Gene Therapy (2007) 14, 1555–1563& 2007 Nature Publishing Group All rights reserved 0969-7128/07 $30.00REVIEWProgress and Prospects: Gene Therapy ClinicalTrials (Part 2)This is the second part of a review summarizing progresstranscarbamylase deficiency, a -antitrypsin deficiency, haemo-and prospects in gene therapy clinical research. Twenty keydiseases/strategies are succinctly describe
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Ovid: schmidt: int j dermat.International Journal of Dermatology 1999 Blackwell Science Ltd.
Tretinoin-iontophoresis in atrophic acne scars[Pharmacology and Therapeutics] Schmidt, Jolanta B. MD; Donath, Peter MD; Hannes, Johanna MD; Perl, Sylvia MD; Neumayer, Renate MD;Reiner, Angelika MD From the Department of Dermatology, Division of Special and Environmental Dermatology and Institute ofClinical Pathology, University of Vienna Medical School, Vienna, AustriaCorrespondence: Jolanta B. Schmidt, MD; Department of Dermatology; University of Vienna MedicalSchool; Währinger Gürtel 18-20; A-1090 Vienna; Austria Clinical documentationHistologic documentation Background: Atrophic acne scars are a frequent problem after acne. Hitherto, mainly invasive treatmentmeasures were possible. In a recent paper, we demonstrated the positive effects of iontophoresis with 0.025%tretinoin gel vs. estriol 0.03%.
Objective: In this further study, the recording of the clinical effects of iontophoresis with 0.025% tretinoin gelin atrophic acne scars was supplemented by immunohistochemistry investigations of collagen I and III,proliferation markers, and the estimation of epidermal thickness.
Methods: The treatment was performed twice weekly in 32 volunteer patients for a period of 3 months byapplication of the substance under a constant direct current of 3 mA for 20 min. Skin biopsies prior to and atthe end of treatment were performed in 32 voluntary patients in order to investigate collagen I/III andproliferation markers by immunohistochemistry methods.
Results: Clinically, at the end of treatment, in 94% of patients a significant decrease in the scar depth wasobserved. Neither epidermal thickness nor proliferation markers revealed a significant increase at the end oftreatment. Furthermore, collagen I and collagen III showed no common trend, as expressed statistically by alack of significance. In some cases, increases in collagen III became evident at the end of treatment.
Conclusions: Tretinoin-iontophoresis is an effective, noninvasive treatment of atrophic acne scars withoutcausing disturbing side-effects.
Acne scars, a widespread problem, represent persisting impairments for patients. While acne keloids areattributed to a genetic disposition, possibly due to an increased response of fibroblasts towards androgenicstimulation, the cause of the development of atrophic or icepack scars, mostly developing spontaneously, stillremains unclear. Patients suffering from multiple atrophic acne scars feel disfigured and are continuouslyreminded of their previous disease.
Hitherto, the following therapeutic methods were available in order to improve atrophic acne scars. Collagenaugmentation is limited to single acne scars and results in only moderate, nonpersisting effects. For multiplescars, dermabrasion can be performed with good success. Moreover, chemopeels and lasers are therapeuticalternatives. The latter methods are invasive, and leave the patients with a crusted face at least for severaldays.
We have developed a new method without any intermediate disfigurement for the patients. In an earlierstudy, iontophoresis was performed in 46 patients with atrophic acne scars with either 0.1% estriol in aqueoussolution or 0.025% tretinoin gel. The treatment showed good clinical results in 93% of patients in the tretinoingroup;1 in a post-treatment survey of 2 years, these results were found to be permanent. In the present study,the clinical findings of iontophoresis treatment with tretinoin gel were supplemented byimmunohistochemistry investigations of collagen I and III distribution, proliferation markers, andmeasurements of skin thickness. Positive effects of tretinoin cream in acne scarring have been described,2and were attributed to the influence of the substance on fibroblast function.
Thirty two patients aged from 15 to 48 years (mean, 25) were treated by iontophoresis with 0.025% tretinoingel. The therapy was performed in 19 women with a mean age of 25 years (15-39) and in 13 men with a meanage of 26 years (18-48).
