Kevin A. Ford

Comparative Metabolism and Pharmacokinetics of Seven Neonicotinoid Insecticides in Spinach

The metabolism of seven commercial neonicotinoid insecticides was compared in spinach seedlings (Spinacia oleracea) using HPLC-DAD and LC-MSD to analyze the large number and great variety of metabolites. The parent neonicotinoid levels in the foliage following hydroponic treatment varied from differences in uptake and persistence. The metabolic reactions included nitro reduction, cyano hydrolysis, demethylation, sulfoxidation, imidazolidine and thiazolidine hydroxylation and olefin formation, oxadiazine hydroxylation and ring opening, and chloropyridinyl dechlorination. The identified phase I plant metabolites were generally the same as those in mammals, but the phase II metabolites differed in the conjugating moieties. Novel plant metabolites were various neonicotinoid-derived O- and N-glucosides and -gentiobiosides and nine amino acid conjugates of chloropyridinylcarboxylic acid. Metabolites known to be active on nicotinic acetylcholine receptors included the desnitro- and descyanoguanidines and olefin derivatives. The findings highlight both metabolites common to several neonicotinoids and those that are compound specific.

Enzymes and Inhibitors in Neonicotinoid Insecticide Metabolism

Neonicotinoid insecticide metabolism involves considerable substrate specificity and regioselectivity of the relevant CYP450, aldehyde oxidase, and phase II enzymes. Human CYP450 recombinant enzymes carry out the following conversions: CYP3A4, 2C19, and 2B6 for thiamethoxam (TMX) to clothianidin (CLO); 3A4, 2C19, and 2A6 for CLO to desmethyl-CLO; 2C19 for TMX to desmethyl-TMX. Human liver aldehyde oxidase reduces the nitro substituent of CLO to nitroso much more rapidly than it does that of TMX. Imidacloprid (IMI), CLO, and several of their metabolites do not give detectable N-glucuronides but 5-hydroxy-IMI, 4,5-diol-IMI, and 4-hydroxythiacloprid are converted to O-glucuronides in vitro with mouse liver microsomes and UDP-glucuronic acid or in vivo in mice. Mouse liver cytosol with S-adenosylmethionine converts desmethyl-CLO to CLO but not desmethyl-TMX to TMX. Two organophosphorus CYP450 inhibitors partially block IMI, thiacloprid, and CLO metabolism in vivo in mice, elevating brain and liver levels of the parent compounds while reducing amounts of the hydroxylated metabolites.

Principles and procedures for implementation of ICH M7 recommended (Q)SAR analyses.

The ICH M7 guideline describes a consistent approach to identify, categorize, and control DNA reactive, mutagenic, impurities in pharmaceutical products to limit the potential carcinogenic risk related to such impurities. This paper outlines a series of principles and procedures to consider when generating (Q)SAR assessments aligned with the ICH M7 guideline to be included in a regulatory submission. In the absence of adequate experimental data, the results from two complementary (Q)SAR methodologies may be combined to support an initial hazard classification. This may be followed by an assessment of additional information that serves as the basis for an expert review to support or refute the predictions. This paper elucidates scenarios where additional expert knowledge may be beneficial, what such an expert review may contain, and how the results and accompanying considerations may be documented. Furthermore, the use of these principles and procedures to yield a consistent and robust (Q)SAR-based argument to support impurity qualification for regulatory purposes is described in this manuscript.

Chemoselective sp2-sp3 cross-couplings: iron-catalyzed alkyl transfer to dihaloaromatics.

The chemoselective functionalization of a range of dihaloaromatics with methyl, cyclopropyl, and higher alkyl Grignard reagents via iron-catalyzed cross-coupling is described. The site selectivity of C-X (X = halogen) activation is determined by factors such as the position of the halogen on the ring, the solvent, and the nucleophile. A one-pot protocol for the chemoselective synthesis of mixed dialkyl heterocycles is achieved solely employing iron catalysis.

