3

J. Photochem. Photobiol. B: Biol., 14 (1992) 3-22

Review

The evolution of photochemotherapy with psoralens U-VA (PUVA): 2000 BC to 1992 AD+ Madhukar

A. Pathak

and Thomas

and

B. Fitzpatrick

Department of Dermatology, Harvard Medical School, Masschusetts General Hospitas Boston, MA 02114 (USA) ‘Historic continuity with the past is not a d&y, it is only a necessity.” Oliver Wendell Holmes, Jr. (Received

November

25, 1991; accepted

January

27, 1992)

Abstract The therapeutic uses of naturally occurring psoralens in modern-day medicine (8-methoxypsoralen (8-MOP), 5-methoxypsoralen (S-MOP), 4,5’,8-trimethylpsqialen, and a few other synthetic psoralens) have evolved through five stages of development. (1) In the historical period (2000 BC to 1930 AD), the pigment-stimulating properties of naturally occurring plants containing psoralens were described anecdotally. (2) The second period (1930-1960) dealing with the chemistry of psoralens involved extraction, identification of their structure, synthesis, and the relationship between chemical structure and their photoreactivity and pigment-stimulating properties. The treatment of vitiligo with oral and topical 8-MOP became popular. (3) In the third period (1960-1974), we witnessed a new beginning and the growth of basic science studies and clinical investigations into various biological properties of psoralens including action spectrum studies, mutagenesis and carcinogenesis studies, in vitro and in vivo photoreactivity studies of various psoralens with DNA, RNA, proteins, and pharmacological and toxicological studies in vitiligo patients undergoing long-term therapy for repigmentation. (4) The fourth period (1974-1988) is recognized as the period of photochemotherapy and the development of the science of photomedicine which established the therapeutic effectiveness of psoralens in combination with newly developed UV irradiation systems that emitted high-intensity UVA radiation in the treatment of severe psoriasis, mycosis fungoides, and over 16 other skin diseases. The effectiveness of PWA (psoralen+ WA) was confirmed by well controlled clinical trials in thousands of patients, both in the USA and in European countries. Combination therapy with oral retinoids and PWA contributed to greater effectiveness and long-term safety of psoralen photochemotherapy. (5) In the fifth period (1989 and beyond), psoralens are DOW emerging as photochemoprotective agents against non-melanoma skin canters and as immunologic modifiers in the management of certain patients with disorders of circulating we would like to dedicate this paper to our friend, Professor Giovanni Rodighiero. Inspired by the late Professor Musajo [l] and his study of the chemistry of coumarins and furocoumarins [2], Giovanni has devoted over 35 years of his scientific interest to synthesizing and elucidating the chemistry and photobiological properties of psoralens [3].

loll-1344/92/$5.00

0 1992 - Elsevier Sequoia. All rights reserved

4 T-cells using new techniques of photopheresis. In the final analysis, perhaps the application of pharmacological and therapeutic concepts and principles of using psoralens in combination with UVA has contributed to the development of a new science of photomedicine in which the interaction between basic scientists, photobiologists, and physicians has produced both basic and new clinical knowledge for the care and control of human suffering.

Keywords: History of psoralens, PUVA, photochemotherapy, photopheresis, photochemoprotection.

psoriasis, vitiligo, retinoids,

1. Early history The introduction of photochemotherapy of generalized psoriasis utilizing an orally administered photoactive drug, 8-methoxypsoralen (methoxsalen or 8-MOP) and exposure of the skin to long-wave UV radiation UVA (320-400 nm) in the early 1970s by a team of dermatologists and pharmacologists at Harvard Medical School and Massachusetts General Hospital, in Boston [4], inaugurated the use of photobiological principles applied to the treatment of a heterogeneous group of diseases. This new subspeciality of medicine which has become known asphotomedicine concentrated on the development of methods for the treatment of skin diseases using non-ionizing electromagnetic radiation (UVR and visible radiation) alone or in combination with endogenous or exogenous photoactive chemicals. Because non-ionizing UV radiant energy has such low penetration compared with ionizing radiation, this new technology has been most beneficial in treating diseases of the skin. This new pharmacological concept was termed photochemotherapy, the combined use of electromagnetic energy and a drug, and although first reported in 1974, is in fact thousands of years old, having been used in Egypt and India since 1200-2000 BC [4, 51. Photochemotherapy for the common disfiguring disease, vitiligo, was practiced in the ancient world by physicians and herbalists who used boiled extracts of leaves, seeds, or the roots of certain umbelliferous plants, e.g. Ammi majus Linnaeus in Egypt or the leguminous plant, Psoralea coryl~olia L. in India. These preparations, which were made from seeds obtained from herbal stores, were either applied to the skin or ingested as an “infusion”; the patient then exposed the skin to the intense Egyptian or Indian sunlight. Vitiligo, a color imperfection of the skin, was and still is a major medical problem in India, inasmuch as it is regarded as “white leprosy”. Victims are social outcasts who often cannot marry or get jobs and rarely may even resort to suicide. The use of psoralen, the active,principle of Psoralea coryrifolia (family Legzuninosae) for the repigmentation of vitiliginous skin is carefully recorded by the Hindus of India in the ancient Ayzuvedic system of Medicine [5]. In India’s sacred book, Atharua-Veda (2000 BC to 1400 BC) and in many other ancient medical writings which date back to 200 AD, the treatment of leukoderma (vitiligo) with the plants vasuchika or bavachee (Psoralia cotylifolia) is also described, and the need for exposure to solar radiation in the form of sun-worshipping prayer is stressed. This plant has now been shown to contain psoralen and several other furocoumarins [5-71. In the Ayurvedic system of Indian medicine, particularly in “Charaka Samhita”, both topical and systemic use of figs is also recommended in conjunction with sun exposure. Figs are now known to contain psoralen and glucoside of psoralen [6, 71. Another plant, known as Ammi Majus Linnaeus, which grows throughout the Nile Valley as a weed, has been employed for centuries, since before the birth of Christ, as a “cure” for leukodenna. Ibn El Bitar, in his famous book of the thirteenth century, Mafiadat Al Adwiya, described

5

the treatment of baras (vitiligo) with the seed Aatrillal (Ammi Muju) and sunlight [5]. He stated that Aatrillal (also referred to as Ammi by Galen) was commonly employed as a remedy for leukoderma by Ben Shoeib, a Berberian tribe dwelling in the northwestern African desert. Aatrillal, a yellowish-brown powder, is still sold by Egyptian native herbalists as a remedy for white spots of leukoderma.

2. Modem history (Table 1) It was Professor Abdel Monem El Mofty of the Department of Dermatology, Cairo University Medical School, Cairo, Egypt, who, in the early 194Os, first used crystalline methoxsalen (8-MOP) followed by exposure to sunlight in the treatment of vitiligo; it had just been isolated from Ammi mu&s L. by the Egyptian pharmacologist Fahmy and his coworkers [5]. It was not until 27 years later, in 1974, that orally administered 8-MOP was first used in the treatment of psoriasis in combination with a new high-intensity, artificial source of long-wave W radiation (320-400 nm), which is the action spectrum for psoralens [4-8]. The new high-intensity WA light source and exposure system was developed by Sylvania engineers in the USA in collaboration with investigators on the staff of the Dermatology Department of Harvard Medical School. This new WA source was initially used in combination with either oral 8MOP or 4,5’,8_trimethylpsoralen for the treatment of vitiligo by the Harvard dermatologists and pharmacologists. Soon Harvard scientists [4] discovered that 8-MOP was also effective in the treatment of psoriasis by using the paired-comparison technique. They exposed one-half of the posterior trunk to a given dose of WA and then administered oral 8-MOP, and two hours later they exposed the other half of the posterior trunk to the same dose of WA. Repeated exposures (18-20 treatments) to WA after oral ingestion of 8-MOP (0.6 mg/kg) in this manner resulted in the disappearance of the psoriatic lesions on the part treated with PUVA and no change in the lesions treated with WA alone. Using the same protocol, Wolff et al. [8] immediately confirmed these results in Vienna, Austria, in 1975. During the years from 1974 to 1982, two teams comprising a dozen or more physician-investigators collaborated to develop PWA photochemotherapy at Harvard and in Austria. The Harvard team included Thomas B. Fitzpatrick, Barbara A. Gilchrest, Ernest0 Gonzalez, John Melski, Khosrow Momtaz, Warwick L. Morison, John A. Parrish, Madhu A. Pathak, Robert Stern, and Lewis Tanenbaum [4, 91. The Austrian group included W. Brenner, P. Fritsch, F. Gschnait, H. Honigsmann, E. Jaschke, K. Konrad, and K. Wolff. Professor Franz Greiter of Vienna, a physiologist, provided support for the development of PWA photochemotherapy in Vienna beginning in 1975 [8]. Three multi-center clinical trials were conducted between 1975 and 1980 with controlled protocols and the accumulation of large databases involving 2000 patients in 25 centers in the USA [9, lo], and nearly 3000 patients in 17 major European centers [8, 111. This comprehensive clinical experience acquired in Europe [ll, 121 and the USA [13] has confirmed the high degree of efficacy of PWA therapy for the treatment of psoriasis and its short-term safety when administered by a standardized method and with a suitable WA irradiator equipped with an appropriate radiometer. In this regard, three monographs on the chemistry, pharmacology, toxicity, and therapeutic effectiveness of psoralens have been published. These publications provide an indepth account on the historical aspects of psoralens between 1959 and 1985 and give a 25-year account of clinical experience [14-161.

