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Treatment of vitiligo with khellin and ultraviolet A
Treatment of vitiligo with khellin and ultraviolet A Bernhard Ortel, M.D., Adrian Tanew, M.D., and Herbert H6nigsmann, M.D. Vienna, Austria Twenty-eight patients with vitiligo were treated with a new photochemotherapeutic regimen using khellin, a furanochromone, as photosensitizer, together with ultraviolet A (UVA) irradiation. Twenty-five patients received khellin orally and three patients were treated with topical khellin. Treatments were given three times weekly. As opposed to psoralens, khellin did not induce skin phototoxicity with UVA but it induced repigmentation similar to psoralens. The treatment success strongly depended on the number of treatments. More than 70% repigmentation was achieved in 41% of the patients who had received 100 to 200 treatments. This success rate is comparable to the rate obtained with psoralens. Seven patients experienced a mild elevation of liver transamlnases within the early treatment phase and their treatments were discontinued. No long-term internal organ or skin toxicity was observed. The major advantage of khellin is that it does not lead to phototoxic skin erythema and thins can be considered safe for home treatment. Because of its photochemistry it may be considered less hazardous than psoralens regarding mutagenicity and carcinogenicity. (J AM ACAD DERMATOL 1988;18:693-701.)
Vitiligo is not a life-threatening disease and does not require treatment unless severe cosmetic disfigurement causes emotional and/or social distress. Therapeutic approaches are directed to reverse the progressive loss of pigment-producing epidermal melanocytes and to reconstitute normal skin coloration. U p to 20% of patients with vitiligo experience transient repigmentation in some skin areas upon exposure to sunlight or artificial ultraviolet radiation. However, only photochemotherapy can induce a permanent, cosmetically acceptable result. 1-3 A n ancient form of photochemotherapy consisting of topical application of plant extracts or ingestion o f seeds of certain psoralen-containing plants and subsequent exposure to sunlight has From the Division of Photobiology, Department of Dermatology I, University of Vienna. Accepted for publication Sept. 18, 1987. Reprint requests to: Dr. Herbert Hrnigsmann, Professor of Dermatology, Division of Photobiology, Department of Dermatology I, University of Vienna, Alserstrasse 4, A-1090 Vienna, Austria.
been in use for more than 3000 years in middle and far eastern countries as a folk remedy to treat "leukoderma." Modern photochemotherapy of vitiligo with psoralens (PUVA) was introduced in 1948 by E1 Mofty in Egypt? Since then detailed information has become available on the efficacy and the limitations of this treatment form with various treatment schedules and different psoralens? ,3,5 In 1982 Abdel-Fattah et al6 reported encouraging results with a new photochemotherapeutic regimen. After oral administration of khellin followed by exposures to sunlight, they observed repigmentation of vitiliginous skin in Egyptian patients. In the present study we have investigated the efficacy and possible side effects of this treatment under standardized conditions with artificial UVA radiation. Khellin (Fig. 1) is a furanochromone that is isolated from the seeds of Ammi visnaga (Arabic "khella"), a plant common in the eastern Meditteranean area. In 1946 Anrep and eoworkers7's ex-
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T a b l e I. Demographic data o f 28 patients
~
0 OCH3
OCH 3
I
II
Female Male Age (mean) Duration of disease Type of vitiligo Generalized Focal/acrofacial
15 13 12-60 yr (30 yr) 1-24 yr (3.8 yr) 23 5
Fig. 1. Chemical structure of 8-methoxypsoralen (I) and khellin (11). plored its p h a r m a c o l o g i c properties and discovered that small doses o f this substance exert a relaxing effect on s m o o t h muscle cells of various organs. Ganglionic blocking was thought to be responsible for this action. 9 In the 1940s and 1950s khellin was widely used as a c o r o n a r y vasodilator in the long-term treatment o f angina pectoris and asthma, s'~~ No longterm side effects were reported at that time. s'1~ Higher effectiveness o f other therapies led to the discontinuation o f the use o f khellin in cardiac disease. 13 T o d a y only a few homeopathic preparations advertised for use in cardiac disease and in bronchial asthma contain khel[in. M A T E R I A L AND M E T H O D S Patients
A total of 28 patients were treated (15 female, 13 male; age range, 12 to 60 years; mean age, 30 years) (Table I). Twenty-three suffered from generalized symmetric vitiligo and 5 had a circumscribed acrofacial type of vitiligo, Mean duration of vitiligo was 3.8 years (range, 1-24 years). Four were of skin type II, 19 of skin type III, and 5 of skin type IV. Twenty-six were treated with oral khellin, and 3 received topical treatment with a khellin solution. Informed consent was obtained. Drug. Khellin (4,9-dimethoxy-7-methyl-5H-furo [3,2-g]-1] benzopyran-5-one) was purchased from Sigma Chemical Co., St. Louis, Mo. For systemic use 50 mg khellin with lactose as excipient were filled into hard gelatin capsules. For topical application a 2% solution in glycerol formol was prepared. Serum levels. Serum levels of khellin were determined by high-pressure liquid chromatography (HPLC) at the Pharmacokinetics Department of Gerot Pharmazeutika, Vienna, Austria. A 25-cm column filled with RPI8 polygosil 10 Ix (Machary-Nagel and Co., Diiren, Federal Republic of Germany) was used with a 60/40 mixture of sodium acetate buffer and acetonitrile. 5-Methoxypsoralen was used as the internal standard.
In 8 patients determination of serum levels was performed 30 minutes, 1, 1.5, 2, 3, 5, 7, and 24 hours after ingestion of a single dose of 100 nag khellin. In three patients blood was taken 21/2 hours after khellin ingestion on several clays. A total of 91 serum samples was evaluated. Double determinations of the coded blood samples were performed in order to monitor the accuracy of the method. Radiation source. UVA was delivered by conventional PUVA units (Waldmann 6000 and 8001; Waldmann Co., Schwenningen, Federal Republic of Germany) equipped with Sylvania FR 90 T12 fluorescent tubes, which emit broad-spectrum UVA (peak emission at 365 ram). Phototoxicity tests. Phototoxicity tests were performed in all patients according to the established testing procedure used in psoralen photochemotherapyl4; 2.5 hours after oral intake of 100 nag khellin, six template areas on the buttocks were exposed to 1.5, 2, 3, 5, 7, and 9 joules/era 2. In addition, five subjects were tested with high-dose UVA (50 to 100 joules/cm 2) before and after khellin ingestion and 1 hour after the application of topical khellin. In these patients the erythemal response to UVA alone and to khellin plus UVA after systemic and topical application was compared in both nomaal and vitiligo skin. Readings were performed by three independent investigators immediately after exposure, at 24, 48, and 72 hours. All test areas were also evaluated for immediate and delayed tanning simultaneously and after 1 week. Treatment schedule. The treatment schedule was essentially similar to the protocols used in our institution for vitiligo treatment with psoralens. The patients were exposed three times weekly to UVA 2.5 hours after ingestion of 100 mg khellin. The drug dose was kept constant at 100 mg in all patients because this dose has been reported to be effective~ and, in addition, the number of patients participating in this study was too low to include variations in the schedule. The time interval of 2.5 hours between drug ingestion and irradiation was chosen because serum level profiles have shown that the peak concentration in the serum is
Volume 18 Number 4, Part 1 April 1988
Khellin-UVA photochemotherapy for vitiligo
695
Wm 200(
O
r
r
3
5
0
0 0
1
a
8
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Fig. 2. Twenty-four-hour serum profile after ingestion of 100 mg khellin. Mean Values after 30 minutes to 24 hours in eight patients.
