ES2254027A1 - Liposomal formulation containing hydrophilic active agent, useful e.g. for topical treatment of cancer of skin and musosa, comprises bilayer comprising neutral and charged lipids - Google Patents

Abstract

Liposomal formulation (A) comprises at least one hydrophilic active agent (I) encapsulated in liposomes containing at least one lipid bilayer formed from a mixture of at least one neutral, saturated phospholipid (II) and at least one charged, saturated lipid (III). An independent claim is also included for a method of preparing (A). ACTIVITY : Dermatological Cytostatic Virucide Antipsoriatic. No details of tests for these activities are given. MECHANISM OF ACTION : None given.

Description

Liposomal formulations

The invention relates to formulations of hydrophilic substances encapsulated in liposomes, to the method of encapsulation of hydrophilic substances in liposomes and at use of said formulations in the prevention and / or treatment of diseases and / or conditions of the human or animal being.

Background of the invention

Liposomes are microscopic vesicles that they have an aqueous central cavity surrounded by a lipid membrane formed by concentric bilayer (s).

The liposomes can be unilamellar (ie having a single lipid bilayer) or oligolamellar or multilamellar (if they have several bilayers). Multilamellar vesicles (MLVs) have a size ranging from 0.2 µm to 10 µm while unilamellar can be large (LUVs) or small (SUVs) with a size between 0.02 and
0.2 µm.

By their structure they have the capacity to incorporate hydrophilic substances (in aqueous interior) or hydrophobic (in the lipid membrane).

The most important structural components of Liposomes are phospholipids (PLs).

To obtain liposomes with properties specific phospholipids with neutral charge can be used, positive or negative and / or with different length and degree of saturation of fatty acid chains.

Likewise, to modify the properties of liposomes, it is possible, for example, to incorporate cholesterol (COL) into the membrane, modify the number of lipid bilayers or covalently bind natural molecules (for example proteins, polysaccharides, glycolipids, antibodies, enzymes) or synthetics. (for example polyethylene glycol) to its super-
ficie.

In sum, the variables to consider at the moment of designing a liposome are multiple and the results are not Always expected.

As vehicles for the administration of drugs, liposomes have, in theory, numerous advantages. In addition to being made up of non-toxic components, generally non-immunogenic, non-irritating and biodegradable, they should be able to encapsulate, retain, transport and yield to target organs a wide variety of therapeutic agents, reducing its side effects (adverse).

However, the goal of developing of optimal liposomal formulations from a technical point of view and Therapeutic is rarely achieved.

In particular, the optimal liposomal vesicles They must be able to:

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encapsulate the active agent in your interior in a high percentage, so that the effectiveness of encapsulation (ratio between amount of encapsulated drug and lipid) is appropriate from the point of view of dosage and of the cost of the formulation;

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be stable, ie retaining the encapsulated active agent during the shelf life of the formulation and, once administered to the patient, until reaching the site of action;

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release the drug at the site of action with a transfer profile appropriate to the therapeutic objective, reducing systemic toxicity.

On the other hand, and in the specific case of topical administration, the lipids that form the liposomal vesicles must confer certain properties to the lipid bilayer so that the resulting liposomes promote the rapid penetration of the active substance through the stratum corneum and its location in the desired site of action (usually in the vicinity of the basal layer of the epidermis).

Although in previous studies it has been achieved encapsulate hydrophilic substances (low molecular weight and polar character) in liposomes, the retention of the active agent in the inside of them remains a technical problem to solve as shown in the following works.

Norley SG et al ., [J. Immunol. 136: 681-685 (1986)], using neutral liposomes composed of egg phosphatidylcholine (PC) and 15: 1 cholesterol prepared by the dehydration-rehydration (DRV) method, obtain a retention of 44% acyclovir after 14 days at 4 ° C and 48% after 24 hours at 37 ° C.

Law SL et al ., [Int.J.Pharm. 161: 253-259 (1998)], using MLV liposomes of egg PC and 1.6: 1 cholesterol, neutral and charged by adding di-methylphosphate or stearylamine in molar ratios between 0.15 and 1.6, achieved at 10 hours at 37 ° C a retention of acyclovir between 75% and less than 25% (depending on the amount of charge) for those with a positive charge, between 90% and 35% (depending on the amount of charge) for those with a negative charge and between 80% and 60% for neutrals.

