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We disclose here the studies that preceded and guided the preparation of the metal-based, redox-active therapeutic Mn(III) meso -tetrakis( N -n-butoxyethylpyridyl)porphyrin, MnTnBuOE-2-PyP 5 + (BMX-001), which is currently in Phase I/II Clinical Trials at Duke University (USA) as a radioprotector of normal tissues in cancer patients. N -substituted pyridylporphyrins are ligands for Mn(III) complexes that are among the most potent superoxide dismutase mimics thus far synthesized. To advance their design, thereby improving their physical and chemical properties and bioavailability/toxicity profiles, we undertook a systematic study on placing oxygen atoms into N -alkylpyridyl chains via alkoxyalkylation reaction. For the first time we show here the unforeseen structural rearrangement that happens during the alkoxyalkylation reaction by the corresponding tosylates. Comprehensive experimental and computational approaches were employed to solve the rearrangement mechanism involved in quaternization of pyridyl nitrogens, which, instead of a single product, led to a variety of mixed N -alkoxyalkylated and N -alkylated pyridylporphyrins. The rearrangement mechanism involves the formation of an intermediate alkyl oxonium cation in a chain-length-dependent manner, which subsequently drives differential kinetics and thermodynamics of competing N -alkoxyalkylation versus in situ N -alkylation. The use of alkoxyalkyl tosylates, of different length of alkyl fragments adjacent to oxygen atom, allowed us to identify the set of alkyl fragments that would result in the synthesis of a single compound of high purity and excellent therapeutic potential. [ABSTRACT FROM AUTHOR]

Ascorbate (Asc) as a single agent suppressed growth of several tumor cell lines in a mouse model. It has been tested in a Phase I Clinical Trial on pancreatic cancer patients where it exhibited no toxicity to normal tissue yet was of only marginal efficacy. The mechanism of its anticancer effect was attributed to the production of tumoricidal hydrogen peroxide (H 2 O 2 ) during ascorbate oxidation catalyzed by endogenous metalloproteins. The amount of H 2 O 2 could be maximized with exogenous catalyst that has optimized properties for such function and is localized within tumor. Herein we studied 14 Mn porphyrins (MnPs) which differ vastly with regards to their redox properties, charge, size/bulkiness and lipophilicity. Such properties affect the in vitro and in vivo ability of MnPs (i) to catalyze ascorbate oxidation resulting in the production of H 2 O 2 ; (ii) to subsequently employ H 2 O 2 in the catalysis of signaling proteins oxidations affecting cellular survival pathways; and (iii) to accumulate at site(s) of interest. The metal-centered reduction potential of MnPs studied, E 1/2 of Mn III P/Mn II P redox couple, ranged from −200 to +350 mV vs NHE. Anionic and cationic, hydrophilic and lipophilic as well as short- and long-chained and bulky compounds were explored. Their ability to catalyze ascorbate oxidation, and in turn cytotoxic H 2 O 2 production, was explored via spectrophotometric and electrochemical means. Bell-shape structure-activity relationship (SAR) was found between the initial rate for the catalysis of ascorbate oxidation, v o (Asc) ox and E 1/2 , identifying cationic Mn(III) N -substituted pyridylporphyrins with E 1/2 >0 mV vs NHE as efficient catalysts for ascorbate oxidation. The anticancer potential of MnPs/Asc system was subsequently tested in cellular (human MCF-7, MDA-MB-231 and mouse 4T1) and animal models of breast cancer. At the concentrations where ascorbate (1 mM) and MnPs (1 or 5 µM) alone did not trigger any alteration in cell viability, combined treatment suppressed cell viability up to 95%. No toxicity was observed with normal human breast epithelial HBL-100 cells. Bell-shape relationship, essentially identical to v o (Asc) ox vs E 1/2 , wa s also demonstrated between MnP/Asc-controlled cytotoxicity and E 1/2 -controlled v o (Asc) ox . Magnetic resonance imaging studies were conducted to explore the impact of ascorbate on T1-relaxivity. The impact of MnP/Asc on intracellular thiols and on GSH/GSSG and Cys/CySS ratios in 4T1 cells was assessed and cellular reduction potentials were calculated. The data indicate a significant increase in cellular oxidative stress induced by MnP/Asc. Based on v o (Asc) ox vs E 1/2 relationships and cellular toxicity, MnTE-2-PyP 5+ was identified as the best catalyst among MnPs studied. Asc and MnTE-2-PyP 5+ were thus tested in a 4T1 mammary mouse flank tumor model. The combination of ascorbate (4 g/kg) and MnTE-2-PyP 5+ (0.2 mg/kg) showed significant suppression of tumor growth relative to either MnTE-2-PyP 5+ or ascorbate alone. About 7-fold higher accumulation of MnTE-2-PyP 5+ in tumor vs normal tissue was found to contribute largely to the anticancer effect. [ABSTRACT FROM AUTHOR]