reached in all patients between 2 and 5 hours (see below). The 2% topical solution was applied 1 hour before UV exposure. The treatment response was documented photographically every 6 months. UVA dosimetry. In preliminary dose finding experiments symmetric lesions in one group of patients received a constant irradiation dose of 5 joules/cm 2 on one side and 10 joules/cm 2 on the other side. In another group the treatment was started with 5 joules/era 2 on both sides, whereby one side remained at 5 joules/cm 2 and the other side was gradually increased by 5 joules/cm 2 every other week up to 20 joules/cm2. As a result effective doses were found to range between 10 and 15 joules/cm 2. The irradiation dose (10 joules/cm 2 for skin types II and II1, 15 joules/cmz for skin type IV) was kept constant in each patient throughout the entire treatment period. L a b o r a t o r y tests. Blood was taken for complete blood count, blood chemistry, and liver and renal function tests (SMAC 24) before initiation of oral treatment and 1, 2, 4, 8, and 12 weeks after initiation of oral treatment. During the later treatment period the tests were repeated every 3 months. Controls. For control purposes two symmetric vitiligo areas were selected. One was shielded during UVA exposure, and the other one was irradiated with the therapeutic UVA dose (10 to 15 joules/cm 2) prior to khellin ingestion. The rest of the body received the actual treatment with khellin plus UVA. Control areas were switched to khellin plus UVA whenever a clearly visible difference in the treated areas was noted, RESULTS Serum levels. The 24-hour serum profiles showed that peak levels ranging from 1275 to 2178 ng/ml were reached 2 to 5 hours after drug inges-
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Fig. 3. Phototoxicity test with oral khellin plus UVA (KUVA) versus UVA alone; reading at 24 hours. Note that there is no difference in erythema formation on both sides with exposure doses of 30 to 80 joules/cm 2. tion (Fig. 2). Twenty-four hours after ingestion no substantial drug concentration was measured. As revealed by multiple determinations the values were found to be most constant 21/a hours after khellin administration, Therefore a time interval of 21/2 hours between drug intake and exposure was chosen for therapy. Phototoxicity. Under the conditions described above khellin did not induce phototoxic skin erythema with doses up to 100 j o u l e s / c m 2. UVA
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Fig. 4. Oral khellin plus UVA. Comparison of 5 versus 15 joules/cm2; note equal extent of follicular repigmentation but different intensity of tan. A and B, Four weeks and 13 weeks, respectively, after initiation of treatment.
Fig. 5. Oral khellin plus UVA. Comparison of 5 versus 10 joules/em 2 before (A) and after (B) 18 weeks of treatment. The 10 joules/cm 2 area exhibits incipient repigmentation whereas 'he 5 ictuSes/c:-:,z area s~ows v!rtual'.y no change.
erythema developed 24 hours after exposures to 50 joules/cm ~-or more in both khellin-treated and untreated areas. A comparison of erythema reactions induced by UVA alone and by khellin plus IJVA after systemic or topical administration in both vitiliginous and normal skin did not reveal any perceptible difference (Fig. 3). In contrast to the phototoxic reaction with psoralens, which peaks as late as 72 hours after exposure, there was no increasing erythema reaction with khellin between 24 and 72 hours. Interestingly, khellin plus UVA also did not induce a more intense immediate and/or delayed pigmentation in normal skin than did UVA alone. UYA d o s i m e t r y and controls. Paired comparison studies in symmetric lesions of two different
dosages, namely, 5 and 10 or 5 and 15 joules/cm 2, respectively, showed that equal repigmentation occurred with respect to the number of repigmented follicular spots in both areas but the intensity of the tan was considerably higher in the high-dose area (Fig. 4). Most patients, however, developed follicular repigmentation only in the 10 joules/cm 2 but not in the 5 joules/cm 2 area (Fig. 5). No pigmentation occurred in vitiligo areas shielded during UVA exposure after khellin intake (Fig. 6, A and B). Also areas that received UVA alone in the same doses as with khellin remained unpigmented (Fig. 6, C and D). Areas adjacent to these control sites exhibited a definite response to khellin plus UVA treatment. Therapeutic results. Four to 8 weeks after the
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Khellin-UVA photochemotherapy for vitiligo 697
Fig. 6. Control areas. A, Area shielded (K) during exposures before and (B) after 11 weeks of treatment; repigmentation is seen only in the khellin plus UVA (KUVA) are~. C, Areas treated with UVA alone before and (D) after 11 weeks. Note the increased t ~ in normal skin but absence of follicular repigmentation.