6

Thus, within the past 18 years, the effectiveness of oral PUVA photochemotherapy has been widely documented in major University Centers of dermatology in the world and has profoundly influenced therapeutic concepts in the evolving science of photomedicine in dermatology. This short era of PWA has stimulated much interest and basic research and has opened up new avenues providing treatment for a diverse number of disorders (Table 1). Although PUVA has become a standard form of therapy and clears about 90% of patients with psoriasis, by 1975 the dermatologists had recognized the long-term risk of developing skin cancer and photoaging changes in the skin of patients receiving PWA therapy for long periods of time [17]. In 1978, a decisive advance and advantage had been achieved by the combination of PWA with oral retinoids in the treatment of disorders of psoriasis [18]. The introduction of synthetic retinoids in the treatment of skin diseases had a significant impact on the treatment of pustular and erythrodermic psoriasis, and this has been confirmed by other investigators [16, l&20], particularly with the aim of reducing the long-term hazards of PWA therapy. Though of limited value if retinoids are used as monotherapy, the combined use of etretinate, an aromatic retinoid, with PWA was found to accelerate the clearance of psoriasis. The therapeutic efficacy of PWA treatment was dramatically potentiated when accompanied by etretinate administration (dose 1.0 mg/kg/day) 10 days prior to initiation of PWA therapy in the clearing phase and is continued throughout the clearing phase treatment. Both the treatment time and the number of treatments necessary for complete clearance were found to TABLE

1

Diseases

responsive

to oral psoralen

photochemotherapy

(PUVA)

PUVA in therapy of disease

PUVA in prevention

Vitiligo Psoriasis Palmoplantar pustolosis Mycosis fungoides (stages IA and IB) Atopic dermatitis Generalized lichen planus Urticaria pigmentosa (cutaneous mastocytosis) Alopecia areata Pityriasis lichenoides’ Lymphomatoid papulosis’ Pityriasis rubra pilaris Generalized granuloma anulare Prurigo nodularis Scleromyxedema Cutaneous graft versus host disease Transient acantholytic dermatitis

Polymorphous light eruption Hydroa vacciniforme+ Solar urticaria’ Persistant light reaction+ Chronic actinic dermatitis’ Actinic reticuloid’ Erythropoietic protoporphyria’ Sun-induced

of disease

non-melanoma

skin cancer’?

Although all of the dermatoses listed have been reported to respond to PUVA therapy, not all represent a true indication for this treatment. In few diseases (‘), the experience is limited to a small number of patients.

7

be reduced by one-third, and the UVA energy required to clear psoriasis was also reduced by 56%. The main advantages of using etretinate in combination with PWA were (a) an acceleration of the response rate of psoriatic lesions in the clearing phase, (b) a reduction in the number of individual treatments to achieve complete clearing, (c) an approximate 50% reduction in the total cumulative dose of WA (in joules per square centimeter) necessary to achieve clearing of psoriasis. The most impressive results were achieved in “poor PWA responders” who could not be cleared completely by PWA alone and were controlled by this regimen. This combination therapy with retinoids, known as chemophotochemotherapy, has now been adopted for the control of psoriasis in many European and American centers [13, 161. 3. Stages in the development

of psoralen photochemotherapy

Thus, the therapeutic uses of psoralens in modern day medicine (e.g. &methoxypsoralen (g-MOP), 5-methoxypsoralen (5-MOP), 4,5’,8-trimethylpsoralen (TMP) and, to a limited extent, other synthetic psoralens), have evolved through five stages of historical development. First, there was the historical period (2000 BC to 1930 AD) in which the pigment-stimulating properties of naturally occurring plants containing psoralens were described anecdotally [5]. The second period dealing with the chemistry of psoralens (1930-1960) involved the extraction of psoralens from natural sources, their structural identification, synthesis of various psoralens, and the relationship between chemical structure and their photoreactivity and pigment-stimulating properties in vitiligo [l, 3, 6, 7, 14-16, 21-351. In this period, chemists isolated, identified, and synthesized psoralens, and physicians recognized the photosensitizing and pigmentstimulating properties of g-MOP, 5-MOP, and other psoralens in vitiligo patients who had lost their normal skin color. In this period (1947-1953), psoralens were originally developed in Egypt and later used in other countries of the world for the treatment of vitiligo [24, 26-291. The first drug to be used was 8-MOP. This therapy was not predictably effective because the basic and clinical research essential for reproducibility of the results and safety of the drug were not systematically carried out according to pharmacological principles of absorption, bioavailability, and the interrelationships between the drug dose and light (UVA) dose for therapeutic effectiveness. In this period, the structure and activity relationship of various psoralens and isopsoralens and their skin photosensitizing properties were also examined and established. The third period (1960-1974) witnessed basic science studies and clinical investigations into various properties of psoralens. These included photosensitizing action spectrum studies, mutagenic studies, carcinogenic studies in mice, in vitro and in vivo photoreactivity studies of psoralens with DNA, RNA and proteins, and several pharmacological and toxicological studies in patients with vitiligo undergoing long-term therapy for repigmentation [24, 34-411. The fourth period (1974-1988) was recognized as the period of photochemotherapy and photomedicine that established the therapeutic effectiveness of psoralens in combination with WA in the treatment of psoriasis and various other skin diseases including vitiligo [4,8-13,18-20,42-571. In this period, it was demonstrated with well controlled double-blind studies and multi-center studies both in the USA and Europe, that oral 8-MOP in combination with newly developed artificial irradiators equipped with fluorescent light tubes that emitted high-intensity WA or long-wave W radiation, were highly effective in the control of psoriasis, mycosis fungoides, and several other skin diseases (Table 1). Combination therapy with oral retinoids and PWA contributed to greater effectiveness and long-term safety of psoralen photochemotherapy.

