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Background: Loxapine inhalation powder delivered by a hand-held device as a thermally generated aerosol (ADASUVE) was recently approved in the United States and European Union for use in the acute treatment of agitation in patients with bipolar disorder or schizophrenia. As smokers comprise a large subpopulation of these patients, and many antipsychotic drugs require dose adjustments for smokers, the objective of this study was to compare the pharmacokinetics of inhaled loxapine administered to smokers and nonsmokers., Methods: Pharmacokinetics and sedation pharmacodynamics using a visual analog scale were studied in 35 male and female adult subjects (18 nonsmokers and 17 smokers) following a single dose of 10 mg of inhaled loxapine. Blood samples were drawn at predose, 30 seconds, 1, 2, 3, 10, 30, and 60 minutes, and 2, 6, 12, and 24 hours after dosing. Loxapine and 8-OH-loxapine were analyzed using reverse-phase liquid chromatography coupled with a tandem mass spectrometer. Pharmacokinetic parameters assessed included Cmax, Tmax, AUCinf, and T1/2 for loxapine and 8-OH-loxapine. Geometric mean ratios (GMRs) were determined for smokers to nonsmokers., Results: Loxapine Cmax was similar in smokers and nonsmokers with a GMR of 99.0%. The median loxapine Tmax was 1.88 and 1.01 minutes for nonsmokers and smokers, respectively. Loxapine AUCinf and AUClast values in nonsmokers were comparable with smokers (GMRs of 85.3% and 86.7%, respectively). A slight decrease in the observed mean terminal half-life values was observed for smokers (6.52 hours for smokers and 7.30 hours for nonsmokers)., Conclusions: Sedation profiles and visual analog scale scores at each time point were similar for nonsmokers and smokers. It was concluded that inhaled loxapine does not require dosage adjustment based on smoking behavior.

The antipsychotic drugs risperidone, paliperidone, olanzapine, quetiapine, aripiprazole, clozapine, haloperidol, and chlorpromazine have been reported to have various degrees of interaction (substrate or inhibitor) with the multidrug resistance transporter, P-glycoprotein (P-gp). An interaction of the antipsychotic drug loxapine with P-gp was recently reported, but an IC50 value was not determined. Loxapine (as the succinate salt) was evaluated as a P-gp substrate, and inhibitor of P-gp mediated transport of digoxin in vitro in Caco-2 cells. Loxapine was not a substrate for P-gp but did exhibit weak-to-moderate inhibition (IC50 = 9.1 μM). Since the typical steady state maximal plasma concentrations of loxapine in clinical use have been reported to be in the nanomolar range, pharmacokinetic interactions due to the inhibition of P-gp activity are not expected.

A highly pure aerosol of the antipsychotic drug, loxapine, can be thermally generated through vaporization from a thin coating of loxapine on a stainless steel substrate with the formation of a condensation aerosol. Because loxapine can exist in two polymorphic forms, the morphological time course from loxapine drug substance to coating on the substrate (intermediate product) and ultimately to the aerosol was investigated using differential scanning calorimetery, X-ray diffraction (XRD), Fourier transform infrared, and Raman spectroscopy. Monoclinic and orthorhombic crystalline forms of loxapine were confirmed by single crystal and powder XRD. A mixture of both loxapine crystalline polymorphs was formed on the substrate, independent of the initial loxapine crystalline morphology, and demonstrated to be stable. The loxapine aerosols generated from the thermal aerosolization process were demonstrated to be amorphous, regardless of the initial polymorph of loxapine active pharmaceutical ingredient used. In humans, the amorphous aerosol was reported to be rapidly absorbed and the particle size resulted in rapid delivery to the deep lung., (Copyright © 2010 Wiley-Liss, Inc.)

