Imiquimod as an antiaging agent

Imiquimod as an antiaging agent

Imiquimod as an antiaging agent Sara Metcalf, MD,a A. Neil Crowson, MD,b Mark Naylor, MD,c Raashid Haque, MD,d and Raymond Cornelison, MDa Oklahoma Ci...

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Imiquimod as an antiaging agent Sara Metcalf, MD,a A. Neil Crowson, MD,b Mark Naylor, MD,c Raashid Haque, MD,d and Raymond Cornelison, MDa Oklahoma City and Tulsa, Oklahoma; and Wichita, Kansas Background: Topical imiquimod therapy has proven to be effective for a variety of infectious, neoplastic, and inflammatory dermatologic diseases. Several published reports have validated the benefit of imiquimod therapy for actinic keratoses and superficial melanoma and nonmelanoma skin cancers. There is, however, limited evidence demonstrating the use of topical imiquimod application as an antiaging treatment. Objectives: We examined the effectiveness of imiquimod 5% cream in the treatment of photoaging by evaluating pretreatment and posttreatment biopsy specimens and documenting the histologic changes. Methods: This study represents an extension of an earlier project in our department in which patients with biopsy-proven lesions of lentigo maligna (LM) were recruited from a university dermatology service, a hospital, and referrals from private practitioners for an open-labeled efficacy trial with daily topical application of 5% imiquimod for 3 months. Biopsy of clinically affected skin was carried out on all patients before and after treatment. Using a semiquantitative method, biopsy specimens were analyzed for changes in the dermal collagen table (solar elastosis vs papillary dermal fibroplasia). Additional parameters analyzed included epidermal changes (atrophy vs acanthosis, melanin content, and hypergranulosis) and inflammatory effects (epidermal and dermal cell populations along with presence of pigment incontinence). Variables were compared using paired Wilcoxan rank sums. Results: Of 26 evaluable patients who completed 3 months of daily application, 24 ([92.3%) showed a significant increase in papillary dermal fibroplasia (P \.0001) with associated reduction in solar elastosis (P = .0036). Other noteworthy findings were restoration of normal epidermal thickness (P = .0073) and melanization (P \ .0001). Limitations: This study only evaluates the effect of imiquimod in the lesional skin of LM. It is not known whether the results are applicable to nonlesional, photoaged skin. Conclusion: Topical imiquimod appears to induce reparative changes to the epidermis and the dermal collagen table in chronically sun-damaged skin associated with LM, indicating its potential use as an antiaging treatment. These findings need to be confirmed in photodamaged skin not associated with LM. ( J Am Acad Dermatol 2007;56:422-5.)

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hotoaging refers to both the clinical and microscopic cutaneous changes that are induced by cumulative exposure to ultraviolet (UV) radiation and superimposed on the background of chronologic aging.1 Although many consider photoaging to be primarily a cosmetic problem, the fact that it constitutes the background for the

From the Department of Dermatology, University of Oklahoma, Oklahoma Citya; Departments of Dermatology, Pathology, and Surgery, University of Oklahoma and Regional Medical Laboratory, St John Medical Center, Tulsab; Department of Dermatology, University of Oklahoma, Tulsac; and Wichita Clinic Northeast.d This is an extension of a previous project supported by a grant from 3M Pharmaceuticals. Disclosure: Drs Naylor and Cornelison each serve as a consultant and investigator for 3M Pharmaceuticals. Presented in poster format at the American Academy of Dermatology Meeting in San Francisco, CA, March 3, 2006.

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Abbreviations used: AP: LM: MMP: TLR: UV:

activator protein lentigo maligna matrix metalloproteinase Toll-like receptor ultraviolet

Accepted for publication October 17, 2006. Reprint requests: A. Neil Crowson, MD, Departments of Dermatology, Pathology, and Surgery, University of Oklahoma and Regional Medical Laboratory, St John Medical Center, 1923 S Utica Ave, Tulsa, OK 74104. E-mail: [email protected] Published online December 30, 2006. 0190-9622/$32.00 ª 2007 by the American Academy of Dermatology, Inc. doi:10.1016/j.jaad.2006.10.034

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Fig 1. Pretreatment lesional photoaged skin (A) and 1 (B), 4 (C), and 16 (D) weeks posttreatment.

development of precancerous and cancerous skin lesions underscores its medical importance. There is a growing body of evidence to support the efficacy of imiquimod in the treatment of such precancerous and cancerous lesions, and this topical immune response modifier is currently indicated for the treatment of actinic keratosis and superficial basal cell carcinoma, in addition to its initial indication for viral-induced genital warts. Our intention was to document the dermatopathologic effects of topical imiquimod therapy in a series of patients previously reported2,3 with lentigo maligna (LM), an in situ form of melanoma typically affecting sun-damaged skin of the head and neck of the elderly.4

