John G. Swales

Simultaneous quantitation of metformin and sitagliptin from mouse and human dried blood spots using laser diode thermal desorption tandem mass spectrometry

A simple, rapid and robust high-throughput assay for the simultaneous analysis of metformin and sitagliptin from mouse and human dried blood spot samples using laser diode thermal desorption interfaced with atmospheric pressure chemical ionization tandem mass spectrometry (LDTD-APCI-MS/MS) was developed for use in a pharmaceutical discovery environment as an alternative to traditional plasma analysis. Analytes were extracted from dried blood spots using a simple punch disc and solvent extract procedure. Details of the method development and optimization of the instrumental parameters are presented. The method was successfully applied to spiked mouse and human dried blood spot samples. Analyte stability was determined in dried blood spots on FTA cards and as extracts of dried blood spots. The method was subsequently used to determine the oral pharmacokinetics of metformin and sitagliptin after dosing to male mice. Metformin and Sitagliptin results are compared to data generated by more traditional liquid chromatography-mass spectrometry methods. Intra-assay and inter-assay accuracy and precision across the analytes and species deviated by less than 30% at all calibration levels and less than 20% at all quality control levels.

Aurora kinase inhibitor nanoparticles target tumors with favorable therapeutic index in vivo

A nanoparticle formulation of an Aurora B kinase inhibitor uses ion pairing to achieve controlled release and efficacious, nontoxic target inhibition in tumors. Accurin nanoparticles dutifully deliver drug A class of drugs, called kinase inhibitors, could stop cancer in its tracks…if only these drugs could reach the tumors, stay for a while, and not be toxic. Hypothesizing that a nanoparticle formulation would solve the inhibitors’ woes, Ashton and colleagues investigated several different compositions of so-called Accurins—polymeric particles that encapsulate charged drugs through ion pairing. An Aurora B kinase, once formulated in Accurins, demonstrated a much-improved therapeutic index and preclinical efficacy compared with its parent molecule, when administered to rats and mice bearing human tumors. The Accurins allowed for sustained release of the drug over days, and did not have the same blood toxicity seen with the parent drug. A phase 1 trial is the next step for this nanomedicine, and additional preclinical studies will reveal whether such nanoformulations can improve the tolerability and efficacy of the broader class of molecularly targeted cancer therapeutics, including cell cycle inhibitors. Efforts to apply nanotechnology in cancer have focused almost exclusively on the delivery of cytotoxic drugs to improve therapeutic index. There has been little consideration of molecularly targeted agents, in particular kinase inhibitors, which can also present considerable therapeutic index limitations. We describe the development of Accurin polymeric nanoparticles that encapsulate the clinical candidate AZD2811, an Aurora B kinase inhibitor, using an ion pairing approach. Accurins increase biodistribution to tumor sites and provide extended release of encapsulated drug payloads. AZD2811 nanoparticles containing pharmaceutically acceptable organic acids as ion pairing agents displayed continuous drug release for more than 1 week in vitro and a corresponding extended pharmacodynamic reduction of tumor phosphorylated histone H3 levels in vivo for up to 96 hours after a single administration. A specific AZD2811 nanoparticle formulation profile showed accumulation and retention in tumors with minimal impact on bone marrow pathology, and resulted in lower toxicity and increased efficacy in multiple tumor models at half the dose intensity of AZD1152, a water-soluble prodrug of AZD2811. These studies demonstrate that AZD2811 can be formulated in nanoparticles using ion pairing agents to give improved efficacy and tolerability in preclinical models with less frequent dosing. Accurins specifically, and nanotechnology in general, can increase the therapeutic index of molecularly targeted agents, including kinase inhibitors targeting cell cycle and oncogenic signal transduction pathways, which have to date proved toxic in humans.

Mass Spectrometry Imaging of Cassette-Dosed Drugs for Higher Throughput Pharmacokinetic and Biodistribution Analysis

