Blackwell Science, LtdOxford, UKIJDInternational Journal ... · International Journal of Dermatology 2002, The International Society of Dermatology

International Journal of Dermatology

2002,

41

, 482–487 © 2002

The International Society of Dermatology

482

Abstract

Background

Low catalase levels and cellular vacuolation in the epidermis of patients with

vitiligo support major oxidative stress in this compartment. There is now

in vivo

evidence for

increased epidermal hydrogen peroxide (H

2

O

2

) accumulation in this patient group by utilizing

noninvasive Fourier Transform Raman spectroscopy (FT Raman). Epidermal H

2

O

2

can be

removed with a topical application of narrow band UVB activated pseudocatalase cream

(PC-KUS). (Mn/EDTA-bicarbonate complex, patent No. EPO 58471 1 A), yielding initiation of

repigmentation. Dead Sea climatotherapy is another successful treatment modality for vitiligo,

but the mode of action has escaped definition so far.

Methods

Epidermal hydrogen peroxide (H

2

O

2

) was assessed

in vivo

before and after 21 days

treatment at the Dead Sea using noninvasive Fourier-Transform Raman spectroscopy. The

effectiveness of repigmentation was followed in 59 patients with vitiligo by comparing Dead Sea

climatotherapy alone with the combination of Dead Sea climatotherapy/pseudocatalase cream

(PC-KUS) as well as Dead Sea climatotherapy/placebo cream. Clinical repigmentation was

documented by standardized black/white photography using non-UV coated bulbs as flashlight

and by color photography.

Results

This study on 59 patients who had vitiligo for an average time of 17 years (range

3–53 years) confirmed

in vivo

H

2

O

2

accumulation in mM concentrations in the epidermis of

untreated patients. Furthermore, we demonstrated a pseudocatalase activity after 15 min of

Dead Sea bathing, but the decrease of epidermal H

2

O

2

levels was significantly less compared

to narrowband UVB activated pseudocatalase cream (PC-KUS). Initiation of repigmentation

was already observed between day 10 and day 16 after a combination of Dead Sea

climatotherapy/pseudocatalase cream compared to conventional pseudocatalase

monotherapy (8–14 weeks) and Dead Sea climatotherapy alone (5–6 weeks).

Conclusion

The results of this study show a significantly faster initiation of repigmentation in

vitiligo after a combination of short-term climatotherapy (21 days) at the Dead Sea in

combination with a pseudocatalase cream (PC-KUS) compared to either conventional

climatotherapy at the Dead Sea alone or with placebo cream in combination with climatotherapy.

This combined therapy is significantly faster in repigmentation than narrowband UVB activated

pseudocatalase cream (PC-KUS) treatment alone.

The results of this study support the necessity of epidermal H

2

O

2

removal as well as the

influence of solar UV-light in the successful treatment of vitiligo.

Blackwell Science, LtdOxford, UKIJDInternational Journal of Dermatology0011-9059Blackwell Science, 200241

Report

Repigmentation in vitiligo and hydrogen peroxide removalSchallreuter et al.

Rapid initiation of repigmentation in vitiligo with Dead Sea climatotherapy in combination with pseudocatalase (PC-KUS)

Karen U. Schallreuter,

MD

, Jeremy Moore,

PhD

, Stephanie Behrens-Williams,

MD

, Angela Panske,

BSc

, and Marco Harari,

MD

From the Department of Biomedical Sciences, Clinical and Experimental Dermatology, University of Bradford, West Yorkshire, BD7 1DP, UK, the Institute for Pigmentary Disorders in association with the Ernst Moritz Arndt University of Greifswald, Greifswald, Germany, and DMZ-Mor Clinic, Ein Bokek, Israel

Correspondence

K U Schallreuter,

MD

Clinical and Experimental Dermatology Department of Biomedical Sciences University of BradfordWest YorkshireBD7 1DPUKE-mail: [email protected]

Introduction

Vitiligo is an acquired depigmentation disorder affecting0.5–4% of the world population.

1

Despite its early recognition,the etiology is still unclear. There are several hypotheses butnone of them can satisfy the entire spectrum of this disfiguringdisorder.

1,2

Almost all the publications describe decreased

numbers of functioning melanocytes or the complete absenceof these cells in the involved skin.

