COMPARATIVE STUDY ON α1-ADRENOCEPTOR ANTAGONIST BINDING IN HUMAN PROSTATE AND AORTA

1. Specific binding of [³H]-prazosin in prostatic and aortic membranes of humans was saturable and of high affinity (prostate: apparent dissociation constant, K_d= 0.35 ± 0.03 nmol/L; aorta: K_d= 0.26 ± 0.03 nmol/L). The density of [³H]-prazosin binding sites (B_max) for prostate and aorta was 546 ± 31 and 61.6 ± 1.6 fmol/mg protein, respectively. 2. Prazosin, YM617, naftopidil and urapidil competed with [³H]-prazosin for the binding sites in a dose-dependent manner in the prostate and aorta of humans. The binding affinities of these antagonists in both tissues were compared, based on the inhibition constant, K_i. Both prazosin and urapidil showed similar affinity to [³H]-prazosin binding sites in human tissue, whereas YM617 and naftopidil showed approximately a 12 and two times higher affinity, respectively, to α₁-adrenoceptor sites of prostate than aorta. 3. The chloroethylclonidine treatment reduced partially the B_max values for specific [³H]-prazosin binding in the prostate and aorta of humans with little effect on the K_d values. 4. These data suggest that YM617 is a relatively selective antagonist of human prostatic α₁-adrenoceptors.     

Efflux Transport of Tolbutamide Across the Blood-brain Barrier

In an attempt to determine the reason for the low brain distribution of tolbutamide, we have demonstrated the transport of tolbutamide from the brain to the blood via a non-P-glycoprotein efflux transport system which is inhibited by sulphonamides. We evaluated the directional transport of tolbutamide across the blood-brain barrier by means of an in-vivo brain-tissue distribution study and experiments on in-vitro transcellular transport and uptake in cultured mouse-brain capillary endothelial cells (MBEC4). The brain-to-unbound-plasma concentration ratio of [¹⁴C]tolbutamide increased in the presence of high concentrations of unlabelled tolbutamide or sulphonamide at steady-state in-vivo. The brain-to-blood concentration ratios of [¹⁴C]tolbutamide were very low compared with that of [³H]propranolol obtained by in-vivo integration plot analysis. From the in-vitro transcellular transport study using a monolayer of MBEC4 cells, we found that the abluminal-to-luminal flux of [¹⁴C] tolbutamide was higher than the reverse flux. Both luminal-to-abluminal and abluminal-to-luminal transport of tolbutamide were saturable. The maximum transport rate (J_max), the half-saturation concentration (K_t), and the first-order rate constant (k_d) were 65.9 ± 29 pmol min^?1 (mg protein)^?1, 7.54 ± 4.4 μM, and 4.89 ± 0.34 μL min^?1 (mg protein)^?1, respectively, for luminal-to-abluminal transport, and 128 ± 66 pmol min^?1 (mg protein)^?1, 5.59 ± 4.2 μM, and 4.43 ± 0.86 μL min^?1 (mg protein)^?1, respectively, for abluminal-to-luminal transport. At therapeutic plasma concentrations of tolbutamide (1–16.9 μM), the efflux rate would be faster than the influx rate. The estimated net efflux was consistent with the very low in-vivo brain distribution of tolbutamide. The efflux process observed in MBEC4 cells was inhibited by sulphonamides such as sulphaphenazole, sulphamethoxazole and sulpha-dimethoxine whereas the steady-state uptake of [¹⁴C]tolbutamide was not affected by either cyclosporin or verapamil, specific inhibitors of P-glycoprotein. These findings suggest that tolbutamide is partly transported from the brain via the non-P-glycoprotein-efflux transport system, which is inhibited by sulphonamides.     