Because of the increased insight into the biological role of hydrogen peroxide (H 2 O 2 ) under physiological and pathological conditions and the role it presumably plays in the action of natural and synthetic redox-active drugs, there is a need to accurately define the type and magnitude of reactions that may occur with this intriguing and key species of redoxome. Historically, and frequently incorrectly, the impact of catalase-like activity has been assigned to play a major role in the action of many redox-active drugs, mostly SOD mimics and peroxynitrite scavengers, and in particular MnTBAP 3– and Mn salen derivatives. The advantage of one redox-active compound over another has often been assigned to the differences in catalase-like activity. Our studies provide substantial evidence that Mn(III) N -alkylpyridylporphyrins couple with H 2 O 2 in actions other than catalase-related. Herein we have assessed the catalase-like activities of different classes of compounds: Mn porphyrins (MnPs), Fe porphyrins (FePs), Mn(III) salen (EUK-8), and Mn(II) cyclic polyamines (SOD-active M40403 and SOD-inactive M40404). Nitroxide (tempol), nitrone (NXY-059), ebselen, and MnCl 2 , which have not been reported as catalase mimics, were used as negative controls, while catalase enzyme was a positive control. The dismutation of H 2 O 2 to O 2 and H 2 O was followed via measuring oxygen evolved with a Clark oxygen electrode at 25 °C. The catalase enzyme was found to have k cat (H 2 O 2 )=1.5×10 6 M –1 s –1 . The yield of dismutation, i.e., the maximal amount of O 2 evolved, was assessed also. The magnitude of the yield reflects an interplay between the k cat (H 2 O 2 ) and the stability of compounds toward H 2 O 2 -driven oxidative degradation, and is thus an accurate measure of the efficacy of a catalyst. The k cat (H 2 O 2 ) values for 12 cationic Mn(III) N -substituted (alkyl and alkoxyalkyl) pyridylporphyrin-based SOD mimics and Mn(III) N,N′ -dialkylimidazolium porphyrin, MnTDE-2-ImP 5+ , ranged from 23 to 88 M –1 s –1 . The analogous Fe(III) N -alkylpyridylporphyrins showed ~10-fold higher activity than the corresponding MnPs, but the values of k cat (H 2 O 2 ) are still ~4 orders of magnitude lower than that of the enzyme. While the k cat (H 2 O 2 ) values for Fe ethyl and n -octyl analogs were 803.5 and 368.4 M –1 s –1 , respectively, the FePs are more prone to H 2 O 2 -driven oxidative degradation, therefore allowing for similar yields in H 2 O 2 dismutation as analogous MnPs. The k cat (H 2 O 2 ) values are dependent on the electron deficiency of the metal site as it controls the peroxide binding in the first step of the dismutation process. SOD-like activities depend on electron deficiency of the metal site also, as it controls the first step of O 2 ● − dismutation. In turn, the k cat ( O 2 ● − ) parallels the k cat (H 2 O 2 ). Therefore, the electron-rich anionic non-SOD mimic MnTBAP 3– has essentially very low catalase-like activity, k cat (H 2 O 2 )=5.8 M –1 s –1 . The catalase-like activities of Mn(III) and Fe(III) porphyrins are at most, 0.0004 and 0.05% of the enzyme activity, respectively. The k cat (H 2 O 2 ) values of 8.2 and 6.5 M –1 s –1 were determined for electron-rich Mn(II) cyclic polyamine-based compounds, M40403 and M40404, respectively. The EUK-8, with modest SOD-like activity, has only slightly higher k cat (H 2 O 2 )=13.5 M –1 s –1 . The biological relevance of k cat (H 2 O 2 ) of MnTE-2-PyP 5+ , MnTDE-2-ImP 5+ , MnTBAP 3– , FeTE-2-PyP 5+ , M40403, M40404, and Mn salen was evaluated in wild-type and peroxidase/catalase-deficient E. coli . [ABSTRACT FROM AUTHOR]

