Process for obtaining liposomes conjugated with amphotericin b and containing dicetyl phosphate, formulation and uses

Abstract

The invention relates to a process for obtaining an amphotericin B (AmB) formulation in liposomes containing dicetyl phosphate (DCP), based on incorporation of the drug into the membrane of preformed liposomes, combining the effects of pH on the solubility of amphotericin B and of temperature on the insertion of the drug into the liposome membrane. The technology also relates to use of the process and of the formulation for the manufacture of medicaments for the treatment of leishmaniasis and sporotrichosis, for oral or topical administration.

Description

[0001] "PROCESS FOR OBTAINING AMPHOTERICIN B-CONJUGATED LIPOSOMES CONTAINING DICETYL PHOSPHATE, FORMULATION AND USES"

[0002]

[0001] The present technology relates to a process for obtaining a formulation of amphotericin B (AmB) in liposomes containing dicetyl phosphate (DCP) based on the incorporation of the drug into the membrane of pre-formed liposomes, which combines the effects of pH on the solubility of amphotericin B and temperature on the insertion of the drug into the liposome membrane. The technology also relates to the use of the process and the formulation for the production of medicines for the treatment of leishmaniasis and sporotrichosis, via oral or topical routes. The use of DCP in the liposome formulation promotes a less aggregated state of amphotericin B, and potentially more bioavailable, leading to a significantly faster release effect of AmB when compared to the formulation with distearoyl-phosphatidylglycerol (DSPG). In addition, the therapeutic efficacy and toxicity of the formulation with DCP were evaluated in a murine model for infection by Leishmania infantum, L.This study investigated the use of DCP in leishmaniasis in Amazonian diseases and for fungal infection by Sporothrix brasiliensis. The results showed that the DCP formulation significantly reduced the parasite load in the liver and spleen, compared to the DSPG formulation, in the treatment of leishmaniasis by oral administration. It also decreased the fungal load on the animals' paws, demonstrating its therapeutic efficacy.

[0003]

[0002] Amphotericin B is a polyene antibiotic, first isolated in 1959 from cultures of the bacterium Streptomyces nodosus, which exhibits highly effective antifungal and antiparasitic activity. However, it causes severe nephrotoxicity, leading to severe renal failure, which limits its use. Due to its side effects, research and development of new formulations, such as emulsions, nanoparticles, and liposomes, have been intensified, with the aim of optimizing drug transport to the desired site, reducing the amount of free AmB in the bloodstream and thus minimizing its toxicity.

[0004]

[0003] Commercial formulations of AmB are available on the market, such as the liposome Ambisome® (Nextar Pharmaceuticals), the colloidal suspensions Amphocil® and Amphotec® (Intermune), the lipid complex Abelcet® (Intermune), and the detergent solution Fungizone®. Among these formulations, the most widely used today is the liposomal formulation Ambisome®, whose related patents expired in 2016 (US 5874104 and US 5965156). This formulation has become the preferred and reference choice for the treatment of severe systemic fungal infections and leishmaniasis, due to its superior safety profile compared to conventional formulations. It is even included in the group of essential medicines and is available in the Unified Health System (SUS) through the Strategic Component of Pharmaceutical Assistance.

[0005]

[0004] Moderate efficacy of AmBisome® has been reported in the treatment of cutaneous leishmaniasis with a cure rate of less than 80%. However, its intravenous administration has significant disadvantages, causing side effects such as fever, chills, bone pain, nephrotoxicity, and thrombophlebitis. Furthermore, the treatment time is extensive, a minimum of 14 days and potentially reaching almost 40 days, increasing the costs associated with treatment. These disadvantages limit its use in developing countries where Leishmania infections occur. This context has stimulated the search for new formulations for optimized AmB delivery, including ways to improve its bioavailability via oral and topical application.

[0005] The state of the art shows different AmB formulations for use in the production of medicines for the treatment of leishmaniasis and sporotrichosis, as discussed below.

[0006]

[0006] Patent document US20100273728A1, entitled “Formulations for the oral administration of therapeutic agents and related methods”, with a priority date of 05 / 23 / 2008, discloses AmB formulations with self-emulsifying systems for the oral treatment of fungal infections, including sporotrichosis, and leishmaniasis. The self-emulsifying systems consist of glycerol esters, fatty acid esters, and PEGylated phospholipids. The main disadvantage of this type of formulation is the side effects associated with the oily components of the formulation, which can cause gastrointestinal disturbances such as nausea and diarrhea, limiting patient adherence, mainly because a prolonged dosage regimen is necessary.

[0007]

[0007] The work of Ishida, K. et al. (Ishida, K. et al. Efficacy of a polyaggregated formulation of amphotericin B in treating systemic sporotrichosis caused by Sporothrix brasiliensis. Medical Mycology, 56, 288-296, 2018) reveals a polyaggregated formulation of AmB with deoxycholate for the treatment via parenteral oral administration of fungal infections caused by Sporothrix brasiliensis. The AmB formulation with deoxycholate, marketed as Fungizone®, presents several disadvantages such as nephrotoxicity, intravenous administration, and high cost. The polyaggregated formulation developed in this work ensures lower toxicity compared to Fungizone®, due to the controlled release of AmB by lipid vesicles. However, it does not cite any studies of oral or topical administration.

