Ana Aguilera ¹, Yilian Bermúdez ¹, Oscar García ², Rolando Páez ¹, Eduardo Martínez ¹

ABSTRACT

In order to obtain a procedure to quantify EGF by ELISA from semisolid formulations different buffers and organic solvents were used, having different polarities and miscibility with water. We studied it from oil-in-water (o/w) cream, water-in-oil (w/o), polyethylene glycol (PEG) ointment, and a jelly. Precision, accuracy and selectivity were determined and variation coefficients were less than 10% after different extraction method with a recovery of 95.54 -108.98%. According to results, it is possible to estimate the EGF concentration with high precision and reproducibility. No statistically significant differences were detected, when compared a placebo extraction solution with the ELISA standard solution demonstrating that placebo does not produce interferences in the quantification of the molecule. To determine the delivery of the active ingredient from different preparations we used a static diffusion cell system to evaluate the release and penetrability of the active ingredient throughout abdominal pig skin. Results showed that release and penetrability of EGF increased proportionally with the hydrosolubility of the vehicle. Therefore, the rate was obtained in jelly, PEG > o/w > w/o. Additionally, PEG ointment and o/w cream allowed the highest distribution of labeled ¹²⁵I EGF in the epidermis and dermis and receptor medium.

Keywords: Epidermal growth factor, release, quantification.

RESUMEN

Palabras clave: Factor de crecimiento epidérmico, liberación, cuantificación.

INTRODUCTION

Wound healing is a complex, integrated and critically orchestrated cascade of events to restore the skin barrier function (1). Growth factors and their receptors regulate key events in this process. Therefore, an effective exogenous delivery of peptides and proteins would be crucial to enhance wound healing (2). One of these factors, the epidermal growth factor (EGF) is a single 53 aminoacid polypeptide, originally isolated from submaxillary glands in mice (3). Topical administration of this macromolecule accelerates regeneration of skin in partial thickness wounds, second-degree burns in pigs or dermatome wounds in humans (4). Successful re-epithelialization was obtained in patients with chronic wounds (5).

However, the EGF administration should meet some pre-requisites to achieve therapeutic efficacy and significantly enhance wound healing. Some of these pre-requisites are prolonged local bioavailability of the active growth factor (6), treatment regimen (7), effective dose (8) and use of a protease inhibitor (9), among others. In this sense, Bukley et al. demonstrated that prolonged exposure to EGF enhances wound healing by its sustained release from pellets implanted in subcutaneous sponges, increasing the formation of granulation tissue when compared to daily injections of EGF into sponges or its topical application to surgical incisions in saline (6). Additionally, formulating EGF in creams also provides a method to release this growth factor to partial thickness wounds for prolonged periods of time. Clinical trials using skin-graft-donor sites have been conducted to determine whether EGF would accelerate the rate of epidermal regeneration in humans. These studies have confirmed that EGF accelerates wound healing (5).

The aim of this study was to evaluate the release profile and the in vitro penetrability of EGF for four semisolid formulations containing it as an active pharmaceutical ingredient.

MATERIALS AND METHODS

Human recombinant EGF (hrEGF) with more than 95% of purity, monoclonal antibodies CB-EGF-1 mouse and CB-EGF-2 peroxidase-conjugated antibody were purchased by the Center for Genetic Engineering and Biotechnology, Havana, Cuba.

Carbopol 940 was purchased from Noveon, Cleveland, USA.

EDTA, PEG 350 and 400, Cetyl alcohol, gelatin, white wax, cholesterol, stearyl alcohol, Chloroform, Carbon tetrachloride, Diethyl ether were purchased from Merk, Darmstadt, Germany.

Composition of the semisolid formulations

Hydrophilic jelly was elaborated using Carbopol 940, EDTA like enzyme inhibitor, water and preservatives.

A polyethylene PEG ointment was elaborated using PEG 350 and 4000 MW.

Emulsion o/w was elaborated using cetyl alcohol, EDTA, ionic emulsifiers, humectation agent, stabilizer, preservatives and water.

Emulsion w/o was elaborated with petrolatum, white wax, cholesterol, stearyl alcohol and water.

Manufacturing processes for semisolid are conventional method for PEG ointment and jelly. In the case of emulsions, fusion method was used (10).

Different organic solvents and buffer solutions were used to extract the active ingredient, depending on the features of the semisolid preparations (Table 1).

Water or PBS were selected as extraction solvents, since the jelly and PEG ointment are water-soluble formulations. Tween 20-0.02% was also used for protein stabilization (11). The w/o and o/w formulations required breaking the emulsion to separate the oil phase, and was achieved by adding chloroform, carbon tetrachloride or diethylether.

EGF was quantified by using an ELISA assay for jelly and PEG ointment after the total dissolution of 0.5 g of these semisolid formulations in either buffer or water.

The extraction of EGF from the other two formulations was accomplished by dissolving 0.5 g of each formulation in the organic solvent given in table 1, followed by adding a buffer solution or water to this suspension.

Each sample was centrifuged for 20 min at 1000 g using a Hitachi, HIMAC SCT5BA centrifuge. The aqueous phase was collected to quantify EGF by ELISA. Accuracy, precision and selectivity of the extraction method were also determined, as described below.

