BRPI0306774B1 - PH-SENSITIVE LIPOSOMES OF CISPLATIN AND OTHER ANTINEOPLASTIC AGENTS AND THEIR OBTAINING PROCESS - Google Patents

Abstract

"Cisplatin ph-sensitive liposomes and other antineoplastic agents and their process for obtaining them." The present invention relates to cisplatin ph-sensitive liposome formulations and other antineoplastic agents included or not included in cyclodextrin and their respective processes for obtaining them. Phisensitive cisplatin liposomes and other antineaplastic agents may allow specific release of chemotherapy at the site affected by the tumor, eliminate and / or reduce its adverse effects and the emergence of treatment resistance as well as allow the adoption of multidrug therapy regimens with incompatible drugs. in unencapsulated form. The sensitive liposomes are composed of non-lamellar phase-forming phospholipids, derived from the cholesteral joint or not with a polyethylene glycol chain-attached phospholipid.

Description

(54) Title: PH-SENSITIVE LIPOSOMES OF CISPLATIN AND OTHER ANTINEOPLASTIC AGENTS AND THEIR PROCESS OF OBTAINING (51) Int.CI .: A61K 9/127; A61P 35/00 (73) Holder (s): FEDERAL UNIVERSITY OF MINAS GERAIS (72) Inventor (s): MÔNICA CRISTINA DE OLIVEIRA; GILSON ANDRADE RAMALDES; MÍRIAM TERESA PAZ LOPES; FERNANDA PIRES VIEIRA; THÉA LUCIANA MESQUITA; VANESSA JÓIA DE MELLO

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“PH-SENSITIVE LIPOSOMES OF CISPLATIN AND OTHER ANTINEOPLASTIC AGENTS AND THEIR PROCESS OF OBTAINING”

The present invention presents new formulations of cisplatin liposomes and other antineoplastic agents as well as the methods of their preparation. These liposome formulations are capable of eliminating or reducing the adverse effects produced by these drugs, leading to greater pharmacological selectivity, eliminating the appearance of resistance to treatment and allowing the adoption of polychemotherapy regimens with the use of a combination of drugs. they are incompatible in the free form.

Currently, cisplatin is one of the most widely used drugs in cancer chemotherapy, presenting a spectrum of action against several solid tumors, including testicular cancer, ovarian carcinoma, head, neck and lung cancer (WILLIAMS, SD, EINHORN, LH Neoplasms of In: CALABRESI, P., SCHEIN, PS, ROSEMBERG,

S.A. Medicai Oncology. New York, 1985, p. 1077-1088; SHIRAZI et al., Cytotoxicity, accumulation, and efflux of cisplatin and its metabolites in human ovarian carcinoma cells. Toxicology and Applied Pharmacology, v. 140, p. 211218, 1996; GUILOT et al., Neoadjuvant chemotherapy in multiple synchronous head and neck and esophagus squamous cell carcinomas. Laryngoscope, v.

102, n. 3, p. 311-319, 1992; LE CHEVALIER et al., Randomized trial of vinorelbine and cisplatin versus vindesine and cisplatin versus vinorelbine alone in advanced non-small-cell lung cancer: Results of a European multicenter trial including 612 patients. Journal of Clinical Oncology, vol. 12, p. 360-367, 1994). However, the use of cisplatin in its free form has some drawbacks, such as: the appearance of clinical resistance; appearance of serious adverse effects, especially severe nephrotoxicity, myelosuppression,

