Methanol adducts leading to the identification of a reactive aldehyde metabolite of CPAQOP in human liver microsomes by ultra-high-performance liquid chromatography/mass spectrometry.

RATIONALE: The incubation of CPAQOP (1-[(2R)-2-[[4-[3-chloro-4-(2-pyridyloxy)anilino]quinazolin-5-yl]oxymethyl]-1-piperidyl]-2-hydroxy) with human liver microsomes generated several metabolites that highlighted the hydroxyacetamide side chain was a major site of metabolism for the molecule. The metabolites were derived predominantly from oxidative biotransformations; however, two unexpected products were detected by liquid chromatography/ultraviolet/mass spectrometry (LC/UV/MS) and identified as methanol adducts. This observation prompted further LC/MS investigations into their formation. METHODS: Three separate incubations of CPAQOP were conducted in human liver microsomes; Naïve, fortified with methoxyamine and fortified with glutathione. Separation was achieved via ultra-high-performance liquid chromatography with either methanol or acetonitrile gradients containing formic acid. MS analysis was conducted by electrospray ionisation LTQ Orbitrap mass spectrometry acquiring accurate mass full scan, data-dependent MS2 and all ion fragmentation. RESULTS: No methanol adducts were detected by MS when acetonitrile was used in the mobile phase instead of methanol, verifying that a metabolite was reacting with methanol on column. Although this reactive metabolite could not be isolated or structurally characterised by LC/MS directly, product ion spectra of the methanol adducts confirmed addition of methanol on the hydroxyacetamide side chain. Additional experiments using methoxyamine showed the disappearance of the two methanol adducts and appearance of a methoxyamine adduct, confirming the presence of an aldhyde. Product ion spectra of the methoxyamine adduct confirmed addition of methoxyamine to the hydroxyacetamide side chain. CONCLUSIONS: The proposed bioactivation of CPAQOP occurred via the reactive aldehyde intermediate, which readily reacted with methanol in the mobile phase to form a pair of isomeric hemiacetal methanol adducts. In acidified methanol the equilibrium favoured the methanol adduct and in acidified acetonitrile it favoured the hydrate; therefore, the reactive aldehyde metabolite was not detected and could not be structurally characterised directly. Copyright © 2016 John Wiley & Sons, Ltd.

Biophysical methods in drug discovery from small molecule to pharmaceutical.

Biophysical methods have become established in many areas of drug discovery. Application of these methods was once restricted to a relatively small number of scientists using specialized, low throughput technologies and methods. Now, automated high-throughput instruments are to be found in a growing number of laboratories. Many biophysical methods are capable of measuring the equilibrium binding constants between pairs of molecules crucial for molecular recognition processes, encompassing protein-protein, protein-small molecule, and protein-nucleic acid interactions, and several can be used to measure the kinetic or thermodynamic components controlling these biological processes. For a full characterization of a binding process, determinations of stoichiometry, binding mode, and any conformational changes associated with such interactions are also required. The suite of biophysical methods that are now available represents a powerful toolbox of techniques which can effectively deliver this full characterization.The aim of this chapter is to provide the reader with an overview of the drug discovery process and how biophysical methods, such as surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), nuclear magnetic resonance, mass spectrometry (MS), and thermal unfolding methods can answer specific questions in order to influence project progression and outcomes. The selection of these examples is based upon the experiences of the authors at AstraZeneca, and relevant approaches are highlighted where they have utility in a particular drug discovery scenario.

ATM kinase inhibition preferentially sensitizes p53-mutant glioma to ionizing radiation.