Renal Excretion and Accumulation Kinetics of 2-Methylbenzoylglycine in the Isolated Perfused Rat Kidney

The effect of protein binding on kidney function has been studied by investigating the renal accumulation and secretion of the hippurate analogue 2-methylbenzoylglycine in the isolated perfused rat kidney in the absence and presence of bovine serum albumin (BSA). Experiments were performed with either 2.5% pluronic or a combination of 2.2% pluronic and 2% BSA as oncotic agents; a wide concentration range (1–190 μg mL^?1) of 2-methylbenzoylglycine was studied. Tubular secretion appeared to be a function of the amount of unbound drug in the perfusate and was best described by a model consisting of a high and low affinity Michaelis-Menten term. Parameters obtained after the analysis of renal excretion data were maximum transport velocity for the high affinity site (T_M,H) = 3.0 ± 2.8 μg min^?1, Michaelis-Menten constant for tubular transport for the high affinity site (K_T,H) = 0.5 ± 0.8 μg mL^?1, maximum transport velocity for the low affinity site (T_M,L) = 250 ± 36 μg min^?1, and Michaelis-Menten constant for tubular transport for the low affinity site (K_T,L) = 62 ± 17 μg mL^?1. The compound accumulated extensively in kidney tissue, ratios up to 175 times the perfusate concentration were reached. Accumulation data were best analysed by a two-site model similar to the model used to describe renal excretion. Calculated parameters were theoretical maximum capacity of the high affinity site (R_M,H) = 26 ± 23 μg g^?1, affinity constant for renal accumulation at the high affinity site (K_A,H) = 0.2 ± 0.4 μg mL^?1, theoretical maximum capacity of the low affinity site (R_M,L)= 1640 ± 1100 μg g^?1 and affinity constant for renal accumulation at the low affinity site (K_A,L) = 60 ± 58 μg mL^?1. The very high accumulation in kidney tissue could be explained by active tubular uptake, mediated by the secretory mechanisms involved, and dependent on the amount of free drug in the perfusate. This study shows that anionic drugs, subject to active secretion, may reach high concentrations in tubular cells even at low plasma concentrations.     

Identification of multiple nitrobenzylthioinosine binding sites in guinea pig platelets: Comparison with binding in guinea pig erythrocytes

The novel existence of multiple binding sites for the potent nucleoside transporter Probe, [³H]nitrobenzylthioinosine, was identified in guinea pig platelet membranes and the binding characteristics compared to those of guinea pig erythrocyte membranes. Scatchard analysis of the binding in platelets reveled two high affinity binding sites with affinity constant (K_D) of 0.94 ± 0.07 nM and 83 ± 13 nM with corresponding maximal binding capacities (B_max) of 21 ± 7 and 110 ± 25 fmol/mg protein, respectively. In comparison, guinea pig erythrocyte membranes revealed a homogeneous population of the binding sites with K_D of 0.17 ± 0.04 nM and a B_max value of 73 ± 11 fmol/mg protein. Biphasic semi-log plots of the binding site heterogeneity in erythrocytes not reveled by Scatchard plots. Determination of the potencies of selected drugs in inhibiting the binding showed evidence of differential interacitons with the binding sites by various agents which may be exploited pharmacologically. © 1993 Wiley-Liss, Inc.     

Cis-resveratrol glucuronidation kinetics in human and recombinant UGT1A sources

1. It was hypothesized that cis-resveratrol glucuronidation contributes to a greater extent to in-vitro disposition of total resveratrol than previously assumed. To this end, the kinetic data for cis-resveratrol glucuronidation are reported.

2. Glucuronidation assays were conducted in human liver and intestinal microsomes and in uridine diphosphate-glucuronosyltransferases (UGTs) UGT1A1, UGT1A6, UGT1A9, and UGT1A10. Kinetic parameters were estimated for the major cis-resveratrol-3-O-glucuronide (cis-R3G). Substrate inhibition was observed with apparent V_max, K_m and K_i of 6.1?±?0.3/27.2?±?1.2 nmol min^?1 mg^?1, 415?±?48.1/989.9?±?92.8 and 789.6?±?76.3/1012?±?55.9?μM in human intestinal microsomes (HIMs) and UGT1A6, respectively (estimate?±?standard error (SE)). Biphasic kinetics were observed in human liver microsomes (HLMs), while sigmoidal kinetics were seen in UGT1A9 (V_max?=?11.92?±?0.2 nmol min^?1 mg^?1; K_m?=?360?μM; n?=?1.27?±?0.07). The 4′-O-glucuronide (cis-R4′G) exhibited atypical kinetics in HLM, HIM, UGT1A1, and UGT1A10. UGT1A9 catalysed cis-R4′G formation at high substrate concentrations (V_max?=?0.33?±?0.015 nmol min^?1 mg^?1; K_m?=?537.8?±?67.8?μM).

