Cutaneous Androgen Metabolism: Basic Research and Clinical Perspectives

REVIEW ARTICLE

Cutaneous Androgen Metabolism: Basic Research and Clinical Perspectives WenChieh Chen, Diane Thiboutot,n and Christos C. Zouboulisw Department of Dermatology, College of Medicine, National Cheng Kung University, Tainan, Taiwan; nDepartment of Dermatology, Milton Hershey Medical Center, Pennsylvania State University, Hershey, PA, U.S.A.; wDepartment of Dermatology, University Medical Center Benjamin Franklin, The Free University of Berlin, Berlin, Germany

The skin, especially the pilosebaceous unit composed of sebaceous glands and hair follicles, can synthesize androgens de novo from cholesterol or by locally converting circulating weaker androgens to more potent ones. As in other classical steroidogenic organs, the same six major enzyme systems are involved in cutaneous androgen metabolism, namely steroid sulfatase, 3b-hydroxysteroid dehydrogenase, 17b-hydroxysteroid dehydrogenase, steroid 5a-reductase, 3a-hydroxysteroid dehydrogenase, and aromatase. Steroid sulfatase, together with P450 side chain cleavage enzyme and P450 17-hydroxylase, was found to reside in the cytoplasm of sebocytes and keratinocytes. Strong steroid sulfatase immunoreactivity was observed in the lesional skin but not in unaffected skin of acne patients. 3b-hydroxysteroid dehydrogenase has been mainly immunolocalized to sebaceous glands, with the type 1 being the key cutaneous isoenzyme. The type 2 17b-hydroxysteroid dehydrogenase isoenzyme predominates in sebaceous glands and exhibits greater reductive activity in glands from facial areas compared with acne nonprone areas. In hair folli-

cles, 17b-hydroxysteroid dehydrogenase was identi¢ed mainly in outer root sheath cells. The type 1 5a-reductase mainly occurs in the sebaceous glands, whereby the type II isoenzyme seems to be localized in the hair follicles. 3a-hydroxysteroid dehydrogenase converts dihydrotestosterone to 3a-androstanediol, and the use of 3a-androstanediol glucuronide serum level to re£ect the hyperandrogenic state in hirsute women may be a reliable parameter, especially for idiopathic hirsutism. In acne patients it is still controversial if 3a-androstanediol glucuronide or androsterone glucuronide could serve as suitable serum markers for measuring androgenicity. Aromatase, localized to sebaceous glands and to both outer as well as inner root sheath cells of anagen terminal hair follicles, may play a ‘‘detoxifying’’ role by removing excess androgens. Pharmacologic development of more potent speci¢c isoenzyme antagonists may lead to better clinical treatment or even prevention of androgen-dependent dermatoses. Key words: acne vulgaris/androgen antagonists/androgenetic alopecia/androgens/ hirsutism. J Invest Dermatol 119:992 ^1007, 2002

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to progesterone by the action of 3b -ol-dehydrogenase/D4,5 -isomerase or to 17a-hydroxypregnenolone by 17a-hydroxylase. The ring system ‘‘sterane’’ of the steroid hormones, carrying a cyclopentanoperhydrophenanthrene nucleus, is a stable structure that cannot be broken down by mammalian cells. Conversion of active hormones to less active or inactive forms involves alteration of ring substituents rather than the ring structure itself. Sex hormones can be easily distinguished by the carbon numbers, C-19 being androgens, C-18 being estrogens, and C-21 being progestenoids. Androgens are derivatives of androstane and contain either a keto group [e.g., dehydroepiandrosterone (DHEA) and androstenedione] or hydroxy group [testosterone and 5a-dihydrotestosterone (5a-DHT)] at position 17 of the ring system. Cholesterol synthesis in mammalian systems is regulated by the key enzyme, 3 -hydroxy-3 -methylglutaryl coenzyme A reductase, and it has been con¢rmed that this process also occurs in the epidermis and the sebaceous glands (Menon et al, 1985; Smythe et al, 1998). Recent work demonstrated the cutaneous expression of steroidogenic acute regulatory protein, cytochrome P450 cholesterol side-chain cleavage (P450scc) and cytochrome P450 17a-hydroxylase (P450c17), suggesting the cutaneously derived cholesterol could be further used

ex hormones, like other steroid hormones, are synthesized from their major parental precursor cholesterol, which undergoes side chain cleavage by the mitochondrial P450 side chain cleavage enzyme (P450scc) to form D5 -pregnenolone, releasing a C6 aldehyde. D5 pregnenolone, the required intermediate compound in the synthesis of all steroid hormones, can be converted intracellularly

Manuscript received February 23, 2002; revised April 20, 2002; accepted for publication May 30, 2002 Reprint requests to: Prof. Christos C. Zouboulis, Department of Dermatology, University Medical Center Benjamin Franklin, The Free University of Berlin, Fabeckstrasse 60- 62, 14195 Berlin, Germany. Email: [email protected] This work is dedicated to Prof. Dr. Prof. h.c. Constantin E. Orfanos and his life-long scienti¢c work. Abbreviations: 3a -Adiol, 5a-androstane-3a, 17b-diol (3a -androstanediol); DHEA, dehydroepiandrosterone; DHEA-S, dehydroepiandrosteronesulfate; 5a-DHT, 5a-dihydrotestosterone; DP, dermal papilla; 3b-D5-HSD, 3b -hydroxysteroid dehydrogenase/D5
4 isomerase; NAD, nicotinamide adenosine dinucleotide; NADP, nicotinamide adenosine dinucleotide pyrophosphatase; ORS, outer root sheath cells

0022-202X/02/$15.00 Copyright r 2002 by The Society for Investigative Dermatology, Inc. 

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Figure 1. Pathways of cutaneous androgen metabolism and the converting enzymes.

as a substrate for de novo steroid hormone synthesis in human epidermis and the sebaceous gland.1,2 On the other hand, steroid hormones inhibit the expression of 3 -hydroxy-3 -methylglutaryl coenzyme A reductase in SZ95 sebocytes at the mRNA level in a negative feedback regulation process (Zouboulis et al, 1999; 2002). In classical ‘‘central’’ steroidogenic organs such as the gonads and the adrenal gland, testosterone can be derived mainly from two pathways: (i) progesterone, 17a-hydroxyprogesterone, D4 -androstenedione, or (ii) 17a-hydroxypregnenolone, DHEA, D5 -androstenediol (Stauss and Pochi, 1969; Witt and Thorneycroft, 1990). Alternatively, in many ‘‘peripheral’’ organs, such as the skin, the potent tissue androgen testosterone results from the conversion of circulating dehydroepiandrosterone sulfate (DHEA-S), a weak but most abundant androgen in higher primates, through the serial action of steroid sulfatase, 3b -hydroxysteroid dehydrogenase/D5
4 isomerase (3b -D5-HSD), and 17b-HSD (Labrie, 1991; Fritsch et al, 2001). Testosterone can be further ‘‘activated’’ to the physiologically most potent tissue androgen 5a-DHT through the action of 5a-reductase or ‘‘inactivated’’ to estradiol by aromatase (Kaufman, 1996). In contrast to classical endocrinology depicting steroid hormone formation and secretion from classical steroidogenic organs, such as gonads and adrenal glands, this peripheral ‘‘on the spot’’ intracellular hormone synthesis/action, now coined as ‘‘intracrinology’’, has been shown to take place in various peripheral, hormone-target tissues such as placenta, prostate, adipose tissue, skin, and skin appendages (Fig 1) (Labrie et al, 2000a; Zouboulis, 2000a). The androgen-sensitive skin appendages (sebaceous gland, hair follicle, and sweat gland) each metabolize androgens in a characteristic 1 Billich A, Rot A, Lam C, Schmidt JB, Schuster I: Immunohistochemical localization of steroid sulfatase in acne lesions: Implications for the contribution of dehydroepiandrosterone sulfate to the pathogenesis of acne. Hormone Res 53:99, 2000 (Abstr.) 2 Thiboutot DM, Sivarajah A, Gilliland K, Cong Z, Clawson G: The skin as steroidogenic tissue: Enzymes and cofactors involved in the initial steps of steroidogenesis are expressed in human skin and rat preputial sebocytes. J Invest Dermatol 117:410, 2001 (Abstr.)

