Process for obtaining liposomes with high levels of encapsulation of water-soluble active ingredients, formulations and uses

Abstract

The invention relates to a process for encapsulating water-soluble active ingredients in preformed liposomes with an encapsulation efficiency ranging from 20 to 63%. It also relates to the use of said process to produce pharmaceutical, cosmetic and nutraceutical compositions. The process consists in permeabilizing liposomes of a calibrated size with bile salt, remotely encapsulating the active ingredient, and subsequently removing the bile salt. The process comprises three steps with a low degree of complexity in terms of execution and scalability, and exhibits 56% encapsulation efficiency for hydrophilic substances, such as angiotensin-(1-7) peptide (Ang-(1-7)). Liposomal compositions comprising an encapsulated Ang-(1-7) proportion of 0.01 g to 0.07 g per g of lipid were obtained. Furthermore, the liposomal composition of Ang-(1-7) derived from the claimed process exhibited a stable level of the encapsulated substance after exposure to extreme acidic pH conditions and exhibited antihypertensive activity when administered orally in a preclinical model of arterial hypertension.

Description

[0001] “PROCESS FOR OBTAINING LIPOSOMES WITH HIGH ENCAPSULATION LEVELS OF WATER-SOLUBLE ACTIVE INGREDIENTS, FORMULATIONS AND USES”

[0001] This technology refers to a process for encapsulating water-soluble active ingredients in pre-formed liposomes with encapsulation efficiency in the range of 20% to 63%. It also refers to the use of this process for the production of pharmaceutical, cosmetic and nutraceutical compositions. The process consists of permeabilizing liposomes of calibrated size with bile salt, remote encapsulation of the active ingredient and subsequent removal of the bile salt. The process is composed of 3 stages with a low degree of complexity for execution and is scalable, and presents an encapsulation efficiency of 56% for hydrophilic substances, such as the Angiotensin-(1-7) peptide (Ang-(1-7)). Liposomal compositions were obtained comprising a ratio of encapsulated Ang-(1-7) of 0.01 g to 0.07 g per g of lipid.Furthermore, the liposomal composition of Ang-(1-7) derived from the claimed process showed stability in the encapsulated substance content after exposure to extreme acidic pH conditions and demonstrated antihypertensive activity when administered orally in a preclinical model of arterial hypertension.

[0002] Liposomes represent one of the most studied and employed carrier nanosystems in pharmaceutical, cosmetic, and nutraceutical compositions. This system stands out for its biocompatibility, versatility in composition, physicochemical characteristics, and ability to accommodate both water-soluble and fat-soluble active substances. The benefits of incorporating active ingredients into liposomes include increased solubility for substances poorly soluble in water, modulation of the aggregation state and pharmacokinetics of the liposome, which can result in increased bioavailability via non-invasive routes such as topical, nasal, pulmonary, and oral administration, and reduced toxicity and increased therapeutic activity.Liposomes can be composed of any mixture of lipids, as long as they are capable of generating vesicular structures. Phospholipids such as soy phosphatidylcholine are often recommended due to their ability to form lipid bilayers and vesicles, as well as their high biocompatibility.

[0003] The main challenges in developing liposomal compositions of water-soluble active substances relate to obtaining high encapsulation levels of these substances, the difficulty of retention in encapsulated form, and the development of inexpensive, simple, and scalable manufacturing processes that preserve the chemical integrity of the substance and lead to the production of liposomes of homogeneous size. Obtaining a high percentage of encapsulated substance allows for the administration of the liposomal formulation with a lower dose of lipids and, consequently, lower treatment costs. A larger fraction of encapsulated active ingredient ensures less loss of active ingredient during the process and lower costs. Controlling the size of the liposomes and their homogeneity in the final composition are also important, as the pharmacokinetics of liposomes and the encapsulated substance strongly depend on the size of the nanoparticle.For safe parenteral administration, liposomes with a diameter of less than 250 nm are recommended.

[0004] The methods for encapsulating active ingredients in liposomes described in the prior art are divided into two types. In the first type, the encapsulation of the active ingredient occurs during the liposome formation step, generally requiring a subsequent step of calibrating the size of the vesicles in the presence of the active ingredient. In the second type of method, a suspension of liposomes of calibrated size is prepared without the presence of the active substance, and the active ingredient is incorporated into the liposomes later, remotely. The advantage of the second type of method is that it does not expose the active substances to the drastic temperature and mechanical energy conditions generally required to obtain calibrated-size lipid vesicles.

[0005] In the case of weak base drugs, remote encapsulation methods in pre-formed liposomes, based on the use of pH gradients or ammonium sulfate, have been successfully employed for over three decades. In the case of polyanionic substances, such as nucleic acids, oligo- or polysaccharides, or certain proteins and peptides, incorporation can be carried out in pre-formed liposomes through electrostatic interactions using cationic surface vesicles. However, these methods are only applicable to restricted groups of active ingredients.

[0006] Several other methods have been developed to increase the encapsulation rate of water-soluble drugs in liposomes, including methods based on reverse-phase evaporation and lyophilization processes. The former involves the use of organic solvents that are difficult to remove and potentially toxic and denaturing to proteinaceous substances, while the latter uses a complex and expensive process. These methods do not allow for fine control of liposome size and do not guarantee their homogeneity in the final composition. Both processes are expensive, difficult to scale up, and difficult to apply on an industrial scale.

[0007] Another method involves injecting an ethanolic lipid solution into an aqueous solution containing the substance to be encapsulated. An alternative method consists of forming mixed micelles composed of a phospholipid and a detergent in an aqueous solution containing the active ingredient, followed by the removal of the detergent either by dilution, dialysis, gel filtration chromatography, or binding to resins. A significant difficulty with these methods is controlling the final size of the liposomes, as it depends on several parameters such as injection flow rate, final lipid concentration, and lipid composition of the vesicles. Furthermore, both of these methods exhibit low encapsulation efficiency of the active substance in liposomes smaller than 200 nm.

[0008] Liposomes co-incorporating phospholipids and bile salts such as cholate and deoxycholate have attracted attention in the last decade due to their biocompatibility and peculiar physicochemical properties, as well as their increased ability to solubilize lipophilic substances and promote their absorption, whether orally or topically. Bile salts are physiological surfactants synthesized in the liver from cholesterol, possessing a specific chemical structure characterized by a large, rigid, and planar hydrophobic steroid nucleus with hydroxyl groups varying in number, position, and orientation, along with a flexible acidic side chain. However, little attention has been paid to the permeabilizing effect of these bile salts on the liposome membrane.

[0009] Therefore, there is a strong demand for new, simpler, more efficient, cheaper, and scalable processes for encapsulating high levels of water-soluble active ingredients in liposomes.

[0010] Patent document EP3922241A1, with a priority date of 01 / 02 / 2013, entitled “REMOTE LOADING OF SPARINGLY WATER-SOLUBLE DRUGS INTO LIPOSOMES”, describes a process for the remote encapsulation of sparingly water-soluble compounds (< 2 mg / mL) through the encapsulation of a precipitate of a solid active agent. The strategy involves preparing an aqueous suspension of liposomes with a buffer that promotes an ionic gradient, then the precipitate with the solid active agent, followed by the formation of a carrier suspension of the liposomal suspension with the precipitate for encapsulation via an ionic gradient. However, this method is limited to the encapsulation of substances with weak acid-base properties. It does not apply to the encapsulation of hydrophilic molecules such as Ang-(1-7).

[0011] Patent document EP1731172B1, with a priority date of 03 / 25 / 2005, entitled “Liposome preparation”, describes a liposome preparation process based on a pH gradient for incorporating a water-soluble substance, and provides for the encapsulation of the substance during or after liposome formation within the pH gradient encapsulation method. The patent process does not include permeabilization of the liposome membrane to achieve drug encapsulation and does not apply to hydrophilic molecules such as Ang-(1-7), but rather to a limited number of weak base drugs.

