Safety and tolerability of topical trametinib in rosacea: Results from a phase I clinical trial

Abstract Purpose Overactivation of the mitogen activated kinase pathway has been associated with rosacea. We hypothesised that inhibitors of this pathway can be repurposed to alleviate rosacea symptoms. Methods In order to test this hypothesis, we designed a double‐blind, randomised, placebo‐controlled phase I clinical trial to assess the safety and tolerability of a first‐in‐kind topical formulation of a MEK kinase inhibitor, trametinib. Subjects applied daily trametinib‐containing cream (0.05 mg in 0.5 mL) to one cheek and cream without inhibitor to the other for consecutive 21 days. Skin irritation scores and blood samples were obtained during visits on days 8, 15 and 22. Results On analysis of high‐performance liquid chromatography, no systemic trametinib absorption was detected during this treatment period. Subjects demonstrated a slight but significant improvement in both cheeks, regardless of drug contents. No adverse effects were reported during this time. Conclusions Topical trametinib was well tolerated at a dose of 0.05 mg per day without meaningful systemic absorption or local adverse events. A dose escalation trial is warranted to determine optimal dosing to treat rosacea while avoiding the adverse effects of systemic treatment.


| INTRODUCTION
4][5][6] Despite this high burden associated with of rosacea, 7 current therapies for this disorder are generally ineffective and associated with significant side effects. 8Historically, a limited understanding of this disease prevented the design of medications specifically targeted to rosacea vpathophysiology.
Recent work provided new insights into the biology of the disease by identifying discrete activation of mitogen-associated protein kinase (mitogen activated kinase pathway [MAPK]) signalling governing its cutaneous inflammation, thus raising hopes for highly targeted therapeutic interventions.Previously, Wladis et al identified increased activation of p38 and ERK in cutaneous specimens of rosacea. 9Similarly, Harden et al demonstrated upregulation of MAPK signalling in explants of papulopustular skin specimens. 10Kan et al reported that cinnamtannin B1 suppressed the cutaneous inflammation of rosacea in a mouse model through inhibition of ERK phosphorylation. 113][14][15] We have previously shown that trametinib limits the acute increases in endothelial permeability in response to the pro-inflammatory cytokine IL-6. 16Together, these findings suggest the inhibition of MAPK may selectively arrest the inflammation associated with rosacea in a specific fashion, thereby providing a meaningful interference of the disease with fewer complications.
The canonical MAPK signalling pathway involves the activation of ERK1 and ERK2 via phosphorylation by MEK1 and MEK2 kinases.Many highly specific inhibitors of this pathway have been developed, with several inhibitors (trametinib, binimetinib, selumetinib, and cobimetinib) being approved by the United States' Food and Drug Administration (FDA) for the treatment of specific cancers. 17,18We thus hypothesised that these drugs could be repurposed for the treatment of rosacea and other inflammatory cutaneous diseases.This trial was performed to assess the safety and tolerability of topical trametinib in patients with rosacea.This approach was employed to interfere with the previously implicated signalling activity that subserves rosacea in a fashion that would minimise systemic absorption and therefore avoid the adverse effects associated with oral intake.

| RESULTS
Twelve patients were identified during the recruitment period.The characteristics of these patients are summarised in Table 1.All patients completed the study, and no patient withdrew from the trial for reasons of adverse events or intolerance of the medication.One patient missed a study visit, due to travel.No statistically significant differences were observed on the baseline scores between the assigned treated and untreated sides (Figure 1a).
The two sides of the face were not statistically significantly different from one another at any time point.We noticed a slight reduction in the Skin Irritation Scoring System (SISS) 19 scores on both sides, which we attributed to a mild effect of applying the ointment every day (Table 1 and Figure 1b).Levels of trametinib in blood remained below the limit of detection by high-performance liquid chromatography (HPLC) (<0.1 ng/mL) as measured from a blood sample obtained at every visit.At the end of the trials the bottles were returned for weighing.Two subjects did not return the bottles.The average weight loss of each bottle was 5.45 g.No statistically significant difference was observed between the placebo and trametinib bottles (5.64 � 2.14 g vs. 5.26 � 2.16 g).