In the present study we have synthesized a novel amphiphilic porphyrin and its Ag(II) complex through modification of water-soluble porphyrinic structure in order to increase its lipophilicity and in turn pharmacological potency. New cationic non-symmetrical meso -substituted porphyrins were characterized by UV–visible, electrospray ionization mass spectrometry (ESI-MS), 1 H NMR techniques, lipophilicity (thin-layer chromatographic retention factor, R f ), and elemental analysis. The key toxicological profile ( i.e. cytotoxicity and cell line- (cancer type-) specificity; genotoxicity; cell cycle effects) of amphiphilic Ag porphyrin was studied in human normal and cancer cell lines of various tissue origins and compared with its water-soluble analog. Structural modification of the molecule from water-soluble to amphiphilic resulted in a certain increase in the cytotoxicity and a decrease in cell line-specificity. Importantly, Ag(II) porphyrin showed less toxicity to normal cells and greater toxicity to their cancerous counterparts as compared to cisplatin. The amphiphilic complex was also not genotoxic and demonstrated a slight cytostatic effect via the cell cycle delay due to the prolongation of S-phase. As expected, the performed structural modification affected also the photocytotoxic activity of metal-free amphiphilic porphyrin. The ligand tested on cancer cell line revealed a dramatic (more than 70-fold) amplification of its phototoxic activity as compared to its water-soluble tetracationic metal-free analog. The compound combines low dark cytotoxicity with 5 fold stronger phototoxicity relative to Chlorin e 6 and could be considered as a potential photosensitizer for further development in photodynamic therapy. [ABSTRACT FROM AUTHOR]

Abstract: Resistance to therapy-mediated apoptosis in inflammatory breast cancer, an aggressive and distinct subtype of breast cancer, was recently attributed to increased superoxide dismutase (SOD) expression, glutathione (GSH) content, and decreased accumulation of reactive species. In this study, we demonstrate the unique ability of two Mn(III) N-substituted pyridylporphyrin (MnP)-based SOD mimics (MnTE-2-PyP5+ and MnTnBuOE-2-PyP5+) to catalyze oxidation of ascorbate, leading to the production of excessive levels of peroxide, and in turn cell death. The accumulation of peroxide, as a consequence of MnP+ascorbate treatment, was fully reversed by the administration of exogenous catalase, showing that hydrogen peroxide is essential for cell death. Cell death as a consequence of the action of MnP+ascorbate corresponded to decreases in GSH levels, prosurvival signaling (p-NF-κB, p-ERK1/2), and in expression of X-linked inhibitor of apoptosis protein, the most potent caspase inhibitor. Although markers of classical apoptosis were observed, including PARP cleavage and annexin V staining, administration of a pan-caspase inhibitor, Q-VD-OPh, did not reverse the observed cytotoxicity. MnP+ascorbate-treated cells showed nuclear translocation of apoptosis-inducing factor, suggesting the possibility of a mechanism of caspase-independent cell death. Pharmacological ascorbate has already shown promise in recently completed phase I clinical trials, in which its oxidation and subsequent peroxide formation was catalyzed by endogenous metalloproteins. The catalysis of ascorbate oxidation by an optimized metal-based catalyst (such as MnP) carries a large therapeutic potential as an anticancer agent by itself or in combination with other modalities such as radio- and chemotherapy. [Copyright &y& Elsevier]

Abstract: The Mn porphyrins of k cat(O2 .-) as high as that of a superoxide dismutase enzyme and of optimized lipophilicity have already been synthesized. Their exceptional in vivo potency is at least in part due to their ability to mimic the site and location of mitochondrial superoxide dismutase, MnSOD. MnTnHex-2-PyP5+ is the most studied among lipophilic Mn porphyrins. It is of remarkable efficacy in animal models of oxidative stress injuries and particularly in central nervous system diseases. However, when used at high single and multiple doses it becomes toxic. The toxicity of MnTnHex-2-PyP5+ has been in part attributed to its micellar properties, i.e., the presence of polar cationic nitrogens and hydrophobic alkyl chains. The replacement of a CH2 group by an oxygen atom in each of the four alkyl chains was meant to disrupt the porphyrin micellar character. When such modification occurs at the end of long alkyl chains, the oxygens become heavily solvated, which leads to a significant drop in the lipophilicity of porphyrin. However, when the oxygen atoms are buried deeper within the long heptyl chains, their excessive solvation is precluded and the lipophilicity preserved. The presence of oxygens and the high lipophilicity bestow the exceptional chemical and physical properties to Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin, MnTnBuOE-2-PyP5+. The high SOD-like activity is preserved and even enhanced: log k cat(O2 .-)=7.83 vs 7.48 and 7.65 for MnTnHex-2-PyP5+ and MnTnHep-2-PyP5+, respectively. MnTnBuOE-2-PyP5+ was tested in an O2 .- -specific in vivo assay, aerobic growth of SOD-deficient yeast, Saccharomyces cerevisiae, where it was fully protective in the range of 5–30μM. MnTnHep-2-PyP5+ was already toxic at 5μM, and MnTnHex-2-PyP5+ became toxic at 30μM. In a mouse toxicity study, MnTnBuOE-2-PyP5+ was several-fold less toxic than either MnTnHex-2-PyP5+ or MnTnHep-2-PyP5+. [Copyright &y& Elsevier]

