Biomedicine & Pharmacotherapy 83 (2016) 771–775
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New effective azelaic acid liposomal gel formulation of enhanced pharmaceutical bioavailability skib , K. Karłowicz-Bodalskac, T. Hanc , S. Hanc E. Burchackaa,* , P. Potaczekb , P. Paduszyn skiego Str. 27, 50-370 Wroclaw, Poland Department of Microbiology and Medicinal Chemistry, Wroclaw University of Technology, Wyspian ska Str. 5, 51-423 Wrocław, Poland Research and Development Center NOVASOME, Olsztyn c Department of Industrial Pharmacy, Wrocław Medical University, Borowska Str. 211, 50-556 Wrocław, Poland a
A R T I C L E I N F O
Article history: Received 9 May 2016 Received in revised form 5 July 2016 Accepted 8 July 2016 Keywords: Azelaic acid Liposomal hydrogel Pharmaceutical formulation
A B S T R A C T
Azelaic acid is a naturally occurring saturated C9-dicarboxylic acid which has been shown to be effective in the treatment of comedonal acne and inﬂammatory acne, as well as hiperpigmentary skin disorders. The aim of the present study is to compare new developed liposomal hydrogel (lipogel) and commercially available product in terms of the active substance—azelaic acid bioavailability. Topical formulations were evaluated for physical parameters, such as pH measurement, organoleptic evaluation and liposome size analysis in lipogel formulation. In addition, studies were performed on in vitro antimicrobial preservation, stability and accumulation in the stratum corneum according to guidelines established by European Pharmacopoeia and International Conferences on Harmonisation. The new formula for liposomal gel with azelaic acid has the stability required for pharmaceutical preparations. Moreover, presented formulation F2 reveals a very high accumulation (187.5 mg/cm2) of an active substance in the stratum corneum, which results in opportunity to decrease of the API content to 10% in comparison to a reference formula: commercially available cream with 20% of azelaic acid. The study reveals that the ﬁnal formula of lipogel F2 with azelaic acid had acceptable physical parameters that showed that they were compatible with the skin and in addition this formulation passed stability studies. In vitro antimicrobial preservation studies showed that the formulated lipogel F2 showed strong antibacterial activity; thus, no preservatives were added to the ﬁnal composition of the preparation. The present study concludes that the formulated lipogel F2 with azelaic acid is stable, efﬁcient in antimicrobial preservation and reveals improved active substance bioavailability. ã 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Azelaic acid is a saturated C9-dicarboxylic acid (Fig. 1) which is produced naturally by certain yeast species (Pityrosporum ovale) living on normal, healthy skin. Presence of its small amounts on human skin helps to prevent the comedo production by inﬂuencing the ﬁnal ceratinisation process of hair follicles, therefore among topical pharmaceuticals, azelaic acid is well known by their anti-inﬂammatory and anti-bacterial properties towards acne bacteria (Propionibacterium acnes) infecting skin pores . Additionally, azelaic acid inhibits free radical production by neutroﬁls especially in melasma and postinﬂammatory hyperpigmentation , what makes it really effective agent in acne vulgaris
* Corresponding author. E-mail address: [email protected]
(E. Burchacka). http://dx.doi.org/10.1016/j.biopha.2016.07.014 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
and rosacea treatment, typically associated with skin discoloration. Clinical research revealed that the azelaic acid is well tolerated by most patients do not exhibit acute nor chronic toxicity and is non-teratogenic and non-mutagenic [3,4]. Can be administered to humans topically, orally, and in the form of the disodium salt by intra-tissue injection or infusion, intra-venously, intra-arterially, and intra-lymphatically, all without local or general ill effects . To maintain a constant level of required active pharmaceutical ingredient level in the infection site, which is one of the main problems of contemporary pharmacy, application of active pharmaceuticals ingredients in gel-formulation such as liposomal hydrogels (lipogels) or hydrogels are highly beneﬁcial. Such kind of gel-formula preparations exhibit a lot of advantages in clinical trials, where better bioavailability seems to be the most proﬁcient in terms of required active pharmaceutical ingredient (API) dose supplementation. Additional, but highly beneﬁcial effects of hydrogels for topical therapeutic usage are:
E. Burchacka et al. / Biomedicine & Pharmacotherapy 83 (2016) 771–775
purchased from AkzoNobel. The dimethicone was purchased from DowCrowing and the phospholipon 50 IP was procured from Lipoid. The sodium hydroxide was purchased from Chempur. 2.1. Preparation of the azelaic acid lipogel formulation Fig. 1. Chemical structure of azelaic acid.