Table II, Treatment results
Number of patients Duration of therapy (x +_ SD; rap) Number of treatments (x • SD) Total irradiation dose (x + SD; joules/era z)
15 5.8 --- 4.5
8 12.5 ~ 4.8
5 21.0 __+ 10.1
57 + 39
111 - 51
157 ___ 44
586 ___ 522
1328 + 619
2290 --_ 1070
initiation of khellin photochemotherapy, beginning repigmentation was heralded by the appearance of pigmented follicular spots in the responding patients. These follicular pigmentations gradually enlarged and coalesced over a period of 8 to 24 months. There was a great interindividual variation in the patients' responses to treatment with regard to the duration of treatment until acceptable reconstitution of skin pigmentation was achieved. Therapy was maintained as long as the repigmentation process continued and was stopped when repigmentation ceased. As has been described for conventional psoralen photochemotherapy, certain skin areas with vitiligo, in particular acral skin, did not respond adequately despite many months of treatment. ~.2,~5
Tables II and III show the results in all 28 patients. Five patients exhibited a cosmetically satisfying result with repigmentation in more than 70% of the original vitiligo areas after a mean treatment dtJration of 21 months, a mean of I57 treatments, and a total UVA dose of 2290 joules/cm z (Fig. 7). Eight patients improved by 30% to 70% after a mean treatment period of 12.5 months. O f this group, two patients who were good responders developed an elevation of liver transaminases after 4 to 6 weeks of treatment and their treatment was discontinued. After normalization of the liver values they were switched to a psoralen protocol and later acquired repigmentation of more than 70% (data not shown). At the time of writing two other responding patients of
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Table III. Extent of repigmentation versus number of treatments Number of patients with repigmentation (%)
Number of treatments
Total
<30
<50 50-99 100-199 >200
10 6 11 1 28
9 3 3 0 15
this group were still under treatment after 12 and 15 months of treatment, respectively; the other four had discontinued treatment after 8, 11, 16, and 21 months because they were not satisfied with the result. In the group of 15 patients with less than 30% repigmentation, 10 patients had been treated for less than 10 months (Table III). In five patients treatment was discontinued because of elevated transaminases, in 8 it was discontinued because of poor response, and 2 were still under treatment at the time of writing and showed gradual improvement. Seven patients were switched to a psoralen protocol, but the poor responders also failed to improve with the PUVA regimen (data not shown). Of the three patients treated with topical khellin, one with generalized vitiligo (Fig. 8) was still under treatment after 12 months, showing slow but continuous progression of repigmentation. The other two patients, one with acrofacial and the other with generalized vitiligo, did not improve more than 30% after 12 months of treatment. They also failed to respond to subsequent PUVA treatment (data not shown). Unrelated to the extent o f repigmentation, the khellin-treated patients noted an increased tolerance to natural sunlight in vitiliginous skin after 2 to 3 months, similar to the observation in patients treated with other forms of photochemotherapy.'S.~6 Side effects S h o r t - t e r m side effects. Six (21%) of the patients ingesting khellin reported episodes of mild nausea and two (7%) reported orthostatic problems. These complaints usually occurred within
,,
30-70
1 3 4 0 8
l
>70
0 0 4 1 5
the first 2 weeks of therapy, subsided spontaneously upon continuation of the treatment, and did not necessitate discontinuation of therapy. In seven (25%) of 28 patients under systemic khellin treatment, a mild elevation of liver transaminases (aspartate aminotransferase, AST, SGOT; alanine aminotransferase, AST, SGPT; and gamma glutamyltransferase, GGT), with values of up to three times the normal limit, was observed within the first 2 months of treatment. These laboratory changes occurred unrelated to systemic symptoms such as nausea, and it has to be mentioned that these patients had normal values at the beginning and showed negative hepatitis antibody screening findings. After discontinuation of khellin administration the values returned to normal within 5 to 12 weeks. A second course of khellin in two of these patients again resulted in a similar increase of transaminases after 4 and 8 weeks, respectively. Patients who did not develop an elevation of transaminases within the initial treatment period retained normal values throughout the entire treatment. Long-term side effects. No long-term side effects were'observed with regard to internal organ or skin toxicity. In particular, no skin changes as reported for long-term PUVA treatment with high cumulative UVA doses, such as lentigines, mottling of the skin, and solar degeneration, were noted. DISCUSSION Khellin-UVA photochemotherapy is effective in restoring normal skin color in more than 70% of the originally involved vitiliginous areas in a sub-
Volume 18 Number 4, Part I April 1988
stantial number of patients with vitiligo after 1 to 2 years of continuous therapy. However, as with psoralen photochemotherapy (PUVA), complete repigmentation is never achieved since certain skin areas, such as the distal dorsal surfaces of the hands and feet, tips of the fingers and toes, areas of bony prominences, palms, soles, and nipples, do not respond at all. Since the only other effective treatment option is PUVA, it appears important to compare the two regimens with regard to efficacy, side effects, and safety. In the early reports on the use of psoralens in vitiligo, the assessment of the actual treatment success is difficult because treatment schedules were different and UV radiometry and radiation sources were not well defined. ~s'~7-2~More recent clinical trials 3,5,2t23 provide for better information on the success rate and permit the conclusion that, at least in whites, khellin may be as effective as psoralens provided treatment is performed for a long enough period of time. An evaluation of our patients indicates that a minimum of 100 exposures is required to reach an overall repigmentation of 70%. If one considers only the group of 12 patients who received 100 to more than 200 treatments (Table III), the success rate (that is, more than 70% repigmentation) was at 41%. Controlled studies in generalized vitiligo 3,5'zta3 with PUVA using 8-methoxypsoralen (8-MOP) and/or trimethylpsoralen (TMP) have yielded comparable success rates ranging from roughly 25% to 40%. The most elaborate study of Pathak et aP in 366 patients from India indicates that the most effective treatment consists of a combination of 8-MOP and TMP, and we are currently investigating whether a combination of khellin and psoralens may increase the therapeutic success. With regard to short-term side effects, khellin has the advantage of being nonphototoxic even after exposure doses of up to 100 joules/era 2. Therefore, severe erythema reactions as may occur in PUVA therapy, particularly in fair-skinned whites, can be avoided. Khellin photochemotherapy can be considered safe with natural sunlight, as a UVA source or also as home treatment with artificial UVA, provided the patients are instructed
Khellin-UVA photochemotherapy for vitiligo 699
Fig. 7. Oral khellin plus UVA treatment. Patient with generalized vitiligo showing more than 70% repigmentation. A, C, and E, Before therapy. B, D, and F, After 16 months of treatment.
appropriately and are monitored regularly. Preliminary results in a few patients indicate that khellin plus sunlight may be effective also in a moderate climate. No serious adverse reactions or long-term side effects have been reported at the time when khellin
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Fig. 8. Topical khellin plus UVA treatment. A, Before therapy. B, Follicular repigmentation after 12 weeks of treatment.
was used in much higher doses (up to 300 mg daily) as a long-term treatment for angina pectoris. 7.sa~ In one large-scale clinical study, shorttime side effects were not encountered more frequently in khellin-treated patients compared to a placebo-treated control group.~~ A more serious complication may be the observed elevation of transaminases in about one third of our patients. In these patients treatment was discontinued and the transaminases returned to normal values. As yet there exists no parameter to predict a predisposition for this drug intolerance reaction. All of our patients had normal values prior to treatment and hepatitis screening showed negative findings. The drug doses used in the past for angina pectoris had ranged from 100 to 300 mg per day and khellin has been administered over several years without any report of hepatic toxicity. However, liver transaminases had not been determined at that time. Although this phenomenon might be interpreted as harmless drug-induced "transaminitis," further investigation of this issue is clearly needed. Patients who remained at normal values during the first weeks after the initiation of khellin did not exhibit an elevation throughout the entire treatment period of up to 2 years. At present there exists no information regarding long-term side effects of khellin plus UVA, but all possible long-term side effects that may arise from an interaction with deoxyribonucleic acid (DNA) have to be considered. Khellin has some structural similarity to 8-MOP and also shares some photochemical properties with psoralens, 24-26 Khellin
forms monofunctional adducts and cross-links with DNA 2v with a maximum action spectrum at 360 rim. However, khellin predominantly forms monoadducts, and the induction of cross-links requires fluences of up to i00 times more than needed for the formation of cross-links with 8-MOP. Compared with 8-MOP, khellin is a very weak producer of reactive oxygen species. 28 Phototoxic and genotoxic properties of khellin have been investigated in various experimental systems. Khellin was shown to be 100- to 1000-fold less active compared to 8-methoxypsoralen?9,3~ Monofunctional psoralens are considered to be less mutagenic than cross-linking analogs. 3~ The very low mutagenic activity of khellin in vitro suggests that potential risks of long-term side effects, such as skin carcinoma formation, may be much lower than with conventional psoralen photochemotherapy. The mode of action of all forms of photochemotherapy for vitiligo is still unknown. Neither khellin alone nor UVA alone can induce repigmentation. The lack of repigmentation in the shielded control areas also excludes a systemic action of the treatment. Remelanization appears to be confined to the interaction of both khellin and UVA. Psoralens dramatically enhance the tanning properties of UVA in normal skin. However, khellin plus UVA is not more melanogenic than UVA alone. This indicates that the stimulation of melanogenesis and the induction of repopulation of vitiliginous skin with melanocytes may be mediated by different mechanisms.
