- Email: [email protected]
Acne – a multifaceted problem
Drug Discovery Today: Disease Mechanisms
DRUG DISCOVERY
TODAY
Vol. 5, No. 2 2008
Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Charles Lowenstein – The John Hopkins School of Medicine, Baltimore, USA
DISEASE Common skin conditions and disorders MECHANISMS
Acne – a multifaceted problem Neelam Muizzuddin1,*, Paolo Giacomoni2, Daniel Maes1 1 2
Este´e Lauder R&D, 125 Pinelawn Road, Melville, NY 11747, USA Clinique Laboratories, 125 Pinelawn Road, Melville, NY 11747, USA
Acne affects teenagers and adults under psychological, environmental or hormonal stresses. It can be treated with benzoyl peroxide or retinoids, but milder agents
Section Editor: Michael Roberts – School of Medicine, University of Queensland, Australia
would be welcome. Exfoliating agents associated with anti-inflammatory,
antimicrobial,
antiseborrheic
agents and stimulators of defensin biosynthesis
Acne: aetiology and treatments
decrease inflammatory lesions by 55%, and noninflam-
Acne is a family of skin disorders that vary greatly in pathogenesis and clinical manifestations. Acne is associated with adolescence but is also observed in concomitance with psychological stress or hormonal imbalance. The pathogenesis of acne is complex, with strong evidence supporting the involvement of sebaceous hyperplasia, follicular hyperkeratinization, bacterial hypercolonization, as well as immune reactions and inflammation. The clinical presentation of acne can range from a mild comedonal form to severe inflammatory cystic acne of the face, chest and back. At the ultrastructural level, follicular keratinocytes in comedones can be seen to possess increased numbers of desmosomes and tonofilaments, which result in ductal hypercornification. The increased activity of sebaceous glands elicited by androgen causes proliferation of P. acnes, an anaerobe present within the retained sebum in the pilosebaceous ducts [6]. The embryologic development of the human sebaceous gland is closely related to the differentiation of the hair follicle and the epidermis. The number of sebaceous glands remains approximately the same throughout life, whereas their size tends to increase with age. The development and function of the sebaceous gland in the fetal and neonatal periods appear to be regulated by maternal androgens and by endogenous steroid synthesis, as well as by other morphogens. The most apparent function of the glands is to excrete sebum. A strong increase in sebum excretion occurs a few hours after birth; this peaks during the first week and slowly subsides thereafter. A new rise takes
matory lesions by 69% in six weeks, an efficacy comparable to the one of 5% benzoyl peroxide.
Introduction Acne is a family of skin disorders that vary greatly in pathogenesis. It can occur in mild, moderate or severe manifestations. Treatments for severe acne are inappropriate for mild acne, which affects the vast majority of patients, insofar as their side effects outweigh the anticipated benefits: benzoyl peroxide and retinoic acid are irritant when topically applied [1,2], and retinoic acid is teratogenic when administered per os [3]. The onset of inflammatory lesions is understood as the consequence of excessive sebum secretion and loosely bound corneocytes which clog pores and create an anaerobic environment where anaerobic micro-organisms multiply and eventually provoke inflammatory reactions [4]. The onset is of noninflammatory lesions is understood as the consequence of follicular keratinocytes failing to differentiate thus producing hypergranulosis, resulting in the formation of microcomedones [5]. A blend of mild ingredients to stimulate exfoliation and defensin biosynthesis, associated with antiinflammatory, antiangiogenic, antimicrobial and antiseborrheic agents affords effects comparable to the ones of 5% benzoyl peroxide. *Corresponding author: N. Muizzuddin ([email protected]) 1740-6765/$ ß 2008 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmec.2008.08.001
e183
Drug Discovery Today: Disease Mechanisms | Common skin conditions and disorders
place at about age 9 years with adrenarche and continues up to age 17 years, when the adult level is reached. The sebaceous gland is an important formation site of active androgens. Androgens are well known for their effects on sebum excretion, whereas terminal sebocyte differentiation is assisted by peroxisome proliferator-activated receptor ligands. Estrogens, glucocorticoids and prolactin also influence sebaceous gland function. In addition, stress-sensing cutaneous signals lead to the production and release of corticotrophin-releasing hormone from dermal nerves and sebocytes with subsequent dose-dependent regulation of sebaceous nonpolar lipids [7]. There is ample clinical evidence suggesting that the nervous system such as emotional stress can influence the course of acne. Immunohistochemical studies revealed that substance P (SP)-immunoreactive nerve fibers were in close apposition to the sebaceous glands, and that neutral endopeptidase (NEP) was expressed in the germinative cells of the sebaceous glands in the skin from acne patients. Nerve growth factor showed immunoreactivity only within the germinative cells. In addition, an increase in the number of mast cells and a strong expression of endothelial leukocyte adhesion molecule-1 on the postcapillary venules were observed in adjacent areas to the sebaceous