moderate cooling sensation, once applied on skin and their ability to evaporation resulting with a drying effect on skin which is especially important in acne infection treatment . The essential functional component of liposomal hydrogels are liposomes—small vesicles composed of various type phospholipids, which have been used for many years to bring active ingredients into the skin [7–12]. They are commonly used as drug carriers controlling release of the medicinal agent, however alternatively, may provide a localized API deposit in the skin, thus minimizing systemic effects or they can be used for targeting delivery to skin appendages . The liposome-skin interactions and their effects on the skin permeability of the drug concentration depend on lipids constituted the liposome membrane and particle size of liposomes [13– 16]. To azelaic acid lipogel composition described in this report liposomes, obtained from soybean phospholipids have been chosen. This kind of liposomal formulations are able to essentially enhance the percutaneous absorption rate of API in the stratum corneum, without uncontrolled penetration of the skin, as it was reported for hydrocortisone-loaded liposomes, which applied topically, might act as a selective drug delivery system of therapeutic efﬁcacy and decreased adverse systemic effects . Moreover, an increased absorption of liposomes after transdermal application was not observed, as reported for corticosteroid, lidocaine, tretinoin, cyclosporin-loaded liposomes [18–21]. Obtained liposomal hydrogel formulations F1 and F2 containing azelaic acid as its active substance provides, as expected excellent API bioavailability and exhibits the stability required for such preparations which allows it to be adopted to various lipogel formulation based on azelaic acid for acne vulgaris, rosacea and skin hyperpigmentation disease treatment Due to this enhanced delivery efﬁcacy liposomal formulation allows to signiﬁcantly reduce the API concentration, to achieve the same therapeutic effect demonstrated by others commercially available formulations. Pharmaceutical effect of reduced concentration (10%) of azelaic acid in F1 and F2 formulations is fully comparable to the reference formula of Skinoren1 cream, containing approximately a double dose (20%) of the same active substance. 2. Materials and methods The size of the liposomes was calculated on a Zeta-sizer Nano ZS Malvern ZEN 3600. HPLC (High-Performance Liquid Chromatography) was performed on an Agilent 1200 system (Agilent Technologis, USA) equipped with ELSD (light scattering detector, Alltech 3300 ECSD) and Zorbax SB (C18) column, 75 4.6 mm (3.5 mm). pH was measured on an ELMETRON 0039/07 pH meter. The accumulation of API in the stratum corneum was estimated with use of a PERME GEAR USA V9-CB Franz Diffusion cell system. The phase separation was provided on Hettich Mikro 22R Centrifugal Devices. The formulation was prepared on a Stephan agitator UMC 5 Universal Machine. All commercially available starting materials, reagents and solvents were used without further puriﬁcation. The azelaic acid was purchased from Utikon. The carbopol was purchased from Noveon. The prophylene glycol was procured from Centrochem. The edetate disodium was
2.1.1. Formulation 1 (F1) The edetate disodium (EDTA) was dissolved in distilled water and stirred at room temperature for 5 min in a Stephan agitator. Then, carbopol was added and the mixture was stirred at room temperature for 1 h in a Stephan agitator under a vacuum to create a gel phase. In a separate container, prophylene glycol with phospholipon 50 IP was stirred at 40 C in a Stephan agitator to create organic phase. After 40 min, azelaic acid was suspended in a mixture and mixed at 40 C for 15 min. The gel phase was transferred to the organic phase with continuous stirring (Stephan agitator, T = 40 C, t = 15 min). Then, 15% NaOH was added and the mixture was mixed at 40 C for 20 min. Finally dimethicone was added and the mixture was stirred at 40 C for 20 min in a Stephan agitator. In the last step, an air was removed from the preparation under vacuum. 