Kinetics and crystal structure of catechol-o-methyltransferase complex with co-substrate and a novel inhibitor with potential therapeutic application

Catechol-O-methyltransferase (COMT; E.C. 2.1.1.6) is a ubiquitous enzyme in nature that plays an important role in the metabolism of catechol neurotransmitters and xenobiotics. In particular, inactivation of drugs such as L-3,4-dihydroxyphenylalanine (L-DOPA) via O-methylation is of relevant pharmacological importance, because L-DOPA is currently the most effective drug used in the treatment of Parkinson's disease. This justified the interest in developing COMT inhibitors as potential adjuncts to L-DOPA therapy. The kinetics of inhibition by BIA 3-335 (1-[3,4-dihydroxy-5-nitrophenyl]-3-(N-3'-trifluormethylphenyl)-piperazine-1-propanone dihydrochloride) were characterized using recombinant rat soluble COMT. BIA 3-335 was found to act as a potent, reversible, tight-binding inhibitor of COMT with a K(i) of 6.0 +/- 1.6 nM and displaying a competitive inhibition toward the substrate binding site and uncompetitive inhibition toward the S-adenosyl-L-methionine (SAM) binding site. The 2.0-A resolution crystal structure of COMT in complex with its cosubstrate SAM and a novel inhibitor BIA 3-335 shows the atomic interactions between the important residues at the active site and the inhibitor. This is the first report of a three-dimensional structure determination of COMT complexed with a potent, reversible, and tight-binding inhibitor that is expected to have therapeutic applications.     

BIA 3-202, a novel catechol-O-methyltransferase inhibitor,reduces the peripheral O-methylation of L-DOPA and enhances its availability to the brain

The present study aims at determining the effects of the catechol-O-methyltransferase (COMT) inhibitor BIA 3-202 [1-(3,4-dihydroxy-5-nitrophenyl)-2-phenyl-ethanone] upon levels of L-3,4-dihydroxyphenylalanine (L-DOPA) and metabolites in peripheral circulation (jugular vein), whole brain, and striatal microdialysates in rats orally treated with L-DOPA plus benserazide. A low dose (3 mg/kg) of BIA 3-202 was relatively selective to inhibit liver COMT, being devoid of major significant inhibitory effects upon brain COMT. By contrast, a high dose (30 mg/kg) of BIA 3-202 markedly inhibited liver and brain COMT. BIA 3-202 (3 and 30 mg/kg) reduced the L-DOPA-induced rise of 3-O-methyl-L-DOPA in the peripheral circulation (jugular vein), brain tissue, and striatal dialysate, but failed to increase the levels of dopamine in striatal dialysates despite the increase in dopamine brain levels. However, the changes in brain levels of L-DOPA, 3-O-methyl-L-DOPA, and dopamine and in striatal dialysate levels of L-DOPA and 3-O-methyl-L-DOPA, obtained with 3 mg/kg BIA 3-202, were not different from those obtained with 30 mg/kg BIA 3-202. In conclusion, inhibition of peripheral COMT by BIA 3-202 may suffice to improve the availability of L-DOPA to the brain.     

Catechol-O-methyltransferase inhibition in erythrocytes and liver by BIA 3-202 (1-[3,4-dibydroxy-5-nitrophenyl]-2-phenyl-ethanone)

The present study evaluated the relationship between the degree of catechol-O-methyltransferase (COMT) inhibition in erythrocytes and liver by BIA 3-202 (1-[3,4-dihydroxy-5-nitrophenyl]-2-phenyl-ethanone) and determined its effects upon the O-methylation of L-DOPA in rats orally treated with L-DOPA plus benserazide. The soluble form of COMT (S-COMT) in erythrocytes was endowed with the same affinity as liver S-COMT for the substrate adrenaline. BIA 3-202 inhibited erythrocytes and liver S-COMT with ED50's of 1.9 (0.7, 3.1) and 1.9 (0.5, 3.2) (95% confidence limits) mg kg(-1), respectively. BIA 3-202 reduced the L-DOPA-induced rise of 3-O-methyl-L-DOPA in the peripheral circulation, striatal dialysate levels and striatum, and increased dopamine striatal levels. In BIA 3-202-treated rats the increase in L-DOPA in peripheral blood and striatal dialysates was significantly greater than in vehicle-treated rats. It is concluded that S-COMT activity in erythrocytes may provide important information on the pharmacodynamic profile of COMT inhibitors. The novel COMT inhibitor BIA 3-202 is a potent COMT inhibitor that enhances the availability of L-DOPA to the brain by reducing its O-methylation, which may prove beneficial in patients with Parkinson's disease treated with L-DOPA.     