glands. These findings suggest that SP may stimulate lipogenesis of the sebaceous glands which may be followed by the proliferation of Propionibacterium acnes, and may yield a potent influence on the sebaceous glands by the provocation of inflammatory reactions via mast cells. Thus, cutaneous neurogenic factors should contribute to onset and/or exacerbation of acne inflammation [8]. Hormonal factors, particularly androgens, appear to be important in the pathogenesis of acne vulgaris. The sebaceous glands in acne are more sensitive to normal blood levels of androgens, and are stimulated to produce more sebum. P. acnes in the sebaceous follicles act on triglycerides in the sebum to form free fatty acids which might alter the process of keratinization in the follicular canal. A microcomedo that can progress to the clinical lesions of acne is formed. Sebum and its components may also be inflammatory if released into the skin. There are, however, still a number of unanswered questions relating to acne pathogenesis. The structural organization and bacteriological profile of follicular casts and early comedones in prepuberal children were investigated. Follicular casts were present in all samples but were not as abundant as usually seen in older individuals with acne. When examined with the light microscope, all casts and comedones were composed of numerous layers of horny cells and sebum, but were devoid of bacteria. Neither follicular casts nor comedones yielded bacteria when cultured. Electron microscopy of the follicular casts revealed the presence of small round, discrete lipid droplets and alternating dense and less dense lamellar configurations within the horny cells. Some horny cells closest to the e184
www.drugdiscoverytoday.com
Vol. 5, No. 2 2008
sebum-filled lumen contained large lipid masses, resulting in ‘balloon-shaped’ regions. Prepuberal follicular horny cells contained all of the abnormalities usually seen in follicular casts and biopsy material from acne patients, and this suggests that these casts are potential comedones. These aberrations occurred in the complete absence of bacteria indicating that bacteria are not essential to the formation of casts or comedones. Furthermore, our findings indicate that bacteria play little if any role in the initial events of pathological keratinization [9]. In adolescents and adults, high sebum concentrations and follicular hyperkeratinization lead to a change of the follicular milieu with consecutive proliferation of bacteria, chiefly P. acnes (Fig. 1). This proliferation leads to further increased production of the proinflammatory cytokines interleukin-1b, IL-8, GM-CSF and tumor necrosis factor a by T cells and keratinocytes, leading to the proliferation of both cell types. Further inflammatory responses lead to the development of increasing degrees of severity in inflammatory forms of acne. Agents like retinoids help reduce the hyperproliferation of keratinocytes, and can inhibit the migration of leucocytes [10,11]. The primary micro-organism associated in the development of inflammatory as well as noninflammatory acne is P. acnes [12]. P. acnes belongs to a family of anaerobic, nonspore-forming gram-positive bacteria. In addition to antibiotics, topical benzoyl peroxide and retinoids have consistently been found to be effective in reducing acne lesions [13,14]. We have undertaken experimental analyses to identify milder agents able to reduce the incidence of lesions with comparable efficacy.
Procedure Subjects A total of 51 females between the ages of 18 and 50 were recruited for the study, out of which 47 completed the study. The panel was divided in five groups of nine to ten each corresponding to the materials listed. The panelists exhibited Grade 2 acne (10–30 papules and comedones over about onefourth of the face) to Grade 4 acne (about half the face has papules, comedones and a few pustules; some lesions are red and inflamed), or Subtypes 1, erythematotelangiectatic and 2, papulopustular, rosacea (National Rosacea Society). Other than that they were healthy subjects with no other visible skin disease. Subjects exhibiting current sunburn, rashes, scratches, burn marks, etc., which might interfere with the evaluation of test results were excluded from the study. Pregnant or lactating females were also excluded. The panelists were questioned about their medical history and were reported to be on no antibiotic, antihistamines, anti-inflammatories or steroid therapy. Written informed consent was obtained from each volunteer before his/her entrance into the study.
Vol. 5, No. 2 2008
Drug Discovery Today: Disease Mechanisms | Common skin conditions and disorders
Figure 1. Three stage of formation of an acneic inflammatory lesion. (1) The pilosebaceous unit is normally producing sebum and differentiating keratinocytes, in the presence of the cutaneous flora. (2) Hyperkeritinization and excess sebum provoke the clogging of the pore. (3) Anaerobic bacteria proliferate, recruit inflammatory cells which release inflammatory signals (IL-1b, TNF a, etc.) (see text).
Clinical
Experiments and results
This was a six-week in-use study where the subjects were examined at baseline (before treatment) and after two and six weeks of treatment with their assigned test materials. At each time point, lesions of the full face were counted by trained personnel. Both inflammatory as well as noninflammatory lesions were counted.