Volume 18 Number 4, Part I April 1988 CONCLUSIONS Khellin p h o t o c h e m o t h e r a p y can be recomm e n d e d as a valuable alternative option to conventional P U V A f o r the treatment o f vitiligo in patients w h o do n o t develop an elevation of liver t r a n s a m i n a s e s . B o t h oral and topical khellin phot o c h e m o t h e r a p y m a y be less hazardous with respect to l o n g - t e r m side effects and can be perf o r m e d e a s i l y with sunlight or as h o m e treatment with c o m m e r c i a l l y available U V A sources.
REFERENCES 1. Ortonne JP, Mosher DB, Fitzpatrick TB. Vitiligo and other hypomelanoses of the skin. New York: Plenum, 1983. 2. Fitzpatrick TB, Parrish JA, Pathak MA. Phototherapy of vitiligo. In: Fitzpatrick TB, Pathak MA, Harber LC, Seiji M, Kukita A, eds. Sunlight and man. Tokyo: Tokyo University Press, 1974:783-91. 3. Parrish JA, Fitzpatrick TB, Shea C, Pathak MA. Photochemotherapy of vitiligo. Arch Dermatol 1976; 112:1531-4. 4. E1 Mofty AM. A preliminary clinical report on the treatment of leukoderma with Ammi majus Linn. J R Egypt Med Assoc 1948;31:651-65. 5. Pathak MA, Mosher DB, Fitzpatrick TB, Parrish JA. Safety and therapeutic effectiveness of 8-methoxypsoralen, 4,5',8-trimethylpsoralen, and psora]en in vitiligo. Natl Cancer Inst Monogr 1984;66:165-73. 6. Abdel-Fattah A, Aboul-Enein MN, Wassel GM, E1Menshawi BS. An approach to the treatment of vitiligo by khellin. Dermatologica 1982;165:136-40. 7. Anrep GV, Barsoum GS, Kenawy MR, Misrahy G. Therapeutic uses of khellin. Method of standardisation. Lancet 1947;252:557. 8. Anrep GV, Kenawy MR, Barsoum GS. The coronary vasodilator action of khellin. Am Heart I 1949;37: 531-42. 9. Vidal-Beretervide K, Monti M. Ganglionic blocking action of piperidino-methyl-khellin. Arzneim Forsch 1965; 15:242. I0. Greiner T, Gold H, Catell M, et al. A method for the evaluation of the effects of drugs on cardiac pain in patients with angina of effort: a study ofkhe]lin (visammin). Am J Med 1950;9:143-55. 11. Ayad H. Khellin in angina pectoris. Lancet 1948;1:305. 12. Armbrust CA, Levine SA. The treatment of angina pectoris with a preparation of khellin. Am I Med Sci 1950;220:127. 13. Benson H, MeCallie DP. Angina pectoris and the placebo effect. N Engl J Med 1979;300:1424-9. 14. Henseler T, H6nigsmann H, Wolff K, Christophers E. Oral 8-methoxypsoralen photochemotherapy of psoriasis. The European PUVA Study: a cooperative study among 18 European centres. Lancet 1981; 1:853-7.
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I5. Lemer A.B, Denton CR, Fitzpatrick TB. Clinical experimental studies with 8-methoxypsoralen in vitiligo. J Invest Dermatol 1953;20:299-314. 16. Cormane RH, Siddiqui AH, Westerhof W, Schutgens RBH. Phenylalanine and UVA light for the treatment of vitiligo. Arch Dermatol Res 1985;277:126-30. 17. Arnold HL. Aul3ere Anwendung von 8-Methoxypsoralen bei Vitiligo. Hautarzt 1960;11:324-6. 18. Dawber RPR. Oral trisoralen therapy in vitiligo. Br J Dermatol 1970;83:386-90. 19. London ID. Evaluation of methoxsalen in the treatment of vitiligo. J Invest Dermatol 1959;32:315-7. 20. Bleehen SS. Vitiligo treatment with trimethylpsoralen. Br J Dermatol 1971;84"186-8. 21. Bleehan SS. Treatment of vitiligo with oral psoralens. Clinically Oriented Symposium of the European Society for Dermatological Research [book of abstracts]. Baden, 1985:67-8. 22. Wennersten G, H/igermark 0. Outdoor and office photochemotherapy of vitiligo: a comparison. Photodermatology 1984;1:91-3. 23. Lassus A, Halme K, Eskelinen A, Ranki A, Puska P, Salo O. Treatment of vitiligo with oral methoxsalen and UVA. Photodermatology 1984; 1:170-3. 24. Beaumont PC, Land EJ, Navaramam S, Parsons BJ, Phillips GO. A pulse radiolysis study of the complexing of furoeoumarins with DNA and proteins. Biochim Biophys Acta 1980;608:182-9. 25. Niccolai N, Bovalini L, Martclli P. The mechanisms of interaction between furanochromones and DNA: a heteronuclear Overhauser effect study on the khellinthymidine model system. Biophys Chem 1986;24: 217-20. 26. Rucheton M, Jeanteur P. Studies on amikhellin. I. Intercalative binding to double-stranded DNA. Biochimie 1973;55:1415-20. 27. Cassuto E, Gross N, Bardwell E, Howard-Flanders P. Genetic effects of photoadducts and photocross-links in the DNA of phage lambda exposed to 360 nm light and tri-methylpsoralen or khellin. Bioehim Biophys Aeta 1977;475:589-600. 28. Martelli P, Bovalini L, Ferri S, Franchi GG, Bari M. Active oxygen forms in photoreaction between DNA and furochromones khellin and visnagin. FEBS Lett 1985; 189:255-7. 29. Abeysekera BF, Abramowski Z, Towers GHN. Genotoxicity of the natural furochromones, khellin and vlsnagin and the identification of a khellin-thymine photoadduct. Photochem Photobiol 1983;38 3:311-5. 30. Hitchins VM, Bockstahler LE, Carney PG, et al. Comparison of khellin and 8-MOP as UVA photosensiti= zers for mammalian cells. Photochem Photobiol 1986; 43[suppl]'275. 3I. Averbeck D. Relationship between lesions photoinduced by mono- and bi-functional furocoumarins in DNA and genotoxicity in diploid yeast. Mutation Res 1985;151: 217-33.