3

A recent paper by Tobin

et al

. showed that melanocytes are still present even in longstanding disease.

4

In addition, altered functionality of Lang-erhans cells has been stressed.

5

Nowadays there is convincingevidence for the presence of oxidative stress in the skin ofaffected individuals.

6–11

Extremely high concentrations of

IJD_1463.fm Page 482 Wednesday, August 14, 2002 8:37 AM

© 2002

The International Society of Dermatology International Journal of Dermatology

2002,

41

, 482–487

483

Schallreuter

et al. Repigmentation in vitiligo and hydrogen peroxide removal

Report

epidermal H

2

O

2

together with low catalase levels have beenidentified

in vivo

and

in vitro

using Fourier Transform Ramanspectroscopy (FT Raman).

12,13

It has been shown that themissing catalase activities can be substituted with a narrow-band activated pseudocatalase cream (PC-KUS) which isbased on a bis manganese EDTA bicarbonate complex(Patent No. EPO 58417 A). This complex successfully removesthe high H

2

O

2

levels from these patients leading to initiationof repigmentation and to recovery from vacuolation in theepidermal cells.

4,12–15

The time course for repigmentation canvary considerably in the patients, but repigmentation can beachieved in long-term vitiligo even after 47 years

12

(Fig. 1).Since the combination of solar radiation and sea waterbathing at the Dead Sea is a successful treatment modality invarious skin diseases, such as psoriasis and atopic eczema, aswell as vitiligo, we wanted to investigate whether we couldenhance the effect of the conventional pseudocatalase cream

(PC-KUS) monotherapy in combination with the Balneo-climatotherapy at the Dead Sea.

16

Until now it is generally believed that the high salt concen-tration (346 g/L) contributes to the efficacy of the therapyby several mechanisms. A release of pro-inflammatory andchemotactic mediators has been proposed.

16,17

Only recentlythe effect of the high magnesium chloride (MgCl

2

) concentra-tion of this water has been further elucidated by Schempps

et al

. who were able to show that MgCl

2

dramatically influ-ences the antigen presenting capacity of Langerhans cells.

18

Furthermore, it is generally believed that the unique solar UVspectrum in the Dead Sea basin contributes to the sea waterbathing. In this context an increased photosensitivity after thesalt bathing has been implicated.

19,20

Solar radiation at theDead Sea is filtered by an additional 400 m below sea levelatmosphere and it has been shown that the shortest part ofUVB does not arrive at the Dead Sea.

21–23

These conditions

Figure 1 Clinical result of narrowband UVB activated pseudocatalase cream (PC-KUS) therapy. Extensive repigmentation of facial vitiligo in a female black patient (photo skin type VI) before (a) and after (b) 14 months treatment with narrowband UVB activated pseudocatalase (PC-KUS) monotherapy twice daily.12–14 N.B. This result can be achieved even after the duration of vitiligo for 47 years



2002,

41

, 482–487 © 2002


484 Report

Repigmentation in vitiligo and hydrogen peroxide removal

Schallreuter

et al.

could foster activation of the inactive manganese-EDTAbicarbonate complex of the conventional pseudocatalasecream (PC-KUS) therapy.

12,13

Patients and methods

Patient selection and study design

The clinical observation was based on a randomised three-arm

study, in order to compare the effect of climatotherapy alone

(group 1/control arm) vs. placebo cream in combination with

climatotherapy (group 2) and pseudocatalase cream (PC-KUS) in

combination with climatotherapy (group 3) at the Dead Sea basin

over 21 days.

The patients were eligible for the study when they had vitiligo

according to a full body clinical examination in association with

Wood’s light.

Fifty-nine English and German patients with vitiligo (14 men and

45 women) with a mean age of 38.6 years (range 18–62 years)

from the Institute for Pigmentary Disorders in Greifswald,

Germany were included in the study. The majority of the group

(86%) had the most common clinical subtype vulgaris and 14%

presented with acrofacial vitiligo. Only patients with photo skin type

III (Fitzpatrick classification) were included in order to exclude any

possible effect of other skin types.

24

The duration of the disease process varied from 3 to 53 years

with a mean age of 16.7 years. Family history was positive in

28/59 patients (47.5%). The patients were otherwise healthy.