3. In conclusion, although the rates of formation of cis-R3G in HLM and UGT1A9 were higher than those for trans-R3G, the contribution to total resveratrol disposition could not be determined fully due to atypical kinetics observed.

     

Pharmacokinetics of a novel retinoid AGN 190168 and its metabolite AGN 190299 after intravenous administration of AGN 190168 to rats

The pharmacokinetics of AGN 190168, a novel synthetic retinoid, and its major metabolite, AGN 190299, in rat blood after intravenous administration was investigated. Approximately 4.4 mg kg^?1 (high dose) or 0.49 mg kg^?1 (low dose) of AGN 190168 was administered to rats via the femoral vein. Blood was collected from the femoral artery at various time points during an 8 h period. Blood concentrations of AGN 190168 and AGN 190299 were determined by a specific and sensitive high-pressure liquid chromatographic (HPLC) method. AGN 190168 was rapidly metabolized in rats. The only detectable drug-related species in the blood was AGN 190299. Therefore, only pharmacokinetics of AGN 190299 were calculated. Elimination of AGN 190299 appeared to be non-linear after administration of the high dose, and linear after administration of the low dose. The maximum elimination rate (V_max) and the concentration at half of the V_max (k_m), as estimated by a Michaelis—Menten one-compartment model, were 7.58 ± 2.42 μg min^?1 (mean ± SD) and 6.10 ± 1.58 μg mL^?1, respectively. The value of the area under the blood concentration time curve (AUC) was 9.54 ± 1.68 μg h mL^?1 after administration of the high dose and 0.594 ± 0.095 μg h mL^?1 after administration of the low dose. The clearance value was 7.79 ± 1.20 mL min^?1 kg^?1 after the high dose, statistically significantly different from that after the low dose (p < 0.05), 14.0 ± 2.2 mL min^?1 kg^?1. The terminal half-life (t_1/2) was 1.25 ± 0.74 h for the high-dose group and 0.95 ± 0.16 h for the low-dose group. Study results demonstrate rapid systemic metabolism of AGN 190168 to AGN 190299, non-linear pharmacokinetics of AGN 190299 after the 4.4 mg kg^?1 dose, and the lack of difference in disposition profiles between sexes after intravenous administration of AGN 190168 to rats.     

Disposition and bioavailability of the β1-adrenoceptor antagonist talinolol in man

In an open randomized crossover study, the pharmacokinetics and bioavailability of the selective β₁-adrenoceptor antagonist talinolol (Cordanum®—Arzneimittelwerk Dresden GmbH, Germany) were investigated in twelve healthy volunteers (five female, seven male; three poor and nine extensive metabolizers of the debrisoquine hydroxylation phenotype) after intravenous infusion (30 mg) and oral administration (50 mg), respectively. Concentrations of talinolol and its metabolites were measured in serum and urine by HPLC or GC-MS. At the end of infusion a peak serum concentration (C_max) of 631 ± 95 ng mL^?1 (mean ± SD) was observed. The area under the serum concentration-time curve from zero to infinity (AUC_0-∞) was 1433 ± 153 ng h mL^?1. The following parameters were estimated: terminal elimination half life (t_1/2), 10.6 ± 3.3 h; mean residence time, 11.6 ± 3.1 h; volume of distribution, 3.3 ± 0.5 L kg^?1; and total body clearance, 4.9 ± 0.6 mL min^?1 kg^?1. Within 36 h 52.8 ± 10.6% of the administered dose was recovered as unchanged talinolol and 0.33 ± 0.18% as hydroxylated talinolol metabolites in urine. After oral administration a C_max of 168 ± 67 ng mL^?1 was reached after 3.2 ± 0.8h. The AUC_0-∞ was 1321 ± 382 ng h mL^?1. The t_1/2 was 11.9 ± 2.4 h. 28.1 ± 6.8% of the dose or 55.0 ± 11.0% of the bioavailable talinolol was eliminated as unchanged talinolol and 0.26 ± 0.17% of the dose as hydroxylated metabolites by kidney. The absolute bioavailability of talinolol was 55 ± 15% (95% confidence interval, 36–69%). Talinolol does not undergo a relevant first-pass metabolism, and its reduced bioavailability results from incomplete absorption. Talinolol disposition is not found to be altered in poor metabolizers of debrisoquine type.     

Pharmacological characterization and autoradiographic localization of a putative dopamine D4 receptor in the heart

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Repeated dose pharmacokinetics of pancopride in human volunteers

The aim of this study was to assess the pharmacokinetic profile of pancopride after repeated oral dose administration of 20 mg pancopride in tablet form once a day for 5 d in 12 healthy male volunteers. Plasma levels were measured by HPLC using a solid phase extraction method and automated injection. The minimum quantification limit of pancopride in plasma was 2 ng mL^?1. The maximum plasma concentration (mean ± SD) after the first dose was 92.5 ± 41.5 ng mL^?1 and t_max was 1.7 ± 0.9 h. The elimination half-life (t_1/2) was 14.3 ± 6.9 h. The area under the concentration-time curve from zero to infinity (AUC) was 997 ± 396 ng h mL^?1. The maximum plasma concentration (mean ± SD) at steady state (day 5) was 101.8 ± 36.9 ng mL^?1 and t_max was 2.2 ± 1.2 h. The elimination half-life (t_1/2) was 16.3 ± 2.7 h and the minimum plasma concentration (C) was 16.6 ± 6.9 ng mL^?1. The area under the concentration-time curve during the dosing interval (AUC) was 995 ± 389 ng h mL^?1. The average plasma concentration at steady state (C) was 43.3 ± 16.1 ng mL^?1 and the experimental accumulation ratio (R_AUC) was 1.34 ± 0.19, whereas the mean theoretical value (R) was 1.40 ± 0.29. The results obtained showed a good correlation between the experimental plasma levels and the expected values calculated using a repeated dose two-compartment model assessed by means of the Akaike value. It is concluded that the pharmacokinetics of pancopride are not modified after repeated dose administration. The safety parameters showed no clinically relevant alterations.     

Alterations in Renal Uptake Kinetics of the Xanthine Derivative Enprofylline in Endotoxaemic Mice

The pharmacokinetics and renal uptake of enprofylline, which is primarily excreted into the urine by an active tubular secretion mechanism, were investigated in endotoxaemic mice by lipopolysaccharide isolated from Klebsiella pneumoniae. Lipopolysaccharide (1 mg kg^?1) was infused 2 h before starting the examination, thereby inducing a decrease in the systemic clearance and an increase in the steady-state volume of distribution of enprofylline while inducing no changes in the urinary recovery (> 90%). The protein binding of enprofylline significantly decreased in the presence of lipopolysaccharide. Both the systemic clearance for unbound enprofylline and glomerular filtration rate decreased in the treated mice. A nonlinear relationship was found in both groups between the steady-state unbound plasma concentration and renal uptake of enprofylline after constant infusion for 1 h. The renal uptake rate of enprofylline decreased in the treated mice. Lipopolysaccaharide caused increases in the apparent maximum capacity for renal uptake (V_max) from 17.3 to 32.2 μg h^?1 g^?1 of kidney and in the Michaelis–-Menten constant (K_m) from 2.7 to 21.7 μg mL^?1 and decrease in the nonsaturable uptake rate constant (K_d) from 0.87 to 0.43 mL h^?1 g^?1 of kidney. These results indicate that lipopolysaccharide decreases the renal tubular secretion of enprofylline by inducing a decrease in the renal uptake ability.     

Andrographolide Liquisolid using Porous-Starch as the Adsorbent with Enhanced Oral Bioavailability in Rats

Andrographolide (AGL) is the major component of Andrographispaniculata. The poor water solubility and low dissolution strongly affect its oral absorption. Liquisolid technology has been used to improve its dissolution and oral bioavailability. Liquisolid powders of AGL (AGL-LS-PSG) were obtained by firstly dissolving AGL in the mixture of NMP, PEG 6000 and Soluplus®, and solidified by absorption of the blend in porous starch. Angle of repose, Carr index and Hauser ratio presented good powder fluidity and compressibility characteristics of AGL-LS-PSG. The results of optical microscopic observation, PXRD and DSC analysis indicated that AGL has been completely adsorbed in porous starch granules and existed in an amorphous or molecularly dispersing state. AGL-LS-PSG can obviously increase the drug dissolution rate compared to commercial guttate pills and raw drug. In vivo pharmacokinetic behavior of AGL-LS-PSG was investigated following a single oral administration to rats. The C_max (0.37 ± 0.06 μg mL^?1) and AUC_0?2h (13.55 ± 2.67 μg h mL^?1) of AGL-LS-PSG were evidently increased compared to commercial guttate pills (C_max = 0.30 ± 0.21 μg mL^?1, AUC_0–2h = 9.88 ± 3.57 μg h mL^?1). This study indicated great potential of liquisolid technology in effectively improving the dissolution and bioavailability of AGL.     

Oral Sustained-release Cisplatin Capsule

An oral sustained-release cisplatin preparation was prepared by combining microporous water-insoluble pharmaceutical polymer, ethylcellulose, a membrane and a gel-forming polymer, poly(acrylic) acid (Carbopol). As cisplatin is an extremely hydrophilic and small compound, it was difficult to control the release rate solely by the micropores on the ethylcellulose capsule. To retain cisplatin within the capsule, gel-forming polymer was formulated inside the capsule. The release rate of cisplatin was dependent both on the number of micropores of the capsule and the formulated amount of Carbopol. The number of micropores ranged from 20 and 30 to 60, and the formulated amount of Carbopol varied from 15 to 100 mg. In-vitro release experiments suggested that the release rate decreased as the formulated amount of Carbopol increased when the pore number was 60 and 30. However, when pore number was decreased to 20, the effect of the amount of Carbopol was not clearly observed. In the in-vivo study using rabbits, the sustained-release cisplatin capsule was evaluated in comparison with solution after oral administration of 20 mg drug. With the pore number of 60, C_max was 0.46 ± 0.02 μg mL^?1 at 4 h and thereafter serum concentrations declined rapidly. When the pore number was 30, serum cisplatin level-time profiles showed long-acting patterns and AUC was reversely correlated with the formulated amount of Carbopol. C_max and t_max were 0.41 ± 0.02 μg mL^?1 and 3.33 ± 0.88 h, respectively and 0.23 ± 0.01 μg mL^?1 was obtained at 24 h after oral administration of capsule having 30 pores and 15 mg of Carbopol. We conclude that the possibility of developing an oral sustained-release cisplatin preparation is feasible.     

The pharmacokinetics and absolute bioavailability of selegiline in the dog

Selegiline is beneficial to Parkinsonian patients as an adjunct to levodopa therapy. Currently no pharmacokinetic data are available for selegiline in the literature, mainly due to lack of analytical methods that can measure concentrations below 10 ng mL^?1 in plasma. A sensitive fluorimetric assay based on inhibition of rat brain monoamine oxidase-B (MAO-B) in vitro has been developed to measure selegiline in plasma as low as 0.25 ng mL^?1. The pharmacokinetics of selegiline were investigated following intravenous and oral administration to four female mongrel dogs. Each dog received 1 mg kg^?1 selegiline in solution via gavage or by an intravenous route separated by one week. The mean terminal half-life, volume of distribution of the central compartment, and systemic clearance of selegiline were 60.24 ± 9.56 min, 6.56 ± 0.56 L kg^?1, and 159.91 ± 19.28 mL min^?1 kg^?1, respectively. After oral administration selegiline appeared to be absorbed rapidly with a t_max and C_max of 25 ± 5.8 min and 5.2 ± 1.36 ng mL^?1, respectively. The absolute bioavailability of selegiline in the dog was 8.51 ± 3.31%.     