8

The use of UVA radiation and visible radiation in conjunction with photosensitizing drugs has opened up new therapeutic possibilities in controlling and curing skin disorders [13, 15, 16, 42-571. In this regard, the use of hematoporphyrin derivatives (HPD or dihematoporphyrin ether or photofrin II) as a photodynamic or photosensitizing agent for the treatment of neoplastic lesions has seen tremendous growth during the past 10 years as an effective alternative treatment modality for a variety of localized tumors including those of the skin, lung, breast, bladder, and brain [58]. The development of the science of photomedicine has also produced increased interactions among clinicians, physicists, chemists, biologists, and biomedical engineers. In the fifth period (1989 and beyond), psoralens are emerging as photochemoprotective agents [59-63] against the development of non-melanoma skin cancers and as immunological modifiers regulated by photopheresis, a treatment process that appears to be efficacious in the management of certain patients with disorders of circulating T-cells [64-66]. In the first concept recognized asphotochemoprotection, there are two developments worth mentioning. (1) The W-protective mechanism, involving the increased production of melanin pigmentation and increased thickness of the epidermis can be promoted by the action of oral PUVA photochemotherapy in normal white-skinned individuals of skin types I, II, and possibly III who sunburn easily and tan poorly or minimally and are at great risk of developing sun-induced basal and squamous cell carcinomas, [59, 601. With the availability of 5-MOP which is less phototoxic than g-MOP, it is possible to obtain melanogenic doses of drug-sunlight combinations without the development of phototoxic painful erythema response. This permits practical use of oral 5-MOP and exposure to sunlight to induce PUVA-tan that is protective against the noxious effects of UVB exposure and thus can minimize the risk of non-melanoma skin cancer. (2) The second development related to the use of psoralen compounds in combination with long-wavelength UV irradiation for the decontamination of blood products in transfusion-associatedviral infections shows promising progress in inactivating viruses [61-631. Selective inactivation of infectious agents and leucocytes in cellular transfusion products, with adequate preservation of red cell and platelet function, is now feasible. These improvements should facilitate the introduction of psoralen-mediated photochemical decontamination technology in the ultimate selection of platelet suspension media which allow maximal viral inactivation (e.g. hepatitis virus, human immuno-deficiency virus (HIV)). The second concept involving the use of extracorporeal photochemotherapy (photopheresis) for the treatment of erythrodermic cutaneous T-cell lymphoma has been found to be useful as an immune response modifier with potentially broad clinical applications in such diseases as progressive systemic sclerosis (sclerodetma), pemphigus vulgaris, AIDS-related complex, and acute Graft versus Host disease [64-66]. A novel therapeutic modality called “extracorporeal photochemotherapy” for the management of disorders caused by abnormal lymphocytes, in hematogeneously disseminated cutaneous T-cell lymphoma (CTCL), was reported by Edelson et al. in 1987 [64]. This involved the use of psoralens and UVA in an extracorporeal flow system referred to and it represents a more recent adaptation of PUVA therapy for as “photopheresis”, the management of disorders caused by abnormal T-lymphocytes. The irradiated blood in the extracorporeal photopheresis system, after reconstitution, is retransfused to the patient, leading to an apparent immunologic reaction against residual CTCL cells [64-66]. These studies reveal that g-MOP + UVA-treated pathogenic T-cells could efficiently vaccinate against themselves and inhibit subsequent induction of the disease

WI*

9

4. Conclusions

(Table 2)

The development of oral PWA photochemotherapy has evolved from the efforts of several dozen ,clinician-investigators in the USA and Europe who conducted tedious clinical trials in over 7500 patients. This database is the standard of reference for all claims of efficacy and acute toxicity treatment of humans with oral g-MOP and WA. Therapy with PWA is effective in an increasing number of cutaneous disorders including a neoplasm that arises in the skin, i.e. T-cell lymphoma or mycosis fungoides. In some disorders (e.g. vitiligo), PWA is the only effective treatment available; in others it is a reasonable option, and in others it is safer than corticosteroids. For repigmentation in patients with vitiligo [50], clearing of psoriasis [8, 131 of the palms and soles (with or without etretinate) and vesicular dermatitis (“dyshidrotic”) eczema [52] of the hands and feet, PWA is safer than systemic corticosteroids; for actinic reticuloid [53], PWA is an effective treatment equal in efficacy to azathioprine. Clinical experience with carefully controlled studies indicates that PWA is also good for treating disabling psoriasis [8, 10, 12,133 compared with UVB or methotrexate, polymorphous light eruptions [54], mycosis fungoides [55] in its early stages, compared with topical nitrogen mustard, and solar urticaria in preference to WA alone. These controlled clinical studies have also revealed that PWA is an alternative therapy to corticosteroids in generalized plaque or papular psoriasis [8] because topical steroids are contraindicated in generalized psoriasis, chronic atopic eczematous dermatitis [56] rather than prolonged topical c&ticosteroids, and pustular psoriasis (von Zumbusch) as compared with systemic corticosteroids or etretinate [57]. After 18 years of experience in thousands of patients with psoriasis and 18 other skin disorders, the carcinogenic risk of PWA is now generally regarded as low with the following exceptions in the following types of patients: (1) previous history of treatment with ionizing radiation, topical nitrogen mustards, or inorganic trivalent arsenic; (2) patients with severe refractory psoriasis who may require continuous treatment for many years. The limits of radiation dosage have not yet been established. Therefore, combination treatments to keep the cumulative radiation to a minimum are desirable (e.g. retinoids + PWA). TABLE 2 Treatment responses with oral PWA

photochemotherapy

Type of disease

Number of treatments required

Patient response

Psoriasis Vitiligo

12-40 100-200 20-60 10-40

Clearing in 90%, maintenance therapy required in 30% Excellent improvement on the head and neck in over 70%, incomplete responses in 30% Prolonged clearing in early stages of disease Improvement in over 90%

20-60

Improvement in over 90%

20-60

Initial clearing in all 15, aggressive therapy and maintenance required

Mycosis fungoides Polymorphous light eruption Palmar, plantar vesicular dermatitis Eczema

10

Acknowledgment This work was supported by PHS Grant ROl-CA-05003-32, awarded by the National Cancer Institute, US Department of Health and Human Services, Bethesda, MD. References

4 5 6 7 8 9 10 11

12 13

14 15

16 17

18

19 20 21

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22 M. A. Pathak and T. B. Fitzpatrick, Relationship of molecular configuration to the activity of furocoumarins which increases the cutaneous responses following longwave ultraviolet radiation, J. Invest. Dermatol., 32 (1959) 255-262. 23 M. A. Pathak, L. R. Worden and K. D. Kaufman, Effect of structural alterations on the photosensitizing potency of furocoumarins: psoralens and related compounds, .Z. Invest. Derrnatol., 32 (1959) 509-518; 48 (1967) 103-118. 24 M. A. Pathak, D. M. Kramer and T. B. Fitzpatrick, Photobiology and photochemistry of furocoumarins. In M. A. Pathak, L. C. Harber, M. Seiji and A. Kukita (eds.), Sunlight and Man, University of Tokyo Press, Tokyo, 1974, pp. 335-365. 25 H. Kuske, Experimentelle untersuchungen zur photosensiliaierung der haut durch pflanzliche wirkstoffe. Lichtensensibilisierung durch furocoumarine als ursache verschiedener phytogener dermatosen, Arch. Dermatol. Syphilol., 178 (1938) 112-123. 26 I. R. Fahmy and H. Abu-Shady, Ammi majus Lmn: pharmacological study and isolation of a crystalline constituent, ammoidin, Q. J. Pharna. Pharmacol., 20 (1947) 281-291. 27 A. M. El Mofty, A preliminary clinical report on the treatment of leukoderma with Ammi majus Linn, J. R. Egypt Med. Assoc., 31 (1948) 651-665. 28 A. B. Lemer, C. R. Denton and T. B. Fitzpatrick, Clinical and experimental studies with 8-methoxypsoralen in vitiligo, J. Invest. Dermatol., 20 (1953) 299-314. 29 T. B. Fitzpatrick, C. E. Hopkins, D. D. Blickenstaff and S. Swift, Augmented pigmentation and other responses of normal human skin to solar radiation following oral administration of 8-methoxypsoralen, Z. Invest. Dermatol., 25 (1955) 187-190. 30 E. L. Oginsky, G. S. Green, D. G. Griffith and W. L. Fowlks, Lethal photosensitization of bacteria with 8-methoxypsoralen to long-wave ultraviolet radiation, .Z. Bacterial., 78 (1959) 821-833. 31 A. C. Griffin, Methoxsalen in ultraviolet carcinogenesis in the mouse, .Z. Invest. Dermatol., 32 (1959) 367-372. 32 0. C. Stegmaier, The use of methoxsalen in sun tanning, J. Znvest. Dennatol., 32 (1959) 345-349. 33 J. D. Imbrie,F. Daniels, L. Bergeron, C. E. Hopkins and T. B. Fitzpatrick, Increased erythema threshold six weeks after a single exposure to sunlight plus oral methoxsalen, Z. Invest. . Dennatol., 32 (1959) 331-337. 34 H. W. Buck, I. A. Magnus and A. D. Porter, The action spectrum of 8-methoxypsoralen for erythema in human skin, Br. J. Dennatol., 72 (1960) 249-255. 35 M. A. Pathak, Mechanism of psoralen photosensitization and in vivo biological action spectrum of 8-methoxypsoralen, J. Invest. DermutoL, 37 (1961) 397-406. 36 L. Musajo, G. Rodighiero and F. Dall’Acqua, Evidences of a photoreaction of the photosensitizing furocoumarins with DNA and with pyrimidine nucleosides and nucleotides, Erperientia, 21 (1965) 24-25. 37 L. Musajo, P. Visentini, F. Baccichetti and M. A. Razzi, Photoinactivation of Ehrlich ascites tumor cells obtained with skin photosensitizing furocoumarins, metier&z, 23 (1967) 335-336. 38 F. Dall’Acqua, S. Marciani and G. Rodighiero, The action spectrum of xanthotoxin and bergapten for the photoreaction with native DNA, Z. Naturforch. B, 24 (1969) 667671. 39 M. A. Pathak and D. M. Kramer, Photosensitization of skin in vivo by furocoumarins (psoralens), Biochim. Biophys. Acta, 195 (1969) 197-206. 40 R. A. Cole, Light-induced cross-linking of DNA in the presence of a furocoumarin (psoralen): studies with a phage lambda, Escherichia coZi and mouse leukemia cells, Biochim. Biophys. Acta, 217 (1970) 30-39. 41 F. Dall’Acqua, S. Marciani and G. Rodighiero, Interstrand cross-linkages occurring in the photoreactions between psoralen and DNA, FEBS Len., 9 (1970) 121. 42 S. A. Mortzawi, Meladinine mit UVA bei Vitiligo, Psoriasis, Parapsoriasis und Acne Vulgaris, Derm. Monatsschr., 158 (1972) 908-909. 43 H. Tronnier and D. Schule, First results of therapy with longwave UV after photosensitization of skin, paper presented at the Sixth Znt. Congress of Photobiology, Bochum, 1972.