Iontophoresis is well known in physical medicine, whereby antiinflammatory substances penetrate into tissues, such as muscles or joints, thus achieving higher local tissue levels but avoiding systemic side-effects.3Our previous findings with estriol iontophoresis had supported the local effect of iontophoresis treatment inadequate concentrations and frequency due to stable estrogen levels during treatment. Using a constant, directcurrent of, in our case, 3 mA, polar substances diffused along a diffusion gradient between anode andcathode. Thus, changes in the electric cell membrane properties are achieved, with a subsequently increasingintracellular accumulation of the substance,3 which seems to be released in a prolonged way from a depot.
For the treatment, tretinoin gel was applied to the cleaned face and covered by a thin cotton cloth. Thereafterthe iontophoresis mask-a metal facial mask covered by a sponge material and with openings for nostrils andmouth-was placed on the face. Under the influence of galvanic current, acid solutions diffused from thecathode towards the anode. The instrument used was a MINISAN low-frequency instrument (Dr. SchuhfriedMedizintechnik, Vienna, Austria). Iontophoresis with tretinoin gel was performed twice weekly for 20 minover a period of 3 months.
Treatment effects were clinically documented by recording (subjective) assessments of scar depths, skinfirmness and elasticity, pore size, and skin moisture according to the parameters: minor, clear, and significantfindings. For this purpose, we used a personal evaluation scheme (Table 1) that was applied for each of theparameters. Changes in effects were only recorded if observed by both monitor and patient. Photographs forclinical documentation were taken at the start and at the end of treatment.
[Help with image viewing] Table 1 Evaluation of clinical effects of treatment on the atrophic scars For the investigation of the epidermal and dermal effects of tretinoin-iontophoresis, epidermal thickness andproliferation markers, as well as collagen I and collagen III, were estimated. The collagen parameters werealso of special interest in comparison with the mode of action of estrogen treatment on skin, where significantcollagen III and procollagen III increases were reported at the end of treatment and paralleled the clinicaleffects in aging skin.4,5 Comparable clinical effects between estriol- and tretinoin-iontophoresis in our previous study raised thequestion of whether influences on collagen, comparable with those achieved with topical estriol, were apossible mode of action for tretinoin-iontophoresis.
In all of the 32 voluntary patients, who had provided written informed consent, skin was obtained in order toperform histology and immunohistochemistry prior to and at the end of the treatment period. Under localanesthesia with 2% Scandicain, two punch biopsy specimens (3 mm in diameter) were taken from theatrophic scar within the treatment areas on the face. Skin biopsies were formalin fixed and paraffinembedded. Histologic sections were prepared according to routine histologic techniques and stained withhematoxylin and eosin.
Collagen I distribution was investigated using a rabbit anti-human collagen polyclonal antibody (ChemiconInt. Inc., Temecula, CA, diluted 1 : 800).
For the detection of collagen III, a rabbit polyclonal antibody (Monosan, The Netherlands, diluted 1 : 10) wasused.
For the immunohistochemical staining of collagen fibres, slides were enzyme pretreated with 0.1% proteasetype IV (Sigma, St Louis, USA) for 10 min. The slides were then incubated with the primary antibody for 1 hat room temperature; further staining was performed according to the immunohistochemical ABC-method.
For the determination of proliferating cell fractions, we used MIB1 (Clone MIB1, Dianova GmbH, Hamburg,Germany, diluted 1 : 50), a mouse monoclonal antibody directed against the proliferation associated Ki-67antigen, which can be used on formalin-fixed, paraffin-embedded tissues. Paraffin sections were incubated incitrate buffer (pH 6) in the microwave oven, first at 120 W for 10 min and then three times for 5 min at 450W. Citrate buffer was refilled several times in order to keep the slides moist. The slides were cooled for 15-20min at room temperature and, after incubation with the primary antibody for 1 h, they were further processedusing the immunohistochemical ABC-method.
