RRx-001: a systemically non-toxic M2-to-M1 macrophage stimulating and prosensitizing agent in Phase II clinical trials.

INTRODUCTION: According to Hanahan and Weinberg, cancer manifests as six essential physiologic hallmarks: (1) self-sufficiency in growth signals, (2) insensitivity to growth-inhibitory signals, (3) evasion of programmed cell death, (4) limitless replicative potential, (5) sustained angiogenesis, and (6) invasion and metastasis. As a facilitator of these traits as well as immunosuppression and chemoresistance, the presence of tumor-associated macrophages (TAMs) may serve as the seventh hallmark of cancer. Anticancer agents that successfully reprogram TAMs to target rather than support tumor cells may hold the key to better therapeutic outcomes. Areas covered: This article summarizes the characteristics of the macrophage-stimulating agent RRx-001, a molecular iconoclast, sourced from the aerospace industry, with a particular emphasis on the cell-to-cell transfer mechanism of action (RBCs to TAMs) underlying its antitumor activity as well as its chemo and radioprotective properties, consolidated from various preclinical and clinical studies. Expert opinion: RRx-001 is macrophage-stimulating agent with the potential to synergize with chemotherapy, radiotherapy and immunotherapy while simultaneously protecting normal tissues from their cytotoxic effects. Given the promising indications of activity in multiple tumor types and these normal tissue protective properties, RRx-001 may be used to treat a broad spectrum of malignancies, if it is approved in the future.

Safety and activity of RRx-001 in patients with advanced cancer: a first-in-human, open-label, dose-escalation phase 1 study.

BACKGROUND: Epigenetic alterations have been strongly associated with tumour formation and resistance to chemotherapeutic drugs, and epigenetic modifications are an attractive target in cancer research. RRx-001 is activated by hypoxia and induces the generation of reactive oxygen and nitrogen species that can epigenetically modulate DNA methylation, histone deacetylation, and lysine demethylation. The aim of this phase 1 study was to assess the safety, tolerability, and pharmacokinetics of RRx-001. METHODS: In this open-label, dose-escalation, phase 1 study, we recruited adult patients (aged >18 years) with histologically or cytologically confirmed diagnosis of advanced, malignant, incurable solid tumours from University of California at San Diego, CA, USA, and Sarah Cannon Research Institute, Nashville, TN, USA. Key eligibility criteria included evaluable disease, Eastern Cooperative Group performance status of 2 or less, an estimated life expectancy of at least 12 weeks, adequate laboratory parameters, discontinuation of all previous antineoplastic therapies at least 6 weeks before intervention, and no residual side-effects from previous therapies. Patients were assigned to receive intravenous infusions of RRx-001 at increasing doses (10 mg/m(2), 16·7 mg/m(2), 24·6 mg/m(2), 33 mg/m(2), 55 mg/m(2), and 83 mg/m(2)) either once or twice-weekly for at least 4 weeks, with at least three patients per dose cohort and allowing a 2-week observation period before dose escalation. Samples for safety and pharmacokinetics analysis, including standard chemistry and haematological panels, were taken on each treatment day. The primary objective was to assess safety, tolerability, and dose-limiting toxic effects of RRx-001, to determine single-dose pharmacokinetics, and to identify a recommended dose for phase 2 trials. All analyses were done per protocol. Accrual is complete and follow-up is still on-going. This trial is registered with ClinicalTrials.gov, number NCT01359982. FINDINGS: Between Oct 10, 2011, and March 18, 2013, we enrolled 25 patients and treated six patients in the 10 mg/m(2) cohort, three patients in the 16·7 mg/m(2) cohort, three patients in the 24·6 mg/m(2) cohort, four patients in the 33 mg/m(2) cohort, three patients in the 55 mg/m(2), and six patients in the 83 mg/m(2) cohort. Pain at the injection site, mostly grade 1 and grade 2, was the most common adverse event related to treatment, experienced by 21 (84%) patients. Other common drug-related adverse events included arm swelling or oedema (eight [32%] patients), and vein hardening (seven [28%] patients). No dose-limiting toxicities were observed. Time constraints related to management of infusion pain from RRx-001 resulted in a maximally feasible dose of 83 mg/m(2). Of the 21 evaluable patients, one (5%) patient had a partial response, 14 (67%) patients had stable disease, and six (29%) patients had progressive disease; all responses were across a variety of tumour types. Four patients who had received RRx-001 were subsequently rechallenged with a treatment that they had become refractory to; all four responded to the rechallenge. INTERPRETATION: RRx-001 is a well-tolerated novel compound without clinically significant toxic effects at the tested doses. Preliminary evidence of activity is promising and, on the basis of all findings, a dose of 16·7 mg/m(2) was recommended as the targeted dose for phase 2 trials. FUNDING: EpicentRx (formerly RadioRx).

