PL220395B1 - Method of increasing the azelaic acid solubility and the possibility of applying the mixture for the formulation of the ready product - Google Patents

Description

The present invention relates to a mixture of azelaic acid and liposomes and a pharmaceutical or cosmetic preparation containing this mixture.

Liposomes are spherical vesicles surrounded by a double lipid membrane. They are formed spontaneously from lipids in the hydration process. They are made of the same ingredients as biological membranes, therefore they are perfectly tolerated by the skin. The ability of liposomes to trap hydrophilic substances inside the liposomes, accumulate lipophilic substances in the bilayer, and amphiphilic substances at the interface has been effectively used in the process of developing and producing new formulations for both the cosmetics and pharmaceutical industries.

Liposomes support the penetration of active substances through the skin by modifying the stratum corneum - the outermost part of the epidermis. This property is particularly important in the case of active substances with hydrophilic properties, for which the lipophilic nature of the outer layer of the epidermis is a natural barrier that protects the body against the loss of endogenous substances and the expansion of the external environment. Moreover, liposomes stabilize unstable medicinal substances, increase the effectiveness of preparations and thus contribute to lowering the concentration of active substances, which is directly related to the reduction of side effects.

The use of liposomal preparations enables not only better transport and distribution of active substances, but also enables direct introduction of water and fatty substances into the stratum corneum, which in turn affects the regulation of the water-fat balance of the epidermis. [Bouwstra J.A., Honeywell-Nguyen P.L. Skin structure and mode of action of vesicles. Adv. Drug Delivery Rev. 2002, 54, S41-S55; Lee S., Lee J., Choi Y.W. Skin permeation enhancement of ascorbyl palmitate by liposomal hydrogel (Lipogel) formulation and electric asistance. Biol. Pharm. Bull. 2007, 30, 393-396; Ren C., Fang L., Li T., Wang M. Effect of permeation enhancers and organic acids on the skin permeation of indapamide. Inter. J. Pharmaceut. 2008, 350, 43-47; Silva C.L., Topgaard D., Kocherbitov V., Sousa J.J.S., Pais A.A.C.C., Sparr E. Stratum corneum hydration: phase transformations and mobility in stratum corneum, extracted lipids and isolated corneocytes. Biochim. Biophys. Acta 2007, 1768, 2647-2659]. In order to obtain the formulation that is optimal in terms of effectiveness, it is important to control the production process so that the ratio of the active substance to the lipid fraction of the liposomes is maximized.

Azelaic acid is a naturally occurring, saturated, aliphatic dicarboxylic acid with nine carbon atoms that is very sparingly soluble in water. It is formed, most likely, in organisms as a result of lipoperoxidation of free and esterified fatty acids. Cereal grains are a natural source of azelaic acid.

As a result of numerous studies conducted on azelaic acid, its wide spectrum of activity has been proven, which can be used in the treatment of numerous, common dermatological diseases, such as: acne, rosacea, and hyperpigmentation. It also has anti-comedogenic and anti-inflammatory properties.

Azelaic acid has an antibacterial effect by inhibiting the synthesis of cellular proteins in both aerobic and anaerobic bacteria. At lower concentrations it has a bacteriostatic effect on Propionibacterium agues and Staphylococcus epidermidis, while at higher concentrations it is bactericidal. In addition, it does not cause bacterial resistance even with long-term treatment, unlike many antibiotics.

Azelaic acid is effective in reducing the amount of free radicals that trigger inflammatory processes in the body. This action is related to the inhibition of the production of free oxygen radicals by neutrophils, as well as the elimination of existing radicals.

Azelaic acid also affects keratinocytes and the keratinization process by reducing excessive proliferation, keratohalin and filaggrin grains. [Graupe K, Cunliffe WJ, Gollnick HP, Zaumseil RP. “Efficacy and safety of topical azelaic acid (20% cream): an overview of results from European clinical trials and experimental reports”. Cutis. 1996, 57, 20-35; Breathnach AS. “Melanin hyperpigmentation of skin: melasma, topical treatment with azelaic acid, and other therapies”. Cutis. 1996, 57, 36-45; Maddin S. “A comparison of topical azelaic acid 20% cream and topical metronidazole 0.75% cream in the treatment of patients with papulopustular rosacea”. J Am Acad Dermatol. 1999,40,961-965; Holland K, Bojar R. "The effect of azelaic acid on cutaneous bacteria". J Dermatol Treat. 1989, 1, 17-19]. One of the first patents to protect azelaic acid as an active compound in the treatment of hyperpigmentation dermatoses or skin hyperpigmentation is the Italian patent IT 1 075 741

PL 219 395 B1 from 1985. Two years later, azelaic acid is protected as an effective active compound in the treatment of rosacea in European patent EP 0 305 407 filed by Nazzaro-Porro.

