XJB-5-131 inhibited ferroptosis in tubular epithelial cells after ischemia-reperfusion injury.

Regulated necrosis has been reported to exert an important role in the pathogenesis of various diseases, including renal ischemia-reperfusion (I/R) injury. Damage to renal tubular epithelial cells and subsequent cell death initiate the progression of acute kidney injury (AKI) and subsequent chronic kidney disease (CKD). We found that ferroptosis appeared in tubular epithelial cells (TECs) of various human kidney diseases and the upregulation of tubular proferroptotic gene ACSL4 was correlated with renal function in patients with acute kidney tubular injury. XJB-5-131, which showed high affinity for TECs, attenuated I/R-induced renal injury and inflammation in mice by specifically inhibiting ferroptosis rather than necroptosis and pyroptosis. Single-cell RNA sequencing (scRNA-seq) indicated that ferroptosis-related genes were mainly expressed in tubular epithelial cells after I/R injury, while few necroptosis- and pyroptosis-associated genes were identified to express in this cluster of cell. Taken together, ferroptosis plays an important role in renal tubular injury and the inhibition of ferroptosis by XJB-5-131 is a promising therapeutic strategy for protection against renal tubular cell injury in kidney diseases.

Possible artefacts of antioxidant assays performed in the presence of nitroxides and nitroxide-containing nanoparticles.

Nitroxides and nitroxide-containing nanoparticles (RNP) are excellent antioxidants. However, they have relatively high reduction potentials, which make them behave like oxidants or show little activity in some antioxidant assays. We found that stable nitroxyl radicals (TEMPO and 4-amino-TEMPO) has low reactivity in the test of scavenging of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical (ABTS•). As a result, supplementation of blood plasma with nitroxides may decrease its total antioxidant capacity assayed with ABTS•. Nitroxides oxidize Fe2+ and in this way interfere with the ferric-Xylenol Orange assay of peroxides. Nitroxides as well as RNP directly oxidize glutathione and fluorogenic probes used for estimation of reactive oxygen species (ROS) (dihydro-2'7'-dichlorofluorescein diacetate, dihydroethidine and dihydrorhodamine 123) and thus produce artefacts in assays of glutathione and ROS in cell-free and cellular systems. These results point to the necessity of careful interpretation of antioxidant assays concerning nitroxides and RNP or performed in their presence.

Development and validation of a rapid LC-MS/MS method for simultaneous quantification of arecoline and its two active metabolites in rat plasma and its application to a pharmacokinetic study.

Arecoline is the primary active and toxic constituent of areca nut. Arecaidine and arecoline N-oxide are two major active metabolites of arecoline. In this work, an accurate and simple high performance liquid chromatography tandem mass spectrometry method for simultaneous quantification of arecoline, arecaidine and arecoline N-oxide in rat plasma was developed and fully validated to study their pharmacokinetic behaviors in rats. After extracted from rat plasma by protein precipitation with methanol and then concentrated, the analytes were chromatographic separated on a Sepax Sapphire C18 analytical column. The mobile phase consisted of methanol and 2 mM ammonium acetate buffer solution containing 0.2% (v/v) formic acid (8:92, v/v) under isocratic elution. The analytes were detected by multiple reaction monitoring (MRM) with an electrospray ionization source in the positive ion mode. The transitions of m/z 156.2 → 53.2, m/z 142.2 → 44.2 and m/z 172.2 → 60.2 were selected for arecoline, arecaidine and arecoline N-oxide, respectively. The method was linear over the concentration range of 0.5-100 ng/mL for arecoline, 5-5000 ng/mL for arecaidine and arecoline N-oxide with no carry-over effect. The accuracies and intra- and inter-batch precisions were all within the acceptance limits. No matrix effect and potential interconversion between the analytes and other metabolites were observed in this method. The validated method was further employed to a preclinical pharmacokinetic study of arecoline, arecaidine and arecoline N-oxide after oral treatment with 20 mg/kg arecoline to rats.

