CRISPR/Cas9-Mediated fech Knockout Zebrafish: Unraveling the Pathogenesis of Erythropoietic Protoporphyria and Facilitating Drug Screening.

Erythropoietic protoporphyria (EPP1) results in painful photosensitivity and severe liver damage in humans due to the accumulation of fluorescent protoporphyrin IX (PPIX). While zebrafish (Danio rerio) models for porphyria exist, the utility of ferrochelatase (fech) knockout zebrafish, which exhibit EPP, for therapeutic screening and biological studies remains unexplored. This study investigated the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated fech-knockout zebrafish larvae as a model of EPP1 for drug screening. CRISPR/Cas9 was employed to generate fech-knockout zebrafish larvae exhibiting morphological defects without lethality prior to 9 days post-fertilization (dpf). To assess the suitability of this model for drug screening, ursodeoxycholic acid (UDCA), a common treatment for cholestatic liver disease, was employed. This treatment significantly reduced PPIX fluorescence and enhanced bile-secretion-related gene expression (abcb11a and abcc2), indicating the release of PPIX. Acridine orange staining and quantitative reverse transcription polymerase chain reaction analysis of the bax/bcl2 ratio revealed apoptosis in fech-/- larvae, and this was reduced by UDCA treatment, indicating suppression of the intrinsic apoptosis pathway. Neutral red and Sudan black staining revealed increased macrophage and neutrophil production, potentially in response to PPIX-induced cell damage. UDCA treatment effectively reduced macrophage and neutrophil production, suggesting its potential to alleviate cell damage and liver injury in EPP1. In conclusion, CRISPR/Cas9-mediated fech-/- zebrafish larvae represent a promising model for screening drugs against EPP1.

Effects of iron supplements in individuals with erythropoietic protoporphyria.

BACKGROUND: Protoporphyrin IX (PPIX) is the final precursor of heme, forming heme when iron is inserted. Individuals with erythropoietic protoporphyrias (EPP) have accumulation of PPIX, causing photosensitivity and increased liver disease risk. Many also have iron deficiency and anemia. We investigated outcomes of oral iron supplements in individuals with EPP. METHODS: A systematic review identified literature on oral iron supplements in EPP patients. Subsequently, we administered iron supplements to EPP patients with iron deficiency. The primary outcome was impact on PPIX level. Secondary outcomes were adverse events and relative differences in hemoglobin and iron parameters. RESULTS: The systematic review found 13 case reports and one uncontrolled clinical trial with uncertain results. From our department 10 patients with EPP and iron deficiency took daily dosages of 330 mg of ferrous fumarate for two months. Five of our patients had anemia at baseline. After 2 months of supplementation seven patients had increased PPIX level compared to baseline, two had decrease, one remained unchanged. The administration of iron led to a rise in ferritin, and in four of the anemic patients also to an improvement in blood hemoglobin. A small transiently elevation in plasma alanine transaminase concentration was observed during supplementation. CONCLUSIONS: Overall, iron supplementation in EPP patients replenished iron stores and elevated erythrocyte PPIX and plasma alanine transaminase. For anemic patients, there was some degree of normalization of the hemoglobin level. If iron therapy is needed for EPP patients, monitoring of photosensitivity, PPIX, hemoglobin, and plasma liver enzymes is advisable.

The effects of cholecalciferol and afamelanotide on vitamin D levels in erythropoietic protoporphyria: a multicentre cohort study.

