**2. Materials and methods**

### **2.1 Drugs and reagents**

Vaniqa 11.5% cream (11.5%, Vaniqa, Batch: 838225): Each gram of cream containing 115 mg of Eflornithine hydrochloride monohydrate as the active ingredient, 47.2 mg of cetostearyl alcohol, 14.2 mg of stearyl alcohol, 0.8 mg of methyl parahydroxybenzoate, and 0.32 mg of propyl parahydroxybenzoate (Almirall Limited, Harman House, UB8 1QQ, UK).

Armenicum paste (Arpimed CJS, Batch: 0019): Each gram of paste containing iodide anion (J2) – 5.93%, calcium iodide (KJ) – 8.89%, lithium chloride (LiCl) – 0.15%, dextrin (C18H32O16) – 74.13%, polyvinyl alcohol (C2H4O) – 2.23%, sodium chloride (NaCl) – 6.67%, and water – 2%.

To get the 100 g "Eflornithine–Armenicum" composition, add 85 g of Armenicum paste and 15 g of Eflornithine hydrochloride monohydrate (Eflornithine hydrochloride is 13.9 g) to the composition of the container. Stir the mixture at room temperature for 10–15 minutes before using.

The standard used for the Eflornithine hydrochloride monohydrate was the RS USP standard (series R06840). For analytic method validation, the L-ornithine hydrochloride was obtained from Sigma (St. Louis, MO, USA; product A-4571, Batch: 19/L), methanol, high-performance liquid chromatography (HPLC) gradient was obtained from grade Carl Toth GmbH + Co (Art. 7342.1), acetic acid HPLC class was obtained from Carl Roth GmbH (Art. 3738.3), high purity (+99%) acetone was

obtained from Carl Roth GmbH (Art. No. 7328.2), and high purity (+99%) dansyl chloride was obtained from Sigma-Aldrich (product –d2625).

1-Heptansulfonic acid sodium salt monohydrate was obtained from Carlo Erba (France Batch No: V8A553138I), distilled water for HPLC (Part No. 5062–8578), Hewlett-Packard, and 0.45-μm nylon filter was obtained from Carl Roth Germany (Carl Roth Germany, Art.7330.1). All chemicals were of analytical grade, and all solvents were of HPLC grade.

### **2.2 Study animals**

A total of 72 non-breed white male rats, bred by the Institute of Fine Organic Chemistry of the National Academy of Science, Yerevan, Armenia, were used in the study. All animals were clinically examined upon arrival and those that showed signs of abnormality or disease were excluded. Animals were kept in the animal house for 10–15 days prior to the commencement of the study under a 12 h/12 h light/dark cycle at 25 to 27°C and 60 to 65% humidity and were offered humidity and were offered standard rat chow ad libitum. Unfit animals were replaced prior to the start of the study, and no animals were replaced after the study began. The initial weights of the study animals were in the range of 160–200 g.

Care of rats and all interventions were performed according to the PHS Guide for the laboratory animals [10] in accordance with requirements of the YSMU Ethics Committee. All experiments were performed during the light phase of the cycle.

### **2.3 Experimental design**

A 4 cm<sup>2</sup> wound was incised on the inner surface of the hind leg of animals by the model of aerobic wounds created according to Oganesyan S.S. methodology [11]. After this trauma, the musculoskeletal tissues were compressed twice by the Kocher clamp. The preparations were investigated 5–7 minutes after the onset of the injury: Eflornithine paste or "Eflornithine–Armenicum" composition was applied to the entire wound surface of the animals, which covered all the upper wounds.

Development of aerobic wounds in animals and further operations were performed under ether anesthesia. The animals were kept in isolation without bandages after the wound was healed.

The animals were divided into 2 groups given below:


At 0, 2.0, 4.0, 8.0, 12.0, and 24.0 h after the application of investigated preparations, six animals from the both group were sacrificed and blood samples taken.

Following decapitation with a laboratory guillotine, blood was collected from each animal in separate heparinized centrifuge tubes that were centrifuged at 600 g for 15 min in order to obtain blood plasma. An aliquot of plasma (1.0 ml) was taken for Eflornithine assay and the remainder of the sample was conserved by closing the centrifuge tube with a cap and storing it in a freezer for 1–3 days prior to further assay. On the day of the assay, plasma was removed from the refrigerator and stored at room temperature for 1 hour.