Development of methods for the bioanalysis of RRx-001 and metabolites.

BACKGROUND: Bioanalytical methods were required to study the novel anticancer drug, RRx-001 preclinically and for clinical pharmacokinetic analysis; however, RRx-001 quickly and completely disappeared on intravenous administration in preclinical species. RESULTS: Quantification of RRx-001 directly or by derivatization was unsuccessful. On exposure to whole blood, RRx-001 formed the glutathione (GSH) adduct very rapidly, suggesting this metabolite as the bioanalyte. However, rapid enzymatic degradation in the blood matrix of RRx-001-GSH posed significant technical problems. Herein, we describe a novel and broadly applicable solution to stabilize GSH conjugates in blood samples by inhibiting the degrading enzyme. Liquid chromatography-tandem mass spectrometry methods for analysis of RRx-001-GSH in rat, dog and human plasma were developed and successfully validated to good laboratory practice standards. CONCLUSION: Extensive breakdown of RRx-001-GSH was effectively stopped by addition of the enzyme inhibitor, acivicin. The developed liquid chromatography-tandem mass spectrometry assay for RRx-001-GSH was validated for use in preclinical toxicology studies and the Phase I first-in-human clinical trial.

Liquid chromatography/mass spectrometry methods for measuring dipeptide abundance in non-small-cell lung cancer.

RATIONALE: Metabolomic profiling is a promising methodology of identifying candidate biomarkers for disease detection and monitoring. Although lung cancer is among the leading causes of cancer-related mortality worldwide, the lung tumor metabolome has not been fully characterized. METHODS: We utilized a targeted metabolomic approach to analyze discrete groups of related metabolites. We adopted a dansyl [5-(dimethylamino)-1-naphthalene sulfonamide] derivatization with liquid chromatography/mass spectrometry (LC/MS) to analyze changes of metabolites from paired tumor and normal lung tissues. Identification of dansylated dipeptides was confirmed with synthetic standards. A systematic analysis of retention times was required to reliably identify isobaric dipeptides. We validated our findings in a separate sample cohort. RESULTS: We produced a database of the LC retention times and MS/MS spectra of 361 dansyl dipeptides. Interpretation of the spectra is presented. Using this standard data, we identified a total of 279 dipeptides in lung tumor tissue. The abundance of 90 dipeptides was selectively increased in lung tumor tissue compared to normal tissue. In a second set of validation tissues, 12 dipeptides were selectively increased. CONCLUSIONS: A systematic evaluation of certain metabolite classes in lung tumors may identify promising disease-specific metabolites. Our database of all possible dipeptides will facilitate ongoing translational applications of metabolomic profiling as it relates to lung cancer.

Metabolomic-derived novel cyst fluid biomarkers for pancreatic cysts: glucose and kynurenine.

BACKGROUND: Better pancreatic cyst fluid biomarkers are needed. OBJECTIVE: To determine whether metabolomic profiling of pancreatic cyst fluid would yield clinically useful cyst fluid biomarkers. DESIGN: Retrospective study. SETTING: Tertiary-care referral center. PATIENTS: Two independent cohorts of patients (n = 26 and n = 19) with histologically defined pancreatic cysts. INTERVENTION: Exploratory analysis for differentially expressed metabolites between (1) nonmucinous and mucinous cysts and (2) malignant and premalignant cysts was performed in the first cohort. With the second cohort, a validation analysis of promising identified metabolites was performed. MAIN OUTCOME MEASUREMENTS: Identification of differentially expressed metabolites between clinically relevant cyst categories and their diagnostic performance (receiver operating characteristic [ROC] curve). RESULTS: Two metabolites had diagnostic significance-glucose and kynurenine. Metabolomic abundances for both were significantly lower in mucinous cysts compared with nonmucinous cysts in both cohorts (glucose first cohort P = .002, validation P = .006; and kynurenine first cohort P = .002, validation P = .002). The ROC curve for glucose was 0.92 (95% confidence interval [CI], 0.81-1.00) and 0.88 (95% CI, 0.72-1.00) in the first and validation cohorts, respectively. The ROC for kynurenine was 0.94 (95% CI, 0.81-1.00) and 0.92 (95% CI, 0.76-1.00) in the first and validation cohorts, respectively. Neither could differentiate premalignant from malignant cysts. Glucose and kynurenine levels were significantly elevated for serous cystadenomas in both cohorts. LIMITATIONS: Small sample sizes. CONCLUSION: Metabolomic profiling identified glucose and kynurenine to have potential clinical utility for differentiating mucinous from nonmucinous pancreatic cysts. These markers also may diagnose serous cystadenomas.