Vitiligo is not a life-threatening disease and does not require treatment unless severe cosmetic disfigurement causes emotional and/or social distress. Therapeutic approaches are directed to reverse the progressive loss of pigment-producing epidermal melanocytes and to reconstitute normal skin coloration. U p to 20% of patients with vitiligo experience transient repigmentation in some skin areas upon exposure to sunlight or artificial ultraviolet radiation. However, only photochemotherapy can induce a permanent, cosmetically acceptable result. 1-3 A n ancient form of photochemotherapy consisting of topical application of plant extracts or ingestion o f seeds of certain psoralen-containing plants and subsequent exposure to sunlight has From the Division of Photobiology, Department of Dermatology I, University of Vienna. Accepted for publication Sept. 18, 1987. Reprint requests to: Dr. Herbert Hrnigsmann, Professor of Dermatology, Division of Photobiology, Department of Dermatology I, University of Vienna, Alserstrasse 4, A-1090 Vienna, Austria.
been in use for more than 3000 years in middle and far eastern countries as a folk remedy to treat "leukoderma." Modern photochemotherapy of vitiligo with psoralens (PUVA) was introduced in 1948 by E1 Mofty in Egypt? Since then detailed information has become available on the efficacy and the limitations of this treatment form with various treatment schedules and different psoralens? ,3,5 In 1982 Abdel-Fattah et al6 reported encouraging results with a new photochemotherapeutic regimen. After oral administration of khellin followed by exposures to sunlight, they observed repigmentation of vitiliginous skin in Egyptian patients. In the present study we have investigated the efficacy and possible side effects of this treatment under standardized conditions with artificial UVA radiation. Khellin (Fig. 1) is a furanochromone that is isolated from the seeds of Ammi visnaga (Arabic "khella"), a plant common in the eastern Meditteranean area. In 1946 Anrep and eoworkers7's ex-
693
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T a b l e I. Demographic data o f 28 patients
~
0 OCH3
OCH 3
I
II
Female Male Age (mean) Duration of disease Type of vitiligo Generalized Focal/acrofacial
15 13 12-60 yr (30 yr) 1-24 yr (3.8 yr) 23 5
Fig. 1. Chemical structure of 8-methoxypsoralen (I) and khellin (11). plored its p h a r m a c o l o g i c properties and discovered that small doses o f this substance exert a relaxing effect on s m o o t h muscle cells of various organs. Ganglionic blocking was thought to be responsible for this action. 9 In the 1940s and 1950s khellin was widely used as a c o r o n a r y vasodilator in the long-term treatment o f angina pectoris and asthma, s'~~ No longterm side effects were reported at that time. s'1~ Higher effectiveness o f other therapies led to the discontinuation o f the use o f khellin in cardiac disease. 13 T o d a y only a few homeopathic preparations advertised for use in cardiac disease and in bronchial asthma contain khel[in. M A T E R I A L AND M E T H O D S Patients
A total of 28 patients were treated (15 female, 13 male; age range, 12 to 60 years; mean age, 30 years) (Table I). Twenty-three suffered from generalized symmetric vitiligo and 5 had a circumscribed acrofacial type of vitiligo, Mean duration of vitiligo was 3.8 years (range, 1-24 years). Four were of skin type II, 19 of skin type III, and 5 of skin type IV. Twenty-six were treated with oral khellin, and 3 received topical treatment with a khellin solution. Informed consent was obtained. Drug. Khellin (4,9-dimethoxy-7-methyl-5H-furo [3,2-g]-1] benzopyran-5-one) was purchased from Sigma Chemical Co., St. Louis, Mo. For systemic use 50 mg khellin with lactose as excipient were filled into hard gelatin capsules. For topical application a 2% solution in glycerol formol was prepared. Serum levels. Serum levels of khellin were determined by high-pressure liquid chromatography (HPLC) at the Pharmacokinetics Department of Gerot Pharmazeutika, Vienna, Austria. A 25-cm column filled with RPI8 polygosil 10 Ix (Machary-Nagel and Co., Diiren, Federal Republic of Germany) was used with a 60/40 mixture of sodium acetate buffer and acetonitrile. 5-Methoxypsoralen was used as the internal standard.
In 8 patients determination of serum levels was performed 30 minutes, 1, 1.5, 2, 3, 5, 7, and 24 hours after ingestion of a single dose of 100 nag khellin. In three patients blood was taken 21/2 hours after khellin ingestion on several clays. A total of 91 serum samples was evaluated. Double determinations of the coded blood samples were performed in order to monitor the accuracy of the method. Radiation source. UVA was delivered by conventional PUVA units (Waldmann 6000 and 8001; Waldmann Co., Schwenningen, Federal Republic of Germany) equipped with Sylvania FR 90 T12 fluorescent tubes, which emit broad-spectrum UVA (peak emission at 365 ram). Phototoxicity tests. Phototoxicity tests were performed in all patients according to the established testing procedure used in psoralen photochemotherapyl4; 2.5 hours after oral intake of 100 nag khellin, six template areas on the buttocks were exposed to 1.5, 2, 3, 5, 7, and 9 joules/era 2. In addition, five subjects were tested with high-dose UVA (50 to 100 joules/cm 2) before and after khellin ingestion and 1 hour after the application of topical khellin. In these patients the erythemal response to UVA alone and to khellin plus UVA after systemic and topical application was compared in both nomaal and vitiligo skin. Readings were performed by three independent investigators immediately after exposure, at 24, 48, and 72 hours. All test areas were also evaluated for immediate and delayed tanning simultaneously and after 1 week. Treatment schedule. The treatment schedule was essentially similar to the protocols used in our institution for vitiligo treatment with psoralens. The patients were exposed three times weekly to UVA 2.5 hours after ingestion of 100 mg khellin. The drug dose was kept constant at 100 mg in all patients because this dose has been reported to be effective~ and, in addition, the number of patients participating in this study was too low to include variations in the schedule. The time interval of 2.5 hours between drug ingestion and irradiation was chosen because serum level profiles have shown that the peak concentration in the serum is
Volume 18 Number 4, Part 1 April 1988
Khellin-UVA photochemotherapy for vitiligo
695
Wm 200(
O
r
r
3
5
0
0 0
1
a
8
,9
a~ h
"
Fig. 2. Twenty-four-hour serum profile after ingestion of 100 mg khellin. Mean Values after 30 minutes to 24 hours in eight patients.
