INTRODUCTION

Cinnarizine (CNZ) is a piperazine derivative histamine H1-antagonist and a selective calcium channel blocker drug.^1,2 It is commonly prescribed for peripheral and cerebral disorders, vertigo, tinnitus, nystagmus, motion sickness and Ménière’s disease. There are only oral formulations in the pharmaceutical market of CNZ. The oral bioavailability of CNZ is low and variable. Many side effects of CNZ have been reported. Side effects of CNZ range from mild to quite severe. Its more common side effects are drowsiness and blurred vision, sweating, dry mouth, headache, skin problems, lethargy, gastrointestinal irritation, hypersensitivity reactions, muscle rigidity and tremor. CNZ can easily pass blood-brain barrier and it displays a sedative activity. Thus, its use by pilots and aircrew who must be dependably alert due to increased levels of drowsiness caused by the medication, is generally limited. Long-term CNZ therapy may cause weight gain, depressive conditions and several extrapyramidal syndromes, including tremor, acute and chronic Parkinsonism. CNZ can cause a tardive dyskinesia similarly to neuroleptic agents.

An alternative route to oral administration can provide an effective drug therapy. The transdermal route can be stated as one of the most reliable routes of application. Transdermal dosage forms are an alternative for the delivery of actives that have low oral bioavailability and systemic side effects. Moreover, transdermal delivery allows for the avoidance of the first-pass metabolism. There are various strategies to accelerate the drug passing through the skin. Thus, immediate and moderate action can also be observed. Penetration enhancers are required to enhance permeation through the skin by different penetration mechanisms for optimization of well-formulating topical products. Thus, to obtain an efficient treatment can be provided via the transdermal route. Penetration enhancers essentially improve transdermal delivery of both lipophilic and hydrophilic actives by decreasing barrier resistance of the skin.^3,4 Polyols, fatty acids and terpenes are commonly used as penetration enhancers. Diethyleneglycol monoethylether [Transcutol^® (Tc)], propylene glycol (PG), glycerol (Gl), oleic acid (OA) and limonene (L) are some of the most generally used penetration enhancers. They can carry drug delivery further through the skin displaying different mechanisms, through upper layers of the skin, mainly the stratum corneum. Tc and PG alter thermodynamic activity of permeants in their vehicles after permeating through tissues themselves at first. Afterwards, permeants diffuse into the skin by modification of driving forces for diffusion.^5,6,7 The activity of PG is also pronounced to result from solvatation of a-keratin within the stratum corneum, herewith promoting permeation by reducing drug-tissue binding. L promotes the permeation of lipophilic and amphiphilic penetrants by increasing their diffusion in the stratum corneum.^8,9 OA, a long-chain fatty acid, enhances percutaneous drug absorption by decreasing the phase transition temperatures of the skin lipids. A polar head group attached to the alkyl chain of OA conducts its potential enhancement function. However, Gl displays its penetration enhancing effect when along with water.¹⁰

In this study, it was prepared topical formulations of CNZ to overcome side effects caused by oral administration of the drug and to provide an alternative therapy in Ménière’s disease and motion sickness. For this purpose, effects of various traditional vehicles and penetration enhancers on the permeation of CNZ were investigated. Topical formulations based on a hydrogel (G), o/w emulsion (E) and oleaginous cream (OC) were prepared and their physical characteristics were determined. Penetration and permeation of CNZ through the stratum corneum and skin were investigated with an ex vivo study. This study was conducted on the abdominal skin of Wistar Albino rats since the rat skin can be used as a model for investigation of transdermal drug delivery through the human skin as reported in earlier studies. In vivo and ex vivo tests on rats have been demonstrated that can be used for searching properties required from actives and/or vehicles for skin delivery.^11,12,13,14

MATERIALS AND METHODS

CNZ was kindly provided from Nobel Ilaç San. ve Tic. A.Ş., Turkey.  Hydroxypropyl methylcellulose (Methocel^TM K15M) (HPMC) was kindly provided by Colorcon (Turkey). PG, OA, polyethylene glycol (PEG 400), Gl and Tween^® 80 were purchased from Merck (Germany). Polyvinylpyrrolidone^® K90 (PVP K90), cetyl alcohol and liquid paraffine were purchased from Sigma-Aldrich (Germany). Stearic acid and glyceryl monostearate were purchased from Doğa İlaç Hammaddeleri Tic. Ltd. Şti. (Turkey). Tc was provided by Gattéfosse (France). All other reagents and chemicals were of analytical grade.

Preparation of topical formulations

The composition of base CNZ formulations (G, E, and OC) is presented in Table 1. Penetration enhancers (Tc, PG, Gl and OA) were added to these formulations (Table 2). L was also added to base formulations at the rate of 5%. However, they went to the phase separation or they lost their homogeneity within one week. Thus, they were excluded from the study. As an addition, Tc was confirmed to be incompatible with the base formulation OC.