BACKGROUND: Patients with erythropoietic protoporphyria experience lifelong painful photosensitivity resulting in a lack of sunlight exposure. Previous studies have shown that 47-63% of patients with EPP suffer from vitamin D deficiency and a high prevalence of osteoporosis. An effective treatment for EPP has been available since 2016: the α-melanocyte stimulating hormone analogue afamelanotide. So far, studies on vitamin D levels in EPP have only investigated patients who have not been treated with afamelanotide. OBJECTIVES: To investigate the effects of afamelanotide treatment on vitamin D levels in EPP. METHODS: A multicentre observational cohort study in adults with EPP from the Erasmus Medical Centre, the Netherlands, and the University Hospital Düsseldorf, Germany, was carried out. Routinely collected vitamin D levels between 2005 and 2021 were used for analysis. Patient exposure to cholecalciferol or afamelanotide was categorized into four treatment groups: untreated, cholecalciferol, afamelanotide and combined treatment. A linear mixed model for longitudinal data was applied to measure the effect of the treatment groups compared with the untreated groups on vitamin D levels. RESULTS: A total of 230 patients and 1774 vitamin D measurements were included. The prevalence of vitamin D deficiency and severe deficiency remained high despite afamelanotide treatment (< 50 nmol L-1 in 71.8% of patients and < 30 nmol L-1 in 48.1%, respectively). Afamelanotide treatment alone did not lead to a significant average increase in vitamin D levels [ß = 0.5, 95% confidence interval (CI) -3.2 to 4.2]. In contrast, cholecalciferol and combined therapy with afamelanotide led to a significant increase in vitamin D levels [ß = 11.6 (95% CI 7.2-15.9) and ß = 15.2 (95% CI 12.3-18.1), respectively]. CONCLUSIONS: Cholecalciferol remains essential for the treatment of vitamin D deficiency in EPP, irrespective of new treatment options like afamelanotide. Afamelanotide treatment did not affect vitamin D levels. We suggest that future guidelines include continuous monitoring of vitamin D and a prescription for cholecalciferol in all patients with EPP, including those treated with afamelanotide.

Erythropoietic protoporphyria (EPP) is a rare inherited condition. People with EPP experience severe pain after their skin has been exposed to sunlight. To avoid this severe pain, people with EPP avoid going out in the sun by limiting outdoor activities or by wearing protective clothing. As sunlight is needed for our skin to produce vitamin D, approximately half of people with EPP in Europe do not have enough of it. In 2016, a new treatment called afamelanotide (SCENESSE®) became available, which allows people with EPP to go outside and expose themselves to sunlight longer without pain. In this study, we looked at how afamelanotide and vitamin D supplements affect vitamin D levels in people with EPP. We included information from patients treated in Rotterdam in the Netherlands and Düsseldorf in Germany and analysed levels of vitamin D in their blood. We also examined electronic patient files and collected questionnaires on the use of vitamin D supplements. In total, information from 230 patients was included. We found that afamelanotide alone did not raise vitamin D levels, but in combination with vitamin D supplements, vitamin D levels did go up. Even though afamelanotide is now available, our findings suggest that people with EPP may need more time to adapt to an outdoor lifestyle, after being conditioned to avoid sunlight since their childhood. Overall, our study demonstrates that vitamin D supplements remain crucial for people with EPP, with or without afamelanotide treatment.

Current trials in erythropoietic protoporphyria: are placebo controls ethical?

A new active substance called "dersimelagon" (MT-7117) is being tested as an alternative treatment option for Erythropoietic protoporphyria (EPP). At the moment, dersimelagon is being tested both in the US and in Europe in a phase III placebo-controlled RCT. However, given the availability of an already approved treatment option for EPP the use of a placebo arm is questionable from an ethics point of view. We analyze the issue and suggest that a noninferiority active-control trial without placebo is an ethically and scientifically more valid design to test the efficacy of dersimelagon as well as other EPP treatments.

Into the Light: Afamelanotide and the Treatment of Erythropoietic Protoporphyria in the United States.