Catechol‐O‐methyltransferase Inhibition in Erythrocytes and Liver by BIA 3‐202 (1‐[3,4‐dibydroxy‐5‐nitrophenyl]‐2‐phenyl‐ethanone)

Abstract: The present study evaluated the relationship between the degree of catechol‐O‐methyltransferase (COMT) inhibition in erythrocytes and liver by BIA 3‐202 (1‐[3,4‐dihydroxy‐5‐nitrophenyl]‐2‐phenyl‐ethanone) and determined its effects upon the O‐methylation of L‐DOPA in rats orally treated with L‐DOPA plus benserazide. The soluble form of COMT (S‐COMT) in erythrocytes was endowed with the same affinity as liver S‐COMT for the substrate adrenaline. BIA 3‐202 inhibited erythrocytes and liver S‐COMT with ED50's of 1.9 (0.7, 3.1) and 1.9 (0.5, 3.2) (95% confidence limits) mg kg^?1, respectively. BIA 3‐202 reduced the L‐DOPA‐induced rise of 3‐O‐methyl‐L‐DOPA in the peripheral circulation, striatal dialysate levels and striatum, and increased dopamine striatal levels. In BIA 3‐202‐treated rats the increase in L‐DOPA in peripheral blood and striatal dialysates was significantly greater than in vehicle‐treated rats. It is concluded that S‐COMT activity in erythrocytes may provide important information on the pharmacodynamic profile of COMT inhibitors. The novel COMT inhibitor BIA 3‐202 is a potent COMT inhibitor that enhances the availability of L‐DOPA to the brain by reducing its O‐methylation, which may prove beneficial in patients with Parkinson's disease treated with L‐DOPA.     

Pharmacokinetic and pharmacodynamic profiles of BIA 3-202, a novel catechol-O-methyltransferase (COMT) inhibitor, during multiple-dose administration to healthy subjects

The tolerability, pharmacodynamics, and pharmacokinetics of BIA 3-202 (50 mg, 100 mg, and 200 mg twice daily and 200 mg thrice daily), a novel catechol-O-methyltransferase (COMT) inhibitor, were investigated in healthy volunteers. BIA 3-202 was administered to four sequential groups of 8 healthy male subjects under a double-blind, randomized, placebo-controlled design. Within each group, 2 subjects were randomized to treatment with placebo. Treatment duration was 9 days: single dose on the first and last days and twice or thrice daily on days 3 to 8. BIA 3-202 was well tolerated at all dose regimens tested. Median maximum plasma BIA 3-202 concentrations were attained at 0.5 to 2.5 hours postdose. Thereafter, concentrations declined with a t1/2 of approximately 2 to 4 hours. The increase in the extent of systemic exposure, as measured by AUC0-tau, was approximately proportional to the administered dose. Steady state of plasma BIA 3-202 concentrations occurred by day 4 in all dose groups. Less than 1% of the total dose administered was excreted in urine up to 48 hours postdose. BIA 3-202 markedly reduced soluble COMT (S-COMT) activity in erythrocytes, with maximum inhibition occurring at 1 to 2 hours postdose; enzyme activity returned to baseline levels by approximately 8 hours. Inhibition of S-COMT activity appeared to increase with increasing doses of BIA 3-202 on both day 1 and day 9. In conclusion, BIA 3-202 was well tolerated in all the oral multiple-dose regimens tested. BIA 3-202 was shown to inhibit S-COMT activity in erythrocytes, and its pharmacokinetics appeared to be linear (i.e., dose independent and time invariant).     

Inhibition of rat liver and duodenum soluble catechol-O-methyltransferase by a tight-binding inhibitor OR-462

The inhibition kinetics of rat liver and duodenum soluble catechol-O-methyltransferase (COMT) with a disubstituted catechol OR-462 was studied. After preincubation of the enzyme and inhibitor in the presence of magnesium and S-adenyosylmethionine, an inhibition about thirty times greater than that without preincubation was observed. Reversible tight-binding inhibition was demonstrated with Ki values of 0.7 nM and 1.0 nM for liver and duodenum enzyme, respectively. Km values of 53.4 microM and 56.9 microM for substrate 3,4-dihydroxybenzoic acid and 23.0 microM and 17.5 microM for S-adenosylmethionine were calculated for liver and duodenum enzyme, respectively. A catalytic number of 24/min for liver soluble COMT was calculated.     

