Dicarboxylic acids inhibit the growth of keratinocytes in vitro

The antiproliferative effect of three straight-chained saturated dicarboxylic acids was examined with neonatal mouse keratinocyte cultures. Adipic acid (C6), azelaic acid (C9), and sebacic acid (C10) were added to the cultures in concentrations ranging from 1 to 50 mmol/l. Proliferation was assayed by liquid-scintillation counting of 3H-thymidine incorporation into DNA and by autoradiography. Fifty percent inhibition of 3H-thymidine incorporation was observed with 50 mmol/l adipic acid, 20 mmol/l azelaic acid, and 10 mmol/l sebacic acid, respectively. The antiproliferative effect was completely reversible after cessation of treatment. Moreover, treated cultures then showed a rebound effect with increased DNA synthesis. These results show that dicarboxylic acids exert reversible antiproliferative effects on keratinocytes.     

Synergistic antiandrogenic effects of topical combinations of 5 alpha-reductase and androgen receptor inhibitors in the hamster sebaceous glands

The androgenic action of dihydrotestosterone (DHT) is antagonized by agents that compete with testosterone for the 5 alpha-reductase enzyme and by agents that block the binding of DHT to its receptor. The topical synergistic effect of 5 alpha-reductase (5 alpha RI) and androgen receptor inhibitors (ARI) was determined by measurement of the sebaceous gland size (SGS) of the ventral ear skin of the intact, sexually mature male Syrian hamsters. Progesterone (P), a 5 alpha RI, and spironolactone (SL), an ARI, produced a dose responsive decrease in SGS at topical concentrations of 0.01% to 5.0%. At concentrations of 1, 3, and 5%, P and SL combinations produced neither an additive nor synergistic inhibition of SGS. At very low concentrations of up to 0.10%, neither P nor SL alone produced any effect on SGS. When combinations of these two steroids were applied at low concentrations, SGS decreased unilaterally to approximately 50%. This synergy occurred best at a P:SL ratio of 1:2. The lower effective concentrations of P may be explained by its greater percutaneous absorption. Synergy was also demonstrated at low concentrations with other antiandrogens: cyproterone acetate, canrenone, hydroxyflutamide, and N-N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane- 17 beta-carboxamide. The use of anti-androgen combinations at low concentrations is of value because of the decreased risk of systemic side effects while maintaining potent topical efficacy.     

The extract of Thujae occidentalis semen inhibited 5alpha-reductase and androchronogenetic alopecia of B6CBAF1/j hybrid mouse

BACKGROUND: The conversion of testosterone to dihydrotestosterone; 5alpha-androstan-17beta-ol-3-one by 5alpha-reductase plays a crucial role in hair baldness and prostatomegaly. Recent approach showed specific inhibitors for 5alpha-reductase type 2 such as finasteride promoted hair growth in male pattern alopecia. OBJECTIVE: In order to search for effective medicinal plant extracts applied topically for androgenetic alopecia, we screened natural plant extracts having inhibitory activities of 5alpha-reductase type 2 and demonstrated its biological function in androgen-related animal models. METHODS: We evaluated the inhibition activities of numerous plant extracts by contact cell based metabolic method using a stable HEK 293 cell line expressing human 5alpha-reductase (type 2). To elucidate the biological activity in vivo, the Thujae occidentalis semen (TOS) extract was topically applied to fuzzy rat and androchronogenetic alopecia (AGA) mouse, respectively. The secreted sebum and the size of sebaceous glands of fuzzy rat were measured after 6 weeks. Also, after the topical treatment with TOS extract and androgen receptor antagonist (cyproterone acetate) simultaneously with subcutaneous injection of testosterone (1 mg/mice/day), hair loss patterns of female B6CBAF1/j hybrid mouse were observed. RESULTS: TOS extract showed higher inhibition activity of 5alpha-reductase type 2(IC(50) value=2.6 microg/ml) than that of gamma-linolenic acid, but lower than that of finasteride. When applied to fuzzy rat, the amount of sebum and sebaceous gland size decreased remarkably. In AGA model, alopecia degrees of two groups, treated with TOS extract (P<0.015) or cyproterone acetate (P<0.01), were lower than that of vehicle (propylene glycol:ethanol=7:3) and there was no difference between above two groups. CONCLUSION: We have demonstrated the inhibitory activity of TOS extract for 5alpha-reductase type 2 and its biological action in two animal models, suggesting that TOS extract would be used as an effective agent for male pattern baldness by modifying androgen conversion.     

