Nitric oxide induces neurotransmitter release from hippocampal slices

Hydroxylamine (1–300 μM), a nitric oxide generator, stimulated the release of [³H]norepinephrine ([³H]NE) and [¹⁴C]acetylchoIine ([¹⁴C]ACh) from rat hippocampal slices in a concentration-dependent manner (EC₅₀ = 30 μM). A maximally effective concentration of hydroxylamine (300 μM) produced a 24-fold increase in the basal [³H]NE and 3.6-fold increase in the in the basal [¹⁴C]ACh efflux. Sodium nitroprusside (SNP), also stimulated the release of [³H]NE, but only at high concentrations (10–30 mM). Calcium-free experimental buffer (1 mM EGTA) abolished the response. Hemoglobin (0.3 μM) inhibited the effect of 100 μM hydroxylamine in a manner which was specific for nitric oxide. In addition, 100 μM hydroxyiarnine increased the efflux of endogenous GABA and glutamate by 3- and 6-fold, respectively.     

Clonidine accumulation in human neuronal cells

After transport across several epithelial barriers including the blood–brain barrier, clonidine interacts with ₂-adrenergic receptors and imidazoline binding sites in the brain. We hypothesized that neuronal cells take up clonidine thereby removing the drug from the extracellular fluid compartment. Uptake of [³H]clonidine into SH-SY5Y neuroblastoma cells was linear for up to 1 min, unaffected by inside directed Na⁺ or Cl⁻ gradients but strongly inhibited by an outside pH of 6.0. The cells accumulated [³H]clonidine 50–70-fold uphill against a concentration gradient. Unlabeled clonidine, guanabenz, imipramine, diphenhydramine, maprotiline, quinine and the endogenous monoamine phenylethylamine (2 mM) strongly inhibited the [³H]clonidine uptake by 60–95%. Tetraethylammonium, choline and N-methyl-4-phenylpyridinium had no effect. The accumulation at pH 7.5 was saturable with an apparent Michaelis–Menten constant (K_t) of 0.7 mM. We conclude that SH-SY5Y cells not only bind clonidine to extracellular receptors but also take up the drug rapidly by a specific and concentrative mechanism.     

Concentration-dependent accumulation of [H]-deltamethrin in sodium channel Nav1.2/β1 expressing Xenopus laevis oocytes

Disruption of neuronal voltage-sensitive sodium channels (VSSCs) by pyrethroid insecticides such as deltamethrin (DLT) has been widely studied using Xenopus laevis oocytes transfected with VSSC. However, the extent of pyrethroid accumulation in VSSC-expressing oocytes is unknown. Therefore, accumulation of [³H]-DLT in non-transfected, sham (water)-transfected and VSSC (Na_v1.2 + β₁)-transfected oocytes after a 1 h exposure was measured using liquid scintillation counting. Successful transfection of Na_v1.2 + β₁ VSSCs in X. laevis oocytes was confirmed by two-electrode voltage-clamp; inward, tetrodotoxin (TTX)-sensitive currents were obtained in 98% of all oocytes examined (n = 60 in nine experiments). DLT (1.0 μM) induced tail currents in all VSSC-transfected oocytes; TTX also blocked these DLT-induced tail currents. In 0.1 μM DLT solution, non-transfected oocytes accumulated 0.098 ± 0.01 ppm [³H]-DLT, sham-transfected oocytes accumulated 0.06 ± 0.01 ppm DLT, and VSSC-transfected oocytes accumulated 0.050 ± 0.009 ppm DLT. In 1.0 μM DLT solution, non-transfected oocytes accumulated 0.62 ± 0.08 ppm DLT, sham-transfected oocytes accumulated 0.60 ± 0.09 ppm DLT, and VSSC-transfected oocytes accumulated 0.51 ± 0.07 ppm DLT. There was a significant difference in DLT accumulation between VSSC-transfected oocytes and non-transfected controls, where the transfected oocytes consistently had less accumulation.     

