Long-term (6 and 12 months) follow-up of two prospective, randomized, controlled phase III trials of photodynamic therapy with BF-200 ALA and methyl aminolaevulinate for the treatment of actinic keratosis.

BACKGROUND: Two phase III trials of photodynamic therapy (PDT) with BF-200 ALA, a recently approved nanoemulsion formulation of 5-aminolaevulinic acid (ALA) demonstrated high clearance rates in mild-to-moderate actinic keratosis (AK). The comparison to a registered methyl aminolaevulinate (MAL) cream demonstrated significantly superior total patient clearance rates. OBJECTIVES: To evaluate long-term efficacy and safety of PDT for AK 6 and 12 months after the last PDT with BF-200 ALA, MAL or placebo. METHODS: The follow-up phase (FUP) was performed with patients of two phase III studies. Both studies compared BF-200 ALA with placebo, one of the studies additionally with MAL. Overall recurrence rates and various subgroups (light source, lesion severity, lesion location, complete responders after first PDT) were assessed 6 and 12 months after the last PDT. RESULTS: Recurrence rates were similar for BF-200 ALA and MAL, with a tendency to lower recurrence rates for BF-200 ALA. The proportion of patients who were fully cleared during PDT and remained completely clear for at least 12 months after PDT were 47% for BF-200 ALA (both studies) and 36% for MAL treatment. The subgroup that was illuminated with narrow wavelength LED lamps reached 69% and 53% for BF-200 ALA (both studies, respectively) and 41% for MAL. No safety concerns were reported. CONCLUSIONS: The FUP data confirmed the high efficacy and safety of PDT with BF-200 ALA. The slightly lower recurrence rates after BF-200 ALA treatment compared with MAL treatment enhanced the better treatment outcome due to the significantly superior efficacy.

Photodynamic therapy with BF-200 ALA for the treatment of actinic keratosis: results of a multicentre, randomized, observer-blind phase III study in comparison with a registered methyl-5-aminolaevulinate cream and placebo.

BACKGROUND: Photodynamic therapy (PDT) with 5-aminolaevulinic acid (ALA) or its methylester [methyl-5-aminolaevulinate (MAL) or 5-amino-4-oxopentanoate] was recently ranked as first-line therapy for the treatment of actinic keratosis (AK) and is an accepted therapeutic option for the treatment of neoplastic skin diseases. BF-200 ALA (Biofrontera Bioscience GmbH, Leverkusen, Germany) is a gel formulation of ALA with nanoemulsion for the treatment of AK which overcomes previous problems of ALA instability and improves skin penetration. OBJECTIVES: To evaluate the efficacy and safety of PDT of AKs with BF-200 ALA in comparison with a registered MAL cream and with placebo. METHODS: The study was performed as a randomized, multicentre, observer-blind, placebo-controlled, interindividual trial with BF-200 ALA, a registered MAL cream and placebo in a ratio of 3:3:1. Six hundred patients, each with four to eight mild to moderate AK lesions on the face and/or the bald scalp, were enrolled in 26 study centres in Germany, Austria and Switzerland. Patients received one PDT. If residual lesions remained at 3months after treatment, PDT was repeated. RESULTS: PDT with BF-200 ALA was superior to placebo PDT with respect to patient complete clearance rate (78·2% vs. 17·1%; P<0·0001) and lesion complete clearance rate (90·4% vs. 37·1%) at 3months after the last PDT. Moreover, superiority was demonstrated over the MAL cream regarding the primary endpoint patient complete clearance (78·2% vs. 64·2%; P<0·05). Significant differences in the patient and lesion complete clearance rates and severity of treatment-related adverse events were observed for the narrow- and broad-spectrum light sources. CONCLUSIONS: BF-200 ALA is a very effective, well-tolerated new formulation for AK treatment with PDT and is superior to a registered MAL medication. Efficacies and adverse events vary greatly with the different light sources used.

5-Methoxypsoralen plus ultraviolet (UV) A is superior to medium-dose UVA1 in the treatment of severe atopic dermatitis: a randomized crossover trial.