[0008]

[0008] Patent document BR102017002582, entitled “Liposomal formulation for the treatment of leishmaniasis”, whose priority date is 07 / 02 / 2017, claims a process for obtaining liposomal formulations of miltefosine and / or meglumine antimoniate with other leishmanicidal drugs, such as AmB. The liposomes disclosed in the document contain the lipid DCP in their compositions, however, their production process presents disadvantages, such as the complexity of the process, the need for several steps, including a lyophilization step and two extrusion steps in the presence of the drugs, which may compromise the stability and encapsulation efficiency of these drugs. Furthermore, it does not present any effective formulation for oral or topical application.

[0009]

[0009] Patent document US20210369615A1, entitled “Solid oral formulations of amphotericin B”, whose priority date is 07 / 31 / 2019, discloses solid formulations of AmB with at least one lipophilic component for the oral treatment of fungal infections, including sporotrichosis, and leishmaniasis. However, the oral formulation has an organizational structure, lipid composition, and characteristics different from the liposomes of the present invention.

[0010]

[0010] Patent document BR102022000089, entitled “Process for obtaining liposomes conjugated to amphotericin B, formulation and uses”, whose priority date is 04 / 01 / 2022, discloses a process for obtaining a liposomal AmB formulation containing distearoyl-phosphatidylglycerol (DSPG) and its respective use in the production of medicines for the treatment of leishmaniasis in humans and dogs. The document does not foresee the obtaining of AmB liposomes containing the anionic lipid DCP nor its use for the production of medicines for the treatment of cutaneous fungal infections such as sporotrichosis. The liposomes with DCP presented a less aggregated state of AmB and potentially more bioavailable, leading to a significantly faster release effect of AmB, when compared to the DSPG formulation of BR102022000089. Furthermore, it showed greater colloidal stability of 72 days, while the formulation with DSPG showed a significant increase in diameter.In a trial for visceral leishmaniasis in hamsters, the liposome with DCP showed greater therapeutic efficacy via oral administration, significantly decreasing the parasite load in the liver and spleen, an effect not observed for the formulation with DSPG from BR102022000089. In a murine model of fungal infection (sporotrichosis), the liposome with DCP significantly decreased the fungal load on the animals' paws, an effect not observed for the formulation with DSPG from BR102022000089.

[0011]

[0011] In the prior art, no technology has been found that comprises a liposomal formulation containing AmB, formed by the lipids phosphatidylcholine (P90H), cholesterol and dicetylphosphate, PEGylated or not, allowing effective oral or topical therapy for the treatment of leishmaniasis or sporotrichosis, as presented in the present invention.

[0012] BRIEF DESCRIPTION OF THE FIGURES

[0013]

[0012] Figure 1 shows the circular dichroism (A) and UV / Vis absorption (B) spectra of AmB in the liposomal compositions LAmB-DCP / COM, LAmB-DCP / PEG, LAmB-DSPG / PEG and AmBisome®.

[0014]

[0013] Figure 2 shows the release kinetics of AmB at 37°C from the LAmB-DSPG / PEG and LAmB-DCP / PEG compositions. Data are shown as means ± SE (n = 3). T1 / 2 represents the release half-life of AmB, assuming a mono-exponential release model.

[0015]

[0014] Figure 3 shows the size stability of the non-lyophilized compositions (A) LAmB-DSPG / PEG and (B) LAmB-DCP / PEG during storage at 4°C. Data are shown as mean ± SE (n = 3).

[0016]

[0015] Figure 4 shows the polydispersity index (PI) of the non-lyophilized compositions (A) LAmB-DSPG / PEG and (B) LAmB-DCP / PEG during storage at 4°C. Data are shown as mean ± SE (n = 3).

[0016] Figure 5 shows the therapeutic efficacy of PEGylated liposome-containing AmB formulations containing DSPG or DCP in a visceral leishmaniasis model in hamsters, based on parasite loads in the liver (A) and spleen (B). Male Mesocricetus auratus hamsters were infected with Leishmania infantum by intraperitoneal injection and, after 84 days of infection, were treated daily for 10 days with the following AmB formulations: LAmB-DSPG / PEG orally or intraperitoneally at a dose of 5 mg AmB / kg / day; LAmB-DCP / PEG was administered orally at a dose of 5 mg AmB / kg / day; the commercial liposomal formulation Ambisome was administered intraperitoneally at a dose of 5 mg AmB / kg / day. The control group remained untreated. Parasite load was determined by qPCR.Data are shown as medians ± 95% confidence interval (n = 6 per group). *p<0.05 and **p<0.01 for comparison with the untreated group using the Kruskal-Wallis test, followed by Dunn's post-hoc test.