Effects of L- and N-type Ca channel antagonists on excitatory amino acid-evoked dopamine release

In thc present study we tested the effect of dihydropyridine (DHP) Ca²⁺ channel antagonists and of ω-conotoxin GVIA on [³H]dopamine (DA) release evoked by the activation of excitatory amino acid (EAA) receptors in cultures of fetal rat ventral mesencephalon, in order to investigate the role of voltage-sensitive L- and N-type Ca²⁺ channels in these EAA-mediated processes. Micromolar concentrations (10–30 μM) of DHP L-type Ca²⁺ channel antagonists inhibited [³H]DA release evoked by N-methyl-D-aspartate (NMDA), kainate, quisqualate or veratridine. [³H]DA release evoked by the L-type Ca²⁺ channel agonist, Bay K 8644, was inhibited by lower concentrations (0.1–1 μM) of the DHP antagonist, nitrendipine, than was the release evoked by EAAs. The DHP antagonist, ( + )-PN 200-110, was more potent than ( − )-PN 200-110 in inhibiting [³H]DA release evoked by Bay K 8644, but the two stereoisomers were equipotent in inhibiting NMDA-evoked release. These results indicate that activation of L-type Ca²⁺ channels is able to evoke [³H]DA release. However activation of L-type channels is not involved in EAA-induced [³H]DA release and therefore inhibition of EAA-induccd [³H]DA release by micromolar concentrations of DHPs must be mediated by actions other than inhibition of L-type Ca²⁺ channels. ω-Conotoxin GVIA (3 μM) had no effect on [³H]DA release evoked by Bay K 8644, indicating that the toxin may selectively inhibit N-type channels in this preparation. ω-Conotoxin GVIA (3 μM) partially inhibited [³H]DA release evoked by NMDA or kainate, suggesting that N-type Ca²⁺ channels could possibly play a role in FAA-mediated responses in these cells.     

Beware the builders: Construction noise changes [C]GABA release and uptake from amygdaloid and hippocampal slices in the rat

The effects of exposure to chronic noise and vibration, produced by construction work, on the release and uptake of [³H]5-hydroxytryptamine ([³H]5-HT) and [¹⁴C]γ-aminobutyric acid ([¹⁴C]GABA) from rat amygdaloid and hippocampal slices were investigated. Noise-exposure resulted in increased release, with no significant change in uptake, of [¹⁴C]GABA from amygdaloid slices. In hippocampal slices, [¹⁴C]GABA release was also increased, but the changes in release were dependent upon the marked decrease in uptake of [¹⁴C]GABA into these slices. There was an increase in peak K⁺-evoked release of [³H]5-HT from hippocampal slices, but no other changes in [³H]5-HT release or uptake in either region were observed following noise-exposure. These findings may have important practical implications for the research carried out in laboratories exposed to construction noise and vibrations.     

Persistent, specific and dose-dependent effects of toluene exposure on dopamine D22 agonist binding in the rat caudate-putamen

Exposure to toluene (40–320 ppm; 4 weeks, 6 h/day, 5 days/ week), followed by a postexposure period of 29–40 days, decreased the wet weight of the caudate-putamen and of the subcortical limbic area (maximal effect of 10% attained at 80 ppm toluene) of the male rat. Furthermore, toluene exposure decreased the IC₅₀ values (significant effects attained at 80 ppm), the K_H, the K_L, and the R_H% values of dopamine on [³H]raclopride-binding in the caudate-putamen. Toluene exposure did not significantly affect either the body weights, the wet weights of the whole brain, the serum prolactin levels, the K_D or the B_max values of [³H]raclopride-binding in the caudate-putamen and the sub-cortical limbic area, or the IC₅₀ values of dopamine at [³H]raclopride-binding sites in the subcortical limbic area. Exposure to xylene or styrene (80 and 40 ppm, respectively; 4 weeks, 6 h/day, 5 days/ week), followed by a postexposure period of 26–32 days, had no effect on the parameters described above (prolactin levels were not analyzed). The present study indicates that long-term exposure to low concentrations of toluene (≥ 80 ppm), but not xylene (80 ppm) or styr-ene (40 ppm), leads to persistent increases in the affinity of dopamine D2₂agonist binding in the rat caudate-putamen.     

