Clascoterone Stability When Combined with Topical Acne Medications In Vitro

Abstract

Introduction

Multimodal topical therapy is recommended by the American Academy of Dermatology for the treatment of acne vulgaris. However, no data are available regarding stability of clascoterone cream 1% when combined with other topical acne medications. The purpose of this study was to evaluate the stability of clascoterone in the presence of tretinoin, adapalene, dapsone, azelaic acid, benzoyl peroxide (BP)/clindamycin, BP/adapalene, and encapsulated BP.

Methods

Clascoterone cream (0.5 mL) was layered over 0.5 mL of each of the other products on an individual microscope slide and incubated for 8 h at 37 °C. Material from the slides was extracted in methanol and tetrahydrofuran for high-pressure liquid chromatography mass spectrometry (1 mL/min flow rate; 30 °C). Combinations of clascoterone with tretinoin and adapalene were reinjected 12 h after the initial injections to confirm consistent clascoterone detection. Percent recovery of clascoterone for each combination was calculated from a standard curve serially diluted in methanol.

Results

The mean percentage of clascoterone recovered after incubation with other drugs ranged from 98% to 119%, with 86% recovered following injection of clascoterone alone. Reinjected clascoterone-tretinoin and clascoterone-adapalene samples yielded clascoterone recovery percentages of 97% and 93%, respectively, both within 8% of those recovered from the initial injections.

Conclusions

Clascoterone demonstrated consistent and reproducible stability in the presence of other topical acne medications.

Key Summary Points

Why carry out this study?

The American Academy of Dermatology recommends topical combination therapy for acne management; however, stability of layered topical medications is understudied

This study aimed to evaluate the stability of clascoterone cream 1% when combined with commonly prescribed topical acne medications in vitro

What was learned from the study?

This study uses a novel in vitro method to demonstrate the stability of topical clascoterone cream 1% in the presence of other topical products, confirming the utility of acne medication layering

This method could be adaptable for testing the stability of other topical medications

Introduction

Acne vulgaris is a common inflammatory skin condition with an estimated global prevalence of 231.2 million as of 2019 [1, 2]. Androgen-induced excess sebum production is a key driver of the acne pathogenesis cascade that triggers multiple downstream events involved in acne development; these include follicular hyperkeratinization, bacterial colonization, and inflammation [3]. Therefore, acne treatment optimally involves a multimodal approach that incorporates a combination of products to target multiple mechanisms of disease [2]. As outlined in the American Academy of Dermatology (AAD) guidelines of care for acne treatment, topical therapy is the mainstay of treatment for mild-to-moderate acne; such therapies include both single-agent (e.g., benzoyl peroxide, topical retinoids, topical antibiotics, azelaic acid, and clascoterone) and fixed-dose combination therapies (e.g., combinations of benzoyl peroxide and topical retinoids or topical antibiotics) [2].

Clascoterone cream 1% is a first-in-class topical androgen receptor inhibitor approved for the treatment of acne vulgaris in patients ≥ 12 years old [4, 5]. Dermatologists may use combinations of topical retinoids, topical antibiotics, and/or benzoyl peroxide with clascoterone cream 1% in the clinical setting to address the multifactorial pathogenesis of acne, as recommended by the AAD [2]. However, in clinical trials, clascoterone cream 1% was not evaluated in combination with other therapies [5, 6], and no studies have reported the drug interaction potential or stability of clascoterone cream 1% when used in combination with other commonly prescribed acne medications. The objectives of this study were to demonstrate the stability of clascoterone following in vitro combination with other topical acne medications and to optimize a method using high-pressure liquid chromatography mass spectrometry (HPLC–MS) to assess the stability of topical products for this and similar studies.

Methods

Ethical Approval

Ethical approval was not required since no human samples were used.