BIA 3-202, a novel catechol-O-methyltransferase inhibitor, enhances the availability of L-DOPA to the brain and reduces its O-methylation.

1-[3,4-Dihydroxy-5-nitrophenyl]-2-phenyl-ethanone (BIA 3-202) is a new long-acting catechol-O-methyltransferase (COMT) inhibitor with limited access to the brain. The present study evaluated the interference of BIA 3-202 upon levels of L-3,4-dihydroxyphenylalanine (L-DOPA) and metabolites in plasma (3-O-methyl-L-DOPA) and brain [3-O-methyl-L-DOPA, dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)] in rats orally treated with L-DOPA (20 mg/kg) plus benserazide (30 mg/kg). At different time points (1, 3 and 6 h) after the administration of BIA 3-202 (0, 3, 10 and 30 mg/kg) or L-DOPA plus benserazide, rats were sacrificed and the right striatum was quickly dissected out and stored for the assay of L-DOPA, 3-O-methyl-L-DOPA, dopamine and amine metabolites. Levels of L-DOPA, 3-O-methyl-L-DOPA, dopamine, DOPAC and HVA in the striatum in L-DOPA plus benserazide-treated rats were higher than in vehicle-treated rats. However, this increase in striatal L-DOPA, dopamine, DOPAC and HVA was, in a dose- and time-dependent manner, even higher (P<0.05) in rats given BIA 3-202 (3, 10 and 30 mg/kg). This effect was accompanied by a marked decrease in 3-O-methyl-L-DOPA levels in the striatum of L-DOPA plus benserazide-treated rats. Increases in levels of L-DOPA and decreases in 3-O-methyl-L-DOPA levels in plasma also accompanied the administration of BIA 3-202. BIA 3-202 did not significantly affect levels of DOPAC and HVA in the striatum in vehicle-treated rats. It is concluded that administration of BIA 3-202 enhances the availability of L-DOPA to the brain by reducing its O-methylation in the periphery, which may prove beneficial in parkinsonian patients treated with L-DOPA plus an aromatic amino acid decarboxylase inhibitor.     

Pharmacokinetic–pharmacodynamic interaction between BIA 3-202, a novel COMT inhibitor, and levodopa/benserazide

BIA 3-202 is a novel catechol-O-methyltransferase (COMT) inhibitor being developed for use as a levodopa-sparing agent in Parkinsons disease. This study investigated the effect of four single oral doses of BIA 3-202 (50 mg, 100 mg, 200 mg, and 400 mg) compared with placebo on plasma concentrations of levodopa and its metabolite 3-O-methyl-levodopa (3-OMD) and on inhibition of erythrocyte COMT in healthy subjects receiving 100 mg of levodopa and 25 mg of benserazide (Madopar 125). This was a single-centre, double-blind, placebo-controlled, randomised, crossover study with five single-dose treatment periods. The washout period between doses was 2 weeks. On each treatment period, a different dose of BIA 3-202 or placebo was administered concomitantly with Madopar 125. Tolerability was assessed by recording adverse events, vital signs, continuous electrocardiogram and clinical laboratory parameters. In the study, 18 subjects (12 male and 6 female) participated. The drug combination was well tolerated. All doses of BIA 3-202 significantly increased the area under the concentration–time curve (AUC) versus placebo, ranging from 39% (95% confidence intervals, 1.06–1.69) with 50 mg to 80% (95% confidence intervals, 1.42–2.22) with 400 mg. No significant change in mean maximum plasma concentrations (C_max) of levodopa was found. Mean C_max and AUC of 3-OMD significantly decreased for all doses tested. BIA 3-202 caused a rapid and reversible inhibition of S-COMT activity, ranging from 57% (50 mg) to 84% (400 mg). In conclusion, the novel COMT inhibitor BIA 3-202 was well tolerated and significantly increased the bioavailability of levodopa and reduced the formation of 3-OMD when administered with standard release levodopa/benserazide.     