Lobeline inhibits nicotine-evoked [H]dopamine overflow from rat striatal slices and nicotine-evoked Rb efflux from thalamic synaptosomes

The present study evaluated the interaction of lobeline with neuronal nicotinic acetylcholine receptors using two in vitro assays, [³H] overflow from [³H]dopamine ([³H]DA)-preloaded rat striatal slices and ⁸⁶Rb⁺ efflux from rat thalamic synaptosomes. To assess agonist interactions, the effect of lobeline was determined and compared to S(−)-nicotine. To assess antagonist interactions, the ability of lobeline to inhibit the effect of S(−)-nicotine was determined. Both S(−)-nicotine (0.1–1 μM) and lobeline (>1.0 μM) evoked [³H] overflow from superfused [³H]DA-preloaded striatal slices. However, lobeline-evoked [³H] overflow is mecamylamine-insensitive, indicating that this response is not mediated by nicotinic receptors. Moreover, at concentrations (<1.0 μM) which did not evoke [³H] overflow, lobeline inhibited S(−)-nicotine (0.1–10 μM)-evoked [³H] overflow, shifting the S(−)-nicotine concentration–response curve to the right. S(−)-Nicotine (30 nM–300 μM) increased (EC₅₀ VALUE=0.2 μM) ⁸⁶Rb⁺ efflux from thalamic synaptosomes. In contrast, lobeline (1 nM–10 μM) did not evoke ⁸⁶Rb⁺ efflux, and the lack of intrinsic activity indicates that lobeline is not an agonist at this nicotinic receptor subtype. Lobeline completely inhibited (IC₅₀ VALUE=0.7 μM) ⁸⁶Rb⁺ efflux evoked by 1 μM S(−)-nicotine, a concentration which maximally stimulated ⁸⁶Rb⁺ efflux. Thus, the results of these in vitro experiments demonstrate that lobeline inhibits the effects of S(−)-nicotine, and suggest that lobeline acts as a nicotinic receptor antagonist.     

Haloperidol treatment differentially regulates [H]DTG and [H](+)-3-PPP labeled σ binding sites

The effect of repeated haloperidol administration on σ binding sites in brain membranes was assessed using [³H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ((+)-3-PPP) and [³H]1,3-di-o-tolylguanidine (DTG). Administration of haloperidol (1 mg/kg, i.p.) to guinea pigs for 14 consecutive days followed by a 4 day drug-free period prior to sacrifice resulted in 75% and 6% decreases in the specific binding of [³H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine and [³H]1,3-di-o-tolylguanidine, respectively, when measured using a single concentration (2 nM) of radioligand. Scatchard analysis revealed a reduction in both the maximum number of [³H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine binding sites and the affinity of these sites for the radioligand; the potency of 1,3-di-o-tolylguanidine to inhibit [³H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine binding was also reduced. In parallel studies, the potency of 1,3-di-o-tolylguanidine to inhibit [³H]1,3-di-o-tolylguanidine binding was unaffected by haloperidol treatment, but the potency of (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine against [³H]1,3-di-o-tolylguanidine was reduced 3-fold. Phenytoin, which increased (10-fold) the potency of dextromethorphan to inhibit [³H]1,3-di-o-tolylguanidine binding in control membranes, had no effect in membranes obtained from haloperidol-treated animals. The reduction in [³H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine binding was dependent upon the duration of the drug-free period and amounted to 73% and 25% in brain membranes prepared from animals sacrificed 14 days and 28 days, respectively, following cessation of drug treatment. Repeated administration of other antipsychotic and σ agents including DTG, dextromethorphan, spiperone, chlorpromazine and clozapine had no effect on [³H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine or [³H]1,3-di-o-tolylguanidine binding. These findings suggest that repeated haloperidol administration selectively regulates [³H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine binding in guinea pig brain membranes. However, the observations that repeated drug treatment resulted in (1) reduced affinity of (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine for [³H]1,3-di-o-tolylguanidine binding sites, and (2) a loss of the ability of phenytoin to increase dextromethorphan's potency against [³H]1,3-di-o-tolylguanidine, suggest that [³H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine and [³H]1,3-di-o-tolylguanidine binding sites are distinct but functionally coupled.     

