BRPI0206723B1 - LIQUID-CRYSTALLINE PHASES (LAMELAR AND CUBIC) AS A RELEASE SYSTEM FOR TOPIC USE OF PRODUCTS: 5-AMINOLEVULINIC ACID (5-ALA) AND ITS METHOD, FUTY, DECTIL, FETHYL, OCTIL AND DECIL FITTED ESTERS ) AND / OR PHOTODIAGNOSIS (FD) - Google Patents

Abstract

"Liquid-crystalline (lamellar and cubic) phases as a delivery system for topical use of prodrugs: 5-aminolevulinic acid (5-ala) and their methyl, butyl, hexyl, octyl and decyl esters, used in photodynamic therapy ( tfd) and / or photodiagnosis (fd) ". The liquid-crystalline, lamellar and cubic phases mentioned in said patent are essentially comprised of: 95 to 60% by weight, based on the total weight of the composition, of a monoglyceride mixture called monolein, containing an 18-carbon acyl chain, and 5 to 40% by weight of water. The present composition has, within the temperature range between 20 and 75Â ° C, the texture of a liquid-crystalline gel preferably having the cubic phase. The prodrugs incorporated into said lamellar and cubic phases are 5-aminolevulinic acid (5-ala) and its methyl, butyl, hexyl, octyl and decyl esters, or any other ester derived from 5-ala, at a ratio of 0.2 2% relative to the total composition or any other composition, so that the presence of the prodrug does not cause any change in the structure of said phases.

Description

(54) Title: LIQUID-CRYSTALLINE PHASES (LAMELLARY AND CUBIC) AS A RELEASE SYSTEM FOR TOPICAL USE OF PRO-DRUGS: 5-AMINOLEVULINIC ACID (5-ALA) AND ITS DERIVED METHODS, BUTY, HEXILE, OIL, OCT, OCT IN PHOTODYNAMIC THERAPY (TFD) AND / OR PHOTODIAGNOSTIC (FD) (51) Int.CI .: A61K 31/16; A61P 17/02 (73) Holder (s): UNIVERSIDADE DE SÃO PAULO - USP (72) Inventor (s): ROZANE DE FÁTIMA TURCHIELLO; FLÁVIA CRISTINA BONILHAVENA; REGILENE STELUTI; ANTÔNIO CLÁUDIO TEDESCO; MARIA VITÓRIA LOPES BADRA BENTLEY

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Descriptive Report of the Invention Patent "Release system for topical use and process of obtaining Release system for topical use".

[01] The present invention is related to the field of drug delivery systems (pro-drugs), of topical application in Photodynamic Therapy (PDT) and / or for Photodiagnosis (PD). In particular, it relates to delivery systems using the lamellar and cubic liquid-crystalline phases to increase the cutaneous absorption of the pro-drugs 5-aminolevulinic acid (5-ALA) and its derivative esters: methyl, butyl, hexyl, octyl and decyl, or any other ester derived from 5-ALA.

[02] Photodynamic Therapy has received special attention and has been intensively investigated in recent years, as it is considered an extremely effective and selective technique in the destruction of dysplasias and carcinomas "in situ '". In comparison with other cancer treatment techniques, Photodynamic Therapy has the great advantage of being less traumatic for the patient, due to its localized action. The treatment involving Photodynamic Therapy is based on the systemic or topical administration of photosensitive compounds that have a selective retention by the cancer cells, and the subsequent illumination with adequate wavelength light. Photodynamic reactions give rise to free radicals, singlet oxygen ( ¹ O2) and other reactive oxygen species that lead to tumor necrosis or apoptosis.

