Protoporphyrin IX Accumulation Research Articles

Photodynamic anticancer activity evaluation of novel 5-aminolevulinic acid and 3-hydroxypyridinone conjugates

5-Aminolevulinic acid (ALA) and its derivatives, serving as the endogenous precursor of the photosensitizer (PS) protoporphyrin IX (PpIX), successfully applied in tumor imaging and photodynamic therapy (PDT). ALA and its derivatives have been used to treat actinic keratosis (AK), basal cell carcinoma (BCC), and improve the detection of superficial bladder cancer. However, the high hydrophilicity of ALA and the conversion of PpIX to heme have limited the accumulation of PpIX, hindering the efficiency and potential application of ALA-PDT. This study aims to evaluate the PDT activity of three rationally designed series of ALA-HPO prodrugs, which were based on enhancing the lipophilicity of the prodrugs and reducing the labile iron pool (LIP) through HPO iron chelators to promote PpIX accumulation. Twenty-four ALA-HPO conjugates, incorporating amide, amino acid, and ester linkages, were synthesized. Most of the conjugates, exhibited no dark-toxicity to cells, according to bioactivity evaluation. Ester conjugates 19a-g showed promoted phototoxicity when tested on tumor cell lines, and this increased phototoxicity was strongly correlated with elevated PpIX levels. Among them, conjugate 19c emerged as the most promising (HeLa, IC50 = 24.25 ± 1.43 μM; MCF-7, IC50 = 43.30 ± 1.76 μM; A375, IC50 = 28.03 ± 1.00 μM), displaying superior photodynamic anticancer activity to ALA (IC50 > 100 μM). At a concentration of 80 μM, the fluorescence intensity of PpIX induced by compound 19c in HeLa, MCF-7, and A375 cells was 18.9, 5.3, and 2.8 times higher, respectively, than that induced by ALA. In conclusion, cellular phototoxicity showed a strong correlation with intracellular PpIX fluorescence levels, indicating the potential application of ALA-HPO conjugates in ALA-PDT.

Alkylated EDTA potentiates antibacterial photodynamic activity of protoporphyrin

Antibiotic resistance has garnered significant attention due to the scarcity of new antibiotics in development. Protoporphyrin IX (PpIX)-mediated photodynamic therapy shows promise as a novel antibacterial strategy, serving as an alternative to antibiotics. However, the poor solubility of PpIX and its tendency to aggregate greatly hinder its photodynamic efficacy. In this study, we demonstrate that alkylated EDTA derivatives (aEDTA), particularly C14-EDTA, can enhance the solubility of PpIX by facilitating its dispersion in aqueous solutions. The combination of C14-EDTA and PpIX exhibits potent antibacterial activity against Staphylococcus aureus (S. aureus) when exposed to LED light irradiation. Furthermore, this combination effectively eradicates S. aureus biofilms, which are known to be strongly resistant to antibiotics, and demonstrates high therapeutic efficacy in an animal model of infected ulcers. Mechanistic studies reveal that C14-EDTA can disrupt PpIX crystallization, increase bacterial membrane permeability and sequester divalent cations, thereby improving the accumulation of PpIX in bacteria. This, in turn, enhances reactive oxygen species (ROS) production and the antibacterial photodynamic activity. Overall, this effective strategy holds great promise in combating antibiotic-resistant strains.Graphical

Abstract B046: Irradiation of patient-derived high-grade gliomas reduces 5-ALA-induced fluorescence utility during re-resection

