Accumulation Of Protoporphyrin Research Articles

CLPP-Null Eukaryotes with Excess Heme Biosynthesis Show Reduced L-arginine Levels, Probably via CLPX-Mediated OAT Activation.

The serine peptidase CLPP is conserved among bacteria, chloroplasts, and mitochondria. In humans and mice, its loss causes Perrault syndrome, which presents with growth deficits, infertility, deafness, and ataxia. In the filamentous fungus Podospora anserina, CLPP loss leads to longevity. CLPP substrates are selected by CLPX, an AAA+ unfoldase. CLPX is known to target delta-aminolevulinic acid synthase (ALAS) to promote pyridoxal phosphate (PLP) binding. CLPX may also influence cofactor association with other enzymes. Here, the evaluation of P. anserina metabolomics highlighted a reduction in arginine/histidine levels. In Mus musculus cerebellum, reductions in arginine/histidine and citrulline occurred with a concomitant accumulation of the heme precursor protoporphyrin IX. This suggests that the increased biosynthesis of 5-carbon (C5) chain deltaALA consumes not only C4 succinyl-CoA and C1 glycine but also specific C5 delta amino acids. As enzymes responsible for these effects, the elevated abundance of CLPX and ALAS is paralleled by increased OAT (PLP-dependent, ornithine delta-aminotransferase) levels. Possibly as a consequence of altered C1 metabolism, the proteome profiles of P. anserina CLPP-null cells showed strong accumulation of a methyltransferase and two mitoribosomal large subunit factors. The reduced histidine levels may explain the previously observed metal interaction problems. As the main nitrogen-storing metabolite, a deficiency in arginine would affect the urea cycle and polyamine synthesis. Supplementation of arginine and histidine might rescue the growth deficits of CLPP-mutant patients.

Application of a Validated Method for Quantifying Circulating Protoporphyrin IX to the Beacon Trial of Bitopertin in Erythropoietic Protoporphyria

Introduction: Accumulation of the photoreactive heme precursor protoporphyrin IX (PPIX) is a signature of the disease pathophysiology for both erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP), and PPIX levels correlate with disease severity and light tolerance. Additionally, an elevated ratio of circulating metal-free PPIX vs. metal-bound PPIX is observed in patients with protoporphyrias, and the extent of metal-free elevation is distinguishable between EPP (85-100%) and XLP (50-85%). Therefore, the accurate determination of metal-free PPIX and metal-bound PPIX is of particular interest in patients with EPP or XLP. Methods: Novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays were developed and validated to quantify circulating metal-free and zinc (Zn)-complexed PPIX in whole blood and plasma. Each of the four assays were developed and validated according to applicable regulatory guidance for bioanalytical assay validation with regard to accuracy, precision, sensitivity, and selectivity. The minimum required sample volume for each assay was 200 µL, compared to >1 mL for most currently available protoporphyria-related assays. During bioanalytical method development, rapid degradation of metal-free PPIX (≥90%) and Zn-PPIX (up to 96%) was observed in plasma samples within 24 hours under ambient light conditions. In contrast, whole blood was less susceptible to photodegradation (i.e., ≤20% for both metal-free PPIX and Zn-PPIX) under the same conditions. The most effective method for maintaining sample integrity was reduction of light exposure during sample collection, handling, and processing. Implementation of light-reduction controls was used during method validation and facilitated improved stability of metal-free PPIX and Zn-PPIX in plasma and whole-blood samples. For the metal-free PPIX plasma assay validation, the effect of hemolysis was evaluated due to the apparent sequestration of metal-free PPIX in red blood cells. The bioanalytical work informed specific sample-collection procedures to mitigate instability and hemolysis, such as the use of opaque consumables for sample collection, controls for plasma aliquoting, and operating under low-light conditions for all sample handling and processing. BEACON is a Phase 2, randomized, open-label, parallel-arm trial (ACTRN12622000799752) of approximately 22 participants who will receive oral, once-daily administration of 20 or 60 mg of bitopertin for 6 months. Baseline light tolerance was assessed using 2 patient-reported outcomes measures during a 2- to 4-week screening period. Participants were asked to conduct weekly sun exposure challenges and time to first prodromal symptom (itching, burning, tingling, etc.) was averaged for the screening period. Results: More than 600 clinical samples from 17 participants in the BEACON study have been collected as of 06 June 2023 and analyzed using the validated porphyrin assays. Measured metal-free PPIX baseline values ranged from 1440 - 34,100 ng/mL (2.6 - 61 µM) in whole blood and 177 - 2560 ng/mL (0.31 - 4.5 µM) in plasma ( n=16). Baseline percentage of intra-patient plasma/whole-blood concentrations ranged from 2.5 - 16%. Each participant's baseline values were converted to erythrocyte metal-free PPIX concentrations using measured hematocrit levels and ranged from 3590 - 88400 ng/mL (6.4 - 157 µM). These values were comparable to data previously reported in the literature (6-139 µM 1). Additionally, higher baseline whole-blood metal-free PPIX concentrations were associated with decreased averages for time to prodrome (Table 1). Conclusion: Fully-validated LC-MS/MS assays to quantify metal-free PPIX and Zn-PPIX in plasma and whole blood were used to analyze clinical samples from the BEACON study evaluating bitopertin in EPP. PPIX values were consistent with previously reported values for patients with EPP. The methods also provided improved accuracy and specificity for metal-free vs. metal-complexed PPIX and reduced sample volume requirements. In addition, higher baseline whole-blood PPIX values were associated with decreased light tolerance within the trial population, supporting its use as a measure of disease severity in EPP.