BACKGROUND: Ultraviolet (UV) A1 and psoralen plus UVA (PUVA) are effective treatment options for severe atopic dermatitis (AD); however, their relative efficacy has not yet been determined in a head-to-head study. OBJECTIVES: To compare UVA1 and oral 5-methoxypsoralen (5-MOP) plus UVA with respect to efficacy, tolerability and duration of response in patients with severe generalized AD. METHODS: Forty patients were included in this randomized observer-blinded crossover trial. The patients received either 15 exposures to medium-dose UVA1 as the first treatment and, in cases of relapse, another 15 exposures to 5-MOP plus UVA as the second treatment, or vice versa. All patients were followed until 12 months after discontinuation of the last treatment. The SCORAD score was determined by a blinded investigator at baseline, after 10 and 15 treatments each and during the follow-up period. In addition, all adverse events were recorded during the whole study period. RESULTS: Twenty-three patients completed the crossover treatment. Both phototherapies resulted in clinical improvement; however, PUVA reduced the baseline SCORAD score to a significantly greater extent than UVA1 (mean +/- SD 54.3 +/- 25.7% vs. 37.7 +/- 22.8%; P = 0.041). The median length of remission was 4 weeks (interquartile range 4-12) after UVA1 and 12 weeks (interquartile range 4-26) after PUVA therapy (P = 0.012). CONCLUSIONS: PUVA provides a better short- and long-term response than medium-dose UVA1 in patients with severe AD.

European S3-guidelines on the systemic treatment of psoriasis vulgaris.

Of the 131 studies on monotherapy or combination therapy assessed, 56 studies on the different forms of phototherapy fulfilled the criteria for inclusion in the guidelines. Approximately three-quarters of all patients treated with phototherapy attained at least a PASI 75 response after 4 to 6 weeks, and clearance was frequently achieved (levels of evidence 2 and 3). Phototherapy represents a safe and very effective treatment option for moderate to severe forms of psoriasis vulgaris. The onset of clinical effects occurs within 2 weeks. Of the unwanted side effects, UV erythema from overexposure is by far the most common and is observed frequently. With repeated or long-term use, the consequences of high, cumulative UV doses (such as premature aging of the skin) must be taken into consideration. In addition, carcinogenic risk is associated with oral PUVA and is probable for local PUVA and UVB. The practicability of the therapy is limited by spatial, financial, human, and time constraints on the part of the physician, as well as by the amount of time required by the patient. From the perspective of the cost-bearing institution, phototherapy has a good cost-benefit ratio. However, the potentially significant costs for, and time required of, the patient must be considered.

Glycocalyx of epidermal cells in vitro: demonstration and enzymatic removal.

Guinea-pig epidermal cells in culture possess a glycocalyx coat similar to that in vivo, as revealed by the ruthenium red stating technique. Trypsin, phospholipase C, and lysozyme do not produce any changes of the glycocalyx, while hyaluronidase and neuraminidase lead to partial and subcomplete removal respectively. Cells stripped of their glycocalyx coat by neuraminidase do not detach from the support and do not show any signs of toxicity. There is complete reconstitution of the glycocalyx within 24 hr.

Immunofluorescence demonstration of type IV collagen and a noncollagenous glycoprotein in thickened vascular basal membranes in protoporphyria.

Specific rabbit antibodies to type IV collagen isolated from a basement membrane producing mouse tumor were used in indirect immunofluorescence tests to study the thickened vascular basement membrane in skin biopsies from patients with erythropoietic porphyria (EPP) and from protoporphyric mice. In addition, rabbit antibodies to a noncollagenous, basement membrane specific glycoprotein also derived from the mouse tumor were tested. It was shown that normal as well as altered vascular basement membranes in both the human and the murine skin specimens react with the anti-type IV collagen and the antiglycoprotein antibodies. A particular strong reaction in the diseases skin indicated that formation of new vascular basement membrane layers involved deposition of the major structural proteins which also constitute normal basement membrane matrices.

Do oral carotenoids protect human skin against ultraviolet erythema, psoralen phototoxicity, and ultraviolet-induced DNA damage?