Cationic Mn(III) porphyrins (MnPs) are among the most efficacious SOD mimics. A lead MnP, BMX-001 is now in two Clinical Trials as a radioprotector of normal tissues with glioma and head&neck cancer patients. Due to the optimized catalytic properties, MnPs couple efficiently with ascorbate (Asc) thereby inflicting significant anticancer effects. The anticancer activity is due to the intratumoral H 2 O 2 production arising from the MnP-driven catalysis of Asc oxidation. We have recently developed a new class of fluorinated MnPs. Herein we report the synthesis, characterization and redox activity of one member of a new class of fluorinated MnPs, Mn(III) meso -tetrakis(2-fluoroethylpyridinium-2-yl) por-phyrin, MnFE. It is a more potent SOD mimic (log k cat (O 2 •‒ ) = 7.91, E 1/2 = +242 mV vs NHE) and more powerful catalyst for Asc oxidation ( v 0 (Asc) ox = 543 nM s -1 ) than the previous lead analogs, MnTE-2-PyP 5+ and BMX-001. MnFE accumulates more in tumor vs normal tissue, which is essential for the differential effects of MnP in tumor vs normal tissues. Further, MnFE does not cause dose-limiting hypotension-based acute toxicity seen with non-fluorinated MnPs. Finally, MnFE has excellent safety/toxicity profile. In combination with Asc, MnFE exerts stronger cytotoxicity to various cancer cell lines than the earlier lead MnPs. MnFE/Asc treatment significantly suppressed tumor growth in BalbC mice bearing triple negative 4T1 mammary flank tumor. Furthermore, MnFE/Asc system was also tested as an adjuvant to radiotherapy (RT) in a same tumor model. While single (MnFE, Asc, RT) and double (MnFE/Asc, MnFE/RT, Asc/RT) treatments exhibited strong anticancer effects, the triple combination (MnFE/Asc/RT) was superior in suppressing tumor growth. Our findings demonstrate that the fluorination of MnPs comprises major modification in the development of Mn porphyrin-based therapeutics. In the light of our results and the fact that Asc is already in Clinical Trials, the MnFE alone or in combination with Asc and/or RT carries outstanding therapeutic potential which warrants its clinical development. [ABSTRACT FROM AUTHOR]

Prostate cancer is the most common cancer in American men, with approximately 30-50% of men developing loss of erectile function after definitive radiotherapy (RT). Currently, no treatment is available that preserves erectile function. Therefore there is a significant reduction of the quality of life of survivors which incurs considerable healthcare costs. Hence, there is an imminent need to protect normal tissues during prostate cancer RT. We have developed Mn porphyrin-based SOD mimics and demonstrated that they radioprotect normal tissues such as brain, salivary glands and mouth mucosa while radio- and chemosensitizing tumors, such as brain, breast and lymphoma. We have also demonstrated and explained the reasoning behind the significant enhancement of tumor radiosensitization by Mn porphyrins when combined with ascorbate (Asc). A lead drug, BMX-001 is now in two Phase I/II clinical trials as a normal tissue radioprotector with glioma and head&neck cancer patients. Herein we have explored the radioprotection of a lower pelvic region by a member of the new class of powerful Mn porphyrin-based SOD mimics – Mn(III) meso -tetrakis(5',5',5' - trifluoropentylpyridinium-2-yl)porphyrin, MnF 3 Pen. The SOD-like activity given by log k cat (O 2 .- ) is 7.45, and the reduction potential of the Mn site, E 1/2 is +288 mV vs NHE. In this study we also aimed to show that the addition of Asc would not diminish radioprotection of a normal tissue by MnF 3 Pen. The rat toxicity and pharmacokinetic studies of MnF 3 Pen/Asc system showed that MnF 3 Pen reached ~100 to 500 nM levels at targeted sites (penis, prostate, bladder, testes and rectum) within few hours after single subcutaneous (sc) 1 mg/kg injection. We subsequently performed the efficacy study. Single radiation dose of 20 Gy was delivered stereotactically to the prostate of Sprague-Dawley 8 week old rats using image-guided small animal irradiator. MnF 3 Pen was given sc at 0.5 mg/kg/day for four weeks and continued twice weekly for the next 5 weeks. Ascorbate was given intraperitoneally at 1 g/kg/day for first 3 days and then twice weekly for the rest of 9 weeks. We showed that MnF 3 Pen/Asc treatment prevented erectile dysfunction observed in RT-treated group. We further showed that MnF 3 Pen/Asc treatment greatly suppressed RT-induced damage to prostate as evaluated by H&E staining. [ABSTRACT FROM AUTHOR]