pattern; however, sweat glands and sebaceous glands account for the vast majority of androgen metabolism in skin (Deplewski and Rosen¢eld, 2000). In postmenopausal women, 100% of the active sex steroids are synthesized in peripheral target tissues from inactive steroid precursors, whereas, in adult men, approximately 50% of androgens are locally made in intracrine target tissues (Labrie et al, 2000b). Not much is known, however, about the extent to which (i) the de novo cutaneous androgenesis from epidermally formed cholesterol, or (ii) the locally active conversion of potent androgens from circulating DHEA contribute to the androgens formed in the skin. Here is a review of the recent advances in the understanding of cutaneous androgen synthesis and metabolism, trying to correlate the expression of di¡erent converting isoenzymes to the pathogenesis of androgen-dependent dermatoses, such as acne vulgaris, hirsutism, and androgenetic alopecia. The current therapeutics and future developments based on this continuously expanding knowledge are also discussed. HISTORICAL REVIEW

The ¢rst recognition of the role of androgens in the pathogenesis of cutaneous disorders probably came from Aristotle as early as the fourth century BC, as he noticed the relation between the occurrence of baldness (androgenetic alopecia) and the gender state or the sexual maturity (Montagna, 1963). It was not until 1942 as Hamilton’s pioneering work on castrates subjected to testosterone injections (Hamilton, 1942), which for the ¢rst time provided the scienti¢c evidence and hence provoked further investigation on the androgen metabolism in the skin (Takashima, 1990). The ¢rst androgen hormone to be characterized was androsterone isolated from the urine of adult men in 1931 (Tausk, 1968). Testosterone was then demonstrated to be the androgen secreted from testis in 1935. Experimental work on the androgen metabolism in vitro began intensively in 1950s by applying large-scale perfusion (Caspi et al, 1953; Caspi and Hechter, 1954) or by incubating animal internal organ homogenates, e.g., rat liver (Schneider and Horstmann, 1951; Taylor, 1954; Kochakian and Stidworthy, 1954)

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with various steroids, such as cortisone, desoxycorticosterone, progesterone, and androstenedione. Metabolites were separated and isolated mainly by thin-layer adsorption chromatography with paper, aluminum or silica gels as stationary phase. During the 1950s and 1960s increasing evidence accumulated that the skin might be one of the sites of peripheral androgen metabolism (Wotiz et al, 1956; Stauss and Pochi, 1969). The presence of di¡erent hydroxysteroid dehydrogenases, including 3a -HSD, 3b HSD, and 17b-HSD, was soon histochemically revealed in the human skin (Baillie et al, 1965), especially in the sebaceous gland (Baillie et al, 1966). Active androgen metabolism was further con¢rmed by incubating human skin with various (radiolabeled) steroids (Wotiz et al, 1956; Gallegos and Berliner, 1967; Gomez and Hsia, 1968); i.e., the activity of 17b-HSD in the skin was demonstrated by showing the cutaneous conversion of androstenedione to testosterone or 5a-androstanedione to 5a-DHT (Gomez and Hsia, 1968). Studies on rat and human prostate showed that 5a-DHT, rather than testosterone, might be the active androgenic hormone at the target site (Farnsworth and Brown, 1963; Bruchovsky and Wilson, 1968) and this conversion could also take place in the skin (Wilson and Walker, 1969; Flamigni et al, 1971; Bingham and Shaw, 1973). Attempts to inhibit 5a-reductase in the skin cells were soon made (Zerhouni et al, 1979; Leshin and Wilson, 1982). Biochemical characterization of the di¡erent androgen converting enzymes, including the enzyme activity, coenzymes, kinetic properties, and intracellular distribution was performed between 1970s and 1980s. Most of these data were based on the studies on congenital adrenal hyperplasia, various virilizing and feminizing syndromes, and hirsutism (New et al, 1981; Mauvais-Jarvis et al, 1981; James and Few, 1985). Because of the variable stability of di¡erent enzymatic activities in broken cell preparations and the di⁄culties in puri¢cation and stabilization of these membrane-bound enzymes, it was not until the 1990s when the introduction of newer molecular biologic methods, such as RNA expression, cloning, and analysis that better elucidation and comparison of the genetic structure, function, ontogeny, and tissue-speci¢c expression of these converting isoenzymes became feasible (Labrie, 1991; Russell and Wilson, 1994). STEROID SULFATASE

The steroid sulfatase is a microsomal enzyme widely distributed in human tissues that catalyzes the hydrolysis of sulfated 3 -hydroxy steroids to the corresponding free active 3 -hydroxy steroids. Early studies in vitro and in vivo have shown that the human steroid sulfatase activity appears after birth, such as in foreskin and infantile abdominal skin (Kim and Herrmann, 1968). Major breakthroughs in the evaluation of cutaneous expression of steroid sulfatase came from the study on its role in X-linked recessive ichthyosis, which has been shown to be associated with the de¢ciency of the steroid sulfatase/arylsulfatase C (Schlammadinger et al, 1987; Herrmann, 1989; Herrmann et al, 1989). The steroid sulfatase gene is localized to the distal short arm of the X chromosome and most X-linked ichthyosis patients present large deletions of the steroid sulfatase gene and £anking markers, whereas a minority show a point mutation or partial deletion of the steroid sulfatase gene (Bonifas and Epstein, 1990; Basler et al, 1992; Alperin and Shapiro, 1997; Morita et al, 1997; Sugawara et al, 2000; Valdes-Flores et al, 2001). Activity of steroid sulfatase was also demonstrated in cultured keratinocytes (Milewich et al, 1988) in which cholesterol sulfate could inhibit sterol esteri¢cation. These data suggest a novel role for cholesterol sulfate as a modulator of cellular lipid biosynthesis (Williams et al, 1987). In patients with X-linked recessive ichthyosis, cholesterol sulfate accumulation rather than cholesterol de¢ciency seemed to be responsible for the barrier abnormality (Zettersten et al, 1998). On the other hand, in the occipital scalp of normal healthy subjects as well as in the beard, the speci¢c activity of steroid sulfatase was signi¢cantly higher in the hair dermal papillae (DP) as com-

pared with that in the connective tissue sheaths or root sheaths (Ho¡mann et al, 2001). 3b -D5-HSD

This enzyme catalyzes an obligatory step in the biosynthesis of androgens, estrogens, mineralocorticoids, and glucocorticoids; the oxidation/isomerization of 3 -b-hydroxy-5-ene steroids (D5 3b -hydroxysteroids) into the corresponding 3 -keto- 4 -ene steroids (D4 -3 -ketosteroids), i.e., the transformation of DHEA into androstenedione and androstenediol into testosterone, respectively. This process could be seen as the ¢rst step to form more potent androgens and to amplify the androgenic e¡ect. 3b -D5HSD is found not only in classic steroidogenic tissues (placenta, adrenal cortex, ovary, and testis), but also in several peripheral tissues, including skin, adipose tissue, breast, lung, endometrium, prostate, liver, kidney, epididymis, and brain (Labrie et al, 1992). This tissue-speci¢c manner of expression involves separate mechanisms of regulation. An important feature in liver and kidney (at least of hamster, mouse, rabbit, and rat) is the sexual dimorphic nature of 3b -D5-HSD. Two types of human 3b -D5HSD cDNA clones have been characterized; the corresponding genes are located in chromosome 1p13.1, containing four exons and three introns with a total length of 7.7^7.8 kbp, and encoding proteins of 371 and 372 amino acids, respectively, which share 93.5% homology and both prefer NAD þ as cofactors (Pelletier et al, 1992; Labrie et al, 1995; Simard et al, 1996). Human type 1 3b -HSD is the almost exclusive mRNA species expressed in the skin, mammary gland, and placenta (syncytial trophoblast), whereas the type 2 isoform is almost exclusively expressed in the adrenal cortex and the gonads. Early studies demonstrated 3b -D5-HSD enzyme activity in ¢broblasts (Gallegos and Berliner, 1967), and that the sebaceous gland possessed the highest activity in the human skin (Itami and Takayasu, 1982; Simpton et al, 1983), which was further con¢rmed by the immunohistochemical localization of 3b -D5-HSD speci¢cally in sebaceous glands (Dumont et al, 1992; Sawaya and Penneys, 1992). In cultured skin cells, 3b -D5-HSD mRNA was only detected in normal and immortalized sebocytes (SZ95), but neither in normal keratinocytes and in HaCaT cells nor in melanoma cells (MeWo melanoma cells) (Fritsch et al, 2001). The type 1 isotype of 3b -D5-HSD could exclusively be detected. By converting androstenediol to testosterone, an intense enzyme activity of 3b -D5-HSD was also revealed in the DP of human terminal hair follicle (Ho¡mann et al, 2001). The enzyme activity did not correlate with sebum excretion rate (Simpton et al, 1983) and the enzyme expression did not vary with body site or sex (Sawaya and Penneys, 1992). 17B-HSD