reached in all patients between 2 and 5 hours (see below). The 2% topical solution was applied 1 hour before UV exposure. The treatment response was documented photographically every 6 months. UVA dosimetry. In preliminary dose finding experiments symmetric lesions in one group of patients received a constant irradiation dose of 5 joules/cm 2 on one side and 10 joules/cm 2 on the other side. In another group the treatment was started with 5 joules/era 2 on both sides, whereby one side remained at 5 joules/cm 2 and the other side was gradually increased by 5 joules/cm 2 every other week up to 20 joules/cm2. As a result effective doses were found to range between 10 and 15 joules/cm 2. The irradiation dose (10 joules/cm 2 for skin types II and II1, 15 joules/cmz for skin type IV) was kept constant in each patient throughout the entire treatment period. L a b o r a t o r y tests. Blood was taken for complete blood count, blood chemistry, and liver and renal function tests (SMAC 24) before initiation of oral treatment and 1, 2, 4, 8, and 12 weeks after initiation of oral treatment. During the later treatment period the tests were repeated every 3 months. Controls. For control purposes two symmetric vitiligo areas were selected. One was shielded during UVA exposure, and the other one was irradiated with the therapeutic UVA dose (10 to 15 joules/cm 2) prior to khellin ingestion. The rest of the body received the actual treatment with khellin plus UVA. Control areas were switched to khellin plus UVA whenever a clearly visible difference in the treated areas was noted, RESULTS Serum levels. The 24-hour serum profiles showed that peak levels ranging from 1275 to 2178 ng/ml were reached 2 to 5 hours after drug inges-
9
.lI
,t
,i
" t
" 9
~'
4
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"
6-o
t,
| 9
. 2i~/:
Fig. 3. Phototoxicity test with oral khellin plus UVA (KUVA) versus UVA alone; reading at 24 hours. Note that there is no difference in erythema formation on both sides with exposure doses of 30 to 80 joules/cm 2. tion (Fig. 2). Twenty-four hours after ingestion no substantial drug concentration was measured. As revealed by multiple determinations the values were found to be most constant 21/a hours after khellin administration, Therefore a time interval of 21/2 hours between drug intake and exposure was chosen for therapy. Phototoxicity. Under the conditions described above khellin did not induce phototoxic skin erythema with doses up to 100 j o u l e s / c m 2. UVA
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Fig. 4. Oral khellin plus UVA. Comparison of 5 versus 15 joules/cm2; note equal extent of follicular repigmentation but different intensity of tan. A and B, Four weeks and 13 weeks, respectively, after initiation of treatment.
Fig. 5. Oral khellin plus UVA. Comparison of 5 versus 10 joules/em 2 before (A) and after (B) 18 weeks of treatment. The 10 joules/cm 2 area exhibits incipient repigmentation whereas 'he 5 ictuSes/c:-:,z area s~ows v!rtual'.y no change.
erythema developed 24 hours after exposures to 50 joules/cm ~-or more in both khellin-treated and untreated areas. A comparison of erythema reactions induced by UVA alone and by khellin plus IJVA after systemic or topical administration in both vitiliginous and normal skin did not reveal any perceptible difference (Fig. 3). In contrast to the phototoxic reaction with psoralens, which peaks as late as 72 hours after exposure, there was no increasing erythema reaction with khellin between 24 and 72 hours. Interestingly, khellin plus UVA also did not induce a more intense immediate and/or delayed pigmentation in normal skin than did UVA alone. UYA d o s i m e t r y and controls. Paired comparison studies in symmetric lesions of two different
dosages, namely, 5 and 10 or 5 and 15 joules/cm 2, respectively, showed that equal repigmentation occurred with respect to the number of repigmented follicular spots in both areas but the intensity of the tan was considerably higher in the high-dose area (Fig. 4). Most patients, however, developed follicular repigmentation only in the 10 joules/cm 2 but not in the 5 joules/cm 2 area (Fig. 5). No pigmentation occurred in vitiligo areas shielded during UVA exposure after khellin intake (Fig. 6, A and B). Also areas that received UVA alone in the same doses as with khellin remained unpigmented (Fig. 6, C and D). Areas adjacent to these control sites exhibited a definite response to khellin plus UVA treatment. Therapeutic results. Four to 8 weeks after the
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Khellin-UVA photochemotherapy for vitiligo 697
Fig. 6. Control areas. A, Area shielded (K) during exposures before and (B) after 11 weeks of treatment; repigmentation is seen only in the khellin plus UVA (KUVA) are~. C, Areas treated with UVA alone before and (D) after 11 weeks. Note the increased t ~ in normal skin but absence of follicular repigmentation.
Table II, Treatment results
Number of patients Duration of therapy (x +_ SD; rap) Number of treatments (x • SD) Total irradiation dose (x + SD; joules/era z)
15 5.8 --- 4.5
8 12.5 ~ 4.8
5 21.0 __+ 10.1
57 + 39
111 - 51
157 ___ 44
586 ___ 522
1328 + 619
2290 --_ 1070
initiation of khellin photochemotherapy, beginning repigmentation was heralded by the appearance of pigmented follicular spots in the responding patients. These follicular pigmentations gradually enlarged and coalesced over a period of 8 to 24 months. There was a great interindividual variation in the patients' responses to treatment with regard to the duration of treatment until acceptable reconstitution of skin pigmentation was achieved. Therapy was maintained as long as the repigmentation process continued and was stopped when repigmentation ceased. As has been described for conventional psoralen photochemotherapy, certain skin areas with vitiligo, in particular acral skin, did not respond adequately despite many months of treatment. ~.2,~5
Tables II and III show the results in all 28 patients. Five patients exhibited a cosmetically satisfying result with repigmentation in more than 70% of the original vitiligo areas after a mean treatment dtJration of 21 months, a mean of I57 treatments, and a total UVA dose of 2290 joules/cm z (Fig. 7). Eight patients improved by 30% to 70% after a mean treatment period of 12.5 months. O f this group, two patients who were good responders developed an elevation of liver transaminases after 4 to 6 weeks of treatment and their treatment was discontinued. After normalization of the liver values they were switched to a psoralen protocol and later acquired repigmentation of more than 70% (data not shown). At the time of writing two other responding patients of
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O r t e l et al
Table III. Extent of repigmentation versus number of treatments Number of patients with repigmentation (%)
Number of treatments
Total
<30
<50 50-99 100-199 >200
10 6 11 1 28
9 3 3 0 15
this group were still under treatment after 12 and 15 months of treatment, respectively; the other four had discontinued treatment after 8, 11, 16, and 21 months because they were not satisfied with the result. In the group of 15 patients with less than 30% repigmentation, 10 patients had been treated for less than 10 months (Table III). In five patients treatment was discontinued because of elevated transaminases, in 8 it was discontinued because of poor response, and 2 were still under treatment at the time of writing and showed gradual improvement. Seven patients were switched to a psoralen protocol, but the poor responders also failed to improve with the PUVA regimen (data not shown). Of the three patients treated with topical khellin, one with generalized vitiligo (Fig. 8) was still under treatment after 12 months, showing slow but continuous progression of repigmentation. The other two patients, one with acrofacial and the other with generalized vitiligo, did not improve more than 30% after 12 months of treatment. They also failed to respond to subsequent PUVA treatment (data not shown). Unrelated to the extent o f repigmentation, the khellin-treated patients noted an increased tolerance to natural sunlight in vitiliginous skin after 2 to 3 months, similar to the observation in patients treated with other forms of photochemotherapy.'S.~6 Side effects S h o r t - t e r m side effects. Six (21%) of the patients ingesting khellin reported episodes of mild nausea and two (7%) reported orthostatic problems. These complaints usually occurred within