Fresta M. et al ., [J.Parm.Pharmacol. 51: 565-576 (1999)] using liposomes composed of DPPC: COL 7: 4 (rigid neutrals), DPPC: COL 7: 4: 1 (rigid negative charge ) and DPPC: Cholesterol: DDAB 7: 4: 1 (rigid positive charge), obtain a retention of acyclovir between 50 and 30% at 8 hours at 37 ° C for OLV (with minimal retention for neutral and negative) and between 65% and 45% at 8 hours at 37 ° C for MLV (with minimal retention for neutrals and negatives).

Sarbolouki MN, Toliat T. PDA, [J Pharm Sci Technol 52: 23-27 (1998)] using liposomes composed of EPC (flexible, neutral), MLV and REV size, they get a 30-day methotrexate retention of 70% at 4 ° C; 40% at 25 ° C and <10% at 37 ° C.

Khopade AJ, et al . [Int. J. Pharm. 232: 157-162 (2002)] using MLV liposomes consisting of HSPC: COL (neutral, rigid) and HSPC: COL: DCP (negative, rigid), obtain a 35% methotrexate retention at 8 hours at 37 ° C .

Norley SG, et.al., [J. Immunol 136: 681-685 (1986)] using liposomes composed of Egg PC (polyunsaturated, flexible) and 15: 1 cholesterol (neutral), prepared by the DRV method, get a retention of iododeoxyuridine 50% at 24 hours at 4 ° C and 20% at 24 hours 37 ° C

Simmons SP, Kramer PA., [J Pharm Sci 66: 984-986 (1977)] using MLV liposomes consisting of sphingomyelin (SM): COL: DCP 4.8: 2.8: 1 (negative), obtain a 70% floxuridine retention at 40 hours a
25 ° C

Maurer N. et al ., [BBA 1374: 9-20 (1998)] using LUV liposomes composed of DPPC: COL 55:45 (neutral, rigid), achieve 20% ciprofloxacin retention at 30 min at 25 ° C.

In sum, although agents have been encapsulated hydrophilic assets in liposomes remains the problem that these are stable, that is, they retain the active substance in its interior.

Summary of the Invention

There is therefore a need to have Pharmaceutical formulations of hydrophilic active ingredients encapsulated in liposomes that have high efficacy of encapsulation, are stable and have an assignment profile of drug suitable for the therapeutic objective and non-toxic for patient.

The inventors of the present have found that the use of a mixture of neutral saturated phospholipids and of saturated lipids loaded in the preparation of liposomes allows achieve these goals

This is how they have managed to obtain formulations stable hydrophilic drugs, in which the active substance remains encapsulated inside the liposomes during time needed for preparation, storage and distribution of the corresponding pharmaceutical formulations until patient administration

Also, and for the specific case of the topical application, have achieved that these formulations have a good diffusion in skin, reaching the epidermis and having there a depot effect, and do not show signs of toxicity (irritation cutaneous) after repeated dermal application. Hopefully also show good diffusion in mucous membranes.

Consequently, the use of a combination of PLs saturated saturated and saturated saturated lipids allows to obtain topical compositions of hydrophilic drugs and low weight molecular that have high stability, efficiency (based on a good cutaneous diffusion of the drug) and safety (due to low systemic absorption).

On the other hand, the use of a mixture of PLs saturated saturated and saturated saturated lipids would allow encapsulate in liposomes not only drugs but also others hydrophilic substances such as diagnostic agents, cosmetics,  food substances, etc.

In accordance with the above, a First aspect of the present invention relates to formulations liposomes comprising at least one hydrophilic active agent encapsulated in liposomes consisting of one or more bilayers lipids formed by a mixture of at least one phospholipid Saturated neutral and at least one saturated lipid with charge.

A second aspect of the present invention is refers to an encapsulation procedure of active substances hydrophilic in liposomes, that is to say the method of preparation of said liposomal formulations.

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A third aspect of the present invention is refers to the use of the aforementioned liposomal formulations in the preparation of medicines for the prevention or treatment of pathologies or conditions of the human or animal being.

A fourth aspect of the present invention is refers to pharmaceutical or veterinary formulations that they contain said liposomal formulations and at least one excipient pharmaceutically acceptable.

Prior to the description of the realizations particular, some specific related terms can be defined to the main aspects of the present invention, for purposes clarifying, not limiting.

