Kenneth A. Koeplinger

Comparison of drug distribution images from whole-body thin tissue sections obtained using desorption electrospray ionization tandem mass spectrometry and autoradiography.

Desorption electrospray ionization tandem mass spectrometry (DESI-MS/MS) and whole-body autoradiography (WBA) were used for chemical imaging of whole-body thin tissue sections of mice intravenously dosed with propranolol (7.5 mg/kg). DESI-MS/MS imaging utilized selected reaction monitoring detection performed on an AB/MDS SCIEX 4000 QTRAP mass spectrometer equipped with a prototype extended length particle discriminator interface. Propranolol images of the tissue sections using DESI-MS/MS were obtained at surface scan rates of 0.1, 0.5, 2, and 7 mm/s. Although signal decreased with increasing scan rate, useful whole-body images for propranolol were obtained from the tissues even at 7 mm/s, which required just 79 min of analysis time. Attempts to detect and image the distribution of the known propranolol metabolites were unsuccessful. Regions of the tissue sections showing the most radioactivity from WBA sections were excised and analyzed by high-performance liquid chromatography (HPLC) with radiochemical detection to determine relative levels of propranolol and metabolites present. Comparison of the DESI-MS/MS signal for propranolol and the radioactivity attributed to propranolol from WBA sections indicated nominal agreement between the two techniques for the amount of propranolol in the brain, lung, and liver. Data from the kidney showed an unexplained disparity between the two techniques. The results of this study show the feasibility of using DESI-MS/MS to obtain useful chemical images of a drug in whole-body thin tissue sections following drug administration at a pharmacologically relevant level. Further optimization to improve sensitivity and enable detection of the drug metabolites will be among the requirements necessary to move DESI-MS/MS chemical imaging forward as a practical tool in drug discovery.

Robust Antiviral Efficacy upon Administration of a Nucleoside Analog to Hepatitis C Virus-Infected Chimpanzees

ABSTRACT Hepatitis C virus (HCV) infects an estimated 170 million individuals worldwide and is associated with an increased incidence of liver fibrosis, cirrhosis, and hepatocellular carcinoma. Currently approved therapies to treat HCV infection consist of combinations of pegylated alpha interferon and ribavirin which result in a sustained viral response in 40 to 60% of patients. Efforts to develop improved therapies include the development of direct inhibitors of virally encoded enzymes such as the viral RNA-dependent RNA polymerase. A nucleoside analog, 2′-C-methyl-7-deaza-adenosine (MK-0608), has been shown to inhibit viral RNA replication in the subgenomic HCV genotype 1b replicon, with a 50% effective concentration (EC50) of 0.3 μM (EC90 = 1.3 μM). To determine efficacy in vivo, MK-0608 was administered to HCV-infected chimpanzees, resulting in dose- and time-dependent decreases in plasma viral loads. In separate experiments, chimpanzees dosed for 7 days with MK-0608 at 0.2 and 2 mg per kg of body weight per day by intravenous administration experienced average reductions in viral load of 1.0 and >5 log10 IU/ml, respectively. Two other HCV-infected chimpanzees received daily doses of 1 mg MK-0608 per kg via oral administration. After 37 days of oral dosing, one chimpanzee with a high starting viral load experienced a reduction in viral load of 4.6 log10, and the viral load in the other chimpanzee fell below the limit of quantification (LOQ) of the HCV TaqMan assay (20 IU/ml). Importantly, viral load remained below the LOQ throughout the duration of dosing and for at least 12 days after dosing ended. The results demonstrate a robust antiviral effect on the administration of MK-0608 to HCV-infected chimpanzees.

Impact of pH on plasma protein binding in equilibrium dialysis.

Many pharmacokinetic analyses require unbound plasma concentrations, including prediction of clearance, volume of distribution, drug-drug interactions, brain uptake analysis, etc. It is most often more convenient to measure the total drug concentration in plasma rather than the unbound drug concentration. To arrive at unbound plasma concentrations, separate in vitro determinations of the plasma protein binding of a drug are usually carried out in serum or in plasma, and the plasma pharmacokinetic results are then mathematically adjusted by this fraction unbound ( f u,p). Plasma protein binding or the drug fraction unbound in plasma ( f u,p) is known to be affected by protein, drug, free fatty acid concentrations, lipoprotein partitioning, temperature, pH, and the presence or absence of other drugs/displacing agents within plasma samples. Errors in f u,p determination caused by lack of adequate pH control in newer assay formats for plasma protein binding (e.g., 96-well equilibrium thin walled polypropylene dialysis plates) will have significant drug-specific impact on these pharmacokinetic calculations. Using a diverse set of 55 drugs and a 96-well equilibrium dialysis plate format, the effect of variable pH during equilibrium dialysis experiments on measured values of f u,p was examined. Equilibrium dialysis of human plasma against Dulbeccos phosphate buffered saline at 37 degrees C under an air or 10% CO 2 atmosphere for 22 h resulted in a final pH of approximately 8.7 and 7.4, respectively. The ratio of f u,p at pH 7.4 (10% CO 2) vs pH 8.7 (air) was >or=2.0 for 40% of the 55 compounds tested. Only one of the 55 compounds tested had a ratio <0.9. Select compounds were further examined in rat and dog plasma. In addition, physicochemical properties were calculated for all compounds using ACD/Labs software or Merck in-house software and compared to plasma protein binding results. Changes in plasma protein binding due to pH increases which occurred during the equilibrium dialysis experiment were not species specific but were drug-specific, though nonpolar, cationic compounds had a higher likely hood of displaying pH-dependent binding. These studies underscore the importance of effectively controlling pH in plasma protein binding studies.