This study was performed in order to (1) assess the magnitude of a possible protective effect of oral carotenoids on ultraviolet B (UVB)-, ultraviolet A (UVA)-, and psoralen ultraviolet A (PUVA)-induced erythema in human skin and (2) to evaluate whether the postulated prevention of skin cancer by prophylactic administration of carotenoids is based on a decrease in UVB-induced DNA damage. Twenty-three healthy volunteers received oral carotenoids (150 mg/day) for 4 weeks. Serum levels were quantitated, and ranged from 390 to 1710 micrograms/dl. Before and after carotenoid administration, the UVA- and UVB-MEDs and the PUVA-MPD were determined by standard phototesting. DNA damage was assessed by autoradiographical measurement of unscheduled DNA synthesis (UDS) following UVB exposure before and after treatment. No statistically significant carotenoid-dependent protection was found against UVA, UVB, and PUVA erythema by comparing the pre- and postcarotenoid erythema doses. Also at the DNA level there was no indication of a protective effect that could be detected with the methods employed: the amount of UVB-induced UDS was not decreased after carotenoid treatment. We conclude that (1) carotenoids do not reduce UVB-, UVA-, or PUVA-induced erythema in human skin; that (2) reactive oxygen species may not be involved in PUVA-erythema production or, alternatively, carotenoids may not quench these radicals sufficiently in vivo; and that (3) carotenoid protection against UVB-induced carcinogenesis does not operate by reducing the number of mutagenic lesions in DNA.

Change of ultraviolet absorbance of sunscreens by exposure to solar-simulated radiation.

Regarding the outdoor behavior of the Caucasian population, modern sunscreens should provide high and broad-spectrum ultraviolet protection in the ultraviolet B as well as in the ultraviolet A range and should be photochemically stable for ultraviolet doses, which can be expected in solar radiation. At present an assessment of the photostability of suncare products is not a general requirement before marketing. In order to evaluate the photostability of suncare products we conducted an in vitro test and measured the spectral absorbance of 16 sunscreens before, and after exposure to increasing biologically weighted standard erythema doses (5, 12.5, 25, 50) of solar-simulated radiation. Seven of 16 suncare products showed a significant dose- and wavelength-dependent decrease of the ultraviolet A protective capacity, whereas the ability to absorb ultraviolet B was not affected. In the ultraviolet A range, the decrease of absorbance (photoinactivation), respectively, the increase of transmission was 12-48% for an ultraviolet exposure of 25 standard erythema dose. Photoinactivation started in the wavelength range between 320 and 335 nm with a maximum above 350 nm. Furthermore, our analysis showed that the behavior of suncare products was not predictable from its individual ingredients. Neither complex combinations of organic filters nor addition of inorganic filters could absolutely prevent photoinactivation. The inclusion of a single photounstable filter did not mean photoinstability of the complete suncare product. Photoinactivation of sunscreens appears to be an underestimated hazard to the skin, first, by formation of free radicals, second, by increased ultraviolet A transmission.

Mouse model for protoporphyria. II. Cellular and subcellular events in the photosensitivity flare of the skin.

Acute phototoxic reactions were induced by long-wave ultraviolet light (UV-A) in mice with griseofulvin-induced protoporphyria. The clinical response was characterized by erythema, pronounced edema, and purpura. Tracer experiments and electron microscopy revealed pronounced vascular damage and leakage of vascular contents, whereas the epidermis and all other dermal components were intact. There was selective destruction of endothelial cells and damage of the basal lamina of the vessels. This striking vascular injury was absent from nonprotoporphyric UV-A-irradiated mice and from protoporphyric and nonprotoporphyric mice exposed to short-wave ultraviolet light (UV-B). Patients with erythropoietic protoporphyria (EPP) exhibit an identical, selective damage of blood vessels when irradiated with UV-A or sunlight but not with UV-B alone. It is hypothesized that in both murine protoporphyria and EPP, endothelial cells are photosensitized by protoporphyrin circulating in the serum and that photosensitized endothelia represent the primary cellular target of the photochemical reaction induced by UV-A.

Herpes gestationis: fine structural pattern of immunoglobulin deposits in the skin in vivo.

IgG and C3 deposits were observed in the basement membrane zone in a case with herpes gestationis. In addition, circulating IgG anti-basement membrane antibodies were found. Electron microscopic immunocytochemical investigations, performed with a peroxidase-antiperoxidase sandwich technique, revealed the in vivo-bound IgG to be localized at the epidermal basal lamina and to exhibit a distribution pattern which is identical to that reported for bullous pemphigoid.

Ultrastructural localization of immunoglobulins in skin of patients with dermatitis herpetiformis.

