The novel drug CS-891 inhibits 5*-reductase activity in freshly isolated dermal papilla of human hair follicles

ARTICLE

The local conversion of testosterone (T) to the more potent androgen dihydrotestosterone (DHT) by 5alpha-reductase (5aR) plays a pivotal role in the development of androgenetic alopecia (AGA) and hirsutism [1-3].

There are at least two isoforms of 5aR, namely type I and II, characterized by distinct molecular, structural and biochemical properties and by different tissue localization [4, 5]. Early studies have shown that individuals lacking 5aR activity have only sparse beard growth and do not develop AGA [6]. It is clear that a mutation within the gene encoding type II 5aR is responsible for this phenotype [7]. These findings plus better experimental evidence that local overexpression of type II 5aR in human hair follicles (HF) might be involved in androgen-mediated effects on susceptible HF [8, 9] suggest that type II 5aR is involved in the development of AGA. Thus the association of DHT with these disorders has generated great interest in 5aR as a therapeutic target.

Recently, the effectiveness of finasteride, a type II 5aR inhibitor, in treating AGA has been documented. Previous studies have shown that circulating DHT is lowered by 60-70% in men taking finasteride [10, 11]. The source of the residual circulating DHT is presumed to be due to type I 5aR activity which is not affected by finasteride.

Several novel compounds with potent dual inhibitory activity on both isoenzymes have been described [12, 13] and CS-891 is one of them. This compound might be likewise effective in the treatment of DHT-dependent disorders such as AGA. As a prerequisite for such an action, CS-891 should be able to inhibit 5aR activity in its target tissue, the dermal papillae (DP) of the HF. Here we report on the capability of CS-891 to inhibit 5aR activity in DP of human HF ex vivo.

Materials and methods

Chemicals and media

Unlabelled dehydroepiandrosterone (DHEA), androstendione (A-dione), androstenediol, T, DHT and L-glutamine, insulin, transferrin and sodium selenite were bought from Sigma (Deisenhofen, Germany). [1,2,6,7-³H(N)]-andost-4-ene-3,17-dione; [1,2,6,7-³H(N)]-andost-4-ene-3,17-diole; [1,2,6,7-³H(N)]-DHEA; [1,2,6,7-3H(N)]-DHT; [1,2,6,7-³H(N)]-T; [1,2,6,7-³H(N)]-androsterone were obtained from NEN Life Science (Boston, MA). Williams E medium was purchased from BioWhittaker/Serva (Heidelberg, Germany).

Preparation of HF and DP

Biopsies were taken from the occipital scalp of three female and seven male healthy volunteers with their written consent according to the regulations of our local ethics board committee. Intact, viable anagen HF were isolated according to published procedures [14]. Briefly, scalp specimens were placed in Williams E medium supplemented with L-glutamine (2 mM), insulin (10 µg/ml), transferrin (10 µg/ml) and sodium selenite (10 ng/ml). Under a stereo-dissecting microscope, a scalpel blade was used to remove the epidermis and upper parts of the corium. Intact HF were isolated from the subcutaneous fat with a watchmaker's forceps by gently gripping the outer root sheath of the HF and subsequent gentle traction. Single HF were then transferred to a new petri dish and the intact DP were microdissected under a stereomicroscope as described [15]. The basal tip of the HF was cut off just above the visible DP, turned inside out, and the DP was then cut off from its capillary stalk. The isolated DP were placed in individual wells of a 24-well culture dish.

Volume measurement of DP by digital imaging

Virtual volumes were calculated for DP immediately after microdissection and shortly before incubation experiments. For this purpose, digital images were taken at 40-fold magnification by use of an inverse microscope, a digital camera and LUCIA M software version 2.995beta (all from Nikon, Duesseldorf, Germany). The software was calibrated with an objective micrometer (Olympus, Tokyo, Japan). The volumes of the DP were calculated with the "Volume EqSphere" profile and these predefined calculation algorithms were provided by the LUCIA M software.

Measurement of 5aR activity

The activity of 5aR was determined with a radiochemical assay. Six DP from one donor were pooled for one measurement and incubated (37° C, in a humidified atmosphere containing 5% CO₂) for 24-39 hrs in supplemented serum-free Williams E medium in the presence of either 50 nM [1,2,6,7-³H]-T (98 Ci/mmol; Amersham, Braunschweig, Germany) alone or in combination with various concentrations of the dual type 5aR inhibitor CS-891 (Sankyo, Tokyo, Japan). The reaction products in the medium were extracted twice with three volumes of chloroform/methanol 2:1. Extracts were evaporated at 45° C and stored at - 70° C until analysis. The residue was taken up in 80 µl methanol and aliquots of 20 µl were analysed by high-performance liquid chromatography using a LC-10AD chromatograph (Shimadzu, Tokyo, Japan) and a Beckman Ultrasphere ODS column (5 µm, 4.6 x 250 mm) eluted with acetonitrile/methanol/water 1:3:3. The radioactive metabolites and the remaining substrate were quantified by use of a Flow Scintillation Analyzer 500TR Series (Canberra Packard, Meriden CT). Peaks were identified by their co-migration with ³H-labeled steroids. The 5aR activities were calculated from the relative amounts of DHT and normalized to the calculated volume of each DP as described [15].

Results

Our results show that CS-891 inhibited 5aR activity in a dose-dependent manner. Even 0.01 nM of CS-891 inhibited 5aR activity in all of the examined DP (Table I). These results were reproducible in all experiments.

Discussion

Finasteride, the first 5aR inhibitor developed [16], is a selective inhibitor of type II 5aR [7]. However, its clinical effectiveness to treat benign prostate hyperplasia has been less satisfactory than initially expected [11], and this has been attributed to an incomplete suppression of circulating DHT, due to the residual DHT synthesis through type I isoenzyme that is not inhibited by the compound. In support of this hypothesis, studies indicate that maximal doses of a type I 5aR inhibitor suppress serum DHT approximately 40% from baseline [17]. Dual 5aR inhibitors, however, were found to suppress circulating DHT by 85-95% [12], which is more than can be achieved by a type I or type II 5aR inhibitor alone [18].

The clinical usefulness of oral finasteride in men with AGA has been proven [19]. In theory, the oral use of a dual type 5aR inhibitor such as CS-891 could be likewise effective or, perhaps, even more effective, if the undesirable side effects remain within acceptable limits. With our results we can show that CS-891 is able to block 5aR activity in DP of human HF in a very effective way. A 90% reduction of 5aR was observed in all specimens exposed to doses as low as 0.1 nM. Using similar conditions in a different test panel [R. Hoffmann, personal communication], finasteride required a 10 times higher concentration (1 nM) in order to reach similar effects while type I inhibitors showed no effect. Our preliminary data seem to indicate that lower concentrations of CS-891 might be effective. The clinical relevance of these data remain, however, speculative. Provided CS-891 shows an acceptable safety profile, this drug could be effectively used in the treatment of AGA.

Article accepted on 19/10/00

CONCLUSION

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