Partially successful approaches to the treatment of cosmetic acne contemplate the topical application of mild exfoliators such as salicylic acid or N-acetyl D-glucosamine together with anti-inflammatory agents (selected among inhibitors of phospholipase A2, cyclooxygenase 2 or phosphodiesterase 4 and antiangiogenic agents) and antiseborrheic agents such as Saw www.drugdiscoverytoday.com
e185
Drug Discovery Today: Disease Mechanisms | Common skin conditions and disorders
Palmetto extracts together with antimicrobial agents targeting P. acnes. The goal of the experiment described in this chapter was to identify agents other than the ones currently used, and to monitor their effects on inflammatory and noninflammatory lesions in a cohort of women exhibiting Grades 2–4 acne. To maintain the experiments within the limit of feasibility, we have added to this blend in a stearate-based emulsion, an inducer of b-defensin, alone or with substances known to improve the barrier function. The use of stimulators of inducers of b-defensin is the consequence of the observation by Philpott [15] who investigated the expression patterns of two antimicrobial peptides, human b-defensin 1 (hBD1) and human b-defensin 2 (hBD2) in healthy human hair follicles as well as in peri- and intralesional skin of acne vulgaris lesions such as comedones, papules and pustules. He found that the majority of acne biopsies displayed a marked upregulation of hBD2 IR in the lesional and perilesional epithelium, particularly in pustules, and a less marked upregulation of hBD1 IR. The upregulation of bdefensins expression in acne vulgaris lesions when compared to controls suggests that b-defensins may be involved in the pathogenesis of acne vulgaris [15,16]. We used lactobacillus extracts were used as agents to upregulate defensin thereby controlling the P. acnes on acne affected skin. Acne therapy is generally directed against four anticipated pathogenic processes in acne: (1) abnormal keratinization (plugging) of the pilosebacous follicle, (2) excessive production of sebum, (3) proliferation of bacteria (P. acnes) in the follicle and (4) inflammatory reactions [17,18]. This study was designed to monitor the effects of a ‘five-pronged approach’ which addressed desquamation, oil control, antimicrobial, antiangiogenic and anti-inflammatory effects, added to the stimulation of the biosynthesis of b-defensins. In another set of experiments, a barrier repair technology aimed at avoiding the colonization of the surface by micro-organism was added as the sixth factor in the attempt of reducing acne lesions.
Figure 2. Percent reduction in inflammatory lesions.
e186
www.drugdiscoverytoday.com
Vol. 5, No. 2 2008
Because we are targeting several aspects of skin physiology, we label our treatments as five-pronged and six-pronged approach, and they are outlined in detail below.
Five-pronged approach: formulation containing ingredients addressing desquamation, oil control, antimicrobial, antiangiogenesis and anti-inflammatory N-acetyl D-glucosamine – 2% (desquamation) [19] Saw palmetto 0.5% – (5a-reductase inhibitor for oil control) [20] Lactobacillus 2% – (b-defensin induction for antimicrobial effects) [21] Acnacidol P 0.1% – (antimicrobial agent targeting P. acnes) [22] Phytosphingosine 0.2% – (PPAR agonist and antiangiogenesic agent) [23] Conjugated linoleic acid 0.5% – (PPAR agonist and anti-inflammatory agent) [24] Hoelen extract 0.1% – (PLA2 inhibitor for anti-inflammatory effects) [25] Resveratrol 0.1% – (COX 2 inhibitor for anti-inflammatory effects) [26] Amentoflavone 0.1% – (PDE4 inhibitor for anti-inflammatory effects) [27]
Six-pronged approach: formulation containing ingredients addressing desquamation, oil control, antimicrobial, antiangiogenesis and antiinflammatory + barrier repair technology N-acetyl D-glucosamine – 2% (desquamation) [19] Saw palmetto 0.5% – (5a-reductase inhibitor for oil control) [20] Lactobacillus 2% – (b-defensin induction for antimicrobial effects) [21] Acnacidol P 0.1% – (antimicrobial agent targeting P. acnes) [22]
Vol. 5, No. 2 2008
Drug Discovery Today: Disease Mechanisms | Common skin conditions and disorders
Figure 3. Percent reduction in noninflammatory lesions.
Phytosphingosine 0.2% – (PPAR agonist and antiangiogenic agent) [23] Conjugated linoleic acid 0.5% – (PPAR agonist for anti-inflammatory effects) [24] Hoelen extract 0.1% – (PLA2 inhibitor for anti-inflammatory effects) [25] Resveratrol 0.1% – (COX 2 inhibitor for anti-inflammatory effects) [26] Amentoflavone 0.1% – (PDE4 inhibitor for anti-inflammatory effects) [27] BR forest (muru muru butter) 0.2% – (to improve Barrier) [28] Cholesterol 0.2% – (to improve Barrier) [29] SCC complex 0.2% – (contains lipids to improve Barrier) The ingredients were formulated in a nonionic, oil-inwater base. The positive control was a commercial formula, clearasil with 5% benzoyl peroxide. The results are reported in Figs 2,3.
Conclusions From the results reported in Figs 1,2, it appears that at week 2, benzoyl peroxide reduces the count of both inflammatory and noninflammatory lesions. Its effects on noninflammatory lesions are much more marked at week 6, whereas no further improvement is observed for the inflammatory lesions. The vehicle seems to have some positive effects at week 6 for both inflammatory and noninflammatory lesions, and the five-pronged formula performs significantly better than the vehicle, reducing the count of the noninflammatory lesions by 69%. The relative long time interval (six weeks) necessary to observe a positive effect in the five-pronged approach is expected because the synthesis of b-defensins occurs in the basal layer and their antimicrobial effects is exerted in the stratum corneum, where they are expected to accumulate four
to five weeks after the triggering of their biosynthesis. The addition of agents able to improve the barrier and to avoid the colonization of the surface by adventitious micro-organisms, in the six-pronged formula, promotes a relatively rapid albeit limited reduction of inflammatory lesions without effect better than the vehicle on the noninflammatory ones. One can conclude that, for noninflammatory lesions, the five-pronged approach described in this experiment has effects comparable to the ones afforded by benzoyl peroxide. The positive results require longer treatment, as expected, but are devoid of irritating side effects. For the inflammatory lesions, benzoyl peroxide and the two multipronged formulas afforded results only slightly better than the ones obtained with the vehicle control.