Quantification of CNZ

The high performance liquid chromatography (HPLC) method was verified for analytical quantification of the drug in samples obtained during experiments. International Council for Harmonisation (ICH) guideline for the method validation procedure was considered for this purpose.¹⁵ Linearity, intra-day and inter-day precision, accuracy, recovery and specifity were determined for verification of the method. Each verification analysis was replicated 6 times.

For this purpose, a HPLC apparatus (Shimadzu LC-20AT) was equipped with an ultraiole/visible detector (Shimadzu SPD-20A) and autosampler (Shimadzu SIL-20A HT). The separation was carried out using a TC-C₁₈ column (5 µm, 4.6x250 mm) (Agilent Tech, Germany) at 40 ^oC. Samples were detected under 1 mL/min flow rate of acetonitrile: ammonium phosphate monobasic solution (pH: 4.5) (6:4, v/v) as the mobile phase at 253 nm. 0.24 mg/mL stock standard solution of CNZ in methanol was prepared to evaluate the linearity of the method under the selected conditions. Drug determination was carried out at six concentrations (4–24 µg/mL) for providing the calibration curve.

Solubility of CNZ in various mediums

The solubility of CNZ was determined in various media according to the method reported in USP XIX.¹⁶ 15 mL of the dissolution medium was placed in four 25-mL flasks for this purpose. A quantity of CNZ was added to each flask that was greater than the quantity expected to dissolve in the medium. Flasks were closed and they were fixed in a constant temperature water bath (Daihan Scientific, Korea) adjusted to 25±1^oC. The apparatus was maintained under 200 rpm continuous agitation. Dispersions were filtered through S & S⁵⁸⁹³ blue ribbon papers (2 µm pore size, Schleicher & Schuell, Germany) after 24 h agitation. Measured portions of clear supernatants were removed and the solubility of CNZ was determined with HPLC.

Partition coefficient

The partition coefficient of CNZ between isopropyl myristate and distilled water was determined using the shake flask method, following the guidelines of the European Chemical Bureau (European-Chemical-Bureau, Dir 92/69/EEC).

In vitro drug release of formulations

0.45 µm cellulose acetate membranes (Sartorius, Germany) were kept in the receptor phase, a physiological saline solution (PSS): PEG 400 mixture (6:4, v/v) over night. Membranes were placed between two halves of Franz-type diffusion cells with 3.15 cm² surface area and 33.2 mL volume containing the receptor phase. 1 g topical formulation was placed on to the membrane in the donor phase. The receptor phase was maintained at 37±0.5°C constant temperature during this study for 6 h. Samples were taken from the receptor phase at certain time intervals. Cumulative amounts of CNZ released (mg/cm²) determined by HPLC after samples were filtered through S & S⁵⁸⁹³ blue ribbon papers. Six replicates were conducted for each formulation. Drug release profiles were obtained by plotting cumulative amounts of the drug as the function of time and release profiles were evaluated  using different kinetic models (zero order, first order and Higuchi square-root model).^17,18 The exponent value (n) of the Korsmeyer-Peppas kinetic model was considered for specifying drug release mechanism well un-known or for more than one type of release mechanisms comprised.

Ex vivo skin penetration and permeation studies

2.5-3 months aged male Wistar Albino rats (200-250 g) were provided from Aziz Sancar Institute of Experimental Medicine. The experimental protocol the Local Ethical Committee approved the experimental protocol of Animal Experiments (17.12.2013, no: 2013/131). Animals were housed in plastic cages at 22±1°C and 60±1% humidity under 12 h light-dark cycle. They were given standard laboratory diet and tap water ad libitum. Precisely shaved full-thickness abdominal rat skins were taken after they were sacrificed for ex vivo skin penetration and permeation assessments. The underlying fatty tissue was removed with  blunt dissection without damaging the epidermal surface. Skins were placed between two halves of Franz-type diffusion cells. 1 g formulation was applied on the skin in the donor chamber of cells. PSS: PEG 400 (6:4, v/v), was used as the receptor phase. This study was continued for 6 h at 37±1^oC constant temperature. The cumulative amount of CNZ permeated was verified in samples collected from the receptor phase at predetermined time intervals by HPLC. Three replicates were conducted for each formulation. The cumulative amount (Q_n, mcg/cm²) of CNZ permeated through the skin was calculated and cumulative drug amounts were plotted as the function of time (t, h).^19-21 The steady state flux of the drug (J_s, mcg/cm²/h) was ascertained from the slope of linear part of plot using the linear regression analysis (r>0.99) and then the efficiency of the penetration enhancers were determined.

