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We have synthesized and evaluated several new ligands for imaging the norepinephrine transporter (NET) system in baboons with positron emission tomography (PET). Ligands possessing high brain penetration, high affinity and selectivity, appropriate lipophilicity (log P = 1.0-3.5), high plasma free fraction and reasonable stability in plasma were selected for further studies. Based on our characterization studies in baboons, including 11C-labeled (R)-nisoxetine (Nis), oxaprotiline (Oxap), lortalamine (Lort) and new analogs of methylreboxetine (MRB), in conjunction with our earlier evaluation of 11C and 18F derivatives of reboxetine, MRB and their individual (R,R) and (S,S) enantiomers, we have identified the superiority of (S,S)-[11C]MRB and the suitability of MRB analogs [(S,S)-[11C]MRB > (S,S)-[11C]3-Cl-MRB > (S,S)-[18F]fluororeboxetine] as potential NET ligands for PET. In contrast, Nis, Oxap and Lort displayed high uptake in striatum (higher than in thalamus). The use of these ligands is further limited by high non-specific binding and relatively low specific signal, as is characteristic of many earlier NET ligands. Thus, to our knowledge (S,S)-[11C]MRB remains by far the most promising NET ligand for PET studies.

Background: The neurokinin NK(1)-receptor antagonist aprepitant has demonstrated efficacy in preventing highly emetogenic chemotherapy-induced nausea and vomiting., Objective: The objective of the present study was to investigate the effects of impaired renal function on the pharmacokinetics and safety of aprepitant., Subjects and Methods: A total of 32 patients and healthy subjects were evaluated in this open-label, two-part study. Pharmacokinetic parameters after a single oral dose of aprepitant 240 mg were measured in eight patients with end-stage renal disease (ESRD) requiring haemodialysis, eight patients with severe renal insufficiency (SRI [24-hour creatinine clearance <30 mL/min/1.73 m(2)]) and 16 healthy and age-, sex- and weight-matched subjects (controls)., Results: In ESRD patients, the area under the plasma concentration-time curve (AUC) from 0 to 48 hours (AUC(48)) for aprepitant was on average approximately 36% lower than that observed in control subjects (ratio [ESRD patients/healthy controls] of geometric means = 0.64), but the 90% confidence interval 0.52, 0.78 for the ratio of true mean AUC(48) fell within the predefined target interval of 0.5, 2.0. Also in ESRD patients, there was no statistically or clinically significant difference in unbound aprepitant AUC (which may be more clinically relevant than total aprepitant AUC) when compared with healthy control subjects. Aprepitant pharmacokinetic parameters in ESRD patients were clinically similar when haemodialysis was initiated at 4 hours or 48 hours after aprepitant administration. In SRI patients, the ratio (SRI patients/healthy controls) of aprepitant AUC from zero to infinity (AUC(infinity)) geometric mean value was 0.79 with a 90% confidence interval of 0.56, 1.10. On average, in SRI patients the principal aprepitant pharmacokinetic parameters (AUC(infinity), initial maximum plasma concentration [C(max)], time to initial C(max), and apparent elimination half-life) were not statistically different from those obtained in healthy control subjects. Aprepitant was generally well tolerated in both ESRD and SRI patients., Conclusion: The results of this study demonstrate that ESRD, haemodialysis and SRI have no clinically meaningful effect on aprepitant pharmacokinetics. Therefore, no dosage adjustment of aprepitant is warranted in SRI or ESRD patients.

A method for the simultaneous determination of Aprepitant, I (5-[[2(R)-[1(R)-(3,5-bistrifluoromethylphenyl)ethoxy]-3(S)-(4-fluorophenyl) morpholin-4-yl]methyl]-2,4-dihydro-[1,2,4]triazol-3-one) and two active metabolites (II and III) in human plasma has been developed. The method was based on high-performance liquid chromatography (HPLC) with atmospheric pressure chemical ionization tandem mass spectrometric (APCI-MS-MS) detection in positive ionization mode using a heated nebulizer interface. The analytes and internal standard (IV) (Fig. 1) were isolated from basified plasma using liquid-liquid extraction. The organic extracts were dried, reconstituted in mobile phase and injected into the HPLC-MS/MS system. The analytes were chromatographed on a narrow bore (50 mm x 2.0 mm, 3 microm) Keystone Scientific's Prism R.P. analytical column, with mobile phase consisting of acetonitrile (ACN):water containing trifluoroacetic acid with pH adjusted to 3 (40:60, v/v) pumped at a flow rate of 0.5 ml/min. The MS-MS detection was performed on a Sciex API 3000 tandem mass spectrometer operated in selected reaction monitoring mode. The precursor-->product ion combinations of m/z 535-->277, 438-->180, 452-->223 and 503-->259 were used to quantify I, II, III, and IV, respectively, after chromatographic separation of the analytes. The assay was validated in the concentration range of 10-5000 ng/ml for I and II and 25-5000 ng/ml for III when 1 ml of plasma was processed. The precision of the assay (expressed as coefficient of variation, CV) was less than 10% at all concentrations within the standard curve range, with adequate assay accuracy. Matrix effect experiments were performed to demonstrate the absence of any significant change in ionization of the analytes when comparing neat standards to analytes in the presence of plasma matrix. This assay was utilized to support a clinical study where multiple oral doses of I were administered to healthy subjects to investigate the pharmacokinetics, safety, and tolerability of Aprepitant. Concentrations of the two most active metabolites, which if present in high concentrations would increase the neurokinin-1 (NK1) receptor occupancy level and therefore potentially contribute to the antiemetic action of Aprepitant, were determined.

Methods based on high-performance liquid chromatography (HPLC) with atmospheric-pressure chemical ionization (APCI) mass spectrometric (MS) detection using either single (MS) or triple (MS/MS) quadrupole mass spectrometric detection for the determination of (2R)-[1(R)-(3,5-bis-trifluoromethylphenyl)ethoxy]-3(S)-(4-fluoro-phenyl)morpholin-4-ylmethyl]-5-oxo-4,5-dihydro-[1,2,4]triazol)methyl morpholine (Aprepitant, Fig. 1) in human plasma has been developed. Aprepitant (I) and internal standard (II, Fig. 1) were isolated from the plasma matrix buffered to pH 9.8 using a liquid-liquid extraction with methyl-t-butyl ether (MTBE). The analytes were separated on a Keystone Scientific's Javelin BDS C-8 2 mm x 4.6 mm 3 microm guard column coupled to BDS C-8 50 mm x 4.6 mm 3 microm analytical column, utilizing a mobile phase of 50% acetonitrile and 50% water containing 0.1% formic acid and 10 mM ammonium acetate delivered at a flow rate of 1 ml/min. The single quadrupole instrument was operated in a single ion monitoring (SIM) mode analyzing the protonated molecules of Aprepitant and II at m/z 535 and 503, respectively. The triple quadrupole mass spectrometer was operated in multiple reaction monitoring mode (MRM) monitoring the precursor --> ion combinations of m/z 535 --> 277 and 503 --> 259 for Aprepitant and II, respectively. The linear calibration range for both single and triple quadrupole detectors was from 10 to 5000 ng/ml of plasma with coefficients of variation less than 8% at all concentrations. Both single and triple quadrupole instruments yielded similar precision and accuracy results. Matrix effect experiments performed on both instruments demonstrated the absence of any significant change in ionization of the analytes when comparing neat standards to analytes in the presence of plasma matrix. Both instruments were used successfully to support numerous clinical trials of Aprepitant.

Aminoethylcysteine ketimine decarboxylated dimer (AECK-DD) is a natural sulphur compound present in human plasma and urine and in mammalian brain. Recently, it has been detected in many common dietary vegetables. The aim of the present study was to evaluate the ability of AECK-DD to affect cellular response of U937 human monocytic cells to tert-butyl hydroperoxide-induced oxidative stress. AECK-DD was incorporated into cells, as confirmed by GC-MS analyses, without any cytotoxic effect. A 24 h treatment with 50 and 250 microM AECK-DD resulted in the incorporation of 0.10 +/- 0.01 and 0.47 +/- 0.08ng AECK-DD x 10(6) cells, respectively. U937 cells pretreated with AECK-DD (in the range 4-100 microM) showed an increased resistance to tert-butyl hydroperoxide-induced necrotic death, as revealed by a higher percent of survival measured at all incubation times with respect to control cells. Moreover, the protective effect exhibited by AECK-DD is significantly stronger with respect to that obtained with other common antioxidants (N-acetyl cysteine and trolox) and comparable, although somewhat higher, to that of vitamin E. This effect seems to be due to the ability of AECK-DD to reduce glutathione depletion and to inhibit lipid peroxidation during tert-butyl hydroperoxide treatment. It can be concluded that AECK-DD protects cultured human monocytic cells against tert-butyl hydroperoxide-induced oxidative stress and subsequent cell death, likely through an antioxidant action inside the cell. Due to its presence in both human plasma and urine, AECK-DD may play a role in the modulation of oxidative processes in vivo.