Discovery of a selective allosteric M1 receptor modulator with suitable development properties based on a quinolizidinone carboxylic acid scaffold.

One approach to ameliorate the cognitive decline in Alzheimers disease (AD) has been to restore neuronal signaling from the basal forebrain cholinergic system via the activation of the M(1) muscarinic receptor. A number of nonselective M(1) muscarinic agonists have previously shown positive effects on cognitive behaviors in AD patients, but were limited due to cholinergic adverse events thought to be mediated by the activation of the M(2) to M(5) subtypes. One strategy to confer selectivity for M(1) is the identification of positive allosteric modulators, which would target an allosteric site on the M(1) receptor rather than the highly conserved orthosteric acetylcholine binding site. Quinoline carboxylic acids have been previously identified as highly selective M(1) positive allosteric modulators with good pharmacokinetic and in vivo properties. Herein is described the optimization of a novel quinolizidinone carboxylic acid scaffold with 4-cyanopiperidines being a key discovery in terms of enhanced activity. In particular, modulator 4i gave high plasma free fractions, enhanced central nervous system (CNS) exposure, was efficacious in a rodent in vivo model of cognition, and afforded good physicochemical properties suitable for further preclinical evaluation.

Quinolizidinone Carboxylic Acids as CNS Penetrant, Selective M1 Allosteric Muscarinic Receptor Modulators

Positive allosteric modulation of the M1 muscarinic receptor represents an approach to treat the cognitive decline in patients with Alzheimers disease. Replacement of a quinolone ring system in a quinolone carboxylic acid series of M1 modulators with a quinolizidinone bearing a basic amine linkage led to a series of compounds with higher free fraction, enhanced CNS exposure, and improved efficacy in rodent in vivo models of cognition.

Sustained Viral Response in a Hepatitis C Virus-Infected Chimpanzee via a Combination of Direct-Acting Antiviral Agents

ABSTRACT Efforts to develop novel, interferon-sparing therapies for treatment of chronic hepatitis C (HCV) infection are contingent on the ability of combination therapies consisting of direct antiviral inhibitors to achieve a sustained virologic response. This work demonstrates a proof of concept that coadministration of the nucleoside analogue MK-0608 with the protease inhibitor MK-7009, both of which produced robust viral load declines as monotherapy, to an HCV-infected chimpanzee can achieve a cure of infection.

Parallel synthesis of N-biaryl quinolone carboxylic acids as selective M1 positive allosteric modulators

An iterative analog library synthesis approach was employed in the exploration of a quinolone carboxylic acid series of selective M(1) positive allosteric modulators, and strategies for improving potency and plasma free fraction were identified.

Heterocyclic fused pyridone carboxylic acid M1 positive allosteric modulators

The phenyl ring in a series of quinolone carboxylic acid M(1) positive allosteric modulators was replaced with a variety of heterocycles in order to reduce protein plasma binding and enhance CNS exposure.

HILIC LC‐MS for the determination of 2′‐C‐methyl‐cytidine‐triphosphate in rat liver

A very accurate and selective LC-MS/MS method was developed and validated for the quantification of 2-C-modified nucleoside triphosphate in liver tissue samples. An efficient pretreatment procedure of liver tissue samples was developed, using a fully automated SPE procedure with 96-well SPE plate (weak anion exchange sorbent, 30 mg). Nucleotide hydrophilic interaction chromatography has been performed on an aminopropyl column (100 mm x 2.0 mm, 3 microm) using a gradient mixture of ACN and ACN/water (5:95 v/v) with 20 mM ammonium acetate at pH 9.45 as mobile phase at 300 microL/min flow rate. The 2-C-modified nucleoside triphosphate was detected in the negative ESI mode in multiple reaction monitoring (MRM) mode. Calibration curve was linear over the 0.05-50 microM concentration range. Satisfying results, confirming the high reliability of the established LC-MS/MS method, were obtained for intraday precision (CV = 2.5-9.1%) and accuracy (92.6-94.8%) and interday precision (CV = 9.6-11.5%) and accuracy (94.4-102.4%) as well as for recovery (82.0-112.6%) and selectivity. The method has been successfully applied for pharmacokinetic studies of 2-C-methyl-cytidine-triphosphate in liver tissue samples.

N-Heterocyclic derived M1 positive allosteric modulators

Replacement of a phenyl ring with N-linked heterocycles in a series of quinolone carboxylic acid M1 positive allosteric modulators was investigated. In particular, a pyrazole derivative exhibited improvements in potency, free fraction, and CNS exposure.