Interim Analyses from the Beacon Trial: A Phase 2, Randomized, Open-Label Trial of Bitopertin in Erythropoietic Protoporphyria

Introduction: Erythropoietic protoporphyria (EPP) is associated with accumulation of photoreactive protoporphyrin IX (PPIX) in the skin and other organs, causing debilitating phototoxic skin reactions following exposure to sunlight, and potentially life-threatening protoporphyric hepatopathy in some patients. Reduction of PPIX is associated with amelioration of disease in the settings of hematopoietic stem cell transplant, pregnancy, and extracorporeal photoinactivation. Glycine transporter 1 (GlyT1) supplies extracellular glycine for the initial step of heme biosynthesis in erythroid cells. Bitopertin is an investigational, orally-administered inhibitor of GlyT1. It is hypothesized that GlyT1 inhibition leads to a decrease in heme pathway intermediates, including PPIX, and can improve light tolerance. Methods: BEACON is a Phase 2, randomized, open-label, parallel-arm trial (ACTRN12622000799752) of 22 participants who will receive oral, once-daily administration of 20 mg or 60 mg of bitopertin for 24 weeks. The trial is being conducted at 2 sites in Australia and includes participants ≥18 years of age with a confirmed diagnosis of EPP. The primary efficacy endpoint is percent change in whole-blood metal-free PPIX. Additional endpoints include daily patient-reported outcomes (PRO) of light tolerance and quality of life, as well as safety and tolerability. Results: As of data cutoff (05 July 2023), a total of 17 subjects had been enrolled. Treatment with bitopertin resulted in mean (SD) decreases in PPIX of -39% ± 17% by Day 43 (n=12), with more pronounced decreases observed with 60 mg compared to 20 mg (Figure 1). Bitopertin also improved multiple measures of light tolerance. A participant randomized to 20 mg bitopertin reported a >80-fold increase in sunlight tolerance on Day 88 of treatment, increasing from 4.5 minutes at baseline to over 6 hours; no prodromes were reported during any sunlight challenge after Day 20. A participant randomized to 60 mg bitopertin reported a >200-fold increase in sunlight tolerance on Day 74 of treatment, increasing from 1.3 minutes at baseline to over 4 hours, and did not report a prodrome during any sunlight challenge after Day 120. Aggregate measures of light tolerance also improved over time, including time to prodrome averaged over a 2-week period and weekly averages of total time in sunlight. The proportion of prodrome-free sunlight challenges increased from 2% during screening to 49% while receiving bitopertin (n=17), and the proportion of days without symptoms (with sun exposure) increased from 26% during screening to 74% while receiving bitopertin (n=17). Improvements in weekly averages of total time in sunlight were consistent and observed in 15/15 participants with post-baseline PPIX values. The greatest increases in weekly averages of total time in sunlight were observed in participants who experienced reductions in PPIX of at least 30% (Figure 2), for whichtotal time in sunlight increased from a weekly average of 339 minutes at baseline to a maximum of 999 minutes with bitopertin (n=11; mean duration of treatment: 133 days [range: 76 to ≥169 days]). Patient-reported phototoxic reactions decreased by 93% while on treatment compared to baseline (n=17). By Day 43, 15/16 participants reported in the Patient Global Impression of Change (PGIC) their EPP was much better or a little better, and 15/16 reported in the Patient Global Impression of Severity (PGIS) their EPP was mild or not at all severe. A majority (13/16) of participants responded in a novel PRO, the EPP Impact Questionnaire (EPIQ), that EPP had little or no impact on their quality of life while receiving bitopertin. No serious adverse events, discontinuations due to adverse events, or dose reductions have been reported. A total of 12 (71%) participants reported treatment-emergent adverse events; only dizziness and headache were reported in more than one subject. No meaningful changes in hemoglobin have been observed. Conclusion: By reducing whole-blood PPIX levels, bitopertin targets the underlying pathophysiology of EPP, resulting in consistent improvements in multiple measures of light tolerance and quality of life. Bitopertin has been well tolerated to date with no changes in hemoglobin, and its safety profile in EPP is consistent with prior studies that enrolled more than 4000 participants. Updated results will be presented at the meeting.

