the present invention also relates to the compounds obtained as well as their use for the development of pharmaceutical or

cosmetics where the cationic characteristic is desirable,

preferably ophthalmic products.

Brief description of the figures:

[020] For a complete and complete view of the object of this invention, reference figures are given, as follows.

[021] Figure 1 graphs the simple linear regression analysis of zeta potential (mV) versus polymyxin B sulfate concentration (IU / mL).

Figure 2 graphs the simple linear regression analysis of mean hydrodynamic diameter versus polymyxin B sulfate concentration (IU / mL) for CLN-Dexa + Poli-Prot.

[023] Figure 3 A-E graphs the linear regression of mean hydrodynamic diameter (DHM) versus (a) polymyxin B sulfate concentration (SP IU / mL); and (B-E) DHM residues, where (B) is the normal probability plot, (C) versus adjusted, (D) histogram, and (E) versus order.

[024] Figure 4 A-E graphs the linear regression of zeta potential (PZ) versus (a) polymyxin B sulfate concentration (SP IU / mL); and (B-E) DHM residues, where (B) is the normal probability plot, (C) versus adjusted, (D) histogram, and (E) versus order.

[025] Figure 5 AF graphs analysis of variance (ANOVA): mean hydrodynamic diameter (DHM) of VP-Dexa + Poly (polymyxin sulfate concentrations: 2,500, 5,000, 7,500 and 10,000 IU / mL) after coating employing 0.15% (w / v) Protasan®, which (a) is the means and confidence interval; (b) the Tukey test and, (C-F) are the residuals, where (C) is the normal probability plot, (D) versus adjusted, (E) histogram, and (F) versus order.

[026] Figure 6 A-F graphically analyzes the variance (ANOVA) of the zeta potential (PZ) of VP-Dexa + Poly

(polymyxin sulfate concentrations: 2,500, 5,000, 7,500 and 10,000 IU / mL) after coating employing 0, 15% (w / v) Protasan®, which (a) is the means and confidence interval; (b) the Tukey test and (CF) are the residuals, where (C) is the normal probability plot, (D) versus adjusted, (E) histogram, and (F) versus order. Detailed Description of the Invention:

[027] The present invention relates to a process for obtaining amphiphilic cationic lipid and amphiphilic cationic polymeric nanostructured compounds.

[028] This process comprises the steps of:

(a) preparing lipid carriers comprising one liquid lipid or two or more liquid lipids, or comprising one solid lipid or two or more solid lipids, or mixture of liquid lipid and one solid lipid or mixture of two or more liquid lipids and solid lipids, comprising at least one hydrophobic or lipophilic drug or active substance;

b) Prepare the solution containing at least one cationic drug or substance (SC);

c) Incorporating SC into the formulation obtained in step "a"; and d) Coat the formulation obtained in step "c" with low molecular weight (50000-400000 g / mol) and high grade chitosan.

deacetylation (> 80%).

Said drug or active substance is selected from the groups consisting of hydrophobic or lipophilic active substances, such as immunosuppressants (cyclosporine); prostaglandins such as latanoprost, bimatoprost and travoprost; antioxidants such as carotenoids, α-, β-, γ-, δ-tocopherol and a-, β-, γ-, δ-tocotrienol, retinoic acid, retinol and their esters, ubiquinol, ubiquinone, glutathione in their reduced form, flavonoids, lipoic acid and active components of biotechnological origin such as etanercept, pegaptanib, ranibizumab and bevacizumab; and cholinergic drugs such as pilocarpine, ezerin and neostigmine; and non-spheroidal and spheroidal anti-inflammatory, preferably dexamethasone acetate.

[030] Said cationic substance (SC) is selected from the group consisting of hydrophilic drugs, cationic peptides such as polymyxin B sulfate, vancomycin, gramicidine, bacitracin and others. cationic antimicrobial peptides; and benzalkonium chloride and other quaternary ammoniums, preferably polymyxin B sulfate.

[031] In step "a", the preparation of lipid carriers is obtained by heating the oil phase and the aqueous phase separately at a temperature ranging from 55 to 90 ° C,

preferably 85 ° C under agitation ranging from 100 to 500 rpm, preferably 300 rpm until complete dissolution and dispersion of the drugs or hydrophobic or lipophilic active substance, respectively.

The oil phase comprises from 0.0001 to 10%,

preferably 0.10% of the lipid-associated hydrophobic or lipophilic active substance or drug, wherein the lipids are selected from the groups consisting of liquid lipid (oil) at a concentration of 1 to 20% or a mixture of liquid lipids in the proportion ranging from 0.01: 1 to 1: 10; and solid lipid at a concentration of 1 to 20% or a mixture of solid lipids in a ratio ranging from 0.01: 1 to 1: 100; or a mixture of liquid and solid lipids ranging from 0.01: 1 to 1: 100 of the total lipid carrier formulation.

[033] Liquid lipids are selected from the group consisting of unsaturated fatty acids, such as caprylic and caprylic acid glycerol triesters. And solid lipids are selected from the group consisting of saturated fatty acids, such as cetyl palmitate.

The aqueous phase comprises at least one nonionic surfactant such as polysorbate 80, soy or egg phospholipids, poloxamer 188, poloxamer 407 and TPGS (d-alpha tocopheryl polyethylene glycol 1000), Kolliphor ELP (macrogolglyceryl ricinoleate - oil). polyoxyl 35 castor), Kolliphor HS 15 (polyoxyl hydroxystearate 15) diluted in ultrapure water. Alternatively, it may contain an anionic surfactant such as sodium dodecyl sulfate.

Preferably, the aqueous phase comprises from 0.05 to 10%, more preferably 2.5% of poloxamer 188 and poloxamer 407; from 0.05 to 10%, more preferably 1.5% polysorbate 80; from 0.001 at 10%, more preferably 0.5% sodium dodecyl sulfate; and qsp 100% ultrapure water from the total formulation of lipid carriers.

Subsequently, the aqueous phase is dispersed in the oil phase under mechanical agitation ranging from 1000 to 25000 rpm, preferably 8000 rpm for a period of time ranging from 1 to 10 min.

preferably 5 minutes to form a preemulsion which is homogenised using 1 to 6 successive cycles, preferably 3 cycles, at a pressure ranging from 200 to 1000 bar, preferably 600 bar.

[037] After preparation of the lipid carriers, in step "b", the cationic solution containing the hydrophilic drugs (SC) is diluted or not in a suitable container with ultrapure water in a ratio ranging from 1: 1 to 1: 5. preferably 1: 2 such that a concentration ranging from 50,000 to 1,000,000 IU / mL of the cationic substance is obtained.

[038] Step "c" incorporates the cationic peptide substance at a concentration of 5 to 25,000 IU / mL,

preferably 6000 IU / ml SC obtained in step "b" in the lipid carriers obtained in step "a" diluted or not in purified water in a ratio ranging from 1: 1 to 1: 10 preferably 1: 5. That

Incorporation is performed slowly, preferably by dripping, and under agitation ranging from 50 to 300 rpm, preferably 100 rpm, for a period of time ranging from 0.5 to 6 hours, preferably 2 hours.

Thus, in step "c" is formed a lipid nanoparticulate compound comprising a hydrophobic or lipophilic active substance or drug incorporated with at least one cationic substance. It is noteworthy that, at this stage, the coating is controlled to produce smaller but still negative modulus zeta potential particles ranging from -30 to -1 mV, depending on the concentration of the cationic substance (s). (s) (example: polymyxin B sulfate and vancomycin) to be associated with nanoparticles. This control is performed by concentrating the cationic substance used. (example: 1 to 1,000,000 IU / mL).