Abstract High grade gliomas HGGs are among the most aggressive primary brain tumors, yielding a median survival time of less than 2 years and a 5-year survival of 2.5%. There has been little in the way of treatments and novel approaches are needed to combat HGG’s poor prognosis. Recent studies have established that HGG cells exhibit metabolic reprogramming to adapt to diverse metabolic gradients within heterogenous tumor microenvironments. Using an unbiased metabolomics approach, we investigated metabolic changes both pre- and post-ionizing radiation across several patient-derived cell lines. Acute dosages of ionizing radiation resulted in significant changes in the synthesis of aminolevulinic acid (ALA), which is known to be converted into the fluorescent metabolite protoporphyrin IX (PpIX) in the porphyrin synthesis pathway. PpIX has been utilized by surgeons in fluorescence-guided tumor resection for clearer visualization of tumor boundaries in recent years. Fractionation of radiation therapy also resulted in dose-dependent changes in the pathway concerning heme synthesis within these cells, especially presenting decreased levels of ALA dehydratase (ALAD) and increased levels of ferrochelatase. As ALAD promotes PpIX accumulation and ferrochelatase catalyzes its conversion to heme, this indicates a decreased favorability for the accumulation of PpIX in the reprogrammed metabolism of HGG cells following radiation. Consequently, we propose that PpIX fluorescence is not a reliable visual aide in surgical re-resection of HGGs post-radiation therapy. Citation Format: Paras S. Minhas, Aarooran Durairaj, Melanie McReynolds, Katrin I. Andreasson, YooJin Jung, Joshua Rabinowitz. Irradiation of patient-derived high-grade gliomas reduces 5-ALA-induced fluorescence utility during re-resection [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B046.

Protoporphyrin IX in serum of high-grade glioma patients: A novel target for disease monitoring via liquid biopsy.

High-grade gliomas (HGG) carry a dismal prognosis. Diagnosis comprises MRI followed by histopathological evaluation of tissue; no blood biomarker is available. Patients are subjected to serial MRIs and, if unclear,surgery for monitoring of tumor recurrence, which is laborious. MRI provides only limited diagnostic information regarding the differentiation of true tumor progression from therapy-associated side effects. 5-aminolevulinic acid (5-ALA)is routinely used for induction of protoporphyrin IX (PpIX) accumulation in malignant glioma tissue, enablingimproved tumor visualizationduring fluorescence-guided resection (FGR). We investigated whether PpIX can also serve as a serum HGG marker to monitor relapse. Patients (HGG: n = 23 primary, pHGG; n = 5 recurrent, rHGG) undergoing FGR received 5-ALA following standard clinical procedure. The control group of eight healthy volunteers (HCTR) also received 5-ALA. Serum was collected before and repeatedly up to 72h after drug administration. Significant PpIX accumulation in HGG was observed after 5-ALA administration (ANOVA: p = 0.005, post-hoc: HCTR vs. pHGG p = 0.029, HCTR vs. rHGG p = 0.006). Separation of HCTR from pHGG was possible when maximum serum PpIX levels were reached (CI95% of tMax). ROC analysis of serum PpIX within CI95% of tMax showed successful classification of HCTR and pHGG (AUCROC 0.943, CI95% 0.884-1.000, p < 0.001); the optimal cut-off for diagnosis was 1275pmolPpIX/ml serum, reaching 87.0% accuracy, 90.5% positive predictive and 84.0% negative predictive value. Baseline PpIX level was similar in patient and control groups. Thus, 5-ALA is required for PpIX induction, which is safe at the standard clinical dosage. PpIX is a new target for liquid biopsy in glioma. More extensive clinical studies are required to characterize its full potential.

Roles of the ABCG2 transporter in protoporphyrin IX distribution and toxicity.

ATP-binding cassette transporter subfamily G member 2 (ABCG2) is a membrane-bound transporter responsible for the efflux of various xenobiotics and endobiotics, including protoporphyrin IX (PPIX), an intermediate in the heme biosynthesis pathway. Certain genetic mutations and chemicals impair the conversion of PPIX to heme and/or increase PPIX production, leading to PPIX accumulation and toxicity. In mice, deficiency of ABCG2 protects against PPIX-mediated phototoxicity and hepatotoxicity by modulating PPIX distribution. In addition, in vitro studies revealed that ABCG2 inhibition increases the efficacy of PPIX-based photodynamic therapy by retaining PPIX inside target cells. In this review, we discuss the roles of ABCG2 in modulating the tissue distribution of PPIX, PPIX-mediated toxicity, and PPIX-based photodynamic therapy. Significance Statement This review summarized the roles of ABCG2 in modulating PPIX distribution and highlighted the therapeutic potential of ABCG2 inhibitors for the management of PPIX-mediated toxicity.