2.1.2. Formulation 2 (F2) The edetate disodium (EDTA) was dissolved in distilled water and stirred at room temperature for 5 min in a Stephan agitator. Then, carbopol was added and the mixture was stirred at room temperature for 1 h in a Stephan agitator under a vacuum to create a gel phase. In a separate container, prophylene glycol with phospholipon 50 IP was stirred at 40 C in a Stephan agitator to create organic phase. After 40 min, 50% of total weight of azelaic acid was suspended in mixture and mixed at 40 C for 15 min. The gel phase was transferred to the organic phase with continuous stirring (Stephan agitator, T = 40 C, t = 15 min). Then, 15% NaOH was added and the mixture was mixed at 40 C for 20 min. After that, the second portion of azelaic acid (50% of total weight) was added to mixture and stirred at 40 C for 15 min in a Stephan agitator. Finally, dimethicone was added and the mixture was stirred at 40 C for 20 min in a Stephan agitator. In the last step, under a vacuum,an air was removed from the preparation. 2.2. Size analysis of liposomes The liposomes were analyzed with a Zeta-sizer Nano S with a dynamic light scattering system. The polydispersity index (PI) was used to measure unimodal size distribution. A small PI value (<0.1) indicates a homogenous population, while a PI (>0.3) indicates a higher heterogeneity. To 0.7 g of the formulation, the 15 mL of Milli-Q water was added. Next, the mixture was stirred and centrifuged at 6000 rpm for 30 min at 25 C. Then, 2 ml of solution was ﬁltrated through a 1.2 mm membrane ﬁlter and measured using the Zeta-sizer Nano S apparatus. 2.3. In vitro release in the stratum corneum In vitro release studies were performed using a Franz diffusion cells system (PermeGear, Hellertown, USA). The skin patches were cut out from the dorsal area of a pig’s ear. They were physically removed from defrosted pig ears using a scalpel. The skin was then cut into smaller patches of approximately 1 cm in diameter, and hair was removed using scissors. The quality and intactness of each skin patch was checked by measuring impedance (kV/cm2) according to the method described in detail in the publication . Those patches for which the value of impedance was greater than or equal to 27 kV (MT 4090 LCR Meter, Motech Instruments, Tainan, Taiwan) were used for further tests. Round skin fragments were gently placed between the donor and receptor chambers of
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the diffusion cell so that the epidermis was facing the donor compartment. The receptor compartment was ﬁlled with PBS and the cells were then incubated at 32 C (which corresponded to the temperature of human skin) using an external water coat. After 30 min of conditioning of the pig skin with receptor solution, approximately 50 mg of the test formulation was applied onto the donor compartment of each cell using a plastic syringe. The receptor solution was continuously stirred throughout the experiment at 400 rpm by a magnetic stirrer. After 20 h of incubation, the diffuse cells were carefully disassembled. Transdermal permeation of azelaic acid was calculated on the basis of its concentration in the acceptor compartment. The accuracy of the calculations was conﬁrmed by determining the total recovery of azelaic acid from each compartment. Each experiment with Franz diffusion cells was performed using 9 cells for each formulation.
for 18–24 h. The culture of C. albicans was incubated at 20–25 C for 48 h, and the culture of A. brasiliensis was incubated at 20–25 C for 1 week. To reduce the microbial count to about 108 CFU/mL, a suspending ﬂuid containing 9 g/L of sodium chloride reagent was used for the bacteria and C. albicans cultures. To harvest the A. brasiliensis culture, a sterile suspending ﬂuid containing 9 g/L of sodium chloride reagent and 0.5 g/L of polysorbate 80 was used and this adjusted the spore count to about 108 CFU/mL. The agar medium was used for initial cultivation of the respective microorganisms in the inoculated products. The volume of the suspension of inoculum did not exceed 1% of the volume of the product. The criteria for evaluation of antimicrobial activity are given in Table 2 .