It is understood by liposomal formulations those in which part or all of the active substance is found encapsulated inside liposomes.

The liposomes may consist of one or Several lipid bilayers and have different size.

The bilayers have a polar surface (interior and outside) and non-polar core.

Phospholipid is understood as that derivative amphiphilic glycerol in which one of its hydroxyl groups is esterified with phosphoric acid and the other two hydroxyls are esterified with long chain fatty acids.

A saturated phospholipid will be one whose acids fatty do not have double bonds carbon-carbon

A neutral phospholipid will generally be that in which another phosphoric acid hydroxyl is esterified by a alcohol substituted with a polar group (usually hydroxyl or amino) and whose net charge is zero.

A phospholipid with charge will generally be that wherein another hydroxyl of the phosphoric acid is esterified by an alcohol substituted by a polar group and whose net charge is positive or negative

It is understood that saturated lipid with charge, in addition to include saturated phospholipids with charge, it includes other amphiphilic compounds whose net charge is non-zero, such as long chain hydrocarbon derivatives, substituted by a polar group (for example amino) and acid derivatives fatty

In the context of the present invention, Hydrophilic drugs are considered to be those that exhibit a log Pow (decimal logarithm of octanol / water partition coefficient) lower of zero.

Likewise, low molecular weight drugs are considered to be those that have a molecular weight lower than
1000 Da.

Detailed description of the invention

In principle, any neutral saturated PL and Any saturated lipid with charge can be used in the Liposomal formulations of the present invention.

In a preferred embodiment, the saturated PL neutral is chosen from the group consisting of derivatives of phosphatidylcholine and mixtures thereof, for example dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimiristoyl phosphatidylcholine (DMPC) and mixtures thereof.

In another preferred embodiment, the PL saturated with cargo is selected from the group consisting of derivatives of phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, acid phosphatidic and mixtures thereof, for example, Diestearoylphosphatidyl glycerol (DSPG) and a mixture of esters of phosphatidylserine (PS) with different saturated fatty acids (for example PS of bovine brain).

In another preferred embodiment, the saturated lipid with load is stearylamine (SA).

In principle, any relationship between neutral PL and loaded lipids can be used in formulations of this invention.

In a preferred embodiment, the molar ratio Neutral / lipid PL with charge will be between 50/50 and 95/5, preferably between 80/20 and 95/5.

Any hydrophilic substance could be encapsulated in the liposomal formulations of this invention. Preferably, said substance is a drug; more preferably It is a low molecular weight hydrophilic drug.

In a preferred embodiment, the molar ratio hydrophilic drug / lipids will be between 40: 1 and 0.01: 1, plus preferably it will be between 2: 1 and 0.1: 1

The lower limit is determined by the lower amount of drug that is practical for making liposomes given its intended use and can be easily determined by an expert in the matter. The upper limit is conditioned by the crystallization concentration of the drug.

The liposomal formulations provided by this invention may also contain other lipid components as sterols (for example cholesterol) or sphingolipids (for example sphingomyelins and glycosphingolipids, in particular gangliosides).

In principle, any hydrophilic drug can be encapsulated in the liposomal formulations of the present invention.

In the case of liposomal formulations of topical application, the hydrophilic drug is preferably of low molecular weight

In preferred embodiments, the agent pharmacologically active is selected from acyclovir, iododeoxyuridine, methotrexate, fluorouracil and ciprofloxacin.

Drugs, PLs and other components of Formulations of the present invention are known to an expert in the matter and can be obtained from commercial sources.

The liposomal formulations of the present invention can be prepared by any of the methods known to a person skilled in the art.

The size of the resulting liposomes is going to depend on the method of preparation used. In one embodiment preferred liposomes are prepared according to the method set forth in Examples 1 and 4.

The resulting liposomes can be dehydrated (lyophilized) for subsequent formulation and / or Storage and distribution.

Also, liposomes can be maintained in suspension in an aqueous solution containing or not drug. At First case, the resulting suspension will contain part of the drug encapsulated inside the liposomes and partly outside thereof.

The liposomal formulations provided by this invention will be particularly useful in the preparation of pharmaceutical or veterinary compositions for prevention and / or the treatment of diseases or conditions of the human being or animal.