The Relationship between Protoporphyrin IX and Platelet Count in the Protoporphyrias

Introduction: Erythropoietic protoporphyria and X-linked protoporphyria, collectively known as the protoporphyrias, are inherited disorders of heme biosynthesis characterized by the accumulation of protoporphyrin IX in the skin and other organs. Clinical manifestations include severe pain with exposure to sunlight, as well as liver disease in a subset of patients. Approximately half of patients with protoporphyria have mild microcytic anemia, which is not well understood but thought to be related either to decreased iron availability or to decreased heme biosynthesis. Several reports have described mild thrombocytopenia in patients with protoporphyria (Delaby et al., 2009, Wensink et al., 2022), although thrombocytosis would be expected with iron deficiency as low iron biases megakaryocyte-erythroid progenitors to the megakaryocyte lineage (Xavier-Ferrucio et al., 2019). We sought to further characterize the relationship between erythrocyte metal-free protoporphyrin IX (PPIX) levels, platelet count, and iron status in patients with protoporphyria. Methods: We conducted a single-center cohort study of adults with protoporphyria treated at the Massachusetts General Hospital Porphyria Center from March 2021 through June 2023. Patient-level data across multiple time points were collected, including platelet count, ferritin, and alanine transaminase (ALT). Generalized Estimating Equation (GEE) models with an identity link and gaussian family distribution were performed to determine the association, accounting for within-participant associations. Results: A total of 38 patients with protoporphyria were included in the analysis. The median PPIX level was 1169.5ug/dl (interquartile range [IQR] 729.50 - 2069.75, reference range <20). The median platelet count was 208 x10 9/L (IQR, 171-243, reference range 150-400). Only 3 patients had a platelet count <150 x10 9/L and none <100 x10 9/L. A 1ug/dl increase in protoporphyrin level was associated with an 0.02 x10 9/L decrease in platelet count on average (p= < 0.00001, Figure). We next sought to evaluate whether the relationship between PPIX and platelet count was affected by iron status (assessed by ferritin) or liver disease (assessed by ALT). When adjusting for ferritin and ALT, the relationship between PPIX and platelet count remained statistically significant (p= < 0.00001). When adjusting for PPIX and ferritin, an increase in platelets was associated with an increase in ALT (p=0.002), but there was no significant relationship between ferritin and PPIX when adjusting for the other variables in the model. Conclusion: In this study of 38 patients with protoporphyria, higher levels of PPIX were associated with lower platelet counts. This association was independent of markers of iron status or hepatic dysfunction. Only 3 patients had thrombocytopenia, which was mild in all cases. Although increased PPIX was not associated with clinically significant thrombocytopenia, the reason for the relationship between PPIX and platelets in this patient population remains unclear. Considering the well-described risk of anemia in protoporphyria, there may exist some unknown effect on hematologic cells and their precursors, either due to the accumulation of PPIX or variants in heme biosynthetic enzymes. Understanding this fundamental biology may pave the way for further treatment targets in protoporphyria and other hematologic conditions.

PD13 Severe cutaneous manifestations in an Indian child with X-linked dominant erythropoietic protoporphyria

Abstract Erythropoietic protoporphyria (EPP) is usually caused by autosomal dominant mutations in FECH: raised protoporphyrin levels cause itching, burning and erythema after sun exposure, with facial scarring and sometimes liver damage. We report a child with the much rarer X-linked dominant form (XLDPP) and consider reasons for his unusually severe features. A 4-year-old boy living in India presented with long-standing erosions and scarring on the face and sides of the feet, and marked distal onycholysis with subungual hyperkeratosis affecting all nails, suggestive of pachyonychia congenita (PC). There was no relevant family history or parental consanguinity. He was anaemic (haemoglobin 7.2 g dL−1). Genetic testing revealed no variants in PC genes, but there was a hemizygous deletion in ALAS2, c.1706_1709del (p.Glu569GlyfsT er24) reported recurrently in XLDPP. Further review revealed a history of photosensitivity, low vitamin D3 and raised liver enzymes. Measurement of erythrocyte protoporphyrin was not available, but XLDPP was diagnosed on the basis of photosensitivity, liver abnormalities, probable photo-onycholysis and genetic testing. ALAS2 encodes 5-aminolaevulinate synthase, the first enzyme in the haem synthetic pathway. Most ALAS2 mutations block haemoglobin production resulting in sideroblastic anaemia. However, specific deletions, including this one, activate the pathway, increasing protoporphyrin to levels higher than what can be used in haematopoiesis. The manifestations of XLDPP therefore resemble EPP: protoporphyrin accumulation is caused in the former by increased production and in the latter by a block in its conversion to haem. Iron deficiency, common in Indian children, reduces haem production and consequently protoporphyrin utilization, potentially exacerbating the protoporphyrin accumulation caused by XLDPP. In a previous patient with XLDPP and anaemia due to gastrointestinal bleeding, iron therapy successfully lowered red cell protoporphyrin levels (Whatley SD, Ducamp S, Gouya L et al. C-terminal deletions in the ALAS2 gene lead to gain of function and cause X-linked dominant protoporphyria without anemia or iron overload. Am J Hum Genet 2008; 83:408–14). Nail dystrophy has not previously been reported in XLDPP. The appearance here is consistent with photo-onycholysis in a child whose feet are always exposed in sandals. The delay in diagnosing photosensitivity and severity of his skin changes can be attributed to the difficulty in interpreting symptoms in young children and to local conditions where sun exposure is unavoidable. Management here involves iron therapy, photoprotection, monitoring of liver function and genetic counselling; female carriers may also have symptoms. We are grateful to national experts for helpful discussion of this case.

