FormalPara Key Summary Points

Why carry out this study?

Optimizing drug delivery from topical vehicles is essential for efficacy in treating dermatologic disease.

This research examined the delivery of betamethasone dipropionate (BDP) to the skin using a combination calcipotriene/betamethasone dipropionate approved drug formulation in an oil-based gelled topical suspension as compared to a new cream formulation utilizing a novel oil-in-water emulsion system.

What was learned from this study?

The newer emulsion-type formulation and drug delivery technology, termed polyaphron dispersion (PAD) technology, encapsulates dispersed oil droplets in a robust multimolecular aqueous film of surfactants, oil, and water enabling a multifold decrease in surfactant concentration.

The newer PAD technology cream formulation delivered more BDP into the upper stratum corneum and lower epidermis than a traditional suspension.

Introduction

The topical delivery of medications to a locally targeted region for the treatment of skin disease is challenging. The drug must be placed on the skin surface in a thin, even aesthetic vehicle film, be released from the vehicle, enter the stratum corneum, diffuse through the epidermis, and ultimately enter the dermis in a biologically active form. This is no small feat, with vehicle design being critical to success. Several strategies have been investigated to overcome the challenge of penetrating the stratum corneum to improve dermal drug delivery. Innovative topical delivery systems, including emulgels for the delivery of diclofenac and foams for the delivery of minoxidil, have been commercially successful. Innovations such as flexible sub-micron vesicular systems based on liposomes have been investigated as carriers for active ingredients [1, 2]; however, there are few commercial systems available outside of cosmetic applications.

The industry standard for assessing these properties typically involves in vitro assays with porcine or cadaveric skin in conjunction with a Franz diffusion cell (Franz cell) as a surrogate for efficacy in living patients [3, 4]. The Franz cell consists of a lower receptor chamber containing receptor buffer with the solubility of the compound being studied and an upper donor chamber where a test formulation can be applied to a skin sample mounted between the two chambers [4]. Skin tissues applied in this surrogate assay do not have enzymatic activity and are often not human tissue; also the assay inherently measures drug penetration through the skin rather than a drug that resides within the dermis to have a dermatologic effect. Consequently, Franz cell assays have inherently limited relevance to drug and formulation efficacy in real patients. Fortunately, recent advances using tape strips and liquid chromatography with mass spectrometry (LC–MS) have enabled non-invasive assessment of a formulation’s efficacy in living, enzymatically active human skin [5]. The development of new vehicles offers the opportunity to take effective drugs and enhance their efficacy with superior delivery, tolerability, and enhanced aesthetics.

Calcipotriene (CAL) and betamethasone dipropionate (BDP) are two drugs combined into a single topical medication (CAL/BDP) with established efficacy in the treatment of psoriasis. One available formulation is an oil-based gelled topical suspension while a new formulation utilizes a novel oil-in-water emulsion system (polyaphron dispersion [PAD] technology; MC2 Therapeutics, Guildford, UK). Typical oil-in-water emulsion systems utilize a surfactant to allow the lipid and lipid-soluble ingredients to be emulsified and dispersed within an aqueous vehicle. In addition to emulsification of the lipids in the oil-in-water formulation, surfactants can also emulsify and extract the intercellular lipids in the stratum corneum, potentially resulting in damage to the skin barrier [6]. PAD technology is a more modern emulsion-type formulation and drug delivery technology that encapsulates dispersed oil droplets in a robust multimolecular aqueous film of surfactants, oil, and water enabling a multifold decrease in surfactant concentration [7] (Fig. 1).

Fig. 1
figure 1

Reprinted with permission from Dermatology and Therapy [7]

Scanning electron microscope images of a cleaved polyaphron dispersion (PAD) technology oil droplets. Left: scanning electron microscope image of a cleaved PAD technology oil droplet showing the central rough region of oil (in which drugs can be solubilized) and the smoother outer shell structure of surfactant, water and oil. Right: scanning electron microscope image of a cleaved PAD technology oil droplet in which the central oil core has been sublimated off during sample preparation, leaving the outer shell structure of surfactant, water and oil.