[03] Currently, the prodrug 5-ALA, a precursor to the photosensitive compound protoporphyrin IX (PpIX), has been widely used for the treatment of neoplastic and non-neoplastic skin lesions, using topical Photodynamic Therapy. Naturally, the production of 5-ALA occurs through the conversion of glycine and succinyl CoA, being controlled by the action of the enzyme 5-ALA synthase. However, heme biosynthesis studies (formed by the complexation of Fe ²⁺ in PpIX by the enzyme

2+ ferroquelatase) revealed that when there is a natural accumulation of free heme (Fe ²⁺ not complexed), the enzyme 5-ALA synthase ceases to synthesize 5-ALA resulting in a negative feedback mechanism.

[04] Topically administered, excess "exogenous" 5-ALA has been shown to inhibit the action of the enzyme ferroquelatase resulting, again, in a negative feedback mechanism, since this enzyme fails to complex Fe ²⁺ in the PpIX. As a consequence, there is an accumulation of endogenous porphyrins, mainly PpIX, which is concentrated in cancerous tissue. Therefore, it can be said that the enzymes 5-ALA synthase and

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2/10 ferroquelatase are species that limit the reaction rate for the formation of 5ALA and heme, respectively. Thus, the treatment of neoplastic and non-neoplastic diseases has received special attention in relation to the use of 5-ALA as a precursor in the biosynthesis of PpIX, and its consequent application in topical Photodynamic Therapy and / or for Photodiagnosis.

[05] A series of studies using the prodrug 5-ALA as a precursor to the production of the photosensitive agent PpIX have shown encouraging results in the palliative treatment of solar keratoses and superficial skin tumors, including basal cell carcinoma (CBC), Bowen's disease, squamous cell carcinoma (SCC) and other skin tumors, obtaining good clinical responses and excellent cosmetic results. Clinical studies have shown a number of advantages in the application of 5-ALA in topical photodynamic therapy over common therapies, as it constitutes a non-invasive therapeutic modality (when compared to traditional cancer treatments such as: cryosurgery, surgical excision, radiotherapy, chemotherapy, curettage or electrodication). In addition, it produces a short period of photosensitization, disappearing within 24 hours after topical application of 5-ALA, limited only to mild inflammation at the application site. For cases of standard Photodynamic Therapy, involving intravenous application of photosensitive compounds, inflammatory and vascular reactions, such as: damage to endothelial cells, aggregation of blood cells, and decreased blood flow, among other symptoms, can occur, in addition to photosensitivity throughout the skin. . Another advantage of using 5ALA is that it can be used to treat multiple injuries in short periods of treatment, as well as injuries located in regions that require special care (eyes, nose and ear), where other treatment modalities can be problematic. .

[06] Despite the advantages of using 5-ALA as a precursor to PpIX production, the fact that it is zwitterionic at physiological pH and, therefore, has low lipid solubility, means that it has a limited penetration capacity through the stratum. horny. As a result, treatment using topical photodynamic therapy using 5ALA is restricted to superficial lesions around 2-3 mm, presenting a low response rate for nodular basal cell carcinoma. Consequently, to achieve clinically relevant levels of PpIX production, it is

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3/10 necessary to administer a high dose of 5-ALA, which led the researchers to study new conditions of application, providing an increase in its penetration and, therefore, in the concentration of PpIX in the tumor tissues. Among the strategies used so far to increase penetration through the stratum corneum and the concentration of the photosensitive compound (PpIX), we can mention the use of esters derived from 5-ALA, or the use of solutions containing dimethyl sulfoxide (DMSO) and acid tetracetic ethylenediamine (EDTA).

[07] In the present patent, we propose the use of a release system to increase the cutaneous absorption of 5-ALA prodrugs and their esters derived in the skin, thereby increasing the “in situ” accumulation of the photosensitive compound, PpIX, aiming greater efficiency in topical treatment through Photodynamic Therapy or for Photodiagnosis, since they are dependent on the concentration of PpIX formed in the target tissue. As a release system, we propose the lamellar and cubic liquid-crystalline phases formed by the monolein and water system.