References 1 Haustein, U.F. et al. (1985) Allergic and irritant potential of benzoyl peroxide. Contact Dermatitis 13, 252–257 2 Mukherjee, S. et al. (2006) Retinoids in the treatment of skin aging: an overview of clinical efficacy and safety. Clin. Interv. Aging 1, 327–348 3 Mills, J.P. et al. (2007) Ingestion of excessive preformed vitamin A by mothers amplifies storage of retinyl esters in early fetal livers of captive Old World monkeys. Comp. Med. 57, 505–511 4 Holland, K.T. et al. (1978) The role of bacteria in acne vulgaris – a new approach. Clin. Exp. Dermatol. 3, 253–257 5 Holmes, R.L. et al. (1972) Pilosebaceous duct obstruction and acne. Br. J. Dermatol. 87, 327–332 6 Toyoda, M. and Morohashi, M. (2001) Pathogenesis of acne. Med. Electron Microsc. 34, 29–40 7 Zouboulis, C.C. (2004) Acne and sebaceous gland function. Clin. Dermatol. 22, 360–366 8 Toyoda, M. and Morohashi, M. (2003) New aspects in acne inflammation. Dermatology 206, 17–23 9 Lavker, R.M. et al. (1981) The relationship between bacteria and the abnormal follicular keratinization in acne vulgaris. J. Invest. Dermatol. 77, 325–330 10 Gollnick, H. (2003) Current concepts of the pathogenesis of acne: implications for drug treatment. Drugs 63, 1579–1596 11 Buchner, S.A. (1990) Acne and its drug treatment. Ther. Umsch. 47, 670– 674 12 Leyden, J.J. (2004) Antibiotic resistance in the topical treatment of acne vulgaris. Cutis 73 (6 Suppl), 6–10 www.drugdiscoverytoday.com
e187
Drug Discovery Today: Disease Mechanisms | Common skin conditions and disorders
13
14
15 16
17 18 19 20
21
Leyden, J.J. (2002) Effect of topical benzoyl peroxide/clindamycin versus topical clindamycin and vehicle in the reduction of Propionibacterium acnes. Cutis 69, 475–480 Kligman, A.M. (1998) The growing importance of topical retinoids in clinical dermatology: a retrospective and prospective analysis. J. Am. Acad. Dermatol. 39 (2 Pt 3), S2–S7 Philpott, M.P. (2003) Defensins and acne. Mol. Immunol. 40, 457–462 Chronnell, C.M. et al. (2001) Human beta defensin-1 and -2 expression in human pilosebaceous units: upregulation in acne vulgaris lesions. J. Invest. Dermatol. 117, 1120–1125 Weiss, J.S. (1997) Current options for the topical treatment of acne vulgaris. Pediatr. Dermatol. 14, 480–488 Wilson, B.B. (1989) Acne vulgaris. Prim. Care 16, 695–712 Bissett, D.L. (2006) Glucosamine: an ingredient with skin and other benefits. J. Cosmet. Dermatol. 5, 309–315 Wadsworth, T.L. et al. (2007) Effects of dietary saw palmetto on the prostate of transgenic adenocarcinoma of the mouse prostate model (TRAMP). Prostate 67, 661–673 Wehkamp, J. et al. (2004) NF-kB- and AP-1-mediated induction of human beta defensin-2 in intestinal epithelial cells by Escherichia coli Nissle 1917: a novel effect of a probiotic bacterium. Infect Immun. 72, 5750–5758
e188
www.drugdiscoverytoday.com
Vol. 5, No. 2 2008
22 23 24
25
26 27 28
29
Maes, D.H., et al. (2003) Combinatorial anti-acne compositions. United States Patent 20030072777, April 17. Kwon, Y.B. et al. (2007) Anti-angiogenic effect of tetra-acetylphytosphingosine. Exp. Dermatol. 16, 311–317 Evans, M. et al. (2001) trans-10, cis-12 conjugated linoleic acid reduces triglyceride content while differentially affecting peroxisome proliferator activated receptor gamma2 and AP2 expression in 3T3-L1 preadipocytes. Lipids 36, 1223–1232 Sekiya, N. et al. (2003) Inhibitory effects of triterpenes isolated from Hoelen on free-radical induced lysis of red blood cells. Phytother. Res. 17, 160–162 Gentilli, M. et al. (2001) Resveratrol decreases hyperalgesia induced by carrageenan in the rat hind paw. Life Sci. 68, 1317–1321 Chaabi, M. et al. (2007) Biflavones of Decussucarpus rospigliosii as phosphodiesterase inhibitors. Planta Med. 73, 1284–1286 Polezel, M.A. Use of fats derived from palm trees to increase dermal hydration. Patent number 7045154, Filing date: May 7, 2002, Issue date: May 16, 2006 De Paepe, K. et al. (2002) Repair of acetone- and sodium lauryl sulphatedamaged human skin barrier function using topically applied emulsions containing barrier lipids. J. Eur. Acad. Dermatol. Venereol. 16, 587–594
DRUG DISCOVERY
TODAY
Vol. 5, No. 2 2008
Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Charles Lowenstein – The John Hopkins School of Medicine, Baltimore, USA
DISEASE Common skin conditions and disorders MECHANISMS
Acne – a multifaceted problem Neelam Muizzuddin1,*, Paolo Giacomoni2, Daniel Maes1 1 2
Este´e Lauder R&D, 125 Pinelawn Road, Melville, NY 11747, USA Clinique Laboratories, 125 Pinelawn Road, Melville, NY 11747, USA
Acne affects teenagers and adults under psychological, environmental or hormonal stresses. It can be treated with benzoyl peroxide or retinoids, but milder agents
Section Editor: Michael Roberts – School of Medicine, University of Queensland, Australia
would be welcome. Exfoliating agents associated with anti-inflammatory,
antimicrobial,
antiseborrheic
agents and stimulators of defensin biosynthesis