A multistep immunocytochemical method utilizing horseradish peroxidase as an immunologically bound marker was used to detect and localize IgA deposits in skin of patients with dermatitis herpetiformis at the ultrastructural level. IgA was found in the upper papillary dermis forming irregular aggregates in seemingly haphazard distribution. These aggregates were associated with microfibrillar bundles and with the microfibrillar component of the elastic tissue. IgA was also detected on anchoring fibrils, but showed no topical relationship to the basal lamina which was always spared. This finding indicates that basal lamina components do not serve as target sites for the immunologic reaction occurring in dermatitis herpetiformis. The selective affinity of IgA deposits to microbfibrillar bundles may be relevant to the hypothesis that the skin pathology in dermatitis herpetiformis is caused by circulating gluten-antigluten complexes, trapped in the skin by reticulin-bound antireticulin antibodies which cross-react with gluten.

Ultraviolet light depletes surface markers of Langerhans cells.

This report defines the influence of ultraviolet light (UV) on Langerhans cells (LC). Human volunteers and hairless mice (Swiss ha/ha) were exposed to various single and/or cumulative doses of either UV-A, UV-B, or UV-A plus small amounts of UV-B (UV-A (+B)). 24 hr after the last irradiation, morphology of the entire epidermis was evaluated by both light and electron microscopy while LC, in addition, were tested for expression of specific histochemical (ATPase) and functional immunological markers (Ia antigens). In both men and mice, cumulative doses of either 80-120 J/cm2 UV-A (+B) or 1-2 X 100 J/cm2 UV-A resulted in a dramatic reduction of cells exhibiting ATPase and Ia-reactivity. In the UV-B spectrum, single doses of 60-80 mJ/cm2 produced a virtually complete elimination of LC membrane markers. By contrast, pemphigus antigens of keratinocytes were unaffected by these energy doses. Electron microscopy revealed cellular damage of some LC after UV-doses which produce a virtually complete abolition of LC membrane markers. At certain dose ranges (15-30 mJ/cm2 UV-B and 1 x 40 to 2 x 100 J/cm2 UV-A) LC were the only epidermal cells to display morphological damage at the ultrastructural level whereas higher doses affected all epidermal cells. The finding that LC surface markers and to a lesser extent the cells themselves are particularly susceptible to UV irradiation has important implications in view of previous findings that LC are potent stimulators of antigen-specific and allogeneic T cell activation. UV-induced alteration of LC plasma membrane integrity may represent a tool to manipulate adverse immune reactions involving the epidermis.

Immediate pigment darkening phenomenon. A reevaluation of its mechanisms.

Proposed mechanisms of immediate pigment darkening (IPD) are controversial. They include photooxidation of "premelanin," changes in the distribution pattern of microfilaments and microtubules, movement of melanosomes to melanocyte dendrites, increased transfer of melanosomes to keratinocytes, and changes in the melanosome distribution pattern in keratinocytes. We investigated the following aspects of IPD: production of IPD by UVA under physiologic and nonphysiologic conditions in fullthickness skin and epidermal sheets; reversibility of IPD in vitro after in vivo and in vitro production; blocking of IPD by disruption of the microfibrillar or microtubular system in vitro; alterations of the cytoskeleton of melanocytes; the melanosome distribution pattern in melanocytes and keratinocytes. The results were as follows: IPD could be elicited in vitro in full-thickness skin and in epidermal sheets. Its production was temperature independent (0 degrees-37 degrees C) and was not inhibited by repeated freezing and thawing, or by formalin fixation. IPD was reversible in vitro under tissue culture conditions but only in viable skin. IPD could not be blocked by substances that disrupt the microfibrillar or microtubular system (cytochalasin B, colcemid, vincristine). As shown with a monoclonal antivimentin antibody, IPD-producing UVA doses did not induce changes in the cytoskeleton of melanocytes. No changes in number and distribution pattern of melanosomes were observed electron-microscopically and by morphometric analysis of EM micrographs. Production of IPD does not depend on the structural and functional integrity of the melanocyte cytoskeletal apparatus and is not confined to viable skin, whereas its reversibility is. The fact that no increased melanosome transfer occurs may explain the lack of a UV protective action.