The last and key step in the formation of androgens and estrogens is catalyzed by members of the family of 17b-HSD, whereas the reduction step by 17b-HSD is essential for the formation of the more active androgens and the oxidative reaction inactivates the potent sex steroids. These events take place in the same cell where synthesis occurs. The 17b-HSD thus provide each cell with the means of precisely controlling the intracellular concentration of each sex steroid according to local needs (Zouboulis, 2000a). To date, seven types of human 17b-HSD have been cloned, sequenced, and characterized, designated types 1^7 in the chronologic order of their isolation (Biswas and Russell, 1997; Labrie et al, 1997; Krazeisen et al, 1999; Pelletier et al, 1999). The type 1 17b-HSD (17b-HSD1), encoded in chromosome 17q21, is a cytosolic enzyme that was found in ovary, placenta, and in breast cancer cells. 17b-HSD2, encoded in chromosome 16q24, is a microsomal enzyme that was found in placenta, endometrium, normal breast cells, prostate, liver, small intestine, kidney, pancreas, and colon. 17b-HSD3, encoded in chromosome 9q22,

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was only detected in testis and its importance in male steroid hormone physiology was underscored by its de¢ciency in a form of male pseudohermaphroditism, the only genetic mutation of these group described until now (Andersson and Moghrabi, 1997). 17bHSD4 is a peroxisomal enzyme with its mRNA mainly expressed in liver, heart, prostate, testis, and prostate cancer cell lines. 17b-HSD5, encoded in chromosome 10p14,15, was found in the placenta, ovary, endometrium, mammary gland, testis, liver, skeletal muscle, and skin (Labrie et al, 1997, 2000b; Pelletier et al, 1999; Qin and Rosen¢eld, 2000). 17b-HSD6, isolated by expression cloning of rat and human prostate, oxidizes 5a-androstane3a, 17b-diol[3a -androstanediol (3a -Adiol)], to androsterone (Biswas and Russell, 1997). It is a member of the short chain dehydrogenase/reductase family and shares 65% sequence identity with retinol dehydrogenase 1 (RoDH1), which catalyzes the oxidation of retinol to retinal. Expression of rat and human RoDH cDNA in mammalian cells is associated with the oxidative conversion of 3a -Adiol to 5a-DHT. Thus, 17b-HSD6 and RoDH play opposing roles in androgen action; 17b-HSD6 inactivates 3a -Adiol by conversion to androsterone and RoDH activates 3a -Adiol by conversion to 5a-DHT (Biswas and Russell, 1997). The multisubstrate nature of these short chain dehydrogenase/reductase enzymes allows for retinoid/steroid interactions (Napoli, 2001). It is unknown if these interactions could partially explain the speci¢c anti-proliferative e¡ect of isotretinoin (13 -cis retinoic acid) on sebocytes, whose growth and di¡erentiation is strongly in£uenced by androgens (Zouboulis et al, 1994; Tsukada et al, 2000). The human 17b-HSD7 shows 78% and 74% amino acid identity with rat and mouse 17b-HSD7, respectively. These enzymes are responsible for estradiol production in the corpus luteum during pregnancy, but are also present in placenta and several steroid target tissues (breast, testis, and prostate) as revealed by reverse transcription^polymerase chain reaction (Krazeisen et al, 1999). Recently, 17b-HSD8, also known as Ke6 gene found in the HLA region, was shown to be able to transform e⁄ciently estradiol to estrone in transfected HEK-293 cells (Luu-The, 2001). On the whole, the type 1, 3, 5, and 7 isoenzymes, using NADPH as cofactor, seem to reduce weak steroid hormones to more potent ones (e.g., DHEA into androstenediol, androstenedione to testosterone, 5a-androstanedione to 5a-DHT, estrone to estradiol), whereas type 2, 4, 6, and 8 isoenzymes, using NAD þ as cofactor, work in the opposite oxidizing direction and seem to play a general role in the peripheral inactivation of androgens (Labrie et al, 1997; Luu-The, 2001). Only the 17b-HSD3, however, is responsible for pseudohermaphroditism in de¢cient boys. The cutaneous expression of 17b-HSD was mainly demonstrated in the pilosebaceous unit and epidermal keratinocytes. In hair follicles, 17b-HSD was histochemically localized to outer root sheath cells (ORS) (Crovato et al, 1973). The fresh plucked anagen hairs mainly containing keratinocytes from the inner root sheath and ORS expressed very high levels of 17b-HSD2 and moderate levels of 17b-HSD1 (Courchay et al, 1996). This is compatible with the early studies showing androstenedione as the major metabolite of cultured human hair follicle keratinocytes incubated with radiolabeled testosterone (Dijkstra et al, 1987; Sonada et al, 1993). The human sebaceous gland possesses the cellular machinery needed to transcribe the genes for the type 1^5 isoenzymes of 17b-HSD (Thiboutot et al, 1998; Labrie et al, 2000b), among them a strong signal of 17b-HSD2 mRNA was detected (Fritsch et al, 2001). At the protein level, the type 2 isoenzyme of greater oxidative activity predominates in intact sebaceous glands, suggesting its protective role against the e¡ects of excessive amounts of potent androgens in vivo (Thiboutot et al, 1998). Greater reductive activity of 17b-HSD was noted in sebaceous glands from facial areas compared with acne non-prone areas, suggesting an increased net production of potent androgens in facial areas (Thiboutot et al, 1998) and human sebocytes but not keratinocytes expressing 17b-HSD3, undersigning the major regulatory role of the sebaceous gland in androgen metabolism in the skin (Fritsch et al, 2001). The speci¢c localization and importance of 17b-HSD2 in the physiology of sebaceous gland seems to

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be circumstantially evidenced by the description of ‘‘normal’’ development of pubertal acne and male distribution of body hair in male pseudohermaphroditism due to 17b-HSD3 de¢ciency syndrome (Saez et al, 1971; Virdis et al, 1978). Noteworthy is the report of a distinct syndrome of alopecia totalis (actually atrichia), ichthyosis and male pseudohermaphroditism due to steroid 17bHSD de¢ciency in an Israeli-Arab newborn infant (Kauschansky et al, 1998). 17b-HSD enzyme activity was also shown in cultured epidermal keratinocytes (Itami and Takayasu, 1981; Dijkstra et al, 1987; Hughes et al, 1997; Fritsch et al, 2001) and in the microdissected apocrine sweat gland (Sonada et al, 1993). In primary cultured keratinocytes, which predominantly converted estradiol to estrone, mRNA expression of the type 1, 2, and 4 17b-HSD isoenzymes was detected and treatment with 1,25-dihydroxyvitamin D3 upregulated the type 2 mRNA (Hughes et al, 1997). In HaCaT cells, a commonly used keratinocyte cell line, however, only the type 2 isoenzyme was detected (Fritsch et al, 2001). 5a-REDUCTASE

5a-reductase is the enzyme that catalyzes the conversion of testosterone to 5a-DHT. The conversion of testosterone to 5aDHT ampli¢es the androgenic signal through two mechanisms: (i) 5a-DHT, unlike testosterone, cannot be aromatized to estrogen, thus its e¡ect remains purely androgenic, and (ii) in vitro 5aDHT binds to the human androgen receptor with greater a⁄nity than testosterone does, and the 5a-DHT/androgen receptor complex appears to be more stable (Anderson and Liao, 1968). Over the last decade, molecular cloning studies have characterized two genes that encode two isoenzymes of 5a-reductase, namely, type 1 and type 2; the former exists predominantly in the skin, whereas the latter in the prostate (Andersson and Russell, 1990; Andersson et al, 1991). Genetically, the type 1 isoenzyme is encoded by the SRD5A1 gene on the distal arm of chromosome 5 (band p15), whereas the type 2 isoenzyme by the SRD5A2 gene on chromosome 2 (band p23) (Russell and Wilson, 1994). Both genes contain ¢ve exons separated by four introns. Both isoenzymes are hydrophobic with approximately 50% identity in their amino acid sequences. Whereas type 1 5a-reductase has a broad alkaline pH optima of 6.0^8.5 and demonstrates relatively moderate a⁄nity for steroid substrates (Km: 1^5 mM), the type 2 5a-reductase has a narrow acidic pH optima of 5.0^6.0 and demonstrates high a⁄nity for substrates (Km: 4^50 nm) (Russell and Wilson, 1994). These di¡erent enzyme kinetics may have some important implications for disease states. Cutaneous distribution of type 1 isoenzyme in vivo was immunohistochemically identi¢ed in sebaceous glands, epidermis, eccrine sweat glands, apocrine sweat glands (in normal ones as well as in people with osmidrosis), and hair follicles (ORS, DP, matrix), as well as in the endothelial cells of small vessels and the Schwann cells of cutaneous myelinated nerves (Luu-The et al, 1994; Eicheler et al, 1995; Courchay et al, 1996; Sato et al, 1998). In the skin the activity of the type 1 5a-reductase is concentrated in sebaceous glands and is signi¢cantly higher in sebaceous glands from the face and scalp compared with nonacne-prone areas (Thiboutot et al, 1995). In vitro, type 1 5a-reductase was detected in the cytoplasm of cultured human sebocytes, keratinocytes and HaCaT cells, ¢broblasts, dermal microvascular endothelial cells, hair DP cells from various body sites, melanocytes, and melanoma cells (Chen et al, 1998a, b; Ando et al, 1999; Fritsch et al, 2001). Northern blot studies revealed most abundant type 1 mRNA in neonatal foreskin keratinocytes, followed by adult facial sebocytes, and stronger expression in DP from occipital hair cells than from beard (Chen et al, 1998a). Within hair follicles, prominent immunostaining of type 2 5a-reductase was localized in the inner layer of ORS, inner root sheath, the infundibulum of hair follicle, and sebaceous ducts (Bayne et al, 1999). Regional studies showed the type 2 mRNA present in beard DP, but absent from occipital scalp and axillary