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PAD technology has demonstrated protective chemical stability of active molecules solubilized in the oil phase as well as excellent physical stability. Furthermore, PAD technology supports efficient skin penetration, causes minimal disturbance of the skin barrier, and presents itself as a highly convenient cream that absorbs into the skin a few minutes after application [7]. CAL/BDP cream is an example of a topical cream that, based on PAD technology, has demonstrated significantly greater clinical efficacy [8] and superior patient-reported outcomes [9] in the treatment of plaque psoriasis compared to CAL/BDP topical suspension (TS). Thus, greater penetration of the active ingredients CAL and BDP into the epidermis and dermis would be expected from the PAD technology cream formulation.

The objective of this research was to compare the ability of CAL/BDP cream to deposit BDP and its major human metabolite betamethasone 17-propionate (B17P) in human skin as compared to CAL/BDP TS utilizing in vitro and in vivo detection methods.

Methods

The amount of BDP released from the CAL/BDP cream and CAL/BDP TS was evaluated using both in vitro Franz cell analysis and in vivo human tape stripping.

In Vitro Franz Cell Analysis

The novel aqueous cream containing CAL 0.005% (w/w) and BDP 0.064% (w/w) (Wynzora®; MC2 Therapeutics) was compared to a traditional topical suspension formulation containing the same concentration of active ingredients (Taclonex® Topical Suspension; LEO Pharma, Ballerup, Denmark) using the Franz cell technique [3].

Human skin samples (University of Bradford, Bradford, UK) were ethically obtained post-abdominoplasty from a 59-year-old Caucasian female. After surgery, the skin sample was stored at − 20 °C until prepared. Subcutaneous fat was removed mechanically to leave full-thickness skin. The dermis was removed from the epidermis using heat separation (water bath at 60 °C for 1 min), and the prepared epidermis was mounted onto a moist filter paper, air dried for 1 h, and stored at − 20 °C. For the in vitro Franz cell analysis, the skin was trimmed, cut into 12-mm-diameter pieces, and mounted on Franz diffusion cells. The lower receptor chamber was filled with receptor phase, and the Franz cells were placed in a water bath preset at 32 °C. Six replicate Franz cells were used for each formulation, each with a receptor phase volume of approximately 2 mL and a diffusion area of 0.64 cm2.

Skin diffusion was followed over a 72-h study period, similar to the methodology used in Simonsen et al. [10]. Saturating doses of approximately 30 mg of each formulation were pipetted onto the skin surface. Additional doses of the formulations were added to the Franz cells after 16, 40, and 64 h to reflect the once-daily application recommended for the products in clinical practice. A 2-mL aliquot of a mixture of 70% w/w phosphate buffer (pH 7.4) and 30% w/w isopropanol was used as the receptor phase in each cell. Receptor phase samples were collected for analysis at 16, 24, 40, 48, 64, and 72 h and replaced each time with fresh buffer. Data for one cell (CAL/BDP TS at 72 h) was excluded due to a suspicion of perforation of the skin.

BDP Franz Cell Analysis Using High-Pressure Liquid Chromatography

A Waters H-Class ultra-performance liquid chromatography system (Waters Corp., Milford, MA, USA) was used for all BDP analyses. Prior to injection into the high-performance liquid chromatography (HPLC) system, all samples and standards were filtered through a polytetra-fluorethylene (PTFE) filter to remove paper fibers from the sample (Millex PTFE: pore size: 0.45 µm; diameter of filter: 13 mm; Millipore, Watford, UK). The column used was a BEH C18 50 × 2.1 mm, 1.7 µm particle size, fitted with a pre-column filter (VanGuard BEH C18 1.7 µm) (Waters Corp.). The column was maintained at 40 °C. An isocratic HPLC method was used; a mobile phase of 35/45/20 (v/v/v) water/acetonitrile/methanol was pumped at a rate of 0.5 mL/min. A 10-µL injection volume was used for all samples. An UV–Vis photodiode array detector with chromatograms extracted at 240 nm was used for detection. A run time of 3 min was used, and a retention time of 0.84 min was observed for BDP.

