Materials and Reagents

Rapamycin powder (an active substance) was provided by Acofarma (Madrid, Spain). The rest of the excipients used in the preparation of the formulations were supplied by Guinama (Valencia, Spain). All reagents and solvents were provided by Scharlab (Valencia, Spain).

Formulation and Preparation

Galenic optimization of the topical formulation of rapamycin at 0.4% based on its excipients was performed. The most widely used classical formulation in the literature is petrolatum as a vehicle. However, other options were formulated based on previous studies.

Four different formulations were developed in the biological safety cabinet, all of them with a rapamycin concentration of 0.4% and with differences in their excipients. The resulting formulations were ointment, emulsion, gel, and liposomes (Table 1). Each formulation was packaged in a bottle protected from ambient light and stored in a cold room at 5 °C ± 3 °C.

Table 1 Composition of rapamycin formulationsChemical StabilityEquipment

An Agilent Technologies 1100 HPLC system (Agilent Technologies Inc., Waldbronn, Karlsruhe, Germany) with a quaternary pump, micro-vacuum degasser, autosampler, thermostatted column compartment, diode array detector, and Agilent Technologies ChemStation for LC 3D Software was used for the analysis.

Chromatographic Conditions

To determine the validity period of each formulation according to its chemical stability, an HPLC method was developed and validated. A C18 Kromasil column (150 mm × 4.6 mm, 5 μm) was used. The mobile phase consisted of a mixture of acetonitrile and water (75/25 v/v). The flow rate was 1 mL/min. The sample injection volume was 10 μL and sample temperature was 25 °C ± 3 °C. The column temperature was 50 °C and the analysis time was 15 min. Rapamycin detection was processed at 280 nm.

Method Validation

The analytical methods were validated according to ICHQ2R1 (International Consensus on Harmonization) [18]. Two calibration curves were developed: one using a mixture of acetonitrile, hexane, and water for injection (WFI) as a diluent (for the chemical stability of ointment, emulsion, and gel formulations) and the other using methanol (for the chemical stability of liposomes). The interday and intraday precision and accuracy of the methods were established using six concentration levels (0.025, 0.05, 0.075, 0.1, 0.125, and 0.15 mg/mL) in duplicate on three different days. The least squares method was used to evaluate linearity, calculating a regression line between concentrations and peak areas of the chromatogram.

Rapamycin Extraction

For the extraction of rapamycin from ointment, emulsion, and gel formulations, 0.1 g of cream was introduced in a glass tube with 4 mL of a mixture of acetonitrile, hexane, and WFI and kept in the vortex for 10 s. The samples were then centrifuged at 3500 rpm for 10 min. The resulting supernatant was removed, and 1 mL was aliquoted for HPLC analysis. For the extraction of rapamycin from liposomes, 0.100 g of cream was introduced in a glass tube with 4 mL of methanol. The samples were kept in the vortex for 10 s, 50 μL was aliquoted, and after 1/20 dilution with methanol, they were analyzed with the HPLC method.

Rapamycin Degradation

Rapamycin quantity per weighed formulation quantity (Q, mg/g) and percentage content of remaining rapamycin (%RC) in each formulation were determined by triplicate at times (t) = 0, 2, 7, 14, 21, 28, 42, and 56 days. T90 was established when %CR was ≤ 90%.

Physical Stability

All pertinent procedures to establish the physical stability of the formulations were carried out following the specifications of the National Drug Formulary [19] and the Guide to Good Practice in the Preparation of Medications in Hospital Pharmacy Services [17].

pH, uniformity, extensibility, absence of crystals, and absence of phase separations were evaluated on a transparent surface according to 3 levels: level 1, the least favorable, and level 3, the most favorable, for 56 days for each formulation.

Liposome Characterization

Only for liposomes mean particle size, zeta potential, and encapsulation efficiency (EE%) were determined by triplicate at t = 0 days. Mean particle size and zeta potential were determined using a particle size analyzer that uses the laser diffraction method (Malvern Instruments, Worcestershire, UK). The EE% was obtained via ultrafiltration, using Amicon® ultracentrifugal filters (Merck Millipore, Ireland) with a molecular weight cutoff of 10,000 Da. An aliquot of 500 μL of the liposomal formulation was added to the sample reservoir and centrifuged for 15 min at 14,000 g. Then, 50 μL was aliquoted in duplicate and, after 1/20 dilution with methanol, was analyzed with HPLC method to determine the concentration of free drug in the filtrate. The following equations were used for the calculations [13, 20]:

$$\mathrm=\frac-W_}}\times100$$

where Wt and Wf represent the total amount of the drug and the free amount of the drug, respectively.

Microbiological Stability

Culture samples in blood-agar media of each formulation were incubated at 37 °C in duplicate at t = 28 and 56 days, according to the microbiological control instructions of the National Drug Formulary.