Pharmacokinetic drivers of toxicity for basic molecules: Strategy to lower pKa results in decreased tissue exposure and toxicity for a small molecule Met inhibitor

Several toxicities are clearly driven by free drug concentrations in plasma, such as toxicities related to on-target exaggerated pharmacology or off-target pharmacological activity associated with receptors, enzymes or ion channels. However, there are examples in which organ toxicities appear to correlate better with total drug concentrations in the target tissues, rather than with free drug concentrations in plasma. Here we present a case study in which a small molecule Met inhibitor, GEN-203, with significant liver and bone marrow toxicity in preclinical species was modified with the intention of increasing the safety margin. GEN-203 is a lipophilic weak base as demonstrated by its physicochemical and structural properties: high LogD (distribution coefficient) (4.3) and high measured pKa (7.45) due to the basic amine (N-ethyl-3-fluoro-4-aminopiperidine). The physicochemical properties of GEN-203 were hypothesized to drive the high distribution of this compound to tissues as evidenced by a moderately-high volume of distribution (Vd>3l/kg) in mouse and subsequent toxicities of the compound. Specifically, the basicity of GEN-203 was decreased through addition of a second fluorine in the 3-position of the aminopiperidine to yield GEN-890 (N-ethyl-3,3-difluoro-4-aminopiperidine), which decreased the volume of distribution of the compound in mouse (Vd=1.0l/kg), decreased its tissue drug concentrations and led to decreased toxicity in mice. This strategy suggests that when toxicity is driven by tissue drug concentrations, optimization of the physicochemical parameters that drive tissue distribution can result in decreased drug concentrations in tissues, resulting in lower toxicity and improved safety margins.

Extending (Q)SARs to incorporate proprietary knowledge for regulatory purposes: A case study using aromatic amine mutagenicity

Statistical-based and expert rule-based models built using public domain mutagenicity knowledge and data are routinely used for computational (Q)SAR assessments of pharmaceutical impurities in line with the approach recommended in the ICH M7 guideline. Knowledge from proprietary corporate mutagenicity databases could be used to increase the predictive performance for selected chemical classes as well as expand the applicability domain of these (Q)SAR models. This paper outlines a mechanism for sharing knowledge without the release of proprietary data. Primary aromatic amine mutagenicity was selected as a case study because this chemical class is often encountered in pharmaceutical impurity analysis and mutagenicity of aromatic amines is currently difficult to predict. As part of this analysis, a series of aromatic amine substructures were defined and the number of mutagenic and non-mutagenic examples for each chemical substructure calculated across a series of public and proprietary mutagenicity databases. This information was pooled across all sources to identify structural classes that activate or deactivate aromatic amine mutagenicity. This structure activity knowledge, in combination with newly released primary aromatic amine data, was incorporated into Leadscopes expert rule-based and statistical-based (Q)SAR models where increased predictive performance was demonstrated.

Characterization of rat liver proteins adducted by reactive metabolites of menthofuran.

Pulegone is the major constituent of pennyroyal oil, a folkloric abortifacient that is associated with hepatotoxicity and, in severe cases, death. Cytochrome P450-mediated oxidation of pulegone generates menthofuran, which is further oxidized to form electrophilic reactive intermediates, menthofuran epoxide and the ring-opened γ-ketoenal, both of which can form adducts to hepatocellular proteins. Modification of hepatocellular proteins by the electrophilic reactive intermediates of menthofuran has been implicated in hepatotoxicity caused by pennyroyal oil. Herein, we describe the identification of several proteins that are the likely targets of menthofuran-derived reactive metabolites. These proteins were isolated from the livers of rats treated with a hepatotoxic dose of menthofuran by two-dimensional gel electrophoresis (2D-gel) separation and detected by Western blot analysis using an antiserum developed to detect protein adducts resulting from menthofuran bioactivation. The antibody-reacting proteins were excised from the 2D-gel and subjected to tryptic digestion for analysis of peptide fragments by LC-MS/MS. Although 10 spots were detected by Western blot analysis, only 4 were amenable to characterization by LC-MS/MS: serum albumin, mitochondrial aldehyde dehydrogenase (ALDH2), cytoplasmic malate dehydrogenase (MDH1), and mitochondrial ATP synthase subunit d. No direct adduct was detected, and, therefore, we complemented our analysis with enzyme activity determination. ALDH2 activity decreased by 88%, and ATP synthase complex V activity decreased by 34%, with no activity changes to MDH1. Although the relationship between these reactive metabolite adducted proteins and hepatotoxicity is not clear, these targeted enzymes are known to play critical roles in maintaining cellular homeostasis.

Safety Lead Optimization and Candidate Identification: Integrating New Technologies into Decision-Making.

