Original Paper Received: April 29, 2014 Accepted after revision: December 13, 2014 Published online: March 11, 2015
Dermatology 2015;230:332–339 DOI: 10.1159/000371876
L-Tryptophan as a Novel Potential
Pharmacological Treatment for Wound Healing via Aryl Hydrocarbon Receptor Activation Neda Barouti a Carlo Mainetti c Lionel Fontao a Olivier Sorg b
Department of Dermatology, University Hospitals of Geneva and b Swiss Center for Applied Human Toxicology, University of Geneva, Geneva, and c Department of Dermatology, Regional Hospital of Bellinzona, Bellinzona, Switzerland
Key Words Aryl hydrocarbon receptor · L-Tryptophan · Ulcers · Wound healing · Topical treatment
Abstract Background: The aryl hydrocarbon receptor has been shown to be involved in wound healing. Objective: The aim of this study was to assess the effect of tryptophan on wound healing in vitro and in a clinical trial. Methods: The ability of tryptophan and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to increase wound healing was assessed in an in vitro scratch wound model in human keratinocytes. Topical tryptophan and vehicle were assessed for 12 weeks in 51 patients with lower limb ulcers that were resistant to conventional therapies. Results: TCDD 0.1 nM and tryptophan 1 μM increased the rate of scratch recovery in a culture model. Topical tryptophan induced stronger pain relief and faster re-epithelialization than its vehicle in patients with lower limb ulcers. Conclusion: Tryptophan shows promising potential as a novel topical treatment for wound healing. © 2015 S. Karger AG, Basel
© 2015 S. Karger AG, Basel 1018–8665/15/2304–0332$39.50/0 E-Mail [email protected] www.karger.com/drm
Introduction
A patient with acute dioxin intoxication (i.e. 5 million times the admitted daily dose in the general population) was admitted to the dermatology department of the University of Geneva [1]. During the medical treatment of this patient, several extensive skin surgery procedures, such as incisions and dermabrasion, were performed to deal with the numerous dermal cysts (metabolizing acquired dioxin-induced skin hamartomas) that spontaneously appeared all over the body [1]. A close follow-up showed that the numerous incisions performed during these interventions healed much faster than expected. As the molecules of the dioxin family, in particular 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which was responsible for the intoxication of the patient, exerted their biological action by activating the cytoplasmic receptor aryl hydrocarbon receptor (AhR) [2], it was hypothesized that the AhR pathway might have mediated the beneficial effect of wound healing. Furthermore, knowing that dioxins have a very long biological half-life (7–10 years for TCDD), which could trigger a detrimental Neda Barouti Department of Dermatology, University Hospitals of Geneva 4, rue Gabrielle-Perret-Gentil CH–1211 Geneva 14 (Switzerland) E-Mail neda.barouti @ hcuge.ch
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a
Dübendorf, Switzerland) on an EnVision Multilabel Plate Reader platform (PerkinElmer, Schwerzenbach, Switzerland). Luminescence was normalized to methylthiazol tetrazolium (MTT) values (to correct for cell toxicity of the compound). MTT Test Yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added at 5 mg/ml to the medium of NHK cells. After 4 h of incubation, the medium was replaced by dimethyl sulfoxide to dissolve purple formazan. Absorbance of solution was quantified at 555 nm and normalized by subtracting background noise measured at 630 nm [11]. Cytochrome P4501A1 Enzymatic Activity Ethoxyresorufin-O-deethylase (EROD) activity corresponds to the O-deethylation of ethoxyresorufin mainly mediated by CYP1A1 enzyme in living HepG2 cells [12]. Briefly, HepG2 cells were incubated for 1 h at 37 ° C in phosphate-buffered saline solution pH 7.4 containing 8 μM ethoxyresorufin and 10 μM dicumarol. At the end of incubation, 100 μl of supernatant were mixed with 130 μl of ethanol (EtOH), and then the fluorescence of resorufin (excitation: 570 nm; emission: 590 nm) was measured in an EnVision Multilabel Plate Reader platform (PerkinElmer).
Cell Culture Immortalized normal human epidermal keratinocytes (NHK) were cultured in keratinocyte serum-free medium (Life Technologies, Zug, Switzerland), and HepG2 hepatocyte cells were cultured in DMEM supplemented with 10% fetal bovine serum and penicillin-streptomycin 100 μg/ml (all from LuBioScience GmbH, Lucerne, Switzerland).
RNA Interference and in vitro Wound Healing Assay NHK were seeded at near confluence (2 × 105 cells/well) in 12well plates and transfected 24 h later with 40 pmol/well of human AhR-siRNA or scrambled siRNA (Life Technologies) according to the manufacturer’s recommendations using Lipofectamine 2000 (Life Technologies). RNA silencing was verified by ECL-Western blot analysis of cell extracts using anti-AhR antibody (Santa Cruz Biotechnologies/LabForce, Muttenz, Switzerland), and the siRNA resulting in a near complete abolition of AhR expression was used in subsequent tests. Twenty-four hours post transfection, cell monolayers were scratch-wounded with a sterile 200-μl pipette tip and washed two times with culture medium [13]. Cells were then treated with either 1% EtOH, 10 μM Trp or 10 nM TCDD for an additional 24 h before further testing. The wound area was photographed under phase contrast microscopy at marked positions prior to and at the end of the challenge. For each condition, scratched areas of three randomly chosen fields per well were measured in quadruplicate with ImageJ software to define surface area recovery. Results were expressed as the percentage of wound closure relative to baseline, and values were compared to control at each time point. To distinguish between cell proliferation and migration, similar experiments were performed with mitomycin Ctreated cells as described elsewhere [14].