The penetration of CNZ was assayed through the skin. A tape stripping study was conducted. For this purpose, abdominal rat skins over receptor chambers of Franz-type diffusion cells were used. Excess formulation in contact with the stratum corneum was carefully expunged using cotton swabs.²² Circular PVC tape strip sticking plaster pieces in 1 cm semidiameter (Ve-ge^®, İzmir, Turkey) was applied with a light pressure over the diffusion area. Then, it was removed with forceps. The first two strips were thrown away, because they collected residue of the formulation within crevices of the skin surface. The next 10 sticking plaster pieces were then applied using uniform firm pressure to obtain the formulation residue deposited within the skin tissue. They were then removed with uniform force rapidly using forceps. All tape strip sticking plaster pieces were collected in a 25 mL flask for extracting the drug content. For extracting CNZ, 10 mL ethanol was added to flasks and all flasks were tightly closed. They were fixed in a water bath at 25±1^oC. The apparatus was adjusted to 160 rpm continuous agitation for 24 h. Flasks’ contents were then filtered through S & S⁵⁸⁹³ blue ribbon papers. Measured portions of clear supernatants were removed from each flask for determination of CNZ content by HPLC. Subsequently, the solubility constraint (σ_sc) of CNZ in the stratum corneum was also calculated [log σ_sc: 1.911 (10³/melting point as Kelvin) - 2.956].²³ The melting point of CNZ was obtained by differential scanning calorimetry (DSC) analysis. For this purpose, a DSC apparatus (Universal V4.5A TA Instruments, U.S.A.) was employed. 9.7-mg sample was weighted into aluminum pans of the apparatus and heated with 10^oC/min heating rate under 50 mL/min N₂ flow. Thermogram of the sample was obtained indicating its melting point and enthalpy.

Statistical analyses

Drug release and permeation profiles of the formulations obtained from in vitro and ex vivo experiments and data obtained from the tape stripping experiment were compared using One-Way ANOVA test and subsequent Tukey post hoc pairwise tests. The Minitab^® 18 Statistical Software was used for this purpose by setting the significance level as a: 0.05.

RESULTS AND DISCUSSION

Analytical quantification

The analytical quantification of CNZ by HPLC was verified according to the instructions of the ICH Tripartite Guideline.¹⁵ The representative linear equation was A=aC + b, where A is the absorbance, a is the slope, C is the concentration and b is the intercept. The regression equation was A=81417.9C+3405.3 (correlation coefficient, r=0.9999). The retention time of CNZ was found as 5.8 min (Figure 1). Limits of detection and quantification of the quantification method were determined as 5.929 ng/mL and 17.968 ng/mL, respectively. Relative standard deviations for accuracy, intra-day and inter-day precision of the methods were below 2%. The recovery of CNZ was found to be 99.87±0.06-100.74±0.03%. Chromatograms of the receptor phase and placebo base formulations demonstrated the specifity of the method (Figure 1B-E).  Chromatograms of the formulations containing penetration enhancers (TC, PG, GL and OA, individually at the 5% rate in the formulations) were also verified the specificity of the method (they are not presented). Peaks of ingredients in the formulations were observed not to interfere with the drug peak.

Solubility of CNZ

The solubility of CNZ in various media at 25°C are represented in Table 3. PSS:PEG 400 mixture (6:4, v/v) was decided to be used as the receptor phase since the solubility of CNZ was found to be the highest in it.

In vitro drug release of formulations

To achieve the sink condition, the receptor phase must have a high capacity to dissolve or carry away the drug. An acceptable sink condition has been reported to be one where the maximum concentration of the drug in the receptor phase reached during the experiment does not exceed 30% of its maximum solubility in the receptor phase.²⁴ It is provided in a volume of the medium that is at least 3-10 times the saturation volume. The solubility of CNZ at 37±1°C was also determined and it was found to be 2.352±0.012 mg/mL. Thus, the volume of the receptor phase allowed to maintain the sink condition.

Permeation characteristics of a drug through the skin can’t be judged with in vitro drug release experiments. But it avail researchers to reckon some reasons for low drug penetration rate, including the affinity of the drug to the vehicle.^25,26 CNZ was confirmed to display the highest affinity to OC compared to G and E (p<0.05). The emulsion formulation E was confirmed to display the highest drug release rate among base formulations up to 6^th hours (p<0.05) (Figure 2, Table 4). G (0.293 mg/cm²/h) and OC (0.151 mg/cm²/h) respectively, followed E (0.326 mg/cm²/h). It was determined that the incorporation of penetration enhancers to the base formulations led to a significant increase in drug release rate of the vehicles in the case of formulations E and G (p<0.05) while an opposite situation was occurring for OC with all penetration enhancers. Tc and Gl addition to formulation E and PG addition to formulation G provided the highest release rate of drug from vehicles (p>0.05). Due to the lipophilic character of CNZ resulted in its retention in the base OC formulation.²⁷ Moreover, the incorporation of penetration enhancers (PG, Gl and OA) to OC statistically insignificantly changed the drug release rate and slower drug release profiles were obtained from formulations OC-OA, OC-Gl, and OC-PG (p>0.05) compared to the emulsion and hydrogel formulations. Thus, it was affirmed that increase in the solubility of the drug in the vehicle resulted in slower drug release.²⁵ Emulsion and hydrogel based formulations displayed anomalous transport of drug release because of kinetic modeling (Table 4). This phenomena can be attributed to two mechanisms conducted drug release, diffusion and polymer relaxation in hydrogel formulations or drug release free from concentration in emulsion formulations.