Cassette dosing of compounds for preclinical drug plasma pharmacokinetic analysis has been shown to be a powerful strategy within the pharmaceutical industry for increasing throughput while decreasing the number of animals used. Presented here for the first time is data on the application of a cassette dosing strategy for label-free tissue distribution studies. The aim of the study was to image the spatial distribution of eight nonproprietary drugs (haloperidol, bufuralol, midazolam, clozapine, terfenadine, erlotinib, olanzapine, and moxifloxacin) in multiple tissues after oral and intravenous cassette dosing (four compounds per dose route). An array of mass spectrometry imaging technologies, including matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI), liquid extraction surface analysis tandem mass spectrometry (LESA-MS/MS), and desorption electrospray ionization mass spectrometry (DESI-MS) was used. Tissue analysis following intravenous and oral administration of discretely and cassette-dosed compounds demonstrated similar relative abundances across a range of tissues indicating that a cassette dosing approach was applicable. MALDI MSI was unsuccessful in detecting all of the target compounds; therefore, DESI MSI, a complementary mass spectrometry imaging technique, was used to detect additional target compounds. In addition, by adapting technology used for tissue profiling (LESA-MS/MS) low spatial resolution mass spectrometry imaging (∼1 mm) was possible for all targets across all tissues. This study exemplifies the power of multiplatform MSI analysis within a pharmaceutical research and development (R&D) environment. Furthermore, we have illustrated that the cassette dosing approach can be readily applied to provide combined, label-free pharmacokinetic and drug distribution data at an early stage of the drug discovery/development process while minimizing animal usage.

Discovery of a Potent, Selective, and Orally Bioavailable Acidic 11β-Hydroxysteroid Dehydrogenase Type 1 (11β-HSD1) Inhibitor: Discovery of 2-[(3S)-1-[5-(Cyclohexylcarbamoyl)-6-propylsulfanylpyridin-2-yl]-3-piperidyl]acetic Acid (AZD4017)

Inhibition of 11β-HSD1 is an attractive mechanism for the treatment of obesity and other elements of the metabolic syndrome. We report here the discovery of a nicotinic amide derived carboxylic acid class of inhibitors that has good potency, selectivity, and pharmacokinetic characteristics. Compound 11i (AZD4017) is an effective inhibitor of 11β-HSD1 in human adipocytes and exhibits good druglike properties and as a consequence was selected for clinical development.

Determination of metformin in mouse, rat, dog and human plasma samples by laser diode thermal desorption/atmospheric pressure chemical ionization tandem mass spectrometry.

A simple, rapid and robust high-throughput assay for the quantitative analysis of metformin in plasma from different species using laser diode thermal desorption interfaced with atmospheric chemical pressure ionization tandem mass spectrometry (LDTD-APCI-MSMS) was developed for use in a pharmaceutical discovery environment. In order to minimize sample preparation a generic protein precipitation method was used to extract metformin from the plasma. Laser diode thermal desorption is a relatively new sample introduction method, the optimization of the instrumental parameters are presented. The method was successfully applied to spiked mouse, rat, dog and human plasma samples and was subsequently used to determine the oral pharmacokinetics of metformin after dosing to male rats in order to support drug discovery projects. The deviations for intra-assay accuracy and precision across the four species were less than 30% at all calibration and quality control levels.

Novel Acidic 11β-Hydroxysteroid Dehydrogenase Type 1 (11β-HSD1) Inhibitor with Reduced Acyl Glucuronide Liability: The Discovery of 4-[4-(2-Adamantylcarbamoyl)-5-tert-butyl-pyrazol-1-yl]benzoic Acid (AZD8329)

Inhibition of 11β-HSD1 is viewed as a potential target for the treatment of obesity and other elements of the metabolic syndrome. We report here the optimization of a carboxylic acid class of inhibitors from AZD4017 (1) to the development candidate AZD8329 (27). A structural change from pyridine to pyrazole together with structural optimization led to an improved technical profile in terms of both solubility and pharmacokinetics. The extent of acyl glucuronidation was reduced through structural optimization of both the carboxylic acid and amide substituents, coupled with a reduction in lipophilicity leading to an overall increase in metabolic stability.

11β-Hydroxysteroid Dehydrogenase Type 1 (11β-HSD1) Inhibitors Still Improve Metabolic Phenotype in Male 11β-HSD1 Knockout Mice Suggesting Off-Target Mechanisms

The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a target for novel type 2 diabetes and obesity therapies based on the premise that lowering of tissue glucocorticoids will have positive effects on body weight, glycemic control, and insulin sensitivity. An 11β-HSD1 inhibitor (compound C) inhibited liver 11β-HSD1 by >90% but led to only small improvements in metabolic parameters in high-fat diet (HFD)–fed male C57BL/6J mice. A 4-fold higher concentration produced similar enzyme inhibition but, in addition, reduced body weight (17%), food intake (28%), and glucose (22%). We hypothesized that at the higher doses compound C might be accessing the brain. However, when we developed male brain-specific 11β-HSD1 knockout mice and fed them the HFD, they had body weight and fat pad mass and glucose and insulin responses similar to those of HFD-fed Nestin-Cre controls. We then found that administration of compound C to male global 11β-HSD1 knockout mice elicited improvements in metabolic parameters, suggesting “off-target” mechanisms. Based on the patent literature, we synthesized another 11β-HSD1 inhibitor (MK-0916) from a different chemical series and showed that it too had similar off-target body weight and food intake effects at high doses. In summary, a significant component of the beneficial metabolic effects of these 11β-HSD1 inhibitors occurs via 11β-HSD1–independent pathways, and only limited efficacy is achievable from selective 11β-HSD1 inhibition. These data challenge the concept that inhibition of 11β-HSD1 is likely to produce a “step-change” treatment for diabetes and/or obesity.