In Vivo Tape Stripping Method

In this study, ten healthy non-pregnant female subjects aged 18+ years with normal skin and minimal hair on the ventral forearms were enrolled. The study was conducted in accordance with Good Clinical Practices 21CFR Part 50 guidelines and in accordance with the Helsinki Declaration of 1964 and its later amendments. All participants signed an IRB-approved informed consent prior to initiation of the study procedures. The study documents and consent and photography consent were approved by the Allendale Institutional Review Board (AIRB; Old Lyme, CT, USA).

Subjects had five 1-inch squares drawn on both forearms with a permanent black marker, for a total of ten squares per subject. CAL/BDP cream (0.1 cc) was rubbed into each of four squares of skin on one randomized forearm by the dermatologist investigator until completely absorbed; the remaining square on the arm served as a control, with no product applied. CAL/BDP TS (0.1 cc) was rubbed into each of the four squares on the opposite randomized forearm until completely absorbed; one square on this arm also served as a control, with no product applied. The sites on each arm were numbered with the time point for sampling. At the appointed sampling time (1, 2, 4, 8 h), D-Squame (Clinical and Derm, LLC, Dallas, TX, USA) 7/8-inch circular tape strips were applied to the application site and held in place for 10 s with a controlled pressure plunger that produced a pressure of 225 g/cm2 to insure adequate and consistent contact between the tape and the skin. The first two tape strips removed were discarded. This procedure was repeated 20 times on the same area with a new tape strip. The tape strips were immediately placed in a freezer at − 20 °C for storage. The odd-numbered tape strips (Nos. 3–19) were measured for this analysis, while the even-numbered tape strips (Nos. 4–20) were kept as backup samples.

Tape Stripping LC–MS Method

An analytical grade BDP dry powder and B17P were weighed and serially diluted to concentrations of 1000, 500, 250, 125, 62.5, and 31.25 ng/mL and 15.63, 7.81, and 3.91 ng/mL, respectively, using Class A volumetric glassware and standard techniques. The standard curves were prepared in triplicate.

Deuterated BDP-d10 (BD-d10) and B17P-d5 standards (Santa Cruz Biotechnology, Paso Robles, CA, USA) were used without further purification. The extraction solution was prepared in duplicate with these standards for quantitation of BDP and B17P from tape strips for a total volume 2 L in MeCN using Class A volumetric glassware and standard analytical techniques. Approximately 1 mg of the deuterated analytical standards were suspended in 2 mL of MeCN and serially diluted to 1 L MeCN solutions of 325.02 ± 6.23 ng/mL BDP-d10 and 390.52 ± 2.78 ng/mL B17P-d5 for samples from subjects 3 to 8 and to 1 L MeCN solutions of 313.71 ± 14.18 ng/mL BDP-d10 and 333.93 ± 9.18 ng/mL for samples from subjects 1, 2, 9, and 10. These concentrations were determined by triplicate injection.

Tape strips previously collected were stored at − 80 °C until analyzed and then thawed to 20 °C prior to use. To each tape strip, 2 mL of MeCN was added in a 12-mL sample vial. The samples were agitated for 2 min and inverted by hand to ensure consistent mixing, following which 1 mL of the solution was filtered through a 0.2-µm nylon filter and added to a 1.5-mL vial with a sealed PTFE septum. The samples were placed in the HPLC auto-sampler carousel and cooled to 4 °C until analysis within 2 h of preparation. LC–MS data were collected on an UltiMate 3000 system consisting of an LPG-3400SD pump, TCC-3000SD column oven, WPS-3000TSL auto-sampler, and DAD-3000 diode array equipped with a Accucore C8 50 × 2.1-mm2, 2.6-µm column (Thermo Fisher Scientific, Waltham, MA, USA) attached to an ISQ EM Single Quadrupole mass spectrometer with atmospheric pressure chemical ionization (Thermo Fisher Scientific).