Ex vivo skin permeation and penetration studies

CNZ permeation from topical formulations through rat skin was ascertained to involve in the polarity of the formulations and type of penetration enhancers as reported earlier studies conducted on skin permeation of lipophilic drugs.²⁷ Polar hydrogel structure provided the highest drug permeation rates among other vehicles (Figure 3, Table 5). This phenomena can be attributed to the high partition coefficient of CNZ (log P: 5.74±0.03 in isopropyl myristate/water). G significantly displayed the highest permeation rate followed by E and OC (p<0.05), respectively. Tc was found as the most effective penetration enhancer compared to PG, GL and OA for hydrogel based formulations (p<0.05). The highest drug permeation rate was obtained in formulation G-Tc (0.110±0 mg/cm²/h) followed by G-Gl (0.062±0.002 mg/cm²/h), G-PG (0.057±0.001 mg/cm²/h) and G-OA (0.050±0.001 mg/cm²/h). Although emulsion based formulations followed G formulations in the same penetration enhancer order, differences between emulsion and oleaginous cream based formulations were insignificant (p>0.05). Tc and PG possibly contributed to their own permeation through tissues and modified the thermodynamic activity of CNZ before modification of driving forces for drug diffusion as reported earlier.^19,28 Solvatation of a-keratin within the stratum corneum by PG was additionally be claimed to improve the permeation of CNZ by reducing drug-tissue binding. OA and Gl enhanced percutaneous absorption of CNZ by decreasing phase transition temperatures of skin lipids and displaying the occulusion effect on the skin, respectively.^19,29 All formulations were affirmed to reach the steady-state flux (Js) at the 1^st hour, except for the base formulation G (3^rd hour).

CNZ percent in the stratum corneum and the receptor phase that was determined for each formulation at the 6^th hour are presented in Figure 4. It was found that amount of CNZ accumulated in the stratum corneum was significantly higher than determined in the receptor compartment. As can be seen in the figure, G-Tc displayed the highest skin penetration of the drug followed by formulations G-Gl, G-OA, G-PG and, o/w emulsion based and oleaginous cream based formulations, respectively (p<0.05). Formulations displayed high drug accumulation in the stratum corneum were expected to exhibit continuous drug permeation of most of the retained drug by steady state flux in time.

The melting point of CNZ was found as 121.94^oC (121.16^oC onset melting temperature and 98.18 J/g melting enthalpy) according to its DSC thermogram indicating a sharp melting peak (Figure 5).

The solubility constraint (s_sc) of CNZ was calculated as 1.88 in the stratum corneum indicating the potential of this compound that forms a reservoir in the stratum corneum. Organic substances with high melting points and enthalpies have lower aqueous solubility in general since solvents cannot pass into the crystalline structure of such molecules to dissolve them.^30,31 Thus, an indirect relationship exists between the melting point and the solubility of a drug.³² In other words, a decrease in the melting point of a drug would lead to an increase in its solubility in the stratum corneum and consequently its penetration and then permeation through the skin.

CONCLUSION

Formulations that were studied in this research introduce various advantages over many transdermal drug delivery systems. They are suitable for large-scale production and cost-effective dosage forms produced with excipients used in pharmaceuticals and cosmetics for years. Ex vivo study conducted on rats gave information about the influence of the polarity of vehicles and penetration enhancer on skin penetration of CNZ. It was concluded that skin penetration increased as the lipophilicity of the vehicle decreased. The hydrogel formulation without a penetration enhancer provided about five times higher drug permeation compared to an o/w emulsion and oleaginous cream. Furthermore, when Transcutol^® was introduced to the HPMC hydrogel, it displayed the highest penetration enhancer activity. As a result, the HPMC hydrogel containing Tc can be suggested as a suitable carrier for CNZ intended to be used topically for relief of various conditions.

ACKNOWLEDGEMENTS

This study was supported by the Research Fund of Istanbul University (project number: 40188) and TUBITAK (The Scientific and Technological Research Council of Turkey) (grant number: TEYDEB 1649B031305845).

Conflict of interest: No conflict of interest was declared by the authors. The authors are solely responsible for the content and writing of this paper.