Dioxin-Responsive Chemical-Activated Luciferase Gene Expression Bioassay The dioxin-responsive chemical-activated luciferase gene expression bioassay used in this study was similar to the one described by Murk et al. [10]. NHK or HepG2 cells stably expressing Photinus pyralis luciferase gene under the control of the rat CYP1A1 gene promoter containing two XRE were generated by lentiviral transduction. Cells were seeded at 25,000 cells/well in 96-well plates. One day later the medium was removed and replaced by fresh medium containing test compounds or vehicle. Luminescence was measured 24 h later using the Luciferase Reporter Assay System (Promega,
Clinical Trial on Human Patients This prospective experimental pseudo-randomized trial, approved by the local ethics committee, was conducted on 60 adult patients to assess the effect of Trp versus vehicle, and vehicle alone, on wound healing. The study population consisted of ambulatory home caretreated adult patients with at least one chronic leg ulcer diagnosed as arterial and/or venous. The selection criteria included no evidence of healing (i.e. increased wound area and/or depth) despite appropriate conventional treatment (including compression therapy adapted to the ankle brachial index and dressings depending on the wound stage) for the last previous 6 months. Cases of non-
Tryptophan for Wound Healing
Dermatology 2015;230:332–339 DOI: 10.1159/000371876
Methods Chemicals Pure TCDD, FICZ and Trp were obtained from Cambridge Isotope Laboratories (Tewksbury, Mass., USA), Biomol International (Enzo Life Sciences, Lausen, Switzerland) and Sigma-Aldrich (Buchs, Switzerland), respectively. Trp 1% in vehicle (hydrophilic polymer) or vehicle were purchased from Pharmacie Internationale (Lausanne, Switzerland). The other chemicals were purchased from Sigma unless otherwise specified.
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cutaneous effect (such as the cutaneous manifestations of dioxin intoxication, the misnomer being chloracne syndrome) [3], evaluation of AhR agonists with a much shorter biological half-life was considered. Such agonists might exert the short-term actions of dioxins without inducing the characteristic cutaneous syndrome. Various molecules unrelated to the dioxin family are potent AhR agonists with a known rapid metabolism as well as elimination out of the organism. One of these AhR agonists is 6-formylindolo(3,2-b)carbazole (FICZ), a light-induced L-tryptophan (Trp) derivative [4]. Since Trp is a natural constituent, it was assumed that its application on the skin would not cause any safety problems for the patients. Even though to our knowledge no clinical trial has been reported on the topical effect of Trp on lower limb wound healing, Trp has been investigated in numerous clinical studies, especially in neuropsychiatric and neuroendocrinologic domains [5–8], as well as in healing of gastric and duodenal ulcers [9]. Here, we combined in vitro experiments with preliminary in vivo evaluation and a clinical trial to define the efficacy and safety of Trp on lower limb wound healing.
compliance with standard treatment, of known allergies to vehicle or Trp, ongoing systemic diseases known to delay wound healing (such as uncontrolled cardiac, renal or hepatic insufficiency, calciphylaxis) were rejected. Additionally, the following conditions were also considered as exclusion criteria: ongoing systemic diseases known to be associated with pyoderma gangrenosum or necrotizing vasculitis, other autoimmune diseases, cryoglobulinemia, concomitant treatment with corticosteroids, immunosuppressive or cytotoxic drugs. An investigator who was unrelated to the subsequent phases of the study assigned patients to a treatment group in a 1:1 ratio (patients were randomized according to their recruitment order number so that all odd numbers received one treatment and all even numbers received the other). Patients received either 1% Trp (50 mM) or vehicle. Noteworthy, both gels showed identical texture, color and smell. Patients within each group followed a similar procedure: wound irrigation with normal saline, application on the wound of the relevant gel (1 cm of gel/cm2/application), covering with paraffin/Vaseline gauze and a bandage associated with an elastic compression bandage if necessary. Furthermore, patients were closely monitored during the study. Ambulatory treatments were performed on a regular basis (i.e. three times a week) by nurses and monthly controls were performed by specialized nurses at the Ambulatory Wound Care Center of the Department of Dermatology, University Hospitals of Geneva. The primary outcome consisted of the wound area reduction at 4, 8 and 12 weeks after initial gel application. Additionally, the percentage of patients with complete wound closure at completion of the study (i.e. at week 12) was quantified. Adverse events were reported, and visual analog scale (VAS) assessment (1–100 mm) [15], which supports the correlation of the use of analgesics with the health status of the patients [16], was performed on day 0 and at week 12 to evaluate ulcer-related pain. Throughout the clinical trial, patients experiencing strong pain, i.e. VAS ≥50 mm, were followed by the Pain Network of the University Hospitals of Geneva [17]. To calculate the ulcer area, standardized pictures collected at weeks 0, 4, 8 and 12 were processed with ImageJ software.
two XRE. TCDD 10 nM, FICZ 10 μM and Trp 500 μM induced 180-fold, 140-fold and 10-fold luciferase expression in HepG2 cells, respectively (fig. 1b), whereas in NHK cells induction was limited to 6.5-fold, 13-fold and 5-fold for TCDD, FICZ and Trp, respectively (fig. 1c).