Background: Aprepitant is a highly selective substance P (neurokinin 1 [NK(1)] receptor) antagonist that significantly improves the pharmacotherapy of acute and delayed highly emetogenic chemotherapy-induced nausea and vomiting, probably through an action in the brain stem region of the central nervous system. Here, we report the use of positron emission tomography imaging with the NK(1) receptor binding-selective tracer [(18)F]SPA-RQC to determine the levels of central NK(1) receptor occupancy achieved by therapeutically relevant doses of aprepitant in healthy humans., Methods: Two single-blind, randomized, placebo-controlled studies in healthy subjects were performed. The first study evaluated the plasma concentration-occupancy relationships for aprepitant dosed orally at 10, 30, 100, or 300 mg, or placebo (n = 12). The second study similarly evaluated oral aprepitant 30 mg and placebo (n = 4). In each study, dosing was once daily for 14 consecutive days. Data from both studies were combined for analyses. The ratio of striatal/cerebellar [(18)F]SPA-RQ (high receptor density region/reference region lacking receptors) was used to calculate trough receptor occupancy 24 hours after the last dose of aprepitant., Results: Brain NK(1) receptor occupancy increased after oral aprepitant dosing in both a plasma concentration-related (r =.97; 95% confidence interval [CI] =.94-1.00, p <.001) and a dose-related (r =.94; 95% CI =.86-1.00, p <.001) fashion. High (> or =90%) receptor occupancy was achieved at doses of 100 mg/day or greater. The plasma concentrations of aprepitant that achieved 50% and 90% occupancy were estimated as approximately 10 ng/mL and approximately 100 ng/mL, respectively., Conclusions: Positron emission tomography imaging with [(18)F]SPA-RQ allows brain NK(1) receptor occupancy by aprepitant to be predicted from plasma drug concentrations and can be used to guide dose selection for clinical trials of NK(1) receptor antagonists in central therapeutic indications.

Norepinephrine transporter (NET) function has a central role in the regulation of synaptic norepinephrine concentrations. Clinical observations in orthostatic intolerance patients suggest a gender difference in NET function. We compared the cardiovascular response to selective NET inhibition with reboxetine between 12 healthy men and 12 age-matched women. Finger blood pressure, brachial blood pressure, and heart rate were measured. The subjects underwent cardiovascular autonomic reflex testing and a graded head-up tilt test. In a separate study, we applied incremental concentrations of tyramine and isoproterenol through subcutaneous microdialysis catheters in eight men and in eight women. NET inhibition elicited a threefold greater increase in supine blood pressure in men than women (P < 0.05). The pressor response was driven by an increased cardiac output. The orthostatic heart rate increase during NET inhibition was greater in men than women (56 +/- 5 beats/min in men, 42 +/- 4 beats/min in women, P < 0.001). In contrast, NET inhibition resulted in a similar suppression in the cold pressor and handgrip response, low-frequency blood pressure oscillations, and venous norepinephrine in the supine position. Men and women were similarly sensitive to the lipolytic effect of isoproterenol and tyramine. We conclude that NET inhibition results in more pronounced changes in cardiac regulation in men than women. Our observations suggest that the NET contribution to cardiac norepinephrine turnover may be decreased in women. The gender difference in NET function may not be expressed in tissues that are less NET dependent than the heart.

Rationale: Recently a new 5-hydroxytryptamine1B (5-HT1B)-receptor antagonist, AR-A000002, was described. It was shown that this compound dose dependently increased 5-HT metabolism and release in guinea pig brain following a single injection., Objectives: The present study investigated effects of 3 weeks twice-daily treatment of guinea pigs with the 5-HT1B-receptor antagonist AR-A000002 on the serotonergic neurons and receptor densities., Methods: Guinea pigs were injected subcutaneously with AR-A000002, citalopram or saline twice daily for 3 weeks. Groups of animals were treated with challenge doses of AR-A000002 or saline 24 h after the last chronic treatment (citalopram group 48 h) and sacrificed 2 h thereafter. The effect on 5-HT metabolism and 5-HT release was assessed. Plasma and brain concentrations of AR-A000002 were analysed. The effects on binding of [3H]8-OH-DPAT to 5-HT1A receptors, [3H]GR125743 to 5-HT(1B/1D) receptors, [3H]ketanserin to 5-HT2A receptors, and [3H]prazosin to alpha1-adrenoceptors were determined., Results: Repeated treatment of guinea pigs with AR-A000002 did not change the 5-HT(1B/1D), 5-HT1A, 5-HT2A or alpha1-adrenergic receptor densities. Following repeated treatment of guinea pigs for 3 weeks with AR-A000002, the 5-HT1B receptors were still receptive to a challenge with the same compound. Thus, an increase in the 5-HIAA/5-HT ratio and 5-HT release was seen following challenge doses of AR-A000002. No difference in the plasma and brain concentrations of AR-A000002 was found between the sub-chronic treated AR-A000002 and saline-treated guinea pigs., Conclusions: It is concluded that AR-A000002 is a 5-HT1B receptor antagonist, which enhances persistently the serotonergic neurotransmission in guinea pig brain.

The absorption, metabolism, and excretion of [14C]aprepitant, a potent and selective human substance P receptor antagonist for the treatment of chemotherapy-induced nausea and vomiting, was evaluated in rats and dogs. Aprepitant was metabolized extensively and no parent drug was detected in the urine of either species. The elimination of drug-related radioactivity, after i.v. or p.o. administration of [14C]aprepitant, was mainly via biliary excretion in rats and by way of both biliary and urinary excretion in dogs. Aprepitant was the major component in the plasma at the early time points (up to 8 h), and plasma metabolite profiles of aprepitant were qualitatively similar in rats and dogs. Several oxidative metabolites of aprepitant, derived from N-dealkylation, oxidation, and opening of the morpholine ring, were detected in the plasma. Glucuronidation represented an important pathway in the metabolism and excretion of aprepitant in rats and dogs. An acid-labile glucuronide of [14C]aprepitant accounted for approximately 18% of the oral dose in rat bile. The instability of this glucuronide, coupled with its presence in bile but absence in feces, suggested the potential for enterohepatic circulation of aprepitant via this conjugate. In dogs, the glucuronide of [14C]aprepitant, together with four glucuronides derived from phase I metabolites, were present as major metabolites in the bile, accounting collectively for approximately 14% of the radioactive dose over a 4- to 24-h period after i.v. dosing. Two very polar carboxylic acids, namely, 4-fluoro-alpha-hydroxybenzeneacetic acid and 4-fluoro-alpha-oxobenzeneacetic acid, were the predominant drug-related entities in rat and dog urine.

(S,S)-2-(alpha-(2-Methoxyphenoxy)benzyl)morpholine (MeNER), an O-methyl analog of the selective and potent norepinephrine transporter (NET) inhibitor, (S,S)-reboxetine, and its less active enantiomer, (R,R)-MeNER, have each been radiolabeled by O-methylation of their corresponding phenolic precursors in good yields from [(11)C]methyl iodide or [(11)C]methyl triflate. Radiochemical purities were >99% and specific radioactivity at time of injection was about 74 GBq/micromol. Autoradiographic examination of (S,S)-[(11)C]MeNER binding to human brain slices post mortem indicated specific binding in a brain region including the locus coeruleus. PET examination of both [(11)C]MeNER enantiomers in a cynomolgus monkey demonstrated a higher specific binding of the (S,S)-enantiomer with ratios of 1.4-1.6 in the lower brainstem, mesencephalon and thalamus to striatum. Pretreatment with the NET ligand, desipramine, decreased the specific binding of (S,S)-[(11)C]MeNER. Labeled metabolites of [(11)C]MeNER were all more polar. (S,S)-[(11)C]MeNER is a good lead compound in the search for a selective radioligand for quantitation of NET in the human brain in vivo.