Acne: aetiology and treatments
decrease inflammatory lesions by 55%, and noninflam-
Acne is a family of skin disorders that vary greatly in pathogenesis and clinical manifestations. Acne is associated with adolescence but is also observed in concomitance with psychological stress or hormonal imbalance. The pathogenesis of acne is complex, with strong evidence supporting the involvement of sebaceous hyperplasia, follicular hyperkeratinization, bacterial hypercolonization, as well as immune reactions and inflammation. The clinical presentation of acne can range from a mild comedonal form to severe inflammatory cystic acne of the face, chest and back. At the ultrastructural level, follicular keratinocytes in comedones can be seen to possess increased numbers of desmosomes and tonofilaments, which result in ductal hypercornification. The increased activity of sebaceous glands elicited by androgen causes proliferation of P. acnes, an anaerobe present within the retained sebum in the pilosebaceous ducts [6]. The embryologic development of the human sebaceous gland is closely related to the differentiation of the hair follicle and the epidermis. The number of sebaceous glands remains approximately the same throughout life, whereas their size tends to increase with age. The development and function of the sebaceous gland in the fetal and neonatal periods appear to be regulated by maternal androgens and by endogenous steroid synthesis, as well as by other morphogens. The most apparent function of the glands is to excrete sebum. A strong increase in sebum excretion occurs a few hours after birth; this peaks during the first week and slowly subsides thereafter. A new rise takes
matory lesions by 69% in six weeks, an efficacy comparable to the one of 5% benzoyl peroxide.
Introduction Acne is a family of skin disorders that vary greatly in pathogenesis. It can occur in mild, moderate or severe manifestations. Treatments for severe acne are inappropriate for mild acne, which affects the vast majority of patients, insofar as their side effects outweigh the anticipated benefits: benzoyl peroxide and retinoic acid are irritant when topically applied [1,2], and retinoic acid is teratogenic when administered per os [3]. The onset of inflammatory lesions is understood as the consequence of excessive sebum secretion and loosely bound corneocytes which clog pores and create an anaerobic environment where anaerobic micro-organisms multiply and eventually provoke inflammatory reactions [4]. The onset is of noninflammatory lesions is understood as the consequence of follicular keratinocytes failing to differentiate thus producing hypergranulosis, resulting in the formation of microcomedones [5]. A blend of mild ingredients to stimulate exfoliation and defensin biosynthesis, associated with antiinflammatory, antiangiogenic, antimicrobial and antiseborrheic agents affords effects comparable to the ones of 5% benzoyl peroxide. *Corresponding author: N. Muizzuddin ([email protected]) 1740-6765/$ ß 2008 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmec.2008.08.001
e183
Drug Discovery Today: Disease Mechanisms | Common skin conditions and disorders
place at about age 9 years with adrenarche and continues up to age 17 years, when the adult level is reached. The sebaceous gland is an important formation site of active androgens. Androgens are well known for their effects on sebum excretion, whereas terminal sebocyte differentiation is assisted by peroxisome proliferator-activated receptor ligands. Estrogens, glucocorticoids and prolactin also influence sebaceous gland function. In addition, stress-sensing cutaneous signals lead to the production and release of corticotrophin-releasing hormone from dermal nerves and sebocytes with subsequent dose-dependent regulation of sebaceous nonpolar lipids [7]. There is ample clinical evidence suggesting that the nervous system such as emotional stress can influence the course of acne. Immunohistochemical studies revealed that substance P (SP)-immunoreactive nerve fibers were in close apposition to the sebaceous glands, and that neutral endopeptidase (NEP) was expressed in the germinative cells of the sebaceous glands in the skin from acne patients. Nerve growth factor showed immunoreactivity only within the germinative cells. In addition, an increase in the number of mast cells and a strong expression of endothelial leukocyte adhesion molecule-1 on the postcapillary venules were observed in adjacent areas to the sebaceous glands. These findings suggest that SP may stimulate lipogenesis of the sebaceous glands which may be followed by the proliferation of Propionibacterium acnes, and may yield a potent influence on the sebaceous glands by the provocation of inflammatory reactions via mast cells. Thus, cutaneous neurogenic factors should contribute to onset and/or exacerbation of acne inflammation [8]. Hormonal factors, particularly androgens, appear to be important in the pathogenesis of