Thus, in step "d", 0.001% to 10%, preferably 0.2% dispersion of 2.0% (w / w) chitosan in ultrapure water is

added to the compound obtained in step "c" slowly

preferably by stirring dripping ranging from 50 to 500 rpm, preferably 100 rpm. After addition of the dispersion, the stirring period continues for a period of time ranging from 0.5 to 8 hours, preferably 2 hours and the preparations obtained are kept at room temperature or refrigerated at 1 to 10 ° C. preferably 5 ° C. Thus, in step "d", the coating is controlled to produce particles with zeta potential between +5 and +65 mV.

Thus, the preparations obtained in step "d" comprise from 0.001 to 10%, preferably 0.2% (w / v%) chitosan, thus obtaining an amphiphilic cationic lipid nanostructured compound with particles of medium hydrodynamic diameter in the sub-micron range, with an average diameter between 100 and 600 nm,

preferably 200 nm, with positive zeta potential.

[042] In an alternative embodiment, the present process relates to obtaining an amphiphilic cationic polymeric nanostructured compound, wherein to this end, step "a" is to prepare polymeric vesicles comprising one liquid lipid or two or more liquid lipids, or liquid mixture of liquid lipid and a solid lipid, or liquid mixture of two or more liquid lipids and solid lipids, comprising at least one hydrophobic or lipophilic active substance or drug.

[043] Thus, to obtain the vesicles

comprising the hydrophobic or lipophilic drug or substance, the preformed polymer precipitation method consisting of the addition of the organic phase comprising the polymer, one or more lipids, the organic solvent, the nonionic surfactant and the lipophilic drug is used. temperature below 60 ° C, preferably 45 ° C under stirring at 50 to 300 rpm, preferably at 100 rpm, for the time required for complete polymer solubilization, between 15 and 45 minutes, over the aqueous phase. Subsequently, the solvent and part of the water are eliminated using a rotary evaporator and the volume of the suspension is adjusted with the addition of purified water.

Said aqueous phase comprises from 0.1 to 10% of at least one nonionic surfactant such as span 20, span 40, span 60, span 80 (sorbitan esters) polysorbate 80, poloxamer 188 and TPGS (d alpha-tocopheryl polyethylene glycol 1000), Kolliphor ELP (macrogolglyceryl ricinoleate - polyoxyl 35 castor oil), Kolliphor HS 15

(polyoxyl hydroxystearate 15) diluted in purified water.

[045] The organic phase comprises 0.0001 to 10%, preferably 0.10% of the lipid-associated hydrophobic or lipophilic active substance or drug, wherein lipids are selected from the groups consisting of liquid lipid (oil) in the concentration of 1 to 20% or mixture of liquid lipids in the ratio ranging from 0.01: 1 to 1: 10, or a liquid mixture of liquid and solid lipids in the ratio ranging from 0.01: 1 to 1: 100; from 0.01 to 20% of polymers selected from the group consisting of lactic polyacid, copolymers derived from lactic and glycolic acids, aliphatic polyanhydrides, polymer derived from lactones, preferably poly (s-caprolactone); from 5 to 50% organic solvent selected from the group consisting of acetone, ethanol and methanol, preferably acetone; from 0.0001 to 10% hydrophobic or lipophilic drug or substance; and from 0.5 to 10% of unsaturated fatty acids such as capric and caprylic acid glycerol triesters and from 0.5 to 10% of nonionic surfactant of the total formulation of polymeric vesicles.

[046] Liquid lipids are selected from the group consisting of unsaturated fatty acids, such as caprylic and caprylic acid glycerol triesters. And solid lipids are selected from the group consisting of saturated fatty acids, such as cetyl palmitate. Surfactants are selected from nonionics such as span 20, span 40, span 60 and span 80 (sorbitan esters).

Thus, in step "c" is formed a polymeric nanoparticulate compound comprising a hydrophobic or lipophilic active substance or drug incorporated with at least one cationic substance. And in step "d" an amphiphilic cationic polymeric nanostructured compound having particles of medium hydrodynamic diameter in the sub-micron range with positive zeta potential is obtained.

Therefore, in addition, the present invention relates to the amphiphilic cationic lipid nanostructured compound obtained according to the process described herein, which comprises at least two drugs, one hydrophobic and one hydrophilic, associated with:

from 0.5 to 20% oil phase;

from 80 to 99.5% aqueous phase; and

from 0.001 to 10%, preferably 0.2% chitosan.

Said hydrophobic drug is selected from the group consisting of hydrophobic or lipophilic active substances such as immunosuppressants (cyclosporine), prostaglandins (latanoprost, bimatoprost and travoprost), non-spheroidal and spheroidal anti-inflammatory agents, preferably dexamethasone acetate; and antioxidants such as carotenoids, α-, β-, γ-, δ-tocopherol and a-, β-, γ-, δ-tocotrienol, retinoic acid, retinol and their esters, ubiquinol, ubiquinone, glutathione in their reduced form, flavonoids, lipoic acid and active components of biotechnological origin such as etanercept, pegaptanib, ranibizumab and bevacizumab; and cholinergic drugs such as pilocarpine, ezerin and neostigmine.

[050] Said hydrophilic drug is selected from the group consisting of hydrophilic cationic substances, cationic peptides, antimicrobial cationic peptides such as polymyxin B sulfate, vancomycin, gramicidine, bacitracin; other cationic antimicrobial peptides; and benzalkonium chloride, preferably polymyxin B sulfate. [051] The oil phase comprises from 0.0001 to 10%,

preferably 0.10% of the lipid solubilized hydrophobic drug, wherein the lipids are selected from the groups consisting of a liquid lipid at a concentration of 0.1 to 20% or a mixture of liquid lipids ranging from 0.001: 1 to 1: 10; and solid lipid at a concentration of 1 to 20% or a mixture of solid lipids in a ratio ranging from 0.01: 1 to 1: 100; or a mixture of liquid and solid lipids ranging from 0.01: 1 to 1: 100 of the total lipid carrier formulation.

[052] Liquid lipids are selected from the group consisting of unsaturated fatty acids, such as caprylic and caprylic acid glycerol triesters. And solid lipids are selected from the group consisting of saturated fatty acids, such as cetyl palmitate.

The aqueous phase comprises 0.05 to 10% of at least one nonionic surfactant such as polysorbate 80, soy or egg phospholipids, poloxamer 188, poloxamer 407, TPGS (d-alpha tocopheryl polyethylene glycol 1000), Kolliphor ELP (macrogolglyceryl ricinoleate - polyoxyl 35 castor oil) and Kolliphor HS 15 (polyoxyl hydroxystearate 15) diluted in ultrapure water. Alternatively, it may contain from 0.001 to 10% anionic surfactant such as sodium dodecyl sulfate.

Preferably, the aqueous phase comprises 2.5% poloxamer 188 and poloxamer 407, 1.5% polysorbate 80, 0.5% sodium dodecyl sulfate, and 100% ultrapure water of the total lipid carrier formulation.

Also, the amphiphilic cationic lipid nanostructured compound has zeta potential ranging from +5 to +65 mV,

preferably +20 mV; and average hydrodynamic diameter (DHM) ranging from 100 to 600 nm, preferably 200 nm.