European patent EP 0 336 880 describes a pharmaceutical formulation with 20% azelaic acid with excipients such as: water, salt, polysorbate, propylene glycol, triacylglycerols and diacylglycerols.

Another composition, containing azelaic acid, used for the treatment of rosacea, senile skin, hyperpigmentation, acne vulgaris and skin irritations is described in European patent EP 1 032 379. Its composition, in addition to the active substance, includes: triacylglycerol, propylene glycol, polyacrylic acid, lecithin soybean and polysorbate, as well as water and salt.

Currently, there are several preparations based on azelaic acid on the Polish market, including: Acne-derm - 20% cream, Hascoderm - 20% cream, Skinoren - 20% cream and Skinoren - 15% gel. In these preparations, azelaic acid is present predominantly in a crystalline form, which significantly reduces the efficiency of its absorption. One way to increase the bioavailability of hydrophobic active substances in dermatological preparations is to increase their solubility by binding them to lipid aggregates, e.g. liposomes [Gregoriadis G, 1999, Liposome Technology, 2nd edition, CRC Press].

DE 4225697 discloses azelaic acid liposomal compositions containing liposomes. This publication does not disclose the pH of the prepared mixtures. The solubility of azelaic acid increases with the pH. Therefore, it should be expected that a formulation with a higher pH will contain more dissolved azelaic acid - more bioavailable azelaic acid, which, in combination with liposomes, should penetrate the stratum corneum to a greater extent, which should directly translate into the accumulation of azelaic acid in the skin . It has surprisingly been found that an increase in the solubility of azelaic acid can be achieved by selecting an appropriate pH range for the mixture of azelaic acid and liposomes.

Azelaic acid is a dicarboxylic acid that has two different dissociation constants: pKa1 = 4.55 and pKa2 = 5.41 [Brown, HC et al., In Braude, EA and FC Nachod Determination of Organic Structures by Physical Methods, Academic Press, New York, 1955]. Accordingly, the pH of the solution (preparation) will determine the degree of dissociation of azelaic acid and its physicochemical properties, such as the standard partition coefficient LogP (n-octanol / water), which characterizes the lyophilicity and affinity of the compound for stratum corneum. Figure 1 shows the structure of azelaic acid as a function of the pH of the solution.

The mixture of azelaic acid and liposomes according to the invention is characterized in that its pH is in the range from 4.0 to 7.4, in particular between 4.5 and 5.4.

Preferably the liposomes used have a net charge of 0.

Preferably the liposomes are composed of 1,2-dioleyl-sn-glycero-3-phosphatidylcholine (DOPC).

Preferably used liposomes have a net positive charge.

Preferably the liposomes are composed of 1,2-dioleyl-sn-glycero-3-phosphatidylcholine (DOPC) or

1.2-dioleyl-3-trimethylaminopropane (DOTAP).

Preferably used liposomes have a net negative charge.

Preferably the liposomes are composed of 1,2-dioleyl-sn-glycero-3-phosphatidylcholine (DOPC) or

1.2-dioleyl-sn-glycero-3-phosphatidylglycerol (DOPG).

The invention also relates to a pharmaceutical preparation in the form of an ointment, cream, gel, emulsion or suspension, which is characterized in that it comprises the mixture according to the invention.

The object of the present invention is to increase the solubility of azelaic acid by appropriate selection of the liposome composition and the conditions (pH) to enable the azelaic acid to be entrapped in the liposomes.

The measurements carried out using the Isothermal Titration Calorimetry (ITC) method show that the dissociation degree of azelaic acid is of key importance for the energetic of the interaction (change in the molar enthalpy of AH association) of this acid with liposomes, which is a measure of the amount of azelaic acid bound to liposomes. The largest change in the molar enthalpy of association with neutral liposomes composed of 1,2-dioleyl-sn-glycero-3-phosphatidylcholine (DOPC) was recorded for the amphiphilic form of azelaic acid (4.5 <pH <5.4). This value is four times higher than that obtained for the dissociated (pH> 5.4) and undissociated (pH <4.5) form of azelaic acid. Fig. 2 shows the dependence of the cumulative heat of the interaction of azelaic acid with neutral liposomes as a function of the degree of acid dissociation (pH