The present liposomal formulations have proved to be particularly useful and advantageous in prevention and / or the treatment of diseases or conditions of skin and / or mucous

Preferably, the liposomal formulations of the present invention will be administered in a vehicle pharmaceutically acceptable, which will be constituted by Less an excipient. Said excipient can be selected from any of those known to a person skilled in the art and of according to the route of administration to be used, which will depend of the therapeutic objective sought.

For parenteral administration, the vehicle usually consists of water and auxiliary substances required to make it compatible with physiological conditions such as pH, tonicity, etc. regulating agents Aqueous solution resulting can be sterilized by conventional methods and packaged for use, or filtered and lyophilized, which can require the use of cryoprotective agents. Lyophilized material it will be combined with a sterile aqueous solution prior to its administration.

For topical administration, the vehicle will be constituted by excipients selected in accordance with the pharmaceutical form that is intended in each case, preferably liquid or semi-solid. Liquid formulations include aqueous solutions, hydroalcoholic solutions, lotions, etc. The semi-solid formulations are going to be mostly ointments, creams or gels.

Any of these formulations may require the use of preservatives, antioxidant agents, which lantes, pH regulating systems, preservatives, bioadhesive polymers, etc.

The preservative agent can be selected between, for example, sodium benzoate, ascorbic acid, parabens and their mixtures The antioxidant can be chosen, by way of example, between butylated hydroxytoluene (BHT), hydroxyanisole and tocopherol and Their derivatives. The agent that can be selected by example, of the group formed by ethylenediaminetetraacetic acid (EDTA), ethylene glycol acid bis- (2-aminoethyl) -tetraacetic acid (EGTA) and citric acid or its salts. The pH regulating system can be chosen, by way of For example, between a phosphate buffer solution and a buffer solution citrate.

In a particularly preferred embodiment, the Pharmaceutical or veterinary compositions of the present invention they will be formulated for topical administration, in skin or mucous

Doses of pharmacologically active agent at administering to the patient will depend on the therapeutic indication of the formulation, of the patient's response to the treatment and of concomitant therapy.

essays

The unexpected and advantageous properties of the formulations of the present invention are manifested through the following "in-vitro" and "in-vivo" non-limiting tests.

A- Preparation procedures and testing of stability

Example one

Stable encapsulation of acyclovir (ACV) in MLV liposomes of DSPC composition: DSPG

DSPC and DSPG broken steam flask are added in a 9: 1 molar ratio and chloroform: methanol in a 2: 1 ratio (v: v). Once the lipids are dissolved in the solvent mixture, the flask is connected to the rotary evaporator, adjusting the bath temperature to 40 ° C, and the solvent is evaporated under reduced pressure (800 mbar) until  form a lipid film on the walls of the flask. Subsequently, the pressure is reduced to 100 mbar and maintained for 1 hour. Finally, the flask is removed from the rotary evaporator and freeze-dried overnight to eliminate possible traces of organic solvent The lipid film thus formed is hydrated. by stirring with 9 ml of an aqueous solution of sodium acyclovir (50 mg / ml) at 65 ° C for 1 hour on the rotary evaporator.

The multilamellar liposomes thus obtained are processed by extrusion through membranes of polycarbonate with pores of 2.0 µm. Next acyclovir not encapsulated is removed by diafiltration, first versus three volumes of a 150 mM NaCl solution, pH 11.5 and then vs. 5-6 volumes of 10 mM pH phosphate buffer 6.0, 150 mM NaCl. For this, Vivaflow 50 membranes are used with a 100 KD cut (Sartorius). The resulting liposomes are diluted with the same buffer, the solution is divided into aliquots that are stored in polypropylene vials at 25 ° C until analysis.

To determine the total LCA present in the liposomal suspension, the lipids of the liposomal membrane and its content is released by dilution of the suspension in methanol. Then the concentration of LCA by spectroscopy at 254 nm against a curve of calibration.

To determine the encapsulated LCA, they settle previously liposomes by centrifugation for 25 minutes at 26,000 g, collecting the supernatant. Subsequently analyze the content of LCA in the supernatant by the procedure previously described.

The difference between the total LCA and the present in the supernatant represents the encapsulated fraction. In the example described, the total ACV concentration was adjusted to 0.555 mg / ml, being 96.3% of the same encapsulated inside the Liposomes at the beginning of the study. The ACV / lipid molar ratio was of 0.83.

