Simultaneous Analysis of Tramadol, O-Desmethyltramadol, and N-Desmethyltramadol Enantiomers in Rat Plasma by High-Performance Liquid Chromatography-Tandem Mass Spectrometry: Application to Pharmacokinetics

Tramadol (T) is available as a racemic mixture of (+)-trans-T and (-)-trans-T. The main metabolic pathways are O-demethylation and N-demethylation, producing trans-O-desmethyltramadol (M1) and trans-N-desmethyltramadol (M2) enantiomers, respectively. The analgesic effect of T is related to the opioid activity of (+)-trans-T and (+)-M1 and to the monoaminergic action of (+/-)-trans-T. This is the first study using tandem mass spectrometry as a detection system for the simultaneous analysis of trans-T, M1, and M2 enantiomers. The analytes were resolved on a Chiralpak (R) AD column using hexane: ethanol (95.5:4.5, v/v) plus 0.1% diethylamine as the mobile phase. The quantitation limits were 0.5 ng/ml for trans-T and M1 and 0.1 ng/ml for M2. The method developed and validated here was applied to a pharmacokinetic study in rats. Male Wistar rats (n = 6 at each time point) received a single oral dose of 20 mg/kg racemic trans-T. Blood samples were collected up to 12 h after drug administration. The kinetic disposition of trans-T and M2 was enantioselective (AUC((+)/(-)) ratio = 4.16 and 6.36, respectively). The direction and extent of enantioselectivity in the pharmacokinetics of trans-T and M2 in rats were comparable to data previously reported for healthy volunteers, suggesting that rats are a suitable model for enantioselective studies of trans-T pharmacokinetics. Chirality 23: 287-293, 2011. (C) 2010 Wiley-Liss, Inc.

Determination of mitragynine in rat plasma by LC-MS/MS: Application to pharmacokinetics

This study used for the first time LC-MS/MS for the analysis of mitragynine (MIT), a mu-opioid agonist with antinociceptive and antitussive properties, in rat plasma. Mitragynine and the internal standard (amitriptyline) were extracted from plasma with hexane-isoamyl alcohol and resolved on a Lichrospher (R) RP-SelectB column (9.80 and 12.90 min, respectively). The quantification limit was 0.2 ng/mL within a linear range of 0.2-1000 ng/mL The method was applied to quantify mitragynine in plasma samples of rats (n = 8 per sampling time) treated with a single oral dose of 20 mg/kg. The following pharmacokinetic parameters were obtained (mean): maximum plasma concentration: 424 ng/mL; time to reach maximum plasma concentration: 1.26 h; elimination half-life: 3.85 h, apparent total clearance: 6.35 L/h/kg, and apparent volume of distribution: 37.90 L/kg. (C) 2009 Elsevier B.V. All rights reserved.

Hemispheric contributions to lexical ambiguity resolution: Evidence from individuals with complex language impairment following left-hemisphere lesions

Nine individuals with complex language deficits following left-hemisphere cortical lesions and a matched control group (n 5 9) performed speeded lexical decisions on the third word of auditory word triplets containing a lexical ambiguity. The critical conditions were concordant (e.g., coin–bank–money), discordant (e.g., river–bank–money), neutral (e.g., day–bank– money), and unrelated (e.g., river–day–money). Triplets were presented with an interstimulus interval (ISI) of 100 and 1250 ms. Overall, the left-hemisphere-damaged subjects appeared able to exhaustively access meanings for lexical ambiguities rapidly, but were unable to reduce the level of activation for contextually inappropriate meanings at both short and long ISIs, unlike control subjects. These findings are consistent with a disruption of the proposed role of the left hemisphere in selecting and suppressing meanings via contextual integration and a sparing of the right-hemisphere mechanisms responsible for maintaining alternative meanings.

Leaflets left in gutter after Ky demonstration Brisbane, Australia

Leaflets in gutter after demonstration of former South Vietnamese vice president Nguyen Cao Ky to Brisbane, Australia in January 1967.

