Mixed liposomal composition of remdesivir and an angiotensinergic peptide, method and use

Abstract

The present technology relates to liposomal compositions, to a method for obtaining said compositions, and to use thereof in the preparation of medicaments for the treatment of respiratory and neurological viral infections. The liposomal composition comprises a mixture of an antiviral or remdesivir, and an anti-inflammatory, an angiotensinergic peptide, both encapsulated in conventional or PEGylated liposomes.

Description

[0001] "Mixed liposomal composition of remdesivir with an angiotensin peptide, process and use"

[0002]

[0001] The present technology deals with liposomal compositions, their production process and use in the preparation of medicines for the treatment of respiratory and neurological viral infections. The liposomal composition comprises a mixture of an antiviral, remdesivir, and an anti-inflammatory, an angiotensin-releasing peptide, both incorporated into conventional or PEGylated liposomes. The therapeutic efficacy and safety of the composition incorporating the two active ingredients in conventional LMix liposomes were evaluated in a murine model for SARS-CoV-2 infection. The composition administered intranasally eliminated the virus in the lungs and brain of the animals, unlike the commercial parenteral formulation of remdesivir, which had no effect on the viral load in the brain and showed toxicity.Systemic immunoprotective response was also evidenced after treatment with LMIX, not observed with the commercial formulation of remdesivir or liposomal compositions containing only one of the two active ingredients, demonstrating the synergism of the remdesivir-angiotensin-(1-7) combination, ensuring therapeutic efficacy and safety in the treatment of viral lung and central nervous system infections.

[0003]

[0002] Viral epidemics or pandemics of acute respiratory infections represent a global threat. Examples include influenza caused by the H1N1 virus in 2009, severe acute respiratory syndrome (SARS) in 2003, and coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 in 2019. In addition to respiratory tract infections, many viruses can invade the central nervous system (CNS). In viral CNS infections (often called aseptic meningitis), the virus can infect the brain by retrograde travel along the axons of the spinal cord or brain. With viral replication in neural cells, cell death and dysfunction occur. The extent of neuronal damage can contribute to the clinical severity of viral meningoencephalitis.

[0004]

[0003] SARS-CoV-2 is an example of a virus that, in addition to causing respiratory infections, has been associated with neurological complications. SARS-CoV-2 and human coronaviruses (HCoV) are enveloped viruses that have a positive-sense single-stranded RNA genome and possess the spike protein (S), nucleocapsid proteins (N), hemagglutinin, membrane protein, and envelope protein, with the S protein being responsible for binding to the host cell mediated by the angiotensin-converting enzyme II (ACE2) receptor. In brain infection, in addition to the interaction of the receptor-binding domain of the S protein (RBD) with ACE2, co-receptors or alternative receptors, including neuronal-expressed Neuropilin-1, can increase SARS-CoV-2 uptake in the CNS.“Long-COVID” is a type of persistent post-acute infection syndrome that is gaining notoriety, with patients reporting persistent manifestations such as hyposmia, hypogeusia, sleep disturbances, and substantial cognitive impairment, the latter affecting approximately one in four COVID-19 cases. These clinical symptoms are supported by extensive evidence of SARS-CoV-2 infectivity in various CNS cell types and significant structural changes in the brains of COVID-19 patients.

[0005]

[0004] Aiming to reduce the serious impact of viral infections on public health, there has been a constant search for new, more potent drugs. Among the antivirals, we can highlight Remdesivir (RDV) or GS-5734, a nucleotide analog prodrug and precursor of GS-441524, which inhibits the replication of RNA viruses. A phase III clinical trial in non-hospitalized COVID-19 patients at high risk of severe disease demonstrated that a three-day course of treatment with RDV resulted in an 87% lower risk of hospitalization or death compared to placebo (Gottlieb, RL et al., Early Remdesivir to Prevent Progression to Severe Covid-19 in Outpatients. New England Journal of Medicine, 386, 305-315, 2022). However, the antiviral can cause kidney and liver damage, as well as cognitive dysfunction.

[0006]

[0005] In addition to antiviral treatment, the use of anti-inflammatory and immunomodulatory substances as therapeutic tools has been investigated due to the exacerbated and frequent inflammatory response in pulmonary viral infections. In this context, the great potential of combined therapy, associating an antiviral with an anti-inflammatory, has also been highlighted. Among the substances with great potential in controlling the inflammatory response to pulmonary viral infections, we can highlight angiotensin-(1-7) (ang-(1-7)). Studies in preclinical models of SARS suggest that ang-(1-7) protects against acute pulmonary failure, reducing inflammation and fibrosis (Li, Y. et al. Angiotensin-converting enzyme 2 prevents lipopolysaccharide-induced rat acute lung injury via suppressing the ERK1 / 2 and NF-KB signaling pathways. Scientific Reports, 6, 27911., 2016).

[0007]

[0006] Liposomes are an emerging option as drug delivery nanosystems for pulmonary administration due to their excellent biocompatibility with alveolar surfactants. Liposomes are spherical structures formed by one or more lipid bilayers separated by an aqueous medium. They possess a high capacity for carrying both hydrophilic and lipophilic drugs, providing sustained release of the active ingredient and reducing its toxicity. This makes them an attractive choice for delivering drugs at effective concentrations to less accessible locations, including the alveoli of the lungs. They also allow for drug delivery to the brain via the olfactory route through intranasal administration, bypassing the blood-brain barrier and first-pass metabolism of intravenous and oral routes.

[0008]

[0007] Studies have shown that liposomes can encapsulate antiviral drugs for the treatment of COVID-19. For example, the drug hydroxychloroquine has several limitations for its use in the treatment of COVID-19, mainly its insufficient concentration in the lungs to eliminate the virus. However, its inhalable liposomal formulation has shown efficacy in preclinical studies in a murine model. The liposomal formulation of hydroxychloroquine provided higher pulmonary concentrations (approximately 30 times) and lower blood exposure compared to the unformulated drug (Tai, Tien-Tzu et al. A strategy to treat COVID-19 disease with targeted delivery of inhalable liposomal hydroxychloroquine: a preclinical pharmacokinetic study. Clinical and translational science, 14,132, 2021).

[0009]

[0008] The use of liposomes for the localized treatment of pulmonary infections has proven to be an effective, efficient, and safe strategy in the treatment of COVID-19. The state of the art shows some liposomal compositions with antivirals, such as remdesivir, and / or anti-inflammatories, such as ang-(1-7), not combined, for the treatment of COVID-19.

[0010]

[0009] LI, Jingjing et al. developed liposomal compositions of remdesivir for pulmonary drug delivery for the treatment of COVID-19 (LI, Jingjing et al. Liposomal remdesivir inhalation solution for targeted lung delivery as a novel therapeutic approach for COVID-19. Asian journal of pharmaceutical sciences, 16, 772-783, 2021). The liposomes improved the solubility and biocompatibility of the drug. In addition to increasing the concentration of remdesivir active metabolite nucleotide triphosphate (NTP) in the lungs of mice.

[0010] Patent document W02021202907A2, with a priority date of 01 / 04 / 2021, entitled “Remdesivir and remdesivir analogs, solutions, and nanoparticle, liposomal, and microparticle compositions for treating viral infections”, describes pharmaceutical compositions including liposomal compositions of remdesivir and its analogs for the treatment of viral infections, such as COVID-19.

[0011]

[0011] Patent document US10925889B2, whose priority date is 23 / 02 / 2022, entitled “Method of treating, reducing, or alleviating a medical condition in a patient”, describes a method for treating inflammatory diseases of the respiratory tract. The method consists of using pharmaceutical compositions of antiviral drugs such as remdesivir, combined or not, incorporated into liposomes.

[0012]

[0012] Patent document BR102021006371 A2, whose priority date is 01 / 04 / 2021, entitled “Pharmaceutical compositions comprising angiotensin-(1-7) for the treatment of early mild, moderate and / or severe forms of coronaviruses and their late symptoms”, describes liposomal compositions of the angiotensin-(1-7) peptide and / or derivatives for the treatment of coronavirus infections.

[0013]

[0013] Patent document US20240123023A1, whose priority date is 04 / 05 / 2022, entitled “Pharmaceutical angiotensin-(1-7) compositions in the treatment of (SARS)-CoV- or (SARS)-CoV-2-infection related diseases”, describes a pharmaceutical composition comprising the angiotensin-(1-7) peptide encapsulated in liposomes for use in the treatment of diseases related to coronavirus infection.

[0014]

[0014] Chen, Chian-Wei et al. developed the encapsulation of remdesivir and dexamethasone in nanostructured lipid carriers (NLCs) for pulmonary administration in the treatment of COVID-19 (Chen, Chian-Wei et al. Pulmonary delivery of remdesivir and dexamethasone encapsulated nanostructured lipid carriers for enhanced inflammatory suppression in lung. Journal of Drug Delivery Science and Technology, 90, 105144, 2023). In this prior art, NLCs are used to encapsulate antiviral and anti-inflammatory drugs, both with lipophilic characteristics. It is worth noting that NLCs do not allow the co-encapsulation of lipophilic and hydrophilic molecules, unlike the liposomes designed in this invention, in which the lipophilic remdesivir was incorporated into the membrane and the hydrophilic peptide encapsulated in the aqueous compartment of the liposomes.Therefore, the liposomes of the present invention allow the encapsulation of molecules with distinct physicochemical characteristics (hydrophilic and lipophilic) in separate compartments, which is technically different from CLNs that encapsulate lipophilic substances in the same compartment. This selective structuring increases the stability of the molecules, allows the controlled release of each active ingredient based on its location in the liposome, and minimizes the degradation and loss of activity of the encapsulated substances.

[0015]

[0015] In the prior art, no liposomal compositions of an antiviral drug, such as remdesivir, associated with an angiotensin-(1-7) peptide have been found. The prior art also does not describe the use of mixed liposomal compositions of the present invention or similar ones in the production of drugs for pulmonary and cerebral delivery in the treatment of respiratory and neurological viral infections.

[0016] BRIEF DESCRIPTION OF THE FIGURES

[0017]

[0016] Figure 1 shows the release kinetics of remdesivir (RDV) from different liposomal formulations under dialysis conditions at 37°C. The formulations comprise liposomes with conventional fluid membranes (IE-CONV) or PEGylated (IE-PEG) prepared by the ethanolic solution injection (IE) of lipids method; the RDV complex with HP- / 3-CD was used as a control in the experiment. Data are shown as mean ± standard error obtained from triplicates. *p< 0.0001.

[0018]

[0017] Figure 2 presents the therapeutic efficacy of liposomal compositions of remdesivir (LRDV), Ang-(1-7) (LANG), and the combined active ingredients in the same formulation (LMix), in relation to viral load in the lungs of K18-hACE2 transgenic mice inoculated intranasally with 6.0 x 10 4pf u SARS-CoV-2, assessed by plaque assay. Treatment was performed intranasally with liposomal compositions of remdesivir (LRDV), Ang-(1-7) (LANG) and the combined actives (LMix) or with PBS as a negative control. The positive control received the commercial formulation of remdesivir (Verklury®) intraperitoneally. Viral load in the lungs was analyzed on the 5th day. o dpi by plaque assay for infectious virus. Data shown as median ± interquartile range (N = 8).

[0019]

[0018] Figure 3 shows the therapeutic efficacy of liposomal compositions of remdesivir (LRDV), Ang-(1-7) (LANG), and the combined active ingredients in the same formulation (LMix), in relation to viral load in the lungs of K18-hACE2 transgenic mice inoculated intranasally with 6.0 x 10 4pfu SARS-CoV-2, assessed by RT-qPCR. Treatment was administered intranasally with liposomal compositions of remdesivir (LRDV), Ang-(1-7) (LANG), and the combined active ingredients (LMix), or with PBS as a negative control. The positive control received the commercial formulation of remdesivir (Verklury®) intraperitoneally. Viral load in the lungs was analyzed on the 5th day. o dpi by RT-qPCR for viral RNA levels. Data shown as mean ± standard error (N = 5).

[0020]

[0019] Figure 4 presents the therapeutic efficacy of liposomal compositions of remdesivir (LRDV), Ang-(1-7) (LANG), and the combined active ingredients in the same formulation (LMix), in relation to viral loads in the brains of K18-hACE2 transgenic mice inoculated intranasally with 6.0 x 10 4Viral load in the brain was assessed using plaque assays for SARS-CoV-2. Treatment was administered intranasally with liposomal compositions of remdesivir (LRDV), Ang-(1-7) (LANG), and the combined active ingredients (LMIX), or with PBS as a negative control (N = 8 per group). The positive control received the commercial formulation of remdesivir (Verklury®) intraperitoneally. Viral load in the brain was analyzed on the 5th day of the study. o dpi by plaque assay for infectious viruses. Data shown as median ± interquartile range.

[0021]

[0020] Figure 5 presents the therapeutic efficacy of liposomal compositions of remdesivir (LRDV), Ang-(1-7) (LANG), and the combined active ingredients in the same formulation (LMix), in relation to viral loads in the kidneys of K18-hACE2 transgenic mice inoculated intranasally with 6.0 x 10 4pf u SARS-CoV-2, assessed by plaque assay. Treatment was performed intranasally with liposomal compositions of remdesivir (LRDV), Ang-(1-7) (LANG) and the combined actives (LMIX) or with PBS as a negative control (N = 8 per group). The positive control received the commercial formulation of remdesivir (Verklury®) intraperitoneally. Viral load in the kidneys was analyzed on the 5th day. o dpi by plaque assay for infectious viruses. Data shown as median ± interquartile range.

[0022]

[0021] Figure 6 shows the inflammatory response in the lungs of K18-hACE2 transgenic mice inoculated intranasally with 6.0 x 10 4pf u SARS-CoV-2, after treatment with liposomal compositions of remdesivir, Ang-(1-7) and the combined active ingredients in the same formulation (LMix), in relation to the levels of the pro-inflammatory cytokine TNF-alpha. Treatment was performed intranasally with liposomal compositions of remdesivir (LRDV), Ang-(1-7) (LANG) and the combined active ingredients (LMix) or with PBS as a negative control (N = 8 per group). The positive control received the commercial formulation of remdesivir (Verklury®) intraperitoneally. TNF-alpha cytokine levels at 5 o Lung pressure (dpi) was analyzed by ELISA. Data are shown as mean ± standard error.

[0023]

[0022] Figure 7 shows the inflammatory response in the lungs of K18-hACE2 transgenic mice inoculated intranasally with 6.0 x 10 4pf u SARS-CoV-2, after treatment with liposomal compositions of remdesivir, Ang-(1-7) and the combined active ingredients in the same formulation (LMix), in relation to the levels of the pro-inflammatory cytokine IL-6. Treatment was performed intranasally with liposomal compositions of remdesivir (LRDV), Ang-(1-7) (LANG) and the combined active ingredients (LMix) or with PBS as a negative control (N = 8 per group). The positive control received the commercial formulation of remdesivir (Verklury®) intraperitoneally. IL-6 cytokine levels at 5 o Lung pressure (dpi) was analyzed by ELISA. Data are shown as mean ± standard error.

[0024]

[0023] Figure 8 shows the recruitment of myeloid cells in the lungs of K18-hACE2 transgenic mice inoculated intranasally with 6.0 x 10 4pf u SARS-CoV-2, after treatment with liposomal compositions of remdesivir, Ang-(1-7) and the combined actives in the same formulation (LMix). Treatment was performed intranasally with liposomal compositions of remdesivir (LRDV), Ang-(1-7) (LANG) and the combined actives (LMix) or with PBS as a negative control (N = 8 per group). The positive control received the commercial formulation of remdesivir (Verklury®) intraperitoneally. Lung myeloid cells were characterized and counted by flow cytometry. (A) macrophages, (B) neutrophils, (C) monocytes, (D) inflammatory monocytes and (E) M2 macrophages. Data are shown as median ± interquartile range.

[0024] Figure 9 shows the lymphoid cell profile in the spleen of K18-hACE2 transgenic mice inoculated intranasally with 6.0 x 10 4pf u SARS-CoV-2, after treatment with liposomal compositions of remdesivir, Ang-(1-7) and the combined active ingredients in the same formulation (LMix). Treatment was performed intranasally with liposomal compositions of remdesivir (LRDV), Ang-(1-7) (LANG) and the combined active ingredients (LMix) or with PBS as a negative control (N = 8 per group). The positive control received the commercial formulation of remdesivir (Verklury®) intraperitoneally. Splenic lymphoid cells were characterized and counted by flow cytometry.

[0025] A: hematopoietic cells (CD45+), B: T lymphocytes (CD3+CD45+), C: helper T lymphocytes (CD4+), D: cytotoxic T lymphocytes (CD8+), E: type 2 helper T lymphocytes (CD4+IL-4+), F: IL-4-producing cytotoxic T lymphocytes (CD8+IL-4+), G: IL-10-producing regulatory T lymphocytes (CD4+IL-10+), H: IL-10-producing cytotoxic T lymphocytes (CD8+IL-10+), I: type 1 helper T lymphocytes (CD4+IFN-γ+), J: IFN-γ-producing cytotoxic T lymphocytes (CD8+IFN-γ+), K: type 17 helper T lymphocytes (CD4+IL-17+), and L: IL-17-producing cytotoxic T lymphocytes (CD8+IL-17+). Data are shown as median ± interquartile range. Figure 10 shows the myeloid cell profile in the spleen of K18-hACE2 transgenic mice that were inoculated intranasally with 6.0 x 10 4pf u SARS-CoV-2, after treatment with liposomal compositions of remdesivir, Ang-(1-7) and the combined active ingredients in the same formulation (LMix). Treatment was performed intranasally with liposomal compositions of remdesivir (LRDV), Ang-(1-7) (LANG) and the combined active ingredients (LMIX) or with PBS as a negative control (N = 8 per group). The positive control received the commercial formulation of remdesivir (Verklury®) intraperitoneally. Splenic myeloid cells were characterized and counted by flow cytometry. A: hematopoietic cells (CD45+), B: F4 / 80+ CD11b+ Gr-1- myeloid cells (macrophages or monocytes), C: F4 / 80+ Gr-1 myeloid cells (macrophages), D: F4 / 80+ Gr-1+ myeloid cells (neutrophils or monocytes), E: F4 / 80+ CD206+ Gr-1- myeloid cells (M2 phenotype-associated macrophages), F: F4 / 80+ CD206+ Gr-1- myeloid cells (M2 phenotype-associated macrophages). Data are shown as median ± interquartile range.

[0026] Figure 11 shows the toxicity of liposomal compositions of remdesivir, Ang-(1-7), and the combined active ingredients in the same formulation, assessed by body weight variation. K18-hACE2 transgenic mice were inoculated intranasally with 6.0 x 10 4 pfu SARS-CoV-2. Treatment was administered intranasally with liposomal compositions of remdesivir (LRDV), Ang-(1-7) (LANG), and the combined active ingredients (LMix), or with PBS as a negative control (N = 8 per group). The positive control received the commercial formulation of remdesivir (Verklury®) intraperitoneally. Body weight was monitored during the 5 days of treatment, showing the variation between time zero and the last treatment time. Data are shown as mean ± standard error.

[0027]

[0025] Figure 12 shows the toxicity of liposomal compositions of remdesivir, Ang-(1-7), and the mixed active ingredients, assessed by body weight variation. K18-hACE2 transgenic mice were inoculated intranasally with 6.0 x 10 4 pfu SARS-CoV-2. Treatment was administered intranasally with liposomal compositions of remdesivir (LRDV), Ang-(1-7) (LANG), and the combined active ingredients in the same formulation (LMix), or with PBS as a negative control (N = 8 per group). The positive control received the commercial formulation of remdesivir (Verklury®) intraperitoneally. Body weight was monitored during the 5 days of treatment, and the % change from baseline was calculated. Data are shown as mean ± standard error.

[0028]

[0026] Figure 13 presents the hepatic toxicity of liposomal compositions of remdesivir, Ang-(1-7), and the combined active ingredients in the same formulation (LMix), evaluated by histological analysis of the liver after treatment. Histological score in each group based on the degree of hydrotopic degeneration. K18-hACE2 transgenic mice were inoculated intranasally with 6.0 x 10 4 pfu SARS-CoV-2. Treatment was performed intranasally with liposomal compositions of remdesivir (LRDV), Ang-(1-7) (LANG), and the combined active ingredients (LMIX) or with PBS as a negative control (N = 8 per group). The positive control received the commercial formulation of remdesivir (Verklury®) intraperitoneally. The Mock group was neither infected nor treated.

[0029]

[0027] Figure 14 shows the liver toxicity of liposomal compositions of remdesivir (LRDV), Ang-(1-7) (LANG), and the combined actives in the same formulation (LMix), assessed by histological analysis of the liver after treatment. Representative H&E micrographs of liver sections. A- Mock (uninfected): B- PBS: C- Veklury: D- LANG: E- LRDV: F- LMIX. 20x magnification. K18-hACE2 transgenic mice were inoculated intranasally with 6.0 x 10 4 pf u SARS-CoV-2. Treatment was performed intranasally with liposomal compositions of remdesivir (LRDV), Ang-(1-7) (LANG), and the combined actives (LMix) or with PBS as a negative control (N = 8 per group). The positive control received the commercial formulation of remdesivir (Verklury®) intraperitoneally. The Mock group was neither infected nor treated.

[0030] DETAILED DESCRIPTION OF THE TECHNOLOGY

[0031]

[0028] The present technology deals with liposomal compositions, their production process and use in the preparation of medicines for the treatment of respiratory and neurological viral infections. The liposomal composition comprises a mixture of an antiviral, remdesivir, and an anti-inflammatory, an angiotensin-releasing peptide, both incorporated into conventional or PEGylated liposomes. The therapeutic efficacy and safety of the composition incorporating the two active ingredients in conventional LMix liposomes were evaluated in a murine model for SARS-CoV-2 infection. The composition administered intranasally eliminated the virus in the lungs and brain of the animals, unlike the commercial parenteral formulation of remdesivir, which had no effect on the viral load in the brain and showed toxicity.Systemic immunoprotective response was also evidenced after treatment with LMIX, not observed with the commercial formulation of remdesivir or liposomal compositions containing only one of the two active ingredients, demonstrating the synergism of the remdesivir-angiotensin-(1-7) combination, ensuring therapeutic efficacy and safety in the treatment of viral lung and central nervous system infections.

[0032]

[0029] The liposomal composition comprises a mixture of remdesivir and an angiotensin-releasing peptide, incorporated into conventional or PEGylated liposomes.

[0033]

[0030] Liposomal composition is characterized by liposomes being formed by at least one of the lipids selected from the group comprising phosphatidylcholine, cholesterol (GOL), 2-dioleoyl-3-trimethylammonium-propane (DOTAP), phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid or other ionizable lipid, and an antioxidant lipid such as alpha-tocopherol.

[0034]

[0031] The PEGylated liposomal composition comprises mixtures of the following lipids: phosphatidylcholine, cholesterol (GOL) and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000) in molar proportions of 76%, 11% and 13%, respectively, relative to the total lipids.

[0035]

[0032] The liposomal composition is characterized by the angiotensin-(1-7) peptide being selected from the group comprising angiotensin-(1-7), alamandine, angiotensin-(1-9), angiotensin-(1-5). SEQ ID No. 1: Angiotensin-(1-7): Asp-Arg-Val-Tyr-lle-His-Pro; SEQ ID No. 2: Alamandine Ala-Arg-Val-Tyr-lle-His-Pro; SEQ ID No. 3: Angiotensin-(1-9): Asp-Arg-Val-Tyr-lle-His-Pro-Phe-His; SEQ ID No. 4: Angiotensin-(1-5): Asp-Arg-Val-Tyr-lle.

[0036]

[0033] The process for obtaining the liposomal composition is characterized by being an ethanol injection method comprising the following steps:

[0037] a. Prepare an ethanolic solution of the lipids with a concentration ranging from 200 g / L to 1,500 g / L, preheated to a temperature in the range of 25 to 65°C;

[0038] b. Add remdesivir to the solution obtained in step “a”, in an antiviral / lipid molar ratio ranging from 0.025 to 0.2; c. Subject the solution obtained in “b” to magnetic stirring at 300 to 800 rpm at a temperature in the range of 20 to 65°C until complete dissolution;

[0039] d. Inject the ethanolic phase obtained in “c” into an aqueous solution of the angiotensin-containing peptide at a peptide / lipid molar ratio between 0.0001 and 0.2, with a final maximum ethanol concentration of 25% (v / v);

[0040] e. Calibrate the size of the liposomes by extrusion, high-pressure homogenization, or microfluidization, in order to obtain a final liposome diameter of less than 250 nm and a polydispersity index of less than 0.3; f. Remove ethanol and unencapsulated peptide by dialysis or tangential filtration, at a temperature ranging from 0 oat 30°C.

[0041]

[0034] The use of liposomal composition can be in the preparation of medicines for the treatment of respiratory and neurological viral infections, such as, but not limited to, viral infections caused by viruses of the Coronaviridae and Orthomyxoviridae families.

[0042]

[0035] The liposomal composition may be used in the form of an aqueous suspension or in powder form in the presence of a cryoprotectant.

[0043]

[0036] The use of liposomal composition may be in the preparation of medicines for administration via intranasal, inhalation, intravenous, intraperitoneal, subcutaneous or intramuscular routes.

[0044]

[0037] The present invention can be better understood through the following examples, which are not limiting.

[0045] EXAMPLE 1 - PREPARATION OF DIFFERENT LIPOSOMAL COMPOSITIONS OF REMDESIVIR WITH OR WITHOUT CONVENTIONAL AND PEGYLATED ANGIOTENSIN-(1-7) PEPTIDE BY THE ETHANOL SOLUTION INJECTION METHOD

[0046]

[0038] Liposomes were prepared by the ethanol injection method (EI). According to the EI method, conventional liposomes (LIE-CONV) were prepared using 73 mg of soy phosphatidylcholine (PH90, Phospholipon90G, Lipoid, Germany) dissolved in 75 µL of ethanol heated to 40°C as the organic phase. The ethanol solution was then withdrawn and added to an Eppendorf tube containing 7.5 mg of remdesivir (RDV-GS-5734, acquired from MedChem Express®, NJ, USA), followed by magnetic stirring until complete dissolution. The phospholipid / drug solution (molar ratio: 7.5:1) was injected into a beaker containing 0.75 mL of PBS (0.15 M NaCl, 10 mM phosphate, pH 7.2), using a 1 mL syringe, under magnetic stirring at a rotation speed of 300 to 800 rpm.

[0047]

[0039] PEGylated liposomes (PEG-LLI) were also prepared using 64 mg of PH90, 11.2 mg of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG), and 9 mg of cholesterol (COL), followed by the addition of 350 µL of ethanol, gently stirred, and heated to 60°C until complete dissolution. Then, the organic phase was added to 8 mg of remdesivir. After dissolution of the drug, the phospholipid / drug solution (molar ratio: 6.5:1) was withdrawn with a 1 mL syringe and injected into the aqueous phase containing 3 mL of PBS under magnetic stirring. Conventional empty liposomes (CONV-EMPTY-LLI) and PEGylated liposomes (PEG-EMPTY-LLI), without the drug, were prepared using the same conditions.

[0048]

[0040] For particle size calibration, the resulting suspensions were extruded (5x) through polycarbonate membranes with gradually reduced pore size (200 and 100 nm), using the liposofast LF-12 device (Avestin®, Canada). Aliquots were separated for characterization and the remaining suspensions were subjected to dialysis in a 15 MWCO membrane (Spectra / Por®), for 4 hours at 4°C against PBS to remove the ethanol. For encapsulation of Ang-(1-7) in conventional liposomes, the peptide was added in the amount of 2 mg to the PBS solution, into which the ethanolic solution of the lipid with or without remdesivir was injected. EXAMPLE 2 - CHARACTERIZATION OF LIPOSOMAL COMPOSITIONS OF REMDESIVIR WITH OR WITHOUT ANGIOTENSIN-(1-7) PEPTIDE

[0049]

[0041] The particle size distribution and zeta potential were evaluated using a ZS90 zeta-sizer from Malvern Panalytical (Malvern, UK). Remdesivir assay in the formulations was performed by ultraviolet spectrophotometry (Synergy™ HTX, BioTek®, USA), as previously described by AKBEL & ERTEN (2021) and LAMA et al. (2023). During the process, it is assumed that the unencapsulated drug is retained during extrusion or eliminated during dialysis or centrifugation. The drug encapsulation efficiency was determined as:

[0050]

[0042] %EE = 100 x (AFinal - Ao) / (Alnicial - Ao), where Alnicial and AFinal are the absorbance values ​​at 260 nm determined respectively before and after separation. Ao represents the absorbance value of the blank liposome (prepared without the drug).

[0051]

[0043] For the quantification of Ang-(1-7), the intrinsic fluorescence of the peptide was explored, as described by SILVA-BARCELLOS et al, (2001), in which a 50 pL aliquot was removed and added to 1950 pL of methanol to break the liposomes. The amount of Ang-(1-7) encapsulated was then determined using intrinsic fluorescence of Ang-(1-7) (maximum absorption, 278 nm; maximum emission, 315 nm).

[0052]

[0044] As shown in Table 1, all liposomal formulations presented a polydispersity index (PI) lower than 0.2, indicating monodisperse vesicles and an average diameter (MD) in the range of 100-134 nm. The compositions obtained by the IE method containing remdesivir (LIE-CONV and LIE-PEG) showed PI and diameter values ​​lower than those of the formulations obtained by the DR method. Remdesivir encapsulation resulted in a slight increase in average diameter in the case of IE and DR liposomes and in PI in the case of DR liposomes. The incorporation of the drug also resulted in a slight increase in the negative zeta potential (ZP) only in the case of PEGylated liposomes (Table 1). Regarding the remdesivir encapsulation rate, IE liposomes showed an encapsulation efficiency (EE) of around 60%, while DR liposomes showed a drug encapsulation rate of around 35%. The Ang-(1-7) peptide showed an encapsulation rate of around 16% in IE liposomes.

[0053] Table 1 - Particle size distribution, zeta potential, and encapsulation efficiency of remdesivir in liposomal compositions prepared by IE methods.

[0054] Liposomes DM (nm) IP PZ (mV) EE (%)

[0055] 59.3 ±

[0056]

[0057] 4.4* LIE-CONV 118.4 ± 3.2* 0.047 ± 0.024* -2.14 ± 0.18*

[0058] (n = 5) 57.2 ± 7.2* LIE-PEG 110.1 ± 1.8* 0.026 ± 0.005* -8.97 ± 0.05*

[0059] (n = 2) LIE-EMPTY- 102.7 ± 1.7* 0.056 ± 0.036* -3.55 ± 0.07* CONV LIE-EMPTY- 104.2 ± 2.1* 0.098 ± 0.016* -2.55 ± 0.09* PEG LANG-(1-7) 99.94 ± 1.7 0.018 ± 0.00 -2.96 ± 0.04 16% EMPTY 101.1 ± 2.7 0.016 ± 0.01 -5.45 ± 0.08

[0060]

[0045] In conclusion, it was possible to obtain different compositions of remdesivir with conventional and PEGylated liposomes, with the drug incorporated either into the membrane or into the aqueous compartment of the liposomes. These compositions showed satisfactory particle size distribution and a high drug encapsulation rate, compatible with their use as a nanostructured drug. Using the IE method, it was possible to obtain a remdesivir composition with liposomes co-incorporating the Ang-(1-7) peptide, with lipophilic remdesivir incorporated into the membrane and the hydrophilic peptide encapsulated in the aqueous compartment of the liposomes.

[0061] EXAMPLE 3 - RELEASE KINETICS OF REMDESIVIR FROM DIFFERENT LIPOSOMAL COMPOSITIONS

[0046] The release study of remdesivir from the different compositions prepared as per example 1 was performed with a dialysis device, using HP- / 3-CD to ensure sink conditions (refers to a situation where the amount of substance dissolved or released from a dosing system - for example, from a tablet or a liposomal formulation - into the dissolution medium or the body is very small relative to the medium's capacity to hold the substance in solution). Immediately after removing the ethanol residue from compositions IE, HP- / 3-CD was added at a final concentration of 0.01 M.The resulting suspensions were divided into triplicates of 100 pL and added to Slide-A-Lyzer mini 10 kDa MWCO dialysis devices (Thermo Scientific®, BR) coupled to an Eppendorf tube containing 1.5 mL of PBS solution supplemented with 0.01 M HP- / 3-CD under agitation in a ThermoMixer (Eppendorf®, BR) at 300 rpm / 37 °C. A total of 2.5 pL of the dialyzed suspension (or 7.5 pL in the case of PEG-LSL) was withdrawn at intervals of 0, 0.5, 1, 2, 3, 4, 5, 6, and 24 h and diluted with 250 pL of EtOH for absorbance reading at 260 nm in a Synergy™ HTX spectrophotometer, with replacement of 2.5 pL of PBS solution containing 0.01 M HP- / 3-CD after collection of each sample (or 7.5 pL in the case of PEG-LSL). Blank liposomes were dialyzed under the same conditions.

[0062]

[0047] The kinetics of the released drug as a function of time are shown in Figure 1. The kinetics showed a good fit with the monoexponential release model (R 2>0.94). In the drug release study, LIE-CONV showed greater retention (half-life = 5.34 h [4.86 - 5.87]) when compared to LIE-PEG (2.54 h [2.27 - 2.84]). Therefore, this data indicates that the LIE-CONV composition is capable of promoting a more sustained release of remdesivir. EXAMPLE 4 - STABILITY OF LIPOSOMAL COMPOSITIONS DURING THE NEBULIZATION PROCESS

[0063]

[0048] In a first study, remdesivir compositions prepared by IE with conventional and PEGylated liposomes, as described in Example 1, were evaluated for their size stability and encapsulation rate when subjected to the nebulization process. Empty liposome suspensions were also used as a control. For this, 200 pL of the liposomal suspension was nebulized in a 50 mL Falcon tube containing 1 mL of PBS, using a preclinical nebulizer (flow rate: >0.1 mL / min, particle size: VMD between 2.5 - 4.0 µm, residual volume: <0.2 mL). The size distribution was analyzed using a zeta-sizer before and after nebulization. To evaluate the encapsulation rate, 100 pL of the nebulized formulations were subjected to ultrafiltration in an Amicon 50 kDa device. The drug was quantified by UV absorption in the initial composition and in the ultrafiltrate.As shown in Table 2, nebulization did not significantly alter the average diameter or zeta potential of conventional liposomes. On the other hand, PEGylated liposomes showed an increase in diameter and PI after the nebulization process. A minimum drug release of less than 3% was observed during the aerosolization process, with both conventional and PEGylated liposomes retaining more than 92% of the encapsulated active ingredient.

[0064] Table 2 - Stability of conventional IE liposomes containing remdesivir during nebulization.

[0065] PZ %RDV Liposomes DM (nm) IP %RDV

[0066] (mV) encapsulated before nebulization

[0067] 102.3 ±

[0068] LIE-CONV 0.062 ± 0.081 -2.58 ± 0.18 - 96

[0069] 3.2LIE-EMPTY- 100.6 ±

[0070] 0.053 ± 0.00 -3.64 ± 0.15

[0071] CONV 2.0

[0072] 96.8 ±

[0073] LIE-PEG 0.026 ± 0.025 -3.53 ± 0.37 97

[0074] 1.8

[0075] LIE-EMPTY- 108.5 ±

[0076] 0.062 ± 0.008 -3.53 ± 0.37

[0077] PEG 2.9

[0078] After nebulization

[0079] 101.5 ±

[0080] LIE-CONV 0.118 ± 0.021 -3.42 ± 0.16 -7.15 ± 0.35 92

[0081] 3.5

[0082] LIE CONV- 98.1 ±

[0083] 0.054 ± 0.006 -3.58 ± 0.05

[0084] EMPTY 1.5

[0085] 115.9 ±

[0086] LIE-PEG 0.330 ± 0.002 -3.43 ± 0.20 -4.05 ± 0.53 96

[0087] 1.7

[0088] LIE-EMPTY- 111.7 ±

[0089] 0.198 ± 0.006 -3.95 ± 0.28

[0090] PEG 1.8

[0091] Diameter: average hydrodynamic diameter; IP: polydispersity index; and PZ: zeta potential of conventional liposomes subjected to the nebulization process. % RDV (% of unencapsulated RDV); % RDV ENCP (% of encapsulated RDV) (mean ± SD, n = 3).

[0092]

[0049] In a second study, the ability of conventional IE liposomes to retain encapsulated calcein as a fluorescent marker of hydrophilic drugs was evaluated. As shown in Table 3, in both independent assays, nebulization resulted in a slight reduction in the encapsulation rate, corresponding to a variation of only 3 to 6% of the encapsulated content.

[0093] Table 3 - Effect of nebulization of IE liposome formulation containing the fluorescent probe calcein as a hydrophilic drug model on the marker encapsulation rate.

[0094] Condition % encapsulated calcein

[0095] Study 1 After nebulization 73.7

[0096] Before nebulization 77.1

[0097] Study 2

[0098] After nebulization 71.6

[0099] Before nebulization 76.8

[0100] PBS fluorescence (FO) was used as a “blank” for subtraction. % Encapsulated calcein = (FTritonxlOO) — (Formulation) / (FTritonx100 — FO) X 100. (mean ± *SD, n = 3).

[0101]

[0050] In conclusion, the good stability of IE liposomes, especially conventional ones, was verified in the nebulization process, both in terms of particle size and the ability to retain hydrophilic and lipophilic drugs. This shows that IE liposomes are good carriers of hydrophilic and lipophilic drugs when aerosolized, and therefore, have great potential for pulmonary drug delivery.

[0102] EXAMPLE 5 - THERAPEUTIC EFFICACY IN A MURINE MODEL OF COVID-19 AND SAFETY OF TREATMENT WITH A LIPOSOMAL COMPOSITION CO-INCORPORATING REMDESIVIR AND ANGIOTENSIN-(1-7) VIA THE INTRANASAL ROUTE, COMPARED TO COMPOSITIONS CONTAINING REMDESIVIR OR ANGIOTENSIN-(1-7) AND TO THE COMMERCIAL FORMULATION OF REMDESIVIR VIA THE PARENTERAL ROUTE.

[0103]

[0051] The efficacy of conventional liposomal compositions (IE) containing remdesivir, Ang-(1-7) or a mixture thereof, prepared as described in Example 1, was evaluated.

[0104]

[0052] The experimental COVID-19 model used consists of transgenic C57BL / 6 mice, which express the human ACE2 receptor driven by the cytokeratin-18 (K18) gene promoter (K18-hACE2). Infection of k18-hACE mice (males and females aged 11 to 16 weeks) was performed intranasally with 6x10 4PFU / mL of SARS-CoV-2 (Gamma strain - P. 1) in an appropriate biological safety cabinet following the experimental protocol described by Seibert et al. (2021), the animals are anesthetized with isoflurane and intranasally (in) challenged with 30 µl of phosphate-buffered saline (PBS) or 6x10 4PFU / mL, resulting in high levels of viral infection in the lungs and brain, with dissemination to other organs. Five groups of infected animals were used (N = 8 / group), with treatment initiated 12 hours after infection and carried out for 5 days with one dose every 12 hours: 1) LRDV group, treated intranasally (15 uL in each nostril) with IE liposomes containing remdesivir at a dose of 10 mg / kg / dose; 2) LMIX group, treated intranasally with IE liposomes co-incorporating remdesivir (10 mg / kg / dose) and Ang-(1-7) (360 ug / kg / dose); 3) LANG group, treated intranasally with liposomes containing Ang-(1-7) (360 ug / kg / dose); 4) Veklury® group, treated intraperitoneally with Veklury® at a remdesivir dose of 20 mg / kg / dose; 5) PBS group, treated intranasally with PBS vehicle.

[0105]

[0053] The animals' body weight was monitored throughout the treatment. At the end of the treatment, the animals were euthanized for evaluation of the viral load in the lungs, brain, and kidneys, as well as the immune and inflammatory responses in the lungs and spleen.

[0106]

[0054] Treatment with intraperitoneal Veklury® and those with intranasal LRDV and LMIX at a dose 2 times lower resulted in complete elimination of SARS-CoV-2 in the lungs, as confirmed by the plate dilution assay (Figure 4) and RT-qPCR (Figure 5). In the kidneys, only the PBS control group showed viral titer (Figure 7). There was also no detectable infectious virus recovered from the brain in animals treated with LRDV and LRDV / ANG, while the Veklury® group showed a high viral titer equivalent to that of PBS (Figure 6). Treatment with the intranasal compositions LRDV, LANG, and LMIX exerted a significant anti-inflammatory action in the lungs, as shown by the significant reduction in TNF-α (Figure 8) and IL-6 (Figure 9) levels compared to the PBS control. Inhibition of myeloid cell recruitment in the lung, including neutrophils and monocytes, was also evidenced, mainly after treatment with LANG and LRDV (Figure 10).It is worth noting that the inhibition of pro-inflammatory cytokines was more intense after intranasal liposomal compositions than after intraperitoneal Veklury®. These results imply that this anti-inflammatory action in the lung could generate a favorable prognosis for patients with exacerbated inflammation.