In addition, the present invention also relates to the amphiphilic cationic polymeric nanostructured compound obtained as an alternative embodiment of the present process, which comprises at least least two drugs, one hydrophobic and one hydrophilic, associated with: 0.5 to 20% organic phase (without the organic solvent to be evaporated);

from 80 to 99.5% aqueous phase; and

from 0.001 to 10%, preferably 0.2% chitosan;

[056] Said hydrophobic drug is selected from the group consisting of hydrophobic or lipophilic active substances such as immunosuppressants (cyclosporine), prostaglandins (latanoprost, bimatoprost and travoprost), non-spheroidal and spheroidal anti-inflammatory agents, preferably dexamethasone acetate; antioxidants such as carotenoids, α-, β-, γ-, δ-tocopherol and a-, β-, γ-, δ-tocotrienol, retinoic acid, retinol and its esters, ubiquinol, ubiquinone, glutathione in their reduced form, flavonoids lipoic acid and active components of biotechnological origin such as etanercept, pegaptanib, ranibizumab and bevacizumab; and cholinergic drugs such as pilocarpine, ezerin and neostigmine.

Said hydrophilic drug is selected from the group consisting of hydrophilic cationic substances, cationic peptides, cationic antimicrobial peptides such as polymyxin B sulfate, vancomycin, gramicidine, bacitracin; other cationic antimicrobial peptides; and benzalkonium chloride, preferably polymyxin B sulfate.

[058] The organic phase comprises from 0.0001 to 10%,

preferably 0.10% of the lipid-associated hydrophobic or lipophilic active substance or drug, wherein the lipids are selected from the groups consisting of liquid lipid (oil) at a concentration of 1 to 20% or a mixture of liquid lipids in the proportion ranging from 0.01: 1 to 1: 10, or a liquid mixture of liquid and solid lipids in a ratio ranging from 0.01: 1 to 1: 100; from 0.01 to 20% polymer selected from the group consisting of lactic polyacid, copolymers derived from lactic and glycolic acids, aliphatic polyanhydrides, polymer derived from lactones, preferably poly (s-caprolactone; from 0.01 to 10% nonionic surfactants such as sorbitan monostearate; from 0.5 to 10% unsaturated fatty acids such as caprylic and caprylic acid glycerol triesters; 0.0001 to 5% hydrophilic drug, and 5 to 30% organic solvent selected from the group consisting of acetone, ethanol and methanol, preferably acetone.

[059] Liquid lipids are selected from the group consisting of unsaturated fatty acids, such as caprylic and caprylic acid glycerol triesters. And solid lipids are selected from the group consisting of saturated fatty acids, such as cetyl palmitate.

The aqueous phase comprises from 0.1 to 10% of at least one nonionic surfactant such as polysorbate 80, poloxamer 188 and TPGS (d-alpha tocopheryl polyethylene glycol 1000), Kolliphor ELP (macrogolglyceryl ricinoleate - polyoxyl castor 35), Kolliphor HS 15

(polyoxyl hydroxystearate 15) diluted in purified water.

[061] Also, the amphiphilic cationic polymeric nanostructured compound has zeta potential ranging from +15 to +55 mV,

preferably +30 mV; and average hydrodynamic diameter (DHM) ranging from 150 to 950 nm, preferably 300 nm.

Therefore, furthermore, the present invention relates to the use of said amphiphilic cationic lipid and amphiphilic cationic polymeric nanostructured compounds as a carrier of at least two drugs, one hydrophilic and the other hydrophobic, for the development of pharmaceutical or cosmetic products in which they are present. cationic characteristic is desirable, preferably ophthalmic products.

[063] For example, the present invention may be used for the development of medicaments for neglected diseases.

For a better understanding of the invention, the present invention is explained in more detail by the following examples, but is not limited by said examples.

Examples of the invention: Nanostructured lipid carriers comprising dexamethasone acetate, polymyxin B and guitosan chloride coated (CLN-Dexa-Poly-Prot):

- Preparation of nanostructured lipid carriers (CLN):

Amount equal to 100.0g of nanostructured lipid carriers (CLN) was first obtained by transferring exact 6.0g of cetyl palmitate (saturated fatty acid); 4.0 g caprylic and caprylic acid glycerol triesters (fatty acid

unsaturated); and 0.25 g of soy lecithin to a 150 ml beaker. The mixture (Oily Phase) was heated to 85 ° C using magnetic stirrer at 300 rpm until complete dissolution.

To another 150 ml beaker, 2.5 g of Pluronic ^® F68 and 1.5 g of Tween 80 ^® and 85.8 g of Milli-Q ^® ultrapure water (Aqueous Phase) were transferred. This solution was also heated to 85 ° C and

homogenized on shaker at 300 rpm until complete dispersion of surfactants.

The aqueous phase was dispersed in the lipid phase under mechanical agitation (8000 rpm) using a stirrer for 5 minutes to form a pre-emulsion which was subjected to high pressure homogenization using four successive cycles at 600 bar.

Table 1 - Nanostructured lipid carrier (CLN-Dexa) formula containing anionic surfactant (sodium dodecyl sulfate):

Components% (P / P)

Glycerol triesters of capric acids

4.00 and caprylic (unsaturated fatty acid)

Phase

Cetyl Palmitate (fatty acid

Oily 6.00

saturated)

Soy Lecithin 0.25

Dexamethasone Acetate 0, 10

Phase Tween ^® 80 1, 50 Pluronic ^® F68 2.50

Sodium Dodecyl Sulphate 0.50

Milli-Q ^® qsp 100 Ultrapure Water

Table 2 - Nanostructured lipid carrier formula (CLN-Dexa) containing nonionic surfactants (tween ^® 80 and pluronic f68 ^® ).

Components% (P / P)

Glycerol triesters of capric acids

Phase 4.00

and caprylic

Oily

Cetyl Palmitate 6.00

Dexamethasone Acetate 0, 10

Tween 80 ^® 1,50

Phase

Pluronic ^® F68 2.50 Aqueous

Milli-Q ^® qsp 100 Ultrapure Water

- Preparation of polymyxin B sulfate solutions:

Subsequently, three stock solutions of polymyxin B sulfate were prepared by transferring 123.37 mg, 92.52 mg and 61.68 mg (polymyxin B sulfate potency: 8106 Ul / mg) to volumetric flasks. 10.0 mL, the volume being supplemented with Milli-Q ^® ultrapure water. In this way, final concentrations were obtained,

respectively 100,000 IU / mL (Solution A), 75,000 IU / mL (Solution B) and 50,000 IU / mL (Solution C).

- Preparation of CLN-Dexa + Poly:

Assay matrix for incorporation of polymyxin B sulfate into nanostructured lipid carriers containing dexamethasone acetate are shown in Table 1. The variables

Independent variables were polymyxin B sulfate concentration (three levels: 5,000, 7,500 and 1,000 IU / mL) and incubation time, in hours, of CLN-Dexa in polymyxin B sulfate solution (three levels: 4, 6 and 8 hours). Table 3 - Assay matrix for incorporation of polymyxin sulfate

B in CLN-Dexa. Concentration of

Preparation Polymyxin Sulphate Time (hours)

B (IU / mL)

1 7,500 6

2 5,000 8

3 10,000 8

4 1 1,000 6

5 5,000 4

6 10,000 4

7 7,500 6

9 7.500 3h 10min

10 7.500 8h 50min

12 4,000 6

13 0 0

First, a 1: 5 dilution of previously prepared CLN-Dexa was made in MilliQ® ultrapure water.