[0012] The article by Szoka F & Papahadjopoulos D, published on September 15, 1978, entitled “Procedure for Preparation of Liposomes with Large Internal Aqueous Space and High Capture by Reverse-Phase Evaporation”, in the journal Proceedings of the National Academy of Sciences of the United States of America, 75, 4194-4198.10.1073 / pnas.75.9.4194, describes a reverse-phase evaporation method. However, it does not allow for fine control of liposome size and uses organic solvents that are difficult to remove and potentially toxic and denaturing to proteinaceous substances, in addition to being difficult to scale up and apply on an industrial scale.

[0013] The articles by Philippot and colleagues in 1983 and 1985 (Biochimica et Biophysica Acta 734, 137-143, 1983; Biochimica et Biophysica Acta 821, 79-84, 1985) describe methods for preparing compositions by detergent removal, based primarily on the formation of mixed micelles consisting of a phospholipid and a detergent in the presence of the substance to be encapsulated, followed by the subsequent removal of the detergent. One of the articles reports encapsulation efficiencies of water-soluble protein ranging from 11% to 47%, with the lowest encapsulation efficiency obtained with liposomes with a diameter close to 240 nm and the highest efficiency with liposomes with a diameter close to 1000 nm. The other article reports a calcein encapsulation efficiency of at most 20%. Therefore, the detergent removal method does not allow for high encapsulation efficiency (> 25%) in small liposomes (diameter < 250 nm), unlike the process reported in the present technology.

[0014] The article by Kirby C & Gregoriadis G, published on November 1, 1984, entitled “Dehydration-Rehydration Vesicles: A Simple Method for High Yield Drug Entrapment in Liposomes”, in the journal Nature Biotechnology, 2, 979–984, https: / / doi.org / 10.1038 / nbt1184-979, describes the dehydration-rehydration method for encapsulating water-soluble drugs of protein and non-protein nature. The process consists of mixing a suspension of pre-formed liposomes with an aqueous solution of the active ingredient, followed by lyophilization. Rehydration of the lyophilized product with a reduced volume of water allows for high encapsulation rates, typically in the range of 30 to 50%, but with large (>250 nm) and highly polydisperse liposomes. An improvement on this method was presented by Zadi and Gregoriadis in 2000 (Zadi B & Gregoriadis G. A Novel Method for High-Yield Entrapment of Solutes into Small Liposomes. Journal of Liposome Research 10, 73-80, 2000. 10.(3109 / 08982100009031096) involves the addition of a cryoprotective sugar to the liposome-drug mixture before lyophilization. The presence of the cryoprotectant allows for better control of the liposome size distribution without compromising encapsulation efficiency. On the other hand, dehydration-rehydration processes are expensive, complex, and difficult to scale up and apply on an industrial scale.

[0015] The article by Mendes et al., published in October 2024 (Intranasal liposomal angiotensin-(1-7) administration reduces inflammation and viral load in the lungs during SARS-CoV-2 infection in K18-hACE2 transgenic mice. Antimicrobial Agents Chemotherapy 2024; 68(12):e00835-24. doi: 10.1128 / aac.00835-24), reports the preparation of a liposomal formulation of Ang-(1-7) by the ethanolic lipid solution injection method followed by a particle size calibration step by extrusion with a 200 nm pore polycarbonate membrane. In this article, the encapsulation efficiency using this process was 13.4% and the encapsulation content was equal to 0.0014 g of encapsulated peptide per g of lipid.

[0016] Patent document PI0105509-7, with a priority date of 05 / 11 / 2001, entitled “FORMULATIONS OF ANGIOTENSIN-(1-7) PEPTIDE USING CYCLODEXTRINS, LIPOSOMES AND PLGA POLYMER”, describes the encapsulation of Ang-(1-7) in liposomes using the dehydration-rehydration method, followed by an extrusion step with a 200 nm and 100 nm pore polycarbonate membrane for vesicle size calibration. However, the encapsulation efficiency using this process does not exceed 15%, and the encapsulation content was 0.03 g of encapsulated peptide per g of lipid. Therefore, the process claimed here differs from the prior art by allowing an encapsulation efficiency of Ang-(1-7) of up to 56% and obtaining a liposomal composition with an encapsulation content of up to 0.07 g of peptide per g of lipid.

[0017] Patent document BR102013030151-5, with a priority date of 11 / 25 / 2023, entitled “TOPICAL FORMULATIONS FOR THE PREVENTION AND TREATMENT OF ALOPECIA”, describes a topical formulation of Ang-(1-7) and the process of encapsulating the molecule in soy phosphatidylcholine liposomes containing sodium cholate, using the ethanol injection technique as an initial step. Patent BR1020130301515 presents a classic process in which peptide encapsulation occurs passively during vesicle formation, and not remotely after vesicle formation. To obtain liposomes of calibrated size, an additional step of extruding the liposome suspension with the drug was performed using a 100 nm pore polycarbonate membrane.In contrast, the technology claimed here includes an initial step of forming liposomes of calibrated size and permeabilized with bile salt, followed by a subsequent incubation step with the active ingredient and, finally, the removal of the bile salt from the membrane either by dialysis or dilution. Therefore, the active ingredient is not subjected to the vesicle size calibration process, avoiding loss or compromise of the peptide's chemical integrity. Furthermore, the final removal of the potentially toxic bile salt by dialysis reduces the risk of toxicity of the liposomal composition when administered to humans or animals. While the encapsulation efficiency of Ang-(1-7) was 8.4% using the process in BR102013030151-5, the process claimed here presents an encapsulation efficiency of 48.3%.

[0018] Patent document BR1020210068205, with a priority date of 09 / 04 / 2021, entitled “PROCESS FOR OBTAINING AMPHOTERICIN B CONJUGATED LIPOSOMES, FORMULATION AND USES”, describes the process of incorporating the water-soluble substance amphotericin B into liposomes formed by hydrogenated soy phosphatidylcholine (HSPC), cholesterol (COL), distearoyl phosphatidylglycerol (DSPG), with or without PEGylated lipid. This is a process involving multiple steps, including the solubilization of lipids in the organic solvent chloroform, hydration of the lipid film, freeze-thaw cycles, extrusion and addition of the amphipathic active substance and pH neutralization in the mixture, in addition to heating steps. Furthermore, the process is restricted to the drug amphotericin B.

[0019] Patent document CN102188378A, with a priority date of 03 / 18 / 2021, entitled “PREPARATION METHOD OF LIPOSOME FOR COATING AND CARRYING WATER-SOLUBLE DRUGS”, describes a method for coating and carrying water-soluble compounds through two steps: dissolution and solubilization of the phospholipid in salt form and active substance in an organic solvent to form the organic phase, followed by injection of the organic phase into an aqueous solution with a different pH to reduce the solubility of the substance and facilitate coating and carrying in liposomes. Unlike the technology claimed here, this method applies only to weak base or weak acid drugs. It does not apply to peptidic substances such as Ang-(1-7) or non-peptidic substances such as calcein.

[0020] Patent document CN106214641A, with a priority date of 22 / 08 / 2024, entitled: “LIPIDOSOME APPLICABLE TO WATER-SOLUBLE DRUGS, AND PREPARATION METHOD OF LIPIDOSOME”, describes a method for preparing liposomes applicable to water-soluble substances through a dehydration-rehydration method. The method is based on the steps of dissolving: phospholipid, stabilizing agent, water-soluble substance, and carrier in a suspension medium, followed by removal of the dispersion medium by vacuum drying, hydration of the precursor liposome, and filtration. The present technology does not include the step of forming permeabilized liposomes separate from the encapsulation step of the active substance and does not guarantee size control of the liposomes.

[0021] Patent document CN102935068A, with a priority date of 10 / 19 / 2012, entitled “PREPARATION METHOD OF LIPOSOME ENTRAPPING WATER-SOLUBLE MEDICINES”, describes a liposome preparation method applicable to water-soluble substances with low thermal stability, through repeated lyophilization steps. The method is based on the steps of dissolving a water-soluble active substance in a colloidal suspension of hydrophilic polymer (containing amino acids in the structure such as gelatin, collagen, and albumin), followed by the addition of a triblock copolymer of polyoxyethylene and polyoxypropylene (to provide thermostability to the composition) and lyophilization, followed by the transfer of lyophilized powder to an organic solvent (tert-butanol solution) containing lipid materials, followed by a further lyophilization process.The present technology does not include the step of permeabilizing liposomes with bile salt, nor does it involve performing the step of forming permeabilized liposomes separately from the step of encapsulating the active substance.