Solution). A similar dependence of the amount of azelaic acid associated with liposomes as a function of pH was demonstrated for positively and negatively charged liposomes. Fig. 3 shows the dependence of the cumulative heat of interaction of azelaic acid with positively charged liposomes, while Fig. 4 - with negatively charged liposomes as a function of the degree of acid dissociation (solution pH). The highest value of the enthalpy of association of azelaic acid with the model lipid bilayer was obtained for the amphiphilic form of this acid with a positively charged bilayer, thus showing the importance of the surface electrostatic charge of the bilayer. The introduction of 20 mole% of positively charged 1,2-dioleyl-3-trimethylaminopropane chloride (DOTAP) increased the molar association enthalpy change by 28%. The introduction of 20 mole% of negatively charged 1,2-dioleyl-sn-glycero-3-phosphatidylglycerol (DOPG) reduced the value of the molar change in association enthalpy by 6% compared to the neutral DOPC. Fig. 5 shows the dependence of the cumulative heat of the interaction of the amphiphilic form of azelaic acid with liposomes as a function of the surface charge of the liposomes and in the presence of salt ions, which shows the lack of influence of the ionic strength of the solution on the degree of association of azelaic acid with neutral liposomes.

As the results presented above show, the closing efficiency is the highest in the pH range where azelaic acid takes the amphiphilic form.

P r z k ł a d 1

To 1.00 ml of a 10 mM liposome suspension obtained by hydration of a dry lipid film with 10 mM phosphate buffer, pH = 4.0, 0.20 ml of a 5.00 mM azelaic acid solution in phosphate buffer, pH = 4 was added. 0. The values of molar changes in the enthalpy of association of azelaic acid with liposomes (AH) were determined by the isothermal titration calorimetry method. The results of the measurements showing the changes in the molar association enthalpy (AH) of azelaic acid with liposomes at pH = 4.0 are presented in Table 1.

Example DOPC [mm] DOTAP [mM] DOPG [mm] Resultant load and 10 0 0 feeding b 8 2 0 positive c 8 0 2 negative

T a b e l a 1

Example DOPC [mm] DOTAP [mM] DOPG [mm] AHass [kJ / mol] 1a 10 0 0 -0.8110.09 1b 8 2 0 -1.29 ± 0.05 1c 8 0 2 -2.7610.11

P r z k ł a d 2

To 1.00 ml of a 10 mM liposome suspension obtained by hydration of a dry lipid film with 10 mM phosphate buffer, pH = 5.0, 0.20 ml of a 6.40 mM azelaic acid solution in phosphate buffer, pH = 5 was added, 0. The values of molar changes in the enthalpy of association of azelaic acid with liposomes (AH) were determined by the isothermal titration calorimetry method. The results of measurements showing changes in the molar enthalpy of association (AH) of azelaic acid with liposomes at pH = 5.0 are presented in Table 2.

T a b e l a 2

Example DOPC [mm] DOTAP [mM] DOPG [mm] AHass [kJ / mol] 2a 10 0 0 -3.68 ± 0.12 2b 8 2 0 -4.70 ± 0.18 2c 8 0 2 -3.46 ± 0.19

PL 219 395 B1

P r z k ł a d 3

To 1.00 ml of a 10 mM liposome suspension obtained by hydration of a dry lipid film with 10 mM phosphate buffer pH = 7.4 was added 0.20 ml of 16.67 mM azelaic acid solution in phosphate buffer pH = 7, 4. The values of molar changes in the enthalpy of association of azelaic acid with liposomes (ΔΗ) were determined by the isothermal titration calorimetry method. The results of the measurements showing the changes in the molar enthalpy of association (ΔΗ) of azelaic acid with liposomes at pH = 7.4 are presented in Table 3.

Claims (8)

A mixture of azelaic acid and liposomes, characterized in that its pH is in the range from 4.0 to 7.4, in particular between 4.5 and 5.4. 2. The mixture according to claim The method of claim 1, characterized in that the liposomes used have a net charge equal to 0. 3. The mixture according to claim The method of claim 2, characterized in that the liposomes are composed of 1,2-dioleyl-sn-glycero-3-phosphatidylcholine (DOPC). 4. The mixture according to claim The method of claim 1, characterized in that the liposomes used have a net positive charge. 5. The mixture according to claim 1 The method of claim 4, characterized in that the liposomes are composed of 1,2-dioleyl-sn-glycero-3-phosphatidylcholine (DOPC) or 1,2-dioleyl-3-trimethylaminopropane (DOTAP). 6. The mixture according to claim 1 The method of claim 1, characterized in that the liposomes used have a net negative charge. 7. The mixture according to claim 1 6. The method of claim 6, characterized in that the liposomes are composed of 1,2-dioleyl-sn-glycero-3-phosphatidylcholine (DOPC) or 1,2-dioleyl-sn-glycero-3-phosphatidylglycerol (DOPG). Pharmaceutical preparation in the form of an ointment, cream, gel, emulsion or suspension, characterized in that it contains the mixture according to claim 1, 2 or 3 or 4 or 5 or 6 or 7.