Aliquot equal to 9 ml of CLN-Dexa was transferred to 25 ml beakers. For preparations number 1, 7, 9 and 10, a volume of 1.0 mL of solution B was used. Similarly, for preparations 2 and 5, 1.0 mL of solution C was used. of preparations 3 and 6, the 1.0 mL volume of solution A was transferred

Solution A was also used for preparation 4, but a volume of 1.1 mL in 8.9 mL of CLN-Dexa was used.

Finally, preparation 12 was obtained by adding 0.8 mL of Solution C to 9.2 mL of CLN-Dexa.

[073] The addition of solutions A, B and C was by

dripping, with stirring using magnetic bar and magnetic stirrer at 200 rpm. After addition of the solutions, the stirring period continued as described in Table 1.

- CLN-Dexa + Poly coating using guitosan chloride: In 9.0 ml aliquots of all preparations obtained were added 1.0 ml Protasan UP CL 1 14 ^® dispersion (chitosan chloride) 2.0% (w / w) in MilliQ ^® ultrapure water , obtaining a final volume of 10.0 mL, by dripping and stirring using a magnetic bar and magnetic stirrer at 200 rpm.

After addition of the dispersion, the stirring period was continued for 2 hours and the preparations were kept in a refrigerator (5 ° C). The final concentration of Protasan UP CL 1 14 ^® in the preparations was 0.2% (w / v). The preparations thus obtained were named CLN-Dexa + Poli-Prot.

[076] Evaluations of the average hydrodynamic diameter (nm) of CLN-Dexa + Poly + Prot were performed immediately after preparation and after 7, 14, 21, 28, 35, 42, 49 and 56 days.

[077] Analysis of the main effects for the mean hydrodynamic diameter (nm) of particulate matter and CLN-Dexa-Poli-Prot, as well as the regression analysis of polymyxin B sulfate concentration as a function of zeta potential was performed by Minitab® 16 software (variable y, mean diameter values, and variable x, polymyxin B sulfate concentration).

Determination of ZN Potential of CLN-Dexa-Poly-Prot:

Following the incorporation of polymyxin B sulfate into the Dexa-CLN, the zeta potential was changed from -18.6 mV (De-CLN) to -2.00 mV (10,000 IU / mL) (Table 4). This result demonstrated the incorporation of cationic surfactant (polymyxin B sulfate) in the carrier. The relationship between polymyxin B sulfate concentration and zeta potential resulted in simple linear regression (Figure 1).

Table 4 - Amphiphilic lipophilic nanostructured compound: average hydrodynamic diameter (DHM), Zeta Potential (mV) and

Polydispersity.

Weeks

Formulation

1 ^o 2 ^o 3 ^o 40 220.2 ± 224.5 ± 218.7 ± 220.0 ±

DHM (nm)

2.150 1.539 1.002 2.205

-4.37 ± -4.93 ± -4.46 ± -6.63 ±

25LP PZ (mV)

0.159 0.186 0.173 0.191

0.135 ± 0.098 ± 0.151 ± 0.120 ±

IP

0.038 0.024 0.003 0.027

219.3 ± 220.4 ± 222.0 ± 218.7 ±

DHM (nm)

3.101 3.534 2.747 0.6658

-3.78 ± -2.96 ± -3.24 ± -4.75 ±

50LP PZ (mV)

0.237 0.0493 0.181 0.212

0.114 ± 0.143 ± 0.123 ± 0.117 ±

IP

0.058 0.017 0.028 0.021

219.8 ± 223.4 ± 219.6 ± 220.1 ±

DHM (nm)

5,163 4,631 2,364 4,258

-2.03 ± -2.37 ± -3.03 ± -3.75 ±

75LP PZ (mV)

0.213 0.457 0.0802 0.229

0.125 ± 0.135 ± 0.118 ± 0.143 ±

IP

0.046 0.008 0.017 0.020

221.4 ± 223.9 ± 218.5 ± 218.7 ±

DHM (nm)

2,762 1,750 2,312 1,852

-2.00 ± -1.94 ± -2.19 ± -2.80 ±

100LP PZ (mV)

0.260 0.275 0.254 0.342

0.127 ± 0.140 ± 0.129 ± 0.127 ±

IP

0.019 0.016 0.019 0.014

223.2 ± 225.8 ± 224.5 ± 225.2 ±

DHM (nm)

4.060 1.680 1.270 2.684

-18.6 ± -16.0 ± -15.7 ± -15.5 ±

White PZ (mV)

0.265 0.945 0.950 0.503

0,136 ± 0,137 ± 0,129 ± 0,135 ±

IP

0.010 0.027 0.007 0.045 LP: lipid nanostructured compound containing dexamethasone acetate and polymyxin B at concentrations 25, 50, 75 and 100 (2,500 IU / mL; 5,000 IU / mL; 7,500 IU / mL and 10,000 IU / mL)

[079] CLN-Dexa + Poli was negatively charged in the concentration range of the antimicrobial agent used (2,500 to 10,000 IU / mL). Such behavior allowed the coating of the carrier (CLN-Dexa + Poly) using cationic polymer (0.15% w / w).

[080] Thus, Table 4 presents the results of dexamethasone acetate-containing nanostructured lipid carriers (CLN-Dexa) prepared after incorporation of polymyxin B sulfate employing concentrations of 2,500, 5,000, 7,500 and 10,000 IU / mL (Table 4). .

[081] Incorporation of the antimicrobial agent (Sulfate of

Polymyxin), which has a surfactant characteristic (cationic surfactant), was confirmed by simple regression analysis (Figure 1). Like polymyxin B sulfate, other cationic surfactants can be used such as benzalkonium chloride. After incorporation of polymyxin B sulfate into the CLN-Dexa, the zeta potential was changed from -18.6 mV (CLN-Dexa) to -2.00 mV (10,000 IU / mL) (Table 4). This result demonstrated the incorporation of cationic surfactant (polymyxin B sulfate) in the carrier.

[082] Thus, it was possible to obtain, by electrostatic interaction between CLN-Dexa + Poly negative and chitosan chloride (Protasan®). Potential zeta values after coating are shown in Table 5. These values were approximately +20 mV and showed no significant differences when different concentrations of polymyxin B sulfate (2,500, 5,000, 7,500 and 10,000 IU / mL) were used (Tables 6 and 7).

Table 5 - Amphiphilic Cationic Lipid Nanostructured Compound: Average Hydrodynamic Diameter (DHM), Zeta Potential (PZ) and Polydispersity Index (IP): 0.15% (w / w) Chitosan Chloride.

0.121 ± 0.138 ± 0.143 ± 0.085 ±

IP

0.040 0.015 0.015 0.008

LP: lipid nanostructured compound containing dexamethasone acetate and polymyxin B at concentrations 25, 50, 75 and 100 (2,500 IU / mL; 5,000 IU / mL; 7,500 IU / mL and 10,000 IU / mL) and 0, 15% (p / p) of

Protasan SR.

Table 6 - Mean and standard deviation (SD) of CLN-zeta potential (mV)

Dexa + Poly-Prot: 0.15% (w / w) chitosan chloride.