Statistical Analyses Data from in vitro experiments were reported as mean ± standard deviation (SD) from at least three independent experiments. Regarding the clinical trial, because of the limited sample size, possible confounding factors among the patient characteristics were assessed using χ2 or Fisher’s exact test, as appropriate, for categorical parameters, and Student’s t test or Mann-Whitney test, as appropriate, for continuous parameters. Differences in wound size area evolution between the two groups of treatment were assessed using generalized estimating equations.
Involvement of AhR in in vitro Wound Healing To better characterize the mechanisms underlying Trp and TCDD stimulation of keratinocyte proliferation and migration, scratch wound assays were performed in NHK that were depleted in AhR using siRNA. As shown in figure 2c, AhR depletion dramatically impaired wound closure stimulation induced by TCDD and Trp. Interestingly, similar observations were made in EtOH-treated cells, which supports the important role that AhR plays in keratinocyte migration.
HepG2 and NHK Express Functional AhR Signaling Pathway The effect of TCDD, FICZ and Trp (fig. 1a) on AhR activation was analyzed on HepG2 (fig. 1b) and NHK (fig. 1c) cells expressing P. pyralis luciferase gene under the control of the rat CYP1A1 gene promoter containing 334
Dermatology 2015;230:332–339 DOI: 10.1159/000371876
TCDD and Trp Stimulate Wound Closure in vitro First we assessed the impact of Trp on wound healing by scratch wound assays using NHK cells. In theses assays, both cell proliferation and migration contributed to wound closure. Twenty-four hours post treatment, a 20% wound closure was found in vehicle-treated cells, whereas in TCDD- and Trp-treated cells, wound closure reached 70% (fig. 2a). We then assessed the contribution of cell migration using mitomycin C-treated cells. As shown in figure 2b, inhibiting cell proliferation significantly reduces, but does not abolish, the stimulation of wound closure induced by TCDD and Trp. Therefore, TCDD and Trp both stimulated cell proliferation and cell migration in the scratch wound assay.
Analysis of Trp Effect on Wound Healing in Humans Globally, no statistically significant difference was observed regarding age, gender, body mass index (BMI), wound etiology, prior skin engraftment, wound size, hypertension, diabetic status, smoking status and reduced mobility status between the two groups (tables 1, 2). Six patients were withdrawn from the control group and three from the Trp group during the study (fig. 3a), but Barouti/Mainetti/Fontao/Sorg
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Results
Induction of CYP1A1 Activity in HepG2 Hepatocytes The enzymatic activity of CYP1A1, the gene of which is a sensitive target of AhR agonists, was assessed using the EROD assay in HepG2 cells (fig. 1d) and NHK (fig. 1e) following application of TCDD, FICZ and Trp. In HepG2 cells, TCDD 10 nM, FICZ 1 μM and Trp 500 μM promoted CYP1A1 activity by 135-fold, 70-fold and 8-fold, respectively (fig. 1d). In NHK, CYP1A1 activity was increased by 119-fold and 99-fold for TCDD and FICZ (fig. 1e).
O Cl
Cl
O
O
H N
O– NH3+
O
Cl
Cl N H
a
TCDD
N H FICZ
Trp
HepG2
NHK
200
15
150
FICZ
AhR activity vs. solvent
AhR signaling activity vs. solvent
FICZ
100 TCDD
50
10
TCDD 5 Trp
Trp –9
–8
–7 –6 –5 log10 concentration (M)
–4
–3
c
0 –12
–11
–10
–9 –8 –7 –6 log10 concentration (M)
HepG2 140
TCDD
EROD activity vs. solvent
EROD activity vs. solvent
TCDD
100
80 FICZ
40
80 FICZ 60 40 20
20
d
–3
NHK
100
0 –12
–4
120
120
60
–5
Trp –11
–10
–9 –8 –7 –6 log10 concentration (M)
–5
–4
–3
e
0 –11
–10
–9 –8 log10 concentration (M)
–7
–6
Fig. 1. Effect of TCDD, FICZ and Trp on the AhR signaling pathway. a Structure of TCDD, FICZ and Trp. b, c Dioxin-responsive chemical-activated luciferase gene expression bioassay on HepG2 cells (b) and NHK cells (c) after 24 h incubation with increasing concentrations of TCDD, FICZ or Trp. d, e EROD activity com-
pared to vehicle assessed in HepG2 cells (d) and NHK cells (e) in the presence of increasing concentrations of TCDD, FICZ or Trp. Each value shown in b–d represents the mean of two separate experiments performed in duplicate.