2/15 ototoxicity and neurotoxicity, as well as the existence of pharmacotechnical incompatibilities during the administration of cisplatin together with other drugs, making it difficult to use polychemotherapy. Some of the problems reported are; (i) precipitation in the presence of etoposide dissolved in sodium chloride solution (STEWART & HAMTON, A. J. Hosp. Pharm., v. 46, p. 1400-1404, 1989); (ii) precipitation in the presence of thiotepa dissolved in 5% glucose (TRISEEL & MARTINEZ, Compatibility of thiotepa (lyophilized) with selected drugs during simulated y-site administration. Am. J. Health-Syst Pharm., v. 53, p 1041-1051, 1996); (iii) degradation of cisplatin due to its interaction with trometamol, an excipient used in the formulation of 5-fluoracil (FOURNIER et al., Modification of the physicochemical and pharmacological properties of anticancer platinum compounds by commercial 5-fluoracil formulations: a comparative study using cisplatin and carboplatin, Cancer Chemotherapy and Pharmacology, v. 29, n. 6, p. 461-466, 1992). Countless scientific researches have been looking for alternatives to improve the selectivity of platinum compounds with antitumor activity, aiming to reduce serious side effects, especially nephrotoxicity. The encapsulation of cisplatin in liposomes is a promising proposal for the improvement of antitumor therapy. Liposomes consisting of hydrogenated soy phosphatidylcholine, distearoylphosphatidylethanolamine associated with molecules of methoxypolyethylene glycol 2000 molecular weight (2000MPEG-DSPE) and cholesterol, in a molar ratio of 51: 5: 44, with a diameter equal to 100 nm, have been prepared to carry molecules of cisplatin (PELEGSHULMAN et al., Characterization of serically stabilized cisplatin liposomes by nuclear magnetic resonance. Biochimica et Biophysica Acta, v. 1510, p. 278-

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291, 2001; Sequus Pharmaceuticals, Inc. , R.  M.  Abra, K.  Reis, Liposomes containing a cisplatin compound.  Int.  A61K 009/127.  U. S.  n.  5,945,122.  22 August 1997, 31 August 1999).  Due to their small size, long blood circulation and reduced interaction with blood constituents, the formulations of these stealthy liposomes have a tissue distribution characteristic of the liposomes and not of the internalized drug (NEWMAN etal. , Comparative pharmacokinetics, tissue distribution, and therapeutic effectiveness of cisplatin encapsulated in long-circulating, pegylated liposomes (SPI-077) in tumor-bearing mice.  Cancer Chemothererapy and Pharmacology, v.  43, p.  1-7, 1999).  Newman and colleagues investigated the pharmacokinetic behavior, biodistribution and therapeutic efficacy of these liposomes compared to cisplatin in non-encapsulated form, using tumor models of colon and lung carcinoma.  The results of this study demonstrated an increase in the concentration of cisplatin in the tumor region, from the encapsulated formulation, with improvement in therapeutic efficacy.  In addition, four times less platinum was detected in the kidneys than in animals treated with free cisplatin.  Harrington and collaborators (HARRINGTON et al. , Pegylated liposomeencapsulated doxorubicin and cisplatin enhance the effect of radiotherapy in a tumor xenograft model.  Clinical Cancer Research, Vol.  6, p.  4939-4949, 2000) evaluated the use of these liposomes in chemotherapy associated with radiotherapy, using animal models implanted with head and neck tumors.  The results of this study demonstrated that these liposomes increase the effect of radiotherapy, without the occurrence of cutaneous or systemic toxicity.  Based on these results, phase I and II clinical studies were carried out by administering these cisplatin liposomes in

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advanced head and neck scaly cancer patients. However, in this study, cisplatin liposomes were inactive. The possible cause for the ineffectiveness of this treatment was the reduced bioavailability of this formulation, requiring further studies regarding the phospholipid composition in order to increase the drug / lipid ratio and its bioavailability.

Therefore, in order to improve the bioavailability of liposomes containing antineoplastic agents, the present invention relates to the development of conventional and stealthy pH-sensitive liposome formulations of cisplatin and other antineoplastic agents, which have the potential to release the drug, preferably in the tumor region, thus increasing its bioavailability at the site of action.

The pH-sensitive liposomes are composed of phospholipids derived from phosphatidylethanolamine (PE), such as, for example, dioleoylphosphatidylethanolamine (DOPE). These derivatives are organized in an aqueous medium, at room temperature, in hexagonal form, not being able to present themselves in the form of vesicles (SIEGEL, DP Inverted micellar intermediates and the transitions between lamellar, cubic, and inverted hexagonal lipid phases-ll Implications for membrane-membrane interactions and membrane fusion (Biophysical Journal, v. 49, p. 1171-1183, 1986). The formation of liposomes with these phospholipids requires the addition of stabilizing agents, usually carboxylated lipids, such as cholesteryl hemisuccinate (CHEMS), which at physiological pH are in ionized form. These stabilizers are able to insert themselves between the phospholipid molecules, and the appearance of electrostatic repulsions between the carboxyl groups, present in the stabilizer, and the phosphate groups of the