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 &amp;lt;20). The median platelet count was 208 x10 9/L (IQR, 171-243, reference range 150-400). Only 3 patients had a platelet count &amp;lt;150 x10 9/L and none &amp;lt;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= &amp;lt; 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= &amp;lt; 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.

BIOM-21. LIQUID BIOPSY IN SERA OF HIGH-GRADE GLIOMA PATIENTS: PROTOPORPHYRIN IX AS A BIOMARKER

Abstract High-grade gliomas (HGG) carry a dismal prognosis. Diagnosis comprises magnetic resonance imaging (MRI) followed by histopathological evaluation of tumor tissue after biopsy collection; no blood biomarker is currently available. Patients are subjected to repeated surgery and MRI for relapse monitoring. Protoporphyrin IX (PpIX) is already in routine use for selectively visualizing HGG tissue during fluorescence-guided resection (FGR). We investigated if it can also serve as serum HGG marker. Patients (HGG: n = 23 primary, pHGG; n = 5 recurrent, rHGG) undergoing FGR received 5-aminolevulinic acid (5-ALA) following standard clinical procedure. The control group of eight healthy volunteers (HCTR) also received 5-ALA. Serum was collected before and repeatedly up to 72 h after drug administration. PpIX was analyzed following liquid-liquid and solid-phase extraction using liquid chromatography coupled to ion trap mass spectrometry. The serum PpIX level before 5-ALA administration was similar in patient and control groups. Significant PpIX accumulation in HGG was observed after 5-ALA administration (ANOVA: p = 0.005, post-hoc: HCTR vs. pHGG p = 0.029, HCTR vs. rHGG p = 0.006). Separation of HCTR from pHGG was possible within CI95% of tMax (AUC: 0.943, CI95% 0.884 - 1.000, p &amp;lt; 0.001); the optimal cut-off for diagnosis was 1275 pmol PpIX/ml serum, reaching 87.0 % accuracy, 90.5 % positive predictive value and 84.0 % negative predictive value. Distinguishing pHGG patients from healthy controls based on serum PpIX is possible. The prodrug 5-ALA is required for PpIX induction, which is safe for the standard clinical dosage. Thus, PpIX is a new target for liquid biopsy in glioma, but larger clinical studies are required to characterize its full potential.

Tumor Regression with 5-Aminolevulinic Acid (5-ALA)-Mediated Radiodynamic Therapy (RDT) Using Different Megavoltage Energies

5-aminolevulinic acid (5-ALA) accumulates in target tumor cells, where it is metabolized to a photosensitizer - protoporphyrin IX (PpIX). Cherenkov light induced by high-energy photon beams effectively activates the PpIX due to the Soret band. The activated photosensitizers lead to cellular toxicities to kill malignant tumor cells by converting surrounding tissue oxygens into singlet oxygens. Radiodynamic therapy (RDT) utilizes cellular damage caused by both radiation dose and activated photosensitizer. The emission of Cherenkov light was observed higher with higher energy irradiation in phantom and ex-vivo tissues. Therefore, using an in-vivo mouse model, this study aimed to investigate the efficacy and energy dependency of RDT combined with 5-ALA and different ranges of megavoltage photon irradiation. In order to investigate individual and synergistic effects of 5-ALA administration and radiation treatment, the tumors (n = 344) were randomized into eight groups: control (untreated), 5-ALA only, 6-, 15-, and 45-MV conventional radiation treatment (RT) only, 6-, 15-, and 45-MV RDT. 4 Gy in a single fraction was delivered to the tumors using three different energy photons for RT only and RDT groups. 5-ALA was systemically injected into 5-ALA only and RDT groups at 100 mg/kg by tail-vein 4 hours before irradiation for endogenous PpIX accumulation in the tumor. Tumor growth was measured using a 1.5 T MR scanner on the day of treatment (prior to the treatment), 3, 7, and 14 days post-treatment. Two-way repeated ANOVA with Bonferroni correction was used to compare each treatment group to determine the statistically significant difference in tumor growth. A total of 45 MV RDT resulted in the most significant decrease in tumor growth by 58.8 ± 3.4 %, 58.0 ± 3.0 %, and 55.0 ± 3.0 % compared to 5-ALA, 45 MV RT, and control group on 7 days post-treatment (P<0.001), respectively. Moreover, a synergistic effect of 45 MV RDT causes a 47.1-54.1 % additional decrease in tumor growth toward the effective treatment outcome. Compared to the different energies, 45 MV RDT resulted in a 52.2 ± 3.1 % and 19.7 ± 7.2 % decrease in tumor growth compared to 6 MV RDT and 15 MV RDT on 7 days post-treatment, respectively (P<0.001), whereas the conventional RT was not. Radiodynamic therapy using 5-ALA administration and 45MV photon beam irradiation resulted in the most significant tumor growth control. A photon energy dependency was observed in radiodynamic therapy. 45MV photon beams showed greater in activating PpIX, improving the synergistic effects of radiation dose and activated photosensitizer than the other energies. The preliminary results provide a foundation for new innovative treatment strategies that have the potential to improve cancer treatment.