Preclinical evaluation of the metabolism and disposition of RRx-001, a novel investigative anticancer agent.

RRx-001 has shown promise as a novel cancer therapeutic agent. The disposition of RRx-001 was evaluated in vitro and after intravenous administration to rats. At both 24 and 168 h after a single intravenous administration of ¹4C-RRx-001 (10 mg/kg), the majority of radiolabel was in the blood. The recovery of label in excreta was quite low, but the major route of radiolabel excretion was via the kidney, with approximately 26% in the urine by the first 8 h and decreasing amounts in all subsequent collections to a total of 36.3% by 168 h. The partitioning of total radioactivity in red blood cells (RBCs) and plasma was determined after in vitro addition to human, rat, dog, and monkey whole blood at 1 and 20 µM. In rat, at 30 min, approximately 75% of the radioactivity is associated with RBCs and 25% with plasma. In human, at 30 min, approximately 25% of the radioactivity is associated with RBCs and 75% with plasma. Analysis by liquid chromatography/radiodetection/mass spectrometry showed that ¹4C-RRx-001 reacted rapidly with whole blood to give four major soluble metabolites: the GSH and Cys adducts of RRx-001 (M1 and M2) and the corresponding mononitro GSH and Cys adducts (M3 and M4). Human Hb was incubated with cold RRx-001 in buffer, and a standard proteomics protocol was used to separate and identify the tryptic peptides. Standard peptide collision-induced fragment ions supported the structure of the peptide GTFATLSELHCDK with the alkylation on the Cys-93 locus of the Hb ß chain.

Metabolic activation of nevirapine in human liver microsomes: dehydrogenation and inactivation of cytochrome P450 3A4.

Nevirapine, a non-nucleoside HIV-1 reverse transcriptase inhibitor, has been associated with incidences of skin rash and hepatotoxicity in patients. Although the mechanism of idiosyncratic hepatotoxicity remains unknown, it is proposed that metabolic activation of nevirapine and subsequent covalently binding of reactive metabolites to cellular proteins play a causative role. Studies were initiated to determine whether nevirapine undergoes cytochrome P450 (P450)-mediated bioactivation in human liver microsomes to electrophilic intermediates. Liquid chromatography-tandem mass spectrometry analysis of incubations containing nevirapine and NADPH-supplemented microsomes in the presence of glutathione (GSH) revealed the formation of a GSH conjugate derived from the addition of the sulfydryl nucleophile to nevirapine. No other GSH conjugates were detected, including conjugates of oxidized metabolites of nevirapine. These findings are consistent with a bioactivation sequence involving initial P450-catalyzed dehydrogenation of the aromatic nucleus with a 4-methyl group in nevirapine to an electrophilic quinone methide intermediate, which is subsequently attacked by glutathione yielding the sulfydryl conjugate. Formation of the nevirapine GSH conjugate was primarily catalyzed by heterologously expressed recombinant CYP3A4 and, to a lesser extent, CYP2D6, CYP2C19, and CYP2A6. In addition, the quinone methide reactive metabolite was a mechanism-based inactivator of CYP3A4, with inactivation parameters K(I) = 31 microM and k(inact) = 0.029 min(-1), respectively. It is proposed that formation of the quinone methide intermediate may represent a rate-limiting step in the initiation of nevirapine-mediated hepatotoxicity.

Screening and characterization of reactive metabolites using glutathione ethyl ester in combination with Q-trap mass spectrometry.