Tryptophan for Wound Healing
Dermatology 2015;230:332–339 DOI: 10.1159/000371876
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b
0 –10
Table 1. Characteristics of the 60 recruited patients
a
EtOH 1%
Trp 1 μM
Trp 10 μM
TCDD 0.1 nM
EtOH 1%
Wound closure (%)
–
b
TCDD 1 nM
Trp (n = 30)
p value
15 (50%) 23 (77%) 8 (27%) 14 (47%) 5 (17%) 13 (43%) 5 (17%) 12 (40%) 23 (77%) 74.47 ± 5.28 24.47 ± 3.46 19.49 ± 8.20
16 (53%) 25 (83%) 4 (13%) 12 (40%) 2 (7%) 16 (53%) 4 (13%) 10 (33%) 18 (60%) 72.97 ± 7.95 23.9 ± 4.29 22.34 ± 7.33
1 0.747 0.333 0.794 0.424* 0.605 1* 0.789 0.267 0.393 0.576 0.163
Table 2. Characteristics of the 51 patients who completed the study –
+
+
–
TCDD 0.1 nM
+
Trp 10 μM
***
70
***
60 50 40
***
30 20 10
0 Scrambled siRNA AhR siRNA
c
Vehicle (n = 30)
Figures are mean ± SD or number with percentage in parentheses. Statistical analysis was done using χ2 test or Fisher’s exact test (*) for categorical parameters and Student’s t test or MannWhitney test for continuous parameters.
***
80 Wound closure (%)
Female Hypertension Diabetes Smoker Reduced mobility Venous ulcer Arterial ulcer Arteriovenous ulcer Prior engraftment Age BMI Wound size
***
***
100 90 80 70 60 50 40 30 20 10 0 MMC
***
***
***
***
Wound closure (%)
100 90 80 70 60 50 40 30 20 10 0
UC
+
UC +
EtOH 1%
+
UC +
TCDD 0.1 nM
+ + Trp 10 μM
Female Hypertension Diabetes Smoker Reduced mobility Venous ulcer Arterial ulcer Arteriovenous ulcer Prior engraftment Age BMI Wound size
Vehicle (n = 24)
Trp (n = 27)
p value
13 (54%) 17 (71%) 8 (33%) 12 (50%) 4 (17%) 12 (50%) 3 (12%) 9 (38%) 18 (75%) 74.92 ± 5.36 24.5 ± 3.79 21.95 ± 7.15
15 (56%) 23 (85%) 3 (11%) 10 (37%) 2 (7%) 14 (52%) 4 (15%) 9 (33%) 16 (59%) 72.59 ± 8.04 23.85 ± 4.27 21.99 ± 7.63
1 0.367 0.113 0.516 0.402* 1 1* 0.986 0.372 0.226 0.568 0.983
Figures are mean ± SD or number with percentage in parentheses. Statistical analysis was done using χ2 test or Fisher’s exact test (*) for categorical parameters and Student’s t test or MannWhitney test for continuous parameters.
Fig. 2. In vitro wound healing with Trp and TCDD. a Percentage
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Dermatology 2015;230:332–339 DOI: 10.1159/000371876
no statistical impact on the patient characteristics at baseline was observed after exclusion of these cases. Only wound size in the control group appeared to be slightly smaller in the 6 excluded patients than in the remaining 24 patients, which made the comparability of the wound size area at baseline even better between the remaining Barouti/Mainetti/Fontao/Sorg
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of wound closure after exposure for 24 h to TCDD 0.1 nM or Trp 10 μM. All conditions compared to EtOH were significantly different (p < 0.001). b Migration assay as a wound healing assay in the presence (+) or absence (–) of 10 μg/ml mitomycin C (MMC). c Wound healing assay after exposure for 24 h to AhR agonists in the presence of AhR siRNA or scrambled siRNA. All experiments were performed in quadruplicate. UC = Untransfected cells. *** p < 0.001.
60 patients randomly assigned
30 allocated to sodium alginate
30 allocated to Trp
6 patients withdrawn: • 2 SAE (bacterial infections) • 3 did not complete all study visits • 1 lost to follow-up
3 patients withdrawn: • 2 did not complete all study visits • 1 lost to follow-up
24 completed treatment
a
27 completed treatment
Alginate
40
Trp
Wound area (cm2)
35 30 25 20 15 10 5 0
0
4 8 Time (weeks)
b
Alginate
12 0
4 8 Time (weeks)
12
Trp
tients through the trial. SAE = Severe adverse event. b Wound area reduction over time for each patient in the control or Trp group. c Mean wound area reduction over time for the control group (darker bars) and the Trp group (lighter bars). * p < 0.05, *** p < 0.001 compared to day 0.
c
*
80
***
60
***
40
***
20 0
Day 0
Week 4
Week 8
Week 12
patients in the two groups. The mean age of the 51 analyzed patients was 73.76 ± 6.70 years, and ulcer etiology showed that on average 51.0% (26 of 51), 13.7% (7 of 51) and 35.3% (18 of 51) of the cases were venous, arterial and arteriovenous, respectively.