Investigating nephrotoxicity of polymyxin derivatives by mapping renal distribution using mass spectrometry imaging.

Colistin and polymyxin B are effective treatment options for Gram-negative resistant bacteria but are used as last-line therapy due to their dose-limiting nephrotoxicity. A critical factor in developing safer polymyxin analogues is understanding accumulation of the drugs and their metabolites, which is currently limited due to the lack of effective techniques for analysis of these challenging molecules. Mass spectrometry imaging (MSI) allows direct detection of targets (drugs, metabolites, and endogenous compounds) from tissue sections. The presented study exemplifies the utility of MSI by measuring the distribution of polymyxin B1, colistin, and polymyxin B nonapeptide (PMBN) within dosed rat kidney tissue sections. The label-free MSI analysis revealed that the nephrotoxic compounds (polymyxin B1 and colistin) preferentially accumulated in the renal cortical region. The less nephrotoxic analogue, polymyxin B nonapeptide, was more uniformly distributed throughout the kidney. In addition, metabolites of the dosed compounds were detected by MSI. Kidney homogenates were analyzed using LC/MS/MS to determine total drug exposure and for metabolite identification. To our knowledge, this is the first time such techniques have been utilized to measure the distribution of polymyxin drugs and their metabolites. By simultaneously detecting the distribution of drug and drug metabolites, MSI offers a powerful alternative to tissue homogenization analysis and label or antibody-based imaging.

Pharmacokinetics and metabolism of midazolam in chimeric mice with humanised livers

The pharmacokinetics and biotransformation of midazolam were investigated following single oral doses of 0.1, 1 and 10 mg/kg to chimeric mice with humanised livers (PXB mice) and to severe combined immunodeficient (SCID) mice used as controls. Pharmacokinetic analysis, on whole blood, revealed rapid absorption of the administered midazolam with a higher Cmax in PXB compared to SCID. The exposure to 1′-hydroxymidazolam was approximately 14-fold greater than to midazolam in the SCID mice and close to equivalent in the PXB mice. The metabolism of midazolam in SCID mice was faster than in the PXB mice such that pharmacokinetic data for midazolam in SCID mice could not be generated from the lowest dose in these animals. Both oxidative and conjugative metabolic pathways were identified in the PXB mice. All the major circulating metabolites observed in humans; 1′-hydroxymidazolam, 4′-hydroxymidazolam, 1′,4′-dihydroxymidazolam and 1′-hydroxymidazolam glucuronide, were detected in the blood of PXB mice. However, 4′-hydroxymidazolam and the 1′-hydroxymidazolam glucuronide were not detected in blood samples obtained from SCID mice. The midazolam metabolite profile in the PXB mouse was similar to that previously reported for human suggesting that the PXB mouse model can provide a model system for predicting circulating human metabolites.

Exemplifying the Screening Power of Mass Spectrometry Imaging over Label-Based Technologies for Simultaneous Monitoring of Drug and Metabolite Distributions in Tissue Sections

Mass spectrometry imaging (MSI) provides pharmaceutical researchers with a suite of technologies to screen and assess compound distributions and relative abundances directly from tissue sections and offer insight into drug discovery–applicable queries such as blood-brain barrier access, tumor penetration/retention, and compound toxicity related to drug retention in specific organs/cell types. Label-free MSI offers advantages over label-based assays, such as quantitative whole-body autoradiography (QWBA), in the ability to simultaneously differentiate and monitor both drug and drug metabolites. Such discrimination is not possible by label-based assays if a drug metabolite still contains the radiolabel. Here, we present data exemplifying the advantages of MSI analysis. Data of the distribution of AZD2820, a therapeutic cyclic peptide, are related to corresponding QWBA data. Distribution of AZD2820 and two metabolites is achieved by MSI, which [14C]AZD2820 QWBA fails to differentiate. Furthermore, the high mass-resolving power of Fourier transform ion cyclotron resonance MS is used to separate closely associated ions.