Vemurafenib-related photosensitivity

Bernadette Eberlein1, Tilo Biedermann1, Rüdiger Hein1, Christian Posch1,2

(1)Department of Dermatology and Allergology, Biederstein, School
of Medicine, Technical University, Munich, Germany
(2)Medical Faculty, Sigmund Freud Private University, Vienna, Austria

Introduction
Summary
Increased photosensitivity is a common cutaneous adverse effect associated with the BRAF inhibitor vemurafenib. Clinically, it presents as an immediate sensation of heat and edematous erythema during sun exposure, as well as a sunburn reaction in terms of a late reaction. Phototesting has shown that the UVA range (320 nm to 400 nm), triggers both the immediate and the late reaction. In terms of pathogenesis, photo- chemical studies have suggested that exposure of vemurafenib to UVA radiation pro- duces an UVA-absorbing photoproduct. In vitro studies on various cell models have also demonstrated that the phototoxic effects of vemurafenib are exclusively caused by UVA irradiation. This latter mechanism is probably responsible for the photosensi- tivity clinically observed in patients receiving vemurafenib.
In addition, vemurafenib is able to inhibit ferrochelatase. The resulting increa- se in protoporphyrin IX has also been observed in some human studies involving the drug. However, it is yet unproven whether porphyrins actually contribute to the immediate skin reactions seen in individuals on vemurafenib, even though the cli- nical presentation is similar to that found in erythropoietic protoporphyria with a comparable pathomechanism.
Other BRAF inhibitors, such as dabrafenib and encorafenib, are associated with significantly lower photosensitivity. It is essential that patients treated with vemura- fenib are informed about immediate and delayed reactions potentially caused even by low doses of UVA. This includes counseling on photoprotective measures.

Clinical characteristics of

By absorbing ultraviolet (UV) radiation, photosensitizing drugs can trigger chemical reactions, which either cause cellular damage (phototoxicity) or, less frequently, produce photoallergens (photoallergy). One such drug is vemurafen- ib. In the European Union, the agent has been approved for the treatment of patients with inoperable or metastatic BRAF V600-mutant melanoma since 2012. Vemurafenib inhibits the oncogene B-Raf, a serine-threonine kinase involved in regulating cell proliferation [1]. Animal models, in vitro tests and biochemical analyses have helped to further elucidate the pathomechanism resulting in increased photosensitivity in individuals on vemurafenib therapy. In vivo studies in hu- mans have primarily used the minimal erythema dose (MED) as a measure of photosensitivity and for determining the ac- tion spectrum. MED is defined as the lowest dose of UV ra- diation that results in a well-defined erythema 24 ± 2 hours after irradiation.
photosensitivity (Table 1)
Even in the first pivotal studies, it was noted that up to 52 % of patients experienced increased photosensitivity [2, 3]. Af- fected individuals reported severe, blistering sunburn reac- tions (Figure 1) as well as an immediate sensation of heat and solar urticaria-like erythema on exposure to sunlight; these reactions also occurred even behind car windows (transmis- sion of UVA radiation, absorption of UVB radiation). Three individuals experienced the same kind of reactions after exposure to fluorescent lamps indoors [4]. Phototesting in five patients showed normal results in the UVB range (285 to 350 nm; peak at 310 to 315 nm) but an action spectrum in the UVA range (330 to 450 nm, peak at 390 to 410 nm). All patients showed a reaction already after ten minutes, as well as intense erythema with severe edema at the test site after 24 hours [5]. Monochromator phototesting in one patient confirmed an action spectrum in the UVA range at

© 2020 The Authors. Journal der Deutschen Dermatologischen Gesellschaft published by John Wiley & Sons Ltd on behalf of Deutsche Dermatologische Gesellschaft. | JDDG | 1610-0379/2020 1
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Figure 1 65-year-old female patient on vemurafenib therapy. Sunburn-like reaction two days after 30-minute sun exposure during lawn mowing. The skin lesions healed within four weeks.