The primary outcome of the study, i.e. wound area, gradually and significantly decreased by re-epithelialization over time, and even at week 4. Trp-treated wounds showed a faster and more potent wound area reduction compared to the control group (fig. 3b, c, table 3). The
Tryptophan for Wound Healing
Dermatology 2015;230:332–339 DOI: 10.1159/000371876
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Fig. 3. Analysis of Trp effect on wound healing in humans. a Flow diagram of pa-
Wound area (% of t0)
100
Time point
Day 0 Week 4 Week 8 Week 12
Surface, cm2 vehicle (n = 24)
Trp (n = 27)
21.95 ± 7.16 21.41 ± 7.62 20.11 ± 7.07 17.83 ± 6.32
22.00 ± 7.63 12.96 ± 7.26 8.00 ± 4.91 4.90 ± 4.26
Mean difference [95% CI]
–0.04 [–4.02; 3.93] 8.49 [5.67; 11.32] 12.15 [9.12; 15.19] 12.97 [9.97; 15.98]
p value
0.982
Dermatology 2015;230:332–339 DOI: 10.1159/000371876
L-Tryptophan as a Novel Potential
Pharmacological Treatment for Wound Healing via Aryl Hydrocarbon Receptor Activation Neda Barouti a Carlo Mainetti c Lionel Fontao a Olivier Sorg b
Department of Dermatology, University Hospitals of Geneva and b Swiss Center for Applied Human Toxicology, University of Geneva, Geneva, and c Department of Dermatology, Regional Hospital of Bellinzona, Bellinzona, Switzerland
Key Words Aryl hydrocarbon receptor · L-Tryptophan · Ulcers · Wound healing · Topical treatment
Abstract Background: The aryl hydrocarbon receptor has been shown to be involved in wound healing. Objective: The aim of this study was to assess the effect of tryptophan on wound healing in vitro and in a clinical trial. Methods: The ability of tryptophan and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to increase wound healing was assessed in an in vitro scratch wound model in human keratinocytes. Topical tryptophan and vehicle were assessed for 12 weeks in 51 patients with lower limb ulcers that were resistant to conventional therapies. Results: TCDD 0.1 nM and tryptophan 1 μM increased the rate of scratch recovery in a culture model. Topical tryptophan induced stronger pain relief and faster re-epithelialization than its vehicle in patients with lower limb ulcers. Conclusion: Tryptophan shows promising potential as a novel topical treatment for wound healing. © 2015 S. Karger AG, Basel
© 2015 S. Karger AG, Basel 1018–8665/15/2304–0332$39.50/0 E-Mail [email protected] www.karger.com/drm
Introduction
A patient with acute dioxin intoxication (i.e. 5 million times the admitted daily dose in the general population) was admitted to the dermatology department of the University of Geneva [1]. During the medical treatment of this patient, several extensive skin surgery procedures, such as incisions and dermabrasion, were performed to deal with the numerous dermal cysts (metabolizing acquired dioxin-induced skin hamartomas) that spontaneously appeared all over the body [1]. A close follow-up showed that the numerous incisions performed during these interventions healed much faster than expected. As the molecules of the dioxin family, in particular 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which was responsible for the intoxication of the patient, exerted their biological action by activating the cytoplasmic receptor aryl hydrocarbon receptor (AhR) [2], it was hypothesized that the AhR pathway might have mediated the beneficial effect of wound healing. Furthermore, knowing that dioxins have a very long biological half-life (7–10 years for TCDD), which could trigger a detrimental Neda Barouti Department of Dermatology, University Hospitals of Geneva 4, rue Gabrielle-Perret-Gentil CH–1211 Geneva 14 (Switzerland) E-Mail neda.barouti @ hcuge.ch
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a
Dübendorf, Switzerland) on an EnVision Multilabel Plate Reader platform (PerkinElmer, Schwerzenbach, Switzerland). Luminescence was normalized to methylthiazol tetrazolium (MTT) values (to correct for cell toxicity of the compound). MTT Test Yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added at 5 mg/ml to the medium of NHK cells. After 4 h of incubation, the medium was replaced by dimethyl sulfoxide to dissolve purple formazan. Absorbance of solution was quantified at 555 nm and normalized by subtracting background noise measured at 630 nm [11]. Cytochrome P4501A1 Enzymatic Activity Ethoxyresorufin-O-deethylase (EROD) activity corresponds to the O-deethylation of ethoxyresorufin mainly mediated by CYP1A1 enzyme in living HepG2 cells [12]. Briefly, HepG2 cells were incubated for 1 h at 37 ° C in phosphate-buffered saline solution pH 7.4 containing 8 μM ethoxyresorufin and 10 μM dicumarol. At the end of incubation, 100 μl of supernatant were mixed with 130 μl of ethanol (EtOH), and then the fluorescence of resorufin (excitation: 570 nm; emission: 590 nm) was measured in an EnVision Multilabel Plate Reader platform (PerkinElmer).
Cell Culture Immortalized normal human epidermal keratinocytes (NHK) were cultured in keratinocyte serum-free medium (Life Technologies, Zug, Switzerland), and HepG2 hepatocyte cells were cultured in DMEM supplemented with 10% fetal bovine serum and penicillin-streptomycin 100 μg/ml (all from LuBioScience GmbH, Lucerne, Switzerland).
RNA Interference and in vitro Wound Healing Assay NHK were seeded at near confluence (2 × 105 cells/well) in 12well plates and transfected 24 h later with 40 pmol/well of human AhR-siRNA or scrambled siRNA (Life Technologies) according to the manufacturer’s recommendations using Lipofectamine 2000 (Life Technologies). RNA silencing was verified by ECL-Western blot analysis of cell extracts using anti-AhR antibody (Santa Cruz Biotechnologies/LabForce, Muttenz, Switzerland), and the siRNA resulting in a near complete abolition of AhR expression was used in subsequent tests. Twenty-four hours post transfection, cell monolayers were scratch-wounded with a sterile 200-μl pipette tip and washed two times with culture medium [13]. Cells were then treated with either 1% EtOH, 10 μM Trp or 10 nM TCDD for an additional 24 h before further testing. The wound area was photographed under phase contrast microscopy at marked positions prior to and at the end of the challenge. For each condition, scratched areas of three randomly chosen fields per well were measured in quadruplicate with ImageJ software to define surface area recovery. Results were expressed as the percentage of wound closure relative to baseline, and values were compared to control at each time point. To distinguish between cell proliferation and migration, similar experiments were performed with mitomycin Ctreated cells as described elsewhere [14].