Sample Preparation

Tretinoin cream 0.025%, adapalene gel 0.3%, dapsone gel 7.5%, azelaic acid 15%, benzoyl peroxide 5%/clindamycin 1%, benzoyl peroxide 2.5%/adapalene 0.1%, and encapsulated benzoyl peroxide 5% (0.5 mL each) were placed separately on individual microscope slides. The acne medication was spread into a thin layer on the slide using the side of a 1-inch 30-gauge needle, after which the slides were allowed to rest for 15 min to replicate the recommended time between application of two topical acne medications. Clascoterone cream 1% (0.5 mL) was then added to each microscope slide and spread into a thin film with the side of a 1-inch 30-gauge needle, and the slides were subsequently incubated for 8 h at 37 °C to mimic the temperature of human skin. Each slide was weighed prior to product application, after application of the first product, and after clascoterone application to determine the amount of each treatment applied. After incubation, the slide was placed in a plastic conical vial with 10 mL of methanol spiked with analytical-grade tazarotene, a compound not part of this research used as an internal calibrant, and 10 mL of tetrahydrofuran. One sample in the condition (clascoterone plus benzoyl peroxide 5%/clindamycin 1%) required extraction with 10 mL of methanol spiked with analytical-grade tazarotene, 10 mL of acetonitrile, and 10 mL of tetrahydrofuran to completely dissolve the material on the slide. The vial was mixed using a vortex mixer until all visible material was removed from the microscope slide. A 1.5-mL sample of the solution containing the acne medications and solvent was collected from the vial and filtered, and 1 mL of the filtered solution was transferred to a vial for HPLC–MS assessment with ultraviolet detection.

Standard Curve Preparation

A clascoterone external standard and tazarotene internal standard were prepared. The tazarotene internal standard was prepared using 2.39 mg of analytical-grade tazarotene diluted with 1 L of methanol. The clascoterone external standard was prepared using 2.66 mg of analytical-grade clascoterone serially diluted in methanol to generate a standard curve, which was measured in triplicate.

HPLC–MS Parameters

HPLC–MS data were collected on an UltiMate 3000 system consisting of an LPG-3400SD pump, a TCC-3000SD column oven, a WPS-3000TSL autosampler, and a DAD-3000 diode array equipped with a Hypersil GOLD C18 4.6 × 150 mm² column (Thermo Fisher Scientific; Waltham, MA), and attached to an ISQ EM Single Quadrupole mass spectrometer with atmospheric pressure chemical ionization (Thermo Fisher Scientific). The HPLC instrument was operated with a flow rate of 1 mL/min and an oven temperature of 30 °C with a binary system consisting of (A) water with 0.1% formic acid and (B) methanol with 0.1% formic acid. The column was pre-equilibrated for 5 min with 65% A and 35% B prior to each injection. The following gradient was used:

• 0–1 min: 65% A, 35% B

• 1–6 min: 65% A, 35% B to 5% A, 95% B

• 6–13 min: 5% A, 95% B

• 13–13.9 min: 5% A, 95% B to 65% A, 35% B

Clascoterone elution was monitored with ultraviolet–visible spectrometry (UV–vis) at 280 nm and by electrospray ionization mass spectrometry (ESI–MS): calculated [M + H]⁺ for C₂₄H₃₅O₅ = 403.25 m/z. The ESI–MS was configured with a vaporizer temperature of 200 °C, an ion transfer tube temperature of 350 °C, a source voltage of 3000 V, a source collision-induced dissociation voltage of 5 V, a sheath gas pressure of 80 psig, an auxiliary gas pressure of 8.7 psig, and a sweet gas pressure of 0 psig. The tazarotene internal standard elution was monitored by UV–vis at 280 nm and by ESI–MS: calculated [M + H]⁺ for C₂₁H₂₂NO₂S = 352.14 m/z. The ESI–MS was configured using identical parameters as those used for clascoterone. All drug combinations were evaluated in duplicate.

Sample Validation

Appropriate validation steps were performed to ensure consistency in clascoterone recovery during the experiment. To determine if any conditions changed from the start to the end of the HPLC–MS run, clascoterone plus tretinoin and clascoterone plus adapalene were reinjected for HPLC–MS assessment of clascoterone recovery 12 h after the original injections. Replicates were not performed for the reinjected samples.

Clascoterone Quantification

Final concentrations were calculated using the clascoterone standard curve. The percent recovery of clascoterone was calculated for each drug combination.

Results

Clascoterone Detection Using HPLC–MS

The clascoterone standard curve is shown in Fig. 1. The linearity of the standard curve demonstrates proper calibration of the system, with sensitive and reproducible detection of clascoterone using HPLC–MS. Sensitivity was > 0.01 mg/mL. All measured samples had clascoterone concentrations within the range of the standard curve on the basis of the counts per min.

Fig. 1.