| DISCUSSION
Multiple therapeutic options are routinely employed to address rosacea, because each modality suffers from incomplete responses and adverse events. 8][22][23][24] Several studies have explored the pathogenesis of rosacea, and our understanding of the cutaneous biology of the disease has evolved considerably. 25pecifically, several biologic aberrancies appear to contribute to the development of rosacea, including tolllike receptors, 26,27 kallikrein-related peptidase 5 and cathelicidin LL-37, 28 nuclear factor κB, 29 myeloid differential factor-88, 30 and multiple genetic factors. 31,32hese steps towards a pathogenetic construct suggest multiple discrete checkpoints that may be amenable to selective manipulation for the treatment of rosacea.
Wladis et al previously implicated p38 and ERK in the pathogenesis of rosacea, 9 suggesting a role for suppression of the MAPK pathway to arrest rosacea.4][35] The current study repurposed an FDA-approved MEK inhibitor as a topical preparation to target rosacea.To minimise systemic absorption, we suspended trametinib in a highly hydrophobic base.Consistent with minimal absorption, we did not detect any drug in circulation, indicating that patients are very unlikely to experience systemic adverse events.Furthermore, we did not detect any local adverse events, and the active What is already known about this topic?� p38 and ERK are enriched in cutaneous specimens of rosacea, and a variety of cellular features suggest that interference with the MAP kinase might be an effective treatment strategy to address rosacea.

What does this study add?
� This study demonstrates that a medication to suppress MAP kinase can be delivered as a topical preparation.This therapy was uniformly safe and the medication was not absorbed systemically.As such, this therapeutic approach can safely be tested for efficacy in later-stage trials.
medication was well-tolerated.It is possible that absorption in other skin types may be different.However, rosacea is highly prevalent in populations with fair skin and older, 36,37 and thus this finding remains clinically relevant.Based on these findings, topical trametinib represents a scientifically-sound approach to combat rosacea in a highly targeted fashion without inducing the side effects that are classically associated with our current therapeutic armamentarium.This important proof of principle raises significant questions for future study.Specifically, while this phase one trial underscored the safety and tolerability of MAPK inhibition in patients that suffer from rosacea, the investigation was not specifically powered or of adequate duration to assess the efficacy of this approach.Due to the characteristics of the population at the recruiting center, all patients enroled were Caucasian males.Alternative centers will be required to expand the enrolment to females and other races  and ethnicities.Additionally, only one dose of the medication was employed in this early trial.A doseescalating phase I/II trial must be implemented to determine the optimal concentration of the medication to control the disease and avoid local and systemic toxicities.A modest but significant decrease in scoring was observed in both cheeks.We attributed this improvement to the daily care on skin that otherwise may not have received any cream.][40] Trials designed to test for efficacy in the absence of other skin treatments will be required to better understand this finding.We do not believe this could be due to the effect of the contralateral trametinib application since this would require systemic absorption and trametinib plasma levels remained below the level of detection.It remains unknown if this effect would disappear after prolonged application.Lastly, we cannot discard the possibility that the subjects applied the cream to opposite sides, masking any potential effect.Consequently, future research will need to explore the benefits of MAPK inhibitors and the ideal dosing of this topical preparation.
Several limitations apply to this investigation.Given that this study was designed to explore safety and tolerability, patients were allowed to continue their current treatments for rosacea.Similarly, the pharmacokinetics and dermal penetration will need to be carefully assessed.Lastly, this trial had a short duration and a limited number of subjects consisting of older Causasian males.While a longer trial of this therapeutic approach may uncover local toxicities, the 22-day duration of this trial was chosen based on the guidelines of the FDA.Additionally, the absence of systemic absorption of the topical medication provides reassurance that systemic side effects are unlikely.Current pharmacological approaches require many months of treatment to achieve a clinically meaningful improvement.Our short duration trial was not designed to test the potential effect of longer treatments.The results of this trial provide opportunities to perform a doseescalating phase I/II trial on a more diverse subject population, and, at that point, rosacea therapies should be discontinued to assess the efficacy of MAPK inhibition.
Nonetheless, this study represents a very meaningful advance in our ability to provide targeted therapy for rosacea.Given that this therapeutic approach directly stems from improvements in our understanding of the cutaneous biology of the disease and that this safety study did not detect any adverse events, additional investigations of this very promising technology may be safely performed to provide relief to patients that suffer from this illness.

| Study design
This is a placebo self-controlled, double-blinded, randomised interventional study to assess safety and tolerability of a topical medication.This study was approved by the Institutional Review Board of the institution.It adhered to the tenets of the Declaration of Helsinki and the Health Insurance Portability and Accountability Act.The phase one trial was registered at clinicaltrials.gov.

| Drug formulation
Extraction of trametinib from commercially available tablets and formulation on a hydrophobic Humco Anhydrous Base cat# 6013-01 (Humco) was done following good laboratory practices manufacturing by a FDA-registered compounding pharmacy (Pharmaceutic Labs, Colonie).An analytical test method was developed and validated by Biomed Innova (Albany, NY) for assaying Trametinib topical cream that was used in the study.It was analysed and quantified by HPLC.The results were consistent with the theoretical amount of 0.1 mg/g Trametinib and was therefore approved for the use in this study.All bottles dispensed to the subjects were weighted before and at the end of the trial.