Table 7 - Analysis of variance for zeta potential (mV) of Dexa + Poly + Prot CLN prepared with 2,500 IU / mL (25LP), 5,000 IU / mL (50LP), 7,500 IU / mL (75LP) and 10,000 (IU) / ml) (100LP): 0.15% (w / w) chitosan chloride.

- Determination of the average hydrodynamic diameter (DHM) of CLN-Dexa-Poly-Prot:

Regarding the average hydrodynamic diameter (DHM), an increase of this characteristic was observed after the incorporation

employing polymyxin B sulfate (Table 8). In the stage of

In the protasan® coating, DHM was dependent on the concentration of polymyxin sulfate as shown in Table 9 and Figure 2. Table 8 - Average hydrodynamic diameter (nm) of CLN-Dexa and CLN-Dexa + Poly employing 1,000, 2,000, 3,000,

4,000, 7,500 and 10,000 IU / mL for a period of 56 days kept refrigerated (5 ° C).

Table 9 - Average hydrodynamic diameter (nm) of CLN-Dexa-Prot and CLN-Dexa + Poly-Prot employing 1,000, 2,000,

3,000, 4,000, 7,500 and 10,000 IU / mL for a period of 56 days kept refrigerated (5 ° C).

In addition, the encapsulation efficiency of dexamethasone acetate in the CLN was greater than 90% (w / v) as shown in Table 10.

Table 10 - Amount of free and encapsulated dexamethasone acetate and encapsulation efficiency in CLN-DEXA:

- Determination of the Inhibitory Minimum Concentration (MIC) of the Dexa-Poli-Prot CLN:

Two microplates containing 96 wells were divided into eight columns and twelve rows. Antibiotic culture medium No. 10 was used, which was sterilized using pressurized saturated water vapor at 121 ° C for 15 minutes in a vertical autoclave.

[086] An aliquot of 192 μΙ_ from the medium was transferred to all wells in the first column and 100 μί to the others. 8 μΙ_ of the polymyxin B sulfate standard from the CLN-Dexa + Poly (CNL-Dexa + 5,000, CLN-Dexa + 7,500, CLN-Dexa + 10,000), ultrafiltered (UF-CLN-Dexa +) samples were added. 5,000, UF-CLN-Dexa + 7,500 and UF-CLN-Dexa + 10,000) and from the CLN-Dexa to the first well of each row, totaling 200 μΙ_.

[087] Then 100 μΙ_ were transferred to the next well of each row and so on to obtain serial 1: 2 ratio dilutions. 100 μΙ_ aliquot was discarded from the last well. Polymyxin B sulfate standard concentrations and CLN-Dexa + Poly concentrations were 4 IU / mL to 0.002 IU / mL. Ultrafiltrate concentrations were estimated from the polymyxin B sulfate standard and were used as obtained (undiluted). [088] Negative control was performed by adding 200 μΙ_ of uninoculated culture medium in a row of the microplate; Positive media control was obtained by transferring 100 μΙ_ from the uninoculated culture medium and 100 μί from the inoculated medium to the well. Incubation was incubated at 37 ± 0.5 ° C for 24 hours. After reading this result, 50 μ 50_ of the triphenyl tetrazolium chloride solution was added and incubated for 2 hours in an oven at 37 ± 0,5 ° C. The plates were read visually, observing the appearance of reddish color indicating microbial growth.

[089] Polymyxin B sulfate concentrations in the samples (5,000 IU / mL, 7,500 IU / mL and 10,000 IU / mL) were estimated from the MIC obtained for the polymyxin B sulfate standard.

[090] IMC values were calculated based on

concentration of the last well, without microbial growth, multiplied by the dilution factor.

[091] Thus, with reference to antimicrobial activity, CLN-Dexa + Poli-Prot was approximately 2-fold more effective than free polymyxin sulfate as shown in Table 1 1.

Table 11 - Minimum Inhibitory Concentration (MIC) of Polymyxin B Sulfate and CLN-Dexa + Poly-Prot diluted in 10% (w / v) phosphate buffer pH 6.0 and in ultra pure Milli Q® water against Bordetella bronchiseptica ATCC 4617.

Nanostructured polymeric vesicles comprising dexamethasone acetate, polymyxin B and guitosan chloride-coated (VP-Dexa-Poli-Prot):

- Preparation of polymeric vesicles containing dexamethasone acetate:

[092] To obtain the dexamethasone-containing vesicles, the preformed polymer precipitation method was used which consists, under stirring, of the organic phase containing the poly-ΝοΝ (ε-caprolactone) acid (PCL), acetone, dexamethasone and the capric / caprylic acid triglyceride mixture over an aqueous phase containing polysorbate 80 (Table 12). Solvent and part of the water were removed using a rotary evaporator and the volume of the suspension was adjusted in a volumetric flask.

Table 12 - Formula of dexamethasone acetate-containing polymeric vesicles:

ORGANIC PHASE

Volume (ml_)

Component

10 50 100

Weight (g)

Poly (s-caprolactone) 0.000 0.5000 1, 0000

Sorbitan Monostearate (Span 60) 0.0770 0.3850 0.7770

Glycerol triesters of capric acids and

0.3333 1, 6665 3.3333 caprylic (Myglyol ^® )

Dexamethasone Acetate 0.0100 0.0500 0, 1000

Acetone P.A. (ml) 27.0 135.0 270.0

WATER PHASE

Volume (ml_)

Component

10 50 100

Weight (g)

Polysorbate 80 0.0770 0.3850 0.7770 Purified Water (mL) 53.0 265.0 530.0

- Preparation of polymyxin B sulfate solutions:

Three stock solutions of polymyxin B sulfate were prepared by transferring 123.37 mg, 92.52 mg and 61.68 mg (potency of polymyxin B sulfate: 8106 Ul / mg) to volumetric flasks. 10.0 mL, the volume being supplemented with Milli-Q® ultrapure water. In this way, final concentrations were obtained,

- Incorporation of polymyxin B sulphate in VP-Dexa:

[094] 1: 5 dilution of VP-Dexa prepared according to item A was performed in MilliQ® ultrapure water. 1 mL aliquot of solutions A, B and C were added to 9 mL of diluted VP-Dexa resulting in final polymyxin B sulfate concentrations of 10,000, 7,500 and 5,000 IU / mL respectively. 0.5 mL aliquot of solution C was added to 9 mL of VP-Dexa resulting in final concentrations of 2500 IU / mL

[095] The addition of solutions A, B and C was by

with stirring using magnetic bar and RTC IKA® magnetic stirrer at 200 rpm. After addition of the solutions, the stirring period continued for a period of 60 minutes.

- Coating of vp-dexa + poly using Protasan UP CL

114 ^® :

In 9.0mL aliquots of all preparations obtained as described above were added 1.0 mL of 1.5% (w / w) Protasan UP CL 1 14® (chitosan chloride) dispersion in water. Ultrapure MilliQ®, obtaining a final volume of 10.0 mL by dripping and stirring using a magnetic bar and RTC IKA® magnetic stirrer at 200 rpm. After addition of the dispersion, the stirring period was continued for 30 minutes and the preparations were kept in a refrigerator (5 ° C). The final concentration of Protasan UP CL 14Â® in the preparations was 0.15% (w / v). The preparations thus obtained were named VP-Dexa + Poli-Prot.

[097] Analysis of the main effects for the mean hydrodynamic diameter (nm) values of VP-Dexa-Poli-Prot as well as the regression analysis of polymyxin B sulfate concentration as a function of zeta was performed by means of Minitab ^® 16 software (variable y, mean diameter values, and variable x, polymyxin B sulfate concentration).