Written consent was obtained from the local ethics committee from

each patient prior to this clinical study.

The study took place at the DMZ-Mor Clinic in Ein Bokek, Israel

during the months of June and July

FT-Raman protocol to follow epidermal H

2

O

2

removal

in vivo

in the skin

All patients (

n

= 59) were tested

in vivo

before treatment and at

the end of the 21-day stay at the Dead Sea basin for epidermal

H

2

O

2

concentrations using

in vivo

FT-Raman spectroscopy. The

results were compared to 15 healthy photo skin-type matched

controls.

12

FT-Raman spectra were obtained with a Bruker RFS 100/S

spectrometer equipped with a liquid nitrogen cooled germanium

detector. Sample excitation was accomplished using a Nd

3+

/YAG

laser operating at 1064 nm with a laser power of 400 mW. Each

spectrum was accumulated over 5 min with 300 scans and a

resolution of 4 cm

−

1

. Total H

2

O

2

was visualized as a well defined

peak at 875 cm

−

1

based on the oxygen-oxygen (O-O) stretch

vibration.

12

The study design was approved by the local Ethics

Committees.

Atomic Absorption Spectroscopy

In order to elucidate the content of transition metals in the Dead

Sea water we utilized Atomic Absorption Spectroscopy. Total

transition metal analyzes were carried out using a Perkin-Elmer

model 1100 Atomic Absorption spectrometer. Samples of Dead

Sea water were diluted 1 : 20 with de-ionized distilled water and

made up to 1 L.

Treatment protocol

All patients had to bathe for 15 min in the Dead Sea twice daily.

The bath was followed by a shower to wash off the salt. Group 1

(

n

= 10) then underwent directly total body sun exposure, whereas

group 2 (

n

= 10) and group 3 (

n

= 39) applied their creams to the

entire body surface prior to sun exposure.

The exposure times were slowly increased to a maximum of 1 h.

The treatment took place in the morning between 7.30 am and

10.30 am and in the afternoon between 2.30 pm and 5.30 pm. The

patients had to record the exposure times.

Clinical assessment by standardized photography

For a valid clinical assessment of the repigmentation of the face

and hands we utilized black and white photographs using a fixed

frame assembly holding the camera (Pentax MZ-M with a 90-mm

lens and an Ilford 828 glass filter) and a Kodak T-MAX 400 film. The

flashlight with non-UV coated bulbs was also fixed (Bowens Esprit

500). This technique assures a good quality comparison of

subsequent photographs and allows, even in very fair skin, the

objective evaluation of affected areas. In addition, we used

whole body color photographs with a standard camera and

flash light (Minolta) to follow disease stability during this

treatment period.

Patients were photographed before treatment and at the end of

the therapy at the Dead Sea after 21 days.

Assessment of efficacy

The efficacy of each treatment modality was assessed after

21 days based on the special photographic documentation of the

face and hands. The grading was based on the repigmentation of

affected areas by comparison of the photos before and after

21 days.

Statistical analysis

For statistical analysis we used the Wilcoxon signed rank test for

paired samples. Values

P

< 0.05 were significant.

Results

The Dead Sea water has pseudocatalase activity

The Dead Sea water analysis showed the presence oftransition metals such as manganese (7140 p.p.m.), copper(582 p.p.m.), and iron (1398 p.p.m.), as well as high concen-trations of sodium bicarbonate and calcium chloride. Theseresults were obtained from analysis of the Dead Sea waterusing Atomic Absorption Spectroscopy. Since these transitionmetals can form the chemical base for pseudocatalases, weexpected that the Dead Sea water bathing would act as a“pseudocatalase”.

25–27

In order to test this assumption, we


© 2002


2002,

41

, 482–487

485

Schallreuter

et al. Repigmentation in vitiligo and hydrogen peroxide removal

Report

followed

in vivo

the reduction of H

2

O

2

levels in 16 of 59patients using FT Raman spectroscopy. The results showed asignificant decrease of epidermal H

2

O

2

after a 15-minbathing session (

p

= 0.001). The data of this analysis are pre-sented in Fig. 2. This

in vivo

observation identified the DeadSea water as a pseudocatalase. A comparison of the efficacy ofthe Dead Sea bathing alone (group 1), and pseudocatalasecream (PC-KUS) plus Dead Sea bathing (group 3) showed asignificantly more potent catalase activity for pseudocata-lase cream (PC-KUS)/Dead Sea bathing after 21 days(

p

= 0.0001). The results are presented in Fig. 3. In order torule out a possible contribution of the cream base, we treated

10 patients with this placebo cream plus Dead Sea bathing for21 days (group 2). There was no significant differencebetween the H

2

O

2

reduction after Dead Sea bathing alone(group 1) and the combination of placebo cream/Dead Seabathing (group 2) (

P

> 0.05) (Fig. 3).