When patients do not respond to an initial antidepressant, one clinical approach is to switch to an agent in a different pharmacological class. However, few studies have examined the neurochemical consequences of this practice. To study this, we examined changes in binding sites in rat brain for norepinephrine (NET) and serotonin transporters (SERT), alpha1, alpha2, and beta1 adrenergic receptors after chronic administration of paroxetine (PRX), reboxetine (RBX), or PRX followed by RBX. We also examined the effects of these treatments on mRNA expression for tyrosine hydroxylase (TH). RBX treatment for 3 weeks reduced NET binding significantly, by approximately 40% in terminal field areas, and 6 weeks of RBX reduced it even more, by approximately 60%. RBX treatment for 3 and 6 weeks reduced beta1 adrenergic receptor-binding sites equally, by 50-60%. At no time did RBX treatment reduce SERT-binding sites. PRX treatment had no effect on beta1 adrenergic or NET-binding sites, but reduced SERT-binding sites by 75-80%. Neither treatment altered mRNA for TH, alpha1, or alpha2 adrenergic receptor-binding sites. When 3 weeks of RBX treatment followed 3 weeks of PRX treatment, NET-binding sites were reduced to the same extent as measured after 6 weeks of RBX treatment alone, indicating that PRX pretreatment may have 'primed' the subsequent regulatory effect of RBX on the NET. Thus, pretreatment of rats with PRX actually enhanced at least one regulatory effect of RBX treatment on the noradrenergic system, and did not interfere with any other pharmacological effect caused by RBX treatment.

The pharmacokinetics, metabolism, and brain penetration of the neurokinin 1 (NK1) receptor antagonist (substance P receptor antagonist), aprepitant (MK-0869), were examined in ferrets. This species exhibits human-type NK1receptor pharmacology and is of proven value in the identification of clinically useful drugs for the treatment of chemotherapy-induced nausea and vomiting in humans. After a single p.o. dose of aprepitant at 1 or 2 mg/kg, plasma levels of the compound were between approximately 200 and 270 ng/ml, 24 h after dosing. In the brain cortex, concentrations of aprepitant reached between approximately 80 and 150 ng/g of tissue 24 h after dosing. The predominant radioactive component present in the plasma and the brain of ferrets at 24 or 48 h after a single oral dose of [14C]aprepitant at 3 mg/kg was the parent compound itself. The slow plasma clearance of aprepitant ( approximately 1.5 ml/min/kg) and its abundance in ferret brain were in accord with its efficacy in blocking the retching and vomiting at 24 and 48 h postdose when ferrets were challenged with the emetic anticancer drug, cisplatin. When aprepitant and some of its metabolites were assessed for their in vitro binding affinity to the human NK1receptor, aprepitant demonstrated the highest affinity. Collectively, these data suggested that aprepitant, rather than its metabolites, was responsible, primarily, for the antiemetic activity of this compound in the male ferret.

The chiral compound reboxetine is used as a selective noradrenaline reuptake inhibitor (NARI) for the treatment of major depressive disorders. The pharmacokinetic variability of the enantiomers of the drug (S,S- and R,R-reboxetine) was studied using stereoselective high-performance liquid chromatography with mass spectrometric detection in a controlled clinical monotherapy situation (trial I) and a naturalistic clinical setting (trial II). Trial I included patients receiving racemic reboxetine as 6-month monotherapy for treatment of major depressive disorder. Trough level serum samples in steady state were analyzed for the concentration of the reboxetine enantiomers in study weeks 4, 12, and 24. In a therapeutic drug monitoring setting (trial II), 47 patients on doses ranging from 4 to 16 mg daily, including much polypharmacy, trough level steady-state serum samples were analyzed by the same bioanalytical method. Data from trials I and II were assessed to determine the inter- and intraindividual pharmacokinetic outcomes. The results showed that the median S,S/R,R ratio in steady state was 0.5 and ranged from 0.22 to 0.88. It was also shown that women have an approximately 30% higher S,S/R,R ratio than men. The S,S/R,R ratios of reboxetine were not found to correlate with reboxetine concentrations. To investigate the NARI activity of a circulating serum reboxetine concentration, a recalculation of the determined enantiomeric concentrations to previously demonstrated experimental NARI potencies of the drug enantiomers was performed. This partly novel concept of estimating pharmacodynamic activity showed that the serum NARI activity in women tended to be higher than in men at a given reboxetine concentration. In conclusion, the variability in the NARI activity per nmol/L reboxetine and the variability in the concentration outcome of the reboxetine enantiomers may justify the use of enantioselective drug monitoring in the clinic. The gender aspects of the drug have to be further assessed.

The (R,R) and (S,S) enantiomers of 2-[(2-methoxyphenoxy)phenylmethyl]morpholine (MeNER) have been radiolabelled with carbon-11 in good yield and at high specific activity. These radiotracers are close analogues of reboxetine, a potent and selective ligand for the norepinephrine transporter (NET). They were examined as potential ligands for imaging NET in vivo by positron emission tomography (PET). The in vivo brain distribution of both [(11)C]-labeled enantiomers were evaluated in rats. Following tail-vein injection of the (R,R)-enantiomer regional brain uptake and washout of radioactivity was homogeneous at all time points examined (5-60 min). In contrast, administration of the (S,S)-enantiomer produced a heterogeneous distribution of radioactivity in brain with highest uptake in the hypothalamus, a NET rich region, and lowest uptake in the striatum, a brain region devoid of NET. Hypothalamus to striatum ratios of 2.5 to one were achieved at 60 min post injection of (S,S)-[(11)C]-MeNER. Pre-injection of the norepinephrine reuptake inhibitors, reboxetine or desipramine, reduced hypothalamus to striatum ratios to near unity while reuptake inhibitors of dopamine and serotonin had no significant effect on binding. In vitro autoradiography studies (rat brain slices) with (S,S)-[(11)C]-MeNER produced a regional distribution pattern that was consistent with the reported distribution of NET. (S,S)-[(11)C]-MeNER has the potential to be the first successful PET ligand to image NET.

Brain extracellular fluid (ECF) concentration of YM992, a novel antidepressant, was determined using brain microdialysis to investigate the high partition of this drug to the brain after systemic administration to rats. Plasma, cerebrospinal fluid (CSF), ECF and brain concentrations were determined at the steady-state after intravenous infusion to rats. The concentration ratio of brain to plasma at the total concentration base was 71.3, while those of ECF to plasma and CSF to plasma at the free concentration base were comparable. The distribution volume in brain was 375 ml/g brain and in vitro binding of YM992 to rat brain was 98.1-98.5%, suggesting a high binding in the brain. The carotid artery injection study showed that the brain uptake index of YM992 was 141%, furthermore, the uptake clearance into brain after i.v. dosing to rats was 0.6 ml/min/g brain, indicating a high permeability at the blood-brain barrier (BBB). These findings suggest that the high partition of YM992 to rat brain is attributed to its high level of binding in the brain as well as its high permeability at the BBB., (Copyright 2002 John Wiley & Sons, Ltd.)

Watson-Crick pairing between complementary oligomers is proving to be an effective means for rapidly directing radioisotopes specifically to the exterior surface of cancer cells in vivo. In such pretargeting applications, it is highly desirable that the excess of isotopically labeled oligomers, which do not bind to the cancer cells, be rapidly cleared from the body. In this context, understanding the influence of chain length and base sequence of the radiolabeled oligomers is critical. We had earlier determined that the kidneys are the principal targets of short-chain radiolabeled morpholino oligomers (MORFs). To explain these observations, MORFs consisting of uniform cytosines (Cs), uniform thymines (Ts), uniform adenines (As), and uniform AAG repeat were labeled with technetium-99m (99mTc) and studied in normal mice. In a limited investigation of the influence of oligomer backbone, a 20-mer MORF (MORF20) with a base sequence rich in Cs was compared with a phosphoromonothioate DNA (S-DNA20) of the same sequence. The in vivo behavior of the labeled MORFs was nearly identical in all organs, with the exception of kidneys. The kidney accumulations were about 25- to 80-fold higher for the uniform Cs relative to the other three uniform MORFs at 3 hours. The S-DNA20 rich in Cs showed only modest kidney accumulations compared with the equivalent MORF20, presumably because of preferential clearance of the S-DNA20 through the liver. Urine analysis showed no evidence of intact labeled S-DNA20 in contrast to fully intact labeled MORF20. We conclude that the high kidney levels observed by us previously for MORFs are most likely due largely to the C residues in the base sequence. In the case of S-DNAs, this phenomenon is partly disguised by the increased hepatic excretion and degradation. These results show that the base sequences of MORFs, and probably other oligomers as well, are an important determinant of biodistribution.