[0022] In the state of the art, no liposome encapsulation methodology has been found based on the simple principle of remote internalization of a water-soluble substance, without the use of potentially toxic and denaturing organic solvents for the protein-based substance, comprising only a phospholipid and a bile salt as base components of the liposome encapsulating the water-soluble substance of interest, and carried out in 3 low-cost and scalable encapsulation steps, with the formation of the bile salt-permeabilized liposome step separate from the addition and encapsulation step of the active water-soluble substance, and resulting in an encapsulation content greater than 50% for hydrophilic molecules such as Ang-(1-7).

[0023] This technology proposes a simple, inexpensive, and scalable method for encapsulating a water-soluble substance based on three steps: preparation of a concentrated suspension of pre-formed liposomes permeabilized with bile salt (step 1), followed by remote encapsulation of the water-soluble active ingredient (step 2) and removal of part of the bile salt from the liposomal membrane for impermeabilization and retention of the active ingredient (step 3). In this way, the preparation of permeabilized liposomes occurs independently of the encapsulation step of the water-soluble substance, thus avoiding exposure of the active substance to solvents or processes that could induce degradation, inactivation, or loss. The method allows for achieving high levels and encapsulation efficiency of the water-soluble substance in liposomes calibrated to 100 nm, as demonstrated for the Ang-(1-7) peptide with an encapsulated peptide / lipid ratio between 0.01 and 0.07 (m / m) and encapsulation efficiency reaching 56%.The method was also applied to the encapsulation of human insulin with an encapsulation rate of 63.5 ± 0.2%, demonstrating its applicability to protein nanoencapsulation. In addition, the liposomal formulation with Ang-(1-7) produced by this method showed stability at gastric pH, with a peptide retention capacity of 60% after exposure under these conditions, and therefore represents an alternative for oral administration. The efficacy of oral treatment with the liposomal formulation of Ang-(1-7) was also demonstrated by its prolonged antihypertensive action after a single dose of 100 µg peptide / kg body weight in spontaneously hypertensive rats.

[0024] The high encapsulation efficiency of the active ingredient means less loss of active ingredient during the process and ensures lower manufacturing costs. The high encapsulation content of the active ingredient allows the use of a low-lipid dose formulation, making the formulation and treatment potentially safer. The reduced size of the liposomes, typically less than 150 nm, and the low polydispersity guarantee high effectiveness and reproducibility in efficacy, as well as safety in parenteral administration. The issue of encapsulation stability of the water-soluble substance during storage can be resolved by generating product up to step 2, with step 3 being performed by the user through simple dilution of the product. Another option that ensures stability is to dehydrate the composition obtained in step 3, for example by lyophilization, in the presence of cryoprotective sugar. In dehydrated powder form, the liposomal composition exhibits high stability during storage.BRIEF DESCRIPTION OF THE FIGURES.

[0025] Figure 1 shows a flowchart illustrating the manufacturing process of liposomal compositions and potential routes of administration and applications.

[0026] Figure 2 shows the antihypertensive activity of the liposomal formulation of Ang-(1-7) when administered orally to spontaneously hypertensive rats. (A) Evolution of mean arterial pressure (MAP) in rats receiving LAng-(1-7) or Lvazio (vehicle) by gavage at time zero. (B) Variation of mean arterial pressure at 6h relative to time zero in animals receiving LAng-(1-7) or Lvazio. Data shown as means ^ standard error. A. **p<0.01, ***p<0.001, ****p<0.0001, Two-way ANOVA with Tukey's post-hoc test. B. **p<0.01, Mann-Whitney test. DETAILED TECHNOLOGY DESCRIPTION

[0027] This technology describes a process for encapsulating water-soluble active ingredients in pre-formed liposomes with encapsulation efficiencies ranging from 20 to 63%. It also refers to the use of this process for the production of pharmaceutical, cosmetic, and nutraceutical compositions. The process consists of preparing calibrated-size liposomes permeabilized with bile salt, remotely encapsulating the active ingredient in the permeabilized liposomes, and subsequently removing the bile salt. The process comprises three steps with a low degree of complexity for execution and is scalable, and presents encapsulation efficiencies of up to 56% for water-soluble substances such as the Ang-(1-7) peptide. Liposomal compositions were obtained comprising a proportion of encapsulated Ang-(1-7) of 0.01 g to 0.07 g per g of lipid.Furthermore, the liposomal composition of Ang-(1-7) derived from the claimed process showed stability in the encapsulated substance content after exposure to extreme acidic pH conditions, and demonstrated antihypertensive activity when administered orally in a preclinical model of arterial hypertension.

[0028] The process for obtaining liposomes with high encapsulation levels of water-soluble active ingredients may comprise the following steps: (a) dissolving a lipid or mixture of lipids in ethanol in a ratio ranging from 20 to 200 g of lipid(s) per 100 mL of ethanol, preferably 97 g per 100 mL of ethanol; (b) preparing a buffer solution with a pH in the range of 3 to 10 and dissolving the bile salt in an amount ranging from 0.5 to 5.4 g of bile salt per 100 mL of buffer solution, preferably 2.6 g of bile salt per 100 mL; (c) inject the ethanolic solution obtained in step “a” into the buffer solution obtained in step “b” in a volume ratio varying between 0.025 and 0.3 and in a mass ratio of bile salt to lipids varying between 0.05 and 0.3 (m / m), preferably with a volume ratio equal to 0.2 and a bile salt / lipid ratio equal to 0.134 (m / m), and keep the mixture under agitation until a homogeneous suspension is obtained;(d) optionally, calibrate the size of the liposomes in the resulting suspension obtained in step “c” to obtain liposomes with a diameter between 30 and 400 nm, preferably 100 nm, and a polydispersity index of less than 0.3; (e) add the water-soluble compound to be encapsulated to the suspension of pre-formed liposomes in step “d”, so that the compound / lipid mass ratio is up to 1, incubate the mixture for a duration ranging from 1 minute to 72 hours at a temperature ranging from 4 ºC to 90 ºC and, optionally, store the resulting mixture as an aqueous suspension at a temperature ranging from 4 ºC to room temperature, until the time of use;(f) partially displace the bile salt from the membrane of the liposomal suspension obtained in step “e” to the aqueous phase by dilution at least 2 times in aqueous solution. Optionally, step “f” may be replaced by removing the bile salt from the liposomal suspension by dialysis, gel filtration chromatography, tangential filtration, diafiltration, use of a resin capable of binding to the bile salt, or separation by centrifugation or ultrafiltration followed by resuspension with a buffer without the bile salt.

[0029] The lipids used in step “a” can be selected from the group comprising phosphatidylcholine with purity ranging from 75 to 100%, lecithin, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, stearylamine, dicetyl phosphate, 1,2-dioleoyl-3-trimethylammonium-propane, ionizable lipid, cholesterol, from the group of antioxidant lipids such as alpha-tocopherol, and / or from the group of pegylated lipids. The bile salt of step “b” can be selected from primary bile salts, such as cholate and chenodeoxycholate salts, secondary bile salts, such as deoxycholate, lithocholate, ursodeoxycholate salts, and bile salts conjugated to glycine and taurine, in addition to semi-synthetic keto derivatives. The water-soluble compound in step “e” can be selected from the group comprising salts, metal complexes, inclusion complexes with cyclodextrins, small molecules with a molecular mass up to 1.500 Da, such as calcein, carbon nanoparticles, metallic nanoparticles, macromolecules including oligosaccharides, polysaccharides, nucleic acids, peptides such as Ang-(1-7) and alamandine, and proteins such as insulin. As a buffer solution, any acid-base system can be used, such as phosphate, acetate, citrate, bicarbonate or borate buffer, depending on the desired pH range. For liposome size calibration in step (d), high-pressure homogenization, high-shear homogenization or microfluidization could also be used. Alternatively, steps (c) and (d) could be performed in a single step using a microfluidic device. Optionally, the final product form can be solid or powder, to ensure long-term stability, by means of an additional dehydration step of the liposomal suspension, by lyophilization or spray drying, in the presence of cryoprotective sugar.Cryoprotective sugars can be selected from the group comprising sucrose, glucose, trehalose, lactose, mannitol, and sorbitol.