MATERIALS AND METHODS Institutional review board approval was obtained for an open-label trial of 5% imiquimod cream for the treatment of LM. From a university and a

hospital dermatology clinic, and private practitioners, 28 patients with a diagnosis of LM established by a combination of clinical, dermoscopic, and histopathologic findings, the latter confirmed using standard published criteria4 by the study pathologist (A. N. C.), were enrolled. Individuals suggested clinically or proven at biopsy to have invasive melanoma were excluded. Patients had to have a minimum of 2 cm2 of tumor left to treat after biopsy. Digital photographic documentation of lesional skin was taken in all cases (Fig 1); lesions were photographed and traced on plastic templates that included outlines of other landmarks so that the affected areas could be located after therapy. Patients were instructed in the application of topical imiquimod and were seen again at 1, 4, 8, 12, 16, 24, and 52 weeks. The intent was for each patient to treat daily for 3 months unless rest periods were required because of intolerable irritation or impending ulceration. Treatment was discontinued at week 12. At week 16 a minimum of 4 punch biopsy specimens

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micrometer on a light microscope (BMAX 60, Olympus, Center Valley, Pa) at 320 objective magnification. Four separate counts of melanocytes over 0.5 mm of epidermal transverse diameter were taken independently in blinded fashion by two observers (A. N. C. and S. M.). Other parameters were graded semiquantitatively on a representative pretreatment and posttreatment biopsy specimen in hematoxylineosinestained 5 paraffin sections. Parameters assessed semiquantitatively included: inflammation (mononuclear cells graded as follows: 0 = \10 cells/403high-power field (hpf); 1 = 10-50 cells/hpf; 2 = 51-75 cells/hpf; 3 = [75 cells/hpf); epidermal thickness (graded as 0 = atrophic; 1 = normal; 2 = slight increase [\20%]; 3 = moderate increase (20%-30%); 4 = marked increase [[30%]); granular layer thickness (graded as 0 = reduced; 1 = normal; 2 = slight increase; 3 = moderate increase; 4 = marked increase); solar elastosis (graded as 0 = none; 1 = mild [\25%]; 2 = moderate [25%-50%]; 3 = severe [[50%]); fibrosis (papillary dermis) (graded as 0 = none; 1 = mild [\25%]; 2 = moderate [25%-50%]; 3 = severe [[50%]); melanin content (epidermis) (graded as 0 = none; 1 = basal layer, discontinuous; 2 = basal layer, continuous; 3 = lower third epidermis; 4 = [lower third epidermis); and pigment incontinence (dermal melanophages) (graded as 0 = none; 1 = 1-3/hpf; 2 = 4-5/hpf; 3 = [5/hpf).

Fig 2. A, Pretreatment histopathologic findings reveal solar elastosis and epidermal atrophy in setting of lentigo maligna. B and C, After 12 weeks of therapy with imiquimod, there is fibroplasia of papillary dermis and reduction of solar elastosis. (A to C, Hematoxylin-eosin stain; original magnifications: A, 320; B, 310; C, 320.)

(4 mm) were taken in the previously affected area at locations determined by ascertaining the areas most suggestive of persistent tumor by dermatoscopic and clinical criteria as previously described.2 Moderate to severe visible erythema was present for at least a period of the 3-month therapy in almost all of the patients who responded to treatment. For all pretreatment and one representative posttreatment biopsy specimen, Melan-A immunostaining was performed on an automated immunostainer (Benchmark, Ventana Medical Systems, Tucson, Ariz) using a fast red chromagen, and the number of melanocytes per linear millimeter of epidermis was determined using a calibrated ocular

Statistical analysis Melanocyte density was analyzed using a repeated measures analysis of variance on logged density values to satisfy the equality of variances. All other studied variables were compared using paired Wilcoxan rank sums.

RESULTS Of 26 evaluable patients who completed 3 months of daily application, 24 ([92.3%) showed a significant increase in papillary dermal fibroplasia (P \ .0001) with associated reduction in solar elastosis (P = .0036) (Fig 2). Other noteworthy findings were restoration of normal epidermal thickness (P = .0073) and melanization (P \ .0001). Regarding treatment of the LM, 24 (92.3%) were complete responders and two were treatment failures. All patients showed a striking reduction in melanocyte density from pretreatment biopsy specimens (197.26 [mean] 6 173.20 [SD] vs 10.76 6 29.70 [P \.0001]) (Fig 2).