Figure 1 shows the evolution of the percentage of encapsulated LCA (expressed as a percentage of the initial) to over 4 months at 25 ° C. The analysis of it allows conclude that the liposomes of the present invention, constituted by a mixture of saturated and saturated saturated PLs with charge, They show high stability.

Example 2

Stable encapsulation of acyclovir (ACV) in LUV liposomes of DPPC composition: Cholesterol: DPPG

They are added to a rotary flask by DPPC, DPPG and Cholesterol in molar ratio 60:10:40 and chloroform: methanol in a 2: 1 ratio (v: v). Once the lipids are dissolved in the mixture of solvents, the flask is connected to the rotary evaporator, adjusting the bath temperature at 40 ° C, and the solvent is evaporated under pressure reduced (800 mbar) to form a lipid film in the flask walls Subsequently the pressure is reduced to 100 mbar and it stays for 1 hour. Finally, the flask is removed from the rotavapor and freeze-dried overnight to remove possible traces of organic solvent. The lipid film like this formed is hydrated by stirring with 9 ml of an aqueous solution of acyclovir sodium (50 mg / ml) at 55 ° C for 1 hour in the rotary evaporator

The liposomes thus obtained are processed in a manner analogous to example 1, except that extrusion is performed through 0.2 gm pores membranes.

The analysis of the total and encapsulated LCA is performed by the same procedure as example 1. Liposomes The resulting ACV / lipid molar ratio of 0.52, with a 98.4% of the ACV encapsulated inside the liposomes. I dont know detected variation in the percentage of encapsulated LCA throughout a total of 6 weeks at 25 ° C.

Example 3

Non-stable encapsulation of acyclovir (ACV) in LUV liposomes of composed of palmitoyloleoylphosphatidylcholine (POPC).

Liposomes are prepared as described in the Example 2, replacing lipids with POPC.

The encapsulated LCA analysis is performed following the methodology described in example 2, checking that using a neutral but unsaturated PL, only 27.3% of the initial ACV remains retained inside the liposomes After 24 hours.

Example 4

Non-stable encapsulation of acyclovir (ACV) in LUV liposomes of POPC composition: DPPG

Liposomes are prepared as described in the Example 2, replacing lipids with POPC and DPPG in relation 9: 1 molar.

The encapsulated LCA analysis is performed following the methodology described in example 2, checking that using a mixture containing an unsaturated neutral PL, only 38.1% of the initial LCA remains retained in the inside of the liposomes after 24 hours.

Example 5

Non-stable encapsulation of acyclovir (ACV) in LUV liposomes of DPPC composition: Cholesterol

Liposomes are prepared as described in the example 2, replacing lipids with DPPC and cholesterol in 6: 4 molar ratio.

The encapsulated LCA analysis is performed following the methodology described in example 2, checking than using a lipid mixture that does not contain a saturated PL with load, only 25.4% of the initial LCA remains retained inside the liposomes after 24 hours.

Example 6

Stable iododeoxyuridine encapsulation (IDUR) in liposomes MLV of DSPC composition: DSPG

They are added to a DSPC and DSPG rotary flask in a 9: 1 molar ratio and chloroform: methanol in a 2: 1 ratio (v: v). Once the lipids are dissolved in the solvent mixture, the flask is connected to the rotary evaporator, adjusting the bath temperature to 40 ° C, and the solvent is evaporated under reduced pressure (800 mbar) until  form a lipid film on the walls of the flask. The pressure is subsequently reduced to l00 mbar and maintained for 1 hour. Finally, the flask is removed from the rotary evaporator and freeze-dried overnight to eliminate possible traces of organic solvent The lipid film thus formed is hydrated. by stirring with 50 ml of an aqueous solution of iododeoxyuridine (2 mg / ml) at 65 ° C for 1 hour on the rotary evaporator.

The liposomal suspension formed is extruded to through 2 µm pore polycarbonate filters to homogenize the size of the vesicles formed. Finally the non-encapsulated drug is eliminated by diafiltration versus 6 volumes of a phosphate buffered saline (pH 6; 150 mM NaCl). For it Vivaflow 50 membranes with a 100KD cut are used (Sartorius). The resulting liposomes are diluted with it buffer, aliquot into polypropylene vials and stored at 25 ° C until analysis.

