Expression of 5-HT1A and 5-HT7 receptors in Caco-2 cells and their role in the regulation of serotonin transporter activity.

Serotonin (5-HT) receptors are expressed in the gastrointestinal tract and play an important role in gastrointestinal activity regulation. 5-HT binding to receptors depends on 5-HT availability, which is, in part, modulated by the 5-HT transporter (SERT) expressed in enterocytes. This work concerns the expression of 5-HT(1A) and 5-HT(7) receptors (5-HTR(1A) and 5-HTR(7)) in the human enterocyte-like Caco-2 cell line and their role in SERT activity modulation. The results demonstrate the mRNA and protein expression of 5-HTR(1A) and 5-HTR(7) in these cells. In addition, both receptors are shown to modulate SERT activity; 5-HTR(1A) activation increased 5-HT uptake and 5-HTR(7) activation inhibited it. In both cases, SERT modulation might involve a cAMP/PKA pathway. Effects on SERT disappeared after long-term activation of 5-HTR(1A) and 5-HTR(7), indicating their desensitization. However, in both cases, the desensitization did not show itself to be mediated by a reduction of the amount of receptors in the membrane.

Regulation of the human serotonin transporter mediated by long-term action of serotonin in Caco-2 cells.

AIM: The aim of this study was to determine the effect of long-term serotonin (5-hydroxytryptamine, 5-HT) treatment on the human serotonin transporter (hSERT) function and its expression. METHODS: This study was carried out in the enterocyte-like cell line Caco-2. These cells constitutively express the hSERT and have been shown to be an excellent model for the study of this protein. We measured serotonin transport, levels of mRNA expression and of the SERT protein after treating the cells with serotonin. RESULTS: Serotonin treatment diminished hSERT activity in a concentration and period-dependent way by increasing the K(t) value and reducing V(max). This inhibition was reversible and was not mediated by either the action of 5-HT(2), 5-HT(3) or 5-HT(4) receptors, or by the intracellular second messengers, protein kinase C and cAMP. 5-HT did not seem to affect either the mRNA level of the SERT or the protein transporter measured in either the membrane or the cell lysate. The 5-HT treatment effect was additive to the inhibitory effect of treatment with a low concentration of citalopram and fluoxetine. Nevertheless, 5-HT did not increase the inhibition yielded by treatment with high concentration citalopram. CONCLUSION: The chronic increase in serotonin in the extracellular medium diminishes the function of the SERT. This effect seems to be due to an effect on the transporter molecule itself in the membrane, without altering protein synthesis, intracellular traffic, or its availability.

Molecular characterization and intracellular regulation of the human serotonin transporter in Caco-2 cells.

The serotonin transporter (SERT) has shown itself to be an effective pharmacological target in the treatment of mood disorders and some kinds of gastrointestinal syndromes. Most of the molecular studies of SERT in humans have been carried out using heterologous models. In this work, we have investigated the human enterocyte-like Caco-2 cell line as a potential "in vitro" model to study the human SERT. The results show that these cells express a SERT mRNA identical to the human brain SERT, and a 70 kDa protein immunodetected using a specific antibody. The SERT activity levels in Caco-2 cells increased in correlation with the onset and maintenance of the morphological and functional differentiation of the cells. Caco-2 SERT was also shown to be a high affinity (Kt=0.216 microM) saturable, Na(+) -dependent transporter that was inhibited by fluoxetine (IC(50)=17.6 nM). In addition, SERT activity was inhibited by the intracellular modulators protein kinase C and cAMP, either after short or long-term treatment. In short, the expression and molecular characteristics of the human SERT in Caco-2 cells indicate that this cell line may be an ideal tool to study in vitro the physiology and pharmacology of human SERT.

Expression and molecular characterization of rat renal D-mannose transport in Xenopus oocytes.

Renal reabsorption appears to play a major role in d-mannose homeostasis. Here we show that in rat kidney, the transport of d-mannose by brush border membrane vesicles from tubular epithelial cells involves an uphill and rheogenic Na-dependent system, which is fully inhibited by d-mannose itself, incompletely inhibited by d-glucose, d-fructose, phloridzin, and phloretin, and noninhibited by l-mannose or disaccharides. In addition, this system exhibits both low capacity (112.9+/-15.6 pmol/mg/second) and high affinity (0.18+/-0.04 mm), with a 2:1 stoichiometry for the Na:d-mannose interaction, and low affinity for sodium (16.6+/-3.67 mm). We also show expression of d-mannose transport by Xenopus laevis oocytes injected with rat renal polyA(+) RNA. Kinetic analysis of the expressed transport was performed after RNA enrichment by fractionation through a sucrose density gradient and was shown to be identical to that measured in membrane vesicles. The RNA species encoding the expressed transport has a small mean size, 1 kb approximately, and shows no homology with the SGLT family of Na-dependent d-glucose transporters, as shown by low stringent RT-PCR and northern analysis. The expressed transport is specific for d-mannose, since in spite of a significant inhibition by d-glucose and d-fructose, neither of these two substrates was transported above the level of the water-injected oocytes.