[0017]

[0017] Figure 6 shows the profile of serum markers of liver function, aspartate aminotransferase (A) and pyruvic aminotransferase (B), and renal function, urea (C) and creatinine (D), after treatment of hamsters infected with Leishmania infantum with different liposomal formulations of AmB. Male Mesocricetus auratus hamsters were infected with Leishmania infantum chagasi (BH401) by intraperitoneal injection and, after 84 days of infection, were treated daily for 10 days with the following AmB formulations: LAmB-DSPG / PEG orally or intraperitoneally at a dose of 5 mg AmB / kg / day; LAmB-DCP / PEG orally at a dose of 5 mg AmB / kg / day; commercial liposomal formulation Ambisome intraperitoneally at a dose of 5 mg AmB / kg / day. The control group remained untreated. After euthanasia, serum was collected for the determination of aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, and creatinine levels.AST and ALT results are shown as medians ± 95% confidence interval (n=6 per group). *p<0.05 for comparison with the untreated group using the Kruskal-Wallis test, followed by Dunn's post-hoc test. Urea and creatinine results are shown as means ± SE (n=6 per group). *p<0.05 for comparison with the untreated group using the One-Way ANOVA test, followed by Dunnett's post-hoc test.

[0018]

[0018] Figure 7 shows the therapeutic efficacy of topical treatment with amphotericin B liposomal hydrogel containing DCP in a murine model of cutaneous leishmaniasis. BALB / c mice were infected at the base of the tail with Leishmania amazonensis. Treatment began 70 days after infection and consisted of daily applications for 25 days. Experimental groups received: 50 µL of AmB liposomal hydrogel applied to the lesion site (LAmB-DCP / PEG); 50 µL of PEGylated “empty” liposome hydrogel applied to the lesion site at the same lipid dose as the LAmB-DCP / PEG group (LVazio-DCP / PEG); oral miltefosine at a dose of 10 mg / kg / day (positive control). A control group remained untreated. (A) evolution of lesion size during treatment. (B) parasite load in the lesion of mice infected with Leishmania amazonensis.*p<0.05; **p<0.01; ****p<0.0001 for comparison with the negative control (A, Two-way ANOVA with Dunnett's post-test; B, Brown-Forsythe ANOVA test, with Dunnett's T3 post-test).

[0019]

[0019] Figure 8 shows the fungal load on the paw of male C57 / BL6 mice after 10 days of intraplantar infection with Sporothrix brasiliensis IC189 and treatments initiated 24 hours after infection. The following amphotericin B formulations were used: LAmB-DOPG / PEG orally at a dose of 10 mg / kg / day; LAmB-DCP / PEG orally at a dose of 10 mg / kg / day; commercial formulation Amphotericin B intraperitoneally at a dose of 5 mg / kg / day-alternate. The untreated group remained untreated. Data are shown as mean ± SE (n = 4-5 per group). *p<0.05, **p<0.01, ***p<0.001 for comparison by One-way ANOVA followed by Tukey's post-hoc test.

[0020] DETAILED DESCRIPTION OF THE TECHNOLOGY

[0021]

[0020] The present technology relates to a process for obtaining a formulation of amphotericin B (AmB) in liposomes containing dicetyl phosphate (DCP) based on the incorporation of the drug into the membrane of pre-formed liposomes, which combines the effects of pH on the solubility of amphotericin B and temperature on the insertion of the drug into the liposome membrane. The technology also relates to the use of the process and the formulation for the production of medicines for the treatment of leishmaniasis and sporotrichosis, via oral or topical routes. The use of DCP in the liposome formulation promotes a less aggregated state of amphotericin B, and potentially more bioavailable, leading to a significantly faster release effect of AmB when compared to the formulation with distearoyl-phosphatidylglycerol (DSPG). In addition, the therapeutic efficacy and toxicity of the formulation with DCP were evaluated in a murine model for infection by Leishmania infantum, L.This study investigated the use of DCP in leishmaniasis in Amazonian diseases and for fungal infection by Sporothrix brasiliensis. The results showed that the DCP formulation significantly reduced the parasite load in the liver and spleen, compared to the DSPG formulation, in the treatment of leishmaniasis by oral administration. It also decreased the fungal load on the animals' paws, demonstrating its therapeutic efficacy.

[0022]

[0021] More specifically, the process for obtaining liposomes conjugated to amphotericin B comprises the following steps:

[0023] a. Prepare a vesicle suspension containing phosphatidylcholine (PC), cholesterol (COL), dicetyl phosphate (DCP), with a calibrated size between 30 and 350 nm and a polydispersity index (PI) of less than 0.3; b. Dissolve amphotericin B at a concentration of 1 to 30 mg / mL in an alkaline aqueous solution with a pH ranging from 10 to 13;

[0024] c. Add the liposome suspension obtained in step “a”, pre-heated to a temperature in the range of 25 to 90°C, in a drug / lipid molar ratio ranging from 1:50 to 1:5, to the amphotericin B solution obtained in step “b”, and maintain the mixture under heating at a temperature of 25 to 90°C for 1 to 5 minutes;

[0025] d. Neutralize the pH of the suspension obtained in step “c” to a value between 6 and 8, followed by heating at a temperature of 25 to 90°C for 1 to 180 minutes.

[0026]

[0022] In step “a”, phosphatidylcholine (PC), cholesterol (COL) and dicetylphosphate (DCP) should preferably be in molar proportions of 40 to 95%, 0 to 40% and 5 to 30%, respectively.

[0027]

[0023] Still in step “a”, liposomes can be PEGylated, that is, in addition to the lipids used in conventional liposomes described above, they can incorporate a PEGylated lipid in a molar proportion of 3 to 10% in relation to the total lipids.

[0028]

[0024] PEGylated liposomes are composed, in a preferred configuration, of PC, COL, DCP and distearoyl-phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000) with molar proportions of 50%, 25%, 20% and 5%, respectively, relative to total lipids.

[0029]

[0025] The suspension obtained in step “d” can be lyophilized after adding a cryoprotective sugar solution in a sugar / lipid mass ratio between 1:1 and 4:1, with the cryoprotective sugars selected from the group comprising sucrose, trehalose, lactose, maltose, glucose and cyclodextrin derivatives.

[0030]

[0026] The formulation containing amphotericin B liposomes obtained according to the process defined above comprises amphotericin B incorporated into the membrane of liposomes formed from conventional or PEGylated phosphatidylcholine (P90H), cholesterol (COL), dicetylphosphate (DCP), and pharmaceutically acceptable excipients.

[0031]

[0027] The liposomal formulation of the present technology may comprise excipients for oral, intranasal, inhalation, subcutaneous, intramuscular or topical application.

[0032]

[0028] The liposomal formulation of amphotericin B of the present technology can be used to produce medicaments for the treatment of leishmaniasis in humans and dogs or to produce medicaments for the treatment of sporotrichosis in humans and cats.

[0033]

[0029] The process of the present technology can be used to produce medicaments for the treatment of leishmaniasis in humans and dogs or to produce medicaments for the treatment of sporotrichosis in humans and cats.

[0034]

[0030] The present invention can be better understood through the following examples, which are not limiting.

[0035] EXAMPLE 1 - PREPARATION AND CHARACTERIZATION OF AMPHOTERICIN B COMPOSITIONS WITH CONVENTIONAL AND PEGYLATED LIPOSOMES CONTAINING DICETYL PHOSPHATE

[0036]

[0031] For the preparation of conventional (LAmB-DCP / CON) and PEGylated (LAmB-DCP / PEG) liposome compositions of amphotericin B (AmB), the following lipid compositions were used. For LAmB-DCP / CON, the lipids used were phosphatidylcholine (Phospholipon®90H, P90H), cholesterol (COL), and dicetylphosphate (DCP) in molar ratios of 5.3, 2.7, and 2.0, respectively. For LAmB-DCP / PEG, the lipids used were P90H, COL, DCP, and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) in molar ratios of 5.1, 2.5, 1.9, and 0.5, respectively.

[0037]

[0032] An organic phase was prepared consisting of lipids (P90H; COL; DCP; DSPE-PEG2000) dissolved in ethanol at 65 °C and an aqueous phase consisting of a 0.007M NaOH solution in water heated to 65 °C, kept under stirring. 1.1 mL of the organic phase was added dropwise to 5 mL of the aqueous phase. This system was kept under stirring for 5 min at 65 °C. After this step, the size of the liposomes was calibrated by filtering the suspension using an extruder (Lipex® Extruder, Burnaby, Canada) and polycarbonate membranes with successive pore diameters of 200 and 100 nm. The suspension was dialyzed for 3 hours at 25 °C for ethanol removal using a 10kDa MWCO dialysis membrane. Subsequently, the AmB solution in 0.1 M NaOH (10 mg in 0.8 mL) was added to 2 mL of the calibrated liposome suspension, with a molar ratio of AmB to lipid of 1:10. The resulting mixture was heated to 60°C and protected from light for 2 min.The pH, initially set at 12, was adjusted to 6.5 with an acidic HEPES solution (prepared with 0.1 M HCl) under constant stirring, remaining under these conditions for 5 minutes at 60 °C. Then, sucrose was added to the mixture in a sugar / lipid mass ratio of 3:1, and the resulting suspension was frozen and lyophilized (Liotop L101, São Carlos, Brazil).

[0038]

[0033] After lyophilization, AmB formulations with conventional and PEGylated liposomes (LAmB-DCP / PEG and LAmB-DCP / CON) were characterized with respect to average hydrodynamic diameter, polydispersity index, and zeta potential (Zetasizer Nano ZS 90, Malvern Instruments, Malvern®, UK). Encapsulation efficiency and content through AmB dosage were analyzed by high-performance liquid chromatography (HPLC). AmB quantification was performed on an Agilent 1260 series chromatograph equipped with a diode array detector (DAD). The analyses were performed using a flow rate of 1 mL / min, a Discovery® C18 column (250 × 4.6 mm; 5 μm), and a mobile phase composed of acetonitrile: methanol: citric acid solution (4.2 g / L) pH 6 (48:12:40) with a gradient. AmB detection was performed by measuring absorbance at a wavelength of 405 nm.As shown in Table 1, the formulations present nanoparticles with average hydrodynamic diameters of less than 140 nm and a polydispersity index (PI) around 0.1. A marked reduction in the zeta potential of (LAmB-DCP / PEG) to (LAmB-DCP / CON) from -3.8 to -13.5 mV was observed, due to the coating of the nanoparticle surface with hydrophilic PEG polymer. It is also observed that the zeta potential of LAmB-DCP / CON was less negative than that of the blank liposomes (LAmB-DCP / CON-Br), which suggests the electrostatic interaction of AmB with DCP on the surface of the vesicles. The dosage of AmB showed efficiencies and encapsulation levels above 90% (Table 1).

[0039] Table 1 - Distribution of particle size, Polydispersity Index (PI), zeta potential (ZP), drug encapsulation efficiency (EE%) and total content (TT%) of PEGylated (LAmB-DCP / PEG) and conventional (LAmB-DCP / CON) liposomal formulations.

[0040] Diameter pr z m\n PP / O / A TTVOZ Pnrms ílsrân ínm'1 +DP ip +DP 1.2 rZ. (ITlV) I i / o) rormuiaçao ÍH ±UH ±DP 1 2 ±DP 1 2 ± DP 2

[0041]

[0042] LAmB- 131.0 ± -3.8 ± 96.0 ± 99.8 ± 0.09 ±0.04

[0043] DCP / PEG 8.7 ' 0.6 2.9 4.9 LAmB- 137.0 ± -13.5 ± 99.1 ± 92.9 ± 0.12 ±0.05

[0044] DCP / CON 4.9 3.6 5.2 9.5 LAmb- 97.0 ± -3.3 ±

[0045] 0.05 ±0.09

[0046] DCP / PEG-Br 5.7 0.5

[0047] LAmB- 97.0 ± -25.1 ±

[0048] 0.05 ±0.02

[0049] DCP / CON-Br 3.1 1.2

[0050] 1 SD: standard deviation; 2 Mean and standard deviation of 7 independent batches. The mean hydrodynamic diameter and IP were determined after dilution of the suspension in 5% glucose. The zeta potential was measured after dilution in PBS (150 mM NaCl, 10 mM phosphate, pH 7.2).

[0051]

[0034] As an important result of this study, we show that AmB is efficiently incorporated into liposomes containing DCP, prepared by the ethanol injection method. This preparation process stands out for its greater simplicity and high potential for scaling up, when compared to that used for the preparation of liposomes containing DSPG.

[0052]

[0035] To investigate the aggregation state of AmB in conventional and PEGylated liposomal compositions, UV-Visible (UV-Vis) absorption spectroscopy and circular dichroism techniques were used. In accordance with Ramos et al. (2022), AmBisome®, a commercial amphotericin B, showed a doublet-type DC signal centered at 330 nm, while LAmB-DSPG / PEG showed a much less intense signal in the same region (Figure 1A). The LAmB-DCP / CON and LAmB-DCP / PEG compositions also showed doublet-type DC signals centered at 335 nm. Surprisingly, the doublet was inverted in the DCP compositions compared to the AmBisome® doublet, and was much more intense for LAmB-DCP / PEG than for LAmB-DCP / CON (Figure 1A). The result shows that there was a change in the aggregation state of amphotericin B in the DCP formulation compared to the DSPG formulation.

[0053]

[0036] Comparison of UV / Vis absorption spectra (Figure 1B) shows a shift in the maximum UV absorption peak of the liposomal formulations, compared to AmBisome®. Furthermore, the compositions showed more intense absorption bands at 363–365, 383–384, and 406–409 nm, which are attributed to the monomeric form of AmB (Frézard et al., 2023). It was also observed that these bands are more intense for the compositions containing DCP than for the composition containing DSPG.

[0054]

[0037] Therefore, the spectroscopy data indicate a less aggregated state of AmB in the compositions containing DCP, compared to the formulation containing DSPG. They also show distinct conformations of the aggregated form of AmB between the two compositions with DCP and DSPG.

[0055]

[0038] The in vitro release kinetics of AmB at 37 °C and isotonic buffer pH 7.2 were compared from PEGylated liposomal compositions with DCP and DSPG, under dialysis conditions in the presence of 5% gamma-cyclodextrin, as described by Frézard et al. (Pharmaceutics.2022; 15(1):99. doi: 10.3390 / pharmaceutics15010099). As shown in Figure 2, the composition containing DCP showed significantly faster release of AmB compared to that containing DSPG. This faster release is consistent with the less aggregated state of AmB in the composition with DCP. This characterizes the liposomal composition of AmB with DCP as potentially more bioavailable.

[0056]

[0039] In order to evaluate the behavior of liposomal formulations in an environment similar to the stomach, a stability test was performed using simulated gastric fluid without enzyme (SGF) at a pH adjusted to 1.2 ± 0.1. A comparison was made with the formulation maintained in PBS at pH 7.2. The particle size characterization results are presented in Table 2.

[0057] Table 2 - Distribution of particle size (diameter), Polydispersity Index (PI) and pH of PEGylated (LAmB-DCP / PEG) and conventional (LAmB-DCP / CON) liposomal composition after dilution in FGS and PBS and incubation for 2h at 37°C.

[0058] LAmB-DCP / CON LAmB-DCP / PEG FGS PBS FGS PBS

[0059] Diameter 100.4 ±

[0060] 207.0 ± 3.8 126.9 ± 5.2 106.3 ± 0.1

[0061] (nm) ± SD 1 ' 2 2.9

[0062] 0.098 ± 0.130 ± IP ± DP 1 2 0.53 ± 0.10 0.22 ± 0.02

[0063] 0.02 0.03 pH 1.20 7.22 1.28 7.12 1 SD: standard deviation; 2 Average and standard deviation of 2 independent lots.

[0064]

[0040] The data show minimal differences in diameter and polydispersity index (PDI) of the LAmB-DCP / PEG composition between the FGS and PBS media. On the other hand, the conventional liposome-based composition (LAmB-DCP / CON) shows high diameter and polydispersity index in FGS compared to PBS, indicating the aggregation of lipid vesicles in the FGS medium. Therefore, the size analysis results indicate greater stability of the LAmB-DCP / PEG formulation in FGS compared to the conventional liposome formulation, pointing to the greater potential of the PEGylated composition for oral administration of AmB.

[0065]

[0041] In order to compare the colloidal stability of PEGylated liposomes in compositions with DCP and DSPG, preparation was carried out up to the AmB incorporation step. Subsequently, the aqueous suspensions were stored between 2 and 8°C and the diameter and PI were determined over time.

[0066]

[0042] As shown in Figures 3 and 4, the LAmB-DSPG / PEG composition showed a significant increase in liposome diameter, indicating instability, unlike the LAmB-DCP / PEG composition.

[0067]

[0043] In conclusion, when compared to the DSPG-containing composition prepared according to Ramos et al. (Ramos GS et al. Formulation of amphotericin B in pegylated liposomes for improved treatment of cutaneous leishmaniasis by parenteral and oral routes. Pharmaceutics, 5, 989, 2022), the PEGylated liposomal composition containing DCP prepared by the ethanolic injection method, in addition to allowing a simplified preparation process, showed a lower state of aggregation of AmB, faster release of AmB, and increased colloidal stability in the form of an aqueous suspension under storage conditions. EXAMPLE 2. EVALUATION OF THERAPEUTIC EFFICACY AND TOXICITY OF AN AMPHOTERICIN B COMPOSITION WITH PEGYLATED LIPOSOMES CONTAINING DICETYL PHOSPHATE IN AN EXPERIMENTAL MODEL OF VISCERAL LEISHMANIASIS, AFTER ORAL ADMINISTRATION

[0068]

[0044] The oral therapeutic efficacy study of AmB compositions with PEGylated liposomes containing DCP and DSPG was conducted in a hamster model of visceral leishmaniasis (VL). Male Mesocricetus auratus hamsters aged between 7 and 8 weeks were infected with 10 7Leishmania infantum (MCAN / BR / 2002 / BH401 isolated from hamster spleen) via intraperitoneal (ip) injection. The 10-day treatment was initiated 84 days after infection. The animals were allocated to the following experimental groups (n=6). The experimental groups are identified as follows: Group 1: receiving the AmB composition in PEGylated liposomes (LAmB-DSPG / PEG prepared according to Ramos et al. (2022)) orally at a dose of 5 mg / kg / day; Group 2: receiving the AmB composition in PEGylated liposomes (LAmB-DCP / PEG prepared according to Example 1) orally at a dose of 5 mg / kg / day; Group 3: receiving AmBisome®, via ip injection at a dose of 5.0 mg / kg / day (positive control); Group 4: receiving the AmB composition in PEGylated liposomes (LAmB-DSPG / PEG) via intraperitoneal injection at a dose of 5 mg / kg / day; Group 5: untreated (negative control).

[0069]

[0045] Four days after the end of treatment, the animals were subjected to general anesthesia and euthanasia. Samples of blood, spleen, liver, intestine, bone marrow, and kidneys were removed and stored at -20 °C until use. The spleens and livers were homogenized in PBS and DNA extracted using a commercial kit for parasite load assessment by qPCR, as described by Silva et al. (da Silva, SM et al. Efficacy of combined therapy with liposome-encapsulated meglumine antimoniate and allopurinol in treatment of canine visceral leishmaniasis. Antimicrob Agents Chemother, 56, 2858-2867, 2012). Primer sequences were chosen for amplification of an approximately 120 bp region of Leishmania kDNA. The quantification of the parasite load in the samples was performed using a standard curve established with different dilutions of L. infantum promastigote suspension (BH401).The parasite load, expressed as the number of Leishmania per ng of total DNA, is shown in Figure 5.

[0070]

[0046] Figure 5 shows a significant reduction in parasite load in the liver in the groups treated with intraperitoneal LAmB-DSPG / PEG and orally with LAmB-DCP / PEG, compared to the untreated control group, at a level comparable to the group treated with AmBisome®. In the spleen, we can observe a reduction in parasite load in the groups treated with intraperitoneal LAmB-DSPG / PEG and orally with LAmB-DCP / PEG, compared to the untreated group. On the other hand, the oral LAmB-DSPG / PEG composition did not significantly reduce the parasite load in either the liver or the spleen.

[0071]

[0047] Therefore, this study establishes the superior oral therapeutic efficacy of the LAmB-DCP / PEG composition, compared to the LAmB-DSPG / PEG composition, in a well-established experimental model of visceral leishmaniasis.