Venereal trichomoniasis: role of men.

It has been suggested that high zinc concentrations found in men may prevent Trichomonas vaginalis from being established in the male reproductive tract. In this investigation T vaginalis was readily killed at concentrations of zinc that occur in the prostatic fluid of healthy men (minimum trichomonacidal concentration (MTC) of 6.4 mmol/l). T vaginalis was also shown to be killed by human prostatic extracts as well as by human seminal fluid, even when the zinc content was much lower than the MTC for T vaginalis. It seems likely, therefore, that there are at least two antitrichomonal mechanisms in the male reproductive tract, one being zinc dependent and the other not relating to zinc content. Tritrichomonas foetus, which causes venereal trichomoniasis in cattle, was unaffected by bovine seminal fluid and was killed by zinc only at concentrations far higher than those found in the prostatic fluid in the bull (MTC 200 mmol/l).     

5 alpha-reductase activity in cultured human dermal papilla cells from beard compared with reticular dermal fibroblasts

The activity of 5 alpha-reductase was assessed in cultured human beard dermal papilla cells and reticular dermal fibroblasts to elucidate the mechanism of androgen action in promoting the growth of beards in men. The monolayer was incubated with 50 nM of [1,2-3H]-testosterone. Steroids were extracted from the medium and analyzed by thin layer chromatography. The major metabolite in the beard dermal papilla cells was dihydrotestosterone (DHT), the most potent androgen in the androgen target tissue. By contrast, the amount of DHT formed was similar to that of androstenedione in reticular dermal fibroblasts. The 5 alpha-reductase activity in beard dermal papilla cells was three to five times as high as that in the reticular dermal fibroblasts from the same skin sample. The apparant Michaelis constant of 5 alpha-reductase in the beard dermal papilla cells was 1.0 X 10(-6) M, which was virtually equivalent to that of genital skin fibroblasts, typical androgen target cells. It was 4.0 X 10(-5) M in reticular dermal fibroblasts. By contrast, the activities of 5 alpha-reductase in dermal papilla cells from occipital scalp hair follicles were similar to those of reticular dermal fibroblasts of the same skin samples. These results strongly suggest that the beard dermal papilla cell is an androgen target cell, and that DHT plays a role in the growth of beards in men.     

The in vitro antimicrobial effect of azelaic acid

Various strains of cutaneous micro-organisms were tested in vitro for their survival rates in 0.5 mol/l (8.4% w/v) azelaic acid solution. All bacterial strains exhibited large reductions in viability (at least 40-fold) over a 24 h test period, but little response was noted with Pityrosporum ovale. The bactericidal effect of azelaic acid was reduced considerably in the presence of nutrients. Minimum inhibitory concentrations (MICs) and minimum bactericidal (or fungicidal) concentrations (MBCs) were also determined. MICs varied from 0.03 mol/l to 0.25 mol/l; MBCs were all either 0.25 mol/l or greater.     

Effect of azelaic acid on the growth of melanoma cell cultures in comparison with fibroblast cultures

Azelaic acid, a linear C9 dicarbonic acid, can effect a reduction in the growth of melanoma cells. This was shown in a comparison of two human melanoma cell cultures and cultured human skin fibroblasts. The cultures were observed for 24 days. The reduction in growth effected by azelaic acid showed significant differences concerning the dose-effect ratio in both melanoma cell lines observed. There was no inhibition of cell proliferation in the fibroblast cultures. Morphological and final trypan blue tests suggest that azelaic acid is not cytotoxic but cytostatic in a selective way.     