Composition analysis of two batches of polysorbate 60 using MS and NMR techniques

Batch variation in Tween 60 has shown to influence the rheological properties of semisolid emulsions. MS (LC–MS, GC–MS, MSⁿ) and NMR (¹³C, ¹H, ¹H COSY and HMBC) techniques were used to analyze and compare the composition of two batches of Tween 60 with particular emphasis on the number of POE groups and their distribution within the molecule. Acid and saponification values were also determined. The batches contained different proportions of components (sorbitan polyethoxylates, sorbitan monoester–diester-polyethoxylates and isosorbide monoester–diester-polyethoxylates). The number of POE groups were averaged over the four sites in sorbitan and the two sites in isosorbide molecules. The batches differed from each other in terms of (i) the POE sorbitan stearate/POE sorbitan palmitate ratios (batch 1, 3:2 and batch 2, 4:5), (ii) the ratio of sorbitans to isosorbides (batch 1, 2:3; batch 2, 7:13); and (iii) the acid values (batch 1, 3.1; batch 2, 0). It is concluded that liquid chromatography combined with electrospray mass spectrometry and ion trap separation is a useful tool for establishing the compositional profile of different batches of Tweens. ¹H NMR could provide a simple and rapid pharmacopoeial test for the ratio of sorbitan to isosorbide in Tweens.     

Receptor-mediated internalization of [3H]-neurotensin in synaptosomal preparations from rat neostriatum

Following its binding to somatodendritic receptors, the neuropeptide neurotensin (NT) internalizes via a clathrin-mediated process. In the present study, we investigated whether NT also internalizes presynaptically using synaptosomes from rat neostriatum, a region in which NT1 receptors are virtually all presynaptic. Binding of [³H]–NT to striatal synaptosomes in the presence of levocabastine to block NT2 receptors is specific, saturable, and has NT1 binding properties. A significant fraction of the bound radioactivity is resistant to hypertonic acid wash indicating that it is internalized. Internalization of [³H]–NT, like that of [¹²⁵I]–transferrin, is blocked by sucrose and low temperature, consistent with endocytosis occurring via a clathrin-dependent pathway. However, contrary to what was reported at the somatodendritic level, neither [³H]–NT nor [¹²⁵I]–transferrin internalization in synaptosomes is sensitive to the endocytosis inhibitor phenylarsine oxide. Moreover, treatment of synaptosomes with monensin, which prevents internalized receptors from recycling to the plasma membrane, reduces [³H]–NT binding and internalization, suggesting that presynaptic NT1 receptors, in contrast to somatodendritic ones, are recycled back to the plasma membrane. Taken together, these results suggest that NT internalizes in nerve terminals via an endocytic pathway that is related to, but is mechanistically distinct from that responsible for NT internalization in nerve cell bodies.     

Cholecystokinin-8 increases K-evoked [H]γ-aminobutyric acid release in slices from various brain areas

[³H]γ-Aminobutyric acid (GABA) release was studied in rat brain slices in the absebce or presence of cholecystokinin-8 (CCK-8). [³H]GABA release under the conditions used was Ca²⁺-dependent and insensitive tot he presence of the glial uptake blocker β-alanine. While the basal release of [³H]GABA was not affected by CCK-8, the K⁺-stimulated release of [³H]GABA wasm significantly enhanced by 300 nM of CCK-8 in the caudate putamen, the substantia nigra, the hippocampal formation and the parietofrontal cortex. In the cerebral cortex the CCK-8 enhancement of [³H]GABA release was concentration-dependent and abolished by the CCK_B receptor antagonists PD135,158 (1.0 nM) and L-365,260 (100 nM). A significant counteraction of the CCK-8 action was also found with the CCK_A receptor antagonist L-364,718 (100 nM) but only in concentrations at which both CCK_A and CCK_B receptors are blocked. No CCK-8 effects on [³H]GABA release were observed when tetrodotoxin was superfused 5 min before the K⁺-induced [³H]GABA release. It is suggested that the enhancing actions of CCK-8 on K⁺-stimulated [³H]GABA release is mainly related to an activation of CCK_B receptors.     