Effects of Fe on ion channels: Na channel, delayed rectified and transient outward K channels

The effects of Fe²⁺ on the properties of three types of ion channels were studied in acutely dissociated rat hippocampal pyramidal neurons from area CA1 at postnatal ages of 7–14 days using the whole cell patch clamp technique. The results indicated that: (1) in the existence of Fe²⁺, the activation voltage threshold of transient outward K⁺ currents (I_A) was decreased. The normalized current-voltage curves of activation were well fitted with a single Boltzmann function, and the V_1/2 was 2.44±1.14 mV (n=15) in control, whereas 1.79±1.53 (n=15), −2.96±0.92 (n=14), −5.11±1.31 (n=13), −9.05±1.64 mV (n=12) in 1, 10, 100 and 1000 μ Fe²⁺, respectively. Differences between two groups were significant (P<0.05, n=12–15), except for that between the control and 1 μ (P>0.05, n=15). (2) Fe²⁺ caused a left shift of the current–voltage curves of steady-state inactivation of I_A in a concentration-dependent manner. The curves were well fitted with a single Boltzmann function with similar slope (P>0.05, n=10–13). The V_1/2 were −70.71±1.23 (n=13), −71.14±1.37 (n=13), −78.21±1.17 (n=11), −84.61±1.34 (n=12), and −89.68±2.59 mV (n=10) in control, 1, 10, 100 and 1000 μ Fe²⁺, respectively. Fe²⁺ also shifted the current–voltage curves of Na⁺ channel steady-state inactivation to more negative depolarization potentials in parallel, with V_1/2, −67.37±1.33 mV (n=12) in control, and −67.52±1.28 mV (n=12), −68.24±1.61 mV (n=10), −71.58±1.45 mV (n=10), −76.65±1.76 mV (n=9) in 1, 10, 100 and 1000 μ Fe²⁺ solutions, respectively. (3) In Fe²⁺ solutions, the recovery from inactivation of I_A was slowed. (4) With application of different concentrations of Fe²⁺, the voltage threshold of activation of delayed rectified outward K⁺ currents (I_K) was decreased, while Fe²⁺ showed a little inhibition at more positive depolarization. Briefly, the results demonstrated that Fe²⁺ is a dose- and voltage-dependent, reversible modulator of I_A, I_K and Na⁺ channels. The results will be helpful to explain the mechanism of Fe²⁺ physiological function and Fe²⁺ intoxication in the central nervous system.     

Biochemical and pharmacological properties of a peripherally acting catechol-O-methyltransferase inhibitor entacapone

Summary Entacapone, OR-611, was found to be a potent peripherally acting inhibitor of catechol-O-methyl-transferase (COMT). IC₅₀ values of 10 nmol/1 and 160 nmol/1 were obtained for rat duodenum and liver-soluble COMT, respectively. There were no effects on other catecholamine metabolizing enzymes. Entacapone showed reversible, tight-binding type of inhibition of soluble rat liver COMT with a K; value of 14 nmol/1 and it also caused 50% inhibition of rat duodenal, erythrocyte, liver and striatal COMT activity 1 h after oral dosing with 1.1, 5.4, 6.7 and 24.2 mg/kg, respectively. However, penetration of entacapone into the brain was poor, since the formation of homovanillic acid (HVA), the O-methyl metabolite of dopamine in the striatum, was not reduced, even after the highest dose of 30 mg/kg.In rat blood serum, the concentration of 3-0-methyldopa (3OMD), the O-methylated product of l-dopa, was reduced in a dose-dependent manner, and the concentration of l-dopa was increased after the administration of entacapone (3 - 30 mg/kg p. o.) together with l-dopa + carbidopa. These changes were reflected, in the striatum, by a significant rise in the dopamine concentration and a reduction in the 30MD concentration.Consequently, when entacapone was added to the treatment with l-dopa + carbidopa, the dose of l-dopa could be lowered from 50 mg/kg to 15 mg/kg in order to produce the same striatal dopamine concentrations as with 50 + 50 mg/kg of l-dopa + carbidopa alone.Correspondence to E. Nissinen at the above address     

Investigation of penbutolol—iron(III) complex and its spectrophotometric determination in tablets

It has been established that penbutolol reacts with iron(III) chloride in the presence of ammonium thiocyanate to form a pink complex (2:1) that is soluble in chloroform with a maximum absorbance at 478 nm. By application of the methods of Sommer and Job involving non-equimolar solutions, the conditional stability constant (log k′) of the complex at the optimum pH of 1.5±0.02 and an ionic strength of (μ) 0.14 M, was found to be 5.769. The molar absorptivity at 478 nm was 136 l mol⁻¹ cm⁻¹ at pH 1.5±0.02. The validity of Beer's law has been tested in the concentration range 3–18 × 10⁻⁴ M; the relative standard deviation (n = 8) was 1.52–3.21%. The proposed method was found to be suitable for the accurate, simple and rapid analysis of penbutolol in the bulk drug and in tablets.     