[0030] The process for obtaining liposomes with high encapsulation levels of water-soluble active ingredients using this technology can be employed to produce liposomal formulations encapsulating water-soluble active substances with a lipid-to-substance mass ratio of up to 1, for administration via oral, parenteral, intranasal, inhalation, topical or transdermal application, and local injection.

[0031] The process of the present technology can also be used for the production of liposomal nanoformulations encapsulating water-soluble compounds in solid or powder form, to ensure long-term stability, through an additional dehydration step of the liposomal suspension, by lyophilization or spray drying, in the presence of cryoprotective sugar. The cryoprotective sugar can be selected from the group comprising sucrose, glucose, trehalose, lactose, mannitol and sorbitol.

[0032] The process claimed herein can be used for the production of solid or suspension liposomal compositions encapsulating active water-soluble substances with a substance / lipid mass ratio of up to 1, for administration via oral, intranasal, inhalation, parenteral, topical or transdermal application, or local injection. More specifically, the process can be used for the production of liposomal compositions encapsulating Ang-(1-7).

[0033] The present technology also refers to liposomal formulations of Ang-(1-7), with an encapsulation content of 0.01 g 0.07 g of peptide per g of lipid.

[0034] The technology also refers to the use of Ang-(1-7) formulations for the production of orally administered medications for the treatment of various pathologies in which Ang-(1-7) provides therapeutic benefits, including hypertension, kidney disease, liver disease, heart disease, lung disease, neurological disease, metabolic disease, muscle disease, endometriosis, inflammatory diseases, and bacterial and viral infectious diseases.

[0035] The technology presented can be better understood through the following, but not limited, examples. EXAMPLE 1 – VALIDATION OF THE EFFICIENCY OF THE NEW ENCAPSULATION PROCESS USING A HYDROPHILIC SUBSTANCE OF PEPTIDE ORIGIN

[0036] Unlike the classic encapsulation of target substances, which occurs passively during vesicle formation, as described in patent BR102013030151-5, the encapsulation process claimed here occurs remotely after vesicle formation. The encapsulation process also exploits, in a novel way, the transient permeabilization of the liposomal membrane with bile salt. The proof of concept (PoC) of the new process's viability was established using the heptapeptide angiotensin-(1-7) as a model water-soluble active substance due to the molecule's various pharmacological actions, including vasodilatory, antioxidant, anti-apoptotic, and anti-inflammatory effects, and its broad therapeutic potential. The PoC was performed as described below.

[0037] First, permeabilized liposomes were prepared using the ethanol injection method. To do this, 388 mg of soy phosphatidylcholine (90% purity) was dissolved in 0.4 mL of 96% ethanol. The ethanolic lipid solution was injected into 2 mL of 20 mM phosphate buffer solution, pH 7.2 or pH 5, with or without 0.15 M NaCl, containing or not 52 mg of dissolved bile salt, either sodium cholate or sodium deoxycholate. After 15 min under agitation at room temperature, the resulting suspension was extruded through a 100 nm pore polycarbonate membrane to calibrate the size of the liposomes. 1 mg of peptide was added to 0.25 mL of the permeabilized liposome suspension, and the mixture was incubated for 2 hours at 25°C. o C or 60 oC. To remove bile salt from the liposome membrane and trap the peptide in the liposome's internal aqueous compartment, the liposome mixture containing the peptide was then diluted 4 times with a dilution buffer consisting of 20 mM phosphate, pH 7.2, with or without 0.15 M NaCl. The diluted liposome suspension was then subjected to dialysis against a dialysis buffer consisting of 20 mM phosphate, 0.15 M NaCl, pH 7.2, for 12 hours at 4 oC, using a dialysis tube with a 12 kDa MWCO membrane for the elimination of the unencapsulated peptide. After recovery of the liposome suspension encapsulating the peptide, the peptide was quantified by fluorescence spectroscopy, exploring its intrinsic fluorescence. Fluorescence was measured with excitation at 285 nm and emission at 305 nm of a solution consisting of 2.2 mL of methanol containing 50 µL of the liposomal suspension, before (Ftot) and after (Fencaps) dialysis. The fluorescence of a blank formulation, prepared under the same conditions but without the peptide, was also measured before (Ftot_0) and after dialysis (Fencaps_0). The linearity of the fluorescence curve as a function of peptide concentration in the study range was also confirmed. The encapsulation efficiency (EE%) was then calculated according to the following equation: EE% = 100*[(Fencaps - Fencaps_0) / (Ftot - Ftot_0)].

[0038] To calculate the encapsulation content (EC), which corresponds to the encapsulated peptide / lipid mass ratio, the initial peptide / lipid mass ratio, used in the preparation of the composition, was multiplied by the encapsulation efficiency (EE%).

[0039] The particle size distribution of the final composition was evaluated by dynamic light scattering using a Zetasizer Nano ZS90 (Malvern, UK), by determining the average hydrodynamic diameter of the vesicles and the polydispersity index (PDI).

[0040] The results of characterization of liposomal compositions in relation to peptide content and encapsulation efficiency and particle size distribution are shown in Table 1. Table 1 – Characterization results of liposomal compositions under different preparation conditions, varying the presence or absence of bile salt, the type of bile salt, the incubation temperature, the osmolarity and pH of the buffer solutions used for preparation, first dilution and dialysis. Bile salt Buffer Duration and Incubation Temperature of EE%* TE* Buffer Diameter (m / m) (PDI*) Preparation dilution dialysis (4x) No salt Buffer 2h to 25 o C Buffer Buffer 5 0.001 118 nm bile phosphate 20 phosphate phosphate (0.04) 20 mM 2 mM pH 7.2 20 mM NaCl 150 mM pH 7.2 Cholate Buffer 2h at 25 o C Buffer Buffer 40 0.010 96 nm phosphate 20 phosphate phosphate (0.08) 20 mM 0 mM pH 7.2 20 mM NaCl Desoxic Buffer 2h to 25 oC Buffer Buffer 33 0.008 106 nm olate phosphate 20 phosphate phosphate (0.06) (influence mM pH 7.2 20 mM 20 mM 2 s al bil pH 7.2 NaCl iar) 150 mM pH 7.2 Cholate Ta 2h at 60 o C Buffer 50 103 nm Buffer 0.012 (influence phosphate 20 phosphate phosphate (0.13) 20 mM 4 temperature mM pH 7.2 20 mM NaCl 150 mM pH 7.2 pH 7.2 o 2h a 60 C Colato Buffer 101 nm Buffer 24 0.005 Tampão (influence phosphate phosphate (0.12) phosphate 20 920 mM 20 mM NaCl buffer 150 mM NaCl pH 7.2 d iluição) mM 150 mM pH 7.2 pH 7.2 o 2h a 60 C Colato Tampão Tampão 32 0,007 - Tampão (influence phosphate 20 phosphate phosphate 9 mM NaCl 20 mM 20 mM buffer of 150 mM NaCl 150 NaCl p reparo) pH 5 mM 150 mM pH 7.2 pH 7.2 *EE%: encapsulation efficiency; TE: encapsulation content; PDI: polydispersity index

[0041] The absence of bile salt resulted in a monodisperse population of liposomes with a diameter of around 120 nm, but with very low peptide content (0.0012 m / m) and encapsulation efficiency (5%). On the other hand, the inclusion of the bile salt sodium cholate or sodium deoxycholate resulted in a monodisperse suspension of liposomes with a diameter of around 100 nm and much higher encapsulation content and efficiency, with a peptide / lipid ratio of around 0.01 (m / m) and higher peptide encapsulation efficiency, up to 50%. The marked difference in peptide encapsulation in the presence of bile salt validates the process of this technology based on the use of permeabilized liposomes. oIncreasing the incubation temperature from 25°C to 60°C increased the peptide encapsulation rate by 40 to 50%, showing that temperature is a relevant factor. The composition of the preparation and dilution buffers also influenced the peptide encapsulation rate. Differences in osmolarity between the preparation and dilution buffers resulted in a lower peptide encapsulation rate.