Investigation of Hydrogen Peroxide for 45MV 5-Aminolevulinic Acid-Mediated Radiodynamic Therapy

Cytotoxicity caused by the reactive oxygen species (ROS), such as singlet oxygen species (1O2), superoxide radical (O2-), and hydroxyl radical (HO), is a mechanism for treating cancer cells in radiation therapy. 5-aminolevulinic acid (5-ALA)-mediated radiodynamic therapy (RDT) is more effective in killing tumor cells than conventional radiation therapy. ROS is produced not only by ionizing radiation but also by Cherenkov light-activated protoporphyrin IX (PpIX), which is metabolized endogenously from 5-ALA. Moreover, PpIX also catalyzes hydrogen peroxide to generate 1O2, and an enhanced catalytic yield of 1O2 was observed in X-ray irradiation in vitro. Therefore, using an in-vivo mouse model, this study aimed to investigate the effect of hydrogen peroxide as a coenzyme catalyst on a novel 45MV 5-ALA-mediated RDT. A subcutaneous C57BL/6 mouse model of KP1 cell line was used. The tumors (n = 240) were randomized into six groups, consisting of untreated, conventional radiation treatment (RT), and RDT with or without hydrogen peroxide: 1. control (untreated), 2. hydrogen peroxide, 3. 45MV RT, 4. 45MV RT + hydrogen peroxide, 5. 45MV RDT, 6. 45MV RDT + hydrogen peroxide. For 45MV photon irradiation, a single fraction of 4 Gy was delivered to the tumors. 5-ALA was systemically injected at 100 mg/kg by tail-vein 4 hours before the treatment for endogenous PpIX accumulation in the tumor. Carbamide peroxide was used to deliver hydrogen peroxide to tissue and was administered at 60 mg/kg intratumorally into tumors ∼3-5 min before the treatment. The treatment effect of a single fraction of treatment was measured by calculating tumor growth, measured using a 1.5 T MR scanner on the day of treatment (prior to the treatment), 3 and 7 days post-treatment. Two-way repeated ANOVA with Bonferroni correction was used to compare each treatment group to determine the statistically significant difference in tumor growth. A total of 45MV RDT with hydrogen peroxide was shown to significantly delay the tumor growth for the mouse model and cell line investigated in this work. 45MV RDT with hydrogen peroxide group resulted in a decrease in tumor growth by 51.3 ± 4.1 % and 56.1 ± 5.1 % compared to the control group on 3 and 7 days post-treatment, respectively (P<0.001), and 43.4 ± 0.8 % and 50.9 ± 0.8 % compared to 45MV RT alone on 3 and 7 days post-treatment, respectively. Moreover, the enhancement effect of hydrogen peroxide on 45MV RDT was 2.2-4.7 times greater on 45MV RT alone (P<0.05). Hydrogen peroxide did not contribute to tumor growth when administered alone. A total of 45MV 5-ALA-mediated RDT with hydrogen peroxide resulted in the most significant tumor growth delay compared to the other groups. The catalytic effect of PpIX and hydrogen peroxide was observed in-vivo. These preliminary results demonstrate an effective cancer treatment modality.