Ultrastructural changes in PAM cells after photodynamic treatment with delta-aminolevulinic acid-induced porphyrins or photosan.

Photodynamic therapy (PDT) is the combination of a photosensitizing drug (Ps) with light in the presence of oxygen leading to the generation of reactive molecular species and destruction of cancer cells. In this study we compared PDT with two Ps, the hematoporphyrin derivative Photosan (Ph) and delta-aminolevulinic acid (ALA)-induced endogenous protoporphyrin IX, with respect to mitochondrial function and ultrastructural alterations. The effects of PDT were investigated in PAM 212 cells after different Ps incubation times, light doses, and post-treatment periods. Both Ps induced a light dose-dependent impairment of the mitochondrial function with the dose-response curve being steep for ALA and flat for Ph. The prolongation of the incubation time from 4 to 20 h resulted in an increased reduction of mitochondrial activity after ALA PDT but not after Ph PDT. Treatment with an irradiation dose that decreased mitochondrial activity by 50% (IC50) led to early and profound changes of mitochondrial morphology in ALA photosensitized cells, whereas photosensitization with Ph resulted in more pronounced alterations of lysosomes. We conclude that at bioequivalent sublethal PDT exposures of PAM 212 cells, ALA-induced damage is primarily restricted to mitochondria, whereas Ph-induced cytotoxicity is mediated by damage of the lysosomal system.

Mouse model for protoporphyria. I. The liver and hepatic protoporphyrin crystals.

Outbred albino mice were rendered protoporphyric by a diet containing 2.5% (weight) of griseofulvin. There was a 5-fold increase in liver weight, hepatocellular degeneration and necrosis, cholestasis, ductular proliferation and cirrhosis. Liver protoporphyrin values were elevated and brown pigment granules were present in hepatocytes, Kupffer cells, and bile ducts. The granules showed red fluorescence, birefringence, and, at the ultrastructural level, consisted of aggregates of needle-like crystals. Crystals isolated from such livers showed solubility and absorption characteristics of protoporphyrin; in vitro recrystallization of protoporphyrin, extracted from protoporphyric mouse livers, yielded crystals identical with those observed in vivo, and commercial protoporphyrin exhibited similar morphologic features. The liver pathology and protoporphyrin crystals observed in these animals are identical to the liver pathology and crystals observed in the human disease, erythropoietic protoporphyria. In this mouse model, protoporphyrin crystals are intimately associated with hepatocellular injury and it appears that their accumulation within hepatocytes leads to hepatocellular destruction. A similar pathogenesis is postulated for the hepatic damage that occurs in some cases of erythropoietic protoporphyria.

Mouse model for protoporphyria. III. Experimental production of chronic erythropoietic protoporphyria-like skin lesions.

Albino mice were made protoporphyric with griseofulvin according to an established procedure. Photosensitivity flares were elicited once a week throughout a 10-month period, using black light as a source for 410 nm radiation and the flares were monitored by the intravenous injection of vascular tracers and by light and electron microscopy. Each irradiation led to a selective destruction of the endothelial cells of superficial capillaries which was followed by massive vascular leakage. The basal lamina remained largely intact, providing the scaffold for regenerating endothelial cells which deposited new basal lamina material at their periphery. Subsequent exposures to 410 nm radiation reproduced the endothelial damage and subsequent basal lamina formation; multiple irradiations thus resulted in excessive, concentric, tubelike basal lamina deposits around dermal vessels which light microscopically appeared as PAS-positive hyaline material and clinically gave the skin a thickened, waxy appearance. This model thus reproduced the skin of erythropoietic protoporphyria clinically, microscopically, and at the ultrastructural level.

Serum levels of 8-methoxypsoralen in two different drug preparations: correlation with photosensitivity and UV-A dose requirements for photochemotherapy.

In a quantitative study we have compared the serum levels, the time course and the photosensitizing capacity of a conventional crystalline 8-methoxypsoralen brand and an investigational liquid formula. Evidence is presented showing that the liquid preparation is superior to the crystalline form: it peaks earlier after ingestion, it produces higher and more constant degree of photosensitization, it is eliminated more rapidly from the blood, and it requires a lower UV-A dose for eliciting photosensitivity reactions aiming at a reduction of the total cumulative UV-A dose required for clearing psoriasis.