It has been proposed that norepinephrine plays a critical role in the modulation of cortical excitability, which in turn is thought to influence functional recovery from brain lesions. The purpose of the present experiments was to determine if it is possible to modulate cortical excitability with the selective norepinephrine reuptake inhibitor reboxetine in intact humans. Recruitment curve and intracortical facilitation, assessed by transcranial magnetic stimulation, were increased after oral intake of 8 and 4 mg reboxetine, in the absence of changes in motor threshold, intracortical inhibition, M-response, F-wave or H-reflex. These results demonstrate that reboxetine enhances cortical excitability and raise the possibility that it could act as a plasticity enhancing substance potentially useful in combination with neurorehabilitative strategies geared to enhance neurorehabilitation., (Copyright 2002 Elsevier Science Ltd.)

The pyrazolopyridine stimulators of soluble guanylate cyclase BAY 41-2272 and 41-8543 were oxidised in rats and dogs at their 5-pyrimidinyl-cyclopropyl and -morpholino residue. These metabolites activate the soluble guanylate cyclase, induce vasoelaxation and thereby may contribute to the in vivo activity of BAY 41-2272 and BAY 41-8543.

In conscious, freely-moving, male, Sprague-Dawley rats, the regional haemodynamic responses to the synthetic cannabinoids, WIN-55212-2 and HU 210, were compared. The possible involvement of cannabinoid, CB(1)-receptors, or beta(2)-adrenoceptors in the responses to WIN-55212-2 and HU 210 were investigated using the CB(1)-receptor antagonist, AM 251, or the beta(2)-adrenoceptor antagonist, ICI 118551, respectively. Both WIN-55212-2 (150 microg kg(-1)) and HU 210 (100 microg kg(-1)) had pressor, renal, and mesenteric vasoconstrictor and hindquarters vasodilator actions, although the effects of HU 210 were much more sustained than those of WIN-55212-2. Lower doses of the cannabinoids (WIN-55212-2, 50 microg kg(-1), HU 210, 10 microg kg(-1)) had less consistent actions. All the significant cardiovascular effects of WIN-55212-2 and HU 210 were antagonized by pretreatment with AM 251 (3 mg kg(-1)). Furthermore, pretreatment with the beta(2)-adrenoceptor antagonist, ICI 118551, inhibited the hindquarters vasodilator effects of WIN-55212-2 and of HU 210. On the basis of the present findings, and our earlier work, it is suggested that, in conscious rats, the pressor and vasoconstrictor effects of HU 210 and WIN-55212-2 involve cannabinoid-receptor-mediated increases in sympathetic activity. The accompanying hindquarters vasodilator actions of these agonists are cannabinoid receptor-mediated and appear to involve beta(2)-adrenoceptors.

Reboxetine is a new norepinephrine reuptake inhibitor (NRI) drug recently introduced in the therapy for depressed patients. It is effective in the treatment of severe depression and safer to use than traditional tricyclic antidepressants. In this paper an original high-performance liquid chromatography (HPLC) method with ultraviolet detection for the determination of reboxetine in human plasma is described. It uses a C8 reversed-phase column and a mobile phase composed of acetonitrile and aqueous tetramethylammonium perchlorate. For the analysis of plasma samples containing very low levels of reboxetine, another HPLC method with fluorimetric detection was developed (limit of quantitation, LOQ=11 ng ml(-1); limit of detection, LOD=4 ng ml(-1)). The fluorimetric method is based on precolumn derivatisation of reboxetine with 9-fluorenylmethyl chloroformate. An accurate sample pretreatment of human plasma samples has been implemented by means of solid-phase extraction (SPE) on Oasis HLB (hydrophilic-lipophilic balance) cartridges with very high extraction yields (>95%). Both methods were applied to the analysis of plasma samples from depressed patients undergoing therapy with reboxetine and gave satisfactory results in terms of precision (RSD<4.5%) and accuracy (mean recovery>94%).

The effect of mosapride citrate (mosapride) on plasma levels of gastrointestinal peptides (motilin, gastrin, somatostatin, and secretin) was studied in five healthy volunteers. After a single oral administration of mosapride (15 mg), the plasma mosapride level (85.0+/-13.7 ng/ml) was highest in the 60-min sample after the administration and then the plasma level fell. Peak plasma motilin levels (18.6+/-1.7 pg/ml) were achieved 60 min after administration of mosapride (p<0.01 vs. placebo), and returned to baseline levels within a further 120 min. Plasma gastrin levels (42.4+/-3.6 pg/ml) increased 60 min after administration of mosapride (p<0.01 vs. placebo). Plasma somatostatin and secretin levels did not change significantly. These results suggest that the pharmacological effects of mosapride on gastrointestinal functions are closely related to changes in motilin-immunoreactive substance levels in human plasma.

A method was developed and validated for the quantification of (+/-)-trans-[2-morpholino-1-(1-naphthalene-ethyloxy]cyclohexane monohydrochloride (RSD1070) and its N-dealkyl metabolite in rat plasma and hepatic microsomal incubates. Chromatographic separations were achieved using reversed-phase high-performance liquid chromatography coupled with positive ion electrospray ionization and detection by tandem mass spectrometry. The assay was linear from 2.5 to 100 ng/ml and this range was used for validation. Inter- and intra-assay variability (n=6), extraction recovery, and stability in plasma were assessed. The estimated limit of quantitation was in the range 2.5-3 ng/ml for both analytes in rat plasma. The analytical method was used in a pharmacokinetic study of RSD1070 in rats after a single i.v. bolus of 12 mg/kg.

Numerous studies have shown that a variety of cytokines are involved in the regulation of ovarian function. Interleukin-6 (IL-6), has been found in follicular fluid. In this report we show by immunocytochemical methods the localization of the extracellular domain of the IL-6-receptor and its associated signal transducer glycoprotein gp 130 on the surface of granulosa cells (GCs). The possibility that IL-6 may also influence the basal and FSH-stimulated production of estradiol (E2) and progesterone (Prog) by GCs in vitro was also investigated. GCs were obtained from infertile patients undergoing in vitro fertilization and embryo transfer treatment and cultured for 72 h or were given increasing concentrations of human recombinant IL-6 (8--128 pg/ml) in the absence or presence of FSH (96 U/ml). For the time-course studies, FSH-stimulated GCs were treated in the absence or presence of IL-6 (128 pg/ml) and supernatants were assayed at 24 h intervals (24-96 h) for E2 and Prog productions. The results show that increasing amounts of IL-6 significantly inhibit E2 production in the absence or presence of FSH vs untreated controls (P=0.025 at IL-6=128 pg/ml and P=0.016 at IL-6=16 pg/ml respectively). IL-6 also inhibited FSH-stimulated but not unstimulated Prog release (P=0.038 at IL-6=8 pg/ml). These findings suggest that IL-6 may be one of the factors that plays a local regulatory role in the course of FSH-stimulated E2 and Prog release. The time-course studies of the effect of the absence or presence of IL-6 demonstrated a significant inhibitory effect on both E2 and Prog secretion (P<0.001) by FSH-stimulated GCs. As infections of the female reproductive tract are often accompanied by elevated local IL-6 levels, this factor may be one of the links leading to endocrine reproductive dysfunction during genital infections.