Noninvasive mapping of the redox status of dimethylnitrosamine-induced hepatic fibrosis using in vivo dynamic nuclear polarization-magnetic resonance imaging.

Hepatic fibrosis is a chronic disorder caused by viral infection and/or metabolic, genetic and cholestatic disorders. A noninvasive procedure that enables the detection of liver fibrosis based on redox status would be useful for disease identification and monitoring, and the development of treatments. However, an appropriate technique has not been reported. This study describes a novel method for assessing the redox status of the liver using in vivo dynamic nuclear polarization-magnetic resonance imaging (DNP-MRI) with the nitroxyl radical carbamoyl-PROXYL as a molecular imaging probe, which was tested in dimethylnitrosamine-treated mice as a model of liver fibrosis. Based on the pharmacokinetics of carbamoyl-PROXYL in control livers, reduction rate mapping was performed in fibrotic livers. Reduction rate maps demonstrated a clear difference between the redox status of control and fibrotic livers according to the expression of antioxidants. These findings indicate that in vivo DNP-MRI with a nitroxyl radical probe enables noninvasive detection of changes in liver redox status.

Radiological and Pathological Correlation in Anti-MDA5 Antibody-positive Interstitial Lung Disease: Rapidly Progressive Perilobular Opacities and Diffuse Alveolar Damage.

We herein present the first case of rapidly progressive interstitial lung disease (RP-ILD) with anti-melanoma differentiation-associated protein 5 (MDA5) antibody evaluated by surgical lung biopsy (SLB). High-resolution CT scan revealed perilobular opacities, which rapidly became thicker and formed consolidation, resulting in remarkable loss of lung volume. Specimens taken from SLB revealed membranous organization with alveolar occlusion, dilation of alveolar ducts, and sacs with collapsed alveoli, which are typical features of diffuse alveolar damage (DAD). Rapidly progressive perilobular opacities may be characteristic of RP-ILD with anti-MDA5 antibody and DAD.

Simultaneous determination of nicotine, cotinine, and nicotine N-oxide in human plasma, semen, and sperm by LC-Orbitrap MS.

Nicotine (Nic) distribution in human fluids and tissues has a deleterious effect on human health. In addition to its poisoning profile, Nic may contribute to the particular impact of smoking on human reproduction. Although present in seminal fluid, still nobody knows whether nicotine is available in sperm or not. Herein, we developed and validated a new bioanalytical method, for simultaneous determination of Nic, cotinine (Cot), and nicotine N'-oxide (Nox) in human plasma, semen, and sperm by LC-ESI-orbitrap-MS. Blood and semen samples were collected from 12 healthy smoking volunteers in this study. Sperm bodies were then separated quantitatively from 1 mL of semen samples by centrifugation. The developed method was fully validated for plasma following European and American guidelines for bioanalytical method validation, and partial validation was applied to semen analysis. Plasma, semen, and sperm samples were treated by trichloroacetic acid solution for protein direct precipitation in single extraction step. The established calibration range for Nic and Nox in plasma and semen was linear between 5 and 250 ng/mL, and for Cot between 10 and 500 ng/mL. Nic and Cot were detected in human sperm at concentrations as high as in plasma. In addition, Nox was present in semen and sperm but not in plasma. Graphical abstract Nicotine correlation between plasma and semen a; Nicotine correlation between semen and sperm c; Cotinine correlation between plasma and semen b; Cotinine correlation between semen and sperm d.

Determination of strychnine, brucine, strychnine N-oxide, and brucine N-oxide in plasma samples after the oral administration of processed semen strychni extract by high-performance liquid chromatography with ultrasound-assisted mixed cloud point extraction.