,,
30-70
1 3 4 0 8
l
>70
0 0 4 1 5
the first 2 weeks of therapy, subsided spontaneously upon continuation of the treatment, and did not necessitate discontinuation of therapy. In seven (25%) of 28 patients under systemic khellin treatment, a mild elevation of liver transaminases (aspartate aminotransferase, AST, SGOT; alanine aminotransferase, AST, SGPT; and gamma glutamyltransferase, GGT), with values of up to three times the normal limit, was observed within the first 2 months of treatment. These laboratory changes occurred unrelated to systemic symptoms such as nausea, and it has to be mentioned that these patients had normal values at the beginning and showed negative hepatitis antibody screening findings. After discontinuation of khellin administration the values returned to normal within 5 to 12 weeks. A second course of khellin in two of these patients again resulted in a similar increase of transaminases after 4 and 8 weeks, respectively. Patients who did not develop an elevation of transaminases within the initial treatment period retained normal values throughout the entire treatment. Long-term side effects. No long-term side effects were'observed with regard to internal organ or skin toxicity. In particular, no skin changes as reported for long-term PUVA treatment with high cumulative UVA doses, such as lentigines, mottling of the skin, and solar degeneration, were noted. DISCUSSION Khellin-UVA photochemotherapy is effective in restoring normal skin color in more than 70% of the originally involved vitiliginous areas in a sub-
Volume 18 Number 4, Part I April 1988
stantial number of patients with vitiligo after 1 to 2 years of continuous therapy. However, as with psoralen photochemotherapy (PUVA), complete repigmentation is never achieved since certain skin areas, such as the distal dorsal surfaces of the hands and feet, tips of the fingers and toes, areas of bony prominences, palms, soles, and nipples, do not respond at all. Since the only other effective treatment option is PUVA, it appears important to compare the two regimens with regard to efficacy, side effects, and safety. In the early reports on the use of psoralens in vitiligo, the assessment of the actual treatment success is difficult because treatment schedules were different and UV radiometry and radiation sources were not well defined. ~s'~7-2~More recent clinical trials 3,5,2t23 provide for better information on the success rate and permit the conclusion that, at least in whites, khellin may be as effective as psoralens provided treatment is performed for a long enough period of time. An evaluation of our patients indicates that a minimum of 100 exposures is required to reach an overall repigmentation of 70%. If one considers only the group of 12 patients who received 100 to more than 200 treatments (Table III), the success rate (that is, more than 70% repigmentation) was at 41%. Controlled studies in generalized vitiligo 3,5'zta3 with PUVA using 8-methoxypsoralen (8-MOP) and/or trimethylpsoralen (TMP) have yielded comparable success rates ranging from roughly 25% to 40%. The most elaborate study of Pathak et aP in 366 patients from India indicates that the most effective treatment consists of a combination of 8-MOP and TMP, and we are currently investigating whether a combination of khellin and psoralens may increase the therapeutic success. With regard to short-term side effects, khellin has the advantage of being nonphototoxic even after exposure doses of up to 100 joules/era 2. Therefore, severe erythema reactions as may occur in PUVA therapy, particularly in fair-skinned whites, can be avoided. Khellin photochemotherapy can be considered safe with natural sunlight, as a UVA source or also as home treatment with artificial UVA, provided the patients are instructed
Khellin-UVA photochemotherapy for vitiligo 699
Fig. 7. Oral khellin plus UVA treatment. Patient with generalized vitiligo showing more than 70% repigmentation. A, C, and E, Before therapy. B, D, and F, After 16 months of treatment.
appropriately and are monitored regularly. Preliminary results in a few patients indicate that khellin plus sunlight may be effective also in a moderate climate. No serious adverse reactions or long-term side effects have been reported at the time when khellin
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Fig. 8. Topical khellin plus UVA treatment. A, Before therapy. B, Follicular repigmentation after 12 weeks of treatment.
was used in much higher doses (up to 300 mg daily) as a long-term treatment for angina pectoris. 7.sa~ In one large-scale clinical study, shorttime side effects were not encountered more frequently in khellin-treated patients compared to a placebo-treated control group.~~ A more serious complication may be the observed elevation of transaminases in about one third of our patients. In these patients treatment was discontinued and the transaminases returned to normal values. As yet there exists no parameter to predict a predisposition for this drug intolerance reaction. All of our patients had normal values prior to treatment and hepatitis screening showed negative findings. The drug doses used in the past for angina pectoris had ranged from 100 to 300 mg per day and khellin has been administered over several years without any report of hepatic toxicity. However, liver transaminases had not been determined at that time. Although this phenomenon might be interpreted as harmless drug-induced "transaminitis," further investigation of this issue is clearly needed. Patients who remained at normal values during the first weeks after the initiation of khellin did not exhibit an elevation throughout the entire treatment period of up to 2 years. At present there exists no information regarding long-term side effects of khellin plus UVA, but all possible long-term side effects that may arise from an interaction with deoxyribonucleic acid (DNA) have to be considered. Khellin has some structural similarity to 8-MOP and also shares some photochemical properties with psoralens, 24-26 Khellin
forms monofunctional adducts and cross-links with DNA 2v with a maximum action spectrum at 360 rim. However, khellin predominantly forms monoadducts, and the induction of cross-links requires fluences of up to i00 times more than needed for the formation of cross-links with 8-MOP. Compared with 8-MOP, khellin is a very weak producer of reactive oxygen species. 28 Phototoxic and genotoxic properties of khellin have been investigated in various experimental systems. Khellin was shown to be 100- to 1000-fold less active compared to 8-methoxypsoralen?9,3~ Monofunctional psoralens are considered to be less mutagenic than cross-linking analogs. 3~ The very low mutagenic activity of khellin in vitro suggests that potential risks of long-term side effects, such as skin carcinoma formation, may be much lower than with conventional psoralen photochemotherapy. The mode of action of all forms of photochemotherapy for vitiligo is still unknown. Neither khellin alone nor UVA alone can induce repigmentation. The lack of repigmentation in the shielded control areas also excludes a systemic action of the treatment. Remelanization appears to be confined to the interaction of both khellin and UVA. Psoralens dramatically enhance the tanning properties of UVA in normal skin. However, khellin plus UVA is not more melanogenic than UVA alone. This indicates that the stimulation of melanogenesis and the induction of repopulation of vitiliginous skin with melanocytes may be mediated by different mechanisms.
