William L. Fitch

Alkaloid and predation patterns in colorado lupine populations

SummaryColorado populations of herbaceous perennial lupines show three distinct patterns of amounts, kinds, and individual variability of inflorescence alkaloids. These patterns, interpreted as alternative chemical defense strategies, can be related to the susceptibility of populations to attack by larvae of a small flower-feeding lycaenid butterfly, Glaucopsyche lygdamus.In situations ecologically unfavorable to G. lygdumus, lupine populations have “low” alkaloidal profiles, accumulating relatively low amounts of single, bicyclic alkaloids in their inflorescences, with little individual alkaloidal variability, Lupine populations which are quite available to G. lygdamus, on the other hand, accumulate much higher amounts of inflorescence alkaloids. Of these alkaloidally “high” populations, those which suffer only minor predation by G. lygdamus have individually variable mixtures of three or four inflorescence alkaloids, which are found to be isomers of lupanine and closely related tetracyclic compounds. In contrast, those which suffer heavy predation by G. lygdamus show a mixture of nine diverse alkaloidal components including lupanine, hydroxylupanine, and hydroxylupanine esters which is quite invariant from individual to individual.It is hypothesized that individual variability in alkaloids is an anti-specialist chemical defense mechanism. Such individual variability may be advantageous to plant populations by reducing the possibility of selection for strains of specialist herbivores capable of detoxifying or otherwise withstanding plant defensive compounds.

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 KI = 31 μM and kinact = 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.

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).

Using Chimeric Mice with Humanized Livers to Predict Human Drug Metabolism and a Drug-Drug Interaction

Interspecies differences in drug metabolism have made it difficult to use preclinical animal testing data to predict the drug metabolites or potential drug-drug interactions (DDIs) that will occur in humans. Although chimeric mice with humanized livers can produce known human metabolites for test substrates, we do not know whether chimeric mice can be used to prospectively predict human drug metabolism or a possible DDI. Therefore, we investigated whether they could provide a more predictive assessment for clemizole, a drug in clinical development for the treatment of hepatitis C virus (HCV) infection. Our results demonstrate, for the first time, that analyses performed in chimeric mice can correctly identify the predominant human drug metabolite before human testing. The differences in the rodent and human pathways for clemizole metabolism were of importance, because the predominant human metabolite was found to have synergistic anti-HCV activity. Moreover, studies in chimeric mice also correctly predicted that a DDI would occur in humans when clemizole was coadministered with a CYP3A4 inhibitor. These results demonstrate that using chimeric mice can improve the quality of preclinical drug assessment.

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 β chain.

Identification of metabolites in Withania sominfera fruits by liquid chromatography and high-resolution mass spectrometry

RATIONALE Withania somnifera is a rich source of biologically active secondary metabolites. Our earlier investigations on the fruits of this plant have yielded novel compounds, withanamides, with potent antioxidant activity and protective effect on β-amyloid-induced cytotoxicity in vitro. However, several minor compounds present in the fruits have not been characterized previously which may contribute to the observed biological activities. METHODS Liquid chromatography (LC) coupled with high-resolution mass spectrometry (HRMS) with data-dependent and targeted MS/MS experiments were conducted to elucidate the structure of observed metabolites. RESULTS A total of 62 metabolites identified included 32 withanamides, 22 withanolides, 3 steroidal saponins, 2 lignanamides, feruloyl tyramine, methoxy feruloyl tyramine and a diglucoside of hydroxyl palmitic acid. The structures of these compounds were proposed based on accurate masses of the molecular and fragment ions. Several of these new compounds identified from the profile were derived from withanamides with variations in aliphatic and/or glycosyl moieties. In addition, six new withanolides, a new hydroxy fatty acid diglucoside and several known compounds in the extract were identified. CONCLUSIONS The current study revealed the presence of several new and known compounds in Withania somnifera fruits. High-resolution MS and MS/MS experiments provide an extremely effective approach to derive the structures of plant secondary metabolites including isomeric compounds.

Isolation, identification and quantitation of urinary organic acids

An application of the HISLIB program for the comparison of gas chromatographic-mass spectrometric profiles of urinary organic acids isolated by extraction and ion-exchange methods is described. Ion-exchange methods are clearly superior to solvent extraction in terms of the variety of compounds isolated. However, the former method has practical difficulties which make solvent extraction more attractive for rapid analyses. For the compounds isolated by both methods, the precision of analysis is similar, with standard deviations of relative concentration in the range 10--30% for most compounds.

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.

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

ABSTRACT 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.