[0042] Therefore, the preparation condition that promoted the highest encapsulation efficiency of Ang-(1-7) of 50% was the one that used: (i) 20 mM phosphate preparation buffer pH 7.2 containing dissolved sodium cholate for the preparation of permeabilized liposomes, (ii) a temperature of 60 ºC (2h) to incubate the liposomal solution with added active substance, (iii) 20 mM phosphate dilution buffer pH 7.2 for dilution of liposomes encapsulating part of the active substance and partial removal of bile salt from the liposomal membrane.

[0043] In order to compare the method claimed herein to that described in patent BR102013030151-5, regarding the encapsulation efficiency of Ang-(1-7) in soy phosphatidylcholine liposomes incorporating sodium cholate, without modifying the original lipid composition of the liposomal formulations, we used the ultrafiltration technique to separate the liposomes from the unencapsulated peptide to determine the encapsulation efficiency. For comparison purposes, we also used the same concentrations of lipid and surfactant, as well as the same initial peptide / lipid ratio of 1:40.4 (w / w).

[0044] For the preparation of the formulation using the classic process (BR102013030151-5), 97 mg of SPC were dissolved in 0.1 mL of 96° ethanol. The ethanolic solution of the lipid was injected into 0.5 mL of buffer solution (HEPES 0.02M, NaCl 0.15M, pH 7.4) containing 13 mg of sodium cholate and 2.4 mg of Ang-(1-7). After 10 min of incubation in 25°C, the solution was then injected into the ethanolic solution. oC under stirring with a magnetic stir bar, the suspension was subjected to a particle size calibration process using a 100 nm pore polycarbonate membrane.

[0045] To prepare the formulation using the claimed process, 97 mg of soy phosphatidylcholine was dissolved in 0.1 mL of 96% ethanol. The ethanolic lipid solution was injected into 0.5 mL of 20 mM phosphate buffer solution, pH 7.2, containing 13 mg of sodium cholate. After 15 min of stirring at room temperature, the resulting suspension was extruded through a 100 nm pore polycarbonate membrane to calibrate the size of the liposomes. 1 mg of peptide was added to 0.25 mL of the permeabilized liposome suspension, and the mixture was incubated for 2 hours at 60°C. oC. The liposome mixture containing the peptide was then diluted 4x with a buffer consisting of 20 mM phosphate, 0.15 M NaCl, pH 7.2, to remove the bile salt from the liposome membrane and trap the peptide in the liposome's internal aqueous compartment.

[0046] To separate the liposomes from the external environment, the suspensions were subjected to an ultrafiltration process (Amicon Ultra 0.5mL Ultra filtration cells). ® -50K) for 20 min at 14,000xg and 15 o C. The peptide was then measured in the medium outside the liposomes (Fe) and in the non-ultrafiltered suspension (Ftot) by spectrofluorimetry, as described above. The encapsulation efficiency of the peptide was determined as: EE% = 100x(Ftot-Fe) / Ftot.

[0047] The encapsulation efficiency value was 48.3% for the formulation prepared by the claimed process, while it was only 8.4% for the formulation obtained by the classical method used in BR102013030151-5. This latter experiment clearly demonstrates the superiority of the new claimed encapsulation process compared to the classical passive encapsulation method reported in the prior art.

[0048] The data shown in this example establish proof of concept for the feasibility of the claimed process for encapsulating water-soluble substances in liposomes and highlight critical parameters of this process.

[0049] Figure 1 shows a flowchart of the manufacturing process for liposomal compositions and potential routes of administration and applications. It highlights the possibility of generating a finished product by applying the process up to step 2. In this case, step 3 is performed at the time of product use, with dilution of the product in an aqueous medium. Furthermore, the liposomal composition as obtained in step 2 has the advantage of not promoting a difference in substance concentration between the intra- and extra-vesicular compartments, which guarantees encapsulation stability.

[0050] The process claimed in this patent application involves, as a first step, the preparation of a concentrated suspension of pre-formed liposomes permeabilized with bile salt (step 1), followed by the remote encapsulation of the water-soluble active ingredient (step 2) and the removal of part of the bile salt from the liposomal membrane for impermeabilization and retention of the active ingredient (step 3). The water-soluble substance can be any salts, metal complexes, small molecules, macromolecules, carbon nanoparticles, and metal nanoparticles. The osmolarities and pH of the preparation and dilution media must be chosen so as not to promote the entry of water into the liposomes and consequent release of the encapsulated active ingredient.

[0051] Step 1 can be performed simply by injecting an aqueous phase ethanolic lipid solution containing a bile salt, followed by vesicle size calibration, for example using an extrusion process, high-pressure homogenization, high-shear homogenization, or microfluidization. Alternatively, Step 1 can be performed using a microfluidic device.

[0052] Liposomes can be composed of any mixture of lipids, provided they are capable of generating vesicular structures. Phospholipids are often preferred due to their ability to form bilayers and vesicles and their high biocompatibility. Among other things, liposomes can be conventional, typically formed from phosphatidylcholine with a purity ranging from 75 to 100% or lecithin, and may also contain cholesterol, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), phosphatidylglycerol, phosphatidic acid, or other ionizable lipids. These liposomes may also include antioxidant lipids such as alpha-tocopherol. Liposomes may also have lipids coupled to ethylene glycol (PEG) polymers, in a molar proportion of 3 to 10% relative to the total lipids.To obtain permeabilized liposomes, a bile salt is added, which can be primary, such as cholate and chenodeoxycholate salts; secondary, such as deoxycholate, lithocholate, ursodeoxycholate salts; or a bile salt conjugated to glycine and taurine, in addition to semi-synthetic keto derivatives.

[0053] In the preparation of permeabilized liposomes, the final lipid concentration is typically between 1 and 60% (w / v), the mass ratio of bile salt to lipids is typically between 0.05 and 0.3 (w / w), and the final ethanol content in the aqueous phase typically varies between 5 and 30% (v / v). The substance can be added at a substance / lipid mass ratio of up to 1 (w / w).

[0054] Step 2 is performed by adding the water-soluble substance to the suspension of pre-formed liposomes, followed by incubation for remote permeation of the active ingredient through the membrane, until the active ingredient equilibrates between the intra- and extravesicular compartments. This incubation step can be performed at room temperature, but increasing the temperature may favor encapsulation, depending on the membrane composition and the molar mass of the active ingredient to be encapsulated. The incubation temperature can be chosen from 4 o C and 90 o C, depending on the molar mass and stability of the substance to be encapsulated and the incubation period, which can last up to 72 hours at lower temperatures.

[0055] Step 3 consists of removing part of the bile salt from the membrane of the liposomal suspension containing the water-soluble substance, in order to reverse its permeabilizing effect and trap the active substance in the internal aqueous compartment of the liposomes. This removal can be partial, through simple dilution in an aqueous solution without bile salt, or more complete, by dialysis, gel filtration chromatography, tangential filtration, diafiltration, use of a resin capable of binding the bile salt, or separation by ultrafiltration or centrifugation followed by resuspension with a buffer without bile salt. EXAMPLE 2 – VALIDATION OF THE EFFICIENCY OF THE ENCAPSULATION PROCESS USING A NON-PEPTIDE HYDROPHILIC SUBSTANCE

[0056] The non-peptidic substance calcein was also used as a hydrophilic substance to validate the proposed encapsulation process.

