Reduction in ultraviolet B light–induced erythema by oxymetazoline and brimonidine is mediated by different α‐adrenoceptors

When applied topically, oxymetazoline and brimonidine reduce the persistent facial erythema of rosacea; this effect is mediated by cutaneous vasoconstriction induced by postsynaptic activation of α‐adrenoceptors. We investigated the α‐adrenergic pharmacology of oxymetazoline and brimonidine. Functional activity on α‐adrenoceptors was evaluated in vitro in HEK293 cells stably expressing single receptor subtypes using a fluorometric imaging plate reader Ca2+ influx assay. Oxymetazoline was an α1‐adrenoceptor agonist with partial α2‐adrenoceptor activity, whereas brimonidine was a highly selective full α2‐adrenoceptor agonist. In vivo pharmacology was investigated in a mouse model of ultraviolet B light (UVB)‐induced skin erythema. To selectively inhibit α‐adrenoceptor subtypes, mice were injected with prazosin (an α1‐selective antagonist) or rauwolscine (an α2‐selective antagonist) following UVB exposure. Oxymetazoline cream 1.0%, brimonidine gel 0.33% or vehicle control was applied topically, and erythema was measured using a chromameter. Oxymetazoline and brimonidine reduced UVB–induced erythema compared with vehicle control (P < .01). The effect of oxymetazoline was impaired in prazosin‐pretreated but not rauwolscine‐pretreated mice. Conversely, the effect of brimonidine was impaired in rauwolscine‐pretreated but not prazosin‐pretreated mice. These data suggest that while oxymetazoline and brimonidine produce cutaneous vasoconstriction, they do so through different α‐adrenergic mechanisms, with oxymetazoline primarily acting via α1‐adrenoceptors and brimonidine acting via α2‐adrenoceptors.

Persistent facial erythema involves a complex pathophysiology of neurovascular dysregulation, enhanced immune response and alteration of the superficial cutaneous vasculature. [4] Adrenergic receptors in the sympathetic nervous system play a key role in modulating the cutaneous vasculature, with individual receptor subtypes varying in tissue distribution and function. [4] The α 1 -adrenoceptors are expressed postsynaptically on vascular smooth muscle cells, whereas the α 2 -adrenoceptors may be expressed presynaptically on sympathetic nerve terminals and/or postsynaptically on vascular smooth muscle and the vascular endothelium. [6] The distribution of expression varies depending on the type of blood vessel. Activation of postsynaptic α 1 -and α 2 -adrenoceptors on vascular smooth muscle cells by noradrenaline or other α-adrenoceptor agonists results in vasoconstriction. [6] Activation of presynaptic α 2 -adrenoceptors inhibits noradrenaline release, and activation of α 2 -adrenoceptors in the vascular endothelium triggers nitric oxide production; both mechanisms enable or promote vasodilation and counter the postsynaptic vasoconstrictor effects. [6,7] Patients with rosacea have higher facial skin temperatures compared with healthy individuals, reflecting the persisting facial erythema and flushing. [8] Higher skin temperature reduces α 2 -adrenoceptor-mediated vasoconstrictor responses by reducing noradrenaline release, decreasing surface expression of α 2 -adrenoceptors on smooth muscle cells and increasing endothelial nitric oxide release. [7,[9][10][11] Therefore, for patients with rosacea, who have cutaneous vessels that have diminished α 2adrenoceptor-mediated vasoconstrictor capacity, the effect of an α 2adrenoceptor agonist on erythema associated with rosacea may be impaired, whereas the vasoconstriction effect of an α 1 -adrenoceptor agonist would not be expected to be impeded.
Currently, two α-adrenergic products are approved by the US Food and Drug Administration for the treatment of adults with persistent facial erythema of rosacea, oxymetazoline hydrochloride cream 1.0% (Rhofade™, Allergan plc, Dublin, Ireland) [12] and brimonidine topical gel 0.33% (Mirvaso ® , Galderma Laboratories, Fort Worth, TX). [13] This study compares the pharmacological mechanism of action of oxymetazoline and brimonidine on α-adrenoceptors in vitro and in vivo. For the latter, we used a murine ultraviolet B light (UVB)induced erythema model, recognising that UVB light is a well-known trigger of erythematous rosacea, and induces an acute cutaneous inflammatory reaction that shares many features with rosacea including erythema, cytokine release and leukocyte infiltration. [14,15] 2 | ME THODS