The in vitro anti-cancer synergy of neurokinin-1 receptor antagonist, aprepitant, and 5-aminolevulinic acid in glioblastoma.

Glioblastoma multiforme (GBM) is the most malignant type of cerebral neoplasm in adults with a poor prognosis. Currently, combination therapy with different anti-cancer agents is at the forefront of GBM research. Hence, this study aims to evaluate the potential anti-cancer synergy of a clinically approved neurokinin-1 receptor antagonist, aprepitant, and 5-aminolevulinic acid (5-ALA), a prodrug that elicits fluorescent porphyrins in gliomas on U-87 human GBM cells. We found that aprepitant and 5-ALA effectively inhibited GBM cell viability. The combinatorial treatment of these drugs exerted potent synergistic growth inhibitory effects on GBM cells. Moreover, aprepitant and 5-ALA induced apoptosis and altered the levels of apoptotic genes (up-regulation of Bax and P53 along with downregulation of Bcl-2). Furthermore, aprepitant and 5-ALA increased the accumulation of protoporphyrin IX, a highly pro-apoptotic and fluorescent photosensitizer. Aprepitant and 5-ALA significantly inhibited GBM cell migration and reduced matrix metalloproteinases (MMP-2 and MMP-9) activities. Importantly, all these effects were more prominent following aprepitant-5-ALA combination treatment than either drug alone. Collectively, the combination of aprepitant and 5-ALA leads to considerable synergistic anti-proliferative, pro-apoptotic, and anti-migratory effects on GBM cells and provides a firm basis for further evaluation of this combination as a novel therapeutic approach for GBM.

Afamelanotide Is Associated with Dose-Dependent Protective Effect from Liver Damage Related to Erythropoietic Protoporphyria.

In this retrospective observational study, we included 2933 liver-function tests, 1186 PPIX concentrations and 1659 afamelanotide implant applications in 70 EPP patients. We investigated whether the number of days since the preceding afamelanotide dose or the number of doses during the preceding 365 days had an effect on LFTs and PPIX levels. In addition, we assessed the effect of global radiation. Inter-patient differences exerted the most prominent effect on PPIX and LFTs. In addition, PPIX increased significantly with an increase in the number of days since the last afamelanotide implant (p < 0.0001). ALAT and bilirubin decreased significantly with an increasing number of afamelanotide doses in the preceding 365 days (p = 0.012, p = 0.0299, respectively). Global radiation only influenced PPIX (p = 0.0113). These findings suggest that afamelanotide ameliorates both PPIX concentrations and LFTs in EPP in a dose-dependent manner.

Decreasing photosystem antenna size by inhibiting chlorophyll synthesis: A double-edged sword for photosynthetic efficiency

Improving photosynthetic efficiency has long been considered as an important strategy to increase crop yield. Optimization of antenna size of photosynthetic systems is one strategy to increase plant photosynthetic efficiency. However, applying this strategy to improve photosynthesis received conflicting results, and the reasons behind these conflicts are unclear. In this study, we constructed transgenic rice with amiRNA targeting to YGL1, which encodes a key enzyme of chlorophyll a/b synthesis in chlorophyll biosynthesis pathway, to generate different lines with different leaf chlorophyll contents and antenna sizes to test under what conditions reduction of antenna size can improve photosynthesis. We found that leaf photosynthesis, canopy photosynthesis (Ac), biomass and grain yield of the heterozygote were not significantly different from those of wild type (WT) while the Ac, biomass and yield of the homozygote were lower than those of WT. Further, when the maximal quantum yield of photosystem II (Fv/Fm) was larger than 0.8, decreasing antenna size by reducing chlorophyll biosynthesis didn't affect leaf photosynthesis. In view of this phenomenon, we proposed that the accumulation of protoporphyrin and the reduced photoprotection capacity might be the cause of the decrease in Fv/Fm and Ac. Therefore, this study shows that reduction of antenna size by inhibiting chlorophyll synthesis can lead to improved light distribution and photosystem efficiency, as long as photodamage and photobleaching can be avoided to maintain the photosystem II efficiency. The double-edged sword effect of inhibiting chlorophyll synthesis on photosynthetic efficiency should be considered when the antenna size is manipulated to gain higher photosynthesis.