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Table I. Cutaneous distribution of steroidogenic isozymes

Steroid sulfatase 3b -HSD 17b-HSD 5a-reductase Aromatase 3a -HSD

Isoenzymes/major cutaneous isoenzyme

Encoding genes of the cutaneous isoenzyme

Cutaneous distribution

? Types 1 and 2/Type 1 Types 1-7/Type 2 Types 1-2/Type 1 ? Types 1-3/Type 3?

chromosome Xp22 chromosome 1p13.1 chromosome 16q24 chromosome 5p15 chromosome 15q21 chromosome 10p15

K, F, DP SG SG, K, A SG, K, F, En, M, A, Ec, DP, ORS SG, ORS, K, F SG, K, M, F

? Not known, HSD: hydroxysteroid dehydrogenase, K: keratinocytes, F: ¢broblast, DP: hair dermal papilla, SG: sebaceous glands, A: apocrine sweat gland, Ec: eccrine sweat gland, En: vascular endothelial cells, M: melanocytes, ORS: outer root sheath

DP (Ando et al, 1999). The type 2 isoenzyme in beard DP has three times higher activity than the type 1 5a-reductase present in the occipital scalp and axillary DP (Itami et al, 1994; Eicheler et al, 1998). The speci¢c activity of 5a-reductase in the hair DP exceeded those in other hair follicle compartments (connective tissue sheaths and ORS) by a factor of at least 14 in the scalp and at least 80 in the beard (Eicheler et al, 1998). The beard DP cells appeared to generate more 5a-DHT than those from nonbalding scalp hair follicles (Thornton et al, 1993); however, the individual freshly isolated intact DP was shown to possess considerably di¡erent levels of ex vivo enzyme activities (Niiyama et al, 2000). Taken together, whereas the type 1 5a-reductase has been de¢nitely demonstrated in sebaceous glands, the isoenzyme distribution in hair follicles is less well-de¢ned, probably due to: (i) physiologic variation of the enzyme activity in di¡erent body regions; (ii) utilization of di¡erent polyclonal/monoclonal antibodies; and (iii) inadequate assessment of enough specimens. More evidence is needed to de¢ne better the existence of the type 1 isoenzyme in hair follicles (Luu-The et al, 1994; Eicheler et al, 1995; Chen et al, 1998a; Ando et al, 1999; Bayne et al, 1999) and the precise localization of the type 2 isoenzyme within the hair follicles (in ORS or in DP) (Ho¡mann and Happle, 1999; Bayne et al, 1999). CYTOCHROME P450 19 (AROMATASE)

Aromatase, the product of the CYP19 gene, catalyzes three consecutive hydroxylation reactions converting C19 androgens to C18 estrogens. It belongs to the cytochrome P450 superfamily of enzymes, which contains 36 gene families and over 300 characterized members (Simpson et al, 1997). Recently, aromatase has been reported to be present in various extragonadal tissues and its expression is regulated in part by means of tissue-speci¢c promoters through the alternative splicing mechanism on multiple exons 1 (Simpson, 2000a). At least six variants of exon 1 have been described; exons 1a, 1b, 1c, 1d, 1e, and 1f that are speci¢c for expression in the placenta, skin ¢broblasts/fetal liver/adipose tissue/ vascular tissue, ovary, ovary/prostate, placenta, and fetal brain, respectively (Harada, 1998, 1999). By immunohistochemical examination, aromastase was found in the ORS of anagen, terminal hair follicles, and in sebaceous glands, but rarely in telogen hair follicles. The expression did not vary with body site or sex in normal subjects, but seemed to di¡er in patients with androgenetic alopecia (Sawaya and Penneys, 1992; Sawaya and Price, 1997; see later). Semiquantitative reverse transcription^polymerase chain reaction methods, however, showed poor expression of aromatase in the plucked hair containing ORS and inner root sheath keratinocytes (Courchay et al, 1996). The aromatase enzyme activity has also been demonstrated in keratinocytes cultured in serum-free medium (Hughes et al, 1997), ¢broblasts from both genital and nongenital skin (Svenstrup et al, 1990), and ¢broblasts from adipose tissue (Rink et al, 1996). In most tissues, aromatase is induced by cyclic adenosine monophosphate or factors utilizing cyclic adenosine monophosphate as a second messenger, whereas

androgens and glucocorticoids have been shown to be able to stimulate the expression or activity of aromatase (Stillman et al, 1991; Zhao et al, 1995; Harada, 1999). To understand better the clinical signi¢cance of aromatase in androgen-dependent dermatoses, it would be interesting to study patients a¡ected with aromatase de¢ciency or excess syndromes to observe the occurrence of acne or androgenetic alopecia (Simpson, 1998, 2000b; Bulun et al, 1997; Bulun, 2000).

3a -HSD

Mammalian 3a -HSD regulate steroid hormone levels. Hepatic 3a -HSD inactivate circulating androgens, progestins, and glucocorticoids by catalyzing the conversion of 3 -ketosteroids to 3a -hydroxy compounds, e.g., the transformation of 5a-DHT into 3a -Adiol. In the prostate, it acts as a molecular switch and controls the amount of 5a-DHT that can bind to the androgen receptor, whereas in the brain 3a -HSD can regulate the amount of tetrahydrosteroids that can alter g-aminobutyric acid receptor function (Penning et al, 1997). Molecular cloning indicates that these mammalian 3a -HSD are highly homologous proteins, prefer NADPH as cofactors and belong to the aldo-keto reductase superfamily, including also 17b-HSD5, ovarian 20aHSD as well as the steroid 5b-reductases (Dufort et al, 1996; Penning 1999). The human type 1 and type 3 isoenzymes, sharing 81.7% identity, both e⁄ciently catalyze the transformation of 5b-DHT into 3a -Adiol in intact vector-transfected transformed human embryonic kidney cells (Dufort et al, 1996, 2001). The human type 1 3a -HSD is expressed exclusively in the liver, whereas the type 3 is more widely expressed in the liver, adrenal, testis, brain, prostate, and HaCaT keratinocytes. The expression of the type 2 3a -HSD was shown in the human prostate, and there it inactivated 5a-DHT through its 3 -ketosteroid reductase activity (Lin et al, 1997). Early studies showed the enzyme activity of 3a -HSD in cultured human skin ¢broblasts and the 5a-DHT reduction is three times higher in ¢broblasts from genital areas than from nongenital areas (Sultan et al, 1983). The expression of 3a -HSD mRNA has been demonstrated in sebocytes (SZ95), keratinocytes (HaCaT cells), and melanoma cells (MeWo), but the isotype has not yet been identi¢ed (Fritsch et al, 2001). Keratinocytes were found to inactivate tissue active androgens in a more pronounced manner than sebocytes do by engagement of 3a -HSD (Fritsch et al, 2001). Clinically, it is still controversial if serum levels of 3a-Adiol conjugates (3a-Adiol glucuronide or 3a-Adiol sulfate) serve as reliable indicator for cutaneous 5a-DHT formation (see later, Lookingbill et al, 1988a; Horton, 1992; Vogt et al, 1992), or are just a marker of adrenal steroid production and metabolism (Rittmaster, 1995). Interestingly, rat liver 3a-HSD was found to be a target for nonsteroidal anti-in£ammatory drugs (Pawlowski et al, 1991). Cutaneous distribution of steroidogenic isozymes is summarized in Table I.