acne vulgaris. The sebaceous glands in acne are more sensitive to normal blood levels of androgens, and are stimulated to produce more sebum. P. acnes in the sebaceous follicles act on triglycerides in the sebum to form free fatty acids which might alter the process of keratinization in the follicular canal. A microcomedo that can progress to the clinical lesions of acne is formed. Sebum and its components may also be inflammatory if released into the skin. There are, however, still a number of unanswered questions relating to acne pathogenesis. The structural organization and bacteriological profile of follicular casts and early comedones in prepuberal children were investigated. Follicular casts were present in all samples but were not as abundant as usually seen in older individuals with acne. When examined with the light microscope, all casts and comedones were composed of numerous layers of horny cells and sebum, but were devoid of bacteria. Neither follicular casts nor comedones yielded bacteria when cultured. Electron microscopy of the follicular casts revealed the presence of small round, discrete lipid droplets and alternating dense and less dense lamellar configurations within the horny cells. Some horny cells closest to the e184
www.drugdiscoverytoday.com
Vol. 5, No. 2 2008
sebum-filled lumen contained large lipid masses, resulting in ‘balloon-shaped’ regions. Prepuberal follicular horny cells contained all of the abnormalities usually seen in follicular casts and biopsy material from acne patients, and this suggests that these casts are potential comedones. These aberrations occurred in the complete absence of bacteria indicating that bacteria are not essential to the formation of casts or comedones. Furthermore, our findings indicate that bacteria play little if any role in the initial events of pathological keratinization [9]. In adolescents and adults, high sebum concentrations and follicular hyperkeratinization lead to a change of the follicular milieu with consecutive proliferation of bacteria, chiefly P. acnes (Fig. 1). This proliferation leads to further increased production of the proinflammatory cytokines interleukin-1b, IL-8, GM-CSF and tumor necrosis factor a by T cells and keratinocytes, leading to the proliferation of both cell types. Further inflammatory responses lead to the development of increasing degrees of severity in inflammatory forms of acne. Agents like retinoids help reduce the hyperproliferation of keratinocytes, and can inhibit the migration of leucocytes [10,11]. The primary micro-organism associated in the development of inflammatory as well as noninflammatory acne is P. acnes [12]. P. acnes belongs to a family of anaerobic, nonspore-forming gram-positive bacteria. In addition to antibiotics, topical benzoyl peroxide and retinoids have consistently been found to be effective in reducing acne lesions [13,14]. We have undertaken experimental analyses to identify milder agents able to reduce the incidence of lesions with comparable efficacy.
Procedure Subjects A total of 51 females between the ages of 18 and 50 were recruited for the study, out of which 47 completed the study. The panel was divided in five groups of nine to ten each corresponding to the materials listed. The panelists exhibited Grade 2 acne (10–30 papules and comedones over about onefourth of the face) to Grade 4 acne (about half the face has papules, comedones and a few pustules; some lesions are red and inflamed), or Subtypes 1, erythematotelangiectatic and 2, papulopustular, rosacea (National Rosacea Society). Other than that they were healthy subjects with no other visible skin disease. Subjects exhibiting current sunburn, rashes, scratches, burn marks, etc., which might interfere with the evaluation of test results were excluded from the study. Pregnant or lactating females were also excluded. The panelists were questioned about their medical history and were reported to be on no antibiotic, antihistamines, anti-inflammatories or steroid therapy. Written informed consent was obtained from each volunteer before his/her entrance into the study.
Vol. 5, No. 2 2008
Drug Discovery Today: Disease Mechanisms | Common skin conditions and disorders
Figure 1. Three stage of formation of an acneic inflammatory lesion. (1) The pilosebaceous unit is normally producing sebum and differentiating keratinocytes, in the presence of the cutaneous flora. (2) Hyperkeritinization and excess sebum provoke the clogging of the pore. (3) Anaerobic bacteria proliferate, recruit inflammatory cells which release inflammatory signals (IL-1b, TNF a, etc.) (see text).
Clinical
Experiments and results
This was a six-week in-use study where the subjects were examined at baseline (before treatment) and after two and six weeks of treatment with their assigned test materials. At each time point, lesions of the full face were counted by trained personnel. Both inflammatory as well as noninflammatory lesions were counted.