In-Tumor Biosynthetic Construction of Upconversion Nanomachines for Precise Near-Infrared Phototherapy.

Targeted construction of therapeutic nanoplatforms in tumor cells with specific activation remains appealing but challenging. Here, we design a cancer-motivated upconversion nanomachine (UCNM) based on porous upconversion nanoparticles (p-UCNPs) for precise phototherapy. The nanosystem is equipped with a telomerase substrate (TS) primer and simultaneously encapsulates 5-aminolevulinic acid (5-ALA) and d-arginine (d-Arg). After coating with hyaluronic acid (HA), it can readily get into tumor cells, where 5-ALA induces efficient accumulation of protoporphyrin IX (PpIX) via the inherent biosynthetic pathway, and the overexpressed telomerase prolonged the TS to form G-quadruplexes (G4) for binding the resulting PpIX as a nanomachine. This nanomachine can respond to near-infrared (NIR) light and promote the active singlet oxygen (1O2) production due to the efficiency of Förster resonance energy transfer (FRET) between p-UCNPs and PpIX. Intriguingly, such oxidative stress can oxidize d-Arg into nitric oxide (NO), which relieves the tumor hypoxia and in turn improves the phototherapy effect. This in situ assembly approach significantly enhances targeting in cancer therapy and might be of considerable clinical value.

Virtual-freezing fluorescence imaging flow cytometry with 5-aminolevulinic acid stimulation and antibody labeling for detecting all forms of circulating tumor cells.

Circulating tumor cells (CTCs) are precursors to cancer metastasis. In blood circulation, they take various forms such as single CTCs, CTC clusters, and CTC-leukocyte clusters, all of which have unique characteristics in terms of physiological function and have been a subject of extensive research in the last several years. Unfortunately, conventional methods are limited in accurately analysing the highly heterogeneous nature of CTCs. Here we present an effective strategy for simultaneously analysing all forms of CTCs in blood by virtual-freezing fluorescence imaging (VIFFI) flow cytometry with 5-aminolevulinic acid (5-ALA) stimulation and antibody labeling. VIFFI is an optomechanical imaging method that virtually freezes the motion of fast-flowing cells on an image sensor to enable high-throughput yet sensitive imaging of every single event. 5-ALA stimulates cancer cells to induce the accumulation of protoporphyrin (PpIX), a red fluorescent substance, making it possible to detect all cancer cells even if they show no expression of the epithelial cell adhesion molecule, a typical CTC biomarker. Although PpIX signals are generally weak, VIFFI flow cytometry can detect them by virtue of its high sensitivity. As a proof-of-principle demonstration of the strategy, we applied cancer cells spiked in blood to the strategy to demonstrate image-based detection and accurate classification of single cancer cells, clusters of cancer cells, and clusters of a cancer cell(s) and a leukocyte(s). To show the clinical utility of our method, we used it to evaluate blood samples of four breast cancer patients and four healthy donors and identified EpCAM-positive PpIX-positive cells in one of the patient samples. Our work paves the way toward the determination of cancer prognosis, the guidance and monitoring of treatment, and the design of antitumor strategies for cancer patients.

Cancer therapy by antibody-targeted Cerenkov light and metabolism-selective photosensitization.

Photodynamic therapy (PDT) is an effective cancer treatment option, but it suffers from penetration limit of light, making it available only for superficial and endoscopically accessible cancers. Recently, there have been reports that Cerenkov luminescence originated from radioisotopes can be utilized as an excitation source for PDT without external light illumination. Here, cancer-selective agents, i.e., (1) clinically available 5-aminolevulinic acid (5-ALA), which promotes cancer metabolism-specific accumulation of protoporphyrin IX (PpIX), and (2) 64Cu-DOTA-trastuzumab, which has HER2-expressing cancer selective uptake, are separately applied as a photosensitizer and an in situ radiator, respectively, to potentiate tumor-specific Cerenkov luminescence energy transfer (CLET) from 64Cu to PpIX for high-precision PDT of cancer. It is shown that the combinational administration and tumor colocalization of 5-ALA and 64Cu-DOTA-trastuzumab exert significant in vitro cytotoxicity (cell viability <9%) as well as in vivo antitumor effects (tumor volume ratio of 0.50 on 14days post-injection) on HER2-expressing breast and gastric cancer models. This study proves that high-precision treatment regimen using dual-targeted CLET-based PDT is feasible for HER2-expressing cancers. Furthermore, the results offer great potential for clinical translation to the dual-targeted CLET-based PDT because the treatment regimen uses components, 5-ALA and 64Cu-DOTA-trastuzumab, which are already in clinical uses.

