At the forefront of new synthetic endeavors, such as drug discovery or natural product synthesis, large quantities of material are rarely available and timelines are tight. A miniaturized automation platform enabling high-throughput experimentation for synthetic route scouting to identify conditions for preparative reaction scale-up would be a transformative advance. Because automated, miniaturized chemistry is difficult to carry out in the presence of solids or volatile organic solvents, most of the synthetic "toolkit" cannot be readily miniaturized. Using palladium-catalyzed cross-coupling reactions as a test case, we developed automation-friendly reactions to run in dimethyl sulfoxide at room temperature. This advance enabled us to couple the robotics used in biotechnology with emerging mass spectrometry-based high-throughput analysis techniques. More than 1500 chemistry experiments were carried out in less than a day, using as little as 0.02 milligrams of material per reaction.

Metabolite identification studies involve the detection and structural characterization of the biotransformation products of drug candidates. These experiments are necessary throughout the drug discovery and development process. The use of high-resolution chromatography and high-resolution mass spectrometry together with data processing using mass defect filtering is described for in vitro and in vivo metabolite identification studies. Data collection was done using UPLC coupled with an orthogonal hybrid quadrupole time-of-flight mass spectrometer. This experimental approach enabled the use of MS(E) data collection (where E represents collision energy) which has previously been shown to be a powerful approach for metabolite identification studies. Post-acquisition processing with a prototype mass defect filtering program was used to eliminate endogenous interferences in the study samples, greatly enhancing the discovery of metabolites. The ease of this approach is illustrated by results showing the detection and structural characterization of metabolites in plasma from a preclinical rat pharmacokinetic study.

Peripheral inflammation involves an increase in cyclooxygenase-2 (COX-2)-mediated prostaglandin (PG) synthesis in the central nervous system (CNS), which contributes to allodynia and hyperalgesia. In the present study we have determined the changes in prostanoid tissue levels and in expression of terminal prostanoid synthases in both the CNS and inflamed peripheral tissue during carrageenan-induced paw inflammation in the rat. Prostanoid levels were measured by liquid chromatography-mass spectrometry and enzyme expression at the RNA level by quantitative PCR analysis during both the early (1-6 h) and late (12 and 24 h) phases of the inflammatory response. In the paw, the early phase was associated with increases in PGE 2 and thromboxane (TX)B 2 levels and with a peak of COX-2 expression that preceded that of microsomal prostaglandin-E 2 synthase-1 (mPGES-1). COX-2 and mPGES-1 remained elevated during the late phase, and PGE 2 continued to further increase through 24 h. The cytosolic PGE 2 synthase (cPGES) showed a small transient increase during the early phase, whereas mPGES-2 expression was not affected by inflammation. In the cerebrospinal fluid, elevated levels of PGE 2 , 6-keto-PGF 1␣ , PGD 2 , and TXB 2 were detected during the early phase. PGE 2 levels also increased in the spinal cord and, to a lesser extent, in the brain and remained elevated in both the cerebrospinal fluid and the spinal cord during the late phase. The expression of mPGES-1 was strongly up-regulated in the brain and spinal cord during inflammation, whereas no change was detected for the expression of cPGES, mPGES-2, COX-1, and terminal PGD, TX, or PGI synthases. The results show that the carrageenan-induced edema in the paw elicits an early phase of COX-2 induction in the CNS leading to an increase synthesis in PGD 2 , 6-keto-PGF 1␣ , and TXB 2 in addition to the major PGE 2 response. The data also indicate that the up-regulation of mPGES-1 contributes to COX-2-mediated PGE 2 production in the CNS during peripheral inflammation.Carrageenan-induced inflammation in the rat paw represents a classical model of edema formation and hyperalgesia, which has been extensively used in the development of nonsteroidal anti-inflammatory drugs and selective COX 1 -2 inhibitors. Several lines of evidence indicate that the COX-2-mediated increase in prostaglandin (PG) E 2 production in the central nervous system (CNS) contributes to the severity of the inflammatory and pain responses in this model. COX-2 is rapidly induced in the spinal cord and other regions of the CNS following carrageenan injection in the paw (1). The administration of selective COX-2 inhibitors, but not COX-1 inhibitors, reduces the levels of PGE 2 in the cerebrospinal fluid (CSF) and hyperalgesia (2-5). In addition, it has been shown that the intrathecal administration of PGE 2 potentiates carrageenaninduced inflammation (6) and that the direct microinjection of PGE 2 in the brain causes hyperalgesia (7). Selective COX-2 inhibitors can also inhibit peripheral pain responses when g...

A microdose cocktail containing midazolam, dabigatran etexilate, pitavastatin, rosuvastatin, and atorvastatin has been established to allow simultaneous assessment of a perpetrator impact on the most common drug metabolizing enzyme, cytochrome P450 (CYP)3A, and the major transporters organic anion-transporting polypeptides (OATP)1B, breast cancer resistance protein (BCRP), and MDR1 P-glycoprotein (P-gp). The clinical utility of these microdose cocktail probe substrates was qualified by conducting clinical drug interaction studies with three inhibitors with different in vitro inhibitory profiles (rifampin, itraconazole, and clarithromycin). Generally, the pharmacokinetic profiles of the probe substrates, in the absence and presence of the inhibitors, were comparable to their reported corresponding pharmacological doses, and/or in agreement with theoretical expectations. The exception was dabigatran, which resulted in an approximately twofold higher magnitude for microdose compared to conventional dosing, and, thus, can be used to flag a worst-case scenario for P-gp. Broader application of the microdose cocktail will facilitate a more comprehensive understanding of the roles of drug transporters in drug disposition and drug interactions.