Electrospray ionization (ESI) liquid chromatography-tandem mass spectrometry (LC/MS/MS) assays provide high-throughput and selective methods for quantitation of small molecules. Use of LC/MS/MS assays for macromolecules, like oligonucleotides, is challenging due to lack of sensitivity and low analyte recovery from biomatrixes. Due to this fact, the method of choice for oligonucleotides quantitation remains hybridization-based ligand-binding assays. These biological assays usually possess high sensitivity but low selectivity and narrow dynamic range. They also require optimizing suitable "capture and detection" probes, which can be prohibitively time-consuming and expensive in a drug discovery lead-optimization scenario. In this paper, we present a unique LC/MS/MS assay for a model phosphorothioate backbone oligodeoxynucleotide (ODN) drug (7692 amu) from rat plasma. Multiple analytical challenges were encountered. The strategies used to solve these challenges should prove useful to scientists pursuing mass spectrometry (MS) to quantitate oligonucleotides. The challenges include analyte multiple charging and cation adduction (reduced sensitivity), oxidation of analyte on drying and high protein binding (low recovery), ODN affinity to exposed silica (low chromatographic reproducibility and high carryover), nonspecific binding of analyte to containers (low storage stability), and optimization/synthesis of an appropriate internal standard (interference and cross-talk). A buffer (7 mM triethylamine and 3 mM ammonium formate)/methanol, 50:50 (v/v), was used as an ESI-MS infusion solvent and produced a sharp multiple charge-state distribution. The sample extraction method combined a phenol/chloroform liquid-liquid extraction and solid-phase extraction steps, which improved the absolute recovery to >70%. The method was validated in the range of 5-2000 ng/mL and had precision (percent relative standard deviation)<10.1% and accuracy (percent relative error)<11.4%.

The imidazoquinoline (R)-5,6-Dihydro-N,N-dimethyl-4H-imidazo[4,5,1-ij]quinolin-5-amine [(R)-3] is a potent dopamine agonist when tested in animals but surprisingly shows very low affinity in in vitro binding assays. When incubated with mouse or monkey liver S9 microsomes, (R)-3 is metabolized by N-demethylation and oxidation to (R)-5,6-dihydro-5-(methylamino)-4H-imidazo[4,5,1-ij]quinolin-2(1H) -one [(R)-6], intermediate metabolites, where N-demethylation to the imidazoquinoline (R)-4 and where oxidation to the imidazoquinolinone (R)-5 has taken place, are also observed in these incubates. A cross-species study on the metabolism of (R)-3 in vitro has shown large variations in the extent of metabolism from species to species. Imidazoquinolinones (R)-5 and (R)-6 have comparable activity to (R)-3 in animals and also show good dopaminergic (D2) and serotonergic (5HT1A) activities in binding assays. It is probable that these metabolites account at least in part for the in vivo activity found for (R)-3. Efficient syntheses for compounds 3-6 as single enantiomers from quinoline are presented together with information on the biological activities and metabolic stabilities of these compounds.

Prolongation of the electrocardiogram QT interval by some, but not all drugs, has been associated with increased incidence of sudden cardiac death. Current preclinical regulatory assays cannot discriminate the arrhythmia liability of these drugs. Consequently, many new medications that prolong the QT interval are not developed despite their potential therapeutic benefit. Alternans (action potential duration alternations) is a measure of cardiac instability in humans and animals associated with the onset of ventricular fibrillation. Due to potential arrhythmia risk from observed QT prolongation, alternans was assessed in the anesthetized guinea pig after azithromycin or chloroquine alone and after combination treatment at clinically relevant concentrations proposed for the management of malaria. Chloroquine alone, but not azithromycin, caused a profound increase in action potential duration but with only minimal effects on alternans (approximately 10 ms). Azithromycin alone and in combination with chloroquine showed no increase in alternans beyond vehicle baseline responses indicating no additional arrhythmia liability.

The subject of metabolites in safety testing has had much debate in the recent past and has shown itself to be a complex issue with no simple solutions to providing absolute assurance of drug safety. Much of the attention has focused on the ability to identify metabolites and then demonstrate that their risk has been adequately characterized, either through their exposure in toxicology species or, failing this, by direct safety testing. In this review, we summarize our forward operational strategy that combines the principles summarized in the FDA Guidance, together with discussions at scientific meetings and literature opinions. It is a balance between the primary goal of assuring patient safety with one of reasonable investment. A key principle in striking this balance is to build stepwise information on metabolites through the drug discovery and development continuum. This allows assessments to be made from early nonclinical studies onward as to whether or not metabolite safety is underwritten by exposure in toxicology species. This strategy does not require absolute quantitation of the metabolites in early clinical trials but relies upon comparison of relative exposures between animals and humans using the capabilities of modern analytical techniques. Through this strategy, human disproportionate metabolites can be identified to allow a decision regarding the need for absolute quantitation and direct safety testing of the metabolite. Definitive radiolabeled studies would be initiated following proof of pharmacology or efficacy in humans, and nonclinical safety coverage would be adequately assessed prior to large-scale clinical trials. In cases where metabolite safety is not supported through the parent compound toxicology program, approaches for the direct safety testing of metabolites with regard to general and reproductive toxicology, safety pharmacology, and genetic safety have been defined.