Protoporphyrin IX derived from dual-species anaerobic biofilms of Fusobacterium necrophorum and Porphyromonas levii attenuates bovine neutrophil function

Host immune cells and clinical interventions often fail to eradicate biofilm-mediated infections, resulting in chronic inflammation. The role of the biofilm three-dimensional structure in this tolerant phenotype has been studied extensively; however, the impact of small molecules released from biofilm-bacteria in modulating host immune function is less well understood. A model of mixed-species biofilms composed of Fusobacterium necrophorum and Porphyromonas levii was developed to evaluate bovine neutrophil responses to bioactive molecules released from either biofilm or planktonic bacteria. We hypothesized that different soluble extracellular factors (ECFs) would be released from planktonic and biofilm bacteria, resulting in altered neutrophil function. Neutrophils exposed to ECFs from planktonic bacteria showed significantly elevated levels of reactive oxygen species (ROS). In contrast, biofilm components from these same species of bacteria failed to induce such a response. Size-exclusion filtration of ECFs revealed that the bioactive molecule causing neutrophil ROS responses was below 3 kDa. Intensive heat, nuclease, lipase, or protease treatments of the <3 kDa fractions did not alter neutrophil functional responses. Protoporphyrin IX (PPIX) is an important heme precursor and growth requirement for many anaerobes. Porphyromonas species can accumulate environmental PPIX at the cell surface as a strategy to protect the bacteria from oxidative stress and we investigated the direct interaction of bovine neutrophils with PPIX. In the present study, evidence suggests that the accumulation of protoporphyrin in these dual-species biofilm ECFs attenuates neutrophil ROS production and chemotaxis. The diminished neutrophil response to biofilm ECFs via the action of PPIX may represent a biofilm immune-evasion strategy that could assist in explaining the ineffectual host clearance of biofilm-mediated infections involving these bacteria.

Phase 2, Randomized, Open-Label Trial of Bitopertin in Erythropoietic Protoporphyria: Beacon Trial Design

Background: Erythropoietic protoporphyria (EPP) caused by FECH and ALAS2 mutations results in toxic accumulation of photoreactive protoporphyrin IX (PPIX). High levels of PPIX result in excruciating phototoxic reactions and hepatopathy caused by biliary stasis. Reduction of PPIX is associated with amelioration of disease in the settings of hematopoietic stem cell transplant, pregnancy and extracorporeal photoinactivation. Bitopertin is a small molecule inhibitor of glycine transporter 1 (GlyT1), which imports large amounts of extracellular glycine into erythropoietic precursors. GlyT1 is needed to supply adequate amounts of substrate for the heme synthesis pathway to enable the large amounts of hemoglobin needed for normal red blood cell production. Treatment of EPP mouse models with FECH and ALAS2 mutations resulted in 45-73% reduction in PPIX, as compared to controls (Hong et al. 2021). These data, combined with a well-defined safety profile that has previously been established in over 4,000 patients and healthy volunteers motivates the current study to evaluate this potentially disease-modifying treatment. Study Design and Methods: This is a Phase 2, randomized, open-label, parallel arm trial of 20 and 60 mg bitopertin daily for 24 weeks in EPP patients. The trial is being conducted at 2 sites in Australia. The primary endpoint is percent change in metal-free PPIX. Secondary endpoints include patient reported outcomes of light tolerance, safety and tolerability, and PK parameters. Eligibility criteria include: age ≥18 years with EPP by FECH/ALAS2 genotyping or porphyrin analysis, AST/ALT <2x upper limit of normal, normal bilirubin, and hemoglobin ≥10 g/dL. Up to 22 participants will be randomized and stratified by baseline daily light tolerance (< or ≥ 30 minutes) and study site. Study sample size calculations were not performed for this first in EPP study. Endpoints will be summarized using descriptive statistics. Results: Subject enrollment began July 2022. Initial data will be presented at the meeting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

SURG-24. QUANTITATIVE ANALYSIS OF TUMOR CELL DENSITY AND PROTOPORPHYRIN IX FLOURESCENCE IN GLIOMA PATIENTS USING PAIRED STIMULATED RAMAN HISTOLOGY AND TWO-PHOTON EXCITATION FLUORESCENCE MICROSCOPY