A sensitive and efficient mixed cloud point extraction combined with high-performance liquid chromatography was developed for the simultaneous separation and determination of four alkaloids (strychnine, strychnine N-oxide, brucine, and brucine N-oxide) in plasma after the oral administration of processed semen strychni extract. Tergitol TMN-6 and cetyl-trimethyl ammonium bromide were chosen as the mixed surfactants, and ultrasound was employed to enhance the extraction efficiency. Some important parameters affecting the mixed cloud point extraction efficiency, such as the content of Tergitol TMN-6 and cetyl-trimethyl ammonium bromide, pH, salt effect, extraction temperature, and ultrasound time were studied and optimized. Under optimum conditions, the linear range of four alkaloids was from 1.0 to 1000 ng/mL. All correlation coefficients of the calibration curves were higher than 0.9993. The intraday and interday precision were below 8.65% and the limits of detection for the four alkaloids were less than 1.0 ng/mL (S/N = 3).

Monitoring Plasma Levels of Donepezil, 5-O-Desmethyl-Donepezil, 6-O-Desmethyl-Donepezil, and Donepezil-N-Oxide by a Novel HPLC Method in Patients With Alzheimer Disease.

BACKGROUND: In humans, donepezil (D) is metabolized to 5-O-desmethyl-donepezil (5DD), 6-O-desmethyl-donepezil (6DD), and donepezil-N-oxide (DNox). Although 6DD and DNox are pharmacologically active, the activity of 5DD is unknown. At present, no routine methods are available to detect D and its 3 metabolites simultaneously. In this study, a novel high-performance liquid chromatography method was developed and applied to a population of patients with Alzheimer disease on stable treatment with the drug. METHODS: Liquid-liquid extraction from plasma was accomplished by means of a solvent mixture of n-hexane/dichloromethane/ethylacetate (45:40:15) after sample alkalinization. Disopyramide was the internal standard. After evaporation, the residue was reconstituted in 200 µL of mobile phase (acetonitrile 85%:1% acetic acid 15%) and 50 µL was injected into the high-performance liquid chromatography column (X-Terra, RP8; flow: 1 mL/min). Photometric and fluorimetric detectors were used in tandem, to maximize the sensitivity of fluorescent compounds (D, 5DD, and DNox) and also to reveal nonfluorescent compounds (6DD and internal standard). RESULTS: The method was linear in the 10-100 ng/mL concentration range. Imprecision (coefficient of variation) varied between 3.2% and 12.6% and inaccuracy (% mean absolute error) between 1.3% and 13.3%, depending on the compound, concentration, and detection mode. The quantitation limits were 0.1-0.3 ng/mL for fluorescent compounds and 1.2-4.3 ng/mL for photometric compounds. D, 5DD, 6DD, and DNox through concentrations were measured in 54 patients with Alzheimer disease on treatment with D (10 mg q.d.). No interfering peaks by endogenous compounds or coadministered drugs were noted. Plasma concentrations were quite variable among patients (D: 10-106 ng/mL; 5DD: 0.07-2.8 ng/mL; 6DD: 1.2-36 ng/mL; DNox: 0.5-45.4 ng/mL). Of note, in 6 patients, the plasma concentrations of the 2 active metabolites (6DD and DNox) were higher than those of the parent drug. CONCLUSIONS: The above method proved to be suitable for therapeutic drug monitoring and may be useful in ascertaining the real contribution of metabolites to the therapeutic effects of donepezil.

Ultra-performance liquid chromatography-tandem mass spectrometric assay for the simultaneous determination of brucine, strychnine and brucine N-oxide in rat plasma: application to a pharmacokinetic study.