12 44 F. Bordin, F. Baccichetti and L. Musajo, Inhibition of nucleic acid synthesis in Ehrlich ascites tumor cells by irradiation in vitro in the presence of skin-photosensitizing furocoumarins, Experientia, 28 (1972) 148. 45 R. E. Levin and J. A. Parrish, Phototherapy of vitiligo, Light. Des. AppZ., 5 (1975) 3543. 46 B. A. Gilchrest, J. A. Parrish, L. Tanenbaum, H. A. Haynes and T. B. Fitzpatrick, Oral methoxsalen photochemotherapy of mycosis fungoides, Cuncer, 38 (1976) 683-689. 47 W. B. Reed, G. E. Sugerman and R. A. Mathis, DeSanctis-Cacchione syndrome: a case report with autopsy findings, Arch. Dermatol., 113 (1977) 1561-1563. 48 L. Dubertret, D. Averbeck, F. Zajdela, E. Bisagni and E. Moustacchi, Photochemotherapy (PUVA) of psoriasis using 3carbethoxypsoralen, a noncarcinogenic compound in mice, Br. J. Dermatol., 101 (1978) 379-389. 49 K. Wolff, PUVA 1979, Klinik und Praxis. In 0. Braun-Falco and H. H. Wolff (eds.), Forschritte der Praktischen Dermatologie und Veneralogie, Vol. 9, Springer, Heidelberg, 1979, pp. 129-138. 50 D. B. Mosher, M. A. Pathak and T. B. Fitzpatrick, Vitiligo: etiology, pathogenesis and treatment. In T. B. Fitzpatrick, A. Z. Eisen, K. Wolff, I. M. Freedberg and K. F. Austen (eds.), Update Dermatology in General Medicine, McGraw-Hill, New York, 1983, pp. 205225. 51 W. L. Morison, J. A. Parrish and T. B. Fitzpatrick, Oral methoxsalen photochemotherapy of recalcitrant dermatoses of the palms and soles, Br. J. Dermatol., 99 (1978) 293-302. 52 M. J. Levine, J. A. Parrish and T. B. Fitzpatrick, Oral methoxsalen photochemotherapy (PUVA) of dyshidrotic eczema, Acta Derm. Venereal. (Stockh.), 61 (1981) 570-571. 53 W. L. Morison, H. A. White, E. Gonzalez et al., Oral methoxsalen phtoochemotherapy of uncommon photodermatoses, Actu Denn. Venereal. (Stockh.), 59 (1979) 366-368. 54 J. A. Parrish, M. J. Levine, W. L. Morison et al., Comparison of PUVA 8-methoxypsoralen plus UV light and beta-carotene in the treatment of polymorphous light eruption, Br. J. Dermatol., 100 (1979) 187-192. 55 N. J. Lowe, D. J. Cripps, P. A. Dutton et aZ., Photochemotherapy for mycosis fungoides: a clinical and histological study, Arch. Dermatol., 115 (1979) 50-53. 56 W. L. Morison, J. A. Parrish and T. B. Fitzpatrick, Oral psoralen photochemotherapy of atopic eczema, Jt Invest. Dennatol., 67 (1976) 561. 57 H. Honigsmann, F. Gschnait, K. Konrad et al, Photochemotherapy for pustular psoriasis (von Zumbusch), Br. I: DermatoZ., 97 (1977) 119-126. 58 C. J. Gomer (ed.), Photodynamic therapy, Photochem. Photobioi., 46(5) (1987) l-953. 59 T. B. Fitzpatrick, The psoralen story: photochemotherapy and photochemoprotection. In T. B. Fitzpatrick, P. Forlot, M. A. Pathak and F. Urbach (eds.), Psoralens: Past, Present, and Future of Photochemoprotection and Other Biological Activities, John Libbey Eurotext, Paris, 1989, pp. 5-10. 60 J. D. Imbrie, F. Daniels, Jr., L. Bergeron, C. L. Hopkins and T. B. Fitzpatrick, Increased erythema threshold six weeks after a single exposure to sunlight plus oral methoxsalen, J. Invest. Dermatol., 32 (1959) 331-337. 61 L. Corash and C. V. Hanson, Photoinactivation of viruses and cells for medical applications, Phochem. PhotobioL, 53(S) (1991) 39. 62 G. P. Wisehahn, P. A. Moore, L. Lin and L. Corash, The use of psoralen compounds and longwavelength ultraviolet radiation for the decontamination of blood products, Photochem Photobiol., 53(S) (1991) 40. 63 G. Moroff, S. Wagner, L. Benade and R. Y. Dodd, Inactivation of viruses in platelet concentrates using aminomethyltrimethyl psoralen and ultraviolet A, Photochem. Photobiol., 53(S) (1991) 40. 64 R. Edelson, C. Berger, F. Gasparro et al., Treatment of cutaneous T-cell lymphoma by extracorporeal photochemotherapy, New Engl. J. Med., 316 (1987) 297-303. 65 S. Armus, B. Keyes, C. Cahill, C. Berger et aI., Photopheresis for the treatment of cutaneous T-cell lymphoma, J. Am. Acad. Dermatol., 23 (1990) 898-902. 66 R. L. Edelson, Photopheresis: present and future aspects, 1. Photochem. PhotobioL B: Biol., 10 (1991) 165-171.