Abstract Fluorescence guidance is widely utilized to improve the precision of cancer surgery. 5-aminolevulinic acid, the most widely used fluorophore in glioma surgery, is thought to cause selective accumulation of fluorescent protoporphyrin IX (PpIX) in tumor cells by exploiting pathologic alterations in heme biosynthetic pathways. PpIX-induced fluorescence is highly specific for densely tumor-infiltrated tissue but less effective for visualizing the tumor periphery. To improve clinical detection of PpIX, we developed a microscope to perform paired stimulated Raman histology and two-photon excitation fluorescence microscopy (TPEF) and validated it in 175 fresh, unprocessed core tumor specimens from 75 high-grade glioma patients and three central nervous system lymphoma patients across three institutions. Surprisingly, intracellular PpIX accumulation was observed primarily in cells with histiocytic, rather than neoplastic morphology, and the number of cells concentrating PpIX within the cytoplasm was associated with the abundance of CD163 positive cells (p&amp;lt; 0.02). There was no correlation between the degree of tumor cellularity and the concentration of PpIX across all imaged specimens (R=-0.21). Our findings encourage reconsideration of the existing theory of 5-ALA-induced tumor cell fluorescence in gliomas and demonstrate how 5-ALA and TPEF imaging can provide a window into the immune microenvironment of human gliomas.

Synthetic biology-instructed transdermal microneedle patch for traceable photodynamic therapy

5-Aminolevulinic acid-based photodynamic therapy heavily depends on the biological transformation efficiency of 5-aminolevulinic acid to protoporphyrin IX, while the lack of an effective delivery system and imaging navigation are major hurdles in improving the accumulation of protoporphyrin IX and optimizing therapeutic parameters. Herein, we leverage a synthetic biology approach to construct a transdermal theranostic microneedle patch integrated with 5-aminolevulinic acid and catalase co-loaded tumor acidity-responsive copper-doped calcium phosphate nanoparticles for efficient 5-aminolevulinic acid-based photodynamic therapy by maximizing the enrichment of intratumoral protoporphyrin IX. We show that continuous oxygen generation by catalase in vivo reverses tumor hypoxia, enhances protoporphyrin IX accumulation by blocking protoporphyrin IX efflux (downregulating hypoxia-inducible factor-1α and ferrochelatase) and upregulates protoporphyrin IX biosynthesis (providing exogenous 5-aminolevulinic acid and upregulating ALA-synthetase). In vivo fluorescence/photoacoustic duplex imaging can monitor intratumoral oxygen saturation and protoporphyrin IX metabolic kinetics simultaneously. This approach thus facilitates the optimization of therapeutic parameters for different cancers to realize Ca2+/Cu2+-interferences-enhanced repeatable photodynamic therapy, making this theranostic patch promising for clinical practice.

Effect of a 5-aminolevulinic acid gel and 660 nm red LED light on human oral osteoblasts: a preliminary in vitro study

This study aimed to evaluate the effects of a new photodynamic protocol (ALAD-PDT) on primary human osteoblasts (hOBs). The ALAD-PDT protocol consists of a heat-sensitive gel with 5% 5-delta aminolevulinic acid commercialized as Aladent (ALAD), combined with 630 nm LED. For this purpose, the hOBs, explanted from human mandible bone fragments, were used and treated with different ALAD concentrations (10%, 50%, 100% v/v) incubated for 45 min and immediately afterwards irradiated with a 630 nm LED device for 7 min. The untreated and unirradiated cells were considered control (CTRL). The cellular accumulation of the photosensitizer protoporphyrin IX (PpIX), the proliferation, the alkaline phosphatase (ALP) activity, and the calcium deposition were assessed. All concentrations (10, 50, 100%) determined a significant increment of PpIX immediately after 45 min of incubation (0 h) with the highest peak by ALAD (100%). The consequent 7 min of light irradiation caused a slight decrease in PpIX. At 48 h and 72 h, any increment of PpIX was observed. The concentration 100% associated with LED significantly increased hOB proliferation at 48 h (+ 46.83%) and 72 h (+ 127.75%). The 50% and 100% concentrations in combination to the red light also stimulated the ALP activity, + 12.910% and + 14.014% respectively. The concentration 100% with and without LED was selected for the assessment of calcium deposition. After LED irradiation, a significant increase in calcium deposition was observed and quantified (+ 72.33%). In conclusion, the ALAD-PDT enhanced proliferation, the ALP activity, and mineralized deposition of human oral osteoblasts, highlighting a promising potential for bone tissue regeneration.

Recovery From Severe Systemic Peripheral Neuropathy Secondary to Erythropoietic Protoporphyria by Liver Transplant: A Case Report

Erythropoietic protoporphyria is a rare inherited metabolic disorder involving the heme biosynthesis pathway and leads to the accumulation of protoporphyrin in the erythrocytes or liver. Although peripheral neuropathy is known to develop occasionally in other types of porphyria, it rarely occurs in patients with erythropoietic protoporphyria. A 16-year-old boy was transferred to our hospital due to end-stage liver disease secondary to erythropoietic protoporphyria. Severe systemic peripheral neuropathy, similar to that presented in Guillain-Barré syndrome, developed; it was promptly managed with mechanical ventilation. Electrophysiological assessment of the presented neuropathy showed no responsiveness, indicating severe axonopathy. Six weeks after the transfer, liver transplant was performed.Postoperatively, hepatorenal syndromes improved immediately, and his erythrocyte protoporphyrin level decreased from 6291 to 174 μg/dL red blood cells.The patient started to move his limbs gradually and was weaned from mechanical ventilation 2 months after liver transplant. Eventually, he was discharged from hospital and was able to ambulate with assistance 10 months after liver transplant. To our knowledge, this is the first report detailing the clinical course in a patient with erythropoietic protoporphyria who recovered from severe systemic peripheral neuropathy after liver transplant.