335 ± 27 nm, 365 ± 27 nm and 400 ± 27 nm (no reaction to 305 ± 5 nm [UVB] and 430 ± 27 nm [blue light]) after one month of vemurafenib therapy. Immediate readings revealed no reaction [6].
A study that compared photosensitivity prior to and af- ter two months of vemurafenib therapy showed a decrease in UVA MED (readings after 20 min) in 17 of 18 patients (94.4 %), with a median UVA MED of 12 J/cm2 during vemurafenib treatment (MED < 20 J/cm2 is considered pa- thological). The MED remained unchanged when using a sunlight simulator. In this particular study patients also de- veloped vemurafenib-induced erythema (solar urticaria-like lesions without edema or pruritus) during UVA exposure. The associated burning sensation resolved quickly after UV exposure had been stopped, whereas the erythema persisted for a period of 24 hours and was no longer detectable after seven days. The study showed a significant increase in ery- throcyte porphyrins (predominantly zinc protoporphyrin) and a decrease in vitamin B3 (niacinamide) levels after two months of vemurafenib therapy. These tests were performed based on the fact that the skin reactions clinically resembled erythropoietic protoporphyria (EPP) [7].
In another study, the one patient under investigation showed an increase in total erythrocyte protoporphyrin only after several months of vemurafenib treatment; here, abnor- mal phototest results did not correlate with higher-than-nor- mal porphyrin levels [6].
Yet another study showed similar results: While ele- ven out of twelve patients saw a decrease in UVA MED (UVA light source with an emission spectrum of 330 nm to 450 nm), there were no changes in MED when using a po- lychromatic light source with UVB (305 nm to 750 nm) [8]. Other studies investigated whether photosensitivity might also be triggered by visible light, but phototesting (using a
halogen lamp) before and after two months of vemurafenib treatment was negative. The immediate UVA-related photo- sensitivity disappeared within one to two weeks after dis- continuation of the drug. Again, UVA MED was decreased in 30 of 34 patients (88 %), with a median of 12 J/cm2 after two months of vemurafenib therapy in this study [9].

Studies in animal models

Using an established mouse model (oral UV-local lymph node assay, UV-LLNA), scientists studied the role of various fac- tors in terms of the development of photosensitivity and es- tablished a pharmacokinetic profile. These factors included both the formulation and dose of vemurafenib, the duration of treatment as well as the timing of the readings following UV exposure.
The backs and ears of Balb/c mice were exposed to 10 J/
cm2 of UVA generated by a sunlight simulator with H1 filter (320 nm up to more than 590 nm). Exposure occurred on three consecutive days, two hours after oral administration of various doses of vemurafenib, either in a crystalline form or an amorphous form; alternatively, the mice were expo- sed once after three days of vemurafenib treatment. Biopsies from the apical portion of the ears were taken 24 hours after the last exposure, and the lymph nodes were characterized on a cellular level. In a further approach, the ears were exa- mined 1, 2, 3, 4 and 6 hours after UV exposure.
Crystalline vemurafenib and amorphous vemurafen- ib (corresponding to the commercially available preparati- on) at a dose of 100 mg/kg did not elicit any phototoxici- ty. After oral administration of amorphous vemurafenib at doses of 350, 450 and 800 mg/kg, erythema was observed immediately after UV exposure on the last day of the three- day treatment period. As this effect faded within 15 hours,

examination of the ears and lymph nodes 24 hours after UV exposure revealed no abnormalities. However, six hours af- ter exposure, there was a pronounced erythema and edema of the ears [10].