The present study describes a new analytical approach for the detection and characterization of chemically reactive metabolites using glutathione ethyl ester (GSH-EE) as the trapping agent in combination with hybrid triple quadrupole linear ion trap mass spectrometry. Polarity switching was applied between a negative precursor ion (PI) survey scan and the positive enhanced product ion (EPI) scan. The negative PI scan step was carried out monitoring the anion at m/z 300, corresponding to deprotonated gamma-glutamyl-dehydroalanyl-glycine ethyl ester originating from the GSH-EE moiety. Samples resulting from incubations in the presence of GSH-EE were cleaned and concentrated by solid-phase extraction, followed by the PI-EPI analysis. Unambiguous identification of GSH-EE-trapped reactive metabolites was greatly facilitated by the unique survey scan of the anion at m/z 300, which achieved less background interference, in particular, from endogenous glutathione adducts present in human liver microsomes. Further structural characterization was achieved by analyzing positive MS(2) spectra that featured rich fragments without mass cutoff and were acquired in the same liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The effectiveness and reliability of this approach was evaluated using a number of model compounds in human liver microsomal incubations, including acetaminophen, amodiaquine, carbamazepine, 4-ethylphenol, imipramine and ticlopidine. In addition, iminoquinone reactive metabolites of mianserin were trapped and characterized for the first time using this method. Compared to neutral loss (NL) scanning assays using GSH as the trapping agent, the results have demonstrated superior selectivity, sensitivity, and reliability of this current approach.

Comparison of in vitro bioactivation of flutamide and its cyano analogue: evidence for reductive activation by human NADPH:cytochrome P450 reductase.

Flutamide (FLU), a nonsteroidal antiandrogen drug widely used in the treatment of prostate cancer, has been associated with idiosyncratic hepatotoxicity in patients. It is proposed that bioactivation of FLU and subsequent binding of reactive metabolite(s) to cellular proteins play a causative role. A toxicogenomic study comparing FLU and its nitro to cyano analogue (CYA) showed that the nitroaromatic group of FLU enhanced cytotoxicity to hepatocytes, indicating that reduction of the nitroaromatic group may represent a potential route of FLU-induced hepatotoxicity [Coe et al. (2007) Chem. Res. Toxicol. 20, 1277-1290]. In the current study, we compared in vitro bioactivation of FLU and CYA in human liver microsomes and cryopreserved human hepatocytes. A nitroreduction metabolite FLU-6 was formed in liver microsomal incubations of FLU under atmospheric oxygen levels and, to a greater extent, under anaerobic conditions. Seven glutathione (GSH) adducts of FLU, FLU-G1-7, were tentatively identified in human liver microsomal incubations using liquid chromatography-tandem mass spectrometry (LC/ MS/MS), while CYA formed only four corresponding GSH adducts, CYA-G1-4, under the same conditions. Of particular interest was the formation of FLU-G5-7 from FLU, where the nitroaromatic group of FLU was reduced to an amino group. A tentative pathway is that upon nitroreduction, the para-diamines undergo cytochrome P450 (P450)-catalyzed two-electron oxidations to form corresponding para-diimine intermediates that react with GSH to form GSH adducts FLU-G5-7, respectively. The identities of FLU-G5-7 were further confirmed by LC/MS/MS analyses of microsomal incubations of a synthesized standard FLU-6. In an attempt to identify enzymes involved in the nitroreduction of FLU, NADPH:cytochrome P450 reductase (CPR) was shown to reduce FLU to FLU-6 under both aerobic and anaerobic conditions. Furthermore, the formation of FLU-G5-7 was completely blocked by the addition of a reversible CPR inhibitor, alpha-lipoic acid, to the incubations of FLU under aerobic conditions. In summary, these results clearly demonstrate that nitroreduction of FLU by CPR contributes to bioactivation and potentially to hepatotoxicity of FLU.

Identification of glutathione-derived metabolites from an IP receptor antagonist.

The metabolic fate of three aromatic carboxylic acid analogs under evaluation as prostaglandin I2-preferring receptor antagonists was studied. The initial analog with unsubstituted phenyl groups was subject to a complex set of aromatic oxidative biotransformations. By introduction of one or two fluorines, these pathways were inhibited. All three analogs were metabolized to a wide variety of carboxylic acid conjugates. Among these were several conjugates formed via secondary metabolism and oxidation of acyl glutathione intermediates. Two of the structure classes, represented by the S-methyl-N-cysteinylglycine conjugate and the N-cysteinylglycine disulfide conjugates, have been described only rarely in the literature. The related S-oxide of the S-methyl-N-cysteinylglycine conjugate and the N,S-bis-acyl derivative of cysteinylglycine are here described for the first time as conjugate metabolites of carboxylic drugs.