All quantitative data analyses were completed using Chromeleon 7.2 software (Thermo Fisher Scientific), and the data were exported to Excel (Microsoft Corp., Redmond, WA, USA) for final distribution. The concentrations of BDP-d10 and B17P-d5 were determined using the external standard curves prepared as described above. For detection of BDP with the method described above, the limit of detection and the limit of quantitation were determined to be 0.52 and 1.57 ng/mL, respectively. For detection of B17P with the method described above, the limit of detection and the limit of quantitation were determined to be 11.36 and 34.42 ng/mL, respectively. A t-test was used to analyze the paired data with statistical significance defined as p ≤ 0.05.

Results

Penetration of BDP into or through the epidermis was compared for the marketed dosage forms CAL/BDP cream and CAL/BDP TS by an in vitro Franz skin diffusion study and a tape stripping study in human volunteers.

In Vitro Franz Cell Analysis Results

The Franz skin diffusion study was conducted using epidermis from surgically excised abdominal human skin. CAL/BDP cream and CAL/BDP TS were applied to epidermis after 16, 40, and 64 h to reflect the once-daily application recommended for the products. At all time points, the cumulative amount of BDP that diffused through the epidermis was statistically significantly greater for CAL/BDP cream than for CAL/BDP TS (Table 1). After an initial lag phase, the flux rate of BDP through epidermis was essentially constant over 72 h for CAL/BDP TS, while the flux rate of BDP from CAL/BDP cream was higher and had a slightly increasing trend over time (Fig. 2).

Table 1 Summary of Franz diffusion data by time after first application
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Fig. 2
figure 2

Cumulative betamethasone dipropionate (BDP) Franz cell diffusion. The amount of BDP diffused through human epidermis, determined by the in vitro Franz cell analysis over the first 72 h following first application. Error bars are the standard error of the mean (SE). A Student’s t test was applied to the data to establish a significant difference. Asterisks indicate significant difference at *p < 0.05 and ****p < 0.001. CAL/BDP Calcipotriene and betamethasone dipropionate, TS topical suspension

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Tape Stripping LC–MS Results

The CAL/BDP cream and CAL/BDP TS were applied to defined test areas on the forearms of ten [10] female healthy volunteers. Deposition of BDP and the major human metabolite B17P were measured by LC–MS from tape strips. Tape stripping continued for 20 iterations to allow sampling of the stratum corneum and epidermis down to the level where punctate bleeding occurred, indicating intrusion into the dermis. Insufficient amounts of B17P metabolite below the limit of quantitation were recovered from the tape strips for reliable quantitation (data not shown), so only data for BDP deposition are reported here. Consistently higher amounts of BDP were recovered following CAL/BDP cream application than following CAL/BDP TS application at 1, 2, 4, and 8 h following application (Fig. 3). Assessment of the BDP deposition data by time and skin depth (represented by tape strip number) confirmed the higher penetration of BDP after application of CAL/BDP cream compared to CAL/BDP TS at all investigated time points and skin depths (Fig. 4). The data further suggest rapid delivery from CAL/BDP cream, with optimum delivery at the first time point (1 h), whereas drug delivery from CAL/BDP TS was slightly slower, with the highest level measured 2 h after application. As might be expected, the measured deposition of drug was highest in the outer stratum corneum and declined significantly with skin depth (Fig. 4).

Fig. 3
figure 3

Recovery of BDP following application of CAL/BDP cream and CAL/BDP TS. The amount of BDP (in ng) recovered from odd tape strips nos. 3–19 reflected higher BDP deposition with the CAL/BDP cream compared to the CAL/BDP TS. Asterisks indicate significant difference at *p < 0.05, **p < 0.01, and ****p < 0.001.