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phospholipids favor lamellar organization, enabling the formation of liposomes. The pH-sensitive liposomes can be of the conventional or stealth type. Conventional pH-sensitive liposomes are made up of phospholipids and substances that modify membrane properties, such as cholesterol. These liposomes when administered intravenously undergo adsorption of serum proteins (opsonins), causing their capture by cells of the mononuclear phagocytic system (SFM), especially Kupffer cells, present in the liver, and splenic macrophages (DELATTRE, J „COUVREUR, P „PUISIEUX, F., PHILIPOT, J. R„ SCHUBER,

F. Pharmacocinétique et potentialités thérapeutiques des liposomes. In: DELATTRE, J., COUVREUR, P., PUISIEUX, F., PHILIPOT, J. R „SCHUBER, F. (Ed.) Les Liposomes Aspects Technologiques, Biologiques et Pharmacologiques. Paris: Les éditions INSERM & Editions Medical les Internationales, 1993, p. 179-213). Stealthy pH-sensitive liposomes also contain phospholipids, as the main constituent, but present in the bilayer another compound that makes the surface of the vesicles more hydrophilic, such as, for example, phospholipids coupled to the polyethylene glycol (PEG) chain. These hydrophilic derivatives prevent the adsorption of serum proteins on the surface of the liposomes, reducing their uptake by SFM and, consequently, prolonging their circulation time in the bloodstream and allowing a greater distribution of the encapsulated drug (DU et al., Grafted poly- ( ethylene glycol) on lipid surfaces inhibits protein adsorption and cell adhesion (Biochimica et Biophysica Acta, v. 1326, n. 2 p. 239-248, 1997). Exposure of conventional or stealthy pH-sensitive liposomes to an acidic pH results in protonation of stabilizing agents, with consequent destabilization of the vesicles and the release of material

6/15 • · · · · ···· · ·· • · ·· · ·· ···· · · · · ··· · · · ·· ·· · · · ···· • ·· ········ · · • ··· ·· ·· ·· ·· · · · «encapsulated (OLIVEIRA et al., PH-sensitive liposomes as a carrier for oligonucleotides: a physico-chemical study of the interaction between DOPE and a 15-mer oligonucleotide in quasi-anhydrous samples.Biochimica and Biophysica Acta, v. 1372, n. 2, p. 301-310, 1998; OLIVEIRA et al., Improvement of in vivo stability of phosphodiester oligonucleotide using anionic liposomes in mice, Life Sciences, v. 67, pp. 1625-1637, 2000). A striking feature of tumor tissues is the fact that the extracellular pH is more acidic than that of normal tissues. The intracellular pH of both tissues is relatively similar, due to the need to maintain a favorable environment for the various cytoplasmic activities. Therefore, the carrying of antineoplastic agents in conventional or stealthy pH-sensitive liposomes may allow the specific release of the drug in the tumor region, reflecting in its greater bioavailability at that location.

In order to increase the encapsulation efficiency of cisplatin and other antineoplastic agents in liposomes, leading to a higher drug / lipid ratio, in the present invention the complexation of cisplatin with cyclodextriná was performed, followed by encapsulation of the complex obtained in pH-sensitive liposomes. Cyclodextrins are cyclic oligosaccharides and can present six, seven or eight glucose units, being denominated as α, β and γ cyclodextrin, respectively. The outer face of the cyclodextrins consists of hydroxyls linked to C-2, C-3 and C-6, which allows solvation by water molecules as well as the introduction of substituents without changing the internal cavity (FROMMING, KL & SZETLI, J. Ciclodextrins in Pharmacy, Dordrecht: Klumer Academic Publishers, 1993. 225 p). These oligosaccharides have a trunk-like shape, whose internal part has a hydrophobic character, responsible for the inclusion of molecules

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The present invention described here relates to the development of controlled release formulations of antineoplastic agents, for use in human and veterinary medicine. The inventive formulations comprise pH-sensitive liposomes formed by:

a- an antineoplastic therapeutic agent in its free form or complexed with cyclodextrin derivatives;

b- non-lamellar phase-forming phospholipids, such as phosphatidylethanolamine derivatives;

c- stabilizers of the lipid bilayer containing carboxylated groups; d- hydrophilic agents that modify the lipid bilayer surface;

The formulation of conventional pH-sensitive liposomes, without restriction, comprises for example:

a- an antineoplastic agent, such as free or complex cisplatin with a cyclodextrin derivative, such as hydropropyl-p-cyclodextrin; b- a non-lamellar phase-forming phospholipid, such as dioleoylphosphatidylethanolamine;

c- a lipid bilayer stabilizing agent, such as cholesteryl hemisuccinate.