Comparative study of the effects of salinomycin and monensin on electrophysiologic and contractile properties of canine myocardium

The effects of the monocarboxylic ionophore, salinomycin (K⁺-selective), on isometric twitcches, high K⁺-induced contracture and transmembrane action potentials were compared with those of the monocarboxylic ionophore, monensin (Na⁺-selective), in isolated canine right ventricular muscle. In a concentration (5 × 10⁻⁶) which did not produce changes in resting force, salinomycin increased peak active force ( P₀, + 170 ± 36%, mean % change from control ±S.D., P< 0.01). and relaxation and maximal rates of force development (dP/dt_max, + 123 ± 33%, P < 0.01) and relaxation (−dP/dt_max, + 180 ± 40%, P < 0.01) of the isometric twitch. A similar response pattern was found for 5 × 10⁻⁶ M monensin (P₀, + 90 ± 24%, P < 0.01; dP/dt_max, + 137 ± 19%, P < 0.01; −dP/dt_max, + 145 ± 20%, P < 0.01). In contrast to their effects on isometric twitches, salinomycin reduced peak K⁺ contracture force (P_c, −35 ± 14%, P < 0.01) whereas monensin increased it (P_c, +30 ± 12%, P < 0.02). Ventricular muscle action potential duration was shortened similarly by the ionophores. β-Adrenergic receptor blockade with nadolol diminished salinomycin's effects on the isometric twitch and K⁺ contracture, but not its effect to shorten the action potential. Monensin's actions were unaffected by nadolol. These results suggest that salinomycin's effects arise from both a direct modulation of K⁺ movement and the release of endogenous catecholamine. In contrast, monensin may act to alter intracellular Na⁺ which in turn leads to Na⁺---Ca²⁺ exchange and Ca²⁺ -mediated modulation of K⁺ movement.     

Correlation of endothelium-dependent and -independent vasodilatation with liver function tests during prolonged perfusion of the rat liver

Twelve male Wistar rats were anaesthetized with pentobarbitone (3 mg 100g⁻¹ i.p.), the livers were excised and perfused in vitro through the hepatic artery and portal vein at constant flow rates of 0.32 ± 0.01 (mean ± S.E.) and 0.98 ± 0.03 ml min⁻¹ g liver⁻¹, respectively. The tone of the preparation was raised by methoxamine (7.5×10⁻⁶ M). Responses to mid-range doses of acetylcholine (−11 log mol) and sodium nitroprusside (−9 log mol) produced submaximal degrees of vasodilatation (−log mol ED₅₀ = 12.18 ± 0.08) and (−log mol ED₅₀ = 9.95 ± 0.23), respectively, which did not subside until 5.5 h of perfusion. These did not coincide with the increase in activities of lactic acid dehydrogenase (LDH) and aspartate serine transaminase (AST) activity at 2.5 h, which were indicative of hepatocellular mitochondrial and cytoplasmic damage, respectively. Vascular responses suggested that there was little deterioration in endothelial or smooth muscle function in the hepatic artery up to 5 h perfusion. This model can be reliably used to investigate endothelium-dependent and -independent vasodilators in vascular pharmacological studies of the rat liver although some minimal increases may occur in AST and LDH activity before hemodynamic changes appear at 5.5 h.     

Molecular modeling and metabolic studies of the interaction of catechol-O-methyltransferase and a new nitrocatechol inhibitor.