The action of trelibet, a new antidepressive agent on [H]noradrenaline release from rabbit pulmonary artery

Trelibet, a new antidepressant, used at 10⁻⁷–10⁻⁴ M failed to affect the [³H]noradrenaline ([³H]NA) release evoked from the isolated main pulmonary artery of the rabbit low frequency (2 Hz) nerve stimulation whether the neuronal uptake inhibitor cocaine (3 × 10⁻⁵ M) was present or not. Its metabolite (EGYT-2760) however, potentiated the nerve-evoked release of [³H]NA. In the absence of cocaine both the resting and the stimulation-evoked release of ³H increased in response to EGYT-2760. These effect were accompanied by muscle contraction. The EGYT-2760-potentiated transmitter release was inhibited either by exogenously applied 1-noradrenaline (10⁻⁶ M) or clonidine (10⁻⁶ M), preferential agonists of presynaptic ₂-adrenoceptors. The 1-noradrenaline-induced inhibition of transmitter release potentiated by EGYT-2760 was antagonized by 3 × 10⁻⁷ M yohimbine, a preferential ₂-adrenoceptor inhibitor. In the absence of cocaine, Ca²⁺ removal from the external medium failed to affect the ³H outflow-increasing effect of EGYT-2760 but abolished the nerve-evoked release-potentiating action of this compound. It is concluded that the metabolite of trelibet exerts a ‘yohimbine-like’ action, as well as a ‘tyramine-like’ effect in peripheral sympathetic nerve fibres.     

Fluorine-19 or phosphorus-31 NMR spectroscopy: a suitable analytical technique for quantitative in vitro metabolic studies of fluorinated or phosphorylated drugs

Fluorine-19 or phosphorus-31 NMR (¹⁹F NMR or ³¹P NMR) spectroscopy provides a highly specific tool for identification of fluorine- or phosphorus-containing drugs and their metabolites in biological media as well as a suitable analytical technique for their absolute quantification. This article focuses on the application of in vitro ¹⁹F or ³¹P NMR to the quantitative metabolic studies of some fluoropyrimidine or oxazaphosphorine drugs in clinical use. The first part presents an overview of the advantages (non-destructive and non-selective direct quantitative study of the biological matrices) and limitations (expensive cost of the spectrometers, limited mass or concentration sensitivity) of NMR spectroscopy. The second part deals with the criteria to be considered for successful quantification by NMR (uniform excitation over the entire spectral width of the spectrum, resonance signals properly characterised by taking into account T₁ values and avoiding NOE enhancements, optimisation of the data processing, choice of a suitable standard reference). The third and fourth parts report some examples of quantification of 5-fluorouracil, its prodrug capecitabine, 5-fluorocytosine and their metabolites in bulk solutions (biofluids, tissue extracts, perfusates and culture media) and heterogeneous media (excised tissues and packed intact cells) as well as cyclophosphamide and ifosfamide in biofluids. These two parts emphasise the high potential of in vitro ¹⁹F or ³¹P NMR for absolute quantification, in a single run, of all the fluorine- or phosphorus-containing species in the matrices analysed. The limit of quantification in bulk solutions is 1–3 μM for ¹⁹F NMR and approximately 10 μM for ³¹P NMR. In heterogeneous media analysed with ¹⁹F NMR, it is 2–5 nmol in excised tissues and cell pellets.     

Optical purity determination and (1)H NMR spectral simplification with lanthanide shift reagents - V. Mephenytoin, 5-ethyl-3-methyl-5-phenyl-2,4-imidazolidinedione

The 60-MHz ¹H NMR spectra of racemic mephenytoin, I, have been studied with the achiral shift reagent, tris(6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5-octane-dionato) europium (III), II, and the chiral reagent, tris[3-(trifluoromethylhydroxy-methylene)-d-camphorato] europium (III), III. Moderate values of the enantiomeric shift differences, Δ, were clearly observed for the NCH₃, NH, aryl and CCH₃ resonances in CDCl₃ solution at 28°C with added III. The NCH₃ and NH absorptions could be useful for direct assays of optical purity. Thus, for a 0.34 molal solution of I in CDCl₃ with a molar ratio III:I of 0.138, the value of Δ for the NCH₃ resonance was 3.1 Hz (0.052 ppm) with 32% valley resolution.     