[0072]

[0048] Serum markers of liver function (aspartate aminotransferase or AST; alanine aminotransferase or ALT) and kidney function (urea and creatinine) were also evaluated after the end of treatment. As shown in Figure 6, treatment with the oral compositions did not promote changes in hepatic or renal parameters compared to the untreated group. On the other hand, the LAmB-DSPG / PEG composition given intraperitoneally caused a significant increase in ALT and urea levels, indicating renal and hepatic toxicities. Therefore, the oral compositions of AmB stand out for their lower toxicity compared to parenteral treatment.

[0073]

[0049] Therefore, the composition of AmB with PEGylated liposomes containing DCP stands out for its high oral efficacy, as well as the absence of toxicity.

[0074] EXAMPLE 3. PREPARATION OF A HYDROGEL WITH AMPHOTERICIN B INCORPORATED INTO PEGYLATED LIPOSOMES CONTAINING DICETYL PHOSPHATE AND EVALUATION OF THE THERAPEUTIC EFFICACY OF THIS FORMULATION IN TOPICAL TREATMENT IN A MURINE MODEL OF CUTANEOUS LEISHMANIASIS.

[0075]

[0050] PEGylated liposomes were prepared from the lipids Phospholipon90H (P90H), cholesterol (COL), and dicetyl phosphate (DCP) and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) in molar ratios of 5.1, 2.5, 1.9, and 0.5, respectively. For this, an organic phase was prepared consisting of the lipids (P90H; COL; DCP; DSPE-PEG2000) dissolved in ethanol at 65 °C and an aqueous phase consisting of a 0.007M NaOH solution in water heated to 65 °C, kept under stirring. 1.1 mL of the organic phase was slowly added dropwise to 5 mL of the aqueous phase. This system was kept under stirring for 5 min at 65 °C. After this step, the size of the liposomes was calibrated by filtering the suspension using an extruder (Lipex® Extruder, Burnaby, Canada) employing polycarbonate membranes with successively 200 and 100 nm pore diameters.Subsequently, the AmB solution in 0.1 M NaOH (10 mg in 0.8 mL) was added to 2 mL of the calibrated liposome suspension, with a molar ratio of AmB to lipid of 1:10. The mixture was heated to 60 °C, protected from light, for 2 min. The pH, initially at 12, was adjusted to 6.5 with an acidic HEPES solution (prepared with 0.7 M HCl) under constant stirring, remaining under these conditions for a further 5 min at 60 °C. The liposomal suspension was then gelled with the addition of 10 mg of hydroxyethylcellulose (Natrosol®) to obtain the LAmB-DCP / PEG hydrogel at a final AmB concentration of 0.38% (w / w). A formulation with empty liposomes (LVazio-DCP / PEG) was also prepared, using the same preparation method and omitting only the drug, for use as a control in the efficacy assays.

[0076]

[0051] The therapeutic efficacy of the LAmB-DCP / PEG formulation was evaluated topically in a murine model of cutaneous leishmaniasis. Female BALB / c mice aged between 7 and 8 weeks were infected with 4x10 6Promastigotes of L. amazonensis (IFLA / BR / 67 / PH8) were treated intradermally at the base of the tail. Treatment was initiated after lesion establishment (70 days post-infection) and consisted of daily topical applications of the liposomal formulation for 25 days. Animals were allocated to the following experimental groups (n=10): group receiving 50 µL of AmB liposomal hydrogel applied to the lesion site (LAmB-DCP / PEG); group receiving 50 µL of PEGylated “empty” liposome hydrogel applied to the lesion site at the same lipid dose as the LAmB-DCP / PEG group (LVazio-DCP / PEG); group receiving oral miltefosine at a dose of 10 mg / kg / day (positive control); untreated group (negative control). Therapeutic efficacy was assessed by monitoring lesion size and evaluating parasite load by qPCR 8 days after the end of treatment.

[0077]

[0052] Figure 7 presents the results of lesion size evolution, as well as the parasite load after treatment. The results demonstrate the therapeutic efficacy of topical treatment with the amphotericin B formulation incorporated into PEGylated liposomes containing DCP, in a cutaneous leishmaniasis model, as evidenced by the significantly smaller growth in lesion size and the significant reduction in parasite load, compared to the negative control (untreated).

[0053] This example expands the therapeutic potential of the LAmB-DCP / PEG liposomal composition, by demonstrating its efficacy in the topical treatment of cutaneous leishmaniasis. The topical route is a non-invasive route of administration, an alternative to the oral route, and is highly sought after to increase patient adherence to treatment.

[0078] EXAMPLE 4. EVALUATION OF THE THERAPEUTIC EFFICACY OF AN AMPHOTERICIN B COMPOSITION WITH PEGYLATED LIPOSOMES CONTAINING DICETYL PHOSPHATE IN AN EXPERIMENTAL MODEL OF FUNGAL INFECTION, AFTER ORAL ADMINISTRATION.