Cationic Mn(III) N-substituted pyridylporphyrins (MnPs), such as MnTnBuOE-2-PyP 5+ and MnTE-2-PyP 5+ , initially optimized as powerful mimics of superoxide dismutase, also impact cellular redox-based signaling pathways, most so redox sensitive thiol bearing proteins such as NF-κB, whereby affecting cellular apoptotic and proliferative pathways. Such actions lead to S-glutathionylation of proteins and their subsequent inactivation. In addition to MnP catalyst, both H 2 O 2 and glutathione are required for such process, which includes initial oxidation of Mn porphyrin by H 2 O 2 , followed by oxidation of protein thiols by high-valent MnP in a GPx-like fashion. While tumors already have higher H 2 O 2 levels, they are sensitive to any further increase in H 2 O 2 which strategy is used to induce tumor killing. Mn porphyrins do not differentiate tumor from normal tissue and thereby interact at identical rate constants with small reactive species and proteins in both types of tissues. Yet, the yield of reactions depends upon the MnPs levels and levels of reactive species. In the case of NF-kB, the higher are the levels of reactants (MnPs and reactive species) the larger would be the yield of its oxidative damage. Consequently, the larger would be the inhibition of NF-κB activation and in turn the suppression of anti-apoptotic pathways (for involvement of NF-κB pathways in MnP/Asc anticancer effects see Tovmasyan et al abstract on Redox Proteomics). Using LC-MS/MS we showed the preferential accumulation of 3 different MnPs in tumor over normal tissue in a 4T1 flank mouse tumor model. In such model, the already high tumor H 2 O 2 levels were increased with either radiation (RT) or ascorbate or both. The higher tumor accumulation of MnTE-2-PyP 5+ relative to MnTnBuOE-2-PyP 5+ resulted in larger tumor suppression. Regardless of much higher tumor vs normal tissue accumulation, the redox-inert MnTBAP 3- sensitized tumor neither to ascorbate nor to radiation. As anticipated the tumor GSSG levels were increased only when redox-active MnTE-2-PyP 5+ , but not MnTBAP 3- , was combined with RT/Ascorbate treatment. Grant support NIH 1R03-NS082704-01, 5-P30-CA14236-29, CNPq and CAPES from Brazil. [ABSTRACT FROM AUTHOR]

We have recently demonstrated that cationic Mn(III) N-substituted pyridylporphyrins (MnPs) couple with ascorbate (Asc) thereby inducing cancer cell death. Mechanism of such anticancer effect has been attributed to the tumoricidal H 2 O 2 production as a result of MnP-catalyzed Asc oxidation. Among MnPs explored, that differ vastly with regards to their redox properties, charge, size/bulkiness and lipophilicity, MnTE-2-PyP 5+ (Mn1) was identified as the best catalyst of Asc oxidation and demonstrated the highest ability to promote cancer cell death. Herein we have analyzed oxidative modifications of proteins via mass spectrometry-based redox proteomics in 4T1 mouse breast cancer cells treated with Mn1/Asc system. Cells were treated for 4 hours in a growth medium with 5 µM Mn1 and 1 mM ascorbate - an optimized treatment scheme that had shown the antitumor effect in vivo. Such treatment affected 3605 peptidyl cysteins (Cys) in total, out of which 1577 were oxidized 1.3-fold or higher compared with control, untreated samples. Distribution analyses of Mn1/Asc-induced oxidation of peptidyl Cys show that >50% of oxidized peptidyl Cys bear 1.3-2.0 fold oxidation level. MnP/Asc system affected 1762 proteins in total, among which 942 proteins were associated with >1.3-fold oxidized peptidyl Cys. Analysis of potential functional pathways revealed that Mn1/Asc oxidized peptides are associated with regulation of cytoskeleton rearrangement, transcription – mRNA processing, translation, protein folding, cell cycle and adhesion. As expected, analyses of total thiol levels and GSSG/GSH and Cys/CySS ratios in treated vs control cells revealed significant oxidative burden imposed by Mn1/Asc system. Evaluation of biological networks affected by Mn1/Asc system using Analyze network algorithm revealed NF-κB-associated pathway as a key affected network. When tested in 4T1 flank mouse tumor model, Mn1/Asc treatment alone or even more so in combination with radiation, induced significant tumor growth delay Support: NIH 1R03-NS082704-01, 5-P30-CA14236-29, NIH R01 ES023485 and R21 ES 025632. [ABSTRACT FROM AUTHOR]