PURPOSE: Glioblastoma multiforme (GBM) is the most lethal form of brain cancer with a median survival of only 12 to 15 months. Current standard treatment consists of surgery followed by chemoradiation. The poor survival of patients with GBM is due to aggressive tumor invasiveness, an inability to remove all tumor tissue, and an innate tumor chemo- and radioresistance. Ataxia-telangiectasia mutated (ATM) is an excellent target for radiosensitizing GBM because of its critical role in regulating the DNA damage response and p53, among other cellular processes. As a first step toward this goal, we recently showed that the novel ATM kinase inhibitor KU-60019 reduced migration, invasion, and growth, and potently radiosensitized human glioma cells in vitro. EXPERIMENTAL DESIGN: Using orthotopic xenograft models of GBM, we now show that KU-60019 is also an effective radiosensitizer in vivo. Human glioma cells expressing reporter genes for monitoring tumor growth and dispersal were grown intracranially, and KU-60019 was administered intratumorally by convection-enhanced delivery or osmotic pump. RESULTS: Our results show that the combined effect of KU-60019 and radiation significantly increased survival of mice 2- to 3-fold over controls. Importantly, we show that glioma with mutant p53 is much more sensitive to KU-60019 radiosensitization than genetically matched wild-type glioma. CONCLUSIONS: Taken together, our results suggest that an ATM kinase inhibitor may be an effective radiosensitizer and adjuvant therapy for patients with mutant p53 brain cancers.

The stability of amitriptyline N-oxide and clozapine N-oxide on treated and untreated dry blood spot cards.

Procedures for drug monitoring based on Dried Blood Spot (DBS) sampling are gaining acceptance for an increasing number of clinical and preclinical applications, where ease of use, small sample requirement, and improved sample stability have been shown to offer advantages over blood tube sampling. However, to-date, the vast majority of this work has described the analysis of well characterized drugs. Using amitriptyline, clozapine, and their potentially labile N-oxide metabolites as model compounds, we consider the merits of using DBS for discovery pharmacokinetic (PK) studies where the metabolic fate of test compounds are often unknown. Both N-oxide metabolites reverted to parent compound under standard drying (2hr) and extraction conditions. Card type significantly affected the outcome, with 14% and 22% degradation occurring for clozapine-N-oxide and amitriptyline-N-oxide on a brand of untreated DBS cards, compared to 59 and 88% on a brand of treated DBS cards. Enrichment of the parent compound ex vivo leads to overestimation of circulating blood concentration and inaccurate determination of the PK profile.

Bioactivation of the cannabinoid receptor antagonist rimonabant to a cytotoxic iminium ion metabolite.

The cannabinoid type 1 receptor (CB1r) antagonist rimonabant was approved in 2006 for the treatment of obesity but was withdrawn in 2008 due to serious drug-related psychiatric disorders. In vitro metabolism studies with rimonabant have revealed high levels of reactive metabolite formation, which resulted in irreversible time-dependent P450 3A4 inhibition and in covalent binding to microsomal proteins. In the present study, an in vitro approach has been used to explore whether metabolic bioactivation of rimonabant might result in cell toxicity. A panel of SV40-T-antigen-immortalized human liver derived (THLE) cells that had been transfected with vectors encoding various human cytochrome P450 enzymes (THLE-1A2, 2C9, 2C19, 2D6, and 3A4) or with an empty vector (THLE-Null) were exposed to rimonabant. Cell toxicity and covalent binding to cellular proteins were evaluated, as was metabolite formation. Rimonabant exhibited markedly potentiated dose and time dependent cytotoxicity to THLE-3A4 cells, compared to that of all other THLE cell lines. This was accompanied by high levels of covalent binding of [(14)C]-rimonabant to THLE-3A4 cell proteins (1433 pmol drug equivalents/mg protein) and the formation of several metabolites that were not generated by THLE-Null cells. These included N-aminopiperidine (NAP) and an iminium ion species. However, no toxicity was observed when THLE cells were incubated with NAP. Glutathione depletion did not alter the observed potent cell cytotoxicity of rimonabant to THLE-3A4 cells. Preincubation of THLE-3A4 cells with the cytochrome P450 3A4 inhibitor ritonavir blocked the selective toxicity of rimonabant to these cells. In addition, ritonavir pretreatment blocked the metabolism of the compound in the cells and thereby significantly decreased the covalent binding of [(14)C]-rimonabant to THLE-3A4 cell proteins. We conclude that the potent toxicity of rimonabant in THLE-3A4 cells occurs by a mechanistic sequence, which is initiated by cytochrome P450 3A4 mediated formation of a highly cytotoxic reactive iminium ion metabolite that binds covalently to cellular proteins.