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ANDROGEN-DEPENDENT DERMATOSES Acne vulgaris Decades of investigation have ¢rmly established that the development and secretory activity of sebaceous glands are strikingly in£uenced by hormones, especially androgens, and that the sebaceous glands at the same time dominates cutaneous androgen production (Pochi and Strauss, 1974; Zouboulis et al, 1998, 2002; Zouboulis 2000a). The distribution of various hydroxysteroid dehydrogenases in human sebaceous glands and their strong activities in acne-prone skin in comparison with nonacne-prone skin areas have long since been evaluated (Baillie et al, 1966). Strong steroid sulfatase immunoreactivity in the acne skin, primarily associated with the monocytes in¢ltrating the lesions, but not in una¡ected skin was observed. The enzymatic hydrolysis of DHEA-S to DHEA and of estrone sulfate to estrone in cultured epidermal keratinocytes has been demonstrated (Milewich et al, 1990). There were no di¡erences in the rates of enzymatic hydrolysis of steroid sulfatase in the epidermis of acne-prone and nonacne-prone skin; however, the rate of estrone sulfate hydrolysis was two to eight times greater than that of DHEA-S in all of the tissues evaluated. Signi¢cant di¡erences in the activity of 5a-reductase or 17bHSD in sebaceous glands regarding the presence of acne were neither noted in men nor in women (Thiboutot et al, 1999). The activity of 5a-reductase or 17b-HSD was signi¢cantly greater in sebaceous glands from men than women. Higher serum androgen levels were signi¢cantly higher in women with acne, whereas no di¡erences were noted in men on the basis of the presence of acne. The exclusive predominance of the type 1 5a-reductase in sebaceous glands and the major in£uence of local androgenesis on sebum production were further con¢rmed by two clinical observations: (i) adult males with type 2 5a-reductase de¢ciency had sebum production scores identical to normal age-matched males; (ii) males with benign prostate hyperplasia treated with ¢nasteride (5 mg per day for 1 y) did not decrease the sebum score from baseline values, although the serum 5a-DHT level was lowered (ImperatoMcGinley, 1993). Moreover, the activity of the type 1 5a-reductase exhibits regional di¡erences in isolated sebaceous glands (Thiboutot et al, 1995), which seemed to correlate with the ¢nding that the stimulatory e¡ect of 5a-DHT on cell proliferation, was more prominent on facial than on nonfacial sebocytes (Akamatsu et al, 1992). In normal hair follicles and in open and closed comedones, the type 2 isoenzyme was demonstrated to localize within the companion layer of the follicle (innermost layer of the ORS). In in£ammatory acne lesions, the type 2 isozyme localized to the companion layer of hair follicles and endothelial cells within the surrounding in£ammatory in¢ltrate, but not to sebaceous glands (Thiboutot et al, 2000). As 5a-DHT could enhance 5a-reductase mRNA and enzyme activity in a feed-forward regulation (Russell and Wilson, 1994), and the type 2 isoenzyme has higher a⁄nity for testosterone than the type 1 isoenzyme, the upregulated type 2 isoenzyme in the diseased state might contribute to the aggravation of the pre-existing ‘‘hyperandrogenic’’ condition. The role of 3a -HSD in the pathogenesis of acne has rarely been addressed; the elevated plasma level of 3a -Adiol glucuro-nide might merely re£ect the increased production of 5a-DHT, decreased local enzyme activity of 3a -HSD to metabolize 5a-DHT, or both. It is not known if di¡erent levels of 3a -HSD occur between men and women, between acne and nonacne subjects or between mild (papulopustular) acne and severe (nodulocytic) acne patients. In women with mild to moderate acne, plasma 3a -Adiol glucuronide was suggested to be the most sensitive marker (Lookingbill et al, 1985), whereas the levels were shown to be decreased or within normal range in other studies (Toscano et al, 1993;

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Joura et al, 1996). Exaggerated androsterone metabolism, however, was observed in hype-randrogenic as well as in some normo-androgenic women with acne (Carmina et al, 1991; Carmina and Lobo, 1993), and androsterone glucuronide/sulfate seemed to be a better marker than 3a -Adiol glucuronide in di¡erentiating acne and hirsutism in hyperandrogenic women (Carmina et al, 1991). In young men, a statistically signi¢cant correlation was found between serum 3a -Adiol glucuronide and chest hairiness, acne as well as a combined chest hairiness and acne score (Lookingbill et al, 1988b). Noteworthy was the decreased serum levels of 3a -Adiol glucuronide in 24 acne subjects (15 men and nine women) treated with 1mg isotretinoin per kg bodyweight per day for 20 wk (Lookingbill et al, 1988a). It is also interesting to note that MPV-2213, a novel nonsteroidal competitive inhibitor of aromatase, could lead to the adverse e¡ect of acne formation in healthy male subjects (Ahokoski et al, 1998), which sheds light on the possible role of aromatase in the pathophysiology of acne formation. Androgenetic alopecia Androgenetic alopecia can be de¢ned as a 5a-DHT-dependent process with continuous miniaturization of androgen sensitive hair follicles (Ho¡mann and Happle, 2000). The intrafollicular conversion of testosterone to 5a-DHT seems to play a central part leading to androgenetic hair loss. Because steroid sulfatase plays an important part in androgen metabolism, and elevated levels of DHEA have been reported in young men with androgenetic alopecia, the hypothesis was advanced that men with X-linked recessive ichthyosis do not show androgenetic alopecia or develop only mild forms of common baldness (Happle and Ho¡mann, 1999). A recent clinical survey, however, did not support this hypothesis that Xlinked recessive ichthyosis and androgenetic alopecia are mutually exclusive, in as much as advanced androgenetic alopecia was found among these men (Trueb and Meyer, 2000). Homogenates of sebaceous glands from a balding scalp had greater 3b -HSD activity than those from a hairy scalp (Sawaya et al, 1988). There is no data about the activity of 3b -HSD in hair follicles from hairy vs balding scalp. Isolated intact hair follicles and sebaceous glands from balding frontal scalp, compared with a nonbalding occipital scalp, of patients with androgenetic alopecia demonstrated increased activity of 17bHSD (Sawaya, 1991). Both women and men with androgenetic alopecia have higher levels of androgen receptors and 5a-reductase type 1 and 2 in frontal than in occipital hair follicles, whereas higher levels of aromatase were found in their occipital follicles (Sawaya and Price, 1997). Aromatase content in women’s frontal hair follicles was six times greater than in frontal hair follicles in men. Frontal hair follicles in women had 3 and 3.5 times less 5areductase type 1 and 2, respectively, than frontal hair follicles in men (Sawaya and Price, 1997). Finasteride, a speci¢c competitive inhibitor of type 2 5a-reductase, maximally decreased both scalp skin and serum 5a-DHT at doses as low as 0.2 mg per day (Drake et al, 1999). Minoxidil was lately shown to increase 17b-HSD and 5a-reductase activity of cultured human DP from balding scalp (Sato et al, 1999), the relevant signi¢cance of which remains unclear. A disorder of androgen conjugation, favoring sulfurylation over glucuronidation, has been suggested to be a characteristic feature in men and women with androgenetic alopecia (Legro et al, 1994).Women with female pattern baldness were noted to have a marked increase in the 3a -Adiol glucuronide/sex hormone binding globulin ratio and low serum level of sex hormone binding globulin (De Villez and Dunn, 1986). In summary, the current understanding of androgenetic alopecia focuses on the excessive in situ conversion of testosterone to 5a-DHT, which involves the hyperactivity of 5areductase and 17b-HSD, coupled with hypoactivity of aromatase and overexpression of androgen receptors in balding vs nonbalding scalps (Kaufman, 1996). 5a-DHT was currently

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shown to induce apoptosis in DP in vitro via a bcl-2 related pathway.3 The roles of steroid sulfatase, 3b -D5-HSD and 3a HSD merit further studies. The molecular steps involved in androgen-dependent beard growth vs androgen-dependent hair miniaturization in androgenetic alopecia remain obscure (Ho¡mann and Happle, 2000). The newly emerging interest in the function of sebaceous glands in follicular biology (Sundburg et al, 2000; Zouboulis et al, 2002) may arouse the re-evaluation of the role of sebaceous glands in the peripheral hyperandrogenism occurring in androgenetic alopecia (Dijkstra et al, 1987; Sawaya et al, 1988; Zouboulis et al, 1994). HIRSUTISM