| Functional α-adrenergic receptor cellular assay
A fluorometric image plate reader (FLIPR) Ca 2+ influx assay was used to measure functional activity of the α-adrenoceptor agonists.
A transient calcium response can be induced by activation of α 1adrenoceptors coupled with the G q signalling protein or by activation of α 2 -adrenoceptors coupled with a chimeric G q protein that has five amino acids from the receptor recognition domain of G i (termed Comparisons between oxymetazoline and brimonidine were made using an independent samples t test.

| Murine UVB erythema model
All animal studies were approved by the Institutional Animal Care and Use Committee and were conducted according to the NIH Guide for the Care and Use of Laboratory Animals. Male SKH1 hairless mice aged 3-5 months and weighing 25-30 g were obtained from Charles River Laboratories (Wilmington, MA, USA). To induce skin erythema, the animals were anesthetised by isoflurane inhalation, the left side of the body including head, trunk and limbs was covered with a black rubber shield to protect it from UVB light exposure, and the right side of the body was exposed to 120 mJ/cm 2 of UVB light (wavelength 305-315 nm) generated by a xenon arc lamp-based solar simulator (Oriel SOLIA, Newport Corp, Irvine, CA, USA). At 1.75 hours after UVB exposure, mice were injected intraperitoneally with 0.3 mg/kg of the α 1 -adrenoceptor antagonist prazosin or the α 2 -adrenoceptor antagonist rauwolscine, or they received no α-adrenoceptor antagonist treatment. Fifteen minutes later (ie, 2 hours after UVB exposure), 10 μL of oxymetazoline cream 1%, brimonidine gel 0.33% or vehicle control was applied topically to a 1-cm 2 area of skin (n = 6 per group) ( Figure 1). Erythema was measured at baseline and for up to 6 hours after UVB exposure using a chromameter as described previously [16] and expressed as a mean haemoglobin (Hb) index. The Hb index for normal mouse skin is 1.0 to 1.3, with higher values indicating greater skin erythema. Comparisons between drug vs vehicle treatment were made using a 2-way analysis of variance with Bonferroni t tests; statistical significance was achieved at P < .05.

| Functional α-adrenoceptor activity in vitro
In the FLIPR assay, oxymetazoline induced transient Ca 2+ influx in the HEK293 cells by activating both α 1A -and α 2A -adrenoceptors, whereas brimonidine exhibited selectivity for α 2A -receptors (Table 1). Oxymetazoline was approximately four times more active on α 1A -adrenoceptors (half-maximal effective drug concentration, ie, concentration of a drug that provides half-maximal response [EC 50 ], 14 nM) compared with its activity on α 2A -adrenoceptors (EC 50 , 55 nM) and exhibited greater efficacy on α 1A -than α 2Aadrenoceptors when the maximum response was compared with the response achieved with the full α-adrenoceptor agonist noradrenaline. In this in vitro assay, oxymetazoline was an α 1A -adrenoceptor agonist with partial α 2A -adrenoceptor activity. In contrast, brimonidine was a selective and full α 2A -adrenoceptor agonist with an EC 50 of 5 nM and had only limited α 1A activity at concentrations of 600 to 2400 nM. The α 1A efficacy and potency and the α 2A efficacy of oxymetazoline and brimonidine were significantly different (P < .001).

| Activity in murine UVB erythema model
As the efficacy of partial agonists can be exaggerated in cellular assays in which receptors and their G proteins are overexpressed, we further investigated the α-adrenergic pharmacology of oxymetazoline and brimonidine in a mouse model of UVB-induced erythema.
The maximal erythema, measured as Hb index score, was achieved at 2 hours after UVB irradiation. In animals not exposed to UVB light, the mean Hb index score remained at baseline levels through 6 hours. The difference in mean Hb index values between the UVBexposed and non-UVB-exposed animals was approximately 0.

