Brian Middleton

In vitro approach to assess the potential for risk of idiosyncratic adverse reactions caused by candidate drugs.

Idiosyncratic adverse drug reactions (IADRs) in humans can result in a broad range of clinically significant toxicities leading to attrition during drug development as well as postlicensing withdrawal or labeling. IADRs arise from both drug and patient related mechanisms and risk factors. Drug related risk factors, resulting from parent compound or metabolites, may involve multiple contributory mechanisms including organelle toxicity, effects related to compound disposition, and/or immune activation. In the current study, we evaluate an in vitro approach, which explored both cellular effects and covalent binding (CVB) to assess IADR risks for drug candidates using 36 drugs which caused different patterns and severities of IADRs in humans. The cellular effects were tested in an in vitro Panel of five assays which quantified (1) toxicity to THLE cells (SV40 T-antigen-immortalized human liver epithelial cells), which do not express P450s, (2) toxicity to a THLE cell line which selectively expresses P450 3A4, (3) cytotoxicity in HepG2 cells in glucose and galactose media, which is indicative of mitochondrial injury, (4) inhibition of the human bile salt export pump, BSEP, and (5) inhibition of the rat multidrug resistance associated protein 2, Mrp2. In addition, the CVB Burden was estimated by determining the CVB of radiolabeled compound to human hepatocytes and factoring in both the maximum prescribed daily dose and the fraction of metabolism leading to CVB. Combining the aggregated results from the in vitro Panel assays with the CVB Burden data discriminated, with high specificity (78%) and sensitivity (100%), between 27 drugs, which had severe or marked IADR concern, and 9 drugs, which had low IADR concern, we propose that this integrated approach has the potential to enable selection of drug candidates with reduced propensity to cause IADRs in humans.

Development of a high-throughput brain slice method for studying drug distribution in the central nervous system.

New, more efficient methods of estimating unbound drug concentrations in the central nervous system (CNS) combine the amount of drug in whole brain tissue samples measured by conventional methods with in vitro estimates of the unbound brain volume of distribution (Vu,brain). Although the brain slice method is the most reliable in vitro method for measuring Vu,brain, it has not previously been adapted for the needs of drug discovery research. The aim of this study was to increase the throughput and optimize the experimental conditions of this method. Equilibrium of drug between the buffer and the brain slice within the 4 to 5 h of incubation is a fundamental requirement. However, it is difficult to meet this requirement for many of the extensively binding, lipophilic compounds in drug discovery programs. In this study, the dimensions of the incubation vessel and mode of stirring influenced the equilibration time, as did the amount of brain tissue per unit of buffer volume. The use of casette experiments for investigating Vu,brain in a linear drug concentration range increased the throughput of the method. The Vu,brain for the model compounds ranged from 4 to 3000 ml · g brain–1, and the sources of variability are discussed. The optimized setup of the brain slice method allows precise, robust estimation of Vu,brain for drugs with diverse properties, including highly lipophilic compounds. This is a critical step forward for the implementation of relevant measurements of CNS exposure in the drug discovery setting.

Improved measurement of drug exposure in the brain using drug-specific correction for residual blood

A major challenge associated with the determination of the unbound brain-to-plasma concentration ratio of a drug (Kp,uu,brain), is the error associated with correction for the drug in various vascular spaces of the brain, i.e., in residual blood. The apparent brain vascular spaces of plasma water (Vwater, 10.3 μL/g brain), plasma proteins (Vprotein, 7.99 μL/g brain), and the volume of erythrocytes (Ver, 2.13 μL/g brain) were determined and incorporated into a novel, drug-specific correction model that took the drug-unbound fraction in the plasma (fu,p) into account. The correction model was successfully applied for the determination of Kp,uu,brain for indomethacin, loperamide, and moxalactam, which had potential problems associated with correction. The influence on correction of the drug associated with erythrocytes was shown to be minimal. Therefore, it is proposed that correction for residual blood can be performed using an effective plasma space in the brain (Veff), which is calculated from the measured fu,p of the particular drug as well as from the estimates of Vwater and Vprotein, which are provided in this study. Furthermore, the results highlight the value of determining Kp,uu,brain with statistical precision to enable appropriate interpretation of brain exposure for drugs that appear to be restricted to the brain vascular spaces.

Dynamic contrast-enhanced MRI of vascular changes induced by the VEGF-signalling inhibitor ZD4190 in human tumour xenografts

Dynamic contrast-enhanced magnetic resonance imaging (DCEMRI) was used to examine the acute effects of treatment with an inhibitor of vascular endothelial growth factor (VEGF) signaling. ZD4190 is an orally bioavailable inhibitor of VEGF receptor-2 (KDR) tyrosine kinase activity, which elicits broad-spectrum antitumour activity in preclinical models following chronic once-daily dosing. Nude mice, bearing established (0.5-1.0 mL volume) human prostate (PC-3), lung (Calu-6) and breast (MDA-MB-231) tumor xenografts, were dosed with ZD4190 (p.o.) using a 1 day (0 and 22 h) or 7 day (0, 24, 48, 72, 96,120,144, and 166 h) treatment regimen. DCEMRI was employed 2 h after the last dose of ZD4190, using the contrast agent gadopentetate dimeglumine. Dynamic data were fit to a compartmental model to obtain voxelwise K(trans), the transfer constant for gadopentetate into the tumor. K(trans) was averaged over the entire tumor, and a multi-threshold histogram analysis was also employed to account for tumor heterogeneity. Reductions in K(trans) reflect reductions in flow, in endothelial surface area, and/or in vascular permeability. A vascular input function was obtained for each mouse simultaneously with the tumor DCEMRI data. ZD4190 treatment produced a dose-dependent (12.5-100 mg x kg(-1) per dose) reduction in K(trans) in PC-3 prostate tumors. At 100 mg x kg(-1), the largest concentration examined, ZD4190 reduced K(trans) in PC-3 tumors by 31% following 2 doses (1 day treatment regimen; p < 0.001) and by 53% following 8 doses (7 day regimen; p < 0.001). Comparative studies in the three models using a showed similar reductions in K(trans) for the lung and breast tumors using the histogram analysis, although the statistical significance was lost when K(trans) was averaged over the entire tumor. Collectively these studies suggest that DCEMRI using gadopentetate may have potential clinically, for monitoring inhibition of VEGF signaling in solid tumors.