BACKGROUND: Erythropoietic protoporphyria (EPP) is a rare disease that causes disabling cutaneous photosensitivity with pain and burning sensations. In 2019, afamelanotide, an α-melanocyte-stimulating hormone analogue, was approved in the United States for treatment of EPP. In this study, patients receiving afamelanotide filled out questionnaires assessing the benefit of treatment. Outcomes measured included: return to normal activities, experience of phototoxic reactions, effect on patient confidence, and more. Patients ranked their experience on a descriptive scale ranging from "very much" to "never". RESULTS: Prior to treatment, 75% of patients indicated that EPP affected their lives "very much" or "a lot". This number fell to 11% after the 1st implant and to 0% after each subsequent implant. The number of patients that willingly ventured outside increased with each subsequent implant. CONCLUSION: The results of this study clearly show that afamelanotide treatment can dramatically and positively impact the lives of EPP patients.  Citation: Resnik SR, Targett D, Resnik BI. Into the light: afamelanotide and the treatment of erythropoietic protoporphyria in the United States. J Drugs Dermatol. 2023;22(9):941-949. doi:10.36849/JDD.7126R1.

Dersimelagon in Erythropoietic Protoporphyrias.

BACKGROUND: Erythropoietic protoporphyria and X-linked protoporphyria are inborn errors of heme biosynthesis that cause elevated circulating levels of metal-free protoporphyrin and phototoxicity. Both disorders are characterized by excruciating phototoxic attacks after exposure to visible light. Dersimelagon is a new, orally administered, selective melanocortin 1 receptor agonist that increases levels of skin eumelanin. METHODS: We conducted a randomized, placebo-controlled, phase 2 trial to investigate the efficacy and safety of dersimelagon with respect to the time to onset and the severity of symptoms associated with sunlight exposure in patients with erythropoietic protoporphyria or X-linked protoporphyria. Patients 18 to 75 years of age were randomly assigned in a 1:1:1 ratio to receive placebo or dersimelagon at a dose of 100 or 300 mg once daily for 16 weeks. The primary end point was the change from baseline to week 16 in the time to the first prodromal symptom associated with sunlight exposure. Patients recorded daily sunlight exposure and symptom data in an electronic diary. Quality of life and safety were also assessed. RESULTS: Of the 102 patients (93 with erythropoietic protoporphyria and 9 with X-linked protoporphyria) who underwent randomization, 90% completed the treatment period. The mean daily time to the first prodromal symptom associated with sunlight exposure increased significantly with dersimelagon: the least-squares mean difference from placebo in the change from baseline to week 16 was 53.8 minutes in the 100-mg dersimelagon group (P = 0.008) and 62.5 minutes in the 300-mg dersimelagon group (P = 0.003). The results also suggest that quality of life improved in patients receiving dersimelagon as compared with placebo. The most common adverse events that occurred or worsened during treatment were nausea, freckles, headache, and skin hyperpigmentation. CONCLUSIONS: At both doses evaluated, dersimelagon significantly increased the duration of symptom-free sunlight exposure in patients with erythropoietic protoporphyria or X-linked protoporphyria. (Funded by Mitsubishi Tanabe Pharma; Endeavor ClinicalTrials.gov number, NCT03520036.).

Quality-Adjusted Life Years in Erythropoietic Protoporphyria and Other Rare Diseases: A Patient-Initiated EQ-5D Feasibility Study.

Erythropoietic protoporphyria (EPP) is an ultra-rare inborn error of metabolism characterised by painful phototoxic burn injuries after short exposure times to visible light. Patients with EPP are highly adapted to their condition which makes the quantification of their health-related quality of life (QoL) challenging. In the presented patient-initiated feasibility study, we describe a new approach to assess treatment benefits in EPP by measuring QoL with the generic EQ-5D instrument in five patients under long-term (≥two years) treatment with afamelanotide, the first approved therapy for EPP. For the study, we selected patients with EPP who in addition were affected by an involuntary treatment interruption (caused by a temporary reimbursement suspension) because we hypothesized that individuals who had previously unlearned their adaptation are better able to assess their life without treatment than treatment-naïve patients. QoL under treatment was comparable to the age-matched population norm, and retrospective results for a treatment interruption and phototoxic reaction time point were comparable to the QoL of patients with chronic neuropathic pain and acute burn injuries, respectively. The results were accepted by the National Institute for Health and Care Excellence in England for their evaluation of the cost-effectiveness of afamelanotide, i.e., the calculation of quality-adjusted life years.