Catechol-O-methyltransferase (COMT, EC 2.1.1.6) plays a central role in the metabolic inactivation of neurotransmitters and neuroactive xenobiotics possessing a catechol motif. 1-(3,4-Dihydroxy-5-nitrophenyl)-2-phenyl-ethanone (BIA 3-202) is a novel nitrocatechol-type inhibitor of COMT, the potential clinical benefit of which is currently being evaluated in the treatment of Parkinson's disease. In the present work we characterize the molecular interactions of BIA 3-202 within the active site of COMT and discuss their implication on the regioselectivity of metabolic O-methylation. Unrestrained flexible-docking simulations suggest that the solution structure of this complex is better described as an ensemble of alternative binding modes, in contrast to the well defined bound configuration revealed by the X-ray structures of related nitrocatechol inhibitors, co-crystallized with COMT. The docking results wherein presented are well supported by experimental evidence, where the pattern of in vitro enzymatic O-methylation and O-demethylation reactions are analyzed. We propose a plausible explanation for the paradoxical in vivo regioselectivity of O-methylation of BIA 3-202, as well as of its related COMT inhibitor tolcapone. Both compounds undergo in vivo O-methylation by COMT at either meta or para catechol hydroxyl groups. However, results herein presented suggest that, in a subsequent step, the p-O-methyl derivatives are selectively demethylated by a microsomal enzyme system. The overall balance is the accumulation of the m-O-methylated metabolites over the para-regioisomers. The implications for the general recognition of nitrocatechol-type inhibitors by COMT and the regioselectivity of their metabolic O-methylation are discussed.     

Pharmacokinetics of thrombin-like enzyme from venom of Agkistrodon halys ussuriensis Emelianov determined by ELISA in the rat.

A thrombin-like enzyme (TLE) was separated and purified from the venom of a northeast Chinese snake Agkistrodon halys ussuriensis Emelianov. Experiments were performed in rats to determine the pharmacokinetic parameters following an intravenous (i.v.) or a subcutaneous (s.c.) injection of the thrombin-like enzyme. The plasma levels of TLE were estimated by enzyme-linked immunosorbent assay. The method exhibited high reproducibility and accuracy in correlating optical densities with TLE concentrations (0.2–30 ng ml⁻¹, r=0.99). The plasma concentration-time course after i.v. administration of 50 μg kg⁻¹ TLE was well fitted by a two-compartment open model. The half-life of the -phase was 18.0±3.2 min, and that of the β-phase 3.9±0.7 h. The apparent volume of distribution was 1.8±0.5 l kg⁻¹, and clearance was 5.4±0.5 ml min⁻¹ kg⁻¹. When the TLE was injected s.c. at a dose of 0.75 mg kg⁻¹, the changes in plasma concentration were best described by a two-compartment model with a first-order absorption. The maximal plasma level of 51±2.7 ng ml⁻¹ was reached at 5.2±0.5 h. The absorption rate constant was 0.3±0.03 h⁻¹. The area under the plasma concentration-time curve (AUC) was 2.8±0.8 μg h⁻¹ ml⁻¹.     

A new hydrogel for the extended and complete prednisolone release in the GI tract

The issue of incomplete release of poorly soluble drugs from sustained-release oral formulations is addressed using prednisolone (PDS) as the model drug and a novel highly swelling hydrogel as the rate-controlling material. The hydrogel was formed by heating N-carboxymethylchitosan (CMC) to 80 °C for 24 h. Swelling, alkalimetry, FTIR, DSC, and solid-state NMR studies showed that the treatment produced physical crosslinking, i.e., polymer chain entanglement. A controlled-release system was prepared by coating an inert compacted support of ethylcellulose (50 mg; diameter, 6 mm) with a CMC layer containing dispersed PDS powder (10–50 μm). The system was heated to crosslink the CMC coating, then drug release to simulated GI fluids was studied in vitro. The drug release pattern and term were modulated via the layer mass (LM) (10 or 14 mg cm⁻²) and/or the drug–polymer wt ratio (D/P) (1:5 or 2:5). The rate parameter, K, and the time exponent, n, of the Peppas equation were: K = 26.6 ± 0.3 h⁻ⁿ, n = 0.78 ± 0.02 (LM, 10 mg cm⁻²; D/P, 1:5); K = 24.7 ± 0.7 h⁻ⁿ, n = 0.56 ± 0.02 (LM, 14 mg cm⁻²; D/P, 1:5); K = 20.7 ± 0.3 h⁻ⁿ, n = 0.76 ± 0.01 (LM, 10 mg cm⁻²; D/P, 2:5). Hydrogel swelling was faster than drug release. This was controlled, in a first stage, by drug dissolution–diffusion in the swollen gel, and subsequently, by diffusion. The drug release rate was unaffected by the GI pH variations, and slightly affected by the environmental hydrodynamics. The system promises an extended and complete release of poorly soluble drugs in the GI tract.     