Dioxin-Responsive Chemical-Activated Luciferase Gene Expression Bioassay The dioxin-responsive chemical-activated luciferase gene expression bioassay used in this study was similar to the one described by Murk et al. [10]. NHK or HepG2 cells stably expressing Photinus pyralis luciferase gene under the control of the rat CYP1A1 gene promoter containing two XRE were generated by lentiviral transduction. Cells were seeded at 25,000 cells/well in 96-well plates. One day later the medium was removed and replaced by fresh medium containing test compounds or vehicle. Luminescence was measured 24 h later using the Luciferase Reporter Assay System (Promega,
Clinical Trial on Human Patients This prospective experimental pseudo-randomized trial, approved by the local ethics committee, was conducted on 60 adult patients to assess the effect of Trp versus vehicle, and vehicle alone, on wound healing. The study population consisted of ambulatory home caretreated adult patients with at least one chronic leg ulcer diagnosed as arterial and/or venous. The selection criteria included no evidence of healing (i.e. increased wound area and/or depth) despite appropriate conventional treatment (including compression therapy adapted to the ankle brachial index and dressings depending on the wound stage) for the last previous 6 months. Cases of non-
Tryptophan for Wound Healing
Dermatology 2015;230:332–339 DOI: 10.1159/000371876
Methods Chemicals Pure TCDD, FICZ and Trp were obtained from Cambridge Isotope Laboratories (Tewksbury, Mass., USA), Biomol International (Enzo Life Sciences, Lausen, Switzerland) and Sigma-Aldrich (Buchs, Switzerland), respectively. Trp 1% in vehicle (hydrophilic polymer) or vehicle were purchased from Pharmacie Internationale (Lausanne, Switzerland). The other chemicals were purchased from Sigma unless otherwise specified.
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cutaneous effect (such as the cutaneous manifestations of dioxin intoxication, the misnomer being chloracne syndrome) [3], evaluation of AhR agonists with a much shorter biological half-life was considered. Such agonists might exert the short-term actions of dioxins without inducing the characteristic cutaneous syndrome. Various molecules unrelated to the dioxin family are potent AhR agonists with a known rapid metabolism as well as elimination out of the organism. One of these AhR agonists is 6-formylindolo(3,2-b)carbazole (FICZ), a light-induced L-tryptophan (Trp) derivative [4]. Since Trp is a natural constituent, it was assumed that its application on the skin would not cause any safety problems for the patients. Even though to our knowledge no clinical trial has been reported on the topical effect of Trp on lower limb wound healing, Trp has been investigated in numerous clinical studies, especially in neuropsychiatric and neuroendocrinologic domains [5–8], as well as in healing of gastric and duodenal ulcers [9]. Here, we combined in vitro experiments with preliminary in vivo evaluation and a clinical trial to define the efficacy and safety of Trp on lower limb wound healing.
compliance with standard treatment, of known allergies to vehicle or Trp, ongoing systemic diseases known to delay wound healing (such as uncontrolled cardiac, renal or hepatic insufficiency, calciphylaxis) were rejected. Additionally, the following conditions were also considered as exclusion criteria: ongoing systemic diseases known to be associated with pyoderma gangrenosum or necrotizing vasculitis, other autoimmune diseases, cryoglobulinemia, concomitant treatment with corticosteroids, immunosuppressive or cytotoxic drugs. An investigator who was unrelated to the subsequent phases of the study assigned patients to a treatment group in a 1:1 ratio (patients were randomized according to their recruitment order number so that all odd numbers received one treatment and all even numbers received the other). Patients received either 1% Trp (50 mM) or vehicle. Noteworthy, both gels showed identical texture, color and smell. Patients within each group followed a similar procedure: wound irrigation with normal saline, application on the wound of the relevant gel (1 cm of gel/cm2/application), covering with paraffin/Vaseline gauze and a bandage associated with an elastic compression bandage if necessary. Furthermore, patients were closely monitored during the study. Ambulatory treatments were performed on a regular basis (i.e. three times a week) by nurses and monthly controls were performed by specialized nurses at the Ambulatory Wound Care Center of the Department of Dermatology, University Hospitals of Geneva. The primary outcome consisted of the wound area reduction at 4, 8 and 12 weeks after initial gel application. Additionally, the percentage of patients with complete wound closure at completion of the study (i.e. at week 12) was quantified. Adverse events were reported, and visual analog scale (VAS) assessment (1–100 mm) [15], which supports the correlation of the use of analgesics with the health status of the patients [16], was performed on day 0 and at week 12 to evaluate ulcer-related pain. Throughout the clinical trial, patients experiencing strong pain, i.e. VAS ≥50 mm, were followed by the Pain Network of the University Hospitals of Geneva [17]. To calculate the ulcer area, standardized pictures collected at weeks 0, 4, 8 and 12 were processed with ImageJ software.
two XRE. TCDD 10 nM, FICZ 10 μM and Trp 500 μM induced 180-fold, 140-fold and 10-fold luciferase expression in HepG2 cells, respectively (fig. 1b), whereas in NHK cells induction was limited to 6.5-fold, 13-fold and 5-fold for TCDD, FICZ and Trp, respectively (fig. 1c).