全反式维A酸对UVA诱导人成纤维细胞胶原合成的影响

目的　探讨全反式维A酸 (at RA)对UVA诱导人成纤维细胞胶原生物合成的影响 ,为应用全反式维A酸治疗光老化皮肤病提供实验依据。方法　正常人来源的单层培养的皮肤成纤维细胞 ,经UVA照射后立即加入含不同浓度at RA的培养液 ,共 2天 ,用MTT法检测细胞增殖情况 ,3 H 脯氨酸检测细胞胶原合成情况。结果　在各组之间细胞增殖差别无显著性时 ,at RA浓度为 10 6mmol/L和 10 -5mmol/L的实验组比对照组的细胞胶原合成显著增高 (P <0 .0 5 ,P <0 .0 1) ;而at RA浓度为 10 -4mmol/L的实验组比对照组的细胞胶原合成显著降低 (P <0 .0 1)。结论　在体外细胞培养情况下 ,相对低浓度的at RA(10 -6mmol/L ,10 -5mmol/L)可逆转UVA诱导人成纤维细胞胶原生物合成的降低 ,而相对高浓度 (10 -4mmol/L)则不能     

Comparative diffusion of fusidic acid, oxacillin, and pristinamycin in dermal interstitial fluid after repeated oral administration

OBJECTIVE: The aim of this study was to use the suction bullae technique to compare skin diffusion of 3 antibiotics commonly used for skin infections (fusidic acid, oxacillin, pristinamycin) and to estimate their potential activity at the site of skin infections. SUBJECTS AND METHODS: This comparative open study was conducted in 12 healthy volunteers using a repeated latin square experimental scheme. Antibiotic concentrations in serum and suction bullae fluid were measured by high performance liquid chromatography after 5.5 days of repeated oral administration of fusidic acid (1 g/d), oxacillin (2 g/d), and pristinamycin (2 g/d). RESULTS: Mean antibiotic concentrations in serum and interstitial fluid (suction bullae fluid) were highest for fusidic acid with a Cmax at 91.3 +/- 23.0 mg/l and 45.5 +/- 18.0 mg/l respectively (interstitial fluid/serum ratio=49 +/- 10 p. 100). For oxacillin, Cmax was 8.3 +/- 3.6 mg/l and 0.98 +/- 0.49 mg/l (ratio 13 +/- 5 p. 100). Pristinamycin concentrations were low with a Cmax at 0.51 +/- 0.40 and 0.26 +/- 0.15 mg/l (ratio 73 +/- 57 p. 100). Comparing the area under the interstitial fluid and the serum concentration-time curves showed that the best diffusion was obtained with pristinamycin (114 +/- 61 p. 100), followed by fusidic acid (57 +/- 13 p. 100) and oxacillin (48 +/- 25 p. 100). DISCUSSION: These data were used to calculate indicators of potential efficacy in the interstitial dermal fluid: inhibitor quotient (Cmax/MIC) and AUIC (ASC/MIC), indicator of the time antibiotic concentrations are maintained above the minimal inhibitor concentration (MIC). This showed that fusidic acid was potentially more active against all staphylococci. For streptococci, the observed interstitial concentrations of pristinamycin and of fusidic acid should theoretically inhibit streptococci A growth, but oxacillin was the most adapted antibiotic.     