[08] Glyceryl monolein or monoleate is a polar lipid capable of forming lyotropic liquid-crystalline phase gels when in contact with water. It is an amphiphilic compound that has an 18-carbon carbon chain containing a cis-type unsaturation on carbon 9, and this chain is linked to glycerol by an ester bond.

[09] The binary monolein and water system presents a very wide phase diagram with different liquid-crystalline phases such as the isotropic, lamellar, cubic and hexagonal phases. For proportions of water less than 14% by weight in relation to the total mixture, at room temperature, there is the presence of a lamellar phase structure, consisting of planar lipid bilayers with alternating layers of water, being optically anisotropic when viewed through the polarized light optical microscope. An increase in the proportion of water produces gels with the cubic phase structure, transparent, optically isotropic and viscous, called cubic phase gels. The structure of these gels consists of a lipid bilayer extended in three dimensions and separated by water channels, with a diameter of 5 nm with the system fully hydrated. These characteristics allow the incorporation of water-soluble or fat-soluble compounds as well as amphiphilic substances, without changing the structure of the cubic phase formed. The cubic phase covers a large part of the diagram

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4/10 phase of the binary monolein and water system, occurring in the proportions of 14% to 40% by weight of water in relation to the weight of the total mixture, in the temperature limits between 20 to 75 ^o C. For temperatures above 75 ^o C, the gels form the hexagonal phase structure consisting of cylinders of lipids arranged in a two-dimensional network, optically anisotropic.

[10] Systems formed by monolein and water have several characteristics desired for drug delivery systems and therefore have been proposed for this purpose. These systems are non-toxic, generally recognized as safe and are widely used in the pharmaceutical, food and cosmetic industry. They are also biodegradable, since monolein can be degraded by esterases present in various tissues, being released oleic acid and glycerol, in addition to being considered biocompatible, as they are produced naturally as a final product of lipid digestion.

[11] One of the important characteristics of the cubic phase of monolein and water is its stability at 37 ^o C, as well as its physical stability in excess of water, which allows its administration by several routes, maintaining its structure when in contact with biological fluids .

[12] Regarding the incorporation of drugs, the systems formed by monolein and water are capable of solubilizing drugs with different physical and chemical properties. Due to its amphiphilic character, the system is capable of incorporating both lipophilic and hydrophilic drugs. Hydrophilic drugs are solubilized in the system's water channels, while lipophilic drugs are incorporated into the lipid bilayer. The cubic phase gels of monolein and water were able to promote a slow release of drugs. Drugs of various molecular masses, including peptides and proteins, can also be incorporated into the monolein and water system, without loss of the biological activity of enzymes.

[13] In addition to having favorable characteristics as drug carriers, monolein is considered an absorption promoter, facilitating the permeation of drugs through the skin. It is suggested that monolein is capable of penetrating intercellular domains of the stratum corneum and modifying the structure of lipids, disrupting their organized structure and thus facilitating the diffusion of drugs through the skin. The presence of unsaturation in the carbon chain of monolein can

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5/10 be responsible for its promoting effect, forming spaces that alter the lipid organization of the skin. The monolein and water systems were able to increase the permeation of drugs in membranes and on the skin, in addition to being able to control their release.

[14] In the present patent, we propose the use of the lamellar and cubic liquid-crystalline phases formed by the monolein and water system as a delivery system for topical use of the 5-ALA prodrugs and their methyl, butyl, hexyl, octyl derivatives and decile, or any other ester derived from 5-ALA for application in the treatment of skin lesions through Photodynamic Therapy or for Photodiagnosis. The proposal for the use of the monolein and water system is based on the favorable characteristics presented by the same, as being non-toxic, biodegradable and stable systems at 37 ^o C, being favorable for topical use. For this purpose, the 5-ALA prodrugs and their derivative esters incorporated in the monolein and water gel, constitute an adequate system and with potential application for cases of treatment of neoplastic and non-neoplastic lesions in topical Photodynamic Therapy or for Photodiagnosis. The topical application of 5-ALA or its derivative esters using an adequate release system, as in the case of the monolein and water system, which provides a greater retention of them in the target tissue, aiming at a greater accumulation of prodrugs and consequently a greater production of the photosensitive compound, PpIX, is the proposal of the present patent.