Fentanyl, N-(1-phenethylpiperidin-4-yl)-N-phenylpropionamide is a rapid-acting, powerful opioid analgesic used extensively for anesthesia and chronic pain management. A forced degradation study of fentanyl active pharmaceutical ingredient (API) was performed using light, acid, base, heat and oxidation. Under acidic conditions, fentanyl was shown to degrade to N-phenyl-1-(2-phenylethyl)-piperidin-4-amine (PPA(1)). Fentanyl was stable to light exposure and base treatment with no degradation observed. Oxidation with hydrogen peroxide produced fentanyl N-oxide by rapidly oxidizing the nitrogen on the piperidine ring. Five degradants were formed during thermal degradation of fentanyl. The two known degradants included propionanilide (PRP(2)) and norfentanyl (NRF(3)). The three unknown degradants were first identified by mass using LC/MS, and postulated compounds were synthesized and confirmed by LC/MS and (1)H NMR. These degradants were identified as 1-phenethylpyridinium salt (1-PEP(4)), 1-phenethyl-1H-pyridin-2-one (1-PPO(5)), and 1-styryl-1H-pyridin-2-one (1-SPO(6)). In addition to the seven degradants, three known process impurities, acetyl fentanyl, pyruvyl fentanyl and butyryl fentanyl were also detected by reverse-phase high performance liquid chromatography (HPLC) with UV detection. All degradants and impurities were identified and confirmed using authentic materials. Method validation was performed for the assay of fentanyl and its related compounds in accordance to ICH guideline Q2(R1), and the method was demonstrated to be specific, linear (r>0.999 for fentanyl assay and r>0.996 for related compounds), accurate (recovery>99.6% for fentanyl assay and recovery>91.0 for related compounds), precise (%RSD<0.8% for fentanyl assay and <4.8% for related compounds), sensitive (limit of detection=0.08 microg/mL or 0.016% of nominal concentration), robust and suitable for its intended use. The chemical structures for the degradants and impurities were submitted to three in silico toxicity programs to identify any structural alerts., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Bidirectional transport studies were conducted to determine whether substrates of five intestinal transporters showed carrier-mediated asymmetric transport across MDCK (Madin-Darby canine kidney) cell monolayers grown under standard conditions. Drug concentrations were quantitated using liquid scintillation counting, liquid chromatography/mass spectrometry/mass spectrometry, or liquid chromatography/mass spectrometry. In the presence of a pH gradient, benzoic acid exhibited net apical-to-basolateral transport, with apparent permeability ratios (apical-to-basolateral permeability/basolateral-to-apical permeability) ranging from 14 to 25. The addition of valproic acid reduced the permeability ratio by 70-90%. Cephalexin transport also exhibited net absorption in the presence of a pH gradient, with apparent permeability ratios ranging from 14 to 71, depending on growth conditions. Radiolabeled phenylalanine exhibited a low level of carrier-mediated absorption with an apparent permeability ratio of 1.8 that was reduced to 1.0 in the presence of unlabeled L-phenylalanine. Taurocholic acid did not exhibit carrier-mediated absorption. Cyclosporine and fexofenadine exhibited P-glycoprotein-mediated efflux from both MDCK and Caco-2 cells, which was more sensitive to inhibition in MDCK cells. These results suggest that although MDCK cell monolayers may be a useful model for evaluating transport by the absorptive monocarboxylic acid and peptide transporters and the efflux transporter, P-glycoprotein, they are not useful for predicting large neutral amino acid or bile acid transport in the intestine., (Copyright 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:2622-2635, 2002)

Furosemide permeation across Caco-2 cells was investigated to determine if previously reported directional differences in transport rates are due to a saturable, energy dependent process. In addition, studies were carried out to determine the route of permeation for this drug. By comparing apical (A) to basolateral (B) and B to A directional transport across Caco-2 cells, a saturable, nonlinear component to furosemide transport was observed. Transport in the secretory direction was fit to yield the following apparent parameters K(m) = 63 +/- 28 microM, V(max) = 436 +/- 137 pmol/cm(2)h, and P(app) = 3.7 +/- 0.9 x 10(-7) cm/s. Evidence of energy dependence was demonstrated using both metabolic inhibition, and transport against a diffusion gradient methods. Disruption of tight junctions by use of the calcium chelator, EGTA, caused a significant increase in furosemide transport (twofold and 12-fold increases in B to A and A to B, respectively) indicating the importance of the paracellular route. We conclude that furosemide secretion from Caco-2 cells is the result of saturable active transport and passive diffusion that has a significant paracellular component., (Copyright 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91: 1169-1177, 2002)