The formulation of stealthy pH-sensitive liposomes, without restriction, comprises, for example:

a- an antineoplastic agent, such as free or complex cisplatin with a cyclodextrin derivative, such as hydropropyl-p-cyclodextrin;

b- a non-lamellar phase-forming phospholipid, such as dioleoylphosphatidylethanolamine;

zo

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d- hydrophilic agents that modify the lipid bilayer surface, such as, for example, distearoylphosphatidylethanolamine coupled to polyethylene glycol.

The present invention innovates by presenting conventional and stealthy pH-sensitive liposomes with a particle size equal to or less than 1000 nm, preferably with a size between 80 and 500 nm. The composition of conventional and stealthy pH-sensitive liposomes has dioleoylphosphatidylethanolamine in an amount corresponding to 60 to 75% of the total lipid concentration, more preferably 60%; 25 to 40% cholesteryl hemisuccinate, more preferably 40%. Stealthy pH-sensitive liposomes still contain 5 to 15% of the total lipid concentration corresponding to distearioyl phosphatidylethanolamine coupled to polyethylene glycol, more preferably 5%. Cisplatin is encapsulated in sensitive or conventional stealthy pH15 liposomes after its solubilization in free form in 0.9% NaCI solution (w / v) or after its complexation with hydroxypropyl-βcyclodextrin in 0.9% NaCI solution (w / v). The cisplatin / hydroxypropyl-p-cyclodextrin complex was prepared in the molar ratios of cisplatin / hydroxypropyl-3-cyclodextrin equal to 1.0: 2.0 to 1.0: 20.0, preferably 1.0: 4.0 to 1 : 18.0, even more specifically 1: 11.0.

The invention described here further presents the process for obtaining conventional and stealthy pH-sensitive liposome formulations containing the antineoplastic agent. The usual methods of preparing liposomes are based on the dissolution of lipid components in organic solvents with subsequent elimination of these solvents and hydration of the formed lipid film, or even in the injection of the organic solution in aqueous medium, followed by

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removal of organic solvent (New, R. R. C. Preparation of liposomes. In: NEW, R. R. C. (Ed.) Liposomes a practical approach. United Kingdom: IRL Press, 1990. p. 33-104). In the case of encapsulation of cisplatin and other antineoplastic agents in conventional and stealthy pH-sensitive liposomes, the reverse phase evaporation method was used and in the encapsulation of the cisplatin / hydroxypropyl-p-cyclodextrin complex, the freeze-thaw method was used. In preparing the pH-sensitive liposomes of cisplatin, the cisplatin / hydroxypropyl-p-cyclodextrin complex and other unstable antineoplastic agents in alkaline pH, without restriction, it was necessary to create a specific process that allows obtaining the pH- liposomes sensitive and at the same time preserve the stability of the antineoplastic agent and ensure effective encapsulation. It is important to mention that other methods of knowledge of those skilled in the art can be used to obtain conventional and stealthy pH-sensitive liposomes containing antineoplastic agents.

The present invention concerning the pH-sensitive liposomes of cisplatin and other antineoplastic agents and their respective process of obtaining it is illustrated in detail through the examples presented below. It is necessary to emphasize that the invention is not limited to these examples, but that it also includes variations and modifications within the objectives for which it works.

Example 1 - Encapsulation of cisplatin in conventional and stealthy pH-sensitive liposomes by the zz reverse phase evaporation method

A lipid film was prepared from chloroform aliquots of 25 dioleoylphosphatidylethanolamine (Lipoid GmBH, Ludwisgshafen, Germany) and cholesteryl hemisuccinate (Sigma Chemical Company, St Louis, MO, USA)

10/15 «··· ·« · · * · · • · 9 · ······ · ··· ·· · · · · »· · · · · · ·· ···· * · ·· · · · · * * · · · · · · · · · * * (lipid concentration between 20 to 40 mM, 6: 4 molar ratio). Then, this lipid film was dissolved in ethyl ether alkalinized with NaOH. Subsequently, the cisplatin 2 mg / mL solution (Quiral Química do Brasil SA, Juiz de Fora, MG, Brazil) was added in a 0.9 g% (w / v) NaCi solution, in the proportion of 1: 3 aqueous and ethereal phase, respectively. The mixture obtained was subjected to rapid vortexing, producing an A / O emulsion. In the case of stealthy pH-sensitive liposomes, 5 mol% of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG) (Lipoid GmBH, Ludwisgshafen, Germany) was added to the lipid composition, maintaining the same final lipid concentration.