Directly-coupled HPLC-NMR spectroscopic studies of metabolism and futile deacetylation of phenacetin in the rat

The metabolism and futile deacetylation of phenacetin has been investigated in the rat via ¹H NMR spectroscopic analysis of urine. Animals were dosed with either phenacetin or phenacetin–C²H₃ and urine samples were collected for −24–0 (pre-dosing), 0–8, 8–24, and 24–48 h post-dosing. Drug metabolites of the two compounds were concentrated from the urine using solid-phase extraction prior to the use of directly-coupled HPLC-¹H NMR spectroscopy for separation and identification. Following dosing of phenacetin, the metabolites identified were paracetamol glucuronide, paracetamol and N-hydroxyparacetamol, whilst paracetamol and N-hydroxyparacetamol sulphate were identified following dosing of phenacetin–C²H₃. Quantitatively the percentage futile deacetylation of phenacetin–C²H₃ metabolites was found to be 32% in both paracetamol and N-hydroxyparacetamol sulphate. This study further indicated the importance of futile deacetylation in simple analgesics and the value of directly-coupled HPLC-NMR spectroscopy for the study of this process.     

Method development and validation for isoflavones in soy germ pharmaceutical capsules using micellar electrokinetic chromatography

The separation of six soy isoflavones (Glycitein, Daidzein, Genistein, Daidzin, Glycitin and Genistin) was approached by a 3² factorial design studying MEKC electrolyte components at the following levels: methanol (MeOH; 0–10%) and sodium dodecylsulfate (SDS; 20–70 mmol L⁻¹); sodium tetraborate buffer (STB) concentration was kept constant at 10 mmol L⁻¹. Nine experiments were performed and the apparent mobility of each isoflavone was computed as a function of the electrolyte composition. A novel response function (RF) was formulated based on the productory of the mobility differences, mobility of the first and last eluting peaks and the electrolyte conductance. The inspection of the response surface indicated an optimum electrolyte composition as 10 mmol L⁻¹ STB (pH 9.3) containing 40 mmol L⁻¹ SDS and 1% MeOH promoting baseline separation of all isoflavones in less than 7.5 min.

The proposed method was applied to the determination of total isoflavones in soy germ capsules from four different pharmaceutical laboratories. A 2 h extraction procedure with 80% (v/v) MeOH under vortexing at room temperature was employed. Peak assignment of unknown isoflavones in certain samples was assisted by hydrolysis procedures, migration behavior and UV spectra comparison. Three malonyl isoflavone derivatives were tentatively assigned. A few figures of merit for the proposed method include: repeatability (n = 6) better than 0.30% CV (migration time) and 1.7% CV (peak area); intermediate precision (n = 18) better than 6.2% CV (concentration); recoveries at two concentration levels, 20 and 50 μg mL⁻¹, varied from 99.1 to 103.6%. Furthermore, the proposed method exhibited linearity in the concentration range of 1.6–50 μg mL⁻¹ (r² > 0.9999) with LOQ varying from 0.67 to 1.2 μg mL⁻¹. The capsules purity varied from 93.3 to 97.6%.     

Binding of [H]brofaromine to monoamine oxidase A in vivo: displacement by clorgyline and moclobemide

Short duration of action, displaceability by endogenously released monoamines, and absence of cumulation of effect of the selective inhibitor of monoamine oxidase type A (MAO-A), brofaromine (CGP 11305 A), indicate reversibility of its interaction with the enzyme in vivo. However, its in vitro interaction with the enzyme showed features commonly associated with irreversible inhibition. To clarify this issue, the in vivo binding of [³H]brofaromine to MAO-A in areas of the rat brain, and in rat heart and liver was investigated. Specific binding, defined by pretreatment with the irreversible inhibitor, clorgyline, was between 15 and 75% of total binding depending on the tissue and the time elapsed after injection of radioactivity. In brain and heart tissue, unlabelled brofaromine, another reversible inhibitor of MAO-A, moclobemide and clorgyline were able to displace [³H]brofaromine when administered after the labelled compound with ED₅₀s of 1–3 mg/kg p.o., 3mg/kg p.o. and 0.3–1 mg/kg s.c., respectively. In the liver, brofaromine and moclobemide and the inhibitor of drug-metabolizing enzymes, proadifen (SK & F 525 A), were able to significantly inhibit [³H]brofaromine binding. Clorgyline was only marginally effective, suggesting that, in this organ, [³H]brofaromine binds predominantly to such enzymes. In conclusion, the binding of [³H]brofaromine to MAO-A in rat brain and heart in vivo was found to be displaceable by other MAO inhibitors and is therefore reversible. In the liver, the compound bound predominantly to other sites, probably microsomal drug-metabolizing enzymes.     