In vitro metabolism of the epoxide substructure of cryptophycins by cytosolic glutathione S-transferase: Species differences and stereoselectivity

The enzyme kinetics of the glutathione (GSH) conjugation of cryptophycin 52 (C52, R-stereoisomer) and cryptophycin 53 (C53,?S-stereoisomer) by cytosolic glutathione S-transferases (cGSTs) from human, rat, mouse, dog and monkey liver were studied. V_max, K_m, and CL_int values for glutathione conjugation of C52 (R-stereoisomer) were 0.10?±?0.01?nmol?min^?1?mg^?1, 3.24?±?0.23?µM, and (3.15?±?0.09)?×?10^?2?ml?min^?1?mg^?1, respectively, in human cytosol. Due to limited solubility relative to the K_m, only CL_int values were determined in rat ((7.76?±?0.10)?×?10^?2?ml?min^?1?mg^?1) and mouse ((7.61?±?0.50)?×?10^?2?ml?min^?1?mg^?1) cytosol. Enzyme kinetic parameters could not be determined for C53 (S-stereoisomer). Microsomal GSH conjugation in human, rat, and mouse was attributed to cytosolic contamination. No GSH conjugation was seen in any biological matrix from dog or monkey. There was little GSH conjugation of C53 by cytosol or microsomes from any species. The metabolism of C52 and C53 by epoxide hydrolase was also investigated. No diol product was observed in any biological matrix from any species. Thus, cGSTs are primarily responsible for C52 metabolism.     

Nanosizing of valsartan by high pressure homogenization to produce dissolution enhanced nanosuspension: pharmacokinetics and pharmacodyanamic study

Purpose: The purpose of the present study was to formulate and evaluate nanosuspension of Valsartan (VAL), a poorly water soluble and low bioavailable drug (solubility of 0.18?mg?mL^?1; 23% of oral bioavailability) with the aim of improving the aqueous solubility thus the bioavailability and consequently better anti-hypertensive activity.

Methods: Valsartan nanosuspension (VAL-NS) was prepared using high-pressure homogenization followed by lyophilisation. The screening of homogenization factors influencing nanosuspension was done by 3-factorial, 3-level Box-Behnken statistical design. Model suggested the influential role of homogenization pressure and cycles on drug nanosizing. The optimized formulation containing Poloxamer^?188 (PXM 188) was homogenized for 2 cycles at 500 and 1000?bar, followed by 5 cycles at 1500 bars.

Results: The size analysis and transmission electron microscopy showed nanometric size range and uniform shape of the nanosuspension. The in vitro dissolution showed an enhanced release of VAL from nanosuspension (VAL-NS) compared to physical mixture with PXM 188. Pharmacodynamic results showed that, oral administration of VAL-NS significantly lowered (p?≤?0.001) blood pressure in comparison to non-homogenized VAL (VAL-Susp) in Wistar rat. The level of VAL in rat plasma treated with VAL-NS showed significant difference (p?≤?0.005) in C_max (1627.47?±?112.05?ng?mL^?1), T_max (2.00?h) and AUC_0→24 (13279.2?±?589.426?ng?h?mL^?1) compared to VAL-Susp that was found to be 1384.73?±?98.76?ng?mL^?1, 3.00?h and 9416.24?±?218.48?ng?h?mL^?1 respectively. The lower T_max value, proved the enhanced dissolution rate of VAL.

Conclusion: The overall results proved that newly developed VAL-NS increased the plasma bioavailability and pharmacodyanamic potential over the reference formulation containing crude VAL.     