2.4. Quantitative analysis of azelaic acid
The appropriate amount of formulation and reference were added to tubes and centrifuged for 10 min at 24 C by Centrifugal devices (Hettich Mikro 22R) rpm.
Quantitative analysis of azelaic acid was performed using a high performance liquid chromatography technique (Agilent 1200 System). The mobile phase was MeOH:H2O (630:370 v/v) that contained acetic acid to adjust the pH to 3.6. The ﬂow rate was set at 1 ml/min and a Zorbax SB (C18) column, 75 4.6 mm (3.5 mm) was used. The Azelaic acid concentration was detected by a light scattering detector. The calibration curve was linear in the 37– 87 mg/ml concentration range. The method was validated for speciﬁcity, linearity, precision, accuracy, robustness, limit of detection and quantitation, according to ICH Q2(R1). The average accuracy of the method was 101.83% with RSD = 1.2%. The method was found to be selective, precise, accurate, and reproducible. The results were analysed using Statistica 12 program and statistical tests with conﬁdence level a = 0.05. 2.5. Stability studies The stability studies were carried out in long-term, intermediate and accelerated storage conditions. The formulations were kept at three different temperatures: 25 C, 30 C and 40 C, respectively. The absolute humidity was 60% RH 5, 65% RH 5, 75% RH 5, respectively. The physical evaluation, pH and microbial contamination determined after 6 months, were compared with the initial parameters (ICH Q1A(R2)). 2.6. Organoleptic evaluation of the formulation
2.9. Phase separation
3. Results This report compares novel liposomal gel and commercially available formulation with azelaic acid. The composition of formulations F1 and F2 is reported in Table 1. Table 2 provides the results and criteria for evaluation of antimicrobial activity, in terms of the log reduction in the number of viable microorganisms against the value obtained for the inoculum. The results of antimicrobial preservation tests formulation revealed that the use of preservatives is unnecessary in ﬁnal formulation F2 (Table 2). The antibacterial properties of azelaic acid are sufﬁcient for the production of lipogel with azelaic acid without the need for any preservatives presence in the formulation. In vitro antimicrobial preservation studies reveal that both liposomal gel formulas—F1 and F2 and also commercial product Skinoren had acceptable antimicrobial parameters. The liposomes in F1 and F2 formulations were analyzed by DLS method with use of Zeta-sizer Nano S system. The polydispersity index (PI) was used to measure unimodal size distribution. Obtained PI with aprox. Value of 0.2 indicates a homogenous population. The pH results are summarized in Table 3. The data reveal that pH of the developed formulation, such as F1, F2 are 4.99, 5.10, respectively. The results show that the formulations are compatible with the skin.
The formulations were inspected visually for their color, homogeneity, consistency and phase separation. 2.7. Measurement of pH The pH was measured using a pH meter, which was calibrated before each use with standard buffer solutions at pH 4, 7, 9. An electrode was inserted into the sample 10 min prior to taking the reading at room temperature. Temperature was compensate by thermocouple added to the pH device. The average pH result was calculated from three measurements . 2.8. Antimicrobial preservation The test for efﬁcacy of antimicrobial preservation was prepared according to European Pharmacopoeia . The surface of casein soya bean digested agar and Sabouraud-dextrose agar was inoculated with bacteria strains (Staphyloccocus aureus ATCC6538 and Pseudomonas aeruginosa ATCC 9027) and fungi (Candida albicans ATCC 10231and Aspergillus brasiliensis ATCC 16404), respectively. The bacterial cultures were incubated at 30–35 C
Table 1 Compositions of lipogels F1 and F2 with azelaic acid. Ingredients of lipogel F1 and F2 Gel Phase Puriﬁed water Carbopol Edetate Disodium Organic Phase Methyl Parahydroxybenzoate Prophyl Parahydroxybenzoate Prophylene glycol Phospholipon 50 IP Azelaic acid 15%NaOH Dimethicone Formulation
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Table 2 The results and criteria for evaluation of the antimicrobial activity of formulation F2. Log reduction
Bacteria Staphylococcus aureus ATCC 6538 Pseudomonas aeruginosa ATCC 9027 Criteria of acceptance Fungi Candida albicans ATCC 10231 Aspergillus brasiliensis ATCC 16404 Criteria of acceptance
*NI—no increased. Fig. 2. Accumulation of azelaic acid in the stratum corneum.