The A / O emulsion was subjected to evaporation under vacuum to eliminate the ether, allowing the formation of lipid vesicles. The non-encapsulated cisplatin was separated from the liposomes by ultracentrifugation at 150000 g, at 10 ° C, for 60 minutes to obtain the purified liposomes. The encapsulation rate of cisplatin was determined by high performance liquid chromatography after opening the liposomes purified with 5.0% (v / v) Triton X-100. The diameter of the liposomes was measured using the N4 nanosizer (Coultronics, Margency, France). The rate of encapsulation of cisplatin in liposomes is shown in Table 1.

Table 1 shows the lipid composition and the respective cisplatin encapsulation rate in these compositions.

Example 2 - Determination of the apparent solubility of cisplatin in the cisplatin / hydroxypropyl-B-cyclodextrin association complex

The determination of the solubility of cisplatin compounded with hydroxypropyl-β-cyclodextrin was carried out by adding increasing amounts of hydroxypropyl-β-cyclodextrin (Cerestar USA Inc., Indiana, USA) to a 27 mM cisplatin solution in NaCI 0 solution, 9 g% (w / v). At

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concentrations of hydroxypropyl ^ -cyclodextrin employed are listed in Table 2.

Table 1 - Evaluation of the encapsulation rate of cisplatin in pH-sensitive liposomes

% OF ENCAPSULATION OF CISPLATIN LIPID COMPOSITION Method Evaporation in Reverse Phase * DOPE: CHEMS 6: 4 (20 mM) 4.0 ± 0.3 DOPE: CHEMS: DSPE-PEG 5.7: 3.8: 0.5 (20 mM) 7.0 ± 0.3 DOPE.CHEMS.DSPE-PEG 5.7: 3.8: 0.5 (30 mM) 8.8 ± 0.3 DOPE.CHEMS.DSPE-PEG 5.7: 3.8: 0.5 (40 mM) 18.6 ± 1.3

* The number of samples was equal to 3.

Table 2 - Molar ratios and concentrations of cisplatin and hydroxy propyl-p-cyclodextrin used in the preparation of association complexes.

Molar Reasons CISP / HBPCD [CISP] (mM) [HPBCD] (mM) 1: 2.0 27 54.0 1: 4.0 27 108.0 1: 6.0 27 162.0

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1: 8.0 27 216.0 1: 14.0 27 375.0 1: 16.0 27 428.6 1: 18.0 27 482.1 1: 20.0 27 535.7

The dispersions obtained were kept under stirring for approximately five days at room temperature. Then, the solubilized cisplatin phase was separated from the insoluble phase by centrifugation at 14000 rpm, for 10 minutes. The quantification of the concentration of soluble cisplatin was performed using high performance liquid chromatography. Table 3 shows the cisplatin solubility (CISP) in the presence of hydroxypropyl-p-cyclodextrin (HPBCD).

Table 3 - Solubility of cisplatin complexed with hydroxypropyl-βcyclodextrin

CISP / HBPCD molar ratios [CISP] Solubilized (mM) 5.8 (So) ** 1 2.0 7.1 ± 0.7 1 4.0 7.8 ± 1.2 1 6.0 8.5 ± 0.2 1 8.0 9.1 ± 0.5 1 14.0 9.9 ± 0.9 1 16.0 10.4 ± 0.0 1 18.0 11.0 ± 0.1 1 20.0 11.0 ± 0.1

* The number of samples was equal to 3.