To determine the total IDUR present in the liposomal suspension, the lipids of the liposomal membrane and its content is released by dilution of the suspension in methanol. Then the concentration of IDUR by spectroscopy at 284 nm against a curve of calibration.

To determine the encapsulated IDUR, it is pre-sediment the liposomes by centrifugation for 25 minutes at 26,000 g, collecting the supernatant. The content of IDUR in the supernatant is subsequently analyzed by the procedure described above.

The difference between the total IDUR and the present in the supernatant it represents the encapsulated fraction. At described example, the total IDUR concentration was adjusted to 0.555 mg / ml, 97.8% of it being encapsulated inside the Liposomes at the beginning of the study. The IDUR / lipid molar ratio was of 0.04.

Figure 2 shows the evolution of the percentage encapsulation (expressed as a percentage of the initial) as 2 months long. The analysis of it allows us to conclude that liposomes of the present invention, consisting of a mixture of Saturated neutral and saturated charged PLs show high stability.

B- Skin diffusion

Example 7

Liposomes were prepared according to example 2 and the resulting suspension was adjusted to a concentration of ACV of 1 mg / ml. Simultaneously a reference solution was prepared ACV of 1 mg / ml in the same buffer as liposomes. Both the Liposome suspension as the reference ACV solution is tested in skin diffusion tests as described in continuation.

The diffusion tests are carried out in a team of continuous flow with PermeGear ILC-07 cells 0.69 cm 2 of surface, using dorsal skin of mice without hair (SKH1). For the purpose of standardizing the data, a ACV fixed concentration of 1 mgml-1, diluting to do this properly prepared liposome suspensions according to example 2. A LCA solution is used as a reference In the same buffer. All tests are done by arranging 200 µl of the liposomal solution or suspension in the cell donor

Skin samples from mice once Once subcutaneous fat is removed, they are mounted in diffusion cells. As the receiving liquid (in contact with the dermis) buffer is used 10 mM phosphate, 150 mM NaCl, pH 6. The thermostated buffer at 37 ° C it circulates in the diffusion chamber at a constant flow of 0.5 mlh-1 All samples are hydrated during a minimum of 1 hour before the start of the broadcast, which proceeded for a total of 18 hours. After that time, the skin membranes washed 3 times with pH 6 isotonic phosphate buffer, and dried carefully with a cotton tip. The present LCA is analyzed both in full skin and in epidermis. For the latter, a Once diffusion has been carried out, the skin membranes are washed and dried as described and the stratum corneum is removed by applying and removing strips of adhesive tape 10 consecutive times on the surface of the skin.

Once washed and cut, skin samples or epidermis freeze and thaw repeatedly in nitrogen liquid, and 1 ml of 0.5 N NaOH is added. The suspension obtained Incubate for 16 hours at 50 ° C for solubilization. Finished this time, the samples are centrifuged removing the solution avoiding the surface fat layer and filters by filters 0.2 µm nylon. At this point the samples freeze until processing prior to analysis.

Samples obtained as described in the The previous paragraph is diluted with 0.5 N NaOH and the proteins with 5 N HClO 4. The cells are then centrifuged samples and the supernatant is collected by filtering it with filters 0.2 µm nylon directly in HPLC injection vials. He Sample analysis is performed using HPLC, using a Discovery Amide C16 column 250 x 4.6 mm and 5 particles \ mum (Supelco) with the following chromatographic conditions:

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Eluent: Water

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Flow: 1.5 ml / min

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Detection: 254 nm

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Temperature: 27 ° C

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Injection volume: 50 \ mul

The results are shown in Table 1. The analysis of it allows to conclude that liposomes according to invention have an important promoter effect of diffusion of ACV in skin, reaching ACV encapsulated in them to the layers viable skin depths beyond the stratum corneum in amounts 3 to 4 times higher than those obtained with the drug in solution.

TABLE 1 ACV levels in skin after 18 hours of diffusion (% dose applied ± D.E.)