Discovery toxicology focuses on the identification of the most promising drug candidates through the development and implementation of lead optimization strategies and hypothesis-driven investigation of issues that enable rational and informed decision-making. The major goals are to [a] identify and progress the drug candidate with the best overall drug safety profile for a therapeutic area, [b] remove the most toxic drugs from the portfolio prior to entry into humans to reduce clinical attrition due to toxicity, and [c] establish a well-characterized hazard and translational risk profile to enable clinical trial designs. This is accomplished through a framework that balances the multiple considerations to identify a drug candidate with the overall best drug characteristics and provides a cogent understanding of mechanisms of toxicity. The framework components include establishing a target candidate profile for each program that defines the qualities of a successful candidate based on the intended therapeutic area, including the risk tolerance for liabilities; evaluating potential liabilities that may result from engaging the therapeutic target (pharmacology-mediated or on-target) and that are chemical structure-mediated (off-target); and characterizing identified liabilities. Lead optimization and investigation relies upon the integrated use of a variety of technologies and models (in silico, in vitro, and in vivo) that have achieved a sufficient level of qualification or validation to provide confidence in their use. We describe the strategic applications of various nonclinical models (established and new) for a holistic and integrated risk assessment that is used for rational decision-making. While this review focuses on strategies for small molecules, the overall concepts, approaches, and technologies are generally applicable to biotherapeutics.

An Algorithm for Evaluating Potential Tissue Drug Distribution in Toxicology Studies from Readily Available Pharmacokinetic Parameters

Having an understanding of drug tissue accumulation can be informative in the assessment of target organ toxicities; however, obtaining tissue drug levels from toxicology studies by bioanalytical methods is labor-intensive and infrequently performed. Additionally, there are no described methods for predicting tissue drug distribution for the experimental conditions in toxicology studies, which typically include non-steady-state conditions and very high exposures that may saturate several processes. The aim was the development of an algorithm to provide semiquantitative and quantitative estimates of tissue-to-plasma concentration ratios (Kp ) for several tissues from readily available parameters of pharmacokinetics (PK) such as volume of distribution (Vd ) and clearance of each drug, without performing tissue measurement in vivo. The computational approach is specific for the oral route of administration and non-steady-state conditions and was applied for a dataset of 29 Genentech small molecules such as neutral compounds as well as weak and strong organic bases. The maximum success rate in predicting Kp values within 2.5-fold error of observed Kp values was 82% at low doses (<100 mg/kg) in preclinical species. Prediction accuracy was relatively lower with saturation at high doses (≥100 mg/kg); however, an approach to perform low-to-high dose extrapolations of Kp values was presented and applied successfully in most cases. An approach for the interspecies scaling was also applied successfully. Finally, the proposed algorithm was used in a case study and successfully predicted differential tissue distribution of two small-molecule MET kinase inhibitors, which had different toxicity profiles in mice. This newly developed algorithm can be used to predict the partition coefficients Kp for small molecules in toxicology studies, which can be leveraged to optimize the PK drivers of tissue distribution in an attempt to decrease drug tissue level, and improve safety margins.

Novel mechanism for dehalogenation and glutathione conjugation of dihalogenated anilines in human liver microsomes: evidence for ipso glutathione addition.

The objective of the present study was to investigate the influence of halogen position on the formation of reactive metabolites from dihalogenated anilines. Herein we report on a proposed mechanism for dehalogenation and glutathione (GSH) conjugation of a series of ortho-, meta-, and para-dihalogenated anilines observed in human liver microsomes. Of particular interest were conjugates formed in which one of the halogens on the aniline was replaced by GSH. We present evidence that a (4-iminocyclohexa-2,5-dienylidene)halogenium reactive intermediate (QX) was formed after oxidation, followed by ipso addition of GSH at the imine moiety. The ipso GSH thiol attacks at the ortho-carbon and eventually leads to a loss of a halogen and GSH replacement. The initial step of GSH addition at the ipso position is also supported by density functional theory, which suggests that the ipso carbon of the chloro, bromo, and iodo (but not fluoro) containing 2-fluoro-4-haloanilines is the most positive carbon and that these molecules have the favorable highest occupied molecular orbital of the aniline and the lowest unoccupied orbital from GSH. The para-substituted halogen (chloro, bromo, or iodo but not fluoro) played a pivotal role in the formation of the QX, which required a delocalization of the positive charge on the para-halogen after oxidation. This mechanism was supported by structure-metabolism relationship analysis of a series of dihalogenated and monohalogenated aniline analogues.