Ataxia-telangiectasia mutated (ATM) gene mediates acute phase DNA damage repair pathways. Inhibition of ATM is theorized to amplify the effects of radiotherapy (RT). AZD1390 is a first-in-class, investigational ATM inhibitor. We conducted a combined phase 0/1b study to evaluate AZD1390 pharmacokinetics (PK) and pharmacodynamics (PD) in patients with newly-diagnosed glioblastoma (GBM), graduating patients to a phase 1b study evaluating a therapeutic regimen of AZD1390 with concurrent RT in O6-methylguanine methyltransferase (MGMT) unmethylated tumors exceeding a prespecified PK threshold in non-enhancing tumor. Patients with presumed newly-diagnosed GBM were enrolled in a phase 0 study receiving 3 days of AZD1390 prior to planned resection 4-6 or 23-25 hours following the final dose. Tumor tissue, cerebrospinal fluid (CSF), and plasma were collected. Total and unbound drug concentrations were measured using validated LC-MS/MS methods. ATM inhibition was assessed by quantification of pRAD50 after 5 Gy ex vivo irradiation in surgical tissue compared to non-irradiated control tissue. A PK 'trigger', 2nM unbound drug levels in Gd-non-enhancing tumor, determined eligibility for the therapeutic phase 1b expansion portion of the study. Patients with MGMT-unmethylated tumors exceeding this PK threshold were eligible for expansion phase dosing of AZD1390 with concurrent RT, 60 Gy in 30 fractions using volumetric-modulated arc therapy (VMAT). Patients with MGMT methylated tumors were not eligible for the expansion phase and proceeded with temozolomide (TMZ) plus RT followed by maintenance TMZ. Seventeen patients were enrolled onto the phase 0 portion of the study. Three patients were excluded from the PK analysis due to dosing deviation (n = 1) or non-GBM diagnosis (n = 2). For the 14 patients included in the PK analysis, the mean unbound concentration of AZD1390 in Gd-non-enhancing tumor was 12.4 and 4.4 pmol/g, for the 4-6 hours preop (n = 10), and 23-25 hours preop (n = 4) time cohorts, respectively. The suppression of pRAD50 levels after ex vivo RT was observed in AZD1390- treated patients (median 0.2% for 4-6-hours cohorts and 1.9% for 23-25 hours cohort vs 32.3% for control cohort). One non-serious adverse event of grade 3 hyperammonemia was reported and deemed potentially related to AZD1390. No serious adverse events related to AZD1390 were reported. As of November 2023, 3 patients with MGMT-unmethylated tumors have completed therapeutic dosing of AZD1390 with concurrent radiotherapy with a median clinical follow-up of 2.3 months. AZD1390 achieved pharmacologically-relevant concentrations in non-enhancing, newly- diagnosed GBM tissue and effectively inhibits ATM as measured by suppression of radiation-induced pRAD50 increases. AZD1390 with concurrent RT is well-tolerated with initial follow-up. Updated follow-up will better determine long-term safety and efficacy of this first-in-class combination. [ABSTRACT FROM AUTHOR]

Purpose: To test the effects of a novel Mn porphyrin oxidative stress modifier, Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin (MnBuOE), for its radioprotective and radiosensitizing properties in normal tissue versus tumor, respectively.Methods and Materials: Murine oral mucosa and salivary glands were treated with a range of radiation doses with or without MnBuOE to establish the dose-effect curves for mucositis and xerostomia. Radiation injury was quantified by intravital near-infrared imaging of cathepsin activity, assessment of salivation, and histologic analysis. To evaluate effects of MnBuOE on the tumor radiation response, we administered the drug as an adjuvant to fractionated radiation of FaDu xenografts. Again, a range of radiation therapy (RT) doses was administered to establish the radiation dose-effect curve. The 50% tumor control dose values with or without MnBuOE and dose-modifying factor were determined.Results: MnBuOE protected normal tissue by reducing RT-mediated mucositis, xerostomia, and fibrosis. The dose-modifying factor for protection against xerostomia was 0.77. In contrast, MnBuOE increased tumor local control rates compared with controls. The dose-modifying factor, based on the ratio of 50% tumor control dose values, was 1.3. Immunohistochemistry showed that MnBuOE-treated tumors exhibited a significant influx of M1 tumor-associated macrophages, which provides mechanistic insight into its radiosensitizing effects in tumors.Conclusions: MnBuOE widens the therapeutic margin by decreasing the dose of radiation required to control tumor, while increasing normal tissue resistance to RT-mediated injury. This is the first study to quantitatively demonstrate the magnitude of a single drug's ability to radioprotect normal tissue while radiosensitizing tumor. [ABSTRACT FROM AUTHOR]