An Orthotopic Model of Glioblastoma Is Resistant to Radiodynamic Therapy with 5-AminoLevulinic Acid.

Simple SummaryRadiosensitization of glioblastoma is a major ambition to increase the survival of this incurable cancer. Before surgery, oral administration of 5-aminolevulinic acid leads to the accumulation of fluorescent protoporphyrin IX, preferentially in the cancer cells rather than in the normal brain. This property is used to optimize the resection of the tumor under fluorescent light. Protoporphyrin IX may also carry radiosensitization activity. To test this hypothesis, we designed a robust murine preclinical model of glioblastoma with tumors implanted into the brain of mice treated by fractionated radiotherapy, as for humans. Despite the specific accumulation of porphyrins in glioblastoma, there was no radiosensitization. We confirmed these results in in vitro 3D patient-derived spheroids. Radiosensitization by molecules such as porphyrins needs more exploration for application in glioblastoma treatment.Radiosensitization of glioblastoma is a major ambition to increase the survival of this incurable cancer. The 5-aminolevulinic acid (5-ALA) is metabolized by the heme biosynthesis pathway. 5-ALA overload leads to the accumulation of the intermediate fluorescent metabolite protoporphyrin IX (PpIX) with a radiosensitization potential, never tested in a relevant model of glioblastoma. We used a patient-derived tumor cell line grafted orthotopically to create a brain tumor model. We evaluated tumor growth and tumor burden after different regimens of encephalic multifractionated radiation therapy with or without 5-ALA. A fractionation scheme of 5 × 2 Gy three times a week resulted in intermediate survival [48–62 days] compared to 0 Gy (15–24 days), 3 × 2 Gy (41–47 days) and, 5 × 3 Gy (73–83 days). Survival was correlated to tumor growth. Tumor growth and survival were similar after 5 × 2 Gy irradiations, regardless of 5-ALA treatment (RT group (53–67 days), RT+5-ALA group (40–74 days), HR = 1.57, p = 0.24). Spheroid growth and survival were diminished by radiotherapy in vitro, unchanged by 5-ALA pre-treatment, confirming the in vivo results. The analysis of two additional stem-like patient-derived cell lines confirmed the absence of radiosensitization by 5-ALA. Our study shows for the first time that in a preclinical tumor model relevant to human glioblastoma, treated as in clinical routine, 5-ALA administration, although leading to important accumulation of PpIX, does not potentiate radiotherapy.

Development of approaches to subcellular antiatherosclerotic therapy

Background and Aims : We have studied changes in the accumulation of protoporphyrin IX (PpIX) induced by exposure to 5-aminolevulinic acid (5-ALA). Cytoplasmic hybrids were the object. PpIX, a widely used photosensitizer in clinical practice, is produced by 5-ALA in mitochondria in the heme synthesis reaction cascade. With an increased intake of 5-ALA from the outside, excess PpIX accumulates in cells at an increased concentration. The accumulation of PpIX depends on the metabolic activity of the mitochondria in cells.

Research Progress on Serum Porphyrin and Chronic Liver Disease

Liver cirrhosis is an important cause of chronic liver cancer. At present, a breakthrough has been achieved in the development of chronic hepatitis C treatment, but there is no effective measure to completely cure chronic hepatitis B. Porphyrins are a class of macromolecular heterocyclic compounds formed by interconnecting the ?-carbon atoms of four pyrrole-like substituents through hypomethyl bonds. Porphyrins and their derivatives widely exist in organelles related to energy transfer in organisms. They are mainly involved in the synthesis of heme in human body. Heme is an iron-containing porphyrin compound, 15–20% of which is synthesized and utilized in the liver. Studies have found that porphyrin metabolism disorder can occur in patients with chronic liver disease. The early stage of viral hepatitis is accompanied by increased production of porphyrins and their compounds, increased levels of peripheral circulating porphyrins, porphyrin cholestasis, and biochemical changes of free porphyrins which precede the histological changes. The accumulation of serum and liver protoporphyrins and the oxidative stress can cause liver and extrahepatic damage. On the one hand, porphyrin metabolism disorder can aggravate chronic liver disease, and even progress to liver cirrhosis and liver cancer. On the other hand, chronic liver disease can also aggravate porphyrin metabolism disorder, recurrent attacks, and damage to liver and extrahepatic tissues and organs.