** So is the solubility of CISP in the absence of HPBCD.

Example 3 - Characterization of the cisplatin / hydroxypropyl-B-cyclodextrin association complex

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The cisplatin / hydroxypropyl-p-cyclodextrin association complex was characterized by the use of differential scanning calorimetry. The

Thermograms from different samples were obtained on the DSC 2910 calorimeter (TA Instruments Lukens Drive, New Castle, USA), which was calibrated with indium at a heating speed of 10 ° C / min. Cisplatin, hydroxypropyl-p-cyclodextrin samples , physical mixture of hydroxypropyl-p-cyclodextrin and cisplatin and the lyophilized cisplatin / hydroxypropyl-p-cyclodextrin association complex were precisely weighed (4-6 mg) in semi-hermetic aluminum crucibles and heated under nitrogen flow. Initially, all samples were heated from 30 to 110 ° C, at 10 ° C / min., To eliminate moisture. Subsequently, the samples of cisplatin, hydroxypropyl-βcyclodextrin, physical mixture of hydroxypropyl-3-cyclodextrin and cisplatin and the lyophilized cisplatin / hydroxypropyl-p-cyclodextrin association complex were subjected to the following heating intervals, respectively: 30260 ° C; 30-310 ° C; 30-310 ° C and 30-350 ° C. The thermograms of these different samples are shown in Figure 1. Figure 1 shows the thermograms for cisplatin (1), the cisplatin / hydroxypropyl-βcyclodextrin association complex (2), hydroxypropii-p-cyclodextrin (3) and the physical mixture of cisplatin and hydroxypropyl-p-cyclodextrin (4).

Example 4 - Preparation of pH-sensitive liposomes containing the cisplatin / hydroxypropyl-8-cyclodextrin association complex

A lipid film was obtained from chloroform aliquots of dioleoylphosphatidylethanolamine, cholesteryl hemisuccinate and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (lipid concentration 120 mM, molar ratio 5.7: 3.8: 0.5, respectively), as described in example 1. The lipid film formed was hydrated with a 0.9 g% NaCI solution, and then alkalized to a pH of around 12. The liposomes obtained were calibrated by extruding them through 0.4 polycarbonate filters and

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0.2 μηη (Millipore Corporation, Bedford, MA, USA) (5 times for each cycle). The pH of the liposomal dispersion was adjusted to approximately 9.0. Subsequently, the calibrated liposomes were mixed with the cisplatin / hydroxypropyl-p-cyclodextrin association complex, molar ratio 1:11, in a volume ratio equal to 1: 1.9, respectively. Finally, the obtained mixture was subjected to three cycles of freezing in liquid nitrogen and thawing at 35 ° C.

The non-encapsulated cisplatin / hydroxypropyl-3-cyclodextrin combination complex was separated from the liposomes by ultrafiltration on an Ultrafree-MC membrane (UFC 30 VUOO, Millipore Corporation, Bedford, MA, USA) at 12,000 g, for 30 minutes. The filtered portion obtained was then filtered on an Ultrafree-MC membrane (PLTK 30 K Millipore, Millipore Corporation, Bedford, MA, USA), at 5000 g, for 60 minutes. The rate of encapsulation of cisplatin was calculated by the difference in the concentration of cisplatin present in non-purified liposomes and in the portion obtained after filtration (non-encapsulated cisplatin). The cisplatin assay was performed using the high performance liquid chromatography technique. The diameter of the liposomes was measured using the N4 nanosizer (Coultronics, Margency, France). The encapsulation rate of the cisplatin / hydroxypropyl-p-cyclodextrin complex was 42.8% (number of samples = 3; sd = ± 3.9).

Example 5 - Evaluation of cytotoxic activity in vitro of cisplatin pH-sensitive liposomes

The A459 cell culture in the logarithmic phase of proliferation was trypsinized and seeded in 96-well plates (Nalge Nunc International, Denmark), suspended in 100 µl of the appropriate culture medium enriched with 10% SFB and incubated at 36 ° C. , 5 ° C in a greenhouse (Sanyo Electric Company Ltd., Japan) with a humid atmosphere containing 2.5% (v / v) carbon dioxide. The number of cells / well was 5x10 ³ . After 24 hours of cultivation, the cells were subjected to treatment with free cisplatin and encapsulated in conventional and stealthy pH-sensitive liposomes in a concentration equal to 3.73 x W ⁶ mol / L, corresponding to their IC50. The tf