Clascoterone standard curve

Clascoterone Stability After Layering with Other Acne Medications

The percentage (standard error of the mean [SEM] × 2) of clascoterone recovered after layering with other acne medications ranged from 98% (2%) for benzoyl peroxide encapsulated to 119% (6%) for azelaic acid (Fig. 2), indicating that the other acne medications evaluated did not induce degradation of clascoterone when used in combination. The percentage of clascoterone recovered following injection of clascoterone cream 1% alone was also assessed. Injection of clascoterone alone yielded a mean (SEM × 2) recovery percentage for clascoterone of 86% (11%; Fig. 3); the variability in clascoterone recovery observed with clascoterone alone (11%) was greater than that observed after combination with other drugs (1–6%).

Fig. 2.

Percentage of clascoterone recovered after combination with other acne medications. BP benzoyl peroxide, SEM standard error of the mean

Fig. 3.

Percentage of clascoterone recovered after injection of clascoterone alone. SEM standard error of the mean

Consistency of Clascoterone Recovery Following Reinjection

To ensure the consistency of clascoterone recovery and to determine if any conditions changed from the start to the end of the HPLC–MS run, clascoterone plus tretinoin and clascoterone plus adapalene were reinjected for HPLC–MS assessment of clascoterone recovery 12 h after the original injections. The percentage of clascoterone recovered from the reinjected clascoterone plus tretinoin and clascoterone plus adapalene samples was 97% and 93% (Fig. 4), respectively, which was within 8% of that recovered from the initial injections, indicating consistency within the HPLC–MS run.

Fig. 4.

Percentage of clascoterone recovered following reinjection of clascoterone plus tretinoin and clascoterone plus adapalene

Discussion

This study reports the use of HPLC–MS and the methods described herein to evaluate the interactions between topical acne medications in vitro and support the potential use of this method to evaluate potential chemical interactions between topical therapies on the skin. Clascoterone demonstrated consistent and reproducible stability in the presence of other commonly prescribed topical acne medications, including tretinoin, adapalene, dapsone, azelaic acid, and benzoyl peroxide, none of which induced degradation of clascoterone following simulated combination therapy. Overall, these results confirm that the use of clascoterone cream 1% in multimodal topical combination therapy for acne vulgaris is a viable strategy with respect to drug stability.

Clascoterone was selected for evaluation because it has a novel antiandrogen mechanism for acne treatment and is therefore commonly used with other topical acne medications. This approach to acne treatment is supported by the 2024 AAD guidelines, which recommend combining topical agents with different mechanisms of action to optimize treatment efficacy [2]. The mechanism of action of clascoterone for the treatment of acne is thought to involve competition with dihydrotestosterone for binding to androgen receptors within sebocytes, thereby inhibiting the transcription of genes involved in the androgen-sebum pathway necessary for acne pathogenesis [7]. The other topical medications evaluated in this study address inflammatory, microbial, and/or comedolytic aspects of acne pathogenesis [2]; however, no other currently available topical medications target sebum production (Table 1). Therefore, combinations of clascoterone cream 1% with other topical acne medications can provide an approach to target multiple mechanisms of disease and may thereby provide optimal disease control and resolution.

Table 1.

Topical acne medications evaluated and their mechanisms of action

Treatment	Type	Mechanism of action
Tretinoin	Topical retinoids	Retinoids are vitamin A derivatives that produce comedolytic and anti-inflammatory effects via binding to retinoic acid receptors
Adapalene
Tazarotene
Azelaic acid	Dicarboxylic acid	Azelaic acid is a topical agent with comedolytic, antibacterial, and anti-inflammatory properties
Dapsone	Topical antibiotics	Topical antibiotics inhibit the growth of Cutibacterium acnes within the skin to hinder inflammatory cascades responsible for acne lesions; monotherapy is not recommended due to concern for antibiotic resistance development
Clindamycin
Benzoyl peroxide	Topical antimicrobial	Benzoyl peroxide is an antimicrobial agent that produces mild comedolytic effects
Clascoterone	Topical antiandrogen	Clascoterone decreases androgen-stimulated sebum and inflammatory cytokine production from sebocytes via competition with dihydrotestosterone for binding to androgen receptors

The protocol described is not the only means to examine drug stability in the context of combination topical therapy. Because the evaluation of topical agents in human participants may not always be feasible owing to ethical or other constraints, in vitro, in vivo, and ex vivo skin models are increasingly used for the characterization and assessment of topical therapeutics [8–11]. These models include ex vivo human skin (typically obtained from cadavers or plastic surgery patients), in vivo models using animal skin or chimeric models (i.e., grafting of human skin on mice), artificial membranes, and reconstructed skin models [8]. For example, investigators recently developed a novel in vitro skin permeation test method to assess the permeation of clascoterone in the skin after topical application using human cadaver skin [11]. Clascoterone cream 1% was applied to the skin explants and permeation samples were collected following an incubation period using either a vertical static diffusion cell or a flow-through cell method, and then analyzed using LC–MS [11]. This technique could also be adapted to examine stability in the presence of other agents.