[15] The invention is further substantiated by means of the following Examples: Example 1. Preparation of the liquid-crystalline cubic phase gel from the monolein and water system.

[16] The liquid-crystalline cubic phase gel was initially prepared by heating the monolein to 42 ^o C in a dry bath (70% by weight, based on the binary mixture monolein and water), and adding the corresponding percentage distilled water at the same temperature (30% by weight). The mixture was left to stand for approximately 24 hours until the solution was evenly distributed. For the incorporation of hydrophilic drugs, they were added to the aqueous solution, and for the incorporation of lipophilic drugs, they were added to the melted (liquid) monolein following the addition of the proportion of water. The amount of prodrug used varied from 0.2 to 2% by weight of the total mixture, for 5-ALA and its derivatives,

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6/10 apparently causing no change in the formation and stability of the liquid-crystalline phases.

Example 2. Monitoring the stability of 5-ALA and its derivative esters incorporated in the liquid-crystalline cubic phase gel formed by the monolein and water system.

[17] To monitor the stability of 5-ALA and its derivative esters incorporated in the cubic phase gel (heterogeneous medium), the 5-ALA derivation reaction was carried out. This derivation reaction is necessary, due to the fact that 5-ALA is non-fluorescent and due to the scattering produced in the region of wavelength less than 300 nm, making monitoring difficult through optical absorption measurements. The derivation reaction is based on the modification of the Hantzch reaction, in which amine compounds react with acetyl ketone and formaldehyde, resulting in a fluorescent compound identified as 2,6-diacetyl-1,5-dimethyl-7- (2-carboxyethyl) -3Hpirrolizina.

[18] For the bypass reaction, the cubic phase gel containing 5-ALA or its derivatives was dissolved in methanol, centrifuged for 5 minutes at 5000 rpm and 50 pL of this solution was mixed with a solution previously prepared in a tube with 3.5 mL of acetyl ketone and 0.45 mL of formaldehyde (10%). The solution was mixed, stirred for 3 seconds and then placed in a dry bath at 100 ^o C, protected from light, for approximately 20 minutes. After the sample was cooled, the absorption and fluorescent emission spectra of the compound formed in the different concentrations of 5-ALA and derivatives were obtained. The derivation reaction was also performed for the compounds in methanol (homogeneous medium), constructing the calibration curves and comparing them with the production of the fluorescent derivative incorporated in the cubic phase gel (heterogeneous medium).

[19] Figure 1 shows the absorption spectra (a) and emission of the fluorescent derivative (b), which has a maximum absorption around 378 nm, and a maximum fluorescent emission around 466 nm.

[20] The results of the linearity of the production of the fluorescent derivative as a function of the concentration of 5-ALA, in a homogeneous medium (calibration curves) are shown in Figure 2. Table 1 shows the intensity values

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7/10 fluorescent resulting from the derivation reaction to 4 mM of 5-ALA and its derivatives in homogeneous medium.

[21] After obtaining the calibration curves in methanol (homogeneous medium), we proceeded to study the stability of 5-ALA and its derivative esters incorporated in the cubic phase gel (heterogeneous medium), through the derivation reaction. Initially, 5-ALA and its derivative esters were incorporated into the cubic phase gel at 0.2% by weight in relation to the total mixture. After 7 and 30 days of sample preparation, they were dissolved in methanol, centrifuged for 5 minutes at 5000 rpm, and proceeded as described above for the derivation reaction. As a control, a sample was prepared only with the cubic phase gel and the bypass reaction was performed to ensure that no reaction was taking place with the gel that could interfere with the stability study. It is worth mentioning that the spectroscopic characteristics of the fluorescent derivative, such as maximum absorption and emission, were not altered in the presence of the gel. The only change in the derivation reaction in heterogeneous medium was to increase the reaction time from 20 minutes to 30 minutes, to make sure that the little monolein that was present in the medium did not interfere with the formation of the fluorescent compound.