Volume 18 Number 4, Part I April 1988 CONCLUSIONS Khellin p h o t o c h e m o t h e r a p y can be recomm e n d e d as a valuable alternative option to conventional P U V A f o r the treatment o f vitiligo in patients w h o do n o t develop an elevation of liver t r a n s a m i n a s e s . B o t h oral and topical khellin phot o c h e m o t h e r a p y m a y be less hazardous with respect to l o n g - t e r m side effects and can be perf o r m e d e a s i l y with sunlight or as h o m e treatment with c o m m e r c i a l l y available U V A sources.
REFERENCES 1. Ortonne JP, Mosher DB, Fitzpatrick TB. Vitiligo and other hypomelanoses of the skin. New York: Plenum, 1983. 2. Fitzpatrick TB, Parrish JA, Pathak MA. Phototherapy of vitiligo. In: Fitzpatrick TB, Pathak MA, Harber LC, Seiji M, Kukita A, eds. Sunlight and man. Tokyo: Tokyo University Press, 1974:783-91. 3. Parrish JA, Fitzpatrick TB, Shea C, Pathak MA. Photochemotherapy of vitiligo. Arch Dermatol 1976; 112:1531-4. 4. E1 Mofty AM. A preliminary clinical report on the treatment of leukoderma with Ammi majus Linn. J R Egypt Med Assoc 1948;31:651-65. 5. Pathak MA, Mosher DB, Fitzpatrick TB, Parrish JA. Safety and therapeutic effectiveness of 8-methoxypsoralen, 4,5',8-trimethylpsoralen, and psora]en in vitiligo. Natl Cancer Inst Monogr 1984;66:165-73. 6. Abdel-Fattah A, Aboul-Enein MN, Wassel GM, E1Menshawi BS. An approach to the treatment of vitiligo by khellin. Dermatologica 1982;165:136-40. 7. Anrep GV, Barsoum GS, Kenawy MR, Misrahy G. Therapeutic uses of khellin. Method of standardisation. Lancet 1947;252:557. 8. Anrep GV, Kenawy MR, Barsoum GS. The coronary vasodilator action of khellin. Am Heart I 1949;37: 531-42. 9. Vidal-Beretervide K, Monti M. Ganglionic blocking action of piperidino-methyl-khellin. Arzneim Forsch 1965; 15:242. I0. Greiner T, Gold H, Catell M, et al. A method for the evaluation of the effects of drugs on cardiac pain in patients with angina of effort: a study ofkhe]lin (visammin). Am J Med 1950;9:143-55. 11. Ayad H. Khellin in angina pectoris. Lancet 1948;1:305. 12. Armbrust CA, Levine SA. The treatment of angina pectoris with a preparation of khellin. Am I Med Sci 1950;220:127. 13. Benson H, MeCallie DP. Angina pectoris and the placebo effect. N Engl J Med 1979;300:1424-9. 14. Henseler T, H6nigsmann H, Wolff K, Christophers E. Oral 8-methoxypsoralen photochemotherapy of psoriasis. The European PUVA Study: a cooperative study among 18 European centres. Lancet 1981; 1:853-7.
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I5. Lemer A.B, Denton CR, Fitzpatrick TB. Clinical experimental studies with 8-methoxypsoralen in vitiligo. J Invest Dermatol 1953;20:299-314. 16. Cormane RH, Siddiqui AH, Westerhof W, Schutgens RBH. Phenylalanine and UVA light for the treatment of vitiligo. Arch Dermatol Res 1985;277:126-30. 17. Arnold HL. Aul3ere Anwendung von 8-Methoxypsoralen bei Vitiligo. Hautarzt 1960;11:324-6. 18. Dawber RPR. Oral trisoralen therapy in vitiligo. Br J Dermatol 1970;83:386-90. 19. London ID. Evaluation of methoxsalen in the treatment of vitiligo. J Invest Dermatol 1959;32:315-7. 20. Bleehen SS. Vitiligo treatment with trimethylpsoralen. Br J Dermatol 1971;84"186-8. 21. Bleehan SS. Treatment of vitiligo with oral psoralens. Clinically Oriented Symposium of the European Society for Dermatological Research [book of abstracts]. Baden, 1985:67-8. 22. Wennersten G, H/igermark 0. Outdoor and office photochemotherapy of vitiligo: a comparison. Photodermatology 1984;1:91-3. 23. Lassus A, Halme K, Eskelinen A, Ranki A, Puska P, Salo O. Treatment of vitiligo with oral methoxsalen and UVA. Photodermatology 1984; 1:170-3. 24. Beaumont PC, Land EJ, Navaramam S, Parsons BJ, Phillips GO. A pulse radiolysis study of the complexing of furoeoumarins with DNA and proteins. Biochim Biophys Acta 1980;608:182-9. 25. Niccolai N, Bovalini L, Martclli P. The mechanisms of interaction between furanochromones and DNA: a heteronuclear Overhauser effect study on the khellinthymidine model system. Biophys Chem 1986;24: 217-20. 26. Rucheton M, Jeanteur P. Studies on amikhellin. I. Intercalative binding to double-stranded DNA. Biochimie 1973;55:1415-20. 27. Cassuto E, Gross N, Bardwell E, Howard-Flanders P. Genetic effects of photoadducts and photocross-links in the DNA of phage lambda exposed to 360 nm light and tri-methylpsoralen or khellin. Bioehim Biophys Aeta 1977;475:589-600. 28. Martelli P, Bovalini L, Ferri S, Franchi GG, Bari M. Active oxygen forms in photoreaction between DNA and furochromones khellin and visnagin. FEBS Lett 1985; 189:255-7. 29. Abeysekera BF, Abramowski Z, Towers GHN. Genotoxicity of the natural furochromones, khellin and vlsnagin and the identification of a khellin-thymine photoadduct. Photochem Photobiol 1983;38 3:311-5. 30. Hitchins VM, Bockstahler LE, Carney PG, et al. Comparison of khellin and 8-MOP as UVA photosensiti= zers for mammalian cells. Photochem Photobiol 1986; 43[suppl]'275. 3I. Averbeck D. Relationship between lesions photoinduced by mono- and bi-functional furocoumarins in DNA and genotoxicity in diploid yeast. Mutation Res 1985;151: 217-33.