Determination of paracetamol in mouse, rat and dog plasma samples by laser diode thermal desorption--APCI-MS/MS.

BACKGROUND: Laser diode thermal desorption (LDTD) is a relatively new sample introduction interface for MS. Analysis times are short as the technique does not require time-consuming separation steps, such as conventional HPLC, thus saving on the use of organic solvents, modifiers and cost, relating to their subsequent disposal. This paper compares the merits of LDTD-APCI-MS/MS and LC-MS/MS for the analysis of paracetamol (acetaminophen) in plasma from different species. RESULTS: LDTD-APCI-MS/MS compared favorably with our existing high-throughput generic LC-MS/MS method giving improved data quality. LDTD-APCI-MS/MS assay performance in terms of accuracy and precision in mouse, rat and dog plasma were within our local acceptance criteria (±30%). Run times were reduced approximately tenfold, while saving approximately 200 ml of solvent per 96-well plate. CONCLUSION: A rapid, sensitive and robust assay is reported. The method was successfully used for the analysis of spiked mouse, rat and dog plasma samples and the determination of oral pharmacokinetics. Reductions in electrical power and reagent consumption position LDTD as an environmentally 'green' bioanalytical method.

High-throughput metabolic stability studies in drug discovery by orthogonal acceleration time-of-flight (OATOF) with analogue-to-digital signal capture (ADC).

Screening assays capable of performing quantitative analysis on hundreds of compounds per week are used to measure metabolic stability during early drug discovery. Modern orthogonal acceleration time-of-flight (OATOF) mass spectrometers equipped with analogue-to-digital signal capture (ADC) now offer performance levels suitable for many applications normally supported by triple quadruple instruments operated in multiple reaction monitoring (MRM) mode. Herein the merits of MRM and OATOF with ADC detection are compared for more than 1000 compounds screened in rat and/or cryopreserved human hepatocytes over a period of 3 months. Statistical comparison of a structurally diverse subset indicated good agreement for the two detection methods. The overall success rate was higher using OATOF detection and data acquisition time was reduced by around 20%. Targeted metabolites of diazepam were detected in samples from a CLint determination performed at 1 microM. Data acquisition by positive and negative ion mode switching can be achieved on high-performance liquid chromatography (HPLC) peak widths as narrow as 0.2 min (at base), thus enabling a more comprehensive first pass analysis with fast HPLC gradients. Unfortunately, most existing OATOF instruments lack the software tools necessary to rapidly convert the huge amounts of raw data into quantified results. Software with functionality similar to open access triple quadrupole systems is needed for OATOF to truly compete in a high-throughput screening environment.

Synthesis and evaluation of PEG414, a novel formulating agent that avoids analytical problems associated with polydisperse vehicles such as PEG400.

Polyethylene glycol (PEG) 400 is widely used as a formulating agent for both intravenous and oral studies during drug discovery. It is a polydisperse material containing more than 16 oligomers, which can cause significant problems for high-performance liquid chromatography-mass spectrometry analysis due to ion suppression and isobaric interference. To overcome these difficulties, we have synthesized the single oligomer PEG414. The material has been characterized with a range of diverse drug compounds and shown to be comparable to PEG400 and superior to propylene glycol in terms of its solubilization power. The toxicological and metabolic properties of PEG414 should be similar to PEG400. It suffers none of the analytical problems associated with polydisperse agents and we expect it to be a useful alternative for the formulation of test compounds for intravenous and oral dosing during drug discovery.