Table 1. Comparison of the fluorescent intensity resulting from the derivation reaction for 5-ALA and its derived esters (4 mM), in homogeneous and heterogeneous medium.

Compound Fluoresc. half homog. (10 ⁴ ) Fluoresc. heterogeneous. (10 ⁴ ) 7 days Fluoresc. heterogeneous. (10 ⁴ ) 30 days F7 days / F30 days (%) 5-ALA 8.81 7.15 6.57 8 ALA-hexyl 0.95 0.92 0.82 10 ALA-octil 1.09 0.93 0.79 15 ALA-decile 0.98 1.0 0.95 5

[22] In Figure 2, the respective fluorescent emissions resulting from the production of the fluorescent compound after incorporation in the cubic phase gel of monolein and water for 7 and 30 days are represented for the 5-ALA and its derived esters. At

Table 1 are represented comparatively for 5-ALA and its esters

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8/10 derivatives (4 mM), the fluorescent intensities resulting from the adjustment of the derivation reaction calibration curve in methanol (homogeneous medium) and in the presence of the cubic phase gel (heterogeneous medium).

[23] Comparing the data of the fluorescent intensity resulting from the derivation reaction for 5-ALA and its derivative esters, 4 mM (Table 1), we can observe that for 5-ALA the production of the fluorescent derivative, in methanol (homogeneous medium) ), is approximately an order of magnitude greater in relation to its derived esters. In the presence of the cubic phase gel (heterogeneous medium), this difference is maintained for 5-ALA. A higher fluorescent intensity, resulting from a higher production of the derivative, could be explained by the fact that the formation of the fluorescent compound in the derivation reaction for 5-ALA is more favorable than its esters, due to the absence of the hydrocarbon chain, the that could hinder the derivation reaction in the case of esters. In a heterogeneous environment, the production of the derivative is of the same order as the production in the homogeneous environment, for all compounds. It is worth mentioning that, for the heterogeneous medium, the reaction time at 100 ^o C was increased to 30 minutes, to make sure that the monolein present, after the centrifugation process, did not interfere in the bypass reaction. Comparing the periods of incorporation, 7 and 30 days, of 5-ALA and its derivatives in heterogeneous medium, we can observe a small decrease in the production of the fluorescent derivative for 30 days, compared to 7 days. The percentage of decrease in fluorescent intensity after 7 and 30 days of incorporation is also shown in Table 1. We can observe that for 30 days the decrease in the estimated production of the fluorescent derivative, resulting from the derivation reaction, is 8, 10, 15 and 5%, for 5-ALA, hexyl, octyl and decyl ester, respectively, representing an average decrease of 10% for the period of 30 days of incorporation, considered acceptable, and indicating that the properties of the compounds are maintained in the gel for the period studied for up to 30 days.

Example 3. In vitro skin permeation and retention study of the prodrug 5-ALA using the cubic phase gel formed by the monolein and water system as the release system.

[24] The study of cutaneous retention of 5-ALA using the cubic phase gel of monolein and water in the proportion 70% by weight of monolein as a delivery system in