Biochemical analyses and in vitro tests

During the early development of the drug, it was already no- ted that vemurafenib exhibited phototoxic properties. This clinically relevant feature was shown to be due to a certain chemical structural element (benzophenone chromophore) and an absorption of UV radiation at wavelengths of up to 350 nm [10, 11]. Spectrofluorometric analyses found peaks at 210 nm, 260 nm (UVC spectrum) and 310 nm (UVB spec- trum), both in lyophilized vemurafenib as well as in serum and feces of patients treated with the drug. Hence, it was con- cluded that vemurafenib-related UVA phototoxicity could not be explained by the absorption spectrum of the substance but possibly by the formation of newly generated metabolites [8]. Photochemical analyses suggest that UVA irradiation of vemurafenib produces singlet oxygen and free radicals and, under certain conditions, generates a UVA-absorbing photo- product that might also be responsible for in vivo photosen- sitivity [12].
In vitro studies of murine fibroblast cell lines irradia- ted using a light source emitting predominantly UVA and visible light (320 nm up to > 700 nm) (in vitro 3T3 Neu- tral Red Uptake phototoxicity test) provided evidence of vemurafenib-induced phototoxicity as well [11]. It has also been shown that vemurafenib induces lipid peroxidation in erythrocytes and may thus lead to photohemolysis following UVA irradiation (300 nm up to 410 nm, λmax.: 350 nm) [13]. In another study, cytotoxic effects in HaCaT keratinocy- tes incubated with vemurafenib occurred after exposure to UVA (λmax.: 365 nm), but not when irradiated with visib- le (blue) light (λmax.: 420 nm, no radiation < 400 nm), in contrast to the comparative assay using protoporphyrin IX (PPIX). No intracellular PPIX was detected in the presence of vemurafenib [6].
While vemurafenib did have UVA-induced phototoxic effects on other cell lines, too, these effects were less depen- dent on singlet oxygen compared to other known photosen- sitizers such as fluoroquinolones. Highly toxic combinations of vemurafenib and UVA caused less protein carbonylation but nevertheless had inhibitory effects on nucleotide exci- sion repair and were associated with reduced protection against mutagenic and carcinogenic DNA damage [14]. The fact that no such changes in DNA repair have been observed with dabrafenib suggests that this effect may be specific to vemurafenib [15].
In addition, it has been shown that vemurafenib – unlike dabrafenib, but similar to other kinase inhibitors – inhibits

the enzyme ferrochelatase by blocking the protoporphyrin binding site [16].

Discussion

Vemurafenib is a drug with specific photosensitizing proper- ties, which may be attributed to various pathogenetic causes. While photosensitizing drugs usually (with some exceptions such as chlorpromazine) lead to an increased (delayed) sun- burn reaction, vemurafenib additionally causes an immediate reaction during and directly after UV exposure, characteri- zed by erythema, edema as well as a sensation of burning and heat. Clinically, this immediate reaction resembles EPP, a condition characterized by partial deficiency in the enzyme ferrochelatase, which results in an increase in protoporphy- rin IX (PPIX) [17].
Similar to other phototoxic agents, the action spec- trum for both the immediate and the delayed reaction du- ring vemurafenib therapy seems to be strictly limited to wa- velengths between 320 nm and 400 nm (UVA spectrum). Neither UVB-rich irradiation nor visible light have been shown to be able to elicit these reactions. Remarkably, only immediate-type reactions were reproducible in animal mo- dels. In vitro studies on cell lines and erythrocytes support the drug’s UVA-induced phototoxic mechanism of action, which appears to be the primary cause, at least of the delayed reaction. The same studies also suggest inhibitory effects on DNA damage repair. However, the occurrence of squamous cell carcinoma (SCC), which is known to develop more fre- quently and rapidly in patients treated with BRAF inhibitors, is likely not associated with the phototoxic properties of the- se agents [18].
Apart from the direct phototoxic properties, some pa- tients on long-term vemurafenib therapy have also been shown to have elevated protoporphyrin levels. On a bioche- mical level, it has been demonstrated that vemurafenib is associated with ferrochelatase inhibition by binding to the protoporphyrin site of the enzyme, which may explain the increase in PPIX and the EPP-like immediate reactions. Ho- wever, the action spectrum for the clinical manifestations of EPP is in the visible light spectrum, especially in the Soret band (400 nm to 415 nm), and is therefore different from the action spectrum of vemurafenib-related phototoxicity (wavelengths ≤ 400 nm). Given that phototesting with vi- sible light at higher doses is not standardized (use of slide projector lamps) and given that the monochromator study only included one patient, it is currently impossible to con- clusively determine the actual significance of elevated por- phyrin levels for photosensitivity in humans treated with Vemurafenib.
Other BRAF inhibitors such as dabrafenib and encora- fenib are associated with significantly lower photosensitivity

(1–3 %) and a less pronounced decrease in UVA MED. The- se agents might therefore represent a treatment alternative [9, 19, 20].
It is essential that patients treated with vemurafenib are informed about the immediate and delayed reactions potenti- ally triggered even by low doses of UVA [21]. Adequate coun- seling should be offered with respect to their behavior (UVA radiation even in the evening sun), wearing sun-protective clothing and the use of sunscreens with broad-spectrum UV filters (UVA protection attention to the UVA-label).