| D ISCUSS I ON
The results in the UV light-induced erythema model suggest that selectivity for α 2A -adrenoceptors. [17] The activities of brimonidine as a full α 2 -adrenoceptor agonist and oxymetazoline as a partial α 2 -adrenoceptor agonist have been consistently reported in studies of overexpressed cloned human receptors and rodent receptors. [18][19][20] In our experiments, the partial α 2 agonist activity of oxymetazoline detected in cells overexpressing α 2A receptors and the G qi5 protein was not observed on the endogenous vascular α 2 -adrenoceptors.
Repetitive UVB irradiation of hairless mice has been used previously to evaluate test compounds on skin photoageing, photodamage, inflammation and tumor development. [21][22][23][24] UVB-induced erythema also has been used clinically in healthy volunteers to assess the photoprotective and anti-inflammatory activity of potential therapeutic agents. [25,26] In the latter studies, UVB doses of approximately one to two times the minimal erythema dose were used, with erythema measured by chromameter. In the present study, we determined experimental conditions under which brief exposure to UVB induces skin erythema without causing skin ulcerations or other obvious skin damage. The acute response to UVB exposure shares multiple features with rosacea, including skin inflammation with erythema. [14,15] Whether the mechanisms underlying development of erythema in this murine model mimic those seen in patients with persistent facial erythema associated with rosacea remains to be determined. Nevertheless, the model affords the opportunity to evaluate and compare agents for their ability to produce in vivo vasoconstriction of cutaneous blood vessels. [4] The efficacy and safety of oxymetazoline and brimonidine in patients with moderate to severe persistent facial erythema of rosacea were demonstrated in pivotal phase 3 trials. [27][28][29] The agents appear to offer similar efficacy, but there may be differences in their respective safety profiles, which in turn may be related to differences in their mechanisms of action. However, no head-to-head studies have been performed. Worsening of erythema and flushing were the most common treatment-emergent adverse events in the pivotal clinical trials with brimonidine. [29,30] However, flushing was not observed, and application-site erythema and worsening of erythema after cessation of treatment, or rebound effect, were observed infrequently in patients who received oxymetazoline during the treatment period of the phase 3 studies of oxymetazoline. [31,32] While UVB-induced erythema is not a model of rebound erythema, on the basis of the present experimental findings, one could speculate that flushing and worsening of erythema with brimonidine may be manifestations of its full α 2 -adrenoceptor agonist activity at presynaptic sympathetic nerve terminals or on the vascular endothelium, resulting in vasodilation that counteracts the drug's direct postsynaptic vasoconstrictive effects. [6,7] Reduced surface expression of vascular smooth muscle α 2 -adrenoceptors in warm skin could further shift the balance towards vasodilation. [10] The α 2 -adrenoceptor agonist activity at presynaptic or endothelial sites resulting in vasodilation has also been suggested by Docherty and colleagues as one of several potential mechanisms responsible for the paradoxical erythema seen with brimonidine. [33] These authors suggested that high local concentrations of brimonidine in skin caused by skin barrier dysfunction could contribute to sustained activation of the presynaptic and endothelial α 2 -adrenoceptors or downregulation of the smooth muscle receptors. The authors also suggested activation of α 2 -adrenoceptors on perivascular inflammatory cells could contribute to sustained vasodilation. [33] Accordingly, the release of the inflammatory mediator tumor necrosis factorα. [34] Oxymetazoline, however, functions primarily as an α 1 -agonist, with minimal α 2 -agonist activity that may not be sufficient for producing clinically meaningful α 2 -adrenoceptor-mediated vasodilatory effects. The consequences of this hypothesis for patients with persistent facial erythema associated with rosacea remain to be evaluated in clinical studies.
In summary, both oxymetazoline and brimonidine produce cutaneous vasoconstriction in the murine UVB light-induced erythema model, but act through different α-adrenergic mechanisms.

ACK N OWLED G EM ENTS
Writing and editorial assistance was provided to the authors by

CO N FLI C T O F I NTE R E S T
At the time the study was performed, E. Hsia and M. Tian were employees of Allergan plc, and D. Gil is an employee of Allergan plc, and all may own stock/stock options in that company.

AUTH O R CO NTR I B UTI O N
E. Hsia and D. Gil designed the research study and wrote the paper.
M. Tian performed the research and data analysis.