Protection against visible light by dihydroxyacetone in erythropoietic protoporphyria.

BACKGROUND: Patients with erythropoietic protoporphyria (EPP) are hypersensitive to long wave ultraviolet (UVA) radiation and visible light and they experience severe skin pain by light exposure. The patients have very limited treatment options. Sunless skin tanning with dihydroxyacetone (DHA) is now being investigated as a possible treatment modality of skin photosensitivity in EPP. METHODS: We simulated the theoretical light protection factor provided by DHA application. In addition, we present 19 cases with EPP who were treated at our department with DHA weekly during spring and summer from 2018 to 2021 inclusive. RESULTS: The protection factor against UVA and visible light was estimated to approximately two. Out of the 19 patients with EPP who were treated with DHA in 2018, 11 patients experienced a sustained good effect and continued to use the treatment on a weekly basis in the spring and summer of 2019, 2020, and 2021. CONCLUSION AND PERSPECTIVES: Both the theoretical estimates and the uncontrolled study suggest that sunless tanning with DHA reduces photosensitivity in patients with EPP. Our hypothesis is that skin treated with DHA can tolerate twice the daylight dose compared to untreated skin before onset of skin symptoms. To validate this conclusion, we plan a randomized clinical trial to determine the effect of DHA application to reduce photosensitivity in patients with EPP under controlled clinical conditions. The study protocol for this trial is presented in the paper.

Erythropoietic protoporphyria in the Netherlands: Clinical features, psychosocial impact and the effect of afamelanotide.

Erythropoietic protoporphyria (EPP) patients experience severe burning pain after light exposure, which results in a markedly reduced quality of life. However, there is limited information on the psychosocial aspects of EPP. To investigate the clinical features and social aspects of living with EPP, before and during afamelanotide treatment in the Netherlands. A single-center prospective longitudinal study of adult patients with EPP attending the Erasmus MC Rotterdam. Patients completed questionnaires, comprising demographic, clinical and social details, including two generic (DS-14 and SF-36) and a disease specific (EPP-QoL) QoL questionnaires. 121 adult EPP patients were included. The educational level of EPP patients seemed higher compared to the Dutch population (36% vs. 30% high-education, 42% vs. 37% middle-education). At baseline 5% of the EPP patients were unemployed, none were unemployed during afamelanotide treatment. Full- and part-time employment rate increased from 59.5% to 69.9% on afamelanotide treatment (p > 0.05). EPP-QoL improved from 44% to 75% on afamelanotide treatment (p < 0.001). Type-D personality was present in 27.4% of patients; their social inhibition scores improved significantly on afamelanotide treatment (p = 0.019). EPP patients scored low on the social functioning domain (SF-36) compared to the Dutch population (74.4 ± 27.3 vs. 84.0 ± 22.4; respectively), and improved during afamelanotide treatment (84.3 ± 20.9, p = 0.001). EPP has a significant negative impact on social aspects, with less employment despite a higher education level. Afamelanotide treatment improves quality of life, social functioning and possibly employment rate. It is important to recognize the impact of EPP on social life, although, more research is needed.

Experimental and approved treatments for skin photosensitivity in individuals with erythropoietic protoporphyria or X-linked protoporphyria: A systematic review.

Erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP) are characterized by skin photosensitivity caused by accumulation of protoporphyrin IX. We aimed to review the clinical evidence of efficacy and safety of skin photosensitivity treatments in individuals with EPP or XLP. We systematically searched MEDLINE, Embase, the Cochrane Library, and ClinicalTrials.gov. A total of 40 studies with data on 18 treatment modalities were included. Comprehensive treatment safety data were obtained from the European Medicines Agency and the United States Food and Drug Administration. The studies used different outcome measures to evaluate the sensitivity without a generally accepted method to assess treatment effect on skin photosensitivity. Of the included studies, 13 were controlled trials. Gathered, the trials showed moderate positive effect of inorganic sunscreen application and subcutaneous implant of afamelanotide and no effect of organic sunscreen application, or oral treatment with beta-carotene, cysteine, N-acetylcysteine, vitamin C, or warfarin. Studies without control groups suggested treatment effect of foundation cream, dihydroxyacetone/lawsone cream, narrow-band ultraviolet B phototherapy, erythrocyte transfusion, extracorporeal erythrocyte photodynamic therapy, or oral treatment with zinc sulphate, terfenadine, cimetidine, or canthaxanthin, but the real effect is uncertain. Assessment of treatment effect on photosensitivity in patients with EPP or XLP carries a high risk of bias since experienced photosensitivity varies with both weather conditions, exposure pattern, and pigmentation. Controlled trials of promising treatment options are important although challenging in this small patient population.

Cimetidine Does Not Inhibit 5-Aminolevulinic Acid Synthase or Heme Oxygenase Activity: Implications for Treatment of Acute Intermittent Porphyria and Erythropoietic Protoporphyria.

Acute intermittent porphyria (AIP) is characterized by acute neurovisceral attacks that are precipitated by the induction of hepatic 5-aminolevulinic acid synthase 1 (ALAS1). In erythropoietic protoporphyria (EPP), sun exposure leads to skin photosensitivity due to the overproduction of photoreactive porphyrins in bone marrow erythroid cells, where heme synthesis is primarily driven by the ALAS2 isozyme. Cimetidine has been suggested to be effective for the treatment of both AIP and EPP based on limited case reports. It has been proposed that cimetidine acts by inhibiting ALAS activity in liver and bone marrow for AIP and EPP, respectively, while it may also inhibit the hepatic activity of the heme catabolism enzyme, heme oxygenase (HO). Here, we show that cimetidine did not significantly modulate the activity or expression of endogenous ALAS or HO in wildtype mouse livers or bone marrow. Further, cimetidine did not effectively decrease hepatic ALAS activity or expression or plasma concentrations of the putative neurotoxic porphyrin precursors 5-aminolevulinic acid (ALA) and porphobilinogen (PBG), which were all markedly elevated during an induced acute attack in an AIP mouse model. These results show that cimetidine is not an efficacious treatment for acute attacks and suggest that its potential clinical benefit for EPP is not via ALAS inhibition.

The effect of vitamin D recommendations on serum 25-hydroxyvitamin D level in patients with erythropoietic protoporphyria.

OBJECTIVES: Patients with erythropoietic protoporphyria (EPP) avoid sun exposure owing to photosensitivity. For decades, sun-avoiding Danes have been recommended daily vitamin D supplements all year. We offered our EPP patients serum 25-hydroxyvitamin D (25(OH)D) monitoring, and counseling if their level was low. We aimed to investigate the effect of the general recommendation and counseling on 25(OH)D status in patients attending our clinic. Additionally, the 25(OH)D status of our EPP patients was compared to that of British patients with EPP not taking vitamin D supplements and with that of the general Danish population. METHODS: Forty-six Danish patients with EPP had 25(OH)D measured in 721 blood samples collected between 2003 and 2021. Dates of individual counseling were noted. Data on British patients with EPP and the general Danish population were extracted from previous publications. RESULTS: Our patients had higher 25(OH)D levels than British patients with EPP not taking vitamin D supplements, but the recommendations did not elevate their 25(OH)D levels to that of the general Danish population. Overall, 17.5% of the 25(OH)D measurements in our EPP patients were below 30 nmol/L (deficiency) and 29.4% were between 30 and 50 nmol/L (insufficiency). Patients were monitored for a median of 11 y. Thirty-one patients had a total of 74 vitamin D counseling sessions, providing an increase in 25(OH)D of about 18 nmol/L the year after. However, many patients repeatedly developed insufficiency. CONCLUSIONS: This study documents the positive effect of vitamin D recommendations on serum 25(OH)D in patients with EPP. Follow-up on vitamin D status and recommendations is essential to increase 25(OH)D levels.