BIOCHEMICAL CHARACTERISTICS OF MUSCARINIC CHOLINORECEPTORS IN SWINE TRACHEAL SMOOTH MUSCLE

• 1 The tritiated muscarinic cholinoreceptor antagonist quinuclidinyl benzilate, [³H]QNB, was used to characterize the muscarinic receptors associated with homogenized membrane of the smooth muscle from swine trachea. Based on receptor binding assays, the homogenate had specific, saturable, high-affinity receptors for [³H]QNB.
• 2 Specific binding was time- and temperature-dependent. The association of [³H]QNB with the muscarinic receptor reached equilibrium much sooner at 37°C than 25°C at a [³H]QNB concentration of 180 pM (30 min and 2 h, respectively). Equilibrium at both temperatures was attained within 5 min at a [³H]QNB concentration of 1800 pM. All remaining experiments were performed at 37°C.
• 3 Binding was saturable with respect to [³H]QNB and tissue concentrations. Analysis of binding isotherms yielded an apparent equilibrium dissociation constant (K_D) of 51±20 pM and a maximum receptor density (B_max) of 2.17±0.27 pmole/mg protein. The Hill coefficient for [³H]QNB binding was 1.07±0.16. The association (K₁) and dissociation (K_-1) rate constants were determined to be (5.51±0.16) × 10⁸ M^?1 min^?1 and (1.41±0.18) × 10^?2 min^?1, respectively. K_D calculated from the ratio of K₁ and K_-1 was 26.3±3.8 pM; this value is close to the value of K_D calculated from Scatchard plots of binding isotherms.
• 4 The density of muscarinic receptor binding sites was 10-fold greater in tracheal smooth muscle than in tracheal epithelium (0.20±0.03 pmole/mg protein). There is no difference between weanling and young adult swine in the density of muscarinic receptors in tracheal smooth muscle.
• 5 The nonselective muscarinic antagonists atropine, scopolamine and quinuclidinyl benzilate (QNB) competitively inhibited [³H]QNB binding to the homogenate with Hill coefficients of 0.9-1.0 and inhibition constants (K_i) of nanomolar range.
• 6 Competition with selective muscarinic antagonists pirenzepine and 3-quinuclidinyl xanthene-9-carboxylate (QNX) gave K_i values, 0.26 M and 0.78 nM, respectively, and Hill coefficients of approximately 1. There was a single population of [³H]QNB binding sites of the M₂ subtype for all tested muscarinic antagonists.
• 7 Competition with selective muscarinic agonists pilocarpine and carbachol yielded K_i values of micromolar range, Hill coefficients of less than 1, and revealed the existence of two binding sites (P < 0.01).

     

pH-Dependent and Carrier-mediated Transport of Salicylic Acid Across Caco-2 Cells

Abstract— The transport of monocarboxylic acid drugs such as salicylic acid was examined in the human colon adenocarcinoma cell line, Caco-2 cells that possess intestinal epithelia-like properties. [¹⁴C]Salicylic acid transport was pH-dependent and appeared to follow the pH-partition hypothesis. However, 10 Mm unlabelled salicylic acid significantly reduced the permeability coefficient of [¹⁴C]salicylic acid. Kinetic analysis of the concentration dependence of the permeation rate of salicylic acid across Caco-2 cells showed both saturable (K₁ = 5·28 ± 0·72 Mm J_max = 36·6 ± 3·54 nmol min^?1 (mg protein)^?1) and non-saturable (k_d = 0·37 ± 0·08 μL min^?1 (mg protein)^?1) processes. The permeation rate of [¹⁴C]salicylic acid was competitively inhibited by both acetic acid and benzoic acid, which were demonstrated in our previous studies to be transported in the carrier-mediated-transport mechanism which is responsible for monocarboxylic acids. Furthermore, certain monocarboxylic acids significantly inhibited [¹⁴C]salicylic acid transport, whereas salicylamide and dicarboxylic acids such as succinic acid did not. From these results, it was concluded that the transcellular transport of [¹⁴C]salicylic acid across Caco-2 cells is by the pH-dependent and carrier-mediated transport mechanism specific for monocarboxylic acids.     

Different β-Adrenergic Receptor Density in Different Rat Skeletal Muscle Fibre Types

Abstract: The effects of adrenaline on skeletal muscle differ between fibre types. The aim of the present study was to investigate the β-adrenoceptor density, affinity and subtype in rat skeletal muscles with different fibre type composition. β-Adrenoceptors were determined in cryostat sections to avoid methodological problems with variable recovery, using the non-selective β-adrenoceptor ligand [³H]CGP-12177 and β₁ and β₂-selective cold ligands CGP 20712A and ICI 118,551. In the presence of protease inhibitors [³H]CGP-12177 binding was stable, saturable, reversible, and displaceable. Scatchard analysis of binding saturation data was compatible with a single class of specific binding sites. Binding site density (B_max) was higher (P<0.02) in adult soleus (9.38 ± 1.13 fmolxmg protein^–1) than in adult extensor digitorum longus (4.74 ± 0.39 fmolxmg protein^–1), whereas the dissociation constants (K_d), 0.37® pL 0.05 and 0.31 ± 0.04 nM for soleus and extensor digitorum longus, respectively, were not significantly different. For young rats (5–6 weeks), B_max was 11.21 ± 0.33 and 5.45 ± 0.11 fmolxmg protein^–1 (P<0.05), and K_d was 0.27±0.02 and 0.24±0.04 nM for soleus and epitrochlearis, respectively. These results correspond to a receptor density of 2 and 1 pmolxg w.wt.^–1 in muscles containing mainly type I and type II fibres, respectively. Displacement studies with CGP 20712A and ICI 118,551 were compatible with mainly β₂-adrenoceptors, but 7–10% β₁-adrenoceptors were present in both types of muscle. In conclusion, the receptor density is twice as high in muscles containing mainly type I muscle fibres compared to muscles containing mainly type II fibres, and this may explain some of the different effects of adrenaline between the two muscle fibre types.     