Pharmacokinetics of verapamil in New Zealand white rabbits during ontogeny.

The pharmacokinetics of verapamil during ontogeny in rabbits and in vitro verapamil demethylase activity have been investigated in the liver and whole blood. In vivo experiments revealed that the slope of the postdistributive phase as well as the area under the curve and clearance showed significant differences when newborn and adult rabbits were compared. Other pharmacokinetic parameters, such as volume of distribution and plasma protein binding did not show any statistical differences. Liver microsomal preparation samples from 1-, 8- and 16-day-old rabbits displayed approximately 20% of the activity observed in adults. A significant verapamil demethylase activity in the whole blood of rabbits was also noted. The in vivo results show that newborn rabbits have a capacity to eliminate verapamil that is similar or even higher than that of adults. These findings could not be explained with regard to the in vitro liver metabolism of verapamil, although they could with respect to blood metabolism.

Diltiazem blood pharmacokinetics in the pregnant and non-pregnant rabbit: maternal and foetal tissue levels.

1. The aim was to investigate the pharmacokinetics of diltiazem (DTZ) and its metabolites, deacetyldiltiazem (M1) and N-demethyldiltiazem (MA), in the pregnant rabbit following DTZ intravenous administration. In addition, DTZ tissue distribution in both the non-pregnant and pregnant rabbit and foetuses was also studied. 2. The slope of the alpha- and beta-phases increased slightly in six of the eight pregnant rabbits as compared with the non-pregnant animal, but the other pharmacokinetic parameters that largely determine drug disposition (AUC, V(n), CL) showed no significant differences. 3. MA blood disposition was unaltered by pregnancy. However, all the pharmacokinetic parameters calculated for the deacetylated metabolite of DTZ were significantly modified in the pregnant as compared with the non-pregnant rabbit. 4. DTZ tended to concentrate in most of the tissues examined. Significant differences were observed in the DTZ concentration in the uterus and kidney from the pregnant as compared with the non-pregnant rabbit. 5. The findings suggest that DTZ diffuses easily through the placenta, reaching DTZ blood concentrations equivalent to that observed in maternal blood. However, the concentration of DTZ and its metabolites in the selected foetal tissues was either higher (in brain and muscle) or lower than that observed in maternal tissues, suggesting a different tissue affinity and/or a different metabolic activity in the foetuses as compared with the mothers.

Development of a method for the determination of danofloxacin in plasma by HPLC with fluorescence detection.

A simple and sensitive HPLC method has been developed for the determination of danofloxacin (DAN) in plasma. Sample preparations were carried out by adding phosphate buffer (pH 7.4, 0.1 M), followed by extraction with trichloromethane. DAN and the internal standard, sarafloxacin (SAR), were separated on a reversed-phase column, and eluted with aqueous solution-acetonitrile (80:20 v/v). The fluorescence of the column effluent was monitored at lambda(ex) = 338 and lambda(em) = 425 nm. The retention times were 2.80 and 4. 40 min for DAN and SAR, respectively. The method was shown to be linear from 1 to 1500 ng/mL (r(2) = 0.999). The detection and quantitation limit were 1 and 5 ng/mL, respectively. Mean recovery was determined as 80% by the analysis of plasma standards containing 150, 750 and 1500 ng/mL. Inter- and intra-assay precisions were 4.0% and 2.4%, respectively.

Comparison of the pharmacokinetics of verapamil in the pregnant and non-pregnant rabbit: study of maternal and foetal tissue levels.

1. The comparison of the pharmacokinetics of verapamil (VER) has been studied between the non-pregnant and pregnant rabbit following VER intravenous (i.v.) bolus administration. Also studied has been VER tissue distribution in the non-pregnant and pregnant rabbit and its foetuses following an i.v. infusion of VER. 2. When the pharmacokinetic variables were compared between the pregnant and non-pregnant rabbit, it was observed that t(1/2)lambda2 V1 and V(D) were significantly higher in the non-pