Introduction

Tuberous sclerosis (TS) is an autosomal dominant rare disease, characterized by the multisystem formation of benign non-invasive tumors, called hamartomas and distributed in tissues and organs such as the brain, kidneys, lungs, heart, eyes, and skin [1]. The most prevalent skin condition in TS is facial angiofibromas (FA), which occur due to an alteration of the mTOR (mammalian target of rapamycin) pathway. These facial injuries imply an aesthetic and psychological problem [2, 3], and sometimes, its treatment is not prioritized due to the existence of more serious complications associated with this rare disease.

Multiple treatments have been established for these patients. Physical treatments like radiofrequency, electrocoagulation, cryotherapy, dermabrasion, and laser therapy [4] are invasive and painful and require anesthesia. The interest of mTOR inhibitors focuses pharmacological treatment on everolimus and sirolimus, and recent publications place topical rapamycin (sirolimus) as the most appropriate alternative [5].

We found multiple studies with topical rapamycin in the literature, including four randomized clinical trials [6,7,8,9], and all of them provide favorable data on effectiveness and safety. However, high variability between studies in terms of concentration, vehicle, posology, duration of treatment, and effectiveness assessment forces us to study several formulations to choose the most appropriate.

Several authors have studied different formulations other than the classic formulation (an ointment based on petrolatum) with the aim of achieving a better appearance and consistency of the formulation in order to improve patient compliance with treatment.

Bouguéon et al. [10] studied the physico-chemical stability of a cream formulation with a commercialized excipient called Excipial Hydrocrème®. Additionally, they used diethylene glycol monoethyl ether P (Transcutol®) as a vehicle and demonstrated that rapamycin was ten times more soluble in Transcutol® (fully soluble, 20.2 mg/mL) compared to liquid paraffin (< 2 mg/mL). Le Guyader et al. [11] conducted a physico-chemical and microbiological stability study on a gel-type formulation. These authors also recommend the use of solubilizing agents such as transcutol in combination with gel-type formulations, since they have demonstrated improved permeation, penetration, and release. Ghanbarzadeh et al. formulated rapamycin integrated into a liposomal solution, explaining the methodology in development of rapamycin liposomes, and additionally studied their chemical stability using a validated high-performance liquid chromatography (HPLC) method [12, 13].

Among all the concentrations and dosages reported in the literature, 0.4% concentration, three times per week, is interesting to avoid exposure to the drug every day and thus minimize possible cutaneous adverse events without compromising effectiveness [14, 15].

In addition, the current legal regulations in Spain on magistral formulas establish that the validity period of non-typified magistral formulas corresponds to the full duration of the treatment [16]. Secondly, the Guide to Good Practice of Preparation of Medications in Hospital Pharmacy Services in Spain establishes a validity period of 30 days for ointments and creams, which can be increased if demonstrated with stability studies [17].

The purpose of this study is to improve the classic formulation of topical rapamycin for FA in TS and determine the validity period of the proposed formulations based on chemical, physical, and microbiological stability.

Methods

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 formulations
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Chemical Stability

Equipment

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]:

$$\mathrm{EE\%}=\frac{W_{\mathrm t}-W_{\mathrm f}}{W_{\mathrm t}}\times100$$

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.

Results

Chemical Stability

Method Validation

The calibration curve for the chemical stability of ointment, emulsion, and gel formulations demonstrated linearity with a coefficient of determination (r2) of 0.9998. The limits of detection (LD) and quantification (LQ) were 0.003 and 0.009 mg/mL, respectively. Linearity was also determined for the chemical stability of the liposomal formulation, with a coefficient of determination (r2) of 0.9958, LD = 0.011 mg/mL, and LQ = 0.038 mg/mL.

Rapamycin Degradation

Table 2 shows the rapamycin quantity per weighed formulation quantity (Q, mg/g) and the percentage content of remaining rapamycin (%RC) in each formulation at times (t) = 0, 7, 14, 21, 28, 42, and 56 days. Figure 1 shows ointment, emulsion, gel, and liposome chromatograms.