Tetra-cationic Zn(II) meso-tetrakis(N-alkylpyridinium-2 (or -3 or -4)-yl)porphyrins (ZnPs) with progressively increased lipophilicity were synthesized to investigate how the tri-dimensional shape and lipophilicity of the photosensitizer (PS) affect cellular uptake, subcellular distribution, and photodynamic efficacy. The effect of the tri-dimensional shape of the molecule was studied by shifting the N-alkyl substituent attached to the pyridyl nitrogen from ortho to meta and para positions. Progressive increase of lipophilicity from shorter hydrophilic (methyl) to longer amphiphilic (hexyl) alkyl chains increased the phototoxicity of the ZnP PSs. PS efficacy was also increased for all derivatives when the alkyl substituents were shifted from ortho to meta, and from meta to para positions. Both cellular uptake and subcellular distribution of the PSs were affected by the lipophilicity and the position of the alkyl chains on the periphery of the porphyrin ring. Whereas the hydrophilic ZnPs demonstrated mostly lysosomal distribution, the amphiphilic hexyl derivatives were associated with mitochondria, endoplasmic reticulum, and plasma membrane. A comparison of hexyl isomers revealed that cellular uptake and partition into membranes followed the order para > meta > ortho. Varying the position and length of the alkyl substituents affects (i) the exposure of cationic charges for electrostatic interactions with anionic biomolecules and (ii) the lipophilicity of the molecule. The charge, lipophilicity, and the tri-dimensional shape of the PS are the major factors that determine cellular uptake, subcellular distribution, and as a consequence, the phototoxicity of the PSs. [ABSTRACT FROM AUTHOR]

Abstract: Oxidative stress, a redox imbalance between the endogenous reactive species and antioxidant systems, is common to numerous pathological conditions such as cancer, central nervous system injuries, radiation injury, diabetes etc. Therefore, compounds able to reduce oxidative stress have been actively sought for over 3 decades. Superoxide is the major species involved in oxidative stress either in its own right or through its progeny, such as ONOO-, H2O2, •OH, CO3 • -, and •NO2. Hence, the very first compounds developed in the late 1970-ies were the superoxide dismutase (SOD) mimics. Thus far the most potent mimics have been the cationic meso Mn(III) N-substituted pyridylporphyrins and N,N’-disubstituted imidazolylporphyrins (MnPs), some of them with k cat(O2 .-) similar to the k cat of SOD enzymes. Most frequently studied are ortho isomers MnTE-2-PyP5+, MnTnHex-2-PyP5+, and MnTDE-2-ImP5+. The ability to disproportionate O2 .- parallels their ability to remove the other major oxidizing species, peroxynitrite, ONOO-. The same structural feature that gives rise to the high k cat(O2 .-) and k red(ONOO-), allows MnPs to strongly impact the activation of the redox-sensitive transcription factors, HIF-1α, NF-κB, AP-1, and SP-1, and therefore modify the excessive inflammatory and immune responses. Coupling with cellular reductants and other redox-active endogenous proteins seems to be involved in the actions of Mn porphyrins. While hydrophilic analogues, such as MnTE-2-PyP5+ and MnTDE-2-ImP5+ are potent in numerous animal models of diseases, the lipophilic analogues, such as MnTnHex-2-PyP5+, were developed to cross blood brain barrier and target central nervous system and critical cellular compartments, mitochondria. The modification of its structure, aimed to preserve the SOD-like potency and lipophilicity, and diminish the toxicity, has presently been pursued. The pulmonary radioprotection by MnTnHex-2-PyP5+ was the first efficacy study performed successfully with non-human primates. The Phase I toxicity clinical trials were done on amyotrophic lateral sclerosis patients with N,N’-diethylimidazolium analogue, MnTDE-2-ImP5+ (AEOL10150). Its aggressive development as a wide spectrum radioprotector by Aeolus Pharmaceuticals has been supported by USA Federal government. The latest generation of compounds, bearing oxygens in pyridyl substituents is presently under aggressive development for cancer and CNS injuries at Duke University and is supported by Duke Translational Research Institute, The Wallace H. Coulter Translational Partners Grant Program, Preston Robert Tisch Brain Tumor Center at Duke, and National Institute of Allergy and Infectious Diseases. Metal center of cationic MnPs easily accepts and donates electrons as exemplified in the catalysis of O2 .- dismutation. Thus such compounds may be equally good anti- and pro-oxidants; in either case the beneficial therapeutic effects may be observed. Moreover, while the in vivo effects may appear antioxidative, the mechanism of action of MnPs that produced such effects may be pro-oxidative; the most obvious example being the inhibition of NF-κB. The experimental data therefore teach us that we need to distinguish between the mechanism/s of action/s of MnPs and the effects we observe. A number of factors impact the type of action of MnPs leading to favorable therapeutic effects: levels of reactive species and oxygen, levels of endogenous antioxidants (enzymes and low-molecular compounds), levels of MnPs, their site of accumulation, and the mutual encounters of all of those species. The complexity of in vivo redox systems and the complex redox chemistry of MnPs challenge and motivate us to further our understanding of the physiology of the normal and diseased cell with ultimate goal to successfully treat human diseases. [Copyright &y& Elsevier]