Statistical Analyses Data from in vitro experiments were reported as mean ± standard deviation (SD) from at least three independent experiments. Regarding the clinical trial, because of the limited sample size, possible confounding factors among the patient characteristics were assessed using χ2 or Fisher’s exact test, as appropriate, for categorical parameters, and Student’s t test or Mann-Whitney test, as appropriate, for continuous parameters. Differences in wound size area evolution between the two groups of treatment were assessed using generalized estimating equations.
Involvement of AhR in in vitro Wound Healing To better characterize the mechanisms underlying Trp and TCDD stimulation of keratinocyte proliferation and migration, scratch wound assays were performed in NHK that were depleted in AhR using siRNA. As shown in figure 2c, AhR depletion dramatically impaired wound closure stimulation induced by TCDD and Trp. Interestingly, similar observations were made in EtOH-treated cells, which supports the important role that AhR plays in keratinocyte migration.
HepG2 and NHK Express Functional AhR Signaling Pathway The effect of TCDD, FICZ and Trp (fig. 1a) on AhR activation was analyzed on HepG2 (fig. 1b) and NHK (fig. 1c) cells expressing P. pyralis luciferase gene under the control of the rat CYP1A1 gene promoter containing 334
Dermatology 2015;230:332–339 DOI: 10.1159/000371876
TCDD and Trp Stimulate Wound Closure in vitro First we assessed the impact of Trp on wound healing by scratch wound assays using NHK cells. In theses assays, both cell proliferation and migration contributed to wound closure. Twenty-four hours post treatment, a 20% wound closure was found in vehicle-treated cells, whereas in TCDD- and Trp-treated cells, wound closure reached 70% (fig. 2a). We then assessed the contribution of cell migration using mitomycin C-treated cells. As shown in figure 2b, inhibiting cell proliferation significantly reduces, but does not abolish, the stimulation of wound closure induced by TCDD and Trp. Therefore, TCDD and Trp both stimulated cell proliferation and cell migration in the scratch wound assay.
Analysis of Trp Effect on Wound Healing in Humans Globally, no statistically significant difference was observed regarding age, gender, body mass index (BMI), wound etiology, prior skin engraftment, wound size, hypertension, diabetic status, smoking status and reduced mobility status between the two groups (tables 1, 2). Six patients were withdrawn from the control group and three from the Trp group during the study (fig. 3a), but Barouti/Mainetti/Fontao/Sorg
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Results
Induction of CYP1A1 Activity in HepG2 Hepatocytes The enzymatic activity of CYP1A1, the gene of which is a sensitive target of AhR agonists, was assessed using the EROD assay in HepG2 cells (fig. 1d) and NHK (fig. 1e) following application of TCDD, FICZ and Trp. In HepG2 cells, TCDD 10 nM, FICZ 1 μM and Trp 500 μM promoted CYP1A1 activity by 135-fold, 70-fold and 8-fold, respectively (fig. 1d). In NHK, CYP1A1 activity was increased by 119-fold and 99-fold for TCDD and FICZ (fig. 1e).
O Cl
Cl
O
O
H N
O– NH3+
O
Cl
Cl N H
a
TCDD
N H FICZ
Trp
HepG2
NHK
200
15
150
FICZ
AhR activity vs. solvent
AhR signaling activity vs. solvent
FICZ
100 TCDD
50
10
TCDD 5 Trp
Trp –9
–8
–7 –6 –5 log10 concentration (M)
–4
–3
c
0 –12
–11
–10
–9 –8 –7 –6 log10 concentration (M)
HepG2 140
TCDD
EROD activity vs. solvent
EROD activity vs. solvent
TCDD
100
80 FICZ
40
80 FICZ 60 40 20
20
d
–3
NHK
100
0 –12
–4
120
120
60
–5
Trp –11
–10
–9 –8 –7 –6 log10 concentration (M)
–5
–4
–3
e
0 –11
–10
–9 –8 log10 concentration (M)
–7
–6
Fig. 1. Effect of TCDD, FICZ and Trp on the AhR signaling pathway. a Structure of TCDD, FICZ and Trp. b, c Dioxin-responsive chemical-activated luciferase gene expression bioassay on HepG2 cells (b) and NHK cells (c) after 24 h incubation with increasing concentrations of TCDD, FICZ or Trp. d, e EROD activity com-
pared to vehicle assessed in HepG2 cells (d) and NHK cells (e) in the presence of increasing concentrations of TCDD, FICZ or Trp. Each value shown in b–d represents the mean of two separate experiments performed in duplicate.
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Dermatology 2015;230:332–339 DOI: 10.1159/000371876
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b
0 –10
Table 1. Characteristics of the 60 recruited patients
a
EtOH 1%
Trp 1 μM
Trp 10 μM
TCDD 0.1 nM
EtOH 1%
Wound closure (%)
–
b
TCDD 1 nM
Trp (n = 30)
p value
15 (50%) 23 (77%) 8 (27%) 14 (47%) 5 (17%) 13 (43%) 5 (17%) 12 (40%) 23 (77%) 74.47 ± 5.28 24.47 ± 3.46 19.49 ± 8.20
16 (53%) 25 (83%) 4 (13%) 12 (40%) 2 (7%) 16 (53%) 4 (13%) 10 (33%) 18 (60%) 72.97 ± 7.95 23.9 ± 4.29 22.34 ± 7.33
1 0.747 0.333 0.794 0.424* 0.605 1* 0.789 0.267 0.393 0.576 0.163
Table 2. Characteristics of the 51 patients who completed the study –
+
+
–
TCDD 0.1 nM
+
Trp 10 μM
***
70
***
60 50 40
***
30 20 10
0 Scrambled siRNA AhR siRNA
c
Vehicle (n = 30)
Figures are mean ± SD or number with percentage in parentheses. Statistical analysis was done using χ2 test or Fisher’s exact test (*) for categorical parameters and Student’s t test or MannWhitney test for continuous parameters.