N (each group) Liposomal ACV LCA in solution Difference significance Full skin 12 0.89 ± 0.40 0.21 ± 0.18 p = 0.0131 Epidermis 6 0.37 ± 0.19 0.13 ± 0.7 P <0.0001

Galenic formulations

The liposomes of the present invention can be vehiculized in different pharmaceutical compositions, some of which are set forth below in a non-limiting manner.

a.- Lotion

Suspension of liposomes c.s.p. achieve desired dose of drug Glycerin fifty g Disodium phosphate 0.044 g Monosodium Phosphate 0.124 g Kathon® CG 0.05 g Water c.s.p. 100%

2.- Gel

Suspension of liposomes c.s.p. achieve desired dose of drug Carbopol® 9801 0.5 - lg Triethanolamine c.s.p. pH = 6.5 Kathon® CG 0.05 g Propylene glycol 0.5 g Glycerin fifteen g Water c.s.p. 100% 1 Instead of Carbopol, another one can be used gelling agent how: Poloxamer 407 15-20% Carboxymethyl cellulose one - 10% Hydroxyethyl cellulose one - 5% Hydroxypropyl methylcellulose one - fifteen% Methylcellulose fifteen%

3- Parenteral solution

Liposome suspension

Phosphate buffered saline

Isotonic Sodium Chloride Solution

Claims (21)

1. Liposomal formulations comprising less a hydrophilic active agent encapsulated in liposomes constituted by at least one lipid bilayer formed by a mixture of at least one neutral saturated phospholipid and at least one saturated lipid with charge. 2. Liposomal formulations according to claim 1 characterized in that the neutral saturated phospholipid is chosen from phosphatidylcholine derivatives and mixtures thereof. 3. Liposomal formulations according to claim 2 characterized in that the phosphatidylcholine derivative is selected from DSPC, DPPC and DMPC. 4. Liposomal formulations according to claim 1 characterized in that the saturated lipid with negative charge is chosen from the group consisting of derivatives of phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, phosphatidic acid and mixtures thereof. 5. Liposomal formulations according to claim 4 characterized in that the lipid is selected from DSPG and phosphatidylserine esters with different saturated fatty acids. 6. Liposomal formulations according to claim 1 characterized in that the positively charged saturated lipid is stearylamine. 7. Liposomal formulations according to a any of claims 1 to 6 which may also contain at least one other lipid selected from sterols and derivatives, gangliosides and sphingomyelins. 8. Liposomal formulations according to claim 7 characterized in that the sterol is cholesterol. 9. Liposomal formulations according to claim 1 characterized in that the hydrophilic active agent is a drug selected from acyclovir, iododeoxyuridine, methotrexate and ciprofloxacin. 10. Liposomal formulations according to the claim 9 comprising acyclovir encapsulated in liposomes constituted by DPPC: COL: DPPG. 11. Liposomal formulations according to the claim 9 comprising acyclovir encapsulated in liposomes constituted by DSPC: DSPG. 12. Liposomal formulations according to any one of the preceding claims characterized in that the bilayer lipids are in a neutral ratio saturated neutral PLs / saturated lipids with a charge between 50/50 and 95/5. 13. Liposomal formulations according to any one of the preceding claims characterized in that the hydrophilic active agent / lipid molar ratio is between 40: 1 and 0.01: 1. 14. Liposomal formulations according to a any of the preceding claims that can also contain at least one antioxidant selected from tocopherol and its derivatives, BHT, hydroxyanisole and mixtures thereof. 15. Liposomal formulations according to a any of the preceding claims that also contain at least one agent that before selected from citric acid or its salts, EDTA, EGTA and mixtures thereof. 16. Liposomal formulations according to a any of the preceding claims that also contain at least one buffer system selected from solution phosphate buffer and citrate buffer solution. 17. Pharmaceutical formulations containing liposomal formulations according to any one of the claims 1 to 16 and a pharmaceutically carrier acceptable. 18. Pharmaceutical formulations according to the claim 17 for topical administration of agents pharmacologically active. 19. Use of liposomal formulations of according to any one of claims 1 to 16 in the preparation of a medicine for prevention and / or treatment of diseases or conditions of the human or animal being. 20. Use according to claim 19 in the that the disease or condition affects skin and / or mucous membranes. 21. Method of preparing liposomal formulations according to any one of claims 1 to 16, characterized in that it comprises:

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dissolve lipids in a mixture of organic solvents;

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remove solvents to form a lipid film on the walls of the container that contains;

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hydrate the film by stirring with an aqueous solution of the active agent;

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whether You want to extrude the liposomal suspension formed through filters to select the vesicular size;

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diafiltrate the suspension resulting against a buffer solution;

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whether If desired, dilute the liposomal suspension with a solution tampon.