Niraparib is a potent and orally bioavailable inhibitor of poly (ADP-ribose) polymerase (PARP) with high specificity for isoforms 1 and 2. It has been approved by the U.S. Food and Drug Administration for ovarian cancer maintenance therapy and is currently under development for various cancers, including glioblastoma. To assess central nervous system (CNS) penetration of niraparib in glioblastoma patients, a novel bioanalytical method was developed to measure total and unbound niraparib levels in human brain tumor tissue and cerebrospinal fluid (CSF). The method was validated using plasma as a surrogate matrix over the concentration range of 1–10,000 nM on an LC-MS/MS system. The MS/MS detection was conducted in positive electrospray ionization mode, while chromatography was performed using a Kinetex™ PS C18 column with a total 3.5-minute gradient elution run time. The maximum coefficient of variation for both intra- and inter-day precision was 10.6%, with accuracy ranging from 92.8% – 118.5% across all matrices. Niraparib was stable in human brain homogenate for at least 6 hours at room temperature (RT) and 32 days at −20°C, as well as in stock and working solutions for at least 21 hours (RT) and 278 days (4°C). Equilibrium dialysis experiments revealed the fractions unbound of 0.05 and 0.16 for niraparib in human brain and plasma, respectively. The validated method is currently employed to assess niraparib levels in human glioblastoma tissue, CSF, and plasma in an ongoing trial on newly diagnosed glioblastoma and recurrent IDH1/2(+) ATRX mutant glioma patients (NCT05076513). Initial results of calculated total (K p) and unbound (K p,uu) tumor-to-plasma partition coefficients indicate significant brain penetration ability of niraparib in glioblastoma patients. [Display omitted] • The LC-MS/MS method was validated to measure niraparib levels in human plasma, brain tumor, and CSF. • Unbound fractions of niraparib were established in human plasma, brain, and brain tumor. • The total and unbound levels of niraparib in glioblastoma patients have been evaluated. [ABSTRACT FROM AUTHOR]

Abstract Cationic manganese(III) ortho N -substituted pyridylporphyrins (MnP) act as efficient antioxidants catalyzing superoxide dismutation and accelerating peroxynitrite reduction. Importantly, MnP can reach mitochondria offering protection against reactive species in different animal models of disease. Although an LC-MS/MS-based method for MnP quantitation and subcellular distribution has been reported, a direct method capable of evaluating both the uptake and the redox state of MnP in living cells has not yet been developed. In the present work we applied resonance Raman (RR) spectroscopy to analyze the intracellular accumulation of two potent MnP-based lipophilic SOD mimics, MnTnBuOE-2-PyP5+ and MnTnHex-2-PyP5+ within endothelial cells. RR experiments with isolated mitochondria revealed that the reduction of Mn(III)P was affected by inhibitors of the electron transport chain, supporting the action of MnP as efficient redox active compounds in mitochondria. Indeed, RR spectra confirmed that MnP added in the Mn(III) state can be incorporated into the cells, readily reduced by intracellular components to the Mn(II) state and oxidized by peroxynitrite. To assess the combined impact of reactivity and bioavailability, we studied the kinetics of Mn(III)TnBuOE-2-PyP5+ with peroxynitrite and evaluated the cytoprotective capacity of MnP by exposing the endothelial cells to nitro-oxidative stress induced by peroxynitrite. We observed a preservation of normal mitochondrial function, attenuation of cell damage and prevention of apoptotic cell death. These data introduce a novel application of RR spectroscopy for the direct detection of MnP and their redox states inside living cells, and helps to rationalize their antioxidant capacity in biological systems. Graphical abstract fx1 Highlights • Raman Resonance was utilized to reveal the redox state of Mn-porphyrins (MnP) in cells. • Mn(III)P are readily reduced intracellularly to the Mn(II) state. • Intramitochondrial oxidation of a peroxynitrite-sensitive probe is inhibited by MnP. • The cytotoxicity of peroxynitrite is neutralized by MnP via a catalytic redox cycle. [ABSTRACT FROM AUTHOR]