Evaluation of repigmentation after 21 days

The clinical result was assessed after 21 days by Wood’s lightand by photodocumentation.

None of the patients was fully repigmented at day 21 nordid any patient develop new white patches during this time.The examination with Wood’s light identified more repig-mentation which could not be appreciated by eye alone. Sincethe hands and face were photodocumented under the specialUV-conditions as outlined in the methods section, we there-fore only evaluated the face and the hands. The results onfacial regimentation are presented in Table 1. The repigmen-tation of the hands was not further evaluated because thechanges were too small after 21 days treatment.

The outcome showed that patients treated with placebocream/Dead Sea climatotherapy (

n

= 10) (group 2) andpatients treated with climatotherapy only (

n

= 10) (group 1)had significantly less follicular repigmentation in the facecompared to the patient group treated with pseudocatalasecream (PC-KUS) in combination with climatotherapy (group 3).

One example of extensive follicular repigmentation in theface is shown in Fig. 4.

There was no significant difference between Dead Seaclimatotherapy (group 1) and placebo cream in combinationwith climatotherapy (group 2).

Discussion

The positive healing effect of climatotherapy at the Dead Seabasin has been well established in the treatment of psoriasisand atopic eczema.

28

The treatment protocol for thesediseases usually lasts for 28–42 consecutive days and theresponse rates are described as high as 80%.

29

Studies onUV-intensities at the Dead Sea area showed that 9.4% of UVB

Figure 2 Efficacy of a 15-min Dead Sea water bath on the removal of epidermal H2O2, in patients (n = 16) with vitiligo using in vivo FT-Raman spectroscopy. Measurements were taken before and after the bath, showing a significant reduction of H2O2 (p = 0.001)

Figure 3 A comparison of the in vivo efficacy of Dead Sea water and combination of Dead Sea water and pseudocatalase cream (PC-KUS) on epidermal H2O2 removal in vitiligo using FT-Raman spectroscopy. The results are presented in percentage of H2O2 levels standardized to 100% before treatment. A significantly stronger effect of pseudocatalase cream (PC-KUS)/Dead Sea bath is shown. The pseudocatalase activity of the Dead Sea water alone is much weaker. The placebo cream has no effect on H2O2 removal. Healthy controls have no detectable H2O2 level in their skin (the data are presented ± SEM)

Table 1 Photographic assessment of repigmentation in the face after 21 days

Repigmentation* 3 + 2 + 1 + 0

Group 1 (n = 10) 0 6 4 0Group 2 (n = 10) 0 7 3 0Group 3 (n = 39) 30 9 0 0

*Grading was based on a scale ranging from 0, 1 +, 2 + and 3 + 0. (0 = no signs of repigmentation; 1 + = minimal follicular repigmentation; 2 + = follicular repigmentation < 50% of the involved areas; 3 + > 50% follicular/confluent repigmentation of involved areas).



2002,

41

, 482–487 © 2002


486 Report

Repigmentation in vitiligo and hydrogen peroxide removal

Schallreuter

et al.

and 3.5% of UVA are filtered out yielding a unique environ-ment in this region.

29

The safety aspects of the solar photo-therapy have been investigated and discussed extensively.

30

It has been suggested that the UVB range at the Dead Seacould be a natural narrow-band therapy.

29

In addition, thehigh mineral content of the Dead Sea has been considered amajor factor in this successful treatment modality. The studypresented here identified the Dead Sea water as a “pseudo-catalase”. We were able to follow

in vivo

the reduction ofepidermal H

2

O

2

directly in the skin of patients with vitiligo.This reducing effect was much weaker than a narrowbandUVB activated pseudocatalase cream (PC-KUS) in combina-tion with Dead Sea water bathing. However, whether thisH

2

O

2

reduction was indeed beyond the mM range cannot yetbe concluded due to the limitation in sensitivity of

in vivo

FT Raman spectroscopy.