[0057] Liposomes were prepared using the ethanol injection method. For this, 776 mg of soy phosphatidylcholine was dissolved in 0.8 mL of 96% ethanol. The ethanolic lipid solution was injected into 4 mL of 20 mM phosphate buffer solution, pH 7.2, containing or not 104 mg of sodium cholate. After 15 min under agitation at room temperature, the resulting suspension was extruded through a 100 nm pore polycarbonate membrane for liposome size calibration.

[0058] To 0.25 mL of the suspension of permeabilized (with sodium cholate) or non-permeabilized (without sodium cholate) liposomes, 0.06 mL of a 0.268 M aqueous calcein solution (pH 7.4) was added, corresponding to a calcein / lipid mass ratio of 0.2. The mixture was incubated for 2 hours at 60°C. oC. The liposome suspension containing calcein was then diluted 100 times with 20 mM phosphate buffer, pH 7.2, to remove the bile salt from the liposome membrane and trap the calcein in the aqueous compartment inside the liposome.

[0059] The calcein encapsulation rate was determined by spectrofluorimetry, exploiting the fact that encapsulated calcein has muted fluorescence due to its high intravesicular concentration. In a cuvette containing 2.2 mL PBS, fluorescence (Fo) was measured in kinetic mode at excitation and emission wavelengths of 490 nm and 515 nm, respectively. 2 µL of the 100x diluted formulation was added, and the fluorescence intensity was measured again before (Fm) and after (Ftot) liposome lysis and subsequent release of encapsulated calcein by the addition of 5 µL of Triton X-100 20% (w / v). The encapsulation efficiency was calculated as EE% = 100*(Ftot-Fm) / (Ftot-Fo).

[0060] The characterization results are shown in Table 2. Table 2 – Characterization results of calcein liposomal compositions, prepared in the presence or absence of bile salt. Composition EE%* TE* Liposomal diameter (m / m) (PDI*) Liposomes 1.7% 0.0034 109.9 nm non-permeabilized (0.066) Liposomes 25.1% 0.050 117.2 nm permeabilized (0.138) with cholate *EE%: encapsulation efficiency; TE: encapsulation content; PDI: polydispersity index

[0061] The results demonstrate a calcein encapsulation efficiency of 25.1% in liposomes permeabilized with sodium cholate, while non-permeabilized liposomes showed an EE% of only 1.7%. This shows that remote calcein encapsulation only occurs effectively after liposome permeabilization. The results of this experiment also demonstrate that the claimed process can be more broadly applied to hydrophilic organic substances. EXAMPLE 3 – STABILITY STUDY OF THE FORMULATION IN SIMULATED GASTRIC MEDIUM

[0062] In order to evaluate the potential of formulations resulting from the claimed process for oral administration, we investigated the encapsulation efficiency of the peptide after exposing the formulation to a simulated gastric medium (0.1 M aqueous HCl solution containing 0.034 M NaCl).

[0063] To prepare the formulation, 97 mg of soy phosphatidylcholine was dissolved in 0.1 mL of 96% ethanol. The ethanolic lipid solution was injected into 0.5 mL of 20 mM phosphate buffer solution, pH 7.2, containing 13 mg of sodium cholate. After 15 min of stirring at room temperature, the resulting liposome size was calibrated by extrusion. 1 mg of Ang-(1-7) peptide was added to 0.25 mL of the permeabilized liposome suspension, and the mixture was incubated for 2 hours at 60°C. o C for encapsulation. After cooling to room temperature, the liposome suspension containing the peptide was diluted 8 times, either in 20 mM phosphate buffer, pH 7.2, or in simulated gastric medium, and incubated for 1 hour at 37°C. o C. After incubation, the suspension was subjected to dialysis at 4 o C for 24 hours against phosphate buffer 20 mM NaCl 150 mM, pH 7.2.

[0064] The encapsulation efficiency of the peptide was determined after incubation in simulated gastric medium and in pH 7.2 buffer, by spectrofluorimetric quantification of the peptide, as described in Example 1. Liposomes showed an encapsulation efficiency of 30% of the initial peptide quantity after exposure to the acidic medium, compared to 40% of the peptide encapsulated in pH 7.2 buffer. This data indicates that, in the period of 1 hour at 37 o C, the proportion of peptide released was 20% in the pH 7.2 buffer and 40% in the simulated gastric medium. This study highlights the ability of liposomes to retain the peptide even under the extreme pH conditions of the gastric medium. It also points to the potential of these liposomes for protecting the peptide in the gastric medium and for oral administration. EXAMPLE 4 – EVALUATION OF THE USE OF THE PRESENT TECHNOLOGY PROCESS IN THE PREPARATION OF SOLID PEPTIDE NANOFORMULATION

[0065] The present process was also evaluated for obtaining liposomal nanoformulation in solid form, by subjecting it to a drying step in the presence of cryoprotective sugar.

[0066] To prepare the nanoformulation, 97 mg of soy phosphatidylcholine was dissolved in 0.1 mL of 96% ethanol. The ethanolic solution of the lipid was injected into 0.5 mL of 20 mM phosphate buffer solution, pH 7.2, containing 13 mg of sodium cholate and 51 mg of sucrose. After 15 minutes of stirring at room temperature, the resulting liposome size was calibrated by extrusion with a 100 nm pore membrane. 1 mg of Ang-(1-7) peptide was added to 0.25 mL of the permeabilized liposome suspension, and the mixture was incubated for 2 hours at 60°C. o C for encapsulation. After cooling to room temperature, the liposome suspension containing the peptide was diluted 4 times in 20 mM phosphate buffer, pH 7.2, containing 121 g / L of sucrose, and subjected to dialysis at 4o Incubation of the encapsulated peptide by spectrofluorimetry, performed as described in Example 1, showed an encapsulation rate of 56%. The encapsulated peptide was incubated for 24 hours in 20 mM phosphate buffer (pH 7.2 – without NaCl) containing 121 g / L of sucrose, to eliminate ethanol and unencapsulated peptide.

[0067] To obtain the solid-state nanoformulation, the resulting liposomal suspension was then lyophilized for 24 hours (Liotop freeze dryer). ^ L101) and stored at 4 o W.

[0068] To evaluate the encapsulated peptide fraction after lyophilization, the lyophilized material was reconstituted with deionized water at room temperature (in the same volume as the original suspension), and the resulting suspension was subjected to ultrafiltration (Amicon Ultra 0.5 mL Ultra -50K filtration cells) for 20 min at 14,000 x e. oC. The peptide was then quantified in the medium outside the liposomes (ultrafiltrate) and in the non-ultrafiltered suspension by spectrofluorimetry, as described in Example 1. An efficiency of 56% of the encapsulated peptide was determined, demonstrating good retention of the peptide after the lyophilization process. The evaluation of the particle size distribution of the reconstituted nanoformulation showed an average diameter of 109 nm with a polydispersity index of 0.182, demonstrating the preservation of particle size.

[0069] This example demonstrates that the liposomal nanoformulation prepared according to the process of the present technology can also be obtained in solid or powder form. The solid form ensures long-term stability during storage. Furthermore, it opens the possibility of subsequent administration in powder form, for example orally or by inhalation. EXAMPLE 5 – OBTAINING LIPOSOMAL COMPOSITIONS OF ANGIOTENSIN-(1-7) PEPTIDE WITH HIGH ENCAPSULATION LEVEL

[0070] To prepare the formulation, 97 mg of soy phosphatidylcholine was dissolved in 0.1 mL of 96% ethanol. The ethanolic lipid solution was injected into 0.5 mL of 20 mM phosphate buffer solution, pH 7.2, containing 13 mg of sodium cholate. After 15 min of stirring at room temperature, the resulting liposome size was calibrated by extrusion, as described in Example 1. To 0.25 mL of the permeabilized liposome suspension, 3 or 5 mg of the Ang-(1-7) peptide was added, and the mixture was incubated for 2 hours at 60°C. oC to promote encapsulation. After cooling to room temperature, liposome suspensions exposed to 3 and 5 mg of peptide were diluted 10 and 20 times, respectively, in 20 mM phosphate buffer, pH 7.2. Particle size characterization of the formulations exposed to 3 and 5 mg of peptide by DLS (using a Zetasizer Nano ZS instrument, Malvern Instruments®) showed average diameters of 101.2 nm and 102.8 nm and polydispersity indices of 0.166 and 0.158, respectively.