- Determination of mean hydrodynamic diameter (DHM) and polydispersity index (IP):

The average hydrodynamic particle diameter (DHM) and polydispersity index (IP) of dexamethasone acetate-containing polymeric vesicles (VP-Dexa) were determined by

dynamic light scattering by diluting the suspension samples 1: 800 in freshly prepared Milli-Q ^® water and observing the scattered light at an angle of 90 °. Particle size distribution, polydispersity index and zeta potential were

determined using Zetasizer Nano ZS90 equipment.

[099] Table 13 shows the VP-Dexa + Poly DHM, IP and PZ using different concentrations of polymyxin B sulfate (2,500, 5,000, 7,500 and 10,000 IU / mL) after coating employing 0, 15% (w / v ) from Protasan® (VP-Dexa + Poly + Prot).

Table 13 - Average hydrodynamic diameter (DHM), polydispersity index (IP) and zeta potential (PZ) of the Protasan® (Chitosan) coated VP-Dexa + Poly.

Weeks

Formulations 1 2 3 4

DHM (n 658.0 ± 6.3 689.5 ± 38.0 705.4 ± 22, 1 623.9 ± 29,

VP-Dexa + Polym) + 22.2 ± 1.0 + 24.9 ± 1.7 +24.1 ± 0.76

25P PZ (mV) 0.420 ± 0.1 8 0.390 ± 0.02 + 24.0 ± 0.4 IP 0.427 ± 0.12 0

0.313 ± 0.0 6

685.1 ±

DHM (n 617.7 ± 29.6 720.8 ± 52.3 27.4

781.4 ± 53.6

VP-Dexa + Poly) + 24.2 ± 2.2 + 22.3 ± 0.6 + 27.0 ±

+23.0 ± 1.4

50P PZ (mV) 9 5 1, 37

0.408 ± 0.06

IP 0.438 ± 0.14 0.648 ± 0.09 0.318 ±

0.04

727.4 ±

DHM (n 953.4 ± 54.3 812.7 ± 26.4 23.9

830.1 ± 22.2

VP-Dexa + Polym) + 22.4 ± 0.6 +21.8 ± 0.5 + 23.7 ±

+ 23.6 ± 2.0

75P PZ (mV) 8 7 1, 27

0.530 ± 0.07

IP 0.665 ± 0.08 0.323 ± 0.02 0.454 ±

0.06

740.7 ±

DHM (n 6.15)

992.4 ± 44.3 789.0 ± 1 1, 5 776, 1 ± 30.8

VP-Dexa + Polym) +21.7 ±

+ 23.4 ± 2.4 + 23.5 ± 2.5 +25, 1 ± 2.6

100P PZ (mV) 0.32

0.630 ± 0.15 0.681 ± 0.17 0.393 ± 0.07

IP 0.409 ±

0.07

[100] Figures 3 and 4 show the linear regression between DHM and polymyxin B sulphate concentration and between zeta potential and polymyxin B sulphate concentration of VP-Dexa + Poly,

respectively.

[101] The results revealed that the mean hydrodynamic diameter (DHM) was dependent on the concentration of polymyxin B sulfate employed (2,500, 5,000, 7,500 and 10,000 IU / mL). This relationship

presented linear function in the working range, with adjusted R ² equal to 86.5%. Thus, the higher the sulfate concentration of

polymyxin B, the higher was the observed DHM being 339.8 nm for the lowest concentration (2,500 IU / mL polymyxin B sulfate) and 445,8nm (10,000 IU / mL polymyxin B sulfate). DHM values are stable for 4 weeks under refrigeration.

[102] Similar result was observed for zeta potential (PZ). Similarly, a linear relationship was observed between this electrical characteristic and the concentration of polymyxin B. This ratio showed adjusted R ² equal to 89.9% and the highest PZ value was observed after the coating of the polymeric vesicles containing

dexamethasone (VP-Dexa) employing the highest concentration of polymyxin B sulfate (10,000 IU / mL). Thus, PZ values were changed from -37 mV (without addition of polymyxin B sulfate) to -19 mV (after addition of 10,000 IU / mL polymyxin B sulfate).

[103] Figures 4 and 5 show the analysis of variance for DHM and PZ of VP-Dexa + Poli + Prot, respectively employing 0.15% (w / v) Protasan®.

[104] Regarding the coating of VP-Dexa + Poly with chitosan (Protasan®), no significant differences were observed in the DHM and PZ of VP-Dexa + Poly + Prot employing different

polymyxin B sulfate concentrations (2,500, 5,000, 7,500 and 10,000 IU / mL) (Figures 5 and 6). Thus, it can be inferred that these characteristics are dependent on the concentration of chitosan used in the coating of VP-Dexa + Poly. Thus, the VP-Dexa + Poli DHM values, which ranged from 339.8 to 445.8 nm, for the different concentrations of polymyxin B sulfate (Table 13), after coating using Protasan ^® , presented DHM values. between 623.9 and 740.7 nm (not significantly different DHM).

[105] pH analysis revealed a value between 4.5 and 6.5 for formulations before and after coating using polymyxin B sulfate and Protasan ^® . The encapsulation efficiency of dexamethasone acetate was 97.2% (w / v).

[106] Therefore, from the tests performed, the present invention provides lipid or polymeric nanostructured compounds comprising hydrophilic and hydrophobic drugs, therefore with amphiphilic characteristic, and cationic due to chitosan chloride.

[107] Those skilled in the art will enhance the knowledge presented herein and may reproduce the invention in the embodiments disclosed and in other embodiments within the scope of the appended claims.

Claims

1 . Process for obtaining amphiphilic cationic lipid and amphiphilic cationic polymeric nanostructured compounds characterized by the following steps:

c) Incorporating SC into the formulation obtained in step "a"; and d) Coat the formulation obtained in step "c" with chitosan of molecular weight ranging from 50000 to 400000 g / mol and degree of

deacetylation> 80%.

Process according to Claim 1, characterized in that said drug or active substance is selected from the group consisting of hydrophobic or lipophilic active substances such as immunosuppressants such as cyclosporine; antiangiogenics and prostaglandins such as latanoprost, bimatoprost and travoprost; non-spheroidal and spheroidal anti-inflammatory, preferably dexamethasone acetate; antioxidants such as carotenoids, α-, β-, γ-, δ-tocopherol and a-, β-, γ-, δ-tocotrienol; retinoic acid, retinol and their esters; ubiquinol, ubiquinone, glutathione in their reduced form; flavonoids, lipoic acid and active components of biotechnological origin such as etanercept, pegaptanib, ranibizumab and bevacizumab; and cholinergic drugs such as pilocarpine, ezerin and neostigmine.

Process according to claim 1, characterized in that said cationic substance (SC) is selected from the group consisting of hydrophilic drugs, cationic peptides such as polymyxin B sulfate, vancomycin, gramicidine, bacitracin and other cationic antimicrobial peptides, benzalkonium chloride and other quaternary ammoniums, preferably polymyxin B sulfate.