For all immunohistochemical staining procedures, products from Vector Laboratories (Burlingame, CA) wereused. Briefly, after incubation with the primary antibody and incubation with a biotinylated secondaryantibody, incubation with the ABC-complex for 1 h each followed. The reaction product was developed withdiaminobenzidine-tetrahydrochloride. Finally, slides were counterstained with Harris' hematoxylin. Allincubation steps were carried out at room temperature.
Collagen I and III staining was estimated semiquantitatively; the staining intensity was classified as weak (+),intermediate (++), or strong (+++).
The MIB1 labeling index was obtained by counting a minimum of 1000 cells of the epidermal layer with a 1 cm2 ocular grid under 400× magnification and calculating the percentage of positive cell nuclei. Nucleishowing even brown staining were regarded as positive.
The thickness of the epidermal layer was measured using a measuring ocular (Leitz) which shows a hundredunit scale. The scale was calibrated using a glass slide carrying a 2-mm scale divided into units of 0.01 mmeach (Leitz). For calibration, both scales were adjusted in parallel side by side and matched; the length of oneunit of the measuring ocular scale was then determined. Measurements were carried out using 400×magnification. The result obtained in units of the measuring ocular was multiplied by 0.0032.
The clinical effects of treatment showed improvement by a decrease in scar depth in 94% of patients (Fig. 1).
Only in two patients was no decrease in scarring observed according to subjective patient opinion, theinvestigator's clinical evaluation (Tables 1 and 2) and the photographs. In both men and women the firsteffects on the scars were noted after 8 weeks of treatment and increased from then on. The subjectiveassessments of the skin showed improvements of skin firmness and of pore size diminution in 47% and 55%respectively after a mean of 9 weeks. Skin moisture revealed no common trend during the treatment period.
Both increases (16%) and decreases (38%) were observed.
[Help with image viewing] Figure 1 Atrophic acne scars. (a) Before treatment. (b) After 3 months of tretinoin- [Help with image viewing] Table 2 Clinical effects at the end of treatment Neither epidermal thickness nor the proliferation markers (MIB1) revealed significant differences betweeninitial and final values (Table 3).
[Help with image viewing] Table 3 Mean values of skin thickness and proliferation markers during tretinoin- With regard to the collagen parameters (collagen I and III), no common trend became evident at the end oftreatment. Equal staining, decreases and increases were noted. Stable amounts of collagen I were found in47% and in 25% for collagen III. Increases and decreases in collagen I were observed in 25% and 28%respectively. For collagen III, increases and decreases were both 37.5% (Table 4).
[Help with image viewing] Table 4 Collagen I/III during tretinoin-iontophoresis Flushing of the treated skin occasionally persisted for 30 min after treatment, and was interpreted as aphysiologic response due to the increased microcirculation. Dryness of the skin was reported in 32% of casesand was accompanied by fine scaling in four cases.
Iontophoresis with tretinoin was shown to be an efficient therapy for atrophic acne scars. The present, clearlypositive findings confirm the preliminary results of our previous study.
Interpretation of the positive clinical effects was sought by evaluation of some of the epidermal parameters,such as MIB1 and epidermal thickness. Although our data conflict with studies that describe increases inepidermal thickness by tretinoin in aging skin 6-8 and increases in epidermal mitotic indices,9 it must be keptin mind that these studies were performed in aged, elastotic skin. In acne scars of more juvenile skin, bothparameters may not be responsible for the clinical benefits of treatment. Furthermore, the report of differenteffects of tretinoin at various stages of treatment 10 could serve as a possible explanation for the lack ofsignificant effects of tretinoin on epidermal properties of acne scars at the end of treatment.
According to the investigated collagen parameters, it was shown that collagenous tissue and mainly collagenIII represent an initial point of action in some cases. Increases in collagen stimulation 11,12 andimprovements in elastic fiber properties 13 have been reported with tretinoin, and are combined with anincrease in the number of keratinocytes 14 responsible for the effects of tretinoin in acne and in skin aging.
The various modes of action of the substance have been extensively investigated in both conditions.
Moreover, striae distensae 15 and acne scars 2 also respond positively to topical tretinoin.