Disposition kinetics and urinary excretion of levofloxacin on concomitant administration with meloxicam in cross-bred calves

The disposition kinetics and urinary excretion study of levofloxacin was conducted in 5 male cross-bred calves following its single intravenous administration (4 mg kg⁻¹) concurrently with meloxicam (0.5 mg kg⁻¹). Levofloxacin was estimated by microbiological assay. The drug levels above MIC₉₀ in plasma, were detected up to 10 h. Disposition kinetic parameters were calculated by two-compartment open model. Rapid distribution of levofloxacin was evidenced by a small distribution half-life (0.13 ± 0.01 h) and high K₁₂/K₂₁ ratio (2.21 ± 0.15). High ratio of AUC/MIC (90.2 ± 3.41) indicated good antibacterial activity of levofloxacin. The AUC, Vd_area, elimination half-life, MRT and total body clearance were 9.02 ± 0.34 μg ml⁻¹ h, 1.38 ± 0.05 l kg⁻1, 2.16 ± 0.08 h, 2.58 ± 0.11 h and 0.45 ± 0.02 l kg⁻¹ h⁻¹, respectively. About 38.4% of the administered dose of levofloxacin was excreted in urine within 24 h. A suitable intravenous dosage regimen for levofloxacin would be 1.8 mg kg⁻¹ repeated at 8 h intervals when prescribed with meloxicam in calves.     

In vitro studies with ICI 169,369, a chemically novel 5-HT antagonist

ICI 169,369 is a chemically novel 5-HT antagonist that has higher affinity for the 5-HT₂ binding sites in rat cortex than it has for 5-HT₁ sites (K_i 1.79 × 10⁻⁸ and 1.58 × 10⁻⁶ M, respectively). In isolated tissue preparations ICI 169,369 was shown to be a competitive antagonist of 5-HT on the rabbit aorta, pig coronary artery and rat caudal artery. In the latter preparation it had a similar pA₂ value to ketanserin (pA₂ 8.18 ± 0.5 and 8.42 ± 0.06, respectively). Unlike ketanserin, which was inactive, ICI 169,369 was a non-surmountable antagonist at the rat stomach fundus 5-HT ‘D’ receptor, recently reclassified as 5-HT_IC. It was inactive ( > 10⁻⁶ M) at the 5-HT₃ receptors found in the isolated perfused rabbit heart and the myenteric plexus of the guinea-pig ileum. At receptors other than those for 5-HT (₁, ₂, β₁, β₂, H₁, H₂ and muscarinic), ICI 169,369 was inactive at concentrations of either 10⁻⁶ or 10⁻⁵ M. Thus the profile of ICI 169,369 should make it useful in the analysis of the role of 5-HT in physiological and pathological states.     

Thromboxane A(2)-mediated Cl(-) secretion induced by platelet-activating factor in isolated rat colon

Thromboxane A₂ is a novel endogenous secretagogue of Cl⁻ secretion in the distal colon. Here, we examined if the Cl⁻ secretion caused by platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is mediated by thromboxane A₂ production using isolated mucosae of the rat colon. Furosemide (100 μM) and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB; 300 μM) completely inhibited PAF (10 μM)-induced increase in short-circuit current (Isc) across the mucosa, indicating that PAF caused a Cl⁻ secretion in the rat colon. A selective thromboxane A₂ receptor antagonist (sodium(E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)-ethylidene]-6,11-dihydrobenz[b,e]oxepine-2-carboxylate monohydrate; KW-3635), and a selective thromboxane synthase inhibitor (sodium 4-[-hydroxy-5-(1-imidazolyl)-2-methylbenzyl]-3,5-dimethylbenzoate dihydrate; Y-20811) inhibited the PAF-induced Cl⁻ current in a concentration-dependent manner. The IC₅₀ values of KW-3635 and Y-20811 were 2.1 and 0.5 μM, respectively. 30 μM KW-3635 and 1 μM Y-20811 inhibited the PAF response by 92% and 83%, respectively. These inhibitors did not affect the prostaglandin E₂-induced increase in Isc. A 5-lipoxygenase-activating protein inhibitor (3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-t-butylthioindol-2-yl]-2,2-dimethyl-propanoic acid sodium; MK-886) (5 μM) did not affect the PAF-induced Cl⁻ current. The present study suggests that the PAF-induced Cl⁻ secretion in the rat colonic mucosa is mainly mediated by a release of thromboxane A₂.     

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.