A possible mechanism of action for azelaic acid in the human epidermis

Summary Azelaic acid, and other saturated dicarboxylic acids (C₉-C₁₂), are shown to be competitive inhibitors of tyrosinase (K _I azelaic acid = 2.73×10^–3 M) and of membrane-associated thioredoxin reductase (K _I azelaic acid = 1.25×10^–5 M). The monomethyl ester of azelaic acid does not inhibit thioredoxin reductase, but it does inhibit tyrosinase, although double the concentration is necessary compared with azelaic acid (K _I azelaic acid monomethyl ester = 5.24×10^–3 M). Neither azelaic acid nor its monomethyl ester inhibit tyrosinase when catechol is used as a substrate instead of l-tyrosine. Therefore, the weak inhibitory action of azelaic acid on tyrosinase appears to be due to the competition of a single carboxylate group on this inhibitor for the -carboxylate binding site of the l-tyrosine substrate on the enzyme active site. Based on the inhibitor constant on tyrosinase, at least cytotoxic levels of azelaic acid would be required for the direct inhibition of melanin biosynthesis in melanosomes if this mechanism is responsible for depigmentation in the hyperpigmentation disorders lentigo maligna and melasma. Alternatively only 10^–5 M azelaic acid is required to inhibit thioredoxin reductase. This enzyme is shown to regulate tyrosinase through a feedback mechanism involving electron transfer to intra-cellular thioredoxin, followed by a specific interaction between reduced thioredoxin and tyrosinase. Furthermore, the thioredoxin reductase/thioredoxin system is shown to be a principal electron donor for the ribonucleotide reductases which regulates DNA synthesis. Inhibition of thioredoxin reductase by azelaic acid provides a rationale for both its depigmenting property and the reversible inhibition of DNA synthesis observed in cultured epidermal cells and also in some of the bacteria associated with acne vulgaris.     

Testosterone metabolism in human skin cells in vitro and its interaction with estradiol and dutasteride

Since the limited knowledge of cutaneous drug metabolism can impair the development of specifically acting topical dermatics and transdermal application systems, the cell-type-specific androgen metabolism in human skin and its inhibition by drugs were investigated. Cultured human foreskin and scalp skin keratinocytes and fibroblasts as well as occipital scalp dermal papilla cells (DPC) were incubated with testosterone 10(-6) and 10(-8)M alone and in the presence of 17alpha-estradiol, 17beta-estradiol or dutasteride for 24 h. Androgens extracted from culture supernatants were subjected to thin-layer chromatography and quantified by beta-counting. In keratinocytes and DPC, dihydrotestosterone (DHT) was only formed to a low extent while androstenedione was the main metabolite. In fibroblasts, DHT formation was pronounced following 10(-8)M testosterone. Dutasteride 10(-8)M completely suppressed 5alpha-dihydro metabolite formation. 17alpha-Estradiol and 17beta-estradiol at nontoxic concentrations decreased 17-ketometabolites. Human skin regulates testosterone action by cell-type-specific activation or deactivation. Effects of 17alpha-estradiol in androgenetic alopecia are not due to 5alpha-reductase inhibition. Dutasteride may be useful in acne and androgenetic alopecia.     

Characterization of 5 alpha-reductase in cultured human dermal papilla cells from beard and occipital scalp hair

In order to gain a deeper insight into the role of 5 alpha-reductase in the growth of beards in men, we studied some kinetic properties of the enzyme in cell homogenates of cultured human dermal papilla cells from beard and occipital scalp hair. When cell homogenates were incubated with [3H]-testosterone, the 5 alpha-reductase of beard dermal papilla cells exhibited an optimum activity at pH 5.5, whereas the enzyme of dermal papilla cells from occipital scalp hair showed a broad and low plateau between pH 6.0 and 9.0, without a sharp peak. The apparent Michaelis constant of 5 alpha-reductase was 3.3 x 10(-7) M in dermal papilla cells from beard and 2.4 x 10(-5) M in those cells from occipital scalp hair. The apparent Km of 5 alpha-reductase for NADPH was 2.8 x 10(-5) M and 7.6 x 10(-4) M in beard and occipital scalp hair dermal papilla cells, respectively. There were no significant differences in the substrate specificity between these two types of cells. The 5 alpha-reductase activity was recovered mainly in the nuclear fraction of beard dermal papilla cells. By contrast, it was widely distributed among the individual subcellular fractions of dermal papilla cells from occipital scalp hair. These results strongly suggest that these two kinds of dermal papilla cells have different types of 5 alpha-reductase, and that the enzyme in beard dermal papilla cells is similar in characteristics to that in the androgen target organs such as prostate.     