Mangostin enhances efficacy of aminolevulinic acid-photodynamic therapy against cancer through inhibition of ABCG2 activity

BackgroundAminolevulinic acid-photodynamic therapy (ALA-PDT) is gaining attention as a potential method for treating select cancers due to its high specificity and low side effect feature. ALA enters cancer cells and accumulate as protoporphyrin IX (PpIX), which will then trigger phototoxicity following light irradiation. However, it is reported that some cancer cells have reduced efficacy of ALA-PDT due to high expression of ABCG2, a transporter involved in the PpIX efflux. In this study, we evaluated the effect of mangostin, a natural compound containing anti-tumor property, on the efficacy of ALA-PDT against cancer and the mechanism involved. MethodsWe utilized TMK1 gastric cancer cell line, which has high ABCG2 expression, to evaluate the PpIX accumulation and phototoxicity exerted by ALA and mangostin co-addition. ResultsWe found that co-addition of ALA and mangostin significantly increase the phototoxicity and PpIX accumulation in TMK1 cells. We also investigated the effect of mangostin on porphyrin-heme pathway enzymes and ABCG2 and found that the addition of mangostin reduce the activity of ABCG2, reducing PpIX efflux. ConclusionThese findings suggest that mangostin enhances the efficacy of ALA-PDT in cancer through inhibition of ABCG2 activity.

Smartphone-based dual radiometric fluorescence and white-light imager for quantification of protoporphyrin IX in skin.

The quantification of protoporphyrin IX (PpIX) in skin can be used to study photodynamic therapy (PDT) treatments, understand porphyrin mechanisms, and enhance preoperative mapping of non-melanoma skin cancers. We aim to develop a smartphone-based imager for performing simultaneous radiometric fluorescence (FL) and white light (WL) imaging to study the baseline levels, accumulation, and photobleaching of PpIX in skin. A smartphone-based dual FL and WL imager (sDUO) is introduced alongside new radiometric calibration methods for providing SI-units of measurements in both pre-clinical and clinical settings. These radiometric measurements and corresponding PpIX concentration estimations are applied to clinical measurements to understand mechanistic differences between PDT treatments, accumulation differences between normal tissue and actinic keratosis lesions, and the correlation of photosensitizer concentrations to treatment outcomes. The sDUO alongside the developed methods provided radiometric FL measurements () with a demonstrated sub nanomolar PpIX sensitivity in 1% intralipid phantoms. Patients undergoing PDT treatment of actinic keratosis (AK) lesions were imaged, capturing the increase and subsequent decrease in FL associated with the incubation and irradiation timepoints of lamp-based PDT. Furthermore, the clinical measurements showed mechanistic differences in new daylight-based treatment modalities alongside the selective accumulation of PpIX within AK lesions. The use of the radiometric calibration enabled the reporting of detected PpIX FL in units of with the use of liquid phantom measurements allowing for the estimation of in-vivo molar concentrations of skin PpIX. The phantom, pre-clinical, and clinical measurements demonstrated the capability of the sDUO to provide quantitative measurements of PpIX FL. The results demonstrate the use of the sDUO for the quantification of PpIX accumulation and photobleaching in a clinical setting, with implications for improving the diagnosis and treatment of various skin conditions.

