The biodisposition and hypertrichotic effects of bimatoprost in mouse skin

Studies on bimatoprost were performed with two objectives: (i) to determine whether bimatoprost possesses hair growth-stimulating properties beyond eyelash hypertrichosis and (ii) to investigate the biodisposition of bimatoprost in skin for the first time. Bimatoprost, at the dose used clinically for eyelash growth (0.03%) and given once daily for 14 days, increased pelage hair growth in C57/black 6 mice. This occurred as a much earlier onset of new hair growth in shaved mice and the time taken to achieve complete hair regrowth, according to photographic documentation and visual assessment. Bimatoprost biodisposition in the skin was determined at three concentrations: 0.01%, 0.03% and 0.06%. Dose-dependent Cmax values were obtained (3.41, 6.74, 12.3 μg/g tissue), and cutaneous bimatoprost was well maintained for 24 h following a single dose. Bimatoprost was recovered from the skin only as the intact molecule, with no detectable levels of metabolites. Thus, bimatoprost produces hypertrichosis as the intact molecule.

derived cells were unlikely to be involved in spontaneous skin wound healing.
Sustained systemic and local clodronate depletion affected both circulating monocytes and WAM. While the clodronate treatment resulted mainly in a defect of Ly6c low circulating monocytes regardless of their level of MHCII (Fig. S1), it affected more drastically the MHCII hi WAM populations, regardless of Ly6c level of expression. This dichotomy between circulating and infiltrating populations could be explained by the fact that recruited monocytes (especially Ly6c hi ) may significantly change their phenotype once exposed to the wound's complex environment (10,16). The fact that the clodronate depletion reduced collagen transcription and affects to a larger extent the MHCII hi WAM may suggest that MHCII hi macrophages have a more prominent role in controlling collagen transcription during the early stages of healing, although this remains to be more specifically tested. (10). The molecular mechanism of the control by macrophages of Col 1 a2 transcription in wounds needs to be further investigated. The direct or indirect production or promotion of TGF-beta could be incriminated for Col 1a2 transcription by competent cells (17). However, other factors that may induce profibrotic mediators by intermedi-ate cell types could also be considered (5,(18)(19)(20). Naturally, because of the central role of macrophages in wound healing biology, these hypotheses are not mutually exclusive.
In conclusion, our data reveal that Col 1a2 transcription starts early during the healing process, only 2 days after wounding. Moreover, our data indicate that this early transcription of Col 1a2 can be controlled by macrophage depletion and more specifically by targeting MHCII hi macrophages. Further studies need to elucidate the underlying molecular mechanisms driving the macrophage-mesenchyme crosstalk.

Supporting Information
Additional Supporting Information may be found in the online version of this article: Data S1. Experimental design. Figure S1. Effect of clodronate treatment on circulating monocyte. The biodisposition and hypertrichotic effects of bimatoprost in mouse skin skin for the first time. Bimatoprost, at the dose used clinically for eyelash growth (0.03%) and given once daily for 14 days, increased pelage hair growth in C57/black 6 mice. This occurred as a much earlier onset of new hair growth in shaved mice and the time taken to achieve complete hair regrowth, according to photographic documentation and visual assessment. Bimatoprost biodisposition in the skin was determined at three concentrations: 0.01%, 0.03% and 0.06%. Dose-dependent C max values were obtained (3.41, 6.74, 12.3 lg/g tissue), and cutaneous bimatoprost was well maintained for 24 h following a single dose. Bimatoprost was recovered from the skin only as the intact molecule, with no detectable levels of metabolites. Thus, bimatoprost produces hypertrichosis as the intact molecule.
Key words: bimatoprost -hair -metabolism -prostanoid -skin Accepted for publication 26 November 2012 Background Bimatoprost was originally designed as an ocular hypotensive and has been extensively used for treating glaucoma (1,2). Eyelash hypertrichosis was observed as a side effect, and bimatoprost effects on eyelash growth have now been studied in detail (3)(4)(5). Because bimatoprost effects on other hair types have not been reported, we conducted studies on mouse pelage skin (6,7). The cutaneous biodisposition of bimatoprost was also investigated because the presence of substantially intact bimatoprost would be indicative of prostamide receptor involvement (8)(9)(10)(11)(12)(13).

Experimental design
Pelage hair growth was studied in C57/black 6 mice. The animals were shaved, and hair growth was evaluated by photographic documentation and visual assessment as (1) the time for onset of hair regrowth and (2) the time taken to completely cover the shaved area with new hair. Bimatoprost, at the dose used clinically to treat eyelash hypotrichosis, was given once daily for 14 days. The duration of the experiment was 42 days. Bimatoprost studies on skin biodisposition were conducted at 0.01%, 0.03% and 0.06% doses. Blood samples were also collected for analysis. Two biodisposition studies were performed, one for 24-h duration and the other for 21-day duration.

Results
The effects of once-daily bimatoprost on pelage hair growth are summarised in Fig 1. The time of onset of hair regrowth was essentially halved (Fig. 1a). More importantly, the time taken to cover the shaved back with regrown hair was dramatically and highly significantly reduced (Fig. 1b). Interestingly, bimatoprost appeared to produce a uniform regrowth of hair over the shaved area, rather than radiating out from a central locus as was observed for the control group.
The cutaneous drug levels of graded doses of bimatoprost are depicted in Fig. 2. High concentrations of bimatoprost were rapidly achieved in the skin, and these remained relatively well maintained for 24 hr at about 1 lg/g tissue. In a subchronic 21-day study, no substantial drug accumulation was apparent, but a clear dose-skin concentration relationship was apparent. Supplementary tables provide the C max values, areas under curve and absolute concentrations for both blood and skin. Bimatoprost was found only as the intact molecule in both skin and blood, with no evidence of hydrolytic conversion to 17-phenyl PGF 2a .