***
80 Wound closure (%)
Female Hypertension Diabetes Smoker Reduced mobility Venous ulcer Arterial ulcer Arteriovenous ulcer Prior engraftment Age BMI Wound size
***
***
100 90 80 70 60 50 40 30 20 10 0 MMC
***
***
***
***
Wound closure (%)
100 90 80 70 60 50 40 30 20 10 0
UC
+
UC +
EtOH 1%
+
UC +
TCDD 0.1 nM
+ + Trp 10 μM
Female Hypertension Diabetes Smoker Reduced mobility Venous ulcer Arterial ulcer Arteriovenous ulcer Prior engraftment Age BMI Wound size
Vehicle (n = 24)
Trp (n = 27)
p value
13 (54%) 17 (71%) 8 (33%) 12 (50%) 4 (17%) 12 (50%) 3 (12%) 9 (38%) 18 (75%) 74.92 ± 5.36 24.5 ± 3.79 21.95 ± 7.15
15 (56%) 23 (85%) 3 (11%) 10 (37%) 2 (7%) 14 (52%) 4 (15%) 9 (33%) 16 (59%) 72.59 ± 8.04 23.85 ± 4.27 21.99 ± 7.63
1 0.367 0.113 0.516 0.402* 1 1* 0.986 0.372 0.226 0.568 0.983
Figures are mean ± SD or number with percentage in parentheses. Statistical analysis was done using χ2 test or Fisher’s exact test (*) for categorical parameters and Student’s t test or MannWhitney test for continuous parameters.
Fig. 2. In vitro wound healing with Trp and TCDD. a Percentage
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Dermatology 2015;230:332–339 DOI: 10.1159/000371876
no statistical impact on the patient characteristics at baseline was observed after exclusion of these cases. Only wound size in the control group appeared to be slightly smaller in the 6 excluded patients than in the remaining 24 patients, which made the comparability of the wound size area at baseline even better between the remaining Barouti/Mainetti/Fontao/Sorg
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of wound closure after exposure for 24 h to TCDD 0.1 nM or Trp 10 μM. All conditions compared to EtOH were significantly different (p < 0.001). b Migration assay as a wound healing assay in the presence (+) or absence (–) of 10 μg/ml mitomycin C (MMC). c Wound healing assay after exposure for 24 h to AhR agonists in the presence of AhR siRNA or scrambled siRNA. All experiments were performed in quadruplicate. UC = Untransfected cells. *** p < 0.001.
60 patients randomly assigned
30 allocated to sodium alginate
30 allocated to Trp
6 patients withdrawn: • 2 SAE (bacterial infections) • 3 did not complete all study visits • 1 lost to follow-up
3 patients withdrawn: • 2 did not complete all study visits • 1 lost to follow-up
24 completed treatment
a
27 completed treatment
Alginate
40
Trp
Wound area (cm2)
35 30 25 20 15 10 5 0
0
4 8 Time (weeks)
b
Alginate
12 0
4 8 Time (weeks)
12
Trp
tients through the trial. SAE = Severe adverse event. b Wound area reduction over time for each patient in the control or Trp group. c Mean wound area reduction over time for the control group (darker bars) and the Trp group (lighter bars). * p < 0.05, *** p < 0.001 compared to day 0.
c
*
80
***
60
***
40
***
20 0
Day 0
Week 4
Week 8
Week 12
patients in the two groups. The mean age of the 51 analyzed patients was 73.76 ± 6.70 years, and ulcer etiology showed that on average 51.0% (26 of 51), 13.7% (7 of 51) and 35.3% (18 of 51) of the cases were venous, arterial and arteriovenous, respectively.
The primary outcome of the study, i.e. wound area, gradually and significantly decreased by re-epithelialization over time, and even at week 4. Trp-treated wounds showed a faster and more potent wound area reduction compared to the control group (fig. 3b, c, table 3). The
Tryptophan for Wound Healing
Dermatology 2015;230:332–339 DOI: 10.1159/000371876
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Fig. 3. Analysis of Trp effect on wound healing in humans. a Flow diagram of pa-
Wound area (% of t0)
100
Time point
Day 0 Week 4 Week 8 Week 12
Surface, cm2 vehicle (n = 24)
Trp (n = 27)
21.95 ± 7.16 21.41 ± 7.62 20.11 ± 7.07 17.83 ± 6.32
22.00 ± 7.63 12.96 ± 7.26 8.00 ± 4.91 4.90 ± 4.26
Mean difference [95% CI]
–0.04 [–4.02; 3.93] 8.49 [5.67; 11.32] 12.15 [9.12; 15.19] 12.97 [9.97; 15.98]
p value
0.982