13 study of the photochemical 67 T. Cech, M. A. Pathak and R. K. Biswas, An electron-microscopic cross-linking of DNA in guinea pig epidermis by psoralen derivatives, Biochim. Biophys. Acta, 562 (1979) 342-360. 68 M. P. Mullen, M. A. Pathak, J. D. West, T. J. Harriest and F. Dall’Acqua, Carcinogenic effects of monofunctional and bifunctional furocoumarins. In M. A. Pathak and J. K. Dunnick (eds.), Photobiologic, Toxicologic and Pharmacologic Aspects of Psorakms, National Cancer Institute Monograph 66, publication 89-2692 (US Department of Health and Human Services, National Institutes of Health, National Cancer Institute, Bethesda, MD), 1984, pp. 205-210. 69 P. Fritsch, H. Honigsmann, E. Jaschke and K. Wolff, Photochemotherapie bei Psoriasis: Steigerung der Wirksamkeit durch ein orales aromatisches Retinoid. Klinische Erfahrungen bei 134 Patienten, Dtsch. Med. Wochenschr., 103 (1978) 1731-1736. 70 C. E. Orfanos, H. Pullman, W. Sterry and M. Kanzing, Retinoid-PUVA (RePUVA): Septemische kombenations-bhandburg bei Psoriasis, Hautarzt, 53 (1978) 494-545. 71 H. H. Roenigk, Jr., and S. M. Garrett, Combination therapy for psoriasis. In H. H. Roenigk, Jr., and H. M. Maibach (eds.), Psoriasis, Marcel Decker, New York, 1991, pp. 847-853, 2nd edn. 72 H. Honigsmann, E. Jaschke, V. Nitsche, W. Brenner, W. Rauschmeier and K. Wolff, Serum levels of 8-methoxypsoralen in two different drug preparations. Correlation with photosensitivity and UVA dose requirements for photochemotherapy, J. Invest. Dermatol., 79 (1982) 233-236. 73 P. C. Levins, R. W. Gange, T. K. Momtaz, J. A. Parrish and T. B. Fitzpatrick, A new liquid formulation of 8-methoxypsoralen: bioavailability and the effect of diet, J. Invest. Dermatol., 82 (1984) 185-187. 74 N. J. Lowe, F. Urbach, P. Bailin and D. P. Weingarten, Comparative efficacy of two dosage forms of oral methoxsalen in psoralens plus ultraviolet-A therapy of psoriasis, J. Am. Acad. Dermatol., 16 (1987) 994-998. 75 A. R. Young, C. S. Potten, C. A. Chadwick, G. M. Murphy, J. L. M. Hawk and A. J. Cohen, Photoprotection and 5-MOP photochemoprotection from UVR-induced DNA damage in humans: the role of skin type, J. Invest. Dermatol., 97 (1991) 942-948. 76 H. Honigsmann, E. Jaschke, F. Gschnait, W. Brenner, P. Fritsch and K. Wolff, 5-Methoxypsoralen (bergapten) in photochemotherapy of psoriasis, Br. J. Dermatol, 101 (1979) 369-378. (bergapten) 77 A. Tanew, 0. Bernard, K. Rappersberger and H. Honigsmann, 5-Methoxypsoralen for photochemotherapy, J. Am. Acad. Dermatol., D?(2) (1988) 333-338. 78 B. Berreti, C. Grupper, D. Bermejo, D. Borenstein, A. Charpentier, Y. Edelson, D. ThiolyBensoussan and R. Thriller, PUVA 5-MOP and PUVA 5-MOP + phenylalanine in the treatment of vitiligo study of 125 patients: preliminary results. In T. B. Fitzpatrick, P. Forlot, M. A. Pathak and F. Urbach (eds.), Psoralens: Past, Present, and Future of Photochemoprotection and Other Biological Activities, John Libbey Eurotext, Paris, 1989, pp. 103-108. (bergapten or 79 M. A. Pathak, T. B. Fitzpatrick and D. B. Mosher, Oral 5-methoxypsoralen 5-MOP) in vitiligo, US FDA Investigational ZND 30428, 1989-1991 (studies in progress). 80 T. B. Fitzpatrick (Boston, MA), M. A. Pathak (Boston, MA), N. J.’ Lowe (Los Angeles, CA), R. D. Baughman (Hanover, NH) and J. D. Bernhard (Worcester, MA), Evaluation of oral 5-methoxypsoralen and UVA in the treatment of psoriasis patients, US FDA Investigational IND 32,213, 1989-1991 (studies in progress).

Appendix: Stepping stones in the development of psoralen photochemotherapy We selected a number of the research contributions that form the basis for PUVA photochemotherapy. These facts, developed by both clinical and basic science investigators, are presented in summarized form below in chronological sequence with references provided. The story of the development of PWA photochemotherapy begins with the first identification in 1938 of psoralens as the basis for skin photosensitization induced by

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certain plants [25] and subsequently by the isolation of three psoralens from plants in 1947 [26]. A year later, the first clinical use of &MOP as an oral photoactive chemical for the treatment of vitiligo was made [27]. Eight years later, dose-response studies were done that demonstrated photosensitization in humans by orally administered psoralens followed by exposure to sunlight [28,29]. In 1959, it was clear that psoralens were not photodynamic agents because unlike the action of dyes, such as rose bengal and acridine, the action of psoralen did not require molecular oxygen [30]. At this time, psoralens were shown to be activated by WA [34,35]. The lethal photosensitization of bacteria by psoralens and WA was believed to be based on the capacity of psoralens to bind to DNA in the presence of UVA [30, 39-411. Linear psoralens can form crosslinks (bifunctional adducts) with two strands of DNA, whereas other non-linear psoralens (isopsoralens) form only monofunctional adducts. Clinical studies of the action of psoralens plus artificial UVA on diseases other than vitiligo were begun when 8-MOP, applied topically to the skin, was first used in 1972 to induce remission of plaques of psoriasis [42, 431. Two years later, it was reported that oral S-MOP in combination with newly developed, high-intensity UVA irradiators induced a remission in 20 patients with psoriasis [4]. This combination of drug and photons was termed “photochemotherapy” (specifically, “PWA photochemotherapy”). Several thousand patients with psoriasis were then treated in multi-center clinical trials in the USA and Europe from 1975 to 1979. These clinical trials conducted in university centers provided a large database from which evolved a treatment regiment for oral PWA photochemotherapy. With careful monitoring of the initial 1300 patients, investigators detected a small incidence of carcinoma (2.5%), first reported in 1979. Without a control group of patients with psoriasis who received treatment other than PUVA, they could not prove the photocarcinogenicity of oral PUVA photochemotherapy in humans. Nevertheless, the reversal of the ratio of basal cell carcinoma to squamous cell carcinoma in the population of patients treated with PWA was highly suggestive that this therapy was photocarcinogenic [17]. The selected studies are listed below. (1) Photosensitization of skin by plants as related to the presence of furocoumarins was first described by Kuske [25] in 1938. He identified natural furocoumarins in plants as photosensitizers and isolated bergapten (5-MOP) from the oil of bergamot. This Swiss dermatologist’s pioneer study was probably the first indication that furocoumarins were photoactive agents. (2) In 1947, Fahmy and his student, Abu-Shady, reported [26] the isolation of 8-MOP from Ammi majus L. The Egyptian investigators prepared an alcoholic extract of powdered fruit of Ammi majus L. and gave it in capsule form to patients who were then exposed to Egyptian sunlight for 30 min (approximately 6 J WA cm-‘). Patients developed gastrointestinal symptoms, but those who could tolerate the extract showed repigmentation of vitiligo macules. Later, crystalline &MOP was isolated from this plant, and a dose of 50 mg was given to patients (Fahmy and Abu-Shady obtained their seeds at a herb store in the bazaar in Cairo. This store is still in existence, and one can obtain the seeds of Ammi majus L. complete with an instruction sheet for the preparation of an infusion). This was then ingested and the patient exposed his/ her skin to the sun. This treatment for vitiligo was among the first using psoralen until the mid-1970s. (3) El Mofty [27], a leading Egyptian dermatologist, pioneered the treatment of vitiligo with a crystalline compound, g-MOP. He reported his findings on the effectiveness of (i) topical S-MOP plus exposure to sunlight, (ii) an oral dose of 40-50 mg g-MOP plus exposure to sunlight, and (iii) a combination of topical and oral treatments in