Process according to Claim 1, characterized in that, in step "a", the preparation of the lipid carriers is obtained by heating the oil phase and the aqueous phase separately at a temperature ranging from 55 to 90 ° C,

Process according to Claim 4, characterized in that the oil phase comprises from 0.0001 to 10%,

preferably 0.10% of the lipid-associated hydrophobic or lipophilic active substance or drug, wherein the lipids are selected from the groups consisting of a liquid lipid (oil) or liquid lipid mixture in a ratio ranging from 0.01: 1 to 1: 10, and solid lipid or a mixture of solid lipids in the ratio ranging from 0.01: 1 to 1: 100, a mixture of liquid and solid lipids in the ratio ranging from 0.01: 1 to 1: 100, only one liquid lipid at a concentration of 1 to 20%, or only one solid lipid at a concentration of 1 to 20%; of the total formulation of lipid carriers.

Process according to Claim 5, characterized in that the liquid lipids are selected from the group consisting of unsaturated fatty acids, such as glycerol triesters of capric and caprylic acids; and solid lipids are selected from the group consisting of saturated fatty acids, such as cetyl palmitate.

Process according to Claim 4, characterized in that the aqueous phase comprises at least one nonionic surfactant selected from the group consisting of polysorbate 80, soybean or egg phospholipids, poloxamer 188, poloxamer 407 and TPGS (d- alpha tocopheryl polyethylene glycol 1000), macrogolglyceryl ricinoleate / castor oil polyoxyl 35 or polyoxyethyl hydroxystearate 15, diluted with ultrapure water; alternatively, it contains an anionic surfactant such as sodium dodecyl sulfate.

Process according to Claim 7, characterized in that the aqueous phase preferably comprises from 0.05 to 10%, more preferably 2.5% of poloxamer 188 and poloxamer 407; from 0.05 to 10%, more preferably 1.5% polysorbate 80; from 0.001 to 10%, more preferably 0.5% sodium dodecyl sulfate; and qsp 100% ultrapure water from the total formulation of lipid carriers.

Process according to Claim 4, characterized in that the aqueous phase is dispersed in the oil phase under mechanical agitation ranging from 1000 to 25000 rpm, preferably 8000 rpm for a period of time ranging from 1 to 10 min. preferably 5 minutes to form a preemulsion which is homogenized using 1 to 6 successive cycles,

preferably 3 cycles, the pressure ranging from 200 to 1000 bar preferably 600 bar.

Process according to Claim 1, characterized in that, in step "b", the cationic solution containing the hydrophilic drugs, the SC is diluted or not in a suitable container with ultrapure water in a proportion ranging from 1: 1 to 1: 5, preferably 1: 2, so that a concentration in this solution ranging from 50,000 to 1,000,000 IU / mL of the cationic substance is obtained.

1 1. Process according to Claim 1, characterized in that, in step "c", incorporation of the cationic substance at a concentration of 5 to 25,000 IU / mL, preferably 6000 IU / mL of SC obtained in step "b" is carried out. lipid carriers obtained in step "a" diluted or not in purified water in a ratio ranging from 1: 1 to 1: 10, preferably 1: 5; wherein such incorporation is performed slowly, preferably by dripping, and under agitation ranging from 50 to 300 rpm, preferably 100 rpm, over a period of time. ranging from 0.5 to 6 hours, preferably 2 hours.

Process according to claim 1, characterized in that in step "c" a lipid nanoparticulate compound is formed comprising a hydrophobic or lipophilic active substance or drug incorporated with at least one cationic substance, wherein the coating is controlled to produce particles with zeta potential ranging from -30 to -1 mV, depending on the concentration of the cationic substance (s) to be associated with the nanoparticles, which is accomplished by the concentration of the cationic substance used.

Process according to Claim 1, characterized in that, in step "d", it adds 0.001% to 10%, preferably 0.2% dispersion of 2.0% (w / w) chitosan in water. in the compound obtained in step "c" slowly, preferably by stirring dripping ranging from 50 to 500 rpm,

preferably 100 rpm, wherein after addition of the dispersion, the stirring period proceeds for a period of time ranging from 0.5 to 8 hours, preferably 2 hours; and the preparations obtained are kept at room temperature or refrigerated ranging from 1 to 10 ° C, preferably 5 ° C, wherein the coating is controlled to produce particles with + 5 to + 65 mV zeta potential.

Process according to Claim 1, characterized in that, in an alternative embodiment, step "a" is to prepare polymeric vesicles comprising one liquid lipid or two or more liquid lipids, or liquid mixture of liquid lipid and one. solid lipid, or liquid mixture of two or more liquid lipids and solid lipids, comprising at least one hydrophobic or lipophilic active substance or drug.

Process according to Claim 14, characterized in that the preformed polymer precipitation method comprising the addition of the organic phase comprising polymer, one or more lipids, the organic solvent, the nonionic surfactant and the lipophilic drug at a temperature below 60 ° C,

preferably 45 ° C, under agitation between 50 and 300 rpm,

preferably at 100 rpm, for the time required for complete polymer solubilization, between 15 and 45 minutes, over the aqueous phase, whereupon the solvent and part of the water is removed using a rotary evaporator and the volume of the suspension is adjusted. with addition of purified water.

Process according to Claim 15, characterized in that the aqueous phase comprises from 0.1 to 10% of at least one nonionic surfactant selected from the group consisting of polysorbate 80, poloxamer 188 and TPGS (d-alpha tocopheryl polyethylene glycol 1000), macrogol glyceryl ricinoleate / castor oil polyoxyl 35, polyoxyl hydroxystearate 15 diluted in purified water.

Process according to claim 15, characterized in that the organic phase comprises from 0.0001 to 10%,

preferably 0.10% of the lipid-associated hydrophobic or lipophilic active substance or drug, wherein the lipids are selected from the groups consisting of liquid lipid (oil) at a concentration of 1 to 20% or a mixture of liquid lipids in the proportion ranging from 0.01: 1 to 1: 10, or a liquid mixture of liquid and solid lipids in a ratio ranging from 0.01: 1 to 1: 100; from 0.01 to 20% of polymers selected from the group consisting of lactic polyacid, copolymers derived from lactic and glycolic acids, aliphatic polyanhydrides, polymer derived from lactones, preferably poly (s-caprolactone); from 5 to 50% organic solvent selected from the group consisting of acetone, ethanol and methanol, preferably acetone; from 0.0001 to 10% hydrophobic or lipophilic drug or substance; from 0.5 to 10% unsaturated fatty acids, such as capric and caprylic acid glycerol triesters; and from 0.5 to 10% nonionic surfactant consisting of sorbitan esters, preferably span 20, span 40, span 60 and span 80; of formulation total polymer vesicles.

Process according to Claim 17, characterized in that the liquid lipids are selected from the group consisting of unsaturated fatty acids, such as caprylic and caprylic acid glycerol triesters; and solid lipids are selected from the group consisting of saturated fatty acids, such as cetyl palmitate.

Process according to any one of claims 14 to 18, characterized in that, alternatively in step "c", a polymeric nanoparticulate compound comprising a hydrophobic or lipophilic drug or active substance incorporated with at least one cationic substance is formed.

Amphiphilic cationic lipid nanostructured compound obtained according to the process defined in any one of claims 1 to 13, characterized in that it comprises at least two drugs, one hydrophobic and one hydrophilic, associated with:

from 0.5 to 20% oil phase;

from 80 to 99.5% aqueous phase; and

from 0.001 to 10%, preferably 0.2% chitosan

(w / v%).