Practical use of the regression offset approach for the prediction of in vivo intrinsic clearance from hepatocytes

Systematic under-prediction of clearance is frequently associated with in vitro kinetic data when extrapolated using physiological scaling factors, appropriate binding parameters and the well-stirred model. The present study describes a method of removing this systematic bias through application of empirical correction factors derived from regression analyses applied to the in vitro and in vivo data for a defined set of reference compounds. Linear regression lines were established with in vivo intrinsic clearance (CLint), derived from in vivo clearance data and scaled in vitro intrinsic clearance from isolated hepatocyte incubations. The scaled CLint was empirically corrected to a predicted in vivo CLint using the slope and intercept from a uniform weighted linear regression applied to the in vitro to in vivo extrapolation. Cross validation of human data demonstrated that 66% of the reference compounds had a predicted in vivo CLint within two-fold of the observed value. The average absolute fold error (AAFE) for the in vivo CLint predictions was 1.90. For rat, 54% of the compounds had a predicted value within two-fold of the observed and the AAFE was 1.98. Three AstraZeneca projects are used to exemplify how a two-sided prediction interval, applied to the rat regression corrected reference data, can form the basis for assessing the likelihood that, for a given chemical series, the in vitro kinetic data is predictive of in vivo clearance and is therefore appropriate to guide optimisation of compound metabolic stability.

Optimisation of a simple method to transiently transfect a CHO cell line in high-throughput and at large scale

Transient expression of heterologous proteins in mammalian systems is a powerful way to generate protein reagents quickly. However, it has historically suffered from poor yields in comparison to methods where the recombinant gene is stably integrated into the genome and high expressing clones isolated. Transient methods have been well described for HEK-based systems. In this paper we show the use of a design of experiments (DoE) approach to quickly analyse the effect of a range of different parameters on protein expression from a CHO-based transient system. We show that this system is amenable to a very simple transfection procedure by independent direct addition of DNA and transfection reagent to the culture vessel. In addition we show that expression can be improved by reducing the temperature of the culture conditions post-transfection. The process is demonstrated to be transferrable from 3 ml cultures in deep 24-well plates through cultures in CultiFlask Bioreactors, shake flasks and up to 25 L culture in Wave Bioreactors. Data are shown to illustrate the utility of the system with a number of different classes of protein.

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.

The Measurement of Focal Diurnal Variation in the Femoral Articular Cartilage of the Knee

Our objective was to test the hypothesis that focal diurnal changes occur in the femoral articular cartilage of the knee in asymptomatic young adults. Six volunteers each were scanned early in the morning, and at the end of a working day spent mainly standing. This protocol was repeated on three successive weeks. Femoral cartilage segmentations were obtained using a region-growing algorithm. These segmentations then were regridded onto a 500-pixel template, and differences in the resulting thickness maps were assessed. Analysis of variance showed no significant diurnal variation in mean thickness. There were, however, statistically-significant diurnal changes in the thickness maps. Cartilage thickness decreased during the day in three specific locations which suffer the greatest biomechanical force.

Sensitivity of Disease Parameters to Flexible Budesonide/Formoterol Treatment in an Allergic Rat Model

RATIONALE Clinical studies show that flexible dosing (maintenance and symptom-driven dose adjustments) of budesonide and formoterol (BUD/FORM) improves control of asthma exacerbations as compared to fixed maintenance dosing protocols (maintenance therapy) even when the latter utilize higher BUD/FORM doses. This suggests that dose-response relationships for certain pathobiologic mechanisms in asthma shift over time. Here, we have conducted animal studies to address this issue. OBJECTIVES (1) To test in an animal asthma-like model whether it is possible to achieve the same or greater pharmacological control over bronchoconstriction and airway/lung inflammation, and with less total drug used, by flexible BUD/FORM dosing (upward adjustment of doses) in association with allergen challenges. (2) To determine whether the benefit requires adjustment of both drug components. METHODS Rats sensitized on days 0 and 7 were challenged intratracheally with ovalbumin on days 14 and 21. On days 13-21, rats were treated intratracheally with fixed maintenance or flexible BUD/FORM combinations. On day 22, rats were challenged with methacholine and lungs were harvested for analysis. RESULTS A flexible BUD/FORM dosing regimen (using 3.3 times less total drug than the fixed maintenance high dose regimen), delivered the same or greater reductions of excised lung gas volume (a measure of gas trapped in lung by bronchoconstriction) and lung weight (a measure of inflammatory oedema). When either BUD or FORM alone was increased on days of challenge, the benefit of the flexible dose upward adjustment was lost. CONCLUSIONS Flexible dosing of the BUD/FORM combination improves the pharmacological inhibition of allergen-induced bronchoconstriction and an inflammatory oedema in an allergic asthma-like rat model.