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the repigmentation of vitiliginous macules. Although El Mofty had no controls, his study has some validity because patients with vitiligo rarely spontaneously repigment; furthermore, exposure to sunlight alone is ineffective, as El Mofty detected. (4) Clinical and experimental studies were performed by Lerner et al. [28] who also used 8-MOP for the treatment of vitiligo. Dose-response studies were done that demonstrated photosensitization of skin in humans by orally administered psoralens followed by exposure to sunlight [28, 291. These investigators reported that a dose of 400-600 mg g-MOP was lethal to 50% of the animals treated. In the uncontrolled clinical study, three albino patients given 30 mg &MOP daily with random exposures to sunlight gave testimonial evidence of increased tolerance to sunlight. That the minimal erythema dose (MED) increased in the three patients suggested a decrease in sensitivity to sun, even though they did not tan. This report of the first use of oral psoralens in the USA confirmed the clinical efficacy in the treatment of vitiligo and the safety of 8-MOP in man over a period of several months. However, whether albinos develop increased tolerance when psoralens and exposure to the sun are used has not been established. (5) Fitzpatrick et al. [29] conducted dose-response studies in 1955 in which 63 volunteers were randomized in double-blind and cross-over design trials in Oregon, Idaho, and Arizona. Oral administration of 50 mg S-MOP before exposure to graduated amounts of sunlight demonstrated augmented cutaneous responses (erythema, edema) 44 h after exposure and markedly increased tanning in 1 week. After oral ingestion of 75 mg followed by hourly exposures to sunlight, blistering of the exposed site was observed 2 h after the drug was taken. Results of this controlled study indicated that oral psoralen plus exposure to sunlight was phototoxic and increased the facultative melanin pigmentation considerably. Also established was the fact that the maximum phototoxic effect occurs 1.5-2.0 h after oral administration. (6) The study on isolation, extraction, and purification of furocoumarins, the naturally occurring oxygen-containing aromatic tricyclic constituents of plants, was initiated by the late Professor Luigi Musajo of the University of Padua, Italy, between 1950 and 1954 [l]. His most diligent, thoughtful, young, and creative associate, Giovanni Rodighiero, pioneered and developed the well conceived program of isolation of psoralens from plant sources, identification of their structure, and synthesis of various psoralens and isopsoralens based on the early work of Professor E. Spath of the University of Vienna [2]. Rodighiero’s initial observations in elucidating the mutagenic, photochemical, and skin-photosensitizing properties of various furocoumarins [l] led to basic studies involving the photoreactivity of psoralens and isopsoralens with prokaryotic and eukaryotic DNA [3, 36-381. This basic in vitro and in vivo research in cutaneous photobiology provided ready access to the new field of photomedicine which is now referred to as photochemotherapy [4]. Musajo [l] first described the mutagenic properties of five furocoumarins in onion root tips. At 5 X lo-’ M, 5-MOP induced mitosis with chromosome mutations. Musajo, after experiencing the photosensitizing effect of bergamot oil on his skin while vacationing in Calabria, Italy, began in 1955 a 25-year organized research study of furocoumaiins. In collaboration with Professor Giovanni Rodighiero and other associates, the Padua group demonstrated the structure-activity relationships and the mutagenic properties of some linear psoralens. His laboratory at the University of Padua, now chaired by Professor Francesco Dall’Acqua, is still the leading center for research on the chemistry of psoralens. (7) Subsequently, in 1959, Oginsky and co-workers [30] reported the killing of bacteria by psoralens and UVA. These investigators demonstrated that the lethal photosensitization of bacteria by psoralens plus UVA was not an oxygen-dependent

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photodynamic event. They also indicated that the active wavelengths were in the range 320-400 nm. (8) After daily exposures to 43 J UVA cm-’ for 6 weeks, 8-MOP, given to each mouse in intraperitoneal doses of 0.4 mg/day, was reported to be carcinogenic to the skin of albino mice [31]. However, the light source also contained a small fraction of UVB. Griffin’s study [31] presents certain problems: (i) the radiant energy to which the mice were exposed contained wavelengths of 290-400 nm, i.e. both UVA and UVB, and we know that UVB is carcinogenic when administered without psoralens; (ii) the mice received the equivalent of 700 mg/70 kg (17 times the human therapeutic dose); (iii) they were exposed to large amounts of UVA. Nevertheless, when given intraperitoneally and followed by UVA (but no UVB), 8-MOP is carcinogenic in mice. (9) The feasibility of increased erythema and pigmentation following oral 8-MOP and low-intensity UVA was first demonstrated by Stegmaier [32] in 1959. Patients were given oral doses of 50 mg 8-MOP and were then exposed to seven daily UVA exposures from three “blacklight” 40 W fluorescent lamps for 30 min; the total dose amounted to 3.95 J cm-‘. (El Mofty [27] had shown previously that topical 8-MOP was phototoxic at much shorter exposures to sunlight than when g-MOP is taken orally.) (10) Also in 1959, Imbrie et al. [33] established that oral 8-MOP and a single exposure to sunlight could alter the skin tolerance to UVB (probably by increased facultative melanogenesis and increased thickness of the epidermis), so that double the dose of UVB was required to induce an erythema. Thus, 8-MOP plus sunlight increased the threshold for the MED to UVB. Increased tolerance of exposed skin to subsequent challenge of 170 PW UVB cm-* obtained from a 175 W sunlamp was observed in eight subjects who were exposed to 30 min of sunlight 2 h after ingestion of 30 mg 8-MOP. Imbrie and co-workers noted that twice as much UVB was required to produce erythema on skin areas of the subjects who received the psoralen plus sunlight compared with those given a placebo and sun exposure. These studies are the basis for the concept of photochemoprotection recently proposed by Fitzpatrick

[W (11) Buck and associates [34] localized the action spectrum of 8-MOP to 360 nm after noting that delayed erythema (36 h) resulted after humans were exposed to 360 nm radiant energy. Their studies were extended by other investigators [35, 38, 391 who found that the action spectrum is in the shorter wavelength region, i.e. as short as 320 nm, with the peak wavelength at 330-340 nm. (12) A mechanism of skin photosensitization based on the production of singlet excited and metastable triplet states of psoralens was reported by Pathak [35] in 1961. Pathak demonstrated the existence of reactive singlet and triplet states of psoralens. With supporting experimental evidence, he proposed that the mechanism of biological photosensitization evoked by psoralens involved excitation of the molecule to a singlet excited state, followed by a transition to a metastable triplet state. Psoralens in the triplet state were shown to generate free radicals, which were believed to evoke biological photosensitization. The mechanism of psoralen-induced skin photosensitization has been recently reviewed [13, 151. (13) The first evidence of photobinding of furocoumarin molecules to DNA was provided by Musajo et al. [36]. By examining the modification of the fluorescence spectrum of the psoralen during UV irradiation in the presence of DNA, RNA, nucleosides, and the purine or pyrimidine bases, they concluded that the furocoumarin molecule was covalently photobound to DNA.

17

(14) Additional work reported by Musajo et al. [37] in 1967 indicated that PUVA inhibits the tumor-transmitting capacity of ascites tumor cells. They demonstrated the inactivation of these cells by irradiating them in the presence of psoralen, 8-MOP, and 5-MOP. (15) By 1968, it was evident that psoralens did photoreact with DNA. Dall’Acqua and associates [38] showed that psoralens formed two types of photoadducts at the 3,4- and the 4’,5’-positions in the psoralen molecule. When a frozen aqueous solution in which the psoralen and thymine molecules were immobilized in a matrix was irradiated, both 3,4- and 4’,5’-photoadducts were obtained. This involved the 3,4- or 4’,5’-double bond of the drug and the 5,6-double bond of the pyrimidine. (16) Pathak and Kramer [24, 391 established the in viva photoreaction of psoralen with DNA in 1969. Application of tritiated trioxsalen to the skin and irradiation with 365 nm WA and subsequent extraction of DNA, RNA, and proteins from the irradiated skin showed in viva photoconjugation of psoralen with DNA and RNA. However, photoconjugation of psoralens with epidermal protein fractions was minimal. Fluorescent and non-fluorescent adducts were formed as well. Whether inhibition of DNA synthesis is the only basis for the therapeutic effect of PUVA on psoriasis has not been established [4, 131. (17) Soon after these observations, investigators in the USA and Italy simultaneously demonstrated intercalation of psoralen and subsequent formation of interstrand crosslinks between psoralen and pyrimidine bases of two opposite strands of DNA. The two most important published works on the formation of cross-links by psoralens and WA in DNA are that of Cole [40] and Dall’Acqua et al. [41]. These observations were subsequently confirmed by Cech and Pathak involving electron-microscopic and biochemical evidence of the photochemical cross-linking of DNA in mammalian skin by psoralens [67]. (18) Mortazawi [42], an Iranian dermatologist working with Oberste-Lehn in Wuppertal, FRG, reported on the induction of a remission in psoriasis after topical application of 8-MOP (0.15%) and 20 exposures to WA from a 16-bulb light source. Tronnier and Schule [43], using essentially the same protocol, made the same observations. (19) In their studies of the synthesis of the nucleic acids in Ehrlich ascites tumor cells by irradiation in the presence of skin-photosensitizing and non-photosensitizing furocoumarins, Bordin and associates [44] detected strong inhibition of DNA and RNA synthesis. The most potent compound was 8-MOP. (20) The first controlled study of oral 8-MOP and high-intensity WA in the treatment of psoriasis was reported in 1974 by the Harvard group [4]. This report introduced carefully monitored dosimetry with a new high-intensity UVA source and the term “photochemotherapy”. Paired comparison studies were conducted on 16 patients with generalized psoriasis. Conventional doses of WB were compared with high-intensity WA alone (UVB was excluded with the use of Mylar). Each patient was given 0.6 mg 8-MOP/kg orally and exposed to the radiant energy thrice weekly. Initial exposures of 2.4-4.8 J cm-* were increased at each exposure time by 0.34-0.68 J cm-*. By the end of a week of treatment, the side treated with PUVA was markedly more improved than the side that received UVB. Complete clearing on the PWAtreated side occurred in 12-18 treatments. Using the same protocol and light system, Wolff and his associates noted and published within 6 months identical results in 29 of 30 patients treated in Vienna, Austria [8]. (21) The stimulus for GTE-Sylvania to build a high-intensity WA source was the need for an all-year treatment of vitiligo. Levin, a physicist, and Parrish, a dermatologist, collaborated in the design of the first lighting units that made PWA