Validity of (-)-[H-3]-CGP 12177A as a radioligand for the 'putative beta(4)-adrenoceptor' in rat atrium

1 We have recently suggested the existence in the heart of a 'putative beta(4)-adrenoceptor' based on the cardiostimulant effects of non-conventional partial agonists, compounds that cause cardiostimulant effects at greater concentrations than those required to block beta(1)- and Bz-adrenoceptors. We sought to obtain further evidence by establishing and validating a radioligand binding assay for this receptor with (-)-[H-3]-CGP 12177A ((-)-4-(3-tertiarybutylamino-2-hydroxypropoxy) benzimidazol-2-one) in rat atrium. We investigated (-)-[H-3]-CGP 12177A for this purpose for two reasons, because it is a nonconventional partial agonist and also because it is a hydrophilic radioligand. 2 Increasing concentrations of(-)-[H-3]-CGP 12177A, in the absence or presence of 20 mu M (-)-CGP 12177A to define non-specific binding, resulted in a biphasic saturation isotherm. Low concentrations bound to beta(1)- and beta(2)-adrenoceptors (pK(D) 9.4+/-0.1, B-max 26.9+/-3.1 fmol mg(-1) protein) and higher concentrations bound to the 'putative beta(4)-adrenoceptor' (pK(D) 7.5+/-0.1, B-max 47.7+/-4.9 fmol mg(-1) protein). In other experiments designed to exclude beta(1)- and beta(2)-adrenoceptors, (-)-[H-3]-CGP 12177A (1-200 nM) binding in the presence of 500 nM (-)-propranolol was also saturable (pK(D) 7.6+/-0.1, B-max 50.8+/-7.4 fmol mg(-1) protein). 3 The non-conventional partial agonists (-)-CGP 12177A (pK(i) 7.3+/-0.2), (+/-)-cyanopindolol (pK(i) 7.6+/-0.2), (-)-pindolol (pK(i) 6.6+/-0.1) and (+)-carazolol (pk(i), 7.2+/-0.2) and the antagonist (-)-bupranolol (pK(i) 6.6+/-0.2), all competed for (-)-[H-3]-CGP 12177A binding in the presence of 500 nM (-)-propranolol at the 'putative beta(4)-adrenoceptor', with affinities closely similar to potencies and affinities determined in organ bath studies. 4 The catecholamines competed with (-)-[H-3]-CGP 12177A at the 'putative beta(4)-adrenoceptor' in a stereoselective manner, (-)-noradrenaline (pK(iH) 6.3 +/- 0.3, pK(i), 3.5 +/- 0.1), (-)-adrenaline (pK(iH) 6.5 +/- 0.2, pK(iL) 2.9 +/- 0.1), (-)-isoprenaline (pK(iH) 6.2 +/- 0.5, pK(iL) 3.3 +/- 0.1), (+)-isoprenaline (pK(i) < 1.7), (-)-R0363 ((-)-(1-(3,4-dimethoxyphenethylamino)-3-(3,4-dihydroxyphenoxy)-2-propranol)oxalate, pK(i) 5.5 +/- 0.1). 5 The inclusion of guanosine 5-triphosphate (GTP 0.1 mM) had no effect on binding of (-)-CGP 12177A or (-)-isoprenaline to the 'putative beta(4)-adrenoceptor'. In competition binding studies, (-)-CGP 12177A competed with (-)-[H-3]-CGP 12177A for one receptor state in the absence (pK(i) 7.3 +/- 0.2) or presence of GTP (pK(i) 7.3 +/- 0.2). (-)-Isoprenaline competed with (-)-[H-3]-CGP 12177A for two states in the absence (pK(iH) 6.6 +/- 0.3, pK(iL) 3.5 +/- 0.1; % H 25 +/- 7) or presence of GTP (pK(iH) 6.2 +/- 0.5, pK(iL) 3.4 +/- 0.1; % H 37 +/- 6). In contrast, at beta(1)-adrenoceptors, GTP stabilized the low affinity state of the receptor for (-)-isoprenaline. 6 The specificity of binding to the 'putative beta(4)-adrenoceptor' was tested with compounds active at other receptors. High concentrations of the beta(4)-adrenoceptor agonists, BRL 37344 ((RR + SS)[4-[2-[[2-(3-chlorophenyl)-2-hydroxy -ethyl]amino]propyl]phenoxy]acetic acid, 6 mu M), SR 58611A (ethyl((7S)-7-[(2R)-2-(3-chlorophenyl)-2-hydroxyethylamino]-5,6,7,8-tetrahydronaphtyl-2-yloxy) acetate hydrochloride, 6 mu M), ZD 2079 ((+/-)-1-phenyl-2-(2-4-carboxymethylphenoxy)-ethylamino)ethan-1-ol, 60 mu M), CL 316243 (disodium (R,R)-5-[2-[2-(3-chlorophenyl)-2-hydroxyethyl-amino]propyl]- 1,3-benzodioxole-2,2-dicarboxylate, 60 mu M) and antagonist SR 59230A (3-(2-ethylphenoxy)-1-[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-2S-2-propanol oxalate, 6 mu M) caused less than 22% inhibition of (-)-[H-3]-CGP 12177A binding in the presence of 500 nM (-)-propranolol. Histamine (1 mM), atropine (1 mu M), phentolamine (10 mu M), 5-HT(100 mu M) and the 5-HT4 receptor antagonist SE 207710 ((1-butyl-4-piperidinyl)-methyl 8-amino-7-iodo-1 ,4-benzodioxan-5-carboxylate, 10 nM) caused less than 26% inhibition of binding. 7 Non-conventional partial agonists, the antagonist (-)-bupranolol and catecholamines all competed for (-)-[H-3]-CGP 12177A binding in the absence of (-)-propranolol at beta(1)-adrenoceptors, with affinities (pK(i)) ranging from 1.6-3.6 log orders greater than at the 'putative beta(4)-adrenoceptor'. 8 We have established and validated a radioligand binding assay in rat atrium for the 'putative beta(4)-adrenoceptor' which is distinct from beta(1)-, beta(2)- and beta(3)-adrenoceptors. The stereoselective interaction with the catecholamines provides further support for the classification of the receptor as 'putative beta(4)-adrenoceptor'.

The effect of protein binding on the hepatic first pass of O-acyl salicylate derivatives in the rat

In this work the in-situ perfused rat liver has been used to examine the effect of changing the protein content of the perfusate on the hepatic extraction of O-acyl esters of salicylic acid. The hepatic availability (F) of these solutes was studied at a flow-rate of 30 mt min(-1) with perfusate albumin concentrations of 0, 2, and 4% w/v. The hepatic availability of the esters was shown to decrease with increasing carbon-chain length in the O-acyl group; for all the esters the hepatic availability increased with increasing albumin concentration in the perfusate. The dispersion-model-derived efficiency number (R-N) Of the esters was shown to increase with increasing lipophilicity and decrease with increasing albumin concentration in the perfusate. The unbound fraction (f(u),) of the esters decreased with lipophilicity. R-N/f(u), for acetylsalicylic acid remained relatively constant as the albumin concentration was increased. However, R-N/f(u), for n-pentanoyl- and n-hexanoylsalicylic acids increased significantly as albumin concentration increased from 0% to 4%. Thus, for the more lipophilic solutes (n-pentanoyl- and n-hexanoylsalicylic acids) the presence of albumin apparently facilitates the uptake of unbound solute relative to acetylsalicylic acid.

Morphine has a dual concentration-dependent effect on K+-evoked substance P release from rat peripheral airways

Our previous investigations of possible lung mechanisms underlying the effectiveness of nebulized morphine for the relief of dyspnoea, have shown a high density of non-conventional opioid binding sites in rat airways with similar binding characteristics (opioid alkaloid-sensitive, opioid peptide-insensitive) to that of putative mu(3)-opioid receptors on immune cells. To investigate whether these lung opioid binding sites are functional receptors, this study was designed to determine (using superfusion) whether morphine modulates the K+-evoked release of the pro-inflammatory neuropeptide, substance P (SP), from rat peripheral airways. Importantly, K+-evoked SP release was Ca2+-dependent, consistent with vesicular release. Submicromolar concentrations of morphine (1 and 200 nM) inhibited K+-evoked SP release from rat peripheral airways in a naloxone (1 mu M) reversible manner. By contrast, 1 mu M morphine enhanced K+-evoked SP release and this effect was not reversed by 1 mu M naloxone. However, 100 mu M naloxone not only antagonized the facilitatory effect of 1 mu M morphine on K+-evoked SP release from rat peripheral airways but it inhibited release to a similar extent as 200 nM morphine. It is possible that these latter effects are mediated by non-conventional opioid receptors located on mast cells, activation of which causes naloxone-reversible histamine release that in turn augments the release of SP from sensory nerve terminals in the peripheral airways. Clearly, further studies are required to investigate this possibility. (C) 1997 Academic Press Limited.