#### **2.4 Racemic quantification of Eflornithine**

Racemic Eflornithine was quantified using precolumn derivatization, followed by high-performance liquid chromatography (HPLC) and ultraviolet (UV) detection according to published methods [8, 9], modified as described below. The HPLC system consisted Shimadzu LC/UV/MS instrument (LC-20 AD/T, Shimadzu Corporation, Kyoto, Japan) consisting of an autosampler (LC-20 AD/SIL-20A), a UV-diode array detector (SPD-M20A IVDD), and data acquisition and analysis software (Lab Solutions, Version 3.40.299. Shimadzu) set at 330 nm.

Plasma samples (0.5 ml) were precipitated with ice-cold methanol (3 ml) containing an internal standard (DL-4-amino-3-hydroxybutyric acid) at a concentration of 20 ng/ml. The samples were placed on a vortex mixer for approximately 10 s, centrifuged for 10 min at 12,000 g, and thereafter kept at 37°C for 10 minutes. The supernatants were transferred to new tubes and evaporated to dryness at 65°C under a gentle stream of air. The dried samples were dissolved in 100 μl phosphate buffer (0.1 M; pH 7.5). The derivatization mixture was prepared daily by mixing o-phthalaldehyde (20 mg), ethanol (1 ml), nitriloacetic acid (4 mg), mercaptoethanol (100 μl), and 10 ml phosphate buffer (0.1 M; pH 7.5).

Prior to injection, the samples were mixed by adding two 50-μl volumes of derivatization mixture (i.e., o-phthalaldehyde) to the 48-well-plate autoinjector.

The temperature for the autoinjector was kept constant at 20°C. The plasma samples containing the derivatization mixture were programmed to stand in the autoinjector for 2.00 min prior to injection. Eflornithine was separated on a Chromolith Performance RP-18e 100-mm by 4.6-mm-ID column (VWR International, Darmstadt, Germany) protected by a ChromSep Guard SS 10-mm by 2-mm-ID column (Varian, Palo Alto, CA), using a gradient program. Mobile phase A consisted of 92% phosphate buffer (0.1 M; pH 7.5), 5% methanol, and 3% acetonitrile. Mobile phase B consisted of 80% methanol, 10% acetonitrile, and 10% water. The flow rate was set to 2 ml/min, using the following gradient program: t = 0 to 6.75 min, linear decrease of A from 80–40%; t = 6.75 to 8.0 min, 40% A; and t = 8.0 to 10.0 min, linear increase of A from 40–80%. The typical retention times for the internal standard and Eflornithine were 5.5 ± 0.3 min and 7.3 ± 0.2 min, respectively.

Calibration curves, constructed using Eflornithine standards, were linear in the range 1.00–10.0 ng/ml with correlation coefficients (r) of 0.9991, for the target signals at 330 nm. The limit of detection (at a signal/noise ratio of 3) was 0.80 ng/ml and the lower limit of quantification (LLOQ ) was 1.5 ng/ml. The LLOQ for racemic Eflornithine was set to 1.5 ng/ml at which level precision and accuracy were < 12%. The experimental plasma samples for method validation were prepared at three concentrations (1.5, 3, and 6 ng/ml) and were analyzed in duplicate at each level during the analytical runs to ensure that experimental samples were accurately and precisely determined. Validation experiments demonstrated that the accuracy was 96.9 ± 3.5%, the precision (coefficient of variation) 3.6%, and the recovery 93.94 ± 2.9%.

#### **2.5 Pharmacokinetic analysis**

The pharmacokinetic parameters were calculated using Kinetica 4.4.1 software (Thermo Electron Corporation, 2004, USA).

The measured parameters were as follows: maximum plasma concentration (Cmax, ng/ml); elimination rate constant (kel, h<sup>−</sup><sup>1</sup> );

elimination half-life (t1/2, h);

area under concentration versus time curve extrapolated to infinity (AUC0-∞, hng/ml); absorption rate constant (ka, h<sup>−</sup><sup>1</sup> );

the time to Cmax (tmax, h).

#### **2.6 Statistical analysis**

Statistical analyses were performed using GraphPad PRISM software (version 2.0, 1996; GraphPad Software, San Diego, USA). The statistical significance of differences between the pharmacokinetics profiles of Eflornithine released from Eflornithine cream and "Eflornithine–Armenicum" composition over time were assessed using two-way between/within ANOVA wherein an interaction effect indicates a different response over time between the two dosage forms.