The loss of collagen fibers and the degradation of elastic fibers are found in photodamaged skin,13 and thusare comparable to some extent with the histologic features observed in atrophic scars. The stimulatory effectsof tretinoin on collagen synthesis 4 and on increases in fibroblast collagen 16 may be considered as thetheoretical background of the possible action of tretinoin on acne scarring. In our investigation,immunohistochemistry revealed collagen III changes by tretinoin-iontophoresis. It is beyond the scope of this study to determine the mechanisms by which tretinoin-iontophoresis exerts its effect. This will be the subjectof further investigations.
In the present study, the clinical effects of tretinoiniontophoresis, as confirmed by post-treatment controlsafter 6 and 12 months, were quite impressive and, as reported previously, permanent. This observationdefinitely excludes edema as a possible mechanism for the decrease in depth of the scars. Preliminaryexperience with the effects of iontophoresis with estrogen in skin aging in comparison with those of topicalcream treatment in identical concentration 17 have shown that the clinical efficacy of iontophoresis treatmentis significantly superior to that of daily treatment with cream only.
A 3-month treatment period with iontophoresis with tretinoin gel is therefore not comparable with topicalapplication of a cream of the same concentration for the same time period. By iontophoresis, increased tissueconcentrations are achieved,3 and the properties of cellular membranes and the intracellular uptake ofsubstances are influenced in an as yet undetermined manner. Although an important part of treatment inphysical therapy, dermatologists have not paid adequate attention to this method until recently.18 According to the "flip-flop" gating model-one proposed mechanism for the mode of action ofiontophoresis-pore formation in the stratum corneum is induced by iontophoretic currents. Pores are formedbetween neighboring keratin helices as a result of molecular realignment and repulsion of neighboringdipoles.19 Hair follicles and sweat gland ducts act as diffusion shunts, offering paths of reduced resistance foriontophoretic transport.20 The explanation of the extent and persistence of the effects of treatment istherefore not only due to the effects of tretinoin alone, but also to those of iontophoresis itself.
The method of iontophoresis should therefore be investigated further. Up to now, in dermatology, only tapwater iontophoresis has been widely used for the treatment of hyperhidrosis.21 According to the previous 1 and present findings, iontophoresis can be used in order to improve the effects ofsubstances in comparison with those achieved by topical application of compounds of the sameconcentration. With changing conditions, e.g. increased tretinoin concentration or increased frequency oftreatment, the systemic action of tretinoin cannot be excluded and thus blood determinations are required.
These should be performed with regard to the teratogenicity of the substance. Although no experimental dataare available on the magnitude of the increase in concentration in skin tissue after iontophoresis, we concludefrom the grade and persistence of the improvements that the above-mentioned mechanisms 3 contribute todepot formation in the skin.
Our first report on the positive effects of tretinoin-iontophoresis in acne scars has been corroborated by theclinical findings and photographs in the present study. The mode of action is not clear. Immunohistochemistrydata conflict to some extent with previous findings on tretinoin effects in solar atrophy. Further investigationsshould therefore be performed with regard to the cellular effects of iontophoresis.
Clinically, iontophoresis with tretinoin gel was confirmed to improve acne scars effectively without anyintermediate impairment of the appearance of the patients. According to the present findings, iontophoresisitself seems to be a promising method for enhancing the effects of a substance, in our case tretinoin.
One explanation for the positive clinical effects may be the initial point of action of tretinoin on skin collagen.
Further explanations for the extent of improvement should involve an investigation of the method ofiontophoresis.