A rapid, simple and sensitive UHPLC-MS/MS method was developed and validated for the simultaneous determination of brucine, strychnine and brucine N-oxide in rat plasma using huperzine A as an internal standard (IS) after protein precipitation with methanol. The analytes were separated on a Purospher® STAR RP18 UHPLC column (2 µm, 2.1 × 100 mm) by gradient elution using a mobile phase composed of methanol and water (containing 0.1% formic acid) at a flow rate of 0.3 mL/min. Brucine, strychnine, brucine N-oxide and IS were detected in positive ion multiple reaction monitoring mode by means of an electrospray ionization interface (m/z 395.2 → 324.1, m/z 335.2 → 184.1, m/z 411.2 → 394.2, m/z 243.1 → 226.1). The calibration curve was linear over the range of 1-500 ng/mL for brucine and strychnine and 0.2-50 ng/mL for brucine N-oxide. The intra- and inter-day precisions of these analytes were all within 15% and the accuracy ranged from 85 to 115%. The stability experiment indicated that the plasma samples at three concentration levels were stable under different conditions. The developed method was successfully applied for the first time to pharmacokinetic studies of brucine, strychnine and brucine N-oxide following a single oral and intravenous administration of modified total alkaloid fraction in rats. Copyright © 2016 John Wiley & Sons, Ltd.

Enantioselective separation of mirtazapine and its metabolites by capillary electrophoresis with acetonitrile field-amplified sample stacking and its application.

A simple, rapid and sensitive chiral capillary zone electrophoresis coupled with acetonitrile-field-amplified sample stacking method was developed that allows the simultaneous enantioselective separation of the mirtazapine, N-demethylmirtazapine, 8-hydroxymirtazapine and mirtazapine-N-oxide. The separation was achieved on an uncoated 40.2 cm×75 µM fused silica capillary with an applied voltage of 16 kV. The electrophoretic analyses were carried out in 6.25 mM borate-25 mM phosphate solution at pH 2.8 containing 5.5 mg/mL carboxymethyl-ß-cyclodextrin. The detection wavelength was 200 nm. Under these optimized conditions, satisfactory chiral separations of four pair enantiomers were achieved in less than 7 min in vitro. After one step clean-up liquid-liquid extraction using 96-well format, sample was introduced capillary zone electrophoresis with acetonitrile-field-amplified sample stacking to enhance the sensitivity of enantiomers. The method was validated with respect to specificity, linearity, lower limit of quantitation, accuracy, precision, extraction recovery and stability. The lower limit of quantification was 0.5 ng/mL with linear response over the 0.5-50 ng/mL concentration range for each mirtazapine, N-demethylmirtazapine and 8-hydroxymirtazapine enantiomer. The developed and validated method has been successfully applied to the enantioselective pharmacokinetic studies in 12 healthy volunteers after oral administration of rac- mirtazapine.

Age-related differences in the disposition of nicotine and metabolites in rat brain and plasma.

INTRODUCTION: Studies have evaluated the behavioral and neurochemical impact of nicotine administration in rodents. However, the distribution of nicotine and metabolites in rat brain and plasma as a function of age has not been investigated. This is a significant issue because human adolescents have a greater risk for developing nicotine addiction than adults, and reasons underlying this observation have not been fully determined. Thus, in this present study, we evaluated the impact of the transition from adolescence (postnatal day [PND 40]) to adulthood (PND 90) on nicotine distribution in rats. METHODS: PND 40, 60, and 90 rats received a single injection of (-) nicotine (0.8 mg/kg, subcutaneously). Liquid chromatography tandem-mass spectrometry was used to measure concentration of nicotine and metabolites in selected biological matrices. RESULTS: Nicotine, cotinine, and nornicotine were detected in rat striata and frontal cortex 30 min, 1 hr, 2 hr, and 4 hr after a single administration. These and several additional metabolites (nicotine-1'-oxide, cotinine-N-oxide, norcotinine, and trans-3'-hydroxycotinine) were also detected in plasma at these same timepoints. The mean concentration of nicotine in brain and plasma was lower in PND 40 versus PND 90 rats. In contrast, the mean concentration of nornicotine was higher in the plasma and brain of PND 40 versus PND 90 rats. CONCLUSIONS: Nicotine and metabolite distribution differs between adolescent and adult rats. These data suggest that adolescent rats metabolize nicotine to some metabolites faster than adult rats. Further studies are needed to investigate the potential correlation between age, drug distribution, and nicotine addiction.