Two new lanostane triterpenoids from Poria cocos

Two new lanostane triterpenoids, 29-hydroxypolyporenic acid C (4) and 25-hydroxypachymic acid (5), together with three known compounds, ergosta-7,22-dien-3β-ol (1), polyporenic acid C (2) and pachymic acid (3), were isolated from the 95% ethanolic extract of the sclerotium of Poria cocos (Schw.) Wolf. Their structures were determined by extensive spectroscopic analyses, including IR, UV, ESITOF-MS, HRESI-MS, 1D and 2D NMR data (¹H NMR, ¹³C NMR, ¹H-¹H COSY, NOESY, HSQC and HMBC).     

Development, validation and utility of an in vitro technique for assessment of potential clinical drug–drug interactions involving P-glycoprotein

Regulatory interest is increasing for drug transporters generally and P-glycoprotein (Pgp) in particular, primarily in the area of drug–drug interactions. To aid in both identifying and discharging the potential liabilities associated with drug–transporter interactions, the pharmaceutical industry has a growing requirement for routine and robust non-clinical assays. An assay was designed, optimised and validated to determine the in vitro inhibitory potency of new chemical entities (NCEs) towards human Pgp-mediated transport. [³H]-Digoxin was established as a suitable probe substrate by investigating its characteristics in the in vitro system (MDCKII-MDR1 cells grown in 24-multiwell inserts). The inhibitory potencies (apparent IC₅₀) of known Pgp inhibitors astemizole, GF120918, ketoconazole, itraconazole, quinidine, verapamil and quinine were determined over at least a 1000-fold concentration range. Validation was carried out using manual and automatic techniques. [³H]-Digoxin was found to be stable and have good mass balance in the system. In contrast to [A → B] transport, [³H]-digoxin [B → A] transport rates were readily measured with good reproducibility. There was no evidence of saturation of transport up to 10 μM digoxin and 30 nM digoxin was selected for routine assay use, reflecting clinical therapeutic concentrations. IC₅₀ values ranged over approximately 100-fold with excellent reproducibility. Results from manual and automated versions were in close agreement. This method is suitable for routine use to assess the in vitro inhibitory potency of NCEs on Pgp-mediated digoxin transport. Comparison of IC₅₀ values against clinical interaction profiles for the probe inhibitors indicated the in vitro assay is predictive of clinical digoxin–drug interactions mediated via Pgp.     

SB-616234-A (1-[6-(cis-3,5-dimethylpiperazin-1-yl)-2,3-dihydro-5-methoxyindol-1-yl]-1-[2'methyl-4'-(5-methyl-1,2,3-oxadiazol-3-yl)biphenyl-4-yl]methanone hydrochloride): a novel, potent and selective 5-HT1B receptor antagonist

SB-616234-A possesses high affinity for human 5-HT_1B receptors stably expressed in Chinese hamster ovary (CHO) cells (pK_i 8.3 ± 0.2), and is over 100-fold selective for a range of molecular targets except h5-HT_1D receptors (pK_i 6.6 ± 0.1). Similarly, affinity (pK_i) for rat and guinea pig striatal 5-HT_1B receptors is 9.2 ± 0.1. In [³⁵S]-GTPγS binding studies in the human recombinant cell line, SB-616234-A acted as a high affinity antagonist with a pA₂ value of 8.6 ± 0.2 whilst providing no evidence of agonist activity in this system. In [³⁵S]-GTPγS binding studies in rat striatal membranes, SB-616234-A acted as a high affinity antagonist with an apparent pK_B of 8.4 ± 0.5, again whilst providing no evidence of agonist activity in this system. SB-616234-A (1 μM) potentiated electrically stimulated [³H]-5-HT release from guinea pig and rat cortical slices (S₂/S₁ ratios of 1.8 and 1.6, respectively). SB-616234-A (0.3–30 mg kg⁻¹ p.o.) caused a dose-dependent inhibition of ex vivo [³H]-GR125743 binding to rat striatal 5-HT_1B receptors with an ED₅₀ of 2.83 ± 0.39 mg kg⁻¹ p.o. Taken together these data suggest that SB-616234-A is a potent and selective 5-HT_1B autoreceptor antagonist that occupies central 5-HT_1B receptors in vivo following oral administration.