Evaluation of the lipogel with azelaic acid formulation F1 and F2 in comparison with the reference material 20% azelaic acid Skinoren cream is summarized in Table 3. The active substance content in of the ﬁnal concentration was 10%. The level of azelaic acid in the ﬁnal formulation described here twice lower than the reference preparation of 20% Skinoren with azelaic acid. The accumulation in stratum corneum of azelaic acid from formulation F1 and F2 (10% of AA) is signiﬁcantly higher (120.0 mg/ cm2 and 187.5 mg/cm2, respectively) than that of the reference 20% Skinoren (52.3 mg/cm2). Lower concentration of the active ingredient – 10% azelaic acid – in F1 and F2 formulations reveals improved pharmaceutical bioavailability when compared with that of the 20% Skinoren available at the pharmaceutical market (Fig. 2). Formulation F2 of the preparation was selected for a scale-up process. The main difference between formulations F1 and F2 was the way of adding the azelaic acid during the technological process. In F1 formulation, the azelaic acid was added in one dose to the organic phase. However, 50% of the azelaic acid was added to the organic phase and the other 50% of the azelaic acid was added to the formulation (before the dimethicone addition step) in the F2 formulation. Method F2 of process design enabled optimization and enhanced production yield. The stability studies on the F2 formulation were carried out in long term, intermediate and accelerated storage conditions (Table 4). The tests were prepared according to European Pharmacopoeia requirements and the results were compatible with European Pharmacopoeia and internal speciﬁcation acceptance criteria for new formula medicament to be introduced onto the commercial market.
The developed formulation described in this report was used to scale-up process from 1 kg to 30 kg. A Kates reactor was used for semi-production scale. Then, the process was optimized and the formulation F2 was selected for the next phase of scaling-up (scaleup process to 10% of commercial scale). However, the scale-up process of lipogel with azelaic acid is currently in progress and will be published separately. 4. Discussion From the present study, it was concluded that the composition and formulation of lipogel with azelaic acid reveals enhanced pharmaceutical bioavailability compared to reference 20% Skinoren cream. The formulated preparation showed acceptable physical properties, and hence, was compatible with the skin. In addition, the formulation satisﬁed long, intermediate and shortterm stability requirements according to European Pharmacopoeia indicating the physical and chemical stability of the product. Moreover, the formulation reveals acceptable results in antimicrobial preservation tests; thus, it does not require preservatives. Hence, the formulation is an efﬁcient API carrier. The bioavailability results of obtained formulations (F1 and F2) indicates that the quality of the product depends on technological process of formulations. Additionally, further investigation clariﬁed the importance of technological process on chemical release in the stratum corneum is currently underway and will be published separately in due course.
Table 3 Evaluation of the lipogel with azelaic acid formulation F1 and F2 in comparsion with reference material 20% skinoren. Performance analysis
good white, smooth, homogenous no evidence 5.02 52.3 6.25 ND* ND* acceptable
good white, smooth, homogenous no evidence 4.99 120.00 9.1 211.2 0.215 acceptable
good white, smooth, homogenous no evidence 5.10 187.5 11.3 211.3 0.244 acceptable
Phase separation pH Cumulative [mg/cm2] drug release in skin Size analysis of liposome [nm] PDI (polydispersity of liposome population) Antimicrobal preservation tests *
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Table 4 Stability studies on F2 formulation in accelerated test conditions.
Organoleptic properties pH Microbiology purity: General number of oxygen microorganism in 1 g Candidia albicans in 0.1 g Staphylococus auresus in 0.1 g Pseudomonas aeruginosa in 0.1 g
Results for F2 formulation 0 months
no more than 103 absent absent absent
<10 absent absent absent
<10 absent absent absent
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