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Fig. 4
figure 4

Comparison of BDP recovery. The recovery of BDP (in ng) by hour after application from tape strips showed higher BDP delivery from CAL/BDP cream compared to the CAL/BDP TS

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Discussion

This research utilized both in vitro and in vivo methods for the detection of BDP skin penetration, comparing two marketed formulations, namely, an oil-in-water cream based on PAD technology and an oil-based topical suspension. The standard method used for testing the delivery of topical drugs is the Franz cell. In the present study, pieces of epidermis from excised human skin were mounted on the Franz cell containing collection fluid. The topical medication was applied to the skin in a given amount for a given period of time, and the amount present in the collection fluid represents the amount of drug flux through the epidermis. This in vitro method has several drawbacks. First, the skin is not as biologically active as intact living skin. Second, only flux through epidermis is measured with this method, without evaluation of deposition of the compound in the epidermis or the dermis. Third, the collection fluid has sink conditions for BDP, which may result in a different result than penetration achieved in vivo. Rather, drugs applied topically must stay in the skin to optimally deliver their intended pharmacologic effect. Nevertheless, the Franz cell remains the standard delivery testing methodology recognized by the US Food and Drug Administration [11]. The Franz cell study presented in this research demonstrated a significantly greater delivery of CAL/BDP cream as compared to CAL/BDP TS.

An in vivo tape stripping technique conducted on healthy female volunteers was conducted as a more accurate measure of delivery of the drug from the formulations to human skin. The tape stripping methodology quantitatively assessed the delivery of drug into living skin, but not the amount of drug that passed through the skin. This more biologically relevant method for assessing drug deposition in the skin also demonstrated a significantly higher and faster delivery of BDP from CAL/BDP cream as compared to CAL/BDP TS. The greater ability of CAL/BDP cream to deliver the drug into the skin can probably be assigned to its use of PAD technology, which allows full solubilization of the drugs and efficient release of drugs into the skin through a unique physical–chemical structure and mix of oil, surfactants, and other excipients that empirically have demonstrated high levels of skin penetration [7].

The BDP skin penetration profile from the formulations aligns well with the phase III clinical trial findings that led to the approval of CAL/BDP cream for the treatment of psoriasis in adults [8]. In the phase III studies, CAL/BDP cream was compared head-to-head with CAL/BDP TS and found to deliver significantly greater improvements in psoriasis resolution. In the pooled phase III data set, 43.2% of patients treated with CAL/BDP cream achieved physician global assessment (PGA) treatment success at week 8 compared to 31.9% for CAL/BDP TS (p < 0.0001) [8]; also, 36.0% of the patients receiving CAL/BDP cream achieved a minimum improvement of 1 grade in the PGA as early as after the first week of treatment [12]. Likewise, patient-reported outcomes were rated significantly better in favor of CAL/BDP cream compared to the CAL/BDP TS, including the proportion of patients achieving a Dermatology Life Quality Index (DLQI) score of 0 or 1 at week 8 (43.8% vs. 34.2%; p = 0.0005) and the proportion of subject global assessment (SGA) success defined by an improvement of 2 grades to clear or very mild disease at week 8 (44.2% vs. 27.9%; p < 0.0001) [9]. Thus, the results of the in vivo tape stripping skin deposition study in human female volunteers presented herein and the clinical trial outcomes in psoriasis patients demonstrate coherent and statistically significant data in favor of CAL/BDP cream compared to CAL/BDP TS. Moreover, it is reassuring that the outcomes of the Franz skin flux study and the deposition study demonstrate a similar penetration advantage in favor of CAL/BDP cream relative to CAL/BDP TS and that the higher exposure to active drug in the skin also is translated to better clinical efficacy.

This research has several limitations. The study was conducted in a limited sample size of ten subjects, although a significant number of tape strips (200) were generated from each subject. Second, only the delivery of BDP was analyzed from the CAL and BDP fixed dose combination formulations. Another set of Franz cell and tape stripping studies would need to be conducted to better understand the delivery of CAL. An evaluation of CAL penetration was not attempted as part of this research since CAL is difficult to detect due to its very low strength in the formulations. Nevertheless, it is probable that CAL penetration is very similar to that of BDP. It is interesting to note that the analysis for the main metabolite of BDP, B17P, was below the limit of detection, indicating that BDP is predominantly metabolized to B17P in plasma or that this conversion appears to be relatively slow in the skin compared to plasma; however, further research would be required to support this supposition.

Conclusion

CAL/BDP cream utilizing a novel emulsion technology delivered more BDP into the upper stratum corneum and lower epidermis than CAL/BDP TS.