The prevalence of hirsuties is di⁄cult to assess. Earlier studies estimated hirsutism a¡ects between 5 and 10% of women (Azziz et al, 2000). Racial as well as social factors, and nowadays the media, however, greatly determine the threshold level for normality of hair growth (Dawber and Sinclair, 2001). Hirsutism is often seen in endocrine disorders characterized by hyperandrogenesis as the result of abnormalities of either the ovaries or adrenal glands (Dawber and Sinclair, 2001). The diagnosis of idiopathic hirsutism, when strictly de¢ned as hirsuteness with normal ovulatory function and circulating androgen levels, will include less than 20% of all hirsute women (Azziz et al, 2000). Earlier studies showed in adult women the higher activity of 5a-reductase in the genital skin as compared with the dorsal skin of the fetus as well as the abdominal skin, respectively (Flamigni et al, 1971), and in hirsute women the increased 5a-reductase activity in genital skin ( Jenkins and Ash, 1973; Sera¢ni and Lobo, 1985; Sera¢ni et al, 1985). Expression of both type 1 and type 2 5areductase mRNA was demonstrated in genital skin as well as in pubic skin ¢broblasts, whereas the type 2 isoenzyme appeared to predominate in pubic skin of normal men, normal women, and hirsute patients (Mestayer et al, 1996). The e¡ectiveness of ¢nasteride (5 mg per day) in the treatment of idiopathic and polycystic ovary syndrome-associated hirsutism further signi¢es the role of type 2 5a-reductase in the pathophysiology of hirsutism (Petrone et al, 1999). As the circulating androgens are known to increase peripheral 5a-reductase activity (Azziz et al, 2000), and as previous studies mostly utilized whole skin tissue or cultured ¢broblasts, but not the individual hair follicle components, however, more work is needed to de¢ne better the precise roles of the di¡erent isoenzymes and their activities in the development of hirsutism, especially the idiopathic hirsutism. 3a -Adiol conjugates (glucuronide or sulfate) were elevated in hirsute compared with nonhirsute women (Carmina et al, 1991; Toscano et al, 1993). Plasma (serum) concentrations of 3a -Adiol glucuronide appeared to re£ect hirsutism most accurately (Carmina et al, 1995a) and were elevated in polycystic ovary syndrome patients with/without hirsutism and in patients with idiopathic hirsutism (Kirschner et al, 1987; Falsetti et al, 1998). In idiopathic hirsutism, the speci¢city of serum 3a -Adiol glucuronide in re£ecting the peripheral 5a-reductase activity has been questioned (Vermeulen and Giagulli, 1991; Gilad et al, 1994; Joura et al, 1997). In women, levels of 3a -Adiol glucuronide essentially re£ect adrenal precursor levels as well as 5a-reductase activity in peripheral tissues (Vermeulen and Giagulli, 1991). The correlation between the 3a -Adiol glucuronide and hirsutism score was signi¢cant only in hirsute women with increased adrenal androgen secretion (increased DHEA/DHEA-S) and in women with idiopathic hirsutism. The correlation was not signi¢cant in hirsute women with increased ovarian testosterone secretion (Pang et al, 1992). In women with facial hirsutism, serum 3a -Adiol glucuronide concentrations had no correlation with 3 Wro¤bel A, Mandt N, Hossini A, Seltmann H, Zouboulis ChC, Orfanos CE, Blume-Peytavi U: 5a-Dihydrotestosterone and testosterone induce apoptosis in human dermal papilla cells by downregulation of the bcl-2 pathway. J Invest Dermatol 115:581, 2000 (Abstr.)

Table II. Enzyme activity in androgen-dependent dermatoses

Steroid sulfatase 3b -HSD 17b-HSD 5a-reductase Aromatase 3a -HSD

Acne

Androgenetic alopecia

Hirsutism

m (Mo) - (K) ? -(SG) -(SG) ? ?

?

?

m (SG, HF) m(HF) m(HF) k(HF) ?

? ? m(F) ? ?

? not known, m: increased, k: decreased, -: no di¡erence, SG: sebaceous gland; K: keratinocytes, F: ¢broblast, Mo: monocytes, HF: hair follicle

degree of facial hirsutism (Salman et al, 1992) and the levels were no more often increased than the other androgen precursors in women with mild to moderate hirsutism (Vermeulen and Giagulli, 1991; Giagulli et al, 1991). Modi¢cations in peripheral androgen activity (presumably through 5a-reductase activity) were shown to be time-dependent, and serum 3a -Adiol glucuronide seemed to re£ect changes after 6 mo of treatment. (Carmina et al, 1995b). Overall, serum levels of 3a -Adiol glucuronide appears to represent either adrenal androgen production or skin 5 a-reductase activity (Paulson et al, 1986; Rittmaster and Thompson, 1990). Although substantial evidence indicated its signi¢cance in hirsute women with polycystic ovary syndrome, the routine measurement of serum 3a -Adiol glucuronide is not recommended in the evaluation of idiopathic hirsutism or in other hirsute patients (Azziz et al, 2000), as this is currently conceptualized to be due to peripherally regionalized in situ hyperandrogenism. Androsterone glucuronide/sulfates were also proposed to re£ect peripheral androgen metabolism (Matteri et al, 1989). In women with idiopathic hirsutism or polycystic ovary disease, androsterone glucuronide was found to be elevated and could re£ect an increased production of adrenal androgens, but its level did not correlate with the severity of hirsutism (Thompson et al, 1990). Androsterone sulfate, being the most abundant 5a-reduced androgen metabolite in serum, however, was not recommended as a marker of either adrenal androgen production or hirsutism (Zwicker and Rittmaster, 1993; Azziz et al, 2000). Table II summarizes the alterations of enzyme activity in androgen-dependent dermatoses. PHARMACOLOGIC DEVELOPMENT OF STEROIDOGENIC ENZYME INHIBITORS Steroid sulfatase inhibitors As androgens and estrogens may be synthesized inside the cells (or cancer cells) utilizing the circulating systemic precursors DHEA-S and estrone sulfate, therapeutic agents targeted to inhibit steroid sulfatase activity may have therapeutic potential for androgen-sensitive and estrogen-sensitive diseases. Many novel compounds have been developed and evaluated mainly for the treatment of breast cancer (Billich et al, 2000; Chetrite and Pasqualini, 2001). Steroidal inhibitors may include 17a-substituted benzyl-estradiols, 3-O-sulfamate estrone, 2-methoxyestrone-3-O-sulfamate, the combination of two substituents at positions C3 and C17a of estradiol 3-O-sulfamate, e.g., 17a-benzyl (or 40 -tert-butylbenzyl) estra-1,3,5(10)-trienes and 17a-derivatives of estradiol, 17b-(Nalkylcarbamoyl)-estra-1,3,5(10)-trien-3-O-sulfamates, and 17b-(Nalkanoyl)-estra-1,3,5(10)-trien-3-O-sulfamates (Woo et al, 1998; Li et al, 1998; Ciobanu et al, 1999; Boivin et al, 2000; Purohit et al, 2001). Nonsteroidal sulfamates such as ( p-O-sulfamoyl)-Nalkanoyl-tyramines, tricyclic coumarin sulfamates, tricyclic oxepin sulfamate, and substituted chromenone sulfamates (e.g., sulfamic acid 2-t-butyl-4 -oxo-4H-chromen-6-yl ester) have been described (Billich et al, 2000; Purohit et al, 2001).