Effects of retinoic acid on adult human epidermis in whole skin organ culture

Summary Adult human skin was cultured in wholeskin organ culture under chemically defined conditions. Retinoic acid was added to the culture at final concentrations of 5×10^-7 and 5×10^-6 M. Both concentrations elicited cell death in the upper epidermal layers and prevented the terminal differentiation of the cells to mature corneocytes. The inhibition of terminal differentiation was not permanent, as the corneocytes produced later during the culture showed no signs of inhibition. The upper vital cells in epidermis cultured with retinoic acid were very flattened and contained reduced amounts of cytoskeleton components. Fine, granular material not present in normal epidermis was oberved in both the intercellular spaces and the intracytoplasmic vesicles of retinoid-treated epidermis. The present results indicate that the response of normal human skin to retinoic-acid treatment involves the same kind of modulation of the epidermal structure previously described in embryonic avian and diseased human skin.     

Follicular concentrations of azelaic acid after a single topical application

Follicular concentrations of azelaic acid (AzA) were determined in vivo using a rapid, non-invasive method, after a single topical application of 20% (w/w) AzA cream, in order to establish whether the in vitro antimicrobial effects observed in previous studies are relevant in vivo. Preweighed amounts of 20% (w/w) AzA cream were applied over demarcated areas on the forehead and back of nine young adults, and samples were taken over a period of 5 h. AzA was removed from the skin surface by washing with acetone, and follicular casts were collected using cyanacrylate gel. The samples were centrifuged to remove particulate matter, and the supernatants derivatized for analysis by HPLC. Although the results showed wide-ranging variability, the follicular concentration increased as the amount present on the surface declined. The maximum follicular concentrations of AzA attained ranged from 7.5 to 52.5 ng (μg of follicular casts)^?1 and 0–5 to 23–4 ng (μg of follicular casts)^?1 in samples taken from the back and forehead, respectively. Assuming an average density of follicular material of 0.9 g ml^?1, the mean maximum follicular concentration attained on the back was between 36 and 251 mmol/l, and on the forehead was between 2 and 112 mmol/l, and indicates that the concentration of AzA attained in follicular casts after a single topical application is comparable with the concentration required to inhibit the growth of Propionibacterium acnes and Staphylococcus epidermidis, in vitro.     

Qualitative and quantitative analysis of cytosol retinoid binding proteins in human skin

The distribution of the cytosol retinol and retinoic acid binding proteins are known to vary greatly within the different layers of the eye, a retinoid target organ. We have analyzed the cytosol retinoid binding from adult human skin, another retinoid target organ, and examined the relative contribution of the epidermis and dermis to the total retinoid binding. The mean specific activity of [3H]retinol (0.52 +/- 0.06 pmol/mg protein) and [3H]retinoic acid (3.20 +/- 0.45 pmol/mg protein) binding to cytosol preparations from different specimens of adult human skin was determined. On the average these skins bound 7-fold more retinoic acid than retinol. When skin was treated with EDTA and separated into epidermal and dermal fractions, [3H]retinol and [3H]retinoic acid binding was found in the cytosol derived from epidermis (0.36 +/- 0.03 pmol/mg protein, 3.69 +/- 0.13 pmol/mg protein, respectively) but not from dermis. To confirm that the absence of dermal binding was not due to loss during the EDTA separation, we assayed skin keratomed at 0.1, 0.2, and 0.3 mm. The skin obtained at 0.1 mm was upper epidermis and exhibited binding for both retinol and retinoid acid. The 0.2 mm skin, which added lower epidermis but little dermal contamination, had higher specific activities for both retinol and retinoic acid binding. The 0.3 mm skin which added primarily dermis, had lower specific activities for binding both retinoids. This is consistent with the concept that the epidermis is responsible for the majority of retinoid binding in adult human skin obtained from the lower limb.     

Inhibition of DNA synthesis of melanoma cells by azelaic acid

Azelaic acid was successfully used in the clinical treatment of 7 cases of lentigo maligna in that remission of the lesions was observed in all our patients. In order to elucidate mechanism(s) of the beneficial clinical effects, we studied the effect of azelaic acid on cultured melanoma cells. Cell numbers recovered from melanoma cell cultures grown for several days in the presence of 10 mM azelaic acid were 50-70% less than those recovered from control cultures or from cultures containing 10 mM adipic acid. This reduction of cell numbers was not due to a simple cytotoxic or cytolytic effect of azelaic acid but rather due to a dose-dependent inhibition of DNA synthesis. Interestingly, nontoxic concentrations of azelaic acid, which significantly reduced DNA synthesis of cultured melanoma cells, had no overt effect on the protein synthesis of these cells. It is conceivable that inhibition of DNA synthesis is one of the mechanisms by which azelaic acid prevents growth and proliferation of abnormal melanocytes.     