12

A significantly faster initiation of repigmentation com-pared to conventional pseudocatalase cream (PC-KUS)monotherapy or climatotherapy alone was observed usingthe combined protocol Dead Sea/pseudocatalase cream (PC-KUS)/solar phototherapy twice daily in the early morning andlate afternoon.

12–14

It should be noted that the treatment pro-tocol in the study presented here used much less sun exposurethan the conventional treatment recommendation at theDead Sea for vitiligo. (2 h compared to 7–8 h per day). In con-clusion, from the results of this placebo controlled study, itappears that the combination of climatotherapy and conven-tional pseudocatalase cream (PC-KUS) initiates a faster repig-mentation in all patients studied. A comparison of our ownresults using narrow-band UVB activated pseudocatalase

cream (PC-KUS) and the combination therapy revealed thatthe combination therapy is much more effective in the onsetof repigmentation.

12–14

This fast repigmentation continues forat least up to 3 months (unpublished results).

This study did not investigate the contribution of theunique solar spectrum to the initiation of repigmentation invitiligo. Since all patients (groups 1, 2, and 3) underwent theirtreatment at the same time of the year and during the samesun hours of the day, we believe that the data of this clinicalobservation are valid and support once more the importanceof epidermal H

2

O

2

removal and also underline the significantcontribution of UV-light to initiating a successful repigmenta-tion in this disease.

12–14

Acknowledgments

We are grateful for the dedication of all patients. This workwas supported by Deutscher Vitiligo Verein e.V. Hamburg,Germany, Bruker Karlsruhe, Germany and Stiefel Interna-tional. Susan Shergill typed the manuscript.

References

1 Ortonne JP, Bose SK. Vitiligo. Where do we stand?

Pigment Cell Res

1993;

8

: 61–72.2 LePoole IC, Das PK, van den Wijngaard RM,

et al.

Review of the etiopathomechanism of vitiligo: a convergence theory.

Exp Dermatol

1992;

2

: 146–153.3 LePoole IC, van den Wijngaard RM, Westerhof W,

et al.

Presence or absence of melanocytes in vitiligo lesions: an

Figure 4 Follicular repigmentation after 21 days. Initiation of extensive follicular repigmentation in the face of a female patient (photo skin type III) after only 21 days combination therapy (climatotherapy/pseudocatalase cream (PC-KUS)) twice per day following the protocol as described in methods. (a) before treatment, (b) after 21 days. (For details of photographic documentation see methods)


© 2002


2002,

41

, 482–487

487

Schallreuter

et al. Repigmentation in vitiligo and hydrogen peroxide removal Report

immunohistochemical investigation. J Invest Dermatol 1993; 100: 816–822.

4 Tobin DJ, Swanson NN, Pittelkow MR, et al. Melanocytes are not absent in lesional skin of long duration vitiligo. J Pathol 2000; 191: 407–416.

5 Nordlund JJ, Ortonne JP. Vitiligo and depigmentation. Current Problems Dermatol 1992; 4: 3–30.

6 Moellman G, Klein-Angerer S Scollay DA, et al. Extracellular granular material and degeneration of keratinocytes in normally pigmented epidermis of patients with vitiligo. J Invest Dermatol 1982; 79: 321–330.

7 Bhawan J, Bhutani LK. Keratinocyte damage in vitiligo. J Cutaneous Path 1983; 10: 207–212.

8 Yohn JJ Norris DA, Yrastorza G, et al. Disparate antioxidant enzyme activities in cultured human cutaneous fibroblasts, keratinocytes and melanocytes. J Invest Dermatol 1991; 97: 405–409.

9 Schallreuter KU, Wood JM, Berger J. Low catalase levels in the epidermis of patients with vitiligo. J Invest Dermatol 1991; 97: 1081–1085.

10 Maresca V, Roccella M, Roccella F, et al. Increased sensitivity to peroxidative agents as a possible pathogenic factor of melanocyte damage in vitiligo. J Invest Dermatol 1997; 109: 310–313.