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Estrone-3-O-sulfamate was shown to inhibit the steroid sulfatase enzyme activity in a concentration-dependent manner in human hair DP ex vivo (Ho¡mann et al, 2001). 3b-HSD inhibitors Known inhibitors used clinically in dermatology include the gestagens cyproterone acetate, norgestrel, norethisterone, which exhibit a dual activity by parallel binding to a androgen receptor (Dumont et al, 1992;

999

Zouboulis et al, 1999). Trilostane (4a -5-epoxy-17b-hydroxy-3 oxo-5a-androstan-2-carbonitrile) and cyanoketone (2acyano-17b-hydroxy- 4,4,17a-trimethylandrost-5-en-3 -one) are two classical steroidal inhibitors of type 1 3b -HSD (Cooke, 1996). Trilostane can attenuate the preovulatory gonadotropin surge by inhibiting progesterone synthesis (Mahesh and Brann, 1998). It has also been clinically tried to treat Cushing’s syndrome (Engelhardt and Weber, 1994). Iso£avonoids such as genistein and daidzein were also shown to exert an anti-3b -

Table III. Speci¢c inhibitors of steroidogenic isoenzymes Steroidogenic enzymes

Isoforms

Steroid sulfatase

3b -hydroxysteroid dehydrogenase

17b-hydroxysteroid dehydrogenase

Type 1

Type 2 Type 1

Type 2

Type 3 5a-reductase

Type 1

Type 2

Type 1/2

Characterized inhibitors Steroidal

Non-steroidal

17a-substituted benzylestradiols Estrone-3 -O-sulfamate 2-methoxyestrone-3 -O-sulfamate 17a-benzyl (or 40 -tert-butylbenzyl)estra-1,3,5(10)-trienes 17b-(N-alkylcarbamoyl)-estra-1,3,5(10)-trien-3 -O-sulfamates

tricyclic coumarin sulfamates tricyclic oxepin sulfamate substituted chromenone sulfamates

trilostane cyproterone acetate norgestrel norethindrone

cyanoketone iso£avonoids (genistein)

thiazolidinediones 16 -(bromoalkyl)-estradiols £avonoids, iso£avonoids, lignans tri£uoromethylacetylenic secoestradiol 6b -(thiaheptanamide) derivatives of estradiol estrone containing a spiro-gamma-lactone at position 17 7a-thioalkyl and 7a-thioaryl derivatives of spironolactone N-butyl-N-methyl-11-(30 -hydroxy-210, 170 carbolactone-190 -nor-170 a-pregna-10,30, 50 (100 )trien-70 a-yl)-undecanamide £avonoids, iso£avonoids, lignans 1,4 -androstadiene-1,6,17- trione androsterone 3b -substituted derivatives 4 -azasteroids (MK386) 6 -azasteroidal 17b-carboxamide triaryls)

¢nasteride turosteride MK- 434 MK-963 dihydro¢nasteride chlormadinone acetate TZP- 4238 epristeride (SK&F 105657, ONO-9302) 17a-estradiol 17-(50 -isoxazolyl)androsta- 4,16 -dien-3 -one N-(1,1,1,3,3,3 -hexa£uorophenyl-propyl)-3 -oxo4 -aza-5a-androst-1-ene-17b-carboxamide (PNU 157706) dutasteride oxendolone (TSAA-291: 16 b -ethyl17b-hydroxy- 4 -estren-3 -one) 19-nor-10-azasteroids progesterone-based steroids bearing an oxime group connected to the steroidal D-ring

8-chloro- 4 -methyl-1,2,3,4,4a,5,6,10boctaahydro-benzo[f] quinolin-3(2 H)one (LY 191704) 6 -[4 -(N,N-diisopropylcarbamoyl)phenyl]N-methyl-quinolin-2-one 5) benzo[c]quinolizin-3 -ones epicatechin-3 -gallate, epigallocatechin-3 -gallate suramin zinc azelaic acid 6 -[4 -(N,N-diisopropylcarbamoyl)phenyl]1H-quinolin-2-one 4 4 -[3 -[5-benzyl-8-(2-methyl)propyl10,11-dihydrodibenz[b,f]azepine2-carboxamido]phenoxy]butyric acid

benzoquinolinone Serenoa repens extract permixon Artocarpus incisus iso£avonoids and lignans alizarin and curcumin phenazine derivatives myristoleic acid g-linolenic acid 4 -[3 -[3 -[bis(4 -isobutylphenyl)methylamino]benzoyl]-1H-indol-1-yl] butyric acid (FK 143)

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HSD e¡ect (Le Bail et al, 2000). Thiazolidinediones was recently shown to inhibit directly the steroidogenic enzymes P450c17 and type 2 3b -HSD (Arlt et al, 2001). 17b-HSD inhibitors As the 17b-HSD system plays a key part in the formation or inactivation of several active androgens and estrogens from circulating precursors, these isoenzymes can regulate tumor cell proliferation in androgen- and estrogendependent cancers. Endocrine therapies for the treatment and prevention of breast cancer are presently under intensive clinical trials with the intention of developing dual-action compounds to

block estrogen action optimally by antagonizing the estrogen receptor as well as inhibiting the estradiol biosynthesis (Blomquist, 1995; Tremblay and Poirier, 1998). As stated before, various 17b-HSD isoenzymes possess di¡erent catalytic reductive or oxidative activity, thus design of speci¢c antagonist should aim at binding to hydrophilic and cofactor-depending sites at the active center of the isoenzyme (Krazeisen et al, 2001). Prior to better characterization of the molecular genetics and physiology of di¡erent isoenzymes, the exploration and development of speci¢c enzyme inhibitors were performed depending on the selected tissues (liver, ovary,

Figure 2. Chemical structures of some representative steroidogenic enzyme inhibitors.

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Figure 2. (continued).

placenta, testis or prostate, etc.) from di¡erent species under examination (human vs rat, mouse or porcine). Speci¢city of the compounds based on the analysis of these data seems to be confounded by the ¢nding that the same organ/tissue may contain more than one 17b-HSD isoenzyme (Luu-The, 2001).

Well characterized 17b-HSD1 inhibitors include estradiol derivatives containing a bromopropyl/or iodopropyl group at position 16a such as 16 -(bromoalkyl)-estradiols (Luu-The et al, 1995; Tremblay et al, 1995), phytoestrogens such as £avonoids (including £avone, £avanone, and iso£avone), iso£avonoids, and lignans (Evans et al, 1995; Makela et al, 1995, 1998; Le Bail

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et al, 1998), tri£uoromethylacetylenic secoestradiol (Lawate and Covey, 1990), and 6b -(thiaheptanamide) derivatives of estradiol (Poirier et al, 1998). Compounds belonging to this group might also include those described to inactivate ovarian 17b-HSD isoenzyme, such as glycyrrhizin and glycyrrhetinic acid (Sakamoto and Wakabayashi, 1988; Armanini et al, 1999) or to inhibit the enzyme activity in breast cancer cell lines, such as medrogestone (Chetrite et al, 1999). Well characterized 17b-HSD2 inhibitors containing a spiro-gslactone at position 17 (Luu-The et al, 1995), 7a-thioalkyl and 7a-thioaryl derivatives of spironolactone such as 3 -Oxo-17apregna- 4 -ene-7a-{4 -[2-(1-pipendiryl)ethoxy]-benzylthio} 21,17carbolactone (Tremblay et al, 1999), N-butyl-N-methyl-11-(30 hydroxy-210, 170 -carbolac-tone-190 -nor-170 a-pregna-10,30,50 (100 )trien-70 a-yl)-undecanamide (Sam et al, 2000), £avonoids, iso£avonoids, and lignans (Evans et al, 1995; Makela et al, 1998; Le Bail et al, 1998). Based on the characterized tissue distribution of the isoenzymes, included in this group might be compounds that are able to work on: 1 The placenta: danazol; ethinylestradiol (Blomquist et al, 1984); unsaturated fatty acids, such as oleic, arachidonic, linoleic, and linolenic acid (Blomquist, 1985); periodate-oxidized NADP þ (Mendoza-Hernandez et al, 1987); 14,15-secoestra-1,3,5(10)-trien15-ynes (Auchus, 1989); spiro-gp-lactones containing the C-18 nucleus (Sam et al, 1995); and chalcones, such as naringenin chalcone and 4 -hydroxychalcone (Le Bail et al, 2001). 2 Human benign prostatic hyperplasia tissue: testolactone (Bartsch et al, 1987). 3 Liver microsomal enzymes: nonsteroidal anti-in£ammatory agents and nonsteroidal estrogens, such as hexestrol, dienstrol, diethylstilbestrol, and zearalenone (Hasebe et al, 1987); and retinoids (13 -cis retinoic acid 49-cis retinoic acid 4 all-trans retinoic acid) (Murray et al, 1994). Well-characterized 17b-HSD3 inhibitors include 1,4 -androstadiene-1,6,17-trione (Luu-The et al, 1995) and androsterone 3b -substituted derivatives (Ngatcha et al, 2000). Included might also be those reported to inhibit testicular 17b-HSD enzyme activity, such as licorice (glycyrrhizin and glycyrrhetinic acid) (Sakamoto and Wakabayashi, 1988; Armanini et al, 1999), losulazine (Ray et al, 1994), amphetamine (Tsai et al, 1997), methotrexate (Badri et al, 2000), and S-petasine (like a sesquiterpene ester, being an antiin£ammatory analgesic component of the butterbur, Petasites hybridus; Lin et al, 2000). 5a-reductase inhibitors Development of speci¢c 5a-reductase inhibitors began soon after 5a-DHT was reported to be the major androgen acting in the periphery (Liang et al, 1983, 1984). Great progress has since been made and continuing interests grow in developing more potent and speci¢c 5a-reductase inhibitors. As 5a-DHT, being two to 10 times stronger than testosterone in androgenicity, was supposed to play a more important part than testosterone in many androgen-dependent diseases (benign prostata hyperplasia, prostatic carcinoma, acne, androgenetic alopecia, hirsutism; Chen et al, 1996; Bartsch et al, 2000), whereas speci¢c inhibition of its formation would bring the advantage of sparing the anti-virilizing side-e¡ects of general androgen receptor blockers (e.g., £utamide). Inhibitors can be classi¢ed based on the chemical structures of steroidal vs nonsteroidal inhibitors or according to the isoenzyme speci¢city as type 1, type 2 and type-1/2 dual inhibitors (Chen et al, 1996; Zouboulis, 2000b). It was noticed that the Ki values vary depending on the species examined (human vs rat) and the cell/tissue origin tested (e.g., testis vs prostate or primary culture vs cell lines). Except for ¢nasteride, most of these synthetic chemicals or phytotherapeutic agents are still undergoing in vitro tests, animal studies, or clinical trials (Zouboulis, 2000b). Finasteride, a speci¢c type 2 5a-reductase competitive inhibitor, is the ¢rst systemic drug approved for clinical use and has been shown to be e¡ective for the treatment of androgenetic alopecia in young to middle-aged men as well as aged men between 53 and 76 y, but