Kinetic analysis of vascular marker distribution in perfused rat livers after regeneration following partial hepatectomy

Background/Aims: Liver clearance models are based on information (or assumptions) on solute distribution kinetics within the microvasculatory system, The aim was to study albumin distribution kinetics in regenerated livers and in livers of normal adult rats, Methods: A novel mathematical model was used to evaluate the distribution space and the transit time dispersion of albumin in livers following regeneration after a two-thirds hepatectomy compared to livers of normal adult rats. Outflow curves of albumin measured after bolus injection in single-pass perfused rat livers were analyzed by correcting for the influence of catheters and fitting a long-tailed function to the data. Results: The curves were well described by the proposed model. The distribution volume and the transit time dispersion of albumin observed in the partial hepatectomy group were not significantly different from livers of normal adult rats. Conclusions: These findings suggest that the distribution space and the transit time dispersion of albumin (CV2) is relatively constant irrespective of the presence of rapid and extensive repair. This invariance of CV2 implies, as a first approximation, a similar degree of intrasinusoidal mixing, The finding that a sum of two (instead of one) inverse Gaussian densities is an appropriate empirical function to describe the outflow curve of vascular indicators has consequences for an improved prediction of hepatic solute extraction.

Ethanol inhibition of brain ornithine decarboxylase activity in the postnatal rat

The purpose of this study was to determine the relationship between ornithine decarboxylase activity (ODC; a marker for perturbed cell development), the blood alcohol level, and alcohol-induced microencephaly in the developing rat brain after binge treatment with ethanol vapour. By manipulating ethanol flow we were able to adjust vapour concentrations (24-65 mg ethanol/l air) such that an acute exposure of ethanol vapour for 3 h resulted in a range of blood alcohol levels (2.3-5.5 mg/ml). Acute studies showed that ethanol dose-dependently inhibited rat hippocampal and cerebellar ODC activity at PND4-PND10. There was a significant correlation between the blood alcohol level and degree of inhibition at all ages tested. Chronic treatment from PND4 to PND9 caused a significant decrease in both brain to body weight ratio and in hippocampal and cerebellar ODC activities at PND10. These results indicate that ethanol-induced disruption in ODC could play a significant role in ethanol's teratogenic effects during early postnatal development. (C) 1998 Elsevier Science Inc.

Nitric oxide inhibition of the rat olfactory cyclic nucleotide-gated cation channel

The effects of nitric oxide (NO) and other cysteine modifying agents were examined on cyclic nucleotide-gated (CNG) cation channels from rat olfactory receptor neurons. The NO compounds, S-nitroso-cysteine (SNC) and 3-morpholino-sydnonomine (SIN-1), did not activate the channels when applied for up to 10 min. The cysteine alkylating agent, N-ethylmaleimide (NEM), and the oxidising agent, dithionitrobensoate (DTNB), were also without agonist efficacy. Neither SNC nor DTNB altered the cAMP sensitivity of the channels. However, 2-min applications of SIN-1, SNC and DTNB inhibited the cAMP-gated current to approximately 50% of the control current level. This inhibition showed no spontaneous reversal for 5 min but was completely reversed by a 2-min exposure to DTT. The presence of cAMP protected the channels against NO-induced inhibition. These results indicate that inhibition is caused by S-nitrosylation of neighboring sulfhydryl groups leading to sulfhydryl bond formation. This reaction is favored in the closed channel state. Since recombinantly expressed rat olfactory alpha and beta CNG channel homomers and alpha/beta heteromers are activated and not inhibited by cysteine modification, the results of this study imply the existence of a novel subunit or tightly bound factor which dominates the effect of cysteine modification in the native channels. As CNG channels provide a pathway for calcum influx, the results may also have important implications for the physiological role of NO in mammalian olfactory receptor neurons.