Partially successful approaches to the treatment of cosmetic acne contemplate the topical application of mild exfoliators such as salicylic acid or N-acetyl D-glucosamine together with anti-inflammatory agents (selected among inhibitors of phospholipase A2, cyclooxygenase 2 or phosphodiesterase 4 and antiangiogenic agents) and antiseborrheic agents such as Saw www.drugdiscoverytoday.com
e185
Drug Discovery Today: Disease Mechanisms | Common skin conditions and disorders
Palmetto extracts together with antimicrobial agents targeting P. acnes. The goal of the experiment described in this chapter was to identify agents other than the ones currently used, and to monitor their effects on inflammatory and noninflammatory lesions in a cohort of women exhibiting Grades 2–4 acne. To maintain the experiments within the limit of feasibility, we have added to this blend in a stearate-based emulsion, an inducer of b-defensin, alone or with substances known to improve the barrier function. The use of stimulators of inducers of b-defensin is the consequence of the observation by Philpott [15] who investigated the expression patterns of two antimicrobial peptides, human b-defensin 1 (hBD1) and human b-defensin 2 (hBD2) in healthy human hair follicles as well as in peri- and intralesional skin of acne vulgaris lesions such as comedones, papules and pustules. He found that the majority of acne biopsies displayed a marked upregulation of hBD2 IR in the lesional and perilesional epithelium, particularly in pustules, and a less marked upregulation of hBD1 IR. The upregulation of bdefensins expression in acne vulgaris lesions when compared to controls suggests that b-defensins may be involved in the pathogenesis of acne vulgaris [15,16]. We used lactobacillus extracts were used as agents to upregulate defensin thereby controlling the P. acnes on acne affected skin. Acne therapy is generally directed against four anticipated pathogenic processes in acne: (1) abnormal keratinization (plugging) of the pilosebacous follicle, (2) excessive production of sebum, (3) proliferation of bacteria (P. acnes) in the follicle and (4) inflammatory reactions [17,18]. This study was designed to monitor the effects of a ‘five-pronged approach’ which addressed desquamation, oil control, antimicrobial, antiangiogenic and anti-inflammatory effects, added to the stimulation of the biosynthesis of b-defensins. In another set of experiments, a barrier repair technology aimed at avoiding the colonization of the surface by micro-organism was added as the sixth factor in the attempt of reducing acne lesions.
Figure 2. Percent reduction in inflammatory lesions.
e186
www.drugdiscoverytoday.com
Vol. 5, No. 2 2008
Because we are targeting several aspects of skin physiology, we label our treatments as five-pronged and six-pronged approach, and they are outlined in detail below.
Five-pronged approach: formulation containing ingredients addressing desquamation, oil control, antimicrobial, antiangiogenesis and anti-inflammatory N-acetyl D-glucosamine – 2% (desquamation) [19] Saw palmetto 0.5% – (5a-reductase inhibitor for oil control) [20] Lactobacillus 2% – (b-defensin induction for antimicrobial effects) [21] Acnacidol P 0.1% – (antimicrobial agent targeting P. acnes) [22] Phytosphingosine 0.2% – (PPAR agonist and antiangiogenesic agent) [23] Conjugated linoleic acid 0.5% – (PPAR agonist and anti-inflammatory agent) [24] Hoelen extract 0.1% – (PLA2 inhibitor for anti-inflammatory effects) [25] Resveratrol 0.1% – (COX 2 inhibitor for anti-inflammatory effects) [26] Amentoflavone 0.1% – (PDE4 inhibitor for anti-inflammatory effects) [27]
Six-pronged approach: formulation containing ingredients addressing desquamation, oil control, antimicrobial, antiangiogenesis and antiinflammatory + barrier repair technology N-acetyl D-glucosamine – 2% (desquamation) [19] Saw palmetto 0.5% – (5a-reductase inhibitor for oil control) [20] Lactobacillus 2% – (b-defensin induction for antimicrobial effects) [21] Acnacidol P 0.1% – (antimicrobial agent targeting P. acnes) [22]
Vol. 5, No. 2 2008
Drug Discovery Today: Disease Mechanisms | Common skin conditions and disorders
Figure 3. Percent reduction in noninflammatory lesions.
Phytosphingosine 0.2% – (PPAR agonist and antiangiogenic agent) [23] Conjugated linoleic acid 0.5% – (PPAR agonist for anti-inflammatory effects) [24] Hoelen extract 0.1% – (PLA2 inhibitor for anti-inflammatory effects) [25] Resveratrol 0.1% – (COX 2 inhibitor for anti-inflammatory effects) [26] Amentoflavone 0.1% – (PDE4 inhibitor for anti-inflammatory effects) [27] BR forest (muru muru butter) 0.2% – (to improve Barrier) [28] Cholesterol 0.2% – (to improve Barrier) [29] SCC complex 0.2% – (contains lipids to improve Barrier) The ingredients were formulated in a nonionic, oil-inwater base. The positive control was a commercial formula, clearasil with 5% benzoyl peroxide. The results are reported in Figs 2,3.
Conclusions From the results reported in Figs 1,2, it appears that at week 2, benzoyl peroxide reduces the count of both inflammatory and noninflammatory lesions. Its effects on noninflammatory lesions are much more marked at week 6, whereas no further improvement is observed for the inflammatory lesions. The vehicle seems to have some positive effects at week 6 for both inflammatory and noninflammatory lesions, and the five-pronged formula performs significantly better than the vehicle, reducing the count of the noninflammatory lesions by 69%. The relative long time interval (six weeks) necessary to observe a positive effect in the five-pronged approach is expected because the synthesis of b-defensins occurs in the basal layer and their antimicrobial effects is exerted in the stratum corneum, where they are expected to accumulate four
to five weeks after the triggering of their biosynthesis. The addition of agents able to improve the barrier and to avoid the colonization of the surface by adventitious micro-organisms, in the six-pronged formula, promotes a relatively rapid albeit limited reduction of inflammatory lesions without effect better than the vehicle on the noninflammatory ones. One can conclude that, for noninflammatory lesions, the five-pronged approach described in this experiment has effects comparable to the ones afforded by benzoyl peroxide. The positive results require longer treatment, as expected, but are devoid of irritating side effects. For the inflammatory lesions, benzoyl peroxide and the two multipronged formulas afforded results only slightly better than the ones obtained with the vehicle control.