1 Schmidt JB, Binder M, Macheiner W, et al. New treatment of acne scars by iontophoresis with estriol and tretinoin. Int J Dermatol 1995; 34: 53-57. Bibliographic Links [Context Link] 2 Harris DW, Buckley CC, Ostlere LS, et al. Topical retinoic acid in the treatment of fine acne scarring. Br JDermatol 1991; 125: 81-82 (letter). [Context Link] 3 Pratzel HG, Macheus R, Dittrich P. Iontophorese zur forcierten Hautresorption von iontomethacin undSalicylsäure. Rheumatol 1986; 45: 74-78. [Context Link] 4 Varila A, Rantala I, Oikarinen A, et al. The effects of topical oestradiol on skin collagen of postmenopausalwomen. Br J Dermatol 1995; 102: 985-989. [Context Link] 5 Schmidt JB, Binder M, Demschik G, et al. Treatment of skin aging with topical estrogens. Int J Dermatol1996; 35: 669-674. Ovid Full Text Bibliographic Links [Context Link] 6 Kligman AM, Grove GL, Hirose R, et al. Topical tretinoin for photoaged skin. J Am Acad Dermatol 1986;15: 836-859. Bibliographic Links [Context Link] 7 Lever L, Kumar P, Marks R. Topial retinoic acid for treatment of solar damage. Br J Dermatol 1990; 122:91-98. Bibliographic Links [Context Link] 8 Weiss JS, Ellis CN, Headington JT, et al. Topical tretinoin improves photoaged skin. A double-blind vehicle-controlled study J Am Med Assoc 1988; 259: 527-532. [Context Link] 9 Kligman AM. Guideline for the use of topical tretinoin (Retin A) for photoaged skin. J Am Acad Dermatol1989; 21: 650-654. Bibliographic Links [Context Link] 10 Yamamoto O, Bhawan J, Hara M, et al. Keratinocyte degeneration in human facial skin: documentation ofnew ultrastructural markers for photodamage and their improvement during topical tretinoin treatment. ExpDermatol 1995; 4: 9-19. Bibliographic Links [Context Link] 11 Schilz JR, Lanigan W, Nabial B. Retinoic acid induces cyclic changes in epidermal thickness and dermalcollagen and glycosaminoglycan biosynthesis rates. J Invest Dermatol 1986; 87: 663-667. Bibliographic Links[Context Link] 12 Kim RY, Stern WH. Retinoids and butyrate modulate fibroblast growth and collagen matrices. InvestOphthalmol Vis Sci 1990; 31: 1183-1186. Bibliographic Links [Context Link] 13 Noble S, Wagstaff A. Tretinoin. A review of its pharmacological properties and clinical efficacy in thetopical treatment of photoaged skin Drugs Aging 1995; 6: 479-496. [Context Link] 14 Tong PH, Horowitz LA, Wheller LA. Transretinoic acid ehances the growth responses of epidermalkeratinocytes to epidermal growth factor and transforming growth factor beta. J Invest Dermatol 1990; 94:126-131. Bibliographic Links [Context Link] 15 Elson ML. Topical tretinoin in the treatment of striae distensae and in the promotion of wound healing: areview. J Dermatol Treat 1994; 5: 163-165. [Context Link] 16 Panizzon RG, Bruckner-Tuderman L, Mindek G, et al. The influence of tretinoin on photodamaged humanskin: a biochemical, cytogenetic and histological evaluation over 9 months. Dermatology 1994; 189: 336.
[Context Link] 17 Schmidt JB. Topica estrogen treatment of skin aging and acne scars. Clinical effects and hormoneparameters. Progress in Cosmetic Dermatology: Science and Safety. IV. International Meeting on Cosmetic Dermatology 30.10.-2.11.1991, Rome. [Context Link] 18 Kassan DG, Lynch AM, Stiller MJ. Physical enhancement of dermatologic drug delivery: iontophoresisand phonophoresis JAMA 1996; 34: 657-666. [Context Link] 19 Phipps JB, Padmanabhan RU, Lattin GA. Iontophoretic delivery of model inorganic and drug ions. JPharm Sci 1989; 78: 365-369. Bibliographic Links [Context Link] 20 Burnette RR, Marrero D. Comparison between the iontopheretic and passive transport of thyrotropinreleasing hormone across excised nude mouse skin. J Pharm Sci 1986; 75: 738-743. Bibliographic Links[Context Link] 21 Levit F. Treatment of hyperhidrosis by tapwater iontophoresis. Cutis 1980; 31: 173-175. [Context Link] Accession Number: 00004342-199902000-00019 Copyright (c) 2000-2003 Ovid Technologies, Inc.
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