18 therapy a practical reality [45]. Initial exposure times (5 min) were reduced from the conventional blacklight sources (40 min) used previously. This lighting system had 48 horizontal lamps aligned in a parallel array on a 4.4 cm center. Plane reflectors of aluminum were located vertically and were perpendicular to the lamps. Only small variations in irradiance occurred over an adequate working distance of about 40.0 cm. Irradiance was 9 mW cm- ’ for the 320-380 nm band with an average exposure time of 5-10 min compared with 0.7 mW cmv2 in the 320-380 nm band for 12 blacklight fluorescent lamps (40 W, 48 inch) aligned horizontally in a parallel array on 15 cm centers with aluminum plane reflectors. The latter system required a minimum exposure time to induce melanogenesis and phototoxicity (following oral psoralen). (22) The treatment of mycosis fungoides with PUVA was found effective in a clinical study conducted by Gilchrest et al. [46]. Of 11 patients, four were erythrodermic and seven were in plaque stage; eight cleared to less than 5% involvement and of three erythrodermic, two improved and one did not. Follow-up results 700-1000 days afterwards of the eight patients who experienced clearing of their disease revealed that five were on maintenance therapy and were controlled, and three, who stopped receiving PUVA therapy for reasons not related to complications of treatment, had recurrence of mycosis fungoides. Inasmuch as sunlight had been known to induce remission in this skin neoplasm, PUVA was a reasonable mode of therapy. Since this article was published, several reports confirmed the effectiveness of PUVA in the early stages of mycosis fungoides [13, 161. The treatment is only palliative, as are all available treatments for this disease (e.g. electron beam therapy, topical nitrogen mustard). (23) The results establishing the efficacy of PUVA in the clearing and remission of psoriasis were reported in 1977 by Melski et al. [9]. This report of cooperative trials conducted in 16 academic dermatology departments in the USA proved that effective PUVA photochemotherapy requires close attention to detail and careful monitoring. Although all the centers used the same protocol, light source, and medication, the percentages of patients who showed clearing varied from center to center; some centers observed a 90% clearing, whereas others stated they had 40% clearing. (24) Between 1975 and 1977, photochemotherapy of psoriasis with oral PUVA became extremely popular. Simultaneously, misleading anecdotal articles started appearing in print, such as that by Reed and associates [47] of a lCyear-old girl with xeroderma pigmentosum (DeSanctis-Cacchione type) who died of pneumonia. The child had had many non-melanoma skin cancers (basal cell and squamous cell carcinomas) and keratoacanthomas before she was 6 years old. At age 13, she was treated for 1 month with trioxsalen (no dose stated) “with very poor results, apparently with a greater number of skin lesions”. This report has no scientific basis because it was completely uncontrolled. It is impossible for one to observe a change in the “number of skin cancers” without a control period. Furthermore, the medication was only administered for 1 month. It is unfortunate that this misleading published report is frequently cited as indicating the carcinogenic action of psoralens in man. (25) Since the time S-MOP was found to be a phototoxic and potentially carcinogenic agent, investigators have been exploring the usefulness of non-phototoxic and noncarcinogenic psoralens. A first report on a non-carcinogenic yet clinically effective psoralen appeared in 1978, the work of Dubertret et al. [48]. They determined that 3carbethoxypsoralen (3-CP) formed only. monofunctional adducts in yeast. Monofunctional adducts are more easily repaired than are cross-links formed by S-MOP. Topical application of 15 mg 3-CP/cm’ to the ears of mice before they were irradiated at a dose rate of 28 J cmw2 s-l resulted in no tumor formation after 196 applications compared with development of tumors in 90% when S-MOP was used topically.

19

Intraperitoneal administration of 0.4 mg per Swiss mouse was followed by WA exposure at 1.68x 104 J cme2. Thirty-six injections of 0.4 mg 3-CP per mouse followed by WA produced neither tumors nor toxicity, but eight mice given g-MOP in the same dosage developed the same percentage of tumors as with topical application. In the clinical studies reported by these authors [48], four of ten patients had good therapeutic results with slow resolution from an average of 33 treatments and a mean total dose of 417 J cmm2. Although 3-CP is non-carcinogenic, it is only weakly effective in treating psoriasis when applied topically and followed by exposure to WA. The clinical studies were inconclusive; perhaps other derivatives of this type may also be carcinogenic and clinically effective, when given topically or orally. Until recently, the carcinogenic activity of psoralens was believed to be due to their skin-photosensitizing potency and their ability to react with DNA to form interstrand cross-links. The monofunctional psoralens were considered to be non-carcinogenic. Studies reported by Mullen et al. [68] disproved this concept and revealed that monofunctional psoralens such as isopsoralen, 5methylangelicin, and 4,5’-dimethylangelicin were as carcinogenic as bifunctional g-MOP. (26) The efficacy of PWA in maintenance of remission in psoriasis was carefully investigated by Wolff et al. 1491 who demonstrated that a period of maintenance for 60 days provides for a prolonged remission (over 1 year). Relapses gradually drop as a function of time but occur during the first 2 months of therapy. With no maintenance treatment, Wolff and associates determined that 11% of their patients were free of disease after 56 weeks; if such therapy was provided for 2-3 months after clearing, more than 66% were still in remission after 33 weeks. They concluded that by discontinuing treatment after 2 months, one could sort out the subset of patients who tend to relapse. Some pertinent data of this study conducted in Innsbruck and Vienna, Austria, are as follows: Number of patients Number of patients cleared Number of exposures to UVA required Duration of treatment (days) Total UVA dose (J cmw2) Number of treatment failures

for clearing

572 534 (93%) 14.7& 8.3 30.4 f 26.6 78.7 f 88.7 37 (6.5%)

(27) The increased incidence of squamous cell carcinoma in certain susceptible patients with psoriasis who were treated with PWA was documented by careful observation at 16 academic and medical centers in the USA [17]. In humans, an increased relative frequency of squamous cell carcinoma in areas not habitually exposed (trunk and lower extremities) to sunlight was observed. The tumors, which occurred most frequently in patients with previous exposure to ionizing radiation, were nonaggressive, and no metastases were observed. This report emphasized a reversal of the ratio of basal cell carcinoma to squamous cell carcinoma, i.e. a greater number of squamous than basal cell carcinomas were observed in patients treated with PWA. Also, the former type of neoplasms was reportedly seen in body sites not normally exposed to sunlight. The overall incidence in 25 months was 2.5% (30 tumors in 1177 patients). The magnitude of this problem is uncertain because some groups with large patient populations and careful follow-up did not experience an increased incidence of skin carcinomas. Ten years of prospective study of the PWA cohort demonstrated dose-dependent increase in the risk of squamous cell carcinoma among patients who developed a first tumor at least 58 months after beginning PUVA therapy. This strong

20

dose-dependent increase in the risk of developing squamous cell carcinoma was noted in patients who had prior substantial exposure to other carcinogens such as coal tar, UVB from sunlight, ionizing radiation, or ingestion of inorganic trivalent arsenic. The important risk of oral PWA photochemotherapy is a low incidence of squamous cell carcinomas largely on the skin of the lower extremities and male genitalia, and it may be seen in patients with skin phototypes I and II or in patients who have a history of previous treatments with mutagenic agents (e.g. X-rays) [13]. (28) A report on the cooperative study among 18 European centers on the efficacy of PWA in remission of psoriasis was published in 1981 [ll]. This multi-center European trial confirmed results of the studies conducted in the USA. Maintenance with PWA had little or no effect on the duration of remission. The data are tabulated as follows. Parameter

Number

Total patients Cleared Improved Moderately improved Unchanged or minimally improved Treatment failures Exposures required for clearing, mean Duration of treatment required for clearing, mean number of weeks Total cumulative dose required for clearing, mean value of J cm-*