Acute toxicity of Zinc pyrithione to human skin cells in vitro

Zinc pyrithione introduced into cultures of rapidly proliferating NCTC 2544 human skin epithelial cells and normal human skin fibroblasts had a rapid cytotoxic effect even at very low concentrations (0.1-0.5 microgram/ml); there was no dose-dependent suppression of cell proliferation and no apparent interference with mitosis. Sodium pyrithione had a similar effect. Zinc oxide and zinc sulphate were at least 100 times better tolerated than zinc pyrithione, but no stimulatory effect on cell growth was detected with low concentrations of either compound. These results suggest that zinc pyrithione's action against dandruff is more likely to arise from a non-specific toxicity for epidermal cells than by an anti-mitotic effect or by remedying a local zinc deficiency.     

Type-1 steroid 5 alpha-reductase is functionally active in the hair follicle as evidenced by new selective inhibitors of either type-1 or type-2 human steroid 5 alpha-reductase

Steroid 5 alpha-reductase catalyzes the reduction of testosterone (T) into the very potent androgen dihydrotestosterone (DHT). The different tissue expression patterns of the two isoforms of 5 alpha-reductase, namely type-1 and type-2 5 alpha-reductase (5 alpha-R1 and 5 alpha-R2, respectively), have prompted studies directed towards the synthesis of selective 5 alpha-R1 or 5 alpha-R2 inhibitors. In this present work, we have performed a structure/activity study on the inhibitory potential of indole carboxylic acids against hair follicle 5 alpha-reductase activity. We have demonstrated that this class of molecules were potent inhibitors of either 5 alpha-R1 or 5 alpha-R2 or both depending on (i) substituents in positions 4, 5 or 6 and (ii) the presence of a free carboxylic group. We have also found that only 5 alpha-R1 or 5 alpha-R1/R2 inhibitors were able to inhibit 5 alpha-reductase activity in plucked hairs from female volunteers or in freshly isolated female hair follicles, selective 5 alpha-R2 inhibitors being inactive.     

Azelaic acid

This review is an update on the literature accumulated over the past 10 years following the original observation that azelaic acid, a naturally occurring and nontoxic C9 dicarboxylic acid, possesses significant biologic properties and a potential as a therapeutic agent. These studies have shown that azelaic acid is a reversible inhibitor of tyrosinase and other oxidoreductases in vitro and that it inhibits mitochondrial respiration. It can also inhibit anaerobic glycolysis. Both in vitro and in vivo it has an antimicrobial effect on both aerobic and anaerobic (Propionibacterium acnes) microorganisms. In tissue culture it exerts a dose- and time-dependent cytotoxic effect on malignant melanocytes, associated with mitochondrial damage and inhibition of deoxyribonucleic acid (DNA) synthesis. Tumoral cell lines not containing tyrosinase are equally affected. Normal cells in culture exposed to the same concentrations of the diacid that are toxic for tumoral cells are in general not damaged. Radioactive azelaic acid has been shown to penetrate tumoral cells at a higher level than normal cells of the corresponding line. Topically applied (a 20% cream), it has been shown to be of therapeutic value in skin disorders of different etiologies. Its beneficial effect on various forms of acne (comedogenic, papulopustular, nodulocystic) has been clearly demonstrated. Particularly important is its action on abnormal melanocytes, which has led to the possibility of obtaining good results on melasma and highly durable therapeutic responses on lentigo maligna. It is also capable of causing regression of cutaneous malignant melanoma, but its role in melanoma therapy remains to be investigated.(ABSTRACT TRUNCATED AT 250 WORDS)