Rationale: Ethnicity can affect the pharmacokinetics and pharmacodynamics of psychopharmacologic drugs., Objectives: Reboxetine disposition differences among Asians, blacks, and Caucasians were examined., Methods: Healthy subjects (12 Asians, 12 blacks, 12 Caucasians) received a single oral dose of one 4-mg reboxetine tablet in an open label, parallel study design. Plasma concentrations of reboxetine enantiomers [R,R(-) reboxetine and predominantly active S,S(+) reboxetine] were quantified using HPLC-MS-MS. Plasma unbound fractions of reboxetine enantiomers were evaluated by equilibrium dialysis. Ethnic group effects on pharmacokinetic parameters were assessed by ANOVA., Results: Mean S,S(+) reboxetine CLPO for blacks was significantly greater, compared to Asians and Caucasians (154+/-82 ml/min, 101+/-19 ml/min and 101+/-18 ml/min, respectively). Mean S,S(+) reboxetine free fractions (fu) were significantly greater for Asians and blacks, compared to Caucasians (3.04+/-1.28%, 2.89+/-0.69%, and 1.99+/-0.58%, respectively). S,S(+) Reboxetine unbound clearance (CLu) was significantly less for Asians, compared to blacks and Caucasians (3742+/-1468 ml/min, 5187+/-2027 ml/min, and 5294+/-1163 ml/min, respectively). S,S(+) Reboxetine mean unbound AUC (AUCu) in these groups were 20.2+/-7.1 ng.h/ml, 14.6+/-5.1 ng.h/ml, and 13.2+/-3.2 ng.h/ml, respectively. AUCu was significantly greater for Asians. CLu and AUCu did not differ significantly between blacks and Caucasians. Ethnic effects of R,R(-) reboxetine were similar to those observed for S,S(+) reboxetine., Conclusions: The AUCu difference between Asian and black and Caucasian subjects was modest. Tolerability differences among groups were not observed. No dosage adjustment is necessary for Asians or blacks.

Central dopaminergic neurons have been suggested to be involved in the pathophysiology of several psychiatric disorders, including depression, and appear to be modulated by noradrenergic activity both at the nerve terminal level and at the somatodendritic level. In recent years reboxetine, a selective noradrenaline reuptake inhibitor that differs from tricyclic antidepressants by its low affinity for muscarinic, cholinergic and alpha(1)-adrenergic receptors, has been introduced clinically. In the present study the effect of reboxetine on the function of the mesolimbocortical dopamine system was investigated by means of single cell recording and microdialysis in rats following administration of reboxetine in doses that appear to yield clinically relevant plasma concentrations. Reboxetine (0.625--20 mg/kg intravenously) induced an increase in burst firing, but not in average firing frequency of dopamine (DA) cells in the ventral tegmental area (VTA). Moreover, reboxetine (0.15--13.5 mg/kg intraperitoneally) caused a significantly enhanced DA output in the medial prefrontal cortex, whereas no effect was observed in the nucleus accumbens. Local administration of reboxetine (333 microM, 60 min), by means of reversed microdialysis into these brain regions, caused a significant increase in DA output in both brain regions. However, local administration of reboxetine into the VTA (333 microM, 60 min) did not affect DA availability in these terminal areas. Our results imply that clinical treatment with reboxetine may result in facilitation of both prefrontal DA output and the excitability of VTA DA neurons, effects that may contribute to its antidepressant action, especially on drive and motivation.

A rapid and sensitive liquid chromatography-electrospray ionisation mass spectrometry (HPLC-ESI-MS) assay has been developed for the measurement of moclobemide and metabolites, Ro12-5637 and Ro12-8095, in human plasma. Sample preparation (0.5 ml plasma) involves solid-phase extraction using C18 cartridges. A Nova-Pak phenyl column (Waters, 4 microm, 150x2 mm I.D.) was employed for analyte separation with a mixture of 0.2 M ammonium formate buffer, pH 3.57 and acetonitrile as the mobile phase. The within- and between-day precisions of the assay were <18% and the limit of quantification for all analytes was 0.01 microg/ml. The total run-time was 6 min. The method described was used to measure moclobemide, Ro12-5637 and Ro12-8095 in human plasma following an oral 300 mg dose.

Rationale: In therapeutic use, amitriptyline, reboxetine and citalopram have all been associated with apparent anticholinergic-like side effects (dry mouth, constipation, etc.), despite the very low antimuscarinic activity of reboxetine and citalopram in vitro., Objectives: We hypothesised that the spectral analysis of heart rate variability (HRV) might detect differences between amitriptyline, citalopram and reboxetine in their anticholinergic activities following a single peroral administration., Methods: In this double-blind, cross-over study, amitriptyline (75 mg), citalopram (20 mg), reboxetine (4 mg) and placebo were randomly given at 1-week intervals to eight healthy male volunteers. Drug and catecholamine concentrations in plasma were determined repeatedly. The drug effect was assessed with periodic recordings of electrocardiogram (ECG) and blood pressure, and with measurements of salivary secretion. The ECG recordings were subjected to spectral analysis of HRV, in which the high frequency (HF) power of R-R interval (RRI) variability was supposed to reflect cardiac parasympathetic tone., Results: Reboxetine increased heart rate and blood pressure and reduced the HF power of RRI and 3,4-dihydroxyphenylglycol (DHPG) plasma concentrations. Amitriptyline diminished salivary secretion and had a prominent sedative action. Measurements after citalopram did not differ significantly from placebo., Conclusions: Reboxetine, despite its low antimuscarinic activity in vitro, had distinct effects on the HF power of RRI, consistent with anticholinergic activity in vivo. Amitriptyline had a measurable anticholinergic effect in the salivary glands, but, surprisingly, not in the heart. We suggest that the sedative effect of amitriptyline could alter cardiac sympathovagal balance and, therefore, counteract the anticholinergic drug effect.

Reboxetine is a new antidepressant drug acting as a potent and selective noradrenaline reuptake inhibitor on the noradrenergic neuronal system. Because of an expected interindividual variability in drug metabolism in the clinical practice the need for therapeutic drug monitoring routines in psychiatry is always a prominent feature. In this application, the preferred bioanalytic methodology was solid phase extraction combined with reversed-phase high-performance liquid chromatography and ultraviolet detection at 210 nm. The technique proved reliable, with interday and intraday variation of less than 5% and a quantification limit for reboxetine and one of its main metabolites O-desethylreboxetine (O-reboxetine) at 5 and 30 nmol/L, respectively. The method was applied on serum samples from 38 patients treated chronically with reboxetine. These samples were drawn as trough levels in steady state with a dosage range of 2-16 mg/day. They evidenced a mean reboxetine concentration that was fairly linear and dose proportional, although the variance in concentration was large between patients, even those taking the same dosage. O-reboxetine was detected in quantifiable amounts in only 1 of the 38 patients (<3%). In conclusion, these results suggest that a routine reboxetine therapeutic drug monitoring service that is robust enough to produce reliable and reproducible results may be introduced into everyday clinical practice.

The goals of the present study were to identify the enzyme responsible for metabolism of itopride hydrochloride (itopride) and to evaluate the likelihood of drug interaction involving itopride. In human liver microsomes, the involvement of flavin-containing monooxygenase in N-oxygenation, the major metabolic pathway of itopride, was indicated by the following results: inhibition by methimazole and thiourea, heat inactivation, and protection against heat inactivation by NADPH. When the effects of ketoconazole on the metabolism of itopride, cisapride, and mosapride citrate (mosapride) were examined using human liver microsomes, ketoconazole strongly inhibited the formation of the primary metabolites of cisapride and mosapride, but not itopride. Other cytochrome P450 (CYP) 3A4 inhibitors, cimetidine, erythromycin, and clarithromycin, also inhibited the metabolism of cisapride and mosapride. In an in vivo study, itopride (30 mg/kg), cisapride (1.5 mg/kg), or mosapride (3 mg/kg) was orally administered to male rats with or without oral pretreatment with ketoconazole (120 mg/kg) twice daily for 2 days. The ketoconazole pretreatment significantly increased the area under the serum concentration curve and the maximum serum concentration of cisapride and mosapride but had no significant effect on the pharmacokinetics of itopride. In addition, itopride did not inhibit five specific CYP-mediated reactions of human liver microsomes. These results suggest that itopride is unlikely to alter the pharmacokinetics of other concomitantly administered drugs.

Objectives: To elucidate pharmacokinetics and pharmacodynamics of landiolol hydrochloride, newer developed ultra-short-acting beta-blocker, in patients with various cardiac tachyarrhythmias., Background: The short duration of action and titratability of landiolol hydrochloride make it ideal for use in patients with a clinical need for beta-blockers., Methods: In a total of 31 examinations we infused the drug in 19 patients (mean age, 55 +/- 14 years). After the persistence of the tachyarrhythmias was confirmed, continuous infusion was started at rates of 0.005, 0.01, 0.02, 0.04, and 0.08 mg/kg/min for 5 minutes (for paroxysmal atrial fibrillation, paroxysmal supraventricular tachycardia, and ventricular tachycardia) or 15 minutes (for ventricular premature complex). We analyzed the pharmacokinetics of 16 examinations. A one-compartment model provided a close fit for each blood concentration-time curve., Results: The maximum blood concentrations obtained clearly showed the dose dependency and revealed very short half-lives (range, 2.3 to 4.0 minutes). Area under the blood concentration-time curves also increased, showing dose dependency. In paroxysmal atrial fibrillation, landiolol hydrochloride reduced the heart rate from 111 +/- 20 to 90 +/- 10/min. Sinus rhythm was restored, without any adverse effects, in three of five patients with paroxysmal supraventricular tachycardia and one patient with ventricular tachycardia. There was no significant change in peripheral blood pressure., Conclusions: Landiolol hydrochloride has a shorter elimination half-life than any other beta-blocker, and it can be administered safely to patients with various tachyarrhythmias.