Kinetics of Bupivacaine After Nicorandil Treatment in Mice

Previous studies have reported interactions between potassium-channel agonists and bupivacaine. This study was designed to document possible changes in the pharmacokinetic behaviour of bupivacaine and its main metabolite, N-desbutylbupivacaine, in mice after a single 1 mg kg^?1 intraperitoneal injection of nicorandil. The kinetic variables of bupivacaine were determined after a single 20 mg kg intraperitoneal dose of bupivacaine in controls (group 1) and in nicorandil-treated mice (group 2). The maximal concentration in the serum (C_max 0.618 ± 0.051 vs 0.408 ± 0.041 μg mL^?1 for group 1 vs 2, P = 0.01) and the area under the concentration curve (AUC 1.039 ± 0.051 vs 0.758 ± 0.072 μg mL^?1 h for group 1 vs 2, P = 0.013) of bupivacaine were significantly lower in nicorandil-treated mice, while CL (0.579 ± 0.025 vs 0.815 ± 0.079 for group 1 vs 2, P = 0.022) and V_d (0.506 ± 0.054 vs 0.981 ± 0.117 for group 1 vs 2, P = 0.1006) were increased in nicorandil-treated animals. The ratio of AUC for N-desbutylbupivacaine to AUC for bupivacaine, which may partially indicate the rate of metabolism, was higher in the presence of nicorandil (1.142 ± 0.017 compared with 0.877 ± 0.013, P = 0.0001). Our data may indicate an increased metabolism of bupivacaine in nicorandil-treated mice. These results do not explain the previously reported enhanced anaesthetic activity of bupivacaine in the presence of nicorandil, but may participate, at least in part, in the relative protective effect of nicorandil against the previously reported bupivacaine-induced toxicity.     

Impaired monoamine oxidase activity in dogs with portacaval shunt

In dogs with portacaval shunt, hypertyraminemia could result from either impaired degradation by monoamine oxidase (MAO) and/or from failure of tyramine to reach this enzyme. MAO activity was evaluated in liver obtained from dogs before and after the construction of an end-to-side portacaval shunt. Diversion of portal blood from the liver by a portacaval shunt resulted in a significant (P < 0.05) decrease in hepatic MAO activity [11.9 ± 4.1 nmoles of 4-hydroxyphenylacetic acid (PHA) formed · (mg protein)^?1 · hr^?1] as compared to controls [28.7 ± 6.3 nmoles PHA formed · (mg protein)^?1 · hr ^?1]. Activity was maximally reduced in shunted dogs with stages II and III hepatic encephalopathy. In addition, more than a 50 per cent reduction in both the V_max and the K_m of hepatic MAO for tyramine was noted in shunted dogs as compared to controls. Similar kinetic abnormalities [post-shunt, K_m 52.63 ± 14.3, V_max 3.8 ± 1.2 vs sham group, K_m 120.1 ± 22.3 μM tyramine, V_max 14.3 ± 4.5 nmoles PHA · (mg protein) ^?1 · hr^?1], as well as decreased MAO activity [post-shunt, 1.85 ± 0.83 vs sham group, 6.7 ± 2.1 nmoles PHA formed · (mg protein)^?1· hr^?1], were found in cerebral cortex from encephalopathic dogs with portacaval shunt. In summary, defective MAO activity may contribute to many of the pathophysiologic events observed in dogs with portacaval anastomosis. Such abnormalities could explain the hypertyraminemia and encephalopathy that have been reported in patients and experimental animals with liver disease.