21 Amphiphilic cationic lipid nanostructured compound according to claim 20, characterized in that the hydrophobic drug is selected from the group consisting of hydrophobic or lipophilic active substances such as immunosuppressants such as cyclosporine; antiangiogenics and prostaglandins such as latanoprost, bimatoprost and travoprost; non-spheroidal and spheroidal anti-inflammatory, preferably dexamethasone acetate; and antioxidants such as carotenoids, α-, β-, γ-, δ-tocopherol and a-, β-, γ-, δ-tocotrienol, retinoic acid, retinol and their esters, ubiquinol, ubiquinone, glutathione in their reduced form. flavonoids, lipoic acid and active components of biotechnological origin such as etanercept, pegaptanib, ranibizumab and bevacizumab; and cholinergic drugs such as pilocarpine, ezerin and neostigmine.

Amphiphilic cationic lipid nanostructured compound according to claim 20, characterized in that the hydrophilic drug is selected from the group consisting of hydrophilic cationic substances, cationic peptides, cationic peptides.

antimicrobials such as polymyxin B sulfate, vancomycin, gramicidine, bacitracin; other cationic antimicrobial peptides; and benzalkonium chloride, preferably polymyxin B sulfate.

Amphiphilic cationic lipid nanostructured compound according to claim 20, characterized in that the oil phase comprises from 0.0001 to 10%, preferably 0.10% of the lipid-soluble hydrophobic drug, wherein the lipids are selected from groups consisting of a liquid lipid in the concentration of 0, 1 to 20% or a mixture of liquid lipids in the proportion ranging from

0.001: 1 to 1: 10; and solid lipid at a concentration of 1 to 20% or a mixture of solid lipids in a ratio ranging from 0.01: 1 to 1: 100; or a mixture of liquid and solid lipids ranging from 0.01: 1 to 1: 100 of the total lipid carrier formulation.

Amphiphilic cationic lipid nanostructured compound according to Claim 23, characterized in that the liquid lipids are selected from the group consisting of unsaturated fatty acids, such as caprylic and caprylic acid glycerol triesters; and solid lipids are selected from the group consisting of saturated fatty acids, such as cetyl palmitate.

Amphiphilic cationic lipid nanostructured compound according to Claim 20, characterized in that the aqueous phase comprises from 0.05 to 10% of at least one nonionic surfactant selected from the group consisting of polysorbate 80, soybean phospholipids. or egg, poloxamer 188, poloxamer 407 and TPGS (d-alpha tocopheryl polyethylene glycol 1000), macroglyceryl ricinoleate / polyoxyl 35 castor oil and polyoxyl hydroxystearate 15 diluted in ultrapure water; where alternatively it contains from 0.001 to 1% anionic surfactant such as sodium dodecyl sulfate.

Amphiphilic cationic lipid nanostructured compound according to Claim 25, characterized in that,

preferably the aqueous phase comprises 2.5% poloxamer 188 and poloxamer 407; 1.5% polysorbate 80; 0.5% sodium dodecyl sulfate; and qsp 100% ultrapure water from the total formulation of lipid carriers.

Amphiphilic cationic lipid nanostructured compound according to any one of claims 20 to 26, characterized in that it has zeta potential ranging from +5 to +65 mV,

Amphiphilic cationic polymeric nanostructured compound obtained according to the process defined in any one of claims 1 to 19, characterized in that it comprises at least two drugs, one hydrophobic and one hydrophilic, associated with:

from 0.5 to 20% organic phase, without organic solvent; from 80 to 99.5% aqueous phase; and

from 0.001 to 10%, preferably 0.2% chitosan

(w / v%).

Amphiphilic cationic polymeric nanostructured compound according to claim 28, characterized in that the hydrophobic drug is selected from the group consisting of hydrophobic or lipophilic active substances such as immunosuppressants such as cyclosporine; antiangiogenics and prostaglandins such as latanoprost, bimatoprost and travoprost; non-spheroidal and spheroidal anti-inflammatory, preferably dexamethasone acetate; and antioxidants such as carotenoids, α-, β-, γ-, δ-tocopherol and a-, β-, γ-, δ-tocotrienol, retinoic acid, retinol and their esters, ubiquinol, ubiquinone, glutathione in their reduced form. , flavonoids, lipoic acid and active components of biotechnological origin such as etanercept, pegaptanib, ranibizumab and bevacizumab; and cholinergic drugs such as pilocarpine, ezerin and neostigmine.

Amphiphilic cationic polymeric nanostructured compound according to Claim 28, characterized in that the hydrophilic drug is selected from the group consisting of hydrophilic cationic substances, cationic peptides, cationic peptides.

antimicrobials, such as polymyxin B sulfate, vancomycin, gramicidine, bacitracin; other cationic antimicrobial peptides; and benzalkonium chloride, preferably polymyxin B sulfate.

31 Amphiphilic cationic polymeric nanostructured compound according to claim 28, characterized in that the organic phase comprises from 0.0001 to 10%, preferably 0.10% of the lipid-associated hydrophobic or lipophilic active substance or drug, wherein the lipids are selected from the groups consisting of liquid lipid (oil) at a concentration of 1 to 20% or a mixture of liquid lipids in a ratio ranging from 0.01: 1 to 1: 10, or a liquid mixture of liquid and solid lipids in a proportion ranging from 0.01: 1 to 1: 100; from 0.01 to 20% polymer selected from the group consisting of lactic polyacid, copolymers derived from lactic and glycolic acids, aliphatic polyanhydrides, polymer derived from lactones, preferably poly (s-caprolactone); from 0.01 to 10% nonionic surfactants such as sorbitan monostearate; from 0.5 to 10% unsaturated fatty acids, such as capric and caprylic acid glycerol triesters; from 0.0001 to 5% hydrophilic drug; and from 5 to 30% organic solvent selected from the group consisting of acetone, ethanol and methanol, preferably acetone.

Amphiphilic cationic polymeric nanostructured compound according to Claim 31, characterized in that the liquid lipids are selected from the group consisting of unsaturated fatty acids, such as caprylic and caprylic acid glycerol triesters; and solid lipids are selected from the group consisting of saturated fatty acids, such as cetyl palmitate.

Amphiphilic cationic polymeric nanostructured compound according to claim 28, characterized in that the aqueous phase comprises from 0.1 to 10% of at least one nonionic surfactant, such as polysorbate 80, poloxamer 188 and TPGS (d- alpha tocopheryl polyethylene glycol 1000), macrogol glyceryl ricinoleate / polyoxyl 35 castor oil and polyoxyl hydroxystearate 15 diluted in purified water.

Amphiphilic cationic polymeric nanostructured compound according to any one of claims 28 to 33, characterized in that it has zeta potential ranging from +15 to +55 mV,

Use of the amphiphilic cationic lipid nanostructured compound as defined in any one of claims 20 to 27, characterized in that it is a carrier of at least two drugs, one hydrophilic and one hydrophobic, in the preparation of pharmaceutical or cosmetic products wherein cationic characteristic is desirable, preferably ophthalmic products.

Use of the amphiphilic cationic lipid nanostructured compound according to claim 35, characterized in that it is in the preparation of a medicament for treating neglected diseases.

Use of the amphiphilic cationic polymeric nanostructured compound as defined in any one of claims 28 to 34, characterized in that it is a carrier of at least two drugs, one hydrophilic and one hydrophobic, in the preparation of pharmaceutical or cosmetic products wherein cationic characteristic is desirable, preferably ophthalmic products.

Use of the amphiphilic cationic polymeric nanostructured compound according to claim 37, characterized in that it is in the preparation of a medicament for treating neglected diseases.