[0079]

[0054] A comparative study of the oral therapeutic efficacy of AmB compositions with PEGylated liposomes containing DCP and dioleylphosphatidylglycerol (LAmB-DOPG / PEG) was conducted in a murine model of sporotrichosis. The LAmB-DCP / PEG and LAmB-DOPG / PEG formulations were prepared as described in Example 1, with the same molar ratio of lipids, replacing only DCP with dioleylphosphatidylglycerol (DOPG) in the LAmB-DOPG / PEG formulation. Male C57BL / 6 mice aged 7 to 8 weeks were infected with 5x10 6The study investigated the presence of Sporothrix brasiliensis IC189 yeast in the plantar region of the right hind paw. Treatment was initiated 24 hours after infection and continued for 10 days with the following experimental groups: Group 1: receiving LAmB-DCP / PEG orally at a dose of 10 mg / kg / day; Group 2: receiving LAmB-DOPG / PEG orally at a dose of 10 mg / kg / day; Group 3: receiving Amphotericin B® intraperitoneally at a dose of 5 mg / kg / day (alternating doses) (positive control); Group 4: untreated (negative control). At the end of the treatment, the animals were euthanized, the infected paw was collected, crushed, and diluted to determine the fungal load.

[0055] As shown in Figure 8, the results of fungal load on the paw of the animals demonstrate the therapeutic efficacy of the LAmB-DCP / PEG formulation at a level equivalent to that of treatment with the commercial injectable formulation of amphotericin B (Amphoricin B).On the other hand, the formulation containing DOPG, unlike the one containing DCP, did not show significant therapeutic efficacy in the experimental model.

[0080]

[0056] The results show that treatment with the LAmB-DCP / PEG composition was superior to that with LAmB-DOPG / PEG and as effective as standard treatment in reducing and eliminating the fungal load in animals infected with S. brasiliensis. This example reinforces the great potential of the LAmB-DCP / PEG composition for the oral treatment of fungal infections.

[0081]

[0057] It is important to emphasize that, despite the similarity in reducing and eliminating the fungal load, treatment administered orally constitutes a great advantage in relation to those administered intraperitoneally, since it eliminates the need for patient hospitalization, significantly improving their quality of life and reducing costs inherent to hospitalization. It is worth noting the lack of an oral formulation of amphotericin B on the market.

Claims

CLAIMS 1. PROCESS FOR OBTAINING LIPOSOMES CONJUGATED TO AMPHOTERICIN B, characterized by comprising the following steps: a. Prepare a vesicle suspension containing phosphatidylcholine, cholesterol, and dicetyl phosphate, with a calibrated size between 30 and 350 nm and a polydispersity index (PI) of less than 0.3; b. Dissolve amphotericin B at a concentration of 1 to 30 mg / mL in an alkaline aqueous solution with a pH ranging from 10 to 13; c. Add the amphotericin B solution obtained in step “b” to the liposome suspension obtained in step “a” pre-heated to a temperature in the range of 25 to 90°C, in a drug / lipid molar ratio ranging from 1:50 to 1:5, and keep the mixture heated at a temperature of 25 to 90°C for 1 to 5 minutes; d. Neutralize the pH of the suspension obtained in step “c” to a value between 6 and 8, followed by heating at a temperature of 25 to 90°C for 1 to 180 minutes.

2. PROCESS FOR OBTAINING AMPHOTERICIN B CONJUGATED LIPOSOMES, according to claim 1, characterized in that, in step “a”, the liposomes are formed by phosphatidylcholine, cholesterol, dicetylphosphate, with molar proportions of 40 to 95%, 0 to 40% and 5 to 30%, respectively.

3. PROCESS FOR OBTAINING AMPHOTERICIN B CONJUGATED LIPOSOMES, according to claim 1, characterized in that, in step “a”, the liposomes incorporate a PEGylated lipid in a molar proportion of 3 to 10% relative to the total lipids.

4. PROCESS FOR OBTAINING LIPOSOMES CONJUGATED TO AMPHOTERICIN B, according to the claim. 1, characterized by the suspension obtained in step “d” being lyophilized after the addition of a cryoprotective sugar solution in a sugar / lipid mass ratio between 1:1 and 4:1, the cryoprotective sugars being selected from the group comprising sucrose, trehalose, lactose, maltose, glucose and cyclodextrin derivatives.

5. Liposomal formulation of amphotericin B, obtained by the process defined in any one of claims 1 to 4, characterized by comprising amphotericin B incorporated into the membrane of liposomes formed from conventional or PEGylated phosphatidylcholine, cholesterol, dicetylphosphate, and pharmaceutically acceptable excipients.

6. Liposomal formulation of amphotericin B, according to claim 5, characterized by comprising excipients for oral, intranasal, inhalation, subcutaneous, intramuscular or topical application.

7. USE of the formulation defined in any one of claims 5 to 6, characterized by being for producing medicaments for the treatment of leishmaniasis in humans and dogs.

8. USE of the process defined in any one of claims 1 to 5, characterized in that it is for producing medicaments for the treatment of leishmaniasis in humans and dogs.

9. USE of the formulation defined in any one of claims 5 to 6, characterized by being for producing medicaments for the treatment of sporotrichosis in humans and cats.

10. USE of the process defined in any one of claims 1 to 5, characterized in that it is for producing medicaments for the treatment of sporotrichosis in humans and cats.

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