Unleashing the potential of 5-Aminolevulinic acid: Unveiling a promising target for cancer diagnosis and treatment beyond photodynamic therapy

The therapeutic properties of 5-aminolevulinic acid (5-ALA) have been extensively studied for cancer detection and treatment using photodynamic therapy (PDT). When administered externally, 5-ALA is converted to protoporphyrin IX (PpIX) in cancer cells, which generates reactive oxygen species (ROS) upon exposure to light. This process enables targeted cell death induction and cancer detection.Given the highly conserved nature of heme biosynthesis over billions of years, we hypothesized that natural mechanisms might exist to prevent excessive accumulation of PpIX or heme resulting from 5-ALA overload. Therefore, we anticipated alterations in protein expression profiles upon exogenous administration of 5-ALA. To understand cellular responses to 5-ALA, we investigated protein expression changes and identified OR1B1 as a promising target in bladder, prostate, lung, and cervical cancer cells. OR1B1 expression was observed only with 5-ALA and ferrous chloride, highlighting the central role of heme in this discovery. Immunofluorescence and electron microscopy confirmed OR1B1's sub-cellular localization. These findings suggest that 5-ALA transformation in cancer cells and OR1B1 expression have potential for enhancing cancer detection and developing alternative treatments, including immunotherapy. This approach overcomes the limitations of PDT and opens new avenues for effective and targeted cancer interventions.

Reactive Oxygen Species Produced by 5-Aminolevulinic Acid Photodynamic Therapy in the Treatment of Cancer.

Cancer is the leading cause of death worldwide and several anticancer therapies take advantage of the ability of reactive oxygen species to kill cancer cells. Added to this is the ancient hypothesis that light alone can be used to kill cancer cells. 5-aminolevulinic acid-photodynamic therapy (5-ALA-PDT) is a therapeutic option for a variety of cutaneous and internal malignancies. PDT uses a photosensitizer that, activated by light in the presence of molecule oxygen, forms ROS, which are responsible for the apoptotic activity of the malignant tissues. 5-ALA is usually used as an endogenous pro-photosensitizer because it is converted to Protoporphyrin IX (PpIX), which enters into the process of heme synthesis and contextually becomes a photosensitizer, radiating a red fluorescent light. In cancer cells, the lack of the ferrochelatase enzyme leads to an accumulation of PpIX and consequently to an increased production of ROS. PDT has the benefit of being administered before or after chemotherapy, radiation, or surgery, without impairing the efficacy of these treatment techniques. Furthermore, sensitivity to PDT is unaffected by the negative effects of chemotherapy or radiation. This review focuses on the studies done so far on 5-ALA-PDT and its efficacy in the treatment of various cancer pathologies.

Near-infrared phototheranostics of tumors with protoporphyrin IX and chlorin e6 photosensitizers

BackgroundThe study aims to develop a method for phototheranostics of tumors in the near-infrared (NIR) range using protoporphyrin IX (PpIX) and chlorin e6 (Ce6) photosensitizers (PSs) Materials and MethodsPhototheranostics includes spectral fluorescence diagnostics of PS distribution and photodynamic therapy (PDT) using a single laser in the red spectral range. PpIX and Ce6 fluorescence were registered in the NIR range. PpIX and Ce6 photobleaching was determined during PDT by the change in PS fluorescence. NIR phototheranostics with PpIX and Ce6 were performed on optical phantoms and tumors of patients with oral leukoplakia and basal cell carcinoma. ResultsNIR spectral fluorescence diagnostics of optical phantoms with PpIX or Ce6 is possible when fluorescence is excited by 635 or 660 nm lasers. Fluorescence intensity of PpIX and Ce6 was measured in the range of 725–780 nm. The highest values of signal-to-noise in the case of phantoms with PpIX were observed at λexc=635 nm, and for phantoms with Ce6 at λexc=660 nm. NIR phototheranostics provides the detection of tumor tissues with PpIX or Ce6 accumulation. The PSs photobleaching in the tumor during PDT occurs according to a bi-exponential law. ConclusionPhototheranostics of tumors containing PpIX or Ce6 allows fluorescent monitoring of PS distribution in the NIR range and measuring PSs photobleaching during light exposure that provides personalization of the photodynamic exposure duration to deeper tumors. Using a single laser for fluorescence diagnostics and PDT reduces patient treatment time.