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9/10 in relation to the total weight and 30% of water, it was carried out with pig ear skin in a modified Franz diffusion cell, Hanson Research. The receptor phase used was the pH 7.2 phosphate buffer solution. The entire system was maintained at 37 ^o C, through a water circulation system. At pre-determined time intervals, aliquots of the receiving phase were collected for the analysis of the permeated 5-ALA. After the 12-hour period, the skins were removed from the diffusion cell and carefully washed with distilled water to remove formulation residues on the skin. After removing excess water with paper, the stratum corneum (EC) was removed by the tape stripping method. The tapes were placed in a tube containing methanol and stored for a period of 10 hours. After that period, the solution was stirred for 1 minute and then filtered. The remaining tissue (viable epidermis + dermis) was weighed, cut into pieces and placed in a pH 7.2 phosphate buffer solution and homogenized in a Turrax apparatus, tissue homogenizer, for 1 minute, followed by sonification for 20 minutes in an ultrasound bath and filtration on filter paper. The amount of 5-ALA extracted from the adhesive tapes, that is, the amount that was retained in the stratum corneum and in the viable epidermis + dermis set, as well as the amount of permeated 5-ALA, were quantified through the derivation reaction for 5 -ALA, as described in Example 2 and expressed in pg of 5-ALA retained per gram of skin (pg 5-ALA / g skin).

[25] The results of the skin permeation study of 5-ALA, incorporated at 1% by weight in relation to the total weight of the ternary mixture, in the cubic phase gel and water are shown in Figure 3. It can be seen that the gel of cubic phase provided less permeation of 5-ALA compared to the control formulation without monolein (phosphate buffer, 7.2). This is a positive result for the cubic phase gel formulation, as it showed that the systemic absorption of 5-ALA will be reduced, which may favor its topical effect, that is, its penetration and retention in the skin.

[26] The results of the skin retention study of 5-ALA, incorporated at 1% by weight, in relation to the total weight of the ternary mixture, in the cubic phase gel of the monolein and water system are represented in Figure 4. We can see that the greatest amount of retained 5-ALA is located mainly in the viable epidermis plus dermis than in the stratum corneum for formulations containing 10% monolein

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10/10 (used as an absorption promoter) and for the cubic phase gel of monolein and water. We can also verify that the gel formed by the monolein and water system showed a higher retention in the viable epidermis + dermis group compared to the 10% monolein formulation.

[27] In the present patent, we show that the cubic phase gel formed by the monolein and water system surpassed the formulation using 10% monolein in the release of 5-ALA; that is, it provided greater skin retention in the skin layer where most of the neoplastic and non-neoplastic skin pathologies develop.

[28] The fact that the cubic phase gel formed by the monolein and water system shows a greater retention of 5-ALA in the skin layers of interest and less permeation, are strong indications that there will be a greater accumulation in vivo of PpIX (photosensitive compound ) in the cutaneous tissue, a condition that will guarantee the efficiency in the treatment through Photodynamic Therapy or for Photodiagnosis. Its future application is not limited to the treatment of neoplastic lesions of the skin (dermatology), but also for the treatment of lesions of the oral cavity or gynecology through Photodynamic Therapy.

[29] In view of the stability results obtained for 5-ALA and its derivative esters when incorporated in the cubic phase gel, formed by the monolein and water system, and mainly of the retention observed in the skin for 5-ALA, this system shows potential application as a topical release system in Photodynamic Therapy, since it can be used to reduce the dose of prodrugs, as well as to optimize their penetration into the skin.

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Claims (2)

1. Delivery system for topical use consisting of monolein and water, characterized by being the transdermal system consisting of 70% by weight of monolein and 30% by weight of water to form the cubic phase and 5 aminolevulinic acid (5-ALA) and its derivatives derived from hexyl, octyl and decyl dispersed in said system ranging from 0.2 to 2% by weight of the total ternary mixture. 2. Process for obtaining a release system for topical use, as defined in claim 1, characterized by comprising: mixing monolein in the range of 70% by weight and 30% by weight of water, the corresponding proportion of water being heated to temperature 42 ^o C and added to the monolein also heated to that same temperature to form the cubic phase, with 5-aminolevulinic acid (5-ALA) being added to the monolein or dissolved in the mixture formed by monolein water. Petition 870170083651, of 10/30/2017, p. 19/20 1/4