References 1 Haustein, U.F. et al. (1985) Allergic and irritant potential of benzoyl peroxide. Contact Dermatitis 13, 252–257 2 Mukherjee, S. et al. (2006) Retinoids in the treatment of skin aging: an overview of clinical efficacy and safety. Clin. Interv. Aging 1, 327–348 3 Mills, J.P. et al. (2007) Ingestion of excessive preformed vitamin A by mothers amplifies storage of retinyl esters in early fetal livers of captive Old World monkeys. Comp. Med. 57, 505–511 4 Holland, K.T. et al. (1978) The role of bacteria in acne vulgaris – a new approach. Clin. Exp. Dermatol. 3, 253–257 5 Holmes, R.L. et al. (1972) Pilosebaceous duct obstruction and acne. Br. J. Dermatol. 87, 327–332 6 Toyoda, M. and Morohashi, M. (2001) Pathogenesis of acne. Med. Electron Microsc. 34, 29–40 7 Zouboulis, C.C. (2004) Acne and sebaceous gland function. Clin. Dermatol. 22, 360–366 8 Toyoda, M. and Morohashi, M. (2003) New aspects in acne inflammation. Dermatology 206, 17–23 9 Lavker, R.M. et al. (1981) The relationship between bacteria and the abnormal follicular keratinization in acne vulgaris. J. Invest. Dermatol. 77, 325–330 10 Gollnick, H. (2003) Current concepts of the pathogenesis of acne: implications for drug treatment. Drugs 63, 1579–1596 11 Buchner, S.A. (1990) Acne and its drug treatment. Ther. Umsch. 47, 670– 674 12 Leyden, J.J. (2004) Antibiotic resistance in the topical treatment of acne vulgaris. Cutis 73 (6 Suppl), 6–10 www.drugdiscoverytoday.com
e187
Drug Discovery Today: Disease Mechanisms | Common skin conditions and disorders
13
14
15 16
17 18 19 20
21
Leyden, J.J. (2002) Effect of topical benzoyl peroxide/clindamycin versus topical clindamycin and vehicle in the reduction of Propionibacterium acnes. Cutis 69, 475–480 Kligman, A.M. (1998) The growing importance of topical retinoids in clinical dermatology: a retrospective and prospective analysis. J. Am. Acad. Dermatol. 39 (2 Pt 3), S2–S7 Philpott, M.P. (2003) Defensins and acne. Mol. Immunol. 40, 457–462 Chronnell, C.M. et al. (2001) Human beta defensin-1 and -2 expression in human pilosebaceous units: upregulation in acne vulgaris lesions. J. Invest. Dermatol. 117, 1120–1125 Weiss, J.S. (1997) Current options for the topical treatment of acne vulgaris. Pediatr. Dermatol. 14, 480–488 Wilson, B.B. (1989) Acne vulgaris. Prim. Care 16, 695–712 Bissett, D.L. (2006) Glucosamine: an ingredient with skin and other benefits. J. Cosmet. Dermatol. 5, 309–315 Wadsworth, T.L. et al. (2007) Effects of dietary saw palmetto on the prostate of transgenic adenocarcinoma of the mouse prostate model (TRAMP). Prostate 67, 661–673 Wehkamp, J. et al. (2004) NF-kB- and AP-1-mediated induction of human beta defensin-2 in intestinal epithelial cells by Escherichia coli Nissle 1917: a novel effect of a probiotic bacterium. Infect Immun. 72, 5750–5758
e188
www.drugdiscoverytoday.com
Vol. 5, No. 2 2008
22 23 24
25
26 27 28
29
Maes, D.H., et al. (2003) Combinatorial anti-acne compositions. United States Patent 20030072777, April 17. Kwon, Y.B. et al. (2007) Anti-angiogenic effect of tetra-acetylphytosphingosine. Exp. Dermatol. 16, 311–317 Evans, M. et al. (2001) trans-10, cis-12 conjugated linoleic acid reduces triglyceride content while differentially affecting peroxisome proliferator activated receptor gamma2 and AP2 expression in 3T3-L1 preadipocytes. Lipids 36, 1223–1232 Sekiya, N. et al. (2003) Inhibitory effects of triterpenes isolated from Hoelen on free-radical induced lysis of red blood cells. Phytother. Res. 17, 160–162 Gentilli, M. et al. (2001) Resveratrol decreases hyperalgesia induced by carrageenan in the rat hind paw. Life Sci. 68, 1317–1321 Chaabi, M. et al. (2007) Biflavones of Decussucarpus rospigliosii as phosphodiesterase inhibitors. Planta Med. 73, 1284–1286 Polezel, M.A. Use of fats derived from palm trees to increase dermal hydration. Patent number 7045154, Filing date: May 7, 2002, Issue date: May 16, 2006 De Paepe, K. et al. (2002) Repair of acetone- and sodium lauryl sulphatedamaged human skin barrier function using topically applied emulsions containing barrier lipids. J. Eur. Acad. Dermatol. Venereol. 16, 587–594