Accuracy determination

Accuracy was determined using semisolid formulations at different concentrations of EGF: 5 µg/g, 10 µg/g, 15 μg/g and 20 µg/g. Six replicas of each concentration were prepared, and the EGF was extracted, as explained above. Linear regression and the null hypothesis slope were equal to 1, and the intercept equal to 0 was tested. Accuracy was determined as the recovery of EGF after its extraction and quantification by ELISA, as compared with the optimal value of 100%.

Precision determination

Specificity analysis

Specificity is the ability to asses unequivocally the analyte in the presence of components which may be expected to be present. Statistical comparison, using a Student’s t test, between the solution obtained after achieving the extraction of placebo and ELISA standard solution (PBS and 0.5% w/v casein) was performed. These samples were analyzed by ELISA.

EGF release profile measurement

To determine the release profile of EGF, we used a diffusion cell described according to Estévez et al. (15). A small teflon-covered magnet, rotating at 600 rpm was used to stir the receptor solution. The receptor chamber was kept at 37 ºC during the experiment. A cellulose acetate membrane with pore diameter of 0.2 mm was used to place the semisolid formulation. It was previously soaked in the receptor solution (phosphate-buffered saline 1X, PBS 1X) during 24 h at 4 ºC to diminish the lag time period.

One gram of the semisolid formulation containing 10 mg of EGF was placed in the receptor compartment, and 150 µL of the buffer solution were sampled at the initial time and after 0.5, 1, 2, 4, 6, 8 and 24 h. After sampling, the same volume of the buffer solution was replaced in the receptor compartment to maintain the total volume. Each sample was further analyzed in duplicates by ELISA.

Penetrability assay

A diffusion cell, assembled with pig abdominal skin punches, was used instead of cellulose acetate membranes, to determine the penetrability on EGF. The skin punches were incubated on receptor medium containing 0.025% gentamicine and PBS 1X during 4 h, at 4 ºC. The skin was placed inside the cell with the epidermis facing the donor chamber. The EGF used in this experiment was labeled with ¹²⁵I following essentially the chloramines-T method (16).

The percentage of EGF in the receptor medium, epidermis, dermis and semisolid was determined by measuring the remaining radioactivity in a gamma counter Wizard, Model 1470, Wallac.

ELISA

After washing, 100 mL of a standard EGF, control and unknown samples previously diluted in PBS, and 0.5% w/v casein were added to each well. Plates were incubated for 1 h at 37 ºC and then washed.

Eighty microliters of a peroxidase-conjugated antibody against the recombinant human EGF (CBEGF2) were added to the plates and incubated for 1h at room temperature. After that time, plates were washed six times. The reaction was developed for 10 min by adding 100 mL of the substrate solution containing 5.5 µL 30% H₂O₂; 5.5 µg O- phenylendiamine and diluted in 0.1 M sodium citrate, pH 5.0. The reaction was stopped by adding 50 µL of 2 M sulfuric acid. Absorbance was measured by using a microtiter plate analyzer Multiscan, Merck-Sensident Scan, Darmstadt, Germany at 492 nm. Positive control and unknown samples concentrations were calculated by interpolating absorbances values in a standard curve generated, when absorbance was plotted against EGF concentration (17).

Statistical analysis

All measurements were expressed as mean ± standard error. Statistical differences were tested using the Student’s t test with a significance of p < 0.05.

RESULTS AND DISCUSSION

EGF determination in semisolid formulations

EGF has been widely used in the treatment of damaged skin in semisolid formulation (6), because of that it is necessary to establish a method to extract EGF for quality control in pharmaceutical research and it is mandatory by many regulatory agencies (18). One of the most commonly used methods to estimate the concentration of EGF is the enzyme-linked immunosorbent assay. In order to obtain a procedure to quantify EGF from semisolid formulations different buffers and organic solvents having different polarities and miscibility with water were used and then EGF was quantified by ELISA.

Figure 1 shows the recovery percent for each semisolid formulation after different extraction procedures described in table 1. The EGF recovery percent was over 90% for PBS 1X-Tween 20- 0.02% solution for jelly and PEG; water/chloroform and water/tetrachloride solutions for o/w; and water/chloroform, water/ tetrachloride, water/diethylether solutions for w/o.

The obtained results were previous step for further experiment.

Accuracy determinations

Accuracy was calculated as the percent of EGF recovery from the selected semisolid formulations, irrespective of the extraction method used. Results obtained are showed in table 2. Extraction methods for PEG and Jelly in PBS 1X-Tween 20-0.02%, o/w in carbon tetrachloride and w/o in chloroform didn’t show statistically significant differences (p > 0.05) in slopes and intercepts respect to values 1 and 0, respectively. Their respective R² values were 0.9893, 0.9908, 0.9845 and 0.9973. Each recovery percent was compared (n = 6) to the total amount of EGF formulated by a Student´s t test, and no significant differences were detected. The recovery range was from 95.5 to 108.9 which confirmed the reliability of the assay. All these indicated that both the extraction and quantitative determination methods were accurate. Similar results were not obtained for the rest of the formulations tested. Our results indicated that ELISA assay and the selective method for EGF extraction fulfill the criterion for measuring it in a semisolid formulation.

Precision determinations