Recent studies in our laboratory have shown that the loop diuretic, furosemide, is actively secreted by Caco-2 cells and rat jejunal tissue. This active secretion could be the result of efflux transporters such as P-gp, MRP1 or MRP2 (cMOAT). To determine if any of these transporters is responsible for the secretion of furosemide, we compared directional permeability in the wild-type cell lines, MDCK strains I and II, and LLC-PK1, vs. cell lines that overexpress a single transporter, in both the presence and absence of various inhibitors, for furosemide as compared to vinblastine. Sulfinpyrazone significantly inhibited the transport of vinblastine in MRP2 expressing cells, but not the wild-type controls. Vinblastine could not be confirmed as a substrate of MRP1. We were also unable to demonstrate that any particular transporter affected furosemide in excess of the background effects of endogenous transporters in the parental cell lines. Furosemide secretion from these kidney-derived cell lines is probably not the primary result of any of the well characterized efflux transporters (P-gp, MRP1 or MRP2), although they may still play a role in the observed Caco-2 secretion. This equivocal result acknowledges the difficulty in trying to determine the effect of a single protein in a complicated expression system., (Copyright 2002 S. Karger AG, Basel)

LC-fluorescence and LC-MS methods have been previously reported for use in decoding bead-based combinatorial libraries. We present the use of capillary electrochromatography (CEC) for highly selective decoding in combination with laser-induced fluorescence (LIF) detection for high sensitivity. The results are compared to prior data obtained using HPLC with fluorescence detection. The use of CEC shows promise for miniaturization and multiplexing for future applications, and the use of LIF detection can allow for detection at sub-pmol amounts.

X-ray crystallographic data to 2.57 A resolution (1 A = 0.1 nm) have been measured for the complex of a peptidyl trifluoromethylketone inhibitor with porcine pancreatic elastase (PPE); R = 0.14. The inhibitor forms a stable complex with the enzyme by means of a covalent attachment to active site Ser195O gamma, resulting in a hemiketal moiety with tetrahedral geometry. The tripeptide protion binds as an antiparallel beta-sheet, with four hydrogen bonds augmenting the active-site covalent linkage, Ki = 9.5 microM. His57 exhibits a bifurcated H-bond to both Ser195O gamma and an F atom of the inhibitor. This study is one of a series which explores the binding geometry of a variety of small substrates and inhibitors to PPE. This peptidyl-PPE complex affords insight into the binding geometry of a novel trifluoromethylketone moiety to a serine proteinase.

The crystal structure of porcine pancreatic elastase (PPE) complexed to carbobenzoxy-alanylisoleucine-boronic acid (ZAIB) is reported to 2.09-A resolution and refined to an R factor of 0.15. This is the first reported structural analysis of PPE with an isoleucine residu in the primary specificity pocket. The results include (1) marked displacement of the inhibitor out of the active site leading to (2) a close (2.2 A) direct contact between B (boron atom of the inhibitor) and N epsilon of His-57 and also (3) covalent bonding (1.5 A) to O gamma of Ser-195. A scheme for the mechanism of inhibition of PPE by ZAIB is proposed. A comparison with a peptidyl difluoromethyl ketone-PPE complex (Ki = 9.5 microns) is made to explain the strong inhibition of PPE by ZAIB (Ki = 0.3 micron). These results lead us to characterize this structure as a time- and space-averaged reaction intermediate, providing fresh insight into the cramped dimensions available in enzymatic catalyses.