Objective: Soluble forms of interleukin-6 (IL-6) receptors are known to modulate biological activities of IL-6. The purpose of the study was to measure circulating levels of IL-6, sIL-6R and sgp130 in patients undergoing coronary artery bypass grafting with cardiopulmonary bypass (CPB group) or without CPB (non-CPB group)., Methods: The CPB group included 19 patients and the non-CPB group 12 patients. Sera levels of IL-6, sIL-6R and sgp130 were measured by specific ELISA at the beginning of the operation (T0, 15 min before skin incision) and 6 h later (T1)., Results: IL-6 sera levels were respectively 9+/-20 pg/ml (mean+/-SD) and 13+/-19 pg/ml at T0 and reached 340+/-250 pg/ml and 965+/-1060 pg/ml at T1 in CPB and non-CPB groups, indicating a significant increase from T0 to T1, but no differences between the two groups. When compared to T0 values, sgp130 levels decreased in both groups (respectively 105+/-37 and 115+/-35 ng/ml at T0 for CPB and non-CPB groups, and 72+/-25 and 84+/-29 ng/ml at T1) while we are not able to detect differences between the groups. Whatever the group or the time, sIL-6R concentrations remained unchanged., Conclusions: We showed that the increase of IL-6 after artery bypass grafting was similar between patients operated with CPB or without CPB. We conclude that the main inductor of IL-6 release is linked to surgical trauma rather than a reaction to CPB. Since it is known that gp130 inhibits IL-6-biological activities, we suggest that the decrease of sgp130 sera levels could further enhance the inflammatory effects of IL-6 in cardiac surgery.

A high-performance liquid chromatographic method with fluorescence detection was developed for the determination of (S)-2-[[(7-fluoro-4-indanyl)oxy]methyl]morpholine monohydrochloride (YM992) in plasma. Plasma samples were extracted with n-hexane under alkali condition. After the organic solvent was evaporated to dryness, the residue was treated with 4-fluoro-7-nitrobezofurazan (NBD-F) in borate buffer (pH 7.5) at room temperature for 20 min. The reaction was terminated with hydrochloric acid and the resultant solution was injected onto HPLC without further purification. No interfering peak was observed at the retention time of YM992 or the internal standard. The calibration curve was linear with the concentration of YM992 up to 200 ng/ml. The limit of quantitation was 1 ng/ml. The intra- and inter-day relative standard deviation was less than 5.6% and 4.1%, respectively, and the intra- and inter-day relative error ranged from -3.0% to 17.2% and 2.8% to 7.5%, respectively. Using the assay, the plasma concentration of YM992 could be determined up to 8 and 10 h after the oral administration of YM992 to rats and dogs, respectively., (Copyright 2000 John Wiley & Sons, Ltd.)

Reboxetine is a new selective norepinephrine reuptake inhibitor (selective NRI) for the short- and long-term treatment of depression that is effective and well tolerated at a dose of 8 to 10 mg/day. This study assessed the pharmacokinetics of reboxetine in volunteers with renal impairment. A single 4 mg dose of reboxetine was administered to a total of 18 volunteers with mild (n = 6), moderate (n = 6), or severe (n = 6) renal impairment (creatinine clearance: 56-64, 26-51, and 9-19 ml/min, respectively), and reboxetine concentrations were measured in plasma by HPLC. Mean AUC infinity increased by 43% (mild vs. severe; p < 0.01) as renal function declined, while renal clearance and total urinary excretion of unchanged reboxetine decreased by 67% and 62%, respectively (mild vs. severe; p < 0.01 for both parameters). tmax and t1/2 were not significantly different between groups. In comparison with historical data from young healthy volunteers, AUC infinity and t1/2 are at least doubled in volunteers with renal impairment, while CLr is halved. This pharmacokinetic study has shown that increasing renal dysfunction leads to increasing systemic exposure to reboxetine, particularly in severe renal insufficiency, although reboxetine was well tolerated by all volunteers. Thus, a reduction of the starting dose of reboxetine to 2 mg twice daily would be prudent in patients with renal dysfunction.

A fully automated method including column-switching and isocratic high-performance liquid chromatography (HPLC) was developed for quantitative analysis of the new antidepressant reboxetine, a noradrenaline reuptake inhibitor. After serum injection into the HPLC system and on-line sample clean-up on a silica C8 (10x4.0 mm I.D.) clean-up column with an eluent consisting of 2.5% acetonitrile in deionized water, the chromatographic separation was performed on an analytical column (Lichrospher CN; 250x4.6 mm I.D.) with an eluent of acetonitrile-aqueous potassium phosphate buffer (0.008 M, pH 6.4) (50:50). The UV detector was set at 273 or 226 nm. The limit of quantification was about 15 ng/ml at 273 nm and about 4 ng/ml at 226 nm. The day-to-day relative standard deviation ranged between 2.7 and 6.7% with recovery rates > or = 90%. Linear regression analyses revealed correlation coefficients > 0.998. The method can be applied to therapeutic drug monitoring of reboxetine as well as pharmacokinetic studies.

A water-soluble phosphoramidate prodrug (L-758,298, compound I) of the potent and selective human Substance P receptor antagonist L-754, 030 (compound II) is under development as an i.v. drug for treatment of emesis, migraine, and chronic pain. Compound I undergoes hydrolysis readily to II under acidic conditions. In the studies reported herein, we investigated the stability of I in blood and hepatic subcellular fractions from rats, dogs, and humans as well as the conversion of I to II in rats and dogs after i.v. dosing. Compound I was converted to II rapidly in rat blood but was stable in dog and human blood. However, the conversion was rapid in liver microsomes prepared from dogs and humans. As expected from the results of in vitro studies, the in vivo conversion of I to II was rapid after i.v. dosing of I to rats and dogs. The relative extent of exposure of II after i.v. dosing of I was estimated by comparing the dose-adjusted area under the plasma concentration versus time curve values of II after i.v. dosing of I with those after i.v. dosing of II. In rats, the extent of exposure was estimated to be approximately 90 and approximately 100% at 1 and 8 mg/kg, respectively; in dogs, that was approximately 59% at 0.5 mg/kg. A nonproportional increase in the area under the concentration versus time curve value of II with dose was observed after i.v. administration of I in dogs from 0.5 to 32 mg/kg, suggesting that the elimination of II might have been saturated at higher doses.

Background: Ketoconazole is a potent inhibitor of the cytochrome P450 3A4 enzyme. Reboxetine, a selective norepinephrine reuptake inhibitor, is metabolized by cytochrome P450 3A4. The potential interaction of reboxetine with this representative from the azole derivative class was examined., Methods: Eleven healthy volunteers received (1) 4 mg reboxetine orally on the second day of a 5-day regimen of 200 mg ketoconazole once daily and (2) 4 mg reboxetine orally in a crossover design. Plasma concentrations of reboxetine enantiomers [R,R(-)-reboxetine and the more active S,S(+)-reboxetine] were measured by high-performance liquid chromatography-tandem mass spectrometry. Effects of ketoconazole on enantiomer pharmacokinetics were assessed by ANOVA., Results: Ketoconazole increased R,R(-)-reboxetine and S,S(+)-reboxetine mean area under the plasma concentration-time curves (AUC) by 58% and 43%, respectively (P < .02). Oral clearance of both enantiomers was consequently decreased 34% and 24%, respectively, by ketoconazole (P < .005). Ketoconazole did not significantly affect maximal plasma concentrations (P > .1). Mean terminal half-life after administration of ketoconazole (21.5 hours and 18.9 hours) was significantly longer than after reboxetine alone (14.8 hours and 14.4 hours; P < or = .005). The AUC ratio for R,R(-)-reboxetine to S,S(+)-reboxetine was reduced by ketoconazole administration (2.76 after ketoconazole versus 2.39; P < .003)., Conclusion: Ketoconazole decreases clearance of both reboxetine enantiomers. Although the adverse effect profile for reboxetine was not altered by ketoconazole, the results of this study suggest that caution should be used and that a reduction in reboxetine dose should be considered when the two are coadministered.