[0107]

[0055] Evaluation of the lymphoid cell profile in the spleen of animals treated with the LMIX composition showed a significant increase in IL-4 (CD4 and CD8) and IL-10 (CD8) secreting lymphocytes with anti-inflammatory action, compared to the PBS control group (Figure 11). LMIX also promoted a specific increase in CD206+ monocytes (Figure 12), a mannose receptor expressed in M2a macrophage subpopulations, mainly responsible for anti-inflammatory responses. This indicates LMIX's action in systemic protection against inflammatory damage. On the other hand, a significant increase in IL-17 secreting CD8 lymphocytes and a high number of IFN-gamma secreting CD8 lymphocytes were also observed in the LMIX group (Figure 11), as well as an increase in inflammatory monocytes (Figure 12), possibly involved in the elimination of infected cells (Figure 11).It is important to highlight that these systemic immunoprotective actions of LMIX were not observed with the other liposomal compositions, demonstrating the synergism of the remdesivir - Ang-(1-7) combination in the same composition.

[0056] The safety assessment of the treatments was performed by monitoring the animals' body weight and by histological evaluation of the animals' livers after treatment. While the groups treated with PBS, Verklury®, and LANG showed a significant reduction in body weight compared to the uninfected Mock group, the groups treated with the liposomal compositions of remdesivir without and with Ang-(1-7) via intranasal administration maintained their body weight (Figure 13 and Figure 14). This data confirms the greater safety of intranasal treatment with the liposomal compositions of remdesivir.We also confirmed the hepatic toxicity of remdesivir in its commercial form (Verklury®) when administered intraperitoneally, as evidenced by the appearance of hydrotopic degeneration in the liver micrograph (Figure 15 and Figure 16). Importantly, the groups treated intranasally with liposomal remdesivir compositions without and with Ang-(1-7) did not show hydrotopic degeneration.

[0108]

[0057] In conclusion, the high efficacy and safety of liposomal remdesivir compositions with and without Ang-(1-7) in the intranasal treatment of murine SARS-CoV-2 infection was demonstrated, particularly in eliminating the virus in the lungs and brain of the animals. It is worth noting that the commercial parenteral formulation of remdesivir had no effect on viral load in the brain and showed toxicity.

[0109]

[0058] The liposomal composition co-incorporating remdesivir and Ang-(1-7) stood out from other compositions for its ability to promote, in addition to a local response, a systemic immunoprotective response.

Claims

CLAIMS 1. LIPOSOMAL COMPOSITION characterized by comprising a mixture of remdesivir with an angiotensin-releasing peptide, incorporated into conventional or PEGylated liposomes.

2. LIPOSOMAL COMPOSITION, according to claim 1, characterized in that the liposomes are formed by at least one of the lipids selected from the group comprising phosphatidylcholine, cholesterol (COL), 2-dioleoyl-3-trimethylammonium-propane (DOTAP), phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid or other ionizable lipid, and an antioxidant lipid such as alpha-tocopherol.

3. LIPOSOMAL COMPOSITION, according to claim 2, characterized in that the liposomes are PEGylated, composed of PH90, cholesterol (COL) and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000) with molar proportions of 76%, 11% and 13%, respectively, relative to the total lipids.

4. LIPOSOMAL COMPOSITION, defined in claim 1, characterized in that the angiotensin-(1-7) peptide compound is selected from the group comprising angiotensin-(1-7), alamandine, angiotensin-(1-9), angiotensin-(1-5). SEQ ID No. 1: Angiotensin-(1-7): Asp-Arg-Val-Tyr-Ile-His-Pro; SEQ ID No. 2: Alamandine Ala-Arg-Val-Tyr-Ile-His-Pro; SEQ ID No. 3: Angiotensin-(1-9): Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His; SEQ ID No. 4: Angiotensin-(1-5): Asp-Arg-Val-Tyr-Ile.

5. PROCESS FOR OBTAINING THE LIPOSOMAL COMPOSITION defined in claim 1, characterized by being an ethanol injection method comprising the following steps: a. Prepare an ethanolic solution of the lipids with a concentration ranging from 200 g / L to 1,500 g / L, preheated to a temperature in the range of 25 to 65°C; b. Add the drug remdesivir to the solution obtained in step “a”, in an antiviral / lipid molar ratio ranging from 0.025 to 0.2; c. Subject the solution obtained in “b” to magnetic stirring at 300 to 800 rpm at a temperature in the range of 20 to 65°C until complete dissolution; d. Inject the ethanolic phase obtained in “c” into an aqueous solution of the angiotensin-containing peptide at a peptide / lipid molar ratio between 0.0001 and 0.2, with a final maximum ethanol concentration of 25% (v / v); e. Calibrate the size of the liposomes by extrusion, high-pressure homogenization, or microfluidization, in order to obtain a final liposome diameter of less than 250 nm and a polydispersity index of less than 0.3; f. Remove ethanol and unencapsulated peptide by dialysis or tangential filtration, at temperatures ranging from 0 o at 30°C.

6. USE OF THE LIPOSOMAL COMPOSITION defined in any one of claims 1 to 4, characterized by being in the preparation of medicaments for the treatment of respiratory and neurological viral infections.

7. USE OF THE LIPOSOMAL COMPOSITION, according to claim 6, characterized by being in the treatment of viral infections caused by viruses of the Coronaviridae and Orthomyxoviridae families.

8. USE OF THE LIPOSOMAL COMPOSITION, according to claim 6, characterized in that the liposomal composition is in the form of an aqueous suspension or in powder form in the presence of a cryoprotectant.

9. USE OF THE LIPOSOMAL COMPOSITION, according to claim 6, characterized in that it is used in the preparation of medicaments for administration via intranasal, inhalation, intravenous, intraperitoneal, subcutaneous or intramuscular routes.

Images