Simultaneous determination of NNK and its seven metabolites in rabbit blood by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

A hydrophilic interaction liquid chromatographic-tandem mass spectrometric (HILIC-MS-MS) method for investigation of the in vivo metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent carcinogen, in rabbit blood has been developed and validated. This method achieved excellent repeatability and accuracy. Recovery ranged from 76.9 to 116.3 % and precision (as RSD) between 0.53 and 6.52 %. Linearity was good for all compounds (R(2)>0.9990) and the limit of detection (LOD) ranged from 0.016 to 0.082 ng mL(-1). Pharmacokinetic analysis indicated that NNK was rapidly eliminated in vivo in rabbit blood and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) was the major metabolite. The hydroxy acid, keto acid, and NNAL-N-oxide were also important metabolites in rabbit blood. It is probable that α-methylene hydroxylation was the major pathway of α-hydroxylation of NNK and NNAL in the rabbit.

Large inter-individual variation of the pharmacodynamic effect of anticoagulant drugs on thrombin generation.

Anticoagulation by a standard dosage of an inhibitor of thrombin generation presupposes predictable pharmacokinetics and pharmacodynamics of the anticoagulant. We determined the inter-individual variation of the effect on thrombin generation of a fixed concentration of direct and antithrombin-mediated inhibitors of thrombin and factor Xa. Thrombin generation was determined by calibrated automated thrombinography in platelet-poor plasma from 44 apparently healthy subjects which was spiked with fixed concentrations of otamixaban, melagatran, unfractionated heparin, dermatan sulfate and pentasaccharide. The variability of the inhibitory effect of the different anticoagulants within the population was determined using the coefficient of variation, i.e. the standard deviation expressed as a percentage of the mean. The inter-individual coefficients of variation of the endogenous thrombin potential and peak height before inhibition were 18% and 16%, respectively and became 20%-24% and 24%-43% after inhibition. The average inhibition of endogenous thrombin potential and peak height (ETP, peak) brought about by the anticoagulants was respectively: otamixaban (27%, 83%), melagatran (56%, 63%), unfractionated heparin (43%, 58%), dermatan sulfate (68%, 57%) and pentasaccharide (25%, 67%). This study demonstrates that the addition of a fixed concentration of any type of anticoagulant tested causes an inhibition that is highly variable from one individual to another. In this respect there is no difference between direct inhibitors of thrombin and factor Xa and heparin(-like) inhibitors acting on the same factors.

Validation of HPLC-MS/MS methods for analysis of loxapine, amoxapine, 7-OH-loxapine, 8-OH-loxapine and loxapine N-oxide in human plasma.

BACKGROUND: Two ESI-LC-MS/MS methods were validated for the quantitative analysis of loxapine, amoxapine, 7-OH-loxapine, 8-OH-loxapine and loxapine N-oxide in human K(2)EDTA plasma. Cation-exchange solid-phase extraction (SPE) was used to extract loxapine, amoxapine and the two hydroxylated metabolites, and organic precipitation was used to quantify loxapine N-oxide. RESULTS: Both methods were shown to be accurate (±13%), intra-assay precision was less than 15%, and inter-assay precision was less than 10% in all instances across the entire dynamic range of the assays (0.0500-50.0 ng/ml for the SPE method and 0.100-25.0 ng/ml for the precipitation method). CONCLUSION: The validated methods for loxapine, amoxapine, 7-OH-loxapine, 8-OH-loxapine and loxapine N-oxide have been used to successfully support clinical trials.

Unusual peroxidase activity of polynitroxylated pegylated hemoglobin: Elimination of H(2)O(2) coupled with intramolecular oxidation of nitroxides.