Although many of these models do not fully recapitulate the conditions of human skin [8], there are recent efforts to develop more realistic experimental models of human inflammatory skin conditions by stimulating human ex vivo skin with inflammatory triggers to activate immune responses and cause disruption of the epidermal barrier [10]. Such models may be useful for evaluating the interactions between topical therapeutics in the context of the pathological changes associated with acne. However, these methods also have some disadvantages; for example, excised human skin may be difficult to obtain and requires ethical approval for experimental use [8]. Therefore, fully in vitro methods such as the one described herein remain useful, especially for preliminary studies.

The variability in the percent recovery of clascoterone was within the expected range of error due to weight measurements, injection variability, product mixing, and detector variability during the experiment. Whereas each source of error is expected to be small individually (1–5%), their combined effect can sufficiently account for the variation observed in the presented data. This study demonstrated reasonable consistency in the percent recovery of clascoterone, except for the samples containing clascoterone plus azelaic acid and clascoterone alone. This may be due to several factors, including differences in sample viscosity and contaminant retention in the sampler needle. For example, the sample containing clascoterone alone formed a harder, thicker film over the microscope slide compared with the other samples, which made it challenging to spread the material into a thin film, leading to residual material left on the slide. It is also possible that a compound within the azelaic acid formulation created a matrix effect that enhanced detection of clascoterone, which would explain the unexpectedly high percent recovery of clascoterone in the sample containing clascoterone plus azelaic acid.

There were several potential limitations to the study design. First, sample conditions could have changed from the start to the end of the HPLC–MS run due to the long run time. However, the percentage of clascoterone recovered remained consistent after rerunning the specimens, which validated the original measurements. Second, the drug material was placed on the microscope slides at room temperature rather than the physiological temperature of the human body, raising the possibility that there may be different interactions in such contexts. However, incubating the slides at 37 °C ensured reproduction of any chemical reactions that would occur at human body temperature. Third, the study was not highly powered since samples were run only in duplicate; however, this limitation is balanced by absence of human variability that would be present in samples recovered from human skin. Finally, some solubility problems were encountered during the study, but changing the solvent system resolved these issues. Future directions could include refining this in vitro method to address challenges encountered during the study in order to optimize this method for future drug stability studies.

These findings confirm the consistent and reproducible stability of clascoterone cream 1% in the presence of other topical acne medications including topical retinoids, topical antibiotics, and benzoyl peroxide. This study utilized an in vitro method that, to the best of our knowledge, has not been used in prior studies to evaluate the layering of multiple topical products on the skin. This method could be adaptable for testing the stability of other topical medications; one possible application could be evaluating the stability of other topical acne products in the presence of clascoterone cream 1%. Studies evaluating clinical outcomes of concomitant treatment with clascoterone cream 1% and other topical acne medications are also needed to expand the efficacy and safety profile of combination topical acne regimens including clascoterone cream 1%.

Conclusions

Clascoterone cream 1% showed consistent and reproducible stability in the presence of other topical acne medications including topical retinoids, topical antibiotics, and benzoyl peroxide. These findings confirm the utility of acne medication layering and support the feasibility of topical combination therapy with clascoterone cream 1% in patients with acne vulgaris.

Author Contributions

All authors (Zoe D. Draelos, Matthew M. Draelos, Kizito Kyeremateng, Nicholas Squittieri) contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Zoe D. Draelos and Matthew M. Draelos. All authors contributed to drafting or revising the manuscript for critically important intellectual content. All authors read and approved the final manuscript.

Funding

Sponsorship for this study and the Rapid Service Fee were funded by Sun Pharma.

Data Availability

All data generated or analyzed during this study are included in this published article.

Declarations

Conflict of Interest

Zoe D. Draelos was an investigator on this study and received a grant from Sun Pharma. She is also an editorial board member of Dermatology and Therapy. Zoe D. Draelos was not involved in the selection of peer reviewers for the manuscript, nor in any of the subsequent editorial decisions. Matthew M. Draelos has no conflicts to disclose. Kizito Kyeremateng and Nicholas Squittieri are employees of Sun Pharmaceutical Industries, Inc.

Ethical Approval

Ethical approval was not required since no human samples were used.