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

not in post-menopausal women (Brenner and Matz, 1999; Whiting et al, 1999). Speci¢c type 1 inhibitors include certain steroidal inhibitors, such as 4 -azasteroids (e.g., MM-386: 4,7b-dimethyl- 4 -aza-5acholestan-3 -one) or 6 -azasteroidal 17b-carboxamide triaryls (Li et al, 1995), and nonsteroidal inhibitors benzoquinolinones (LY 191704: 8-chloro- 4 -methyl-1,2,3,4,4a,5,6,10b-octaahydrobenzo[f]quinolin-3(2H)-one), 6 -[4 -(N,N-diisopropylcarbamoyl) phenyl]-N-methyl-quinolin-2-one 5) (Baston et al, 2000), benzo [c]quinolizin-3 -ones (Guarna et al, 2000), certain plant extracts, such as green tea extract catechins (epicatechin-3 -gallate and epigallocatechin-3 -gallate) (Liao and Hiipakka, 1995), and suramin, zinc, and azelaic acid (Chen et al, 1996). MK-386, an azasteroid that speci¢cally inhibits the type 1 5a-reductase in vitro, was shown to suppress sebum as well as serum 5a-DHT in a concentration-dependent manner (Schwartz et al, 1997; Baston et al, 2000). Speci¢c type 2 inhibitors, in addition to ¢nasteride, include 4 -methyl- 4 -azasteroids (e.g., turosteride: 1-(4 -methyl-3 -oxo4 -aza-5a-androstane-17b-carbonyl)-1,3 -diisopropylurea, or MK963: [5a-23-methyl-4 -aza-21-norchol-1-ene-3,20-dione])4 (Chen et al, 1996), 4 -azasteroids (e.g., MK- 434: 17b-benzoyl- 4 -aza-5aandrost-1-en-3 -one, dihydro¢nasteride)4 (Chen et al, 1996; Zouboulis, 2000b), chlormadinone acetate (Nukui, 1997), osaterone acetate (TZP- 4238: 17a-acetoxy-chloro-2-oxa- 4,6 -pregnadiene3,20-dione) (Takezawa et al, 1992), epristeride (SK&F 105657 or ONO-9302: N-(t-butyl)androst-3,5-diene-17b-carboxamide3 -carboxylic acid,) (Levy et al, 1994), 17a-estradiol (Hevert, 2000), 6 -Methylenesteroidal derivatives (Li et al, 1995), 17(50 -isoxazolyl)androsta- 4,16 -dien-3 -one (L-39) (Nnane et al, 2000), nonsteroidal compounds such as 6 -[4 -(N,N-diisopropylcarbamoyl)phenyl]-1H-quinolin-2-one 4 (Baston et al, 2000), and tricyclic compounds such as 4 -[3 -[5-benzyl-8-(2methyl)propyl-10,11-dihydrodibenz[b,f ]azepine-2-carboxamido] phenoxy]butyric acid (Takami et al, 2000), etc. Several novel compounds with potent dual inhibitory activity on both type 1 and type 2 isoenzymes have been reported. Steroidal antagonists include N-(1,1,1,3,3,3 -hexa£uorophenylpro-pyl)-3-oxo-4 -aza-5a-androst-1-ene-17b-carboxamide (PNU 157706) (di Salle et al, 1998) dutasteride (Frye et al, 1998; Gisleskog et al, 1998), oxendolone (TSAA-291: 16b -ethyl-17bhydroxy- 4 -estren-3 -one) (Sudo et al, 1981; Li et al, 1995), 19-nor10-azasteroids (Guarna et al, 1997), and progesterone-based steroids bearing an oxime group connected to the steroidal D-ring (Hartmann et al, 2000). Nonsteroidal compounds include benzoquinolinone (e.g., LY320236) (McNulty et al, 2000), plant extracts such as Serenoa repens extract permixon (Bayne et al, 2000), Artocarpus incisus (Shimizu et al, 2000), iso£avonoids and lignans (Evans et al, 1995), alizarin and curcumin (Liao et al, 2001), phenazine derivatives (e.g., ribo£avin) (Li et al, 1995), myristoleic acid (Liao et al, 2001) and g-linolenic acid (Liang and Liao, 1992, 1997; Liao and Hiipakka, 1995), indole derivatives such as 4 -[3 -[3 -[bis(4 isobutylphenyl)-methylamino]benzoyl]-1H-indol-1-yl] butyric acid (FK 143) (Katashima et al, 1998), sodium- 4 -[2-(2,3 dimethyl- 4 -and 2 over black square); [1 and 2 over black square]-(4- isobutylphenyl)ethoxy]benzolamino]phenoxy] butyrate (ONO-3805) (Takahashi et al, 1992), indoline and aniline derivatives (Igarashi et al, 2000). g-Linolenic acid was shown to inhibit testosterone-stimulated £ank organ growth but not 5a-DHT-stimulated £ank organ growth (Liang and Liao, 1997). CS-891 inhibits 5a-reductase activity in freshly isolated DP of human hair follicles (Niiyama et al, 2000). It remains to be determined if these dual inhibitors can further decrease the tissue 5a-DHT level and are clinically more e¡ective than ¢nasteride in the treatment of male androgenetic alopecia. 4 Sei¡ert K, Fritsch M, Zoubouls ChC: 5a-reductase inhibitors exhibit distinct e¡ects on human keratinocytes and sebocytes in vitro. J Invest Dermatol 110:550, 1998 (Abstr.)

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Table III and Figure 2 show the representative speci¢c steroidogenic enzyme inhibitors and their chemical structures, respectively.

CONCLUSIONS

Similar to the classical steroidogenic organs, such as gonads and adrenal glands, the skin and its appendages, including hair follicles, sebaceous glands, and eccrine/apocrine glands, are armed with all the necessary enzymes required for androgen synthesis and metabolism. Steroid sulfatase, 3b -HSD1, 17b-HSD3, and the type 1 5a-reductase are the major steroidogenic enzymes responsible for the formation of potent androgens, whereas 17b-HSD2, 3a -HSD, and aromatase seem to inactivate the excess androgens locally in order to achieve androgen homeostasis (Fritsch et al, 2001). Small levels of some isoenzymes found in normal states, may have important implications in disease states, where these factors may be upregulated, contributing to the exaggeration of ‘‘peripheral hyperandrogensim’’. Clari¢cation of the cutaneous expression and tissue-speci¢c regulation of these steroidogenic isoenzymes could help us to understand better their roles in the growth and development of the skin as well as the pathophysiology of androgen-dependent dermatoses. Finasteride is the ¢rst speci¢c isoenzyme inhibitor used for clinical intervention. Rapid pharmacologic advancement in this ¢eld and design for other speci¢c potent antagonists foresees a better control or even chemoprevention of acne, hirsutism, and androgenetic alopecia in the near future.

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