[0071] To evaluate the encapsulated peptide fraction, the resulting suspensions (400 µL) were subjected to ultrafiltration (Amicon Ultra 0.5 mL Ultra -50K ultrafiltration cells) for 40 min at 14,000 x g and 4 oC. The peptide was then quantified in the medium outside the liposomes (ultrafiltrate) and in the non-ultrafiltrate suspension (100%) by spectrofluorimetry. Fluorescence was measured with excitation at 285 nm and emission at 305 nm of a solution consisting of 2.2 mL of methanol containing 50 µL of sample. The fluorescence signal from the 100% solution was subtracted from the signal obtained with a blank formulation (prepared under the same conditions, but without the peptide). The fluorescence signal from the ultrafiltrate, originating from the non-encapsulated peptide, was subtracted from the methanol signal. The absence of interference from the lipid matrix on the peptide's fluorescence was also confirmed, as well as the absence of peptide retention on the ultrafiltration membrane. Encapsulation rates of 48.2% and 52.9% were determined for the suspensions exposed to 3 and 5 mg of peptide.For liposomal compositions exposed to 3 mg and 5 mg of peptide, ratios of 0.036 g and 0.065 g of encapsulated peptide per g of lipid are calculated, which corresponds to the highest content of Ang-(1-7) encapsulated in liposomes reported so far in the state of the art.

[0072] This data demonstrates that the claimed process maintained a very high encapsulation rate of Ang-(1-7) (>50%) in calibrated-size liposomes, even when increasing the amount of peptide added, which allowed achieving a level of encapsulated peptide never before reached in the state of the art. EXAMPLE 6 – OBTAINING THE COMPOSITION OF ANGIOTENSIN-(1-7) PEPTIDE IN PEGYLATED LIPOSOMES

[0073] The novel encapsulation process was evaluated to obtain a formulation of the Ang-(1-7) peptide in PEGylated liposomes. Initially, 388 mg of soy phosphatidylcholine were solubilized in 400 µL of ethanol at 60°C for 10 minutes, followed by the addition of 69 mg of DSPE-PEG2000, with stirring until complete lipid solubilization. In parallel, 52 mg of sodium cholate were dissolved in 2 mL of 20 mM phosphate buffer pH 7.2 and transferred to a beaker. The organic phase was then rapidly injected onto the aqueous phase under intense magnetic stirring for 10 to 15 minutes, promoting liposome formation. The suspension was sequentially extruded through polycarbonate membranes (200 and 100 nm) to control vesicle size, which was evaluated by dynamic light scattering (DLS). Next, 1 mg of Ang-(1-7) was incorporated into the liposome suspension by incubation at 60°C for 2 hours, followed by cooling (4–8°C).

[0074] The formulation was diluted 4 times with 20 mM phosphate buffer pH 7.2 for subsequent characterization. Particle size analysis of the formulation by DLS (using a Zetasizer Nano ZS instrument, Malvern Instruments®) showed an average diameter of 173.7 nm and a polydispersity index of 0.199.

[0075] For evaluation of the encapsulated peptide fraction, the resulting suspension (400 µL) was subjected to ultrafiltration (Amicon Ultra 0.5 mL Ultra -50K ultrafiltration cells) for 40 min at 14,000 x g and 4 oC. The peptide was then quantified in the medium outside the liposomes (ultrafiltrate) and in the non-ultrafiltrate suspension (100%) by spectrofluorimetry. Fluorescence was measured with excitation at 285 nm and emission at 305 nm of a solution consisting of 2.2 mL of methanol containing 50 µL of sample. The fluorescence signal from the 100% solution was subtracted from the signal obtained with a blank formulation (prepared under the same conditions, but without the peptide). And the fluorescence signal from the ultrafiltrate, originating from the non-encapsulated peptide, was subtracted from the methanol signal. In this way, a peptide encapsulation rate of 26% was determined.

[0076] This example demonstrates that the new process claimed here can be applied to the encapsulation of active ingredients in PEGylated liposomes. EXAMPLE 7 – APPLICATION OF THE NEW PROCESS TO THE ENCAPSULATION OF INSULIN WITH A HIGH ENCAPSULATION RATE

[0077] To prepare the formulation, 97 mg of soy phosphatidylcholine was dissolved in 0.1 mL of 96% ethanol. The ethanolic lipid solution was injected into 0.5 mL of 20 mM phosphate buffer solution, pH 7.2, containing 13 mg of sodium cholate. After 15 min of stirring at room temperature, the resulting suspension had its liposome size calibrated by extrusion, as described in Example 1. 1 mg of human insulin was added to 0.25 mL of the permeabilized liposome suspension, and the mixture was incubated for 2 hours at 60°C. o C to promote encapsulation. After cooling to room temperature, the suspension was diluted 4 times in 20 mM phosphate buffer pH 7.2. Particle size characterization of the resulting formulation by DLS (using a Zetasizer Nano ZS instrument, Malvern Instruments®) showed an average diameter of 179.8 nm and a polydispersity index of 0.112.

[0078] To evaluate the encapsulated protein fraction, the resulting suspension (400 µL) was subjected to ultrafiltration (Amicon Ultra 0.5 mL Ultra -50K ultrafiltration cells) for 40 min at 14,000 x g and 4 o C. The protein was then quantified in the medium outside the liposomes (ultrafiltrate) and in the non-ultrafiltrate suspension (100%) by spectrofluorimetry. Fluorescence was measured with excitation at 285 nm and emission at 309 nm of a solution consisting of 2.2 mL of methanol containing 50 µL of sample. The fluorescence signal from the 100% solution was subtracted from the signal obtained with a blank formulation (prepared under the same conditions, but without the protein). And the fluorescence signal from the ultrafiltrate, originating from the non-encapsulated protein, was subtracted from the methanol signal. The absence of interference from the lipid matrix on the protein fluorescence was also confirmed, as well as the absence of protein retention in the ultrafiltration membrane. An encapsulation rate of 63.5 µ0.2% (N=2) was determined.

[0079] This example demonstrates that the new process can be applied to the nanoencapsulation of high molecular weight macromolecules, including proteins such as insulin. EXAMPLE 8 – ANTIHYPERTENSIVE ACTIVITY OF THE LIPOSOMAL FORMULATION OF ANGIOTENSIN-(1-7) ADMINISTERED ORALLY IN SPONTANEOUSLY HYPERTENSIVE RATS

[0080] A liposomal formulation of Ang-(1-7), prepared according to the process of this technology, was evaluated for its antihypertensive activity when administered orally to spontaneously hypertensive rats (SHR).

[0081] To prepare the liposomal formulation of Ang-(1-7) (LAng-(1-7)), 97 mg of soy phosphatidylcholine was dissolved in 0.1 mL of 96% ethanol. The ethanolic lipid solution was injected into 0.5 mL of 20 mM phosphate buffer solution, pH 7.2, containing 13 mg of sodium cholate. After 15 min of stirring at room temperature, the resulting suspension was extruded through a 100 nm pore polycarbonate membrane to calibrate the size of the liposomes. 1 mg of peptide was added to 0.25 mL of the permeabilized liposome suspension, and the mixture was incubated for 2 hours at 60°C. o C. The mixture was then stored at 4 o C until the time of use. A blank formulation (Lvazio) was prepared, using the same process, but without the peptide.

[0082] Shortly before administration to animals, LAng-(1-7) and Lvazio liposome suspensions were diluted 20 times with 20 mM phosphate buffer pH 7.2, to remove bile salt from the liposome membrane and trap the peptide in the liposome's internal aqueous compartment.