11 Boissy R, Liu YY, Medrano EE, Nordlund JJ. Structural aberration of the rough endoplasmic reticulum and melanosome compartmentalisation in long-term cultures of melanocytes from vitiligo patients. J Invest Dermatol 1991; 97: 395–404.

12 Schallreuter KU, Moore J, Wood JM, et al. In vivo and in vitro evidence for hydrogen peroxide accumulation in the epidermis of patients with vitiligo and its successful removal by a UVB-activated pseudocatalase. J Invest Dermatol Symp Proc 1999; 4: 91–96.

13 Schallreuter KU. Successful treatment of oxidative stress in vitiligo. Skin Pharmacol Appl Skin Physiol 1999; 12: 132–138.

14 Schallreuter KU, Moore J, Wood JM, et al. Epidermal H2O2 accumulation alters Tetrahydrobiopterin (6BH4) Recycling in Vitiligo: Identification of a general Mechanism in Regulation of all 6BH4-Dependent Processes? J Invest Dermatol 2001; 116: 167–174.

15 Schallreuter KU, Wood JM, Lemke KR, et al. Treatment of vitiligo with a topical application of pseudocatalase and calcium in combination with short-term UVB exposure: a case study on 33 patients. Dermatol 1995; 190: 223–229.

16 Even-Paz Z, Shani J. The Dead Sea and psoriasis historical and geographical background. Int J Dermatol 1989; 28: 1–9.

17 Abels DJ, Rose T, Bearman JE. Treatment of psoriasis at a Dead Sea Dermatology Clinic. Int J Dermatol 1995; 34: 134–137.

18 Schempps CM, Diltmar HC, Hunnler D, et al. Magnesium ions inhibit the antigen presenting function of human epidermal Langerhans cells in vivo and in vitro. Involvement of ATPase, HLA-DRB7 molecules and cytokines. J Invest Dermatol 2000; 115: 680–686.

19 Wiedow O, Wiese F, Streit V, et al. Lesional elastase activity in psoriasis, contact dermatitis and atopic dermatitis. J Invest Dermatol 1992; 99: 306–309.

20 Boer J, Schotthorst AA, Boom B, et al. Influence of water and salt solutions on UVB irradiation of normal skin and psoriasis. Arch Dermatol Res 1982; 273: 247–259.

21 Even Paz Z. Dermatology at the Dead Sea. Spas Isr J Med Sci 1996; 32: 11–15.

22 Kushelewsky AP, Slifkin MA. Ultraviolet measurements at the Dead Sea and Beer Sheva. Isr J Med Sci 1975; 11: 488–490.

23 Westerhof W, Nieuweboer-Krobotova L. Treatment of vitiligo with UV-B radiation vs topical psoralen plus UV-A. Arch Dermatol 1997; 133: 1525–1528.

24 Fitzpatrick TB. The validity and practicality of sun reactive skin types I through to VI. Arch Dermatol 1988; 124: 869–871.

25 Ya Sychev A, Isak VG, Van Lap D. Catalytic properties of carbonate complexes of manganese (II) in the decomposition of hydrogen peroxide. Zhornal Ficichewskoi Khimii 1977; 51: 363–366.

26 Stadtman ER, Berlett BS, Chock PB. Manganese-dependent disproportionation of hydrogen peroxide in bicarbonate buffer. Proc Natl Acad Sci USA 1990; 87: 384–388.

27 Hage R, Iburg JE, Kerschner J, et al. Efficient manganese catalysts for low–temperature bleaching. Nature 1994; 369: 637–639.

28 Shani J, Seidl V, Hristakieva E, et al. Indications, Contraindications and possible side-effects of climatotherapy at the Dead Sea. Int J Dermatol 1997; 36: 481–492.

29 Harari M, Shani J. Demographic evaluation of successful anti-psoriatic climatotherapy at the Dead Sea (Israel) DMZ Clinic. Int J Dermatol 1997; 36: 304–308.

30 Kushelevsky AP, Harari M, Kudish AI, et al. Safety of solar phototherapy at the Dead Sea. J Am Dermatol 1998; 38: 447–452.


Documents

Blackwell Science, LtdOxford, UKIJDInternational Journal ... · International Journal of Dermatology 2002, The International Society of Dermatology