| Patient recruitment
Patients with erythematotelangiectatic rosacea were identified through the ophthalmology clinic, based on clinical examination.In order to be included, patients were required to meet the standard requirements for a diagnosis of rosacea, including central facial erythema with associated telangiectasias, transient erythema, dry eye disease, or phymomas.Patients were allowed to continue with their current regimens for the treatment of rosacea, although no patient was actively taking a medication to address rosacea at the time of recruitment.Exclusion criteria included age less than 18 and over 80 years, a documented allergy to trametinib, a concurrent condition that would preclude the use of trametinib, pregnancy, heart failure, and the concomitant use of medications that would interact unfavourably with trametinib.Upon identification, informed consent was obtained by the study coordinator.No restrictions were placed on sex, gender, race or ethnicity.Despite the broad criteria, all 12 enroled subjects were Caucasian males, reflecting the majority of the population at the center.All subjects received instruction in person by the investigators at the time of enrolment.

| Study protocol
Medication was dispensed via vials at a controlled pharmacy.Specifically, patients received two 0.5 mL dispensing bottles that were labelled for either the right or left cheek.One container dispensed a suspension vehicle and served as a placebo control.The other bottle contained the same vehicle with 0.01% trametinib.In both cases, the suspension was made on a hydrophobic base to deliver the topical medication while minimising systemic absorption of the drug.The total amount delivered (0.05 mg/day) was determined by dividing the recommended dose of oral trametinib (2 mg/day) by an estimation of the total area to be covered (~1% of the total body surface area).An excess material was delivered to account for cream loss and drug binding to stratum corneum and dead keratinocytes.
Through a computerised random number generator, patients were assigned to apply the contents of the placebo vial to one side of the face and the active medication to the contralateral side once each day over a 22-day period, as suggested by FDA guidance. 19Local skin irritancy was assessed through the SISS on days 1, 8, 15, and 22 of the trial by an observer who was blinded as to which side received the active medication.Briefly, this test analyzes the development of erythema and oedema via a 0-7 scoring mechanism for skin appearance and additional effects ranging from 0 to 3. 19 The scoring criteria is shown in Table 2.
During each visit (days 1, 8, 15, and 22), blood samples (5 mL) from the median cubital vein were collected in EDTA-coated tubes and photographs of the treated areas were taken with a dedicated digital camera.All subjects declared following the procedures when asked at each visit.

| Determination of trametinib in plasma
Plasma was obtained from each blood sample by standard centrifugation (1800 � g for 18 min at room temperature) and maintained at −20 C until use.Trametinib levels were measured trametinib levels were assayed via HPLC by Biomed Innova (Albany, NY).The minimum level of detection was determined to be 0.1 ng/mL by spiking pure trametinib (Selleck Chemicals) in normal plasma (Human Plasma with K3-EDTA obtained from Avantor).
T A B L E 2 Skin appearance scoring system.

Skin appearance Score
No evidence of irritation 0 Minimal erythema, barely perceptible 1 Definite erythema, readily visible; minimal oedema or minimal papular response 2 Erythema and papules 3

Vesicular eruption 6
Strong reaction spreading beyond test site 7

Numerical equivalent
Slight glazed appearance A 0 Marked glazing B 1 Glazing with peeling and cracking C 2 Glazing with fissures F 3 Film of dried serous exudate covering all or part of the patch site G 3 Small petechial erosions and/or scabs H 3 Note: When one or more 'other effects' are observed, the score should be reported as a dermal response number, a letter combination score, and as a numerical total (i.e., numerical 'dermal response' score þ numeric equivalent for the 'other effects' lettered score).For example, the dermal response of 6 with glazing with fissure (F [3]) will equal to the score of 9. When no 'other effects' are observed, score zero should be applied to an observed 'other effects'.Slightly modified from: Assessing the Irritation and Sensitisation Potential of Transdermal and Topical Delivery Systems for ANDAs.Guidance for Industry.DRAFT GUIDANCE.U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), October 2018. 19ADIS ET AL.

| Statistical analysis
Direct comparisons of the SISS scores (Table 1) at each time were performed via a dedicated statistical software package (GraphPad Prism).Baseline levels in treated versus untreated sides were compared by Mann-Whitney U test.The effect during each visit was compared via two-way ANOVA of repeated measurements.p-values of <0.05 were considered statistically significant.

2 F I G U R E 1
Skin irritation scoring system scores.(a) baseline scores in each cheek, separated by stratification.No significant differences were observed (p > 0.8, Mann-Whitney).(b) Mean þ SEM scores in each cheek.Significant differences with time, but not between treatments.*, placebo p < 0.05 versus day 0. #, p < 0.05 trametinib versus day 0. Two-way ANOVA of repeated measurements and Holm-Sidak post-hoc test.
Patient demographic information and Skin Irritation Scoring System scores.
T A B L E 1