3136 2785 179 91 53 28 19.0

Percentage of clearance

Percentage of patients cleared

90-100 SO-90 20-50 O-20

88.8 5.7 2.9 1.7 0.9

5.7

103.3

(29) Systemic retinoids have added another important therapeutic agent to the treatment of disorders of keratinization which includes psoriasis. The combination of PWA with oral retinoids (aromatic retinoid-etretinate) may represent a substantial advance in’combination therapy for psoriasis. Most of the studies have been performed with etretinate (Tegison) which has been most effective in erythrodermic and pustular (localized and generalized) psoriasis. This compound appears to be a potent acceleratory of PWA therapy; the response rate of clearing lesions is accelerated. The retinoid is given 5-7 days prior to PWA therapy in a dosage of 1 mg/lcg body weight, and the dosage is continued until clearing. The main advantages of using etretinate in combination with PWA were: (i) an acceleration of the number of individual treatments to achieve complete clearing; (ii) a reduction in the number of individual treatments to achieve complete clearing; and (iii) an approximate 50% reduction in the total cumulative dose of WA (J cm-‘) necessary to achieve clearing of psoriasis [69-711. (30) The development of liquid formulations of methoxsalen (solubilized form of 8-MOP in soft gelatin capsules) appears to be superior to the crystalline methoxsalen (pulverized or micronized) used in the USA and elsewhere during the 1970s and early 1980s. This has helped to alleviate some of the problems with oral photochemotherapy

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resulting from the poor or unpredictable absorption of 8-MOP, due primarily to its poor or delayed solubility resulting from the crystalline nature of the drug. The liquid methoxsalen preparation in soft gelatin capsules provides a more rapid and higher absorption and more reproducible peak serum level than the crystalline preparation [72-741. The coated liquid preparations of the solubilized drug are unquestionably absorbed faster and yield higher and more constant serum levels than do the old crystalline forms. Furthermore, the original dose requirement of 0.6 mg/kg body weight of 8-MOP per treatment is now reduced to 0.3-0.45 mg/kg [72-741. (31) In 1987, a novel therapeutic modality called “extracorporeal photochemotherapy” for the management of disorders caused by abnormal lymphocytes, in hematogenously disseminated cutaneous T-cell lymphoma (CTCL) was reported by Edelson et al. [64]. This involved the use of psoralens and UVA in an extracorporeal flow system referred to as “photopheresis”, and it represents a more recent adaptation of PWA therapy for the management of disorders caused by abnormal T-lymphocytes. The procedure involves the extracorporeal photoactivation of 8-MOP by passage of blood containing CTCL cells (photopheresis) through a UVA exposure system. Lymphocytes are quite sensitive to the effects of photoactivated 8-MOP; they can be either killed or functionally impaired. Photopheresis entails removing one unit of blood from the patient, and after centrifugation of red cells, the plasma component containing leukocytes is combined with saline containing 8-MOP and passed through a clear plastic channel between twin banks of high-intensity UVA lamps. The irradiated blood, after reconstitution, is retransfused to the patient, leading to an apparent immunological reaction against residual CI’CL cells [64-661. These studies reveal that 8-MOP + WAtreated pathogenic T-cells could efficiently vaccinate against themselves and inhibit subsequent induction of the disease [66]. Encouraging preliminary results, involving patients with other immunological diseases such as systemic sclerosis (scleroderma), psoriatic arthritis, pemphigus vulgaris, rheumatoid arthritis, and AIDS-related complex have been observed [66]. This concept of using psoralen photoactivation as a therapeutic approach to enhancing immune responses against pathogenic clones of T-cells is potentially important in the management of other auto immune diseases in which aberrant T-cells play a role in pathogenesis [65, 661. (32) An interesting aspect of PWA therapy is its use in prevention of diseases. Long-lasting tolerance to sunlight has been achieved (Table 1). Recently, the concept of enhanced tanning as a defense mechanism against W damage has been reactivated [60, 691. The concept of PWA photochemoprotection involves the use of oral or topical psoralens and exposure to UVA obtained from artificial WA irradiators or from sunlight in order to promote and induce epidermal cell hyperplasia (accompanied by thickening of the stratum corneum) and increased epidermal melanin pigmentation manifested by an increase in the population density of melanocytes and an increase in the rate of formation, melanization, and transfer of melanosomes from melanocytes to the increased population of keratinocytes in the epidermis. This PUVA-induced pigmentation functions as a highly effective epidermal-melanin density filter that shields keratinocytes, melanocytes, and dermal components of connective tissue and blood vessels from deleterious IJVR-induced effects. This places the susceptible white population (skin phototypes I, II, and III) in a reduced risk category with respect to the development of dermatolheliosis and W-induced skin cancers. That the PUVA-induced tan is effective in attenuating the damaging effect of sunlight exposure has been recently examined experimentally by Young et al. [75]. Skin damage induced by UVR or 5MOP + WA treatment was assessed by comparing unscheduled DNA synthesis before and after UVB challenge. Sites on previously unexposed buttock skin in 18 human

22

volunteers (skin phototypes I-V) were treated daily for 2 weeks with suberythemogenic doses of solar simulated UVB radiation alone (SSR), SSR+ a UVB sunscreen, and SSR + a UVB sunscreen containing 30 ppm S-MOP. One week later, these skin sites, along with the control untreated sites, were rechallenged with 2-MED doses UVB radiation to determine whether the facultative tanning induced by 5-MOP+SSR is protective against vascular and DNA damage. The investigators concluded the judicious use of S-MOP formulation containing UVB sunscreens resulted in reduced unscheduled DNA synthesis when compared with tanning protocols with SSR alone. S-MOP formulation containing UVB sunscreen was protective, particularly in skin types I and II individuals. With the availability of oral or topical S-MOP which is less phototoxic than g-MOP, it is now possible to obtain melanogenic doses of drug-sunlight combinations without the development of phototoxic erythema as is often seen with g-MOP. This separation of melanogenesis and phototoxic erytherma provides a wider margin of safety that permits a practical approach of oral S-MOP and exposure to sunlight to induce PWA-tan that is protective against potentially harmful effects of UVB exposure. (33) Recently, the use of psoralen compounds and long-wavelength UVA radiation for the decontamination of blood products has been examined with a view to facilitate the introduction of psoralen mediated photochemical inactivation of viruses into clinical practice. Transmission of viral diseases through blood products remains a major problem in transfusion medicine. Using a combination of g-MOP + WA, 4-aminomethyl-4,5’,8trimethylpsoralen (AMT) and 4,5’,8_trimethylpsoralen (IMP), it has been observed that psoralen and WA treatment can inactivate a wide range of animal viruses in proteinacious media (RNA-containing viruses and DNA-containing viruses) in serum or plasma containing clotting factor concentrates without any adverse effects on the biological and biochemical properties and function of platelets. This technique is potentially useful in reducing the risk of viral transmission from platelet transfusion. The transmission of human immunodeficiency virus (HIV-l) by blood or blood products is a major concern in blood banking, and the use of psoralen compounds (g-MOP, AMT, TMP) and long-wavelength UV radiation appears to be very encouraging in inactivation of viruses through oxygen-dependent disruption of membranes [60-631. (34) Recently an alternative g-MOP analog referred to as 5methoxypsoralen (bergapten or S-MOP) has been investigated for the treatment of psoriasis, as well as vitiligo by researchers at Vienna, as well as Boston [76-80], and this psoralen derivative appears to be an effective photochemotherapeutic agent with certain selective advantages to the patient care. It has been observed that a small, but not insignificant, number of patients receiving PWA therapy with oral g-MOP invariably experience the uncomfortable feeling of nausea, malaise, and phototoxicity in the form of painful erythema, edema or vesiculation. Bergapten (S-MOP), on oral administration in adequate doses (1.2 mg/kg), appears to be better than methoxsalen (g-MOP) because of the relative paucity of disturbing side-effects, especially nausea. Our studies, still in progress, reveal that unlike g-MOP which is effective at 0.45-0.6 mg/kg, the dosages of S-MOP required for clearing psoriasis or repigmenting vitiligo are in the range 1.0-1.2 mg/ kg. S-MOP at 1.2 mg/kg dosage, when given thrice weekly with adequate WA exposure dosage (l-10 J cm-‘), is as good as g-MOP as a therapeutic agent for the clearance of psoriasis and for the repigmentation of vitiligo. The decrease or absence of nausea and cutaneous phototoxic reaction is a major advantage and raison d’2tre for oral bergapten in PWA photochemotherapy.