Aminoethylcysteine ketimine decarboxylated dimer (AECK-DD) is a natural sulfur-containing tricyclic compound detected, until now, in human urine and bovine cerebellum. Recently, the antioxidant properties of this compound, and particularly its protective effect on the in vitro oxidation of low-density lipoproteins, have been demonstrated. In this paper, the identification of AECK-DD in human plasma by means of gas chromatography, high-performance liquid chromatography and gas chromatography-mass spectrometry, performed after a simple and fast purification procedure, is reported.

The purpose of this study was to assess the cardiovascular effects of an ultra-short-acting beta-blocker, ONO-1101, by using halothane-anesthetized beagle dogs in comparison with esmolol. ONO-1101 (n = 6) or esmolol (n = 6) was administered at four infusion rates of 0.3, 3, 30, and 300 microg/ kg/min. Each infusion was performed over a 30-min period, and the parameters were measured at 20-30 min after the start of each infusion. ONO-1101 significantly decreased the heart rate, rate-pressure product, left ventricular contraction, cardiac output, and relative refractory period of the right ventricle, suppressed the AV nodal conduction, and increased the effective refractory period of the right ventricle, whereas no significant change was observed in the preload and afterload of the left ventricle, intrinsic sinus nodal automaticity, His-Purkinje-ventricular conduction, and the monophasic action-potential duration of the right ventricle. The cardiovascular effects of esmolol were comparable to those of ONO-1101, except that the preload of the left ventricle was significantly increased, and the ventricular repolarization phase was shortened by 300 microg/kg/min of esmolol infusion. Meanwhile, ONO-1101 as well as esmolol significantly reduced the isoproterenol-induced increase in heart rate and ventricular contraction, but the inhibitory action of ONO-1101 was 6-8 times greater than that of esmolol. These results suggest that the suppressive effects of ONO-1101 on cardiovascular performance are significantly less potent than those of esmolol at equipotent beta-blocking doses.

Reboxetine is a racemic mixture of FCE 22071 and FCE 21684 enantiomers. The pharmacokinetics of the enantiomers of reboxetine were observed to be linear in male healthy subjects (n = 6) after the administration of 1.5, 3, 4.5 mg dose of reboxetine as solutions. Kinetic analysis was based on chiral HPLC assay of the enantiomers in plasma collected up to 72 h after each administration. C(max) and AUC were more than double for FCE 22071 (C(max): 38.3+/-13.5, 76. 6+/-26.3, 99.8+/-24.1 ng/mL and AUC(infinity): 605.8+/-233.2, 1288. 3+/-796.4, 1780.7+/-669.3 ng. h/mL for 1.5, 3, 4.5 mg, respectively) than for FCE 21684 (C(max): 15.2+/-5.3, 34.6+/-14.0, 43.1+/-12.3 ng/mL and AUC(infinity): 247.0+/-103.9, 529.1+/-278.4, 773.0+/-355.3 ng. h/mL), whatever the administered dose. The half-lives of the enantiomers were similar (FCE 22071: 13.1, 11.0, 12.6 h and FCE 21684: 12.8, 11.2, 12.2 h after 1.5, 3, 4.5 mg, respectively) and not substantially affected by the dose level., (Copyright 1999 John Wiley & Sons, Ltd.)

The absolute bioavailability of reboxetine enantiomers was assessed in six male and six female volunteers. In a two-way crossover study, subjects received 1.0 mg reboxetine orally and 0.3 mg reboxetine as an intravenous bolus. The R,R(-) and S,S(+) enantiomers in serial plasma and urine samples were determined by a validated LC-MS-MS method. There were no significant differences between treatments for clearance or dose-corrected AUC(0-infinity) values. The absolute bioavailability was 0.919 and 1.02 for R,R(-) reboxetine and S,S(+) reboxetine, respectively. A secondary objective of the study was to assess gender effects on pharmacokinetics of the enantiomers. Significant differences in volume of distribution between genders were observed, but differences in weight-corrected volumes were not significant. Weight-corrected systemic clearance and oral clearance tended to be lower in males, but this difference reached statistical significance only for weight-corrected oral clearance of R,R(-) reboxetine. C(max) after oral administration was 40 and 48% higher in women than men for R,R(-) reboxetine and S,S(+) reboxetine, respectively. These results indicate that reboxetine enantiomers are well absorbed after oral administration and that little first-pass metabolism occurs. There are no clinically significant effects of gender on the pharmacokinetics of reboxetine enantiomers.

A rapid enantioselective method is described for the quantitation of the reboxetine (R,R)- and (S,S)-enantiomers in plasma utilizing solid-phase extraction, derivatization, normal-phase high-performance liquid chromatography, and fluorescence detection. Plasma samples (0.1 ml) with added internal standard were applied to activated solid-phase extraction discs containing a nonpolar/strong cation mixed-phase, washed, eluted, evaporated to dryness, and derivatized for 5 min with (+)-1-(9-fluorenyl)ethyl chloroformate. After termination of the derivatization reaction, the samples were analyzed by isocratic normal-phase HPLC using a silica column and ethanol-heptane (1:124, v/v) as mobile phase. The derivatized reboxetine peak was column-switched onto cyano and Chiralcel OD-H columns in series using ethanol-heptane (1:49, v/v) as mobile phase to resolve the diastereomeric derivatives of the enantiomers and separate interferences. The column effluent was monitored with fluorescence detection at 260/315 nm. The range of quantitation of each enantiomer was 2-2000 ng/ml. One sample was injected every 18 min.

Steady-state concentrations of S12024, a novel compound for treatment of Alzheimer's disease, were studied to determine the pharmacokinetic parameters of S12024 in Phase IIa patients and to assess the effect of patient characteristics on those pharmacokinetics. A prospective sparse sampling strategy was used to obtain oral repeated data (n = 285) from 89 patients, which were analyzed using a one-compartment model and the NONMEM computer program. The model suggested that apparent clearance of S12024 was influenced by the study and by patient age. In the Spanish study, apparent clearance was increased by 68% and 26% for doses of 100 mg and 300 mg, respectively, and patient age decreased oral clearance by approximately 10% per decade in the patient age range (50 to 90 years). Data from only a few patients in the Spanish study were probably responsible for the observed study influence on apparent clearance of S12024, and no measured covariates could explain this effect. The model provided an excellent characterization of the observed data and it predicted correctly the plasma concentrations from an earlier Phase I trial and a subsequent Phase IIb study. The present model, built from Phase IIa data, provides a basis for examining the influence of patient covariates and the magnitude of their effects on the pharmacokinetics of S12024. The study effect is probably an artefact that will disappear by further expanding of the population model in the future.

CAM 5500 and CAM 5187 are new nonpeptide tachykinin NK3 receptor antagonists with different lipophilicity and solubility. We have developed and validated two separate, simple HPLC methods for quantitation of these two compounds in plasma to support oral pharmacokinetic/bioavailability studies in rats. The two compounds in plasma were extracted on cyano SPE cartridges with different washing schemes to optimize extraction efficiency and chromatographic specificity. The analytes and internal standard in the resulting extracts were chromatographed on a C18 HPLC column, using mobile phases containing different phosphate buffer strengths and acetonitrile concentrations. Both compounds were detected using UV, Peak area ratios were proportional over the concentration range of 50-3000 ng ml-1 for CAM 5500, and 100-1500 ng ml-1 for CAM 5187. Stability profiles of both drugs and internal standard in rat plasma at 37 degrees C and in injection solvent at ambient temperature were good. Assay precision, based on quality controls, was < 5.6% and 13.4% (%RSD) for CAM 5500 and CAM 5187, respectively. Similarly, assay accuracy for both compounds was within +/- 7.1% and +/- 6.0% (%RE), respectively. The HPLC methods were successfully applied to assay samples from two oral bioavailability studies. Oral bioavailability studies were conducted for each compound in rats receiving a PO dose of 20 mg kg-1 or an i.v. dose of 5 mg kg-1. Despite their difference in lipophilicity and solubility, the absolute oral bioavailability of CAM 5500 (5.3 +/- 4.8%) is similar to that of CAM 5187 (8.8% +/-3.2%).