DISCUSSION UV radiation from the sun damages human skin, resulting in a premature aging process called photoaging.5 Significant advances have been made in

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the understanding of the molecular mechanisms of photoaging, creating potential opportunities for therapeutic intervention. It has been shown that UVinduced signal transduction pathways in human skin mediate the clinically apparent damage to connective tissue. Specifically, UV radiation activates growth factor and cytokine receptors on the surfaces of keratinocytes and fibroblasts resulting in signal transduction cascades that induce the transcription factor complex activator protein (AP)-1. In keratinocytes, AP-1 activation results in increased degradation of collagen and other components of the extracellular matrix by increased activity of matrix metalloproteinase (MMP). In fibroblasts, AP-1 activation results in inhibition of procollagen gene expression, thus, reducing collagen synthesis and dermal matrix repair.5 In addition, the levels of certain tissue inhibitors of MMP have been shown to be reduced in both photoaged and chronologically aged skin.5,6 While studying the efficacy of topical imiquimod in biopsy-proven LM, an in situ melanoma that occurs almost exclusively in photodamaged skin, we noted that in addition to the high treatment success rate, imiquimod appeared to reverse some of the histopathologic signs of photoaging. Posttreatment biopsy specimens revealed significant reduction in solar elastosis with normalization of epidermal thickness and papillary dermal fibroplasia compared with pretreatment specimens. The mechanism of this reparative effect of imiquimod on photodamaged skin is unclear, as both UV radiation and imiquimod seem to result in activation of the transcription factors nuclear factorkappa B and AP-1 and the subsequent transcription of proinflammatory cytokine genes, imiquimod doing so by its interaction with the Toll-like receptor (TLR) 7.5,7 However, others have shown a downregulation in imiquimod-treated skin of MMP-9, basic fibroblast growth factor, and vascular endothelial growth factor with a corresponding up-regulation of tissue inhibitor of MMP-1.8-10 The cytokine milieu induced by imiquimod is undoubtedly complex, but these latter findings could explain the reduction in clinical and histologic photoaging by demonstrating an inhibition of dermal matrix degradation, thus, allowing for repair. It is not clear to what degree this response is specific for imiquimodinduced inflammation. Stimulation of TLR 7 by imiquimod is but one of several ways that the cutaneous innate immune response can be activated. It is conceivable that activation of this arm of the immune system by other TLR pathways (eg, TLR 1-bacterial lipoproteins, TLR 2-bacterial peptidoglycans, TLR

3-viral double-stranded RNA, TLR 4-lipopolysaccharide of gram-negative bacteria, TLR 5-bacterial flagellins, or TLR 9-unmethylated viral and bacterial cytidine-guanine DNA)11 or more generally by the common adaptor MyD88, which is thought to be essential for the inflammatory responses mediated by all the TLR family members,12 might also be beneficial to photoaging phenomena.

CONCLUSION This study demonstrates that topical imiquimod appears to induce collagen table repair and restoration of normal epidermal thickness in photodamaged skin in the setting of LM. Additional studies are required to corroborate these findings in photoaged skin not associated with LM, and to elucidate the mechanism of such changes. The statistical analysis was performed by Mary Cheang of the University of Manitoba. Dr Richard Sontheimer kindly reviewed the manuscript.

REFERENCES 1. Stratigos AJ, Katsambas AD. The role of topical retinoids in the treatment of photoaging. Drugs 2005;65:1061-72. 2. Naylor MF, Crowson N, Kuwahara R, Teague K, Garcia C, Mackinnis C, et al. Treatment of lentigo maligna with topical imiquimod. Br J Dermatol 2003;149:66-9. 3. Crowson AN, Metcalf S, Naylor MF, Magro CM, Haque R, Garcia C, Cornelison RL. Lentigo maligna: The dermatopathologic effects of topical Imiquimod therapy. Lab Invest 2004; 84(suppl 1):92A. 4. Crowson AN, Magro CM, Mihm MC Jr. The melanocytic proliferations: a comprehensive textbook of pigmented lesions. New York: John Wiley and Sons; 2001. pp. 281-397. 5. Fisher GJ, Kang S, Varani J, Bata-Csorgo Z, Wan Y, Datta S, et al. Mechanisms of photoaging and chronological skin aging. Arch Dermatol 2002;138:1462-70. 6. Yaar M, Eller MS. Mechanisms of aging. Arch Dermatol 2002; 138:1429-31. 7. Hemmi H, Kaisho T, Takeuchi O, Sato S, Sanjo H, Hoshino K, et al. Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat Immunol 2002;3:196-200. 8. Hesling C, D’Incan M, Mansard S, Franck F, Corbin-Duval A, Chevenet C, et al. In vivo and in situ modulation of the expression of genes involved in metastasis and angiogenesis in a patient treated with topical imiquimod for melanoma skin metastases. Br J Dermatol 2004;150:761-7. 9. Li VW, Li WW, Talcott KE, Zhai AW. Imiquimod as an antiangiogenic agent. J Drugs Dermatol 2005;4:708-15. 10. Lain EL, Carrington PR. Imiquimod treatment of exuberant granulation tissue in a nonhealing diabetic ulcer. Arch Dermatol 2005;141:1368-70. 11. Kang S, Kauls LS, Gaspari AA. Toll-like receptors: applications to dermatologic disease. J Am Acad Dermatol 2006;54:951-83. 12. Takeda K, Akira S. TLR signaling pathways. Semin Immunol 2004;16:3-9.