Correspondence to

Prof. Bernadette Eberlein, MD
Department of Dermatology and Allergology Biederstein Technical University of Munich
Biedersteiner Straße 29 80802 Munich, Germany
E-mail: [email protected]

References
1Flaherty KT, Puzanov I, Kim KB et al. Inhibition of mutated, ac- tivated BRAF in metastatic melanoma. N Engl J Med 2010; 363: 809–19.
2Chapman PB, Hauschild A, Robert C et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med 2011; 364: 2507–16.
3Lacouture ME, Duvic M, Hauschild A et al. Analysis of derma- tologic events in vemurafenib-treated patients with mela- noma. Oncologist 2013; 18: 314–22.
4Boudewijns S, Gerritsen WR, Koornstra RH. Case series: in- door-photosensitivity caused by fluorescent lamps in patients treated with vemurafenib for metastatic melanoma. BMC Can- cer 2014; 14: 967.
5Dummer R, Rinderknecht J, Goldinger SM. Ultraviolet A and photosensitivity during vemurafenib therapy. New Engl J Med 2012; 366; 5: 480–1.
6Woods JA, Ferguson JS, Kalra S et al. The phototoxicity of vemurafenib: An investigation of clinical monochromator phototesting and in vitro phototoxicity testing. J Photochem Photobiol B 2015; 151: 233–8.
7Gelot P, Dutartre H, Khammari A et al. Vemurafenib: an un- usual UVA-induced photosensitivity. Exp Dermatol 2013; 22: 290–301.

8Brugière C, Stefan A, Morice C et al. Vemurafenib skin photo- toxicity is indirectly linked to ultraviolet A minimal erythema dose decrease. Br J Dermatol 2014; 171: 1529–32.
9Gabeff R, Dutartre H, Khammari A et al. Phototoxicity of B-RAF inhibitors: Exclusively due to UVA radiation and rapidly regres- sive. Eur J Dermatol 2015; 25: 452–6.
10Boudon S, Plappert-Helbig U, Odermatt A, Bauer D. Charac- terization of vemurafenib phototoxicity in a mouse model. Toxicol Sci 2014; 137: 259–67.
11Placzek M, Dendorfer M, Przybilla B et al. Photosensitizing properties of compounds related to benzophenone. Acta Derm Venereol 2013; 93: 30–2.
12Morlière P, Boscá F, Silva AM et al. A molecular insight into the phototoxic reactions observed with vemurafenib, a first-line drug against metastatic melanoma. Photochem Photobiol Sci 2015; 14: 2119–27.
13Teixeira A, Morlière P, Ferreira J et al. Interplay between mem- brane lipid peroxidation and photoproduct formation in the ultraviolet A-induced phototoxicity of vemurafenib in skin keratinocytes. Toxicol Sci 2016; 154: 289–95.
14Peacock M, Brem R, Macpherson P, Karran P. DNA repair in- hibition by UVA photoactivated fluoroquinolones and vemu- rafenib. Nucleic Acids Res 2014; 42: 13714–22.
15Kimeswenger S, Mann U, Hoeller C et al. Vemurafenib impairs the repair of ultraviolet radiation-induced DNA damage. Mela- noma Res 2019; 29:134–44.
16Klaeger S, Gohlke B, Perrin J et al. Chemical proteomics reveals ferrochelatase as a common off-target of kinase inhibitors.
ACS Chem Biol 2016; 11: 1245–54.
17Urbanski U, Frank J, Neumann NJ. Erythropoetische Protopor- phyrie. Klinik, Diagnostik und neue therapeutische Möglich- keiten. Hautarzt 2016; 67: 211–5.
18Wu JH, Cohen DN, Rady PL, Tyring SK. BRAF inhibitor-associ- ated cutaneous squamous cell carcinoma: new mechanistic insight, emerging evidence for viral involvement and per- spectives on clinical management. Br J Dermatol 2017; 177: 914–23.
19Hauschild A, Grob JJ, Demidov LV et al. Dabrafenib in BRAF-mutated metastatic melanoma: a multicenter, open-
label, phase 3 randomized controlled trial. Lancet 2012; 380: 358–65.
20Trojaniello C, Festino L, Vanella V, Ascierto PA. Encorafenib in combination with binimetinib for unresectable or metastatic melanoma with BRAF mutations. Expert Rev Clin Pharmacol 2019; 12: 259–66.
21Rinderknecht JD, Goldinger SM, Rozati S et al. RASopathic
skin eruptions during vemurafenib therapy. PLoS One 2013; 8: e58721.LGX818