Conclusions
These studies show, for the first time, the biodisposition of bimatoprost in skin together with its effects on hair growth. The effects of bimatoprost on mouse pelage hair growth were investigated at the same dose as that employed for treating eyelashes (3,4). Cutaneous levels of bimatoprost achieved were dose dependent and were well maintained over a 1-day period. Results from a 21-day study provided no evidence for bimatoprost accumulation on repeated dosing with a 0.01% dose, but some accumulation was apparent for the 0.03% and 0.06% doses. Bimatoprost remained as the intact molecule, indicating that it exerts its effects on hair growth by stimulating prostamide receptors (8)(9)(10)(11)(12)(13).
Bimatoprost essentially remained as the intact molecule in mouse skin; the putative enzymatic hydrolysis product (17-phenyl PGF 2a ) was only detected twice in a total of 270 separate analyses of different skin and blood samples. Previously, in mouse eyes, it was shown that bimatoprost remains intact (14). Similarly, PGF 2a -ethanolamide (prostamide F 2a ) remains without significant hydrolytic degradation in mouse blood (15). On comparing mouse skin and monkey ocular tissue bioavailability (8), it appears that bimatoprost accesses cutaneous tissue more readily and the tissue levels are better maintained than in ocular tissues. These data suggest that once-daily administration to the skin should be adequate to obtain optimal hair growth. This contention presumes that there is a relatively homogeneous distribution of bimatoprost between the hair follicle and the skin layers. A further consideration is that bimatoprost was applied once daily for only 14 days in the hair growth experiment, as an expedient based on limited and overextended manpower resources. It follows that the dosing regimen used in these present studies may have underestimated the effect of bimatoprost on hair growth.
Bimatoprost has long been established as potently effective as the intact molecule, with a pharmacological profile distinct from prostanoid FP receptor agonists and their ester prodrugs (8,9,12,16). The pharmacology of bimatoprost closely resembles that of prostamide F 2a (9)(10)(11). Further pharmacological characterisation has been achieved by designing selective prostamide antagonists (17)(18)(19) and structural elucidation of the prostamide receptor (13). The results herein indicate hair growth as a further prostamide-mediated effect that may be mimicked by bimatoprost.
Bimatoprost was almost invariably found in blood samples from mice that received topical bimatoprost on shaved skin. Blood levels were about one-thousandth of those present in skin. Although bimatoprost was detected in pharmacologically active levels in mouse blood, this would be greatly 'diluted' in the blood by humans as they are about 5000 times heavier/larger. The human scalp area of coverage would be about 10-50 times greater than that of the shaved mouse skin. Presuming similar penetration characteristics for bimatoprost in mouse and human skin, the likely blood concentration in humans would be in the range of 50 pg/ml. Anticipated human blood levels of 10-100 pg/ml are beneath the pharmacologically active levels for bimatoprost (20).
In summary, bimatoprost stimulates the growth of mouse pelage hair. Bimatoprost was found as the intact molecule in mouse skin and blood, indicating that it stimulates hair growth by interacting with prostamide-sensitive receptors (8)(9)(10)(11)(12)(13). These studies indicate that the ability of bimatoprost to stimulate hair growth may extend beyond eyelashes, but effects on human scalp hair growth, for example, cannot necessarily be predicted from mouse pelage hair experiments. The human hair follicle expresses the prostanoid FP receptor (21), but expression of altFP4 (13) is required to predict a positive outcome with bimatoprost. . Furthermore, such molecular differences could result in clinical and histopathological differences among lesions (4,5). Melanomas classified as acral lentiginous melanomas (ALM) develop on volar skin, usually unexposed to UV radiation and are characterized by the presence of an atypical lentiginous proliferation. ALM carry a high number of genomic alterations compared with other melanoma subtypes and most of them account for a smaller proportion of genome (1,3). The molecular hallmarks of ALM are CCND1 amplifications (1,6,7) or somatic mutations in c-KIT (8).
Deletions in the 9p21 region where the CDKN2A gene is located are widely detected (9)(10)(11). However, other genes from this region could be implicated in melanoma because retention of the CDKN2A locus has been found in tumors with deletions at one or both sides of CDKN2A (10). Other reported aberrations include large amplifications of 12q (1,3), 7q or 20q and gains localized at 5p15, 11q13, 11q14 (3) and 22q11-13 (1).

Questions addressed
To characterize acquired molecular genomic alterations in a set of ALM from Spanish patients. The study was focused on specific chromosomal regions where genes involved in signalling pathways, cell cycle progression and apoptosis are located.

Experimental design
Seventeen fresh-frozen histopathologically confirmed primary ALMs based on Clark's classification were included. Sampling was guided by ex vivo dermoscopy (12) and documented by photography without altering the specimen, immediately fixed in formalin and embedded in paraffin (FFPE) for conventional histopathological diagnosis following the step-sectioning protocol for melanoma. Clinical data are described in Table S1. Genomic characterization of the BRAF, NRAS, CDKN2A and MC1R genes was performed by PCR-direct sequencing. Deletions of the 9p21 region and gains of