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020 treatment was carried out on three consecutive days, with the medium being replaced with each new treatment. Controls were performed by replacing cisplatin with an equal volume of the respective culture medium. 72 hours after the initial treatment, cell viability was measured by reducing MTT. First, 10 μΙ_ of the solution was added to 5 mg / mL of the tetrazolium salt in each well of the culture plate. After 4 hours, the MTT crystals were solubilized in 150 μΙ_ of DMSO. The optical density reading was performed at a wavelength of 600 nm (Spectrophotometer Stat Fax 2100, Awariness Technoloy INC, USA). The calculations of the percentage of cell viability for cells treated with free cisplatin and encapsulated in conventional and stealthy pH-sensitive liposomes were performed, based on the mean of the respective absorbances considering the mean absorbance value of the control group as 100% of viability. The comparison between the mean values of% cell inhibition was performed using the Student's t-test. The differences were considered statistically significant at the level of p <0.05. The results are shown in Table 4. Table 4 shows the treatments to which the cells were submitted, the optical density found and the percentage of cell inhibition. Table 4 - Cytotoxic activity of free and encapsulated cisplatin in conventional and stealthy pH-sensitive liposomes

Lineage Cell phone Treatment Density Optics (OF) % cell inhibition A549 (group control) 0.2678 ± 0.038 0 Cisplatin free 0.1198 ± 0.014 55.26 PH- Liposomes sensitive conventional cisplatin 0.0738 ± 0.022 72.44 * Cisplatin stealthy pH-sensitive liposomes 0.0696 ± 0.024 74.01 *

* Cytotoxic activity is significantly greater than that of free cisplatin (p <0.05).

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Claims (7)

R E I V I N D I C A TIONS 1. pH-sensitive liposomes characterized by comprising antineoplastic agent, preferably cisplatin, in free form or complexed with cyclodextrin; a non-lamellar phase-forming phospholipid, 5 preferably dioleoylphosphatidylethanolamine; a lipid bilayer stabilizing agent, preferably cholesteryl hemisuccinate; and optionally a phospholipid coupled to a hydrophilic polymer of molecular weight comprised between 1000 to 5000 Daltons, preferably distearoylphosphatidylethanolamine-polyethylene glycol. 10 Liposomes according to claim 1, characterized in that they have a total lipid concentration comprised between 20 to 40mM, with dioleoylphosphatidylethanolamine in an amount corresponding to 60 to 75% of the total lipid concentration, more preferably 60%; 25 to 40% cholesteryl hemisuccinate, more preferably 40%; and optionally 5 to 15 15% of the total lipid concentration corresponding to distearioylphosphatidylethanolamine coupled to polyethylene glycol, preferably 5%. Liposomes according to claims 1 to 2, characterized in that they have vesicle diameters between 80 to 500 nm. 20 4. Process of encapsulation of antineoplastic agents in the liposomes described in claims 1 to 3, characterized by comprising the following steps: a) dispersion of the solution containing at least one antineoplastic agent, preferably cisplatin, in ethyl ether previously alkalized with 25 NaOH solution containing a non-lamellar phase-forming phospholipid, preferably dioleoylphosphatidylethanolamine; a lipid bilayer stabilizing agent, preferably cholesteryl hemisuccinate; and optionally a phospholipid coupled to a hydrophilic polymer of molecular weight between 1000 to 5000 Daltons, preferably Distearoylphosphatidylethanolamine-polyethylene glycol; b) evaporation of the organic solvent. Petition 870170102378, of 12/27/2017, p. 12/11 1/1 5. Process of encapsulation of antineoplastic agents in the liposomes described in claims 1 to 3, characterized by comprising the following steps: a) encapsulation of the antineoplastic agent, preferably cisplatin, 5 in cyclodextrin, preferably hydroxypropyl-p-cyclodextrin in molar ratios of cisplatin / hydroxypropyl-p-cyclodextrin equal to 1.0: 2.0 to 1.0: 20.0, preferably 1.0: 4.0 to 1: 18.0, even more specifically 1: 11.0; b) previous preparation of white liposomes containing a non-lamellar phase-forming phospholipid, preferably dioleoylphosphatidylethanolamine; 10 a lipid bilayer stabilizing agent, preferably cholesteryl hemisuccinate; and optionally a phospholipid coupled to a hydrophilic polymer of molecular weight between 1000 to 5000 Daltons, preferably distearoylphosphatidylethanolamine-polyethylene glycol, presenting a pH around 12, calibrated in membranes of 15 polycarbonate of varying pore size, with pH adjustment to 9; c) adding the solution obtained in “a” to the liposomes obtained in “b” and performing freezing and thawing in order to promote encapsulation. Petition 870170102378, of 12/27/2017, p. 12/12 1/1 • ·· · · · · · · · ··· · «· · · ·· ·· · · · ···· • · ·· · · · * · · · ··· ·· ·· ·· ·· · · ·