Polynitroxylated hemoglobin (Hb(AcTPO)(12)) has been developed as a hemoglobin-based oxygen carrier. While Hb(AcTPO)(12) has been shown to exert beneficial effects in a number of models of oxidative injury, its peroxidase activity has not been characterized thus far. In the blood stream, Hb(AcTPO)(12) undergoes reduction by ascorbate to its hydroxylamine form Hb(AcTPOH)(12). Here we report that Hb(AcTPOH)(12) exhibits peroxidase activity where H(2)O(2) is utilized for intramolecular oxidation of its TPOH residues to TPO. This represents an unusual redox-catalytic mechanism whereby reduction of H(2)O(2) is achieved at the expense of reducing equivalents of ascorbate converted into those of Hb(AcTPOH)(12), a new propensity that cannot be directly associated with ascorbate.

Comparison of two nitroxide labile esters for delivering electron paramagnetic resonance probes into mouse brain.

In vivo quantitation of O(2) in brain has been hindered by a lack of suitable imaging modalities. Development of low-frequency electron paramagnetic resonance (EPR) spectrometers that can detect free radicals in animals in real time makes it feasible to image paramagnetic oximetry probes such as nitroxides in brain tissue. We have shown that masking the carboxyl group of 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (nitroxide 1) as an esterase-labile acetoxymethyl ester yields 3-acetoxymethoxycarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (nitroxide 2). Nitroxide 2 can cross the blood-brain barrier and is then hydrolyzed in situ by esterases to regenerate nitroxide 1, which becomes entrapped in brain tissue. Seeking to improve the loading of nitroxides into brain, we synthesized the more lipophilic pentanoyloxymethyl ester, 3-pentanoyloxymethoxycarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (nitroxide 3). We report that the higher lipophilicity of nitroxide 3 does not significantly increase its ability to generate EPR signals in the mouse brain. Therefore, irrespective of whether nitroxide 2 or 3 was injected, similar levels of nitroxide were entrapped in brain tissue. These findings suggest that nitroxides 2 and 3 perform comparably well as proimaging agents for measuring O(2) distribution in brain.

The antioxidant tempamine: in vitro antitumor and neuroprotective effects and optimization of liposomal encapsulation and release.

The piperidine nitroxide tempamine (TMN) is a cell-permeable, stable radical having antioxidant, anticancer, and proapoptotic and/or pronecrotic activities, as was demonstrated by us in cell cultures. We also demonstrated synergism between TMN and doxorubicin in doxorubicin-sensitive and doxorubicin-resistant cell lines. Treatment of the C26 mouse colon carcinoma model in vivo also demonstrated synergism between TMN and doxorubicin in sterically stabilized liposomes (SSLs) containing TMN (SSL-TMN) and those containing doxorubicin. The above effects of TMN and SSL-TMN motivated us to develop and optimize the SSL-TMN formulation so that it will be able to reach the disease site with a sufficiently high TMN level and a release rate needed to achieve a therapeutic effect. Because TMN is an amphipathic weak base, it was remote loaded by an intraliposome high/extraliposome low transmembrane ammonium sulfate gradient. The kinetics and level of TMN loading were monitored by cyclic voltammetry (CV) and electron paramagnetic resonance (EPR); the latter also indicates TMN precipitation in the intraliposomal aqueous phase. The regeneration of the original CV and EPR signals by the ionophore nigericin indicates that TMN remained fully intact during loading and release. The cardinal role of the transmembrane ammonium ion gradient in the loading process was proven by the use of the selective ionophores nonactin (for NH4+) and nigericin (for H+). The anion of the ammonium salts affects loading stability and the rate of TMN release, both mediated through the TMN state of aggregation in the intraliposomal aqueous phase. The greater the TMN salt precipitation, the slower the TMN release rate. This was supported by measurement of osmolality, which is inversely related to TMN salt precipitate. Precipitation is in the order SO4(-2)>Cl-1>glucuronate-1. Liposome lipid composition, magnitude of the transmembrane ammonium ion gradient, and type of anion of the ammonium salt determine the amount of TMN loaded and its release rate.