Desferrioxamine Enhances 5-Aminolaevulinic Acid- Induced Protoporphyrin IX Accumulation and Therapeutic Efficacy for Hypertrophic Scar

Hypertrophic scar is a common problem after skin burns or trauma which brings physical, psychological, and cosmetic problems to patients. Photodynamic therapy with 5-aminolevulinic acid (5-ALA) is a promising therapy for hypertrophic scar. However, clinical applications of 5-ALA are limited because of the low permeability of 5-ALA in the skin stratum corneum and the rapid binding of protoporphyrin IX (PpIX) with iron ions, which lead to insufficient PpIX production in target tissues. Herein, a mixture of 5-ALA and DFO (deferoxamine, a special iron chelator) was applied for the treatment of hypertrophic scar. 5-ALA/DFO could efficiently block the biotransformation of PpIX to heme, thus realizing a significant accumulation of photosensitizer. In addition, injection locally into the lesion was applied, which combined with enhanced photodynamic therapy to destroy hypertrophic scar fibroblasts. In vitro experiments showed that 5-ALA/DFO could increase more ROS generation by increasing the accumulation of PpIX, resulting in the apoptosis of hypertrophic scar fibroblasts. Furthermore, 5-ALA/DFO inhibited the proliferation and migration of hypertrophic scar fibroblasts. In vivo study showed that 5-ALA/DFO could effectively inhibit the formation of proliferative scar. Therefore, 5-ALA/DFO has the potential to enhance the photodynamic therapy of 5-ALA and provides a new treatment strategy for hypertrophic scar.

N-Methyl Protoporphyrin IX: An Understudied Porphyrin.

N-Methyl protoporphyrin IX (NmePPIX) is a derivative of protoporphyrin IX (PPIX) and the lattice of heme. Certain xenobiotics strongly induce NmePPIX production in the liver. The existence of endogenous NmePPIX in untreated animal liver has also been reported. The detailed mechanisms of NmePPIX biosynthesis remain unclear, but cytochrome P450 enzymes are thought to be critical in xenobiotic-induced NmePPIX production. High levels of NmePPIX cause PPIX accumulation because NmePPIX is a potent inhibitor (Ki = 7 nM) of ferrochelatase, the last enzyme in the heme biosynthesis pathway that converts PPIX to heme. NmePPIX is also involved in several other physiological processes, including inhibition of nitric oxide production and promotion of lamin aggregation. Compared to the two well-characterized porphyrins, PPIX and heme, NmePPIX is understudied regarding the mechanism of formation, fate, and physiological functions. This Review summarizes the current understanding of NmePPIX and provides perspectives on areas of future research on NmePPIX.

Polymeric iron chelators for enhancing 5-aminolevulinic acid-induced photodynamic therapy.

5-Aminolevulinic acid (5-ALA) is an amino acid that can be metabolized into a photosensitizer, protoporphyrin IX (PpIX) selectively in a tumor cell, permitting minimally invasive photodynamic diagnosis/therapy. However, some malignant tumor cells have excess intracellular labile iron and facilitate the conversion of PpIX into heme, which compromises the therapeutic potency of 5-ALA. Here, we examined the potential of chelation of such unfavorable intratumoral labile iron in photodynamic therapy (PDT) with 5-ALA hydrochloride, using polymeric iron chelators that we recently developed. The polymeric iron chelator efficiently inactivated the intracellular labile iron in cultured cancer cells and importantly enhanced the accumulation of PpIX, thereby improving the cytotoxicity upon photoirradiation. Even in in vivo study with subcutaneous tumor models, the polymeric iron chelator augmented the intratumoral accumulation of PpIX and the PDT effect. This study suggests that our polymeric iron chelator could be a tool for boosting the effect of 5-ALA-induced PDT by modulating tumor microenvironment.