Table 2 Chemical stability results
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Fig. 1
figure 1

Ointment, emulsion, gel, and liposome chromatograms

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T90 was reached during the sampling period for the ointment, emulsion, and gel formulations: T90 = 56, 14, and 21 days, respectively. Only liposomes were stable for 56 days.

Physical Stability

Table 3 exposes the results of the physical stability parameters for the four formulations. The ointment formulation obtained a level 1 score in extensibility property due to its high petrolatum content, similar to the classical formulation. It should be noted that the emulsion formulation resulted in a pre-separation of phases at 14 days and a definitive breakdown of the emulsion at 21 days; therefore, it was assigned a level 1 score in the absence of phase separations property. The score of level 1 in the absence of crystal property for the gel formulation was awarded by the presence of white particles.

Table 3 Physical and microbiological stability results
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Liposome Characterization

The mean particle size of prepared liposomes determined a good quality result of the analysis with a polydispersity index value < 0.2, indicating a homogenous dispersion. The EE% turned out to be very favorable for the liposomal formulation, with a high load of rapamycin per liposome formed.

Microbiological Stability

Culture samples were negative in blood-agar media for each formulation at t = 28 and 56 days (Table 3).

Discussion

This study concludes that only one of the four proposed formulations, liposomes, maintains their chemical, physical, and microbiological stability throughout all the sampling time, awarding them a validity period of 56 days.

For the ointment and gel formulations, the chemical stability was the determining factor for not reaching the validity period of 56 days, with T90 = 56 and 21 days, respectively. In the case of the emulsion formulation, the physical stability was very obviously compromised after pre-separation of phases at 14 days and a definitive breaking up of the emulsion at 21 days. All formulations were microbiologically stable throughout the sampling period. Considering all stability studies, the validity period for each formulation was as follows: ointment = 42 days, emulsion = 7 days, gel = 14 days, and liposomes = 56 days.

Rapamycin is a highly apolar, active substance. This stability study confirms that rapamycin is more comfortable among lipophilic excipients, such as petrolatum and liposomes. In hydrophilic vehicles, it tends to precipitate and lose its physical and chemical stability, as in the case of emulsion and gel formulations.

Regarding excipients, transcutol allows a greater solubility of rapamycin in the vehicle [10], thus avoiding possible precipitations of the active principle and constituting a significant improvement in the formulation.

The liposomal formulation has certain advantages over the ointment formulation: it has a better appearance, is more comfortable, and has better extensibility (Photograph 1). Historically, some patients complained that classic ointment was difficult to apply [21] and the enhanced organoleptic properties of the liposomal formulation would help increase compliance with treatment and patient satisfaction with topical therapy.

Additionally, liposomes have been studied as carriers of molecules for immunosuppressive therapies and chemotherapy [22, 23]. Liposome-encapsulated rapamycin has been shown to have the ability to reach the drug’s site of action more quickly, with antiproliferative properties on T cells [23, 24].

Therefore, liposomes exert their influence on the stratum corneum of the skin, improving the penetration of the active substance and providing an increased bioavailability of rapamycin [20]. This property makes liposomes particularly effective for cosmetic and pharmaceutical use.

In these therapies, the role of the hospital pharmacists is key in the preparation of the formulations, since rapamycin was added to the NIOSH list as a hazardous drug in 2014 [25], which results in greater control in the development of topical formulations [26]. In addition, pharmaceutical care has special relevance in the processes of dispensing, administration, and monitoring, promoting the supervision and control of these patients.

After galenic improvement and confirmation of stability in liposomes, clinical studies are needed to ensure effectiveness, safety, and high patient satisfaction.

Limitations

The authors are aware that there is significant variability in chemical stability results for all formulations. This is due to the fact that during the formulation process, the correct homogenization of the excipients is very difficult, and the extractability of solvents depends on the formulation and its excipients. In any case, this article provides new stability data, allowing support in therapeutic decisions for other authors and clinicians.