In vivo generation of free radicals in the skin of live mice under ultraviolet light, measured by L-band EPR spectroscopy.

Although free radicals may be involved in various types of UV-induced injuries, only a few in vivo studies of the generation of free radicals, including oxygen radicals, during exposure to ultraviolet light (UV) have been reported. In this study, the nitroxyl probe 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl was intravenously injected into hairless mice, and its decay was monitored in the skin with an in vivo EPR spectrometer equipped with a surface-coil-type resonator. The rate of decay of the EPR signal increased during UV (UVA+B) irradiation. This increase in signal decay was suppressed by preadministration of a spin trap, N-tert-butyl-alpha-phenylnitrone (PBN). PBN did not change the rate of signal decay in nonirradiated mice. The correlation between signal decay rate and physiological parameters such as blood velocity, blood mass, or skin temperature was low. The decay rate responded rapidly and reversibly to starting and stopping the UV illumination. Hydroxyl and peroxyl radicals caused reduction of the probe signal in vitro, and PBN inhibited only the peroxyl radical-induced signal reduction. These observations suggest that peroxyl radicals are generated in the skin of live mice during UVA+B irradiation.

Pharmacokinetics of otamixaban, a direct factor Xa inhibitor, in healthy male subjects: pharmacokinetic model development for phase 2/3 simulation of exposure.

The pharmacokinetics of otamixaban was investigated in healthy male subjects over a wide range of intravenous doses, with duration of administration varying between 1-minute infusions (bolus dose) and 24-hour infusions, using noncompartmental and multicompartmental methods. A global compartmental analysis (2 and 3 compartments) generated a single set of pharmacokinetic parameters, regardless of infusion rate and duration, and took into account the 30% decrease in clearance and volume of distribution observed over the dose range. The 2-compartment model was retained to predict bolus plus 3-hour-infusion doses of otamixaban for future phase (2/3) studies. Otamixaban exhibited in healthy subjects several interesting pharmacokinetic features in view of its potential therapeutic use in coronary thrombosis: a rapid plasma distribution and elimination, a well-described dose-exposure relationship, a low intersubject variability in plasma exposure, and a mixed renal and biliary excretion with constant renal clearance.

The toxicity of SCH 351591, a novel phosphodiesterase-4 inhibitor, in Cynomolgus monkeys.

SCH351591, a novel phosphodiesterase-4 inhibitor under investigation as a potential therapeutic for asthma and chronic obstructive pulmonary disease (COPD), was evaluated in a 3-month rising-dose study in Cynomolgus monkeys. Four groups, containing four monkeys/sex, received vehicle control or rising doses up to 12, 24, or 48 mg/kg of SCH351591 daily. Although initial exposure produced clinical signs of emesis, reduced food intake, and reduced body weight, tachyphylaxis to the emesis allowed dose escalation up to 48 mg/kg/day. Two monkeys died and 3 were sacrificed in moribund condition over the course of the study. Early mortality, involving monkeys dosed with 12 or 24 mg/kg, was attributed to sepsis (2 monkeys) or colon inflammation (3 monkeys). Leukocyte function assays on low- and mid-dose group survivors revealed an inhibition of T lymphocyte proliferation for 12 mg/kg group males and 24 mg/kg group monkeys of both sexes. Necropsy findings, unassociated with early mortality, included reduced size and weight of the thymus, depletion of body fat, red discoloration of the gastric mucosa, and perivascular hemorrhage of the stomach and heart. Stomach and heart gross findings were present in the high-dose group only. Histopathologic lesions, in addition to those attributed to concurrent bacterial infection, included thymic atrophy, serous atrophy of fat, myocardial degeneration and acute to chronic inflammation of small to medium-sized arteries in various organs and tissues including the heart, kidneys, stomach, salivary glands, pancreas, esophagus, gallbladder, and mesentery. The findings of this study demonstrate the potential of a PDE4 inhibitor to alter immunologic response as well as to produce arteriopathy in nonhuman primates.