CTNI-13. A FIRST-IN-HUMAN PHASE 0/1 TRIAL OF 5-AMINOLEVULINIC ACID SONODYNAMIC THERAPY (5-ALA SDT) IN RECURRENT GLIOBLASTOMA

Abstract BACKGROUND 5-ALA SDT is not a blood-brain barrier disruption technique, but rather a first-in-class drug-device therapy exploiting the heme synthesis pathway in recurrent glioblastoma (rGBM). Following IV 5-ALA administration, aberrant tumor metabolism results in protoporphyrin-IX (PpIX) accumulation. Activation of PpIX by non-invasive, non-ablative magnetic resonance-guided focused ultrasound (MRgFUS) induces reactive oxygen species and tumor cell death. This first-in-human Phase 0/1 study investigates the feasibility, safety, and biological effects of 5-ALA SDT in rGBM patients. METHODS Six hours prior to SDT, adult patients with rGBM were administered 10mg/kg of an IV formulation of 5-ALA (SONALA-001). Patients were assigned to one of three ascending acoustic energy levels of MRgFUS (200J/400J/800J, measured at transducer surface), followed by a four-day interval prior to planned tumor resection. In each patient, 50% of the enhancing and nonenhancing tumor volume was targeted with MRgFUS with the untreated tumor serving as an internal control. The Optimal Biological Dose (OBD) associated with 5-ALA SDT is the energy level associated with greatest tumor cell death. RESULTS 8 patients were accrued across all energy levels, and none demonstrated drug- or device-related adverse events. Compared to internal control tissue, the apoptosis biomarker, cleaved caspase-3, was elevated in all patients, but most prominently at the 200J energy level. The oxidative stress biomarkers 4-hydroxynonenal, glutathione, cysteine, and thiol were elevated in treated vs. control tissues at all energy levels. The mean Cmax for 5-ALA and PpIX in plasma were 305 µM and 65 nM, respectively. No off-target histological or radiographic alterations were detected in any patient. CONCLUSIONS This first-in-human Phase 0/1 study of a new therapeutic modality for rGBM patients demonstrates that 5-ALA SDT is safe at 200 to 800J and likely induces targeted cell death in rGBM patients via oxidative stress. At 10mg/kg of 5-ALA, the OBD is at or lower than 200J.

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.

Double dose of 5-aminolevulinic acid and its effect on protoporphyrin IX accumulation in low-grade glioma.

Administration of 5-aminolevulinic acid (5-ALA) does not regularly elicit fluorescence in low-grade glioma (LGG) at currently established doses and timing of administration. One explanation may be differences in blood-brain barrier (BBB) integrity compared to high-grade glioma. The authors hypothesized that for a BBB semipermeable to 5-ALA there might be a relationship between plasma 5-ALA concentration and its movement into the brain. A higher dose would elicit more 5-ALA conversion into protoporphyrin IX (PPIX). The authors present a case series of patients harboring LGG who received higher doses of 5-ALA. Patients undergoing surgery for indeterminate glioma later diagnosed as LGG were included in this study. 5-ALA was administered at a standard dose of 20 mg/kg body weight (bw) 4 hours prior to induction of anesthesia. A subgroup of patients received a higher dose of 40 mg/kg bw. Fluorescence was evaluated visually and PPIX concentration (cPPIX) was determined ex vivo by hyperspectral measurements in freshly extracted tissue. All adverse events were recorded. A total of 23 patients harboring diffuse low-grade astrocytomas (n = 19) and oligodendrogliomas (n = 4) were analyzed. Thirteen patients received 20 mg/kg bw, and 10 patients received 40 mg/kg bw of 5-ALA. In the 20 mg/kg group, 30.8% (4 of 13) of tumors harbored areas of visible fluorescence, compared to 60% of cases (n = 6 of 10) with 40 mg/kg bw. The threshold to visibility was 1 μg/ml in both groups. Measured over all biopsies, the mean cPPIX was significantly higher in the double-dose group (1.8 vs 0.45 μg/ml; p < 0.001). In non-visibly fluorescent tissue the mean cPPIX was 0.146 μg/ml in the 20 mg/kg and 0.347 μg/ml in the 40 mg/kg group, indicating an increase of 138% (p < 0.001). These observations demonstrate different regions with different levels of PPIX accumulation in LGG. With higher 5-ALA doses cPPIX increases, leading to more regions surpassing the visibility threshold of 1 μg/ml. These observations can be explained by the fact that the BBB in LGG is semipermeable to 5-ALA. Higher 5-ALA doses result in more PPIX conversion, an observation with implications for future dosing in LGG.