[0083] For direct blood pressure (BP) recording, SHR rats (4-6 months old, n=16; LAng-(1-7), n=11; Lvazio, n=6) were anesthetized with inhaled isoflurane. For the procedure, polyethylene cannulas with PE10 (4 cm for the artery and 3 cm for the vein), heat-polymerized with PE50 (approx. 17 cm) and filled with sterile 0.9% saline solution containing heparin (5000 IU / ml) were inserted into the abdominal aorta via the femoral artery (BP recordings). The catheters were externalized subcutaneously to the interscapular region of the animal and fixed with surgical suture. After 24 hours, cardiovascular parameters were recorded in conscious animals. The arterial catheter was connected to a pressure transducer, and the pulsatile blood pressure was continuously recorded by an A / D data acquisition system (MP100; Biopac Systems, Inc., Santa Barbara, CA, USA).Mean arterial pressure (MAP) was simultaneously derived from arterial pulse waves using software (AcqKnowledge5; Biopac Systems). After a one-hour stabilization period of cardiovascular parameters, either the LAng-(1-7) or Lvazio formulation was administered orally via gavage with the animals connected to the transducer at a dose of 100 µg / kg. Cardiovascular parameters were monitored for 6 hours after injection. MAP data were extracted at 10-minute intervals at each analysis point. Data analysis was performed using a two-way ANOVA with Tukey's post-hoc test for temporal analysis, and the delta after 6 hours of administration was analyzed using the Mann-Whitney test. The software used was Prism 10.3.

[0084] Figure 2 shows the MAP data recorded over time after oral administration of LAng-(1-7) and Lvazio in SHR rats. The group receiving LAng-(1-7) showed a significant reduction in blood pressure compared to baseline, from 3 hours of recording to the last time point of 6 hours, demonstrating the antihypertensive action of the formulation. On the other hand, there was no significant change in the control group receiving the Lvazio vehicle, indicating that the antihypertensive action is due to the Ang-(1-7) peptide.

[0085] The comparison of MAP variation between the LAng-(1-7) and Lvazio groups at 6h (Figure 2, letter B) shows a further significant difference and confirms the antihypertensive action of the LAng-(1-7) formulation.

[0086] The results of this study are consistent with the observed stability of the LAng-(1-7) formulation in simulated gastric juice. This is the first report of a liposomal formulation of Ang-(1-7) to demonstrate oral antihypertensive activity.

[0087] Surprisingly, the process claimed here has made it possible to obtain orally active liposomal formulations.

Claims

1 / 4 CLAIMS 1. PROCESS FOR OBTAINING LIPOSOMES WITH HIGH LEVELS OF ENCAPSULATION OF WATER-SOLUBLE ACTIVE INGREDIENTS, characterized by comprising the following steps: a. Dissolving a lipid or mixture of lipids in ethanol in a proportion ranging from 20 to 200 g of lipid(s) per 100 mL of ethanol; b. Preparing a buffer solution with a pH in the range of 3 to 10 and dissolving bile salt in an amount ranging from 0.5 to 5.4 g of bile salt per 100 mL of buffer solution; c. Inject the ethanolic solution obtained in step “a” into the buffer solution obtained in step “b” at a volume ratio varying between 0.025 and 0.3 and at a mass ratio of bile salt to lipids varying between 0.05 and 0.3 (m / m), and maintain the mixture under agitation until a homogeneous suspension is obtained; d. Optionally, calibrate the size of the liposomes in the resulting suspension obtained in step “c”, to obtain liposomes with a diameter between 30 and 400 nm and a polydispersity index of less than 0.3; e.Add the water-soluble compound to be encapsulated to the suspension of pre-formed liposomes in step “d”, so that the compound / lipid mass ratio is up to 1, incubate the mixture for a duration ranging from 1 minute to 72 hours at a temperature ranging from 4 ºC to 90 ºC and, optionally, store the resulting mixture as an aqueous suspension at a temperature ranging from 4 ºC to room temperature. 2 / 4 f. At the time of use, partially displace the bile salt from the membrane of the liposomal suspension obtained in step “e” to the aqueous phase, by dilution at least 2 times in an aqueous solution without bile salt.

2. PROCESS FOR OBTAINING LIPOSOMES WITH HIGH LEVELS OF ENCAPSULATION OF WATER-SOLUBLE ACTIVE INGREDIENTS, according to claim 1, characterized by replacing step “f” with a step of removing the bile salt from the liposomal suspension, using a process selected from the group comprising dialysis, gel filtration chromatography, tangential filtration, diafiltration, use of a resin capable of binding to the bile salt, separation by centrifugation or ultrafiltration followed by resuspension with an aqueous solution without the bile salt. 3.PROCESS FOR OBTAINING LIPOSOMES WITH HIGH LEVELS OF ENCAPSULATION OF WATER-SOLUBLE ACTIVE INGREDIENTS, according to claim 1, characterized in that, in step “a”, the lipids are selected from the group comprising phosphatidylcholine with purity ranging from 75 to 100%, lecithin, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, stearylamine, dicetylphosphate, 1,2-dioleoyl-3-trimethylammonium-propane, ionizable lipid, cholesterol, from the group of antioxidant lipids such as alpha-tocopherol, and / or from the group of pegylated lipids.

4. PROCESS FOR OBTAINING LIPOSOMES WITH HIGH LEVELS OF ENCAPSULATION OF WATER-SOLUBLE ACTIVE INGREDIENTS, according to claim 1, characterized in that, in step “b”, the bile salt is selected from primary bile salts, such as cholate and chenodeoxycholate salts, secondary bile salts, such as deoxycholate, lithocholate, ursodeoxycholate salts, and so on. 3 / 4 bile salts conjugated to glycine or taurine, in addition to semi-synthetic keto derivatives.

5. PROCESS FOR OBTAINING LIPOSOMES WITH HIGH ENCAPSULATION LEVELS OF WATER-SOLUBLE ACTIVE INGREDIENTS, according to any one of claims 1 to 4, characterized in that, in step “e”, the water-soluble compound is selected from the group comprising salts, metal complexes, inclusion complexes with cyclodextrins, small molecules with a molecular mass up to 1,500 Da, carbon nanoparticles, metal nanoparticles, macromolecules including oligosaccharides, polysaccharides, nucleic acids, proteins and peptides.

6. PROCESS FOR OBTAINING LIPOSOMES WITH HIGH LEVELS OF ENCAPSULATION OF WATER-SOLUBLE ACTIVE INGREDIENTS, according to any one of claims 1 to 5, characterized in that, in step “e”, the water-soluble compound is the angiotensin-(1-7) peptide. 7.PROCESS FOR OBTAINING LIPOSOMES WITH HIGH LEVELS OF ENCAPSULATION OF WATER-SOLUBLE ACTIVE INGREDIENTS, according to any one of claims 1 to 5, characterized in that, in step “e”, the water-soluble compound is insulin.

8. PROCESS FOR OBTAINING LIPOSOMES WITH HIGH LEVELS OF ENCAPSULATION OF WATER-SOLUBLE ACTIVE INGREDIENTS, according to any one of claims 1 to 7, characterized by comprising an additional step of dehydrating the liposomal suspension, by lyophilization or spray drying, in the presence of a cryoprotective sugar selected from the group comprising sucrose, glucose, trehalose, lactose, mannitol and sorbitol. 4 / 4 9. LIPOSOMAL FORMULATION obtained by the process defined in claim 6, characterized by comprising encapsulated angiotensin-(1-7) with an encapsulated peptide / lipid ratio of 0.03 to 0.07 (m / m).

10. USE OF THE LIPOSOMAL FORMULATION defined in claim 9, characterized by being for the production of orally administered medications for the treatment of pathologies in which Ang-(1-7) provides therapeutic benefits, such as hypertension, kidney disease, liver disease, heart disease, lung disease, neurological disease, metabolic disease, muscle disease, endometriosis, inflammatory diseases, bacterial and viral infectious diseases.

11. USE OF THE PROCESS defined in claim 1, characterized by being for the production of solid or suspension liposomal compositions encapsulating active water-soluble substances with a substance / lipid mass ratio of up to 1, for administration via oral, intranasal, inhalation, parenteral, topical or transdermal application or local injection.

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