Simple and precise high-performance liquid chromatographic (HPLC) assays were developed and validated for the determination of a renin inhibitor (RI), 4-cyclohexyl-2-hydroxy-3- (3-methylsulfanyl-2-[2-[(morpholine-4-carbonyl)amino]- 3-phenylpropionylamino]propionylamino)butyric acid isopropyl ester (CP-80,794, I), and its hydrolytic cleavage metabolite, 2-[(morpholine-4-carbonyl)amino]-3-phenylpropionic acid (CP-84,364, II) in dog and human plasma. The internal standard for I, CP-83,092 (III, a carbon-substituted derivative of I) and analyte were extracted by liquid-liquid extraction using n-butyl chloride, cleaved to form a fragment containing a primary amine, and subsequently fluorescently derivatized for measurement by HPLC. Samples were analyzed by reversed-phase HPLC using a Waters C18 column with fluorescence detection at 390 nm excitation/440 nm emission. The quantitation limit of I was 10 ng/ml and the calibration curve was linear over the range of 0.01-1.0 microgram/ml (r2 > 0.99). In dog and human plasma, intra- and inter-assay precision ranged from 2.3 to 16% and 3.0 to 18%, respectively. The average recoveries were similar (> or = 63%) for both I and III and the upper limit of quantification of I can be as high as 4 micrograms/ml. The internal standard for II, CP-96,452 (IV, a methoxy derivative of II) and analyte were extracted by anion-exchange solid-phase extraction and subsequent liquid-liquid extraction using methyl tert.-butyl ether. Samples were analyzed by reversed-phase HPLC using a Waters C18 column with ultraviolet detection at 214 nm. The quantitation limit of II was 20 ng/ml and the calibration curve was linear over the range of 0.02-2.0 micrograms/ml (r2 > 0.99). In dog and human plasma, intra- and inter-assay precision ranged from 2.6 to 13.0% and 1.8 to 20.0%, respectively. The average recoveries were similar (> or = 75%) for both II and IV and the upper limit of quantification of II can be as high as 20 micrograms/ml. The methods described have been successfully applied to the quantification of I and II in about 5000 dog and human plasma samples over a 2 year period.

A high-performance liquid chromatographic (HPLC) assay using ultraviolet (UV) detection was developed and compared with a HPLC method with tandem mass spectrometric (HPLC/MS-MS) detection for the determination of a substance P receptor antagonist 2(S)-((3,5-bis(trifluoromethyl)benzyl)-oxy)-3(S)-phenyl-4-((3-oxo-1,2,4- triazol-5-yl) methyl)morpholine (Fig. 1, Ia, L-742 694) in human plasma and urine. The drug was isolated from the biological matrix through liquid-liquid extraction. In the HPLC/UV method, the samples were initially injected onto a cyano Hypersil column, and the chromatographic region containing the peaks of interest was heart-cut onto an analytical C-18 Hypersil column via a column switching device. The analyte was quantified by monitoring absorbance at 205 nm. The limit of quantification for I extracted from 1 ml of plasma or urine was 2.5 ng ml-1, and the assays were validated in the concentration range 2.5-500 ng ml-1. The HPLC/MS-MS method were validated in the concentration range 0.2-500 ng ml-1. Both assays provided data with precision, measured as coefficient of variation, better than 10% at all points within the standard curve range and with adequate accuracy.

Mosapride citrate, a novel benzamide-type gastroprokinetic agent, is clinically prescribed as a racemate and is metabolized to its des-4-fluorobenzyl structure (M-1). In order to analyze simultaneously the enantiomers of mosapride and M-1 in plasma, a simple and reproducible high-performance liquid chromatographic (HPLC) method has been developed. The enantiomeric separation and determination were successfully achieved using an alpha 1-acid glycoprotein column and gradient elution with a fluorimetric detection (excitation 314 nm/emission 352 nm). Both enantiomers of mosapride and M-1 were well separated between 20 and 22 min at pH 4.4 and between 4 and 7 min at pH 5.0, respectively. Accurate determinations are possible in the concentration ranges of 10-5000 ng ml-1 for mosapride enantiomers and 50-5000 ng ml-1 for M-1 enantiomers. The intra- and inter-day coefficients of variation are satisfactory for the pharmacokinetic study of mosapride.

We conducted a randomized study with four parallel groups to investigate the effect of single and multiple doses of activated charcoal on the absorption and elimination of pholcodine administered in a cough syrup. The first group received 100 mg of pholcodine on an empty stomach with water only (control); the second group took 25 g of activated charcoal immediately after pholcodine; the third group received 25 g of activated charcoal 2 h and the fourth group 5 h after ingestion of the 100-mg dose of pholcodine. In addition, the fourth group received multiple doses (10 g each) of charcoal every 12 h for 84 h. Blood samples were collected for 96 h and urine for 72 h. Pholcodine concentrations were measured by high-performance liquid chromatography. A significant reduction in absorption was found when charcoal was administered immediately after pholcodine; the AUC0-96h was reduced by 91% (p < 0.0005), the Cmax by 77% (p < 0.0005), and the amount of pholcodine excreted into urine by 85% (p < 0.0005). When charcoal was administered 2 h after pholcodine, the AUC0-96h was reduced by 26% (p = 0.002), the Cmax by 23% (p = NS), and the urinary excretion by 28% (p = 0.004). When administered 5 h after pholcodine, charcoal produced only a 17% reduction in the AUC0-96h (p = 0.06), but reduced the further absorption of pholcodine still present in the gastrointestinal tract at the time of charcoal administration, as measured by AUC5-96h (p = 0.006). Repeated administration of charcoal failed to accelerate the elimination of pholcodine. We conclude that activated charcoal is effective in preventing the absorption of pholcodine, and its administration can be beneficial even several hours after pholcodine ingestion.

Reboxetine, (RS)-2-[(RS)-alpha-(2-ethoxyphenoxy)benzyl]morpholine methanesulphonate, is a racemic compound and consists of a mixture of the (R,R)- and (S,S)-enantiomers. The pharmacokinetics of reboxetine enantiomers were determined in a crossover study in three male beagle dogs. Each animal received the following oral treatments, separated by 1-week washout period: 10 mg/kg reboxetine, 5 mg/kg (R,R)- and 5 mg/kg (S,S)-. Plasma and urinary levels of the reboxetine enantiomers were monitored up to 48 h post-dosing using an enantiospecific HPLC method with fluorimetric detection (LOQ: 1.1 ng/ml in plasma and 5 ng/ml in urine for each enantiomer). After reboxetine administration mean tmax was about 1 h for both enantiomers. Cmax and AUC were about 1.5 times higher for the (R,R)- than for the (S,S)-enantiomer, mean values +/- SD being 704 +/- 330 and 427 +/- 175 ng/ml for Cmax and 2,876 +/- 1,354 and 1,998 +/- 848 ng.h/ml for AUC, respectively. No differences between the (R,R)- and (S,S)-enantiomers were observed in t1/2 (3.9 h). Total recovery of the two enantiomers in urine was similar, the Ae (0-48 h) being 1.3 +/- 0.7 and 1.1 +/- 0.7% of the enantiomer dose for the (R,R)- and the (S,S)-enantiomers, respectively. No marked differences in the main plasma pharmacokinetic parameters were found for either enantiomer on administration of the single enantiomers or reboxetine. No chiral inversion was observed after administration of the separate enantiomers, as already observed in humans.

The pharmacokinetics of reboxetine have been investigated in 12 healthy male volunteers after a single 2 mg dose of reboxetine and at steady state, following the last administration of a multiple-dose regimen (2mg twice a day for 5 1/2 d). Reboxetine was analysed in plasma and urine samples collected up to 72 h post-dosing using an HPLC method with UV detection. The urinary excretion rate of 6-beta-hydroxycortisol, used as a marker for cytochrome P450IIIA activity, was also tested, and any possible alteration was correlated to reboxetine plasma levels. The dosing regimen was well tolerated by all subjects. Reboxetine pharmacokinetic parameters, calculated after the single dose using non-compartmental analysis, were in good agreement with those obtained in previous studies. Following the multiple-dosing regimen, no significant deviations from expectation based on linear superposition was observed. The accumulation ratio, based on repeated-dose/single-dose ratios of Cmax, AUC(0-12 h), and C(12 h) was approximately two. A slight rise was recorded for the average excretion rate of 6-beta-hydroxycortisol over 48 h by the end of treatment; however, the difference was not statistically significant and the mean excretion rates were within the normal reference range. Thus a significant induction of P450IIIA was not indicated.