The factors involved in the pathogenesis of acne are depicted in Figure 11 (125), and although the remission of acne remains a mystery (125,126), several technologies have been developed for the topical control of acne identified through single in vitro assays or a combination of different models.
Benzoyl peroxide has been reported to have multiple effects including bacter- iostatic activity, mild comedolytic activity, enhancement of local blood flow, and the release of free radical oxygen. Benzoyl peroxide is an organic peroxide that is bactericidal against P. acnes along with additional anti-inflammatory activity (127). There is a wide range of antimicrobial activity against multiple species and resistance does not occur. Hegemann et al. (127) investigated the release of reactive oxygen species regulated by protein kinase C (PKC) and calmodulin from human neutrophils; although benzoyl peroxide inhibited PKC activity (IC50 of 1.35 mM), there was no activity against calmodulin, suggesting that the
FIGURE 11 Synopsis of factors involved in the pathogenesis of acne. Factors involved in the pathogenesis of acne are increased sebum production, ductal cornification, bacterial colonization, and inflammation. Abbreviations: HLA-DR, human leukocyte antigen-D related; ICAM, intercellular adhesion molecule; IL, interleukin; TNF, tumor necrosis factor. Source: From Ref. 125.
anti-inflammatory activity of benzoyl peroxide was not mediated by PKC or calmodulin.
Babich et al. (128) used a human keratinocyte cell line to evaluate the cytotox- icity of benzoyl peroxide. As determined by uptake of Neutral red, irreversible cell death was evident after a one-hour exposure to 0.15 mM and greater concentrations of benzoyl peroxide, whereas reductions in proliferation were seen at concen- trations from 0.02 to 0.08 mM. Stress and damage as evident by vacuolization and LDH release were seen after four hours at 0.05 mM levels. As a control, the IC50 of benzoic acid was 29.5 mM.
Similar degrees of cytotoxicity were found with other organic peroxides
suggesting that the cytotoxicity of benzoyl peroxide may be related to the gener- ation of reactive oxidative free radicals. King et al. (129) found that benzoyl per- oxide, as a free radical generating compound, acts as a tumor promoter in mouse skin by causing DNA strand breaks and base modifications in cultured murine keratinocytes. Matsui et al. (130) also found that benzoyl peroxide inhibited both human and murine PKC in a cell-free system, although there was no translocation evident from the cytosol to the membrane when tested in cultured human keratino- cytes. Other reports investigating the mechanism of action of benzoyl peroxide include Valacchi et al. (131) who examined the depletion of vitamin E and IL-1a gene expression in HaCaT keratinocytes, Burkhart et al. (132) who reported increases in free radical activity in combination with antibiotics, and Lawrence et al. (133) in that benzoyl peroxide interferes with metabolic cooperation with cultured keratinocytes.
Another compound that has been utilized for the topical control of acne is sal- icylic acid. Salicylic acid reduces inflammation by inhibiting the synthesis of pros- taglandins that are generated in inflamed tissues. Salicylic acid also inhibits the conversion of arachidonic acid to PGE2, which is catalyzed by the enzyme cyclo- oxygenase. Salicylic acid also inhibits comedogenesis by promoting desquamation of the follicular epithelium. Additional agents that have been used to treat acne include sulfur for the treatment of inflammatory acne lesions through keratolysis activity and azelaic acid, a dicarboxylic acid that has both antibacterial and antiker- atinizing activity (134,135).
In addition to the models and assays described in this review, other in vitro models have been developed around the hair follicle. Although these models were not developed to look at acne per se, they may be of interest for specific cell types. Blume et al. (136) reported on the monolayer culture of keratinocytes isolated from terminal hair follicles that express glycoproteins during proliferation (gp38) and differentiation (gp80). These glycoproteins are absent in normal interfollicular keratinocytes. Limat and Noser (137) also reported on the serial cultivation of keratinocytes from the outer root sheath of hair follicles.
Lenoir-Viale et al. (138) documented the effects of retinoic acid on an in vitro model reconstructed from the outer root sheath of human hair follicles, whereas Hoeller et al. (139) divulged an improved method on the construction of in vitro skin equivalents from human hair follicles and fibroblasts. Additionally, Michel et al. (140) has developed a tissue-engineered human skin equivalent containing hair follicles.
The in situ organ culture of the entire pilosebaceous appendage has further been developed by Kealey’s laboratory to monitor hair growth. This model appears to be the most complex, as it contains the entire follicle including the sebaceous gland (141 – 143).
Katz and Taichman (144) reported on the development of a two-chamber culture model in which proteins secreted by keratinocytes could be isolated and characterized. In this model, a fully differentiated keratinocyte epithelium is grown on plastic inserts apart from fibroblasts in the lower chamber.
In conclusion, multiple in vitro models are available for the identification of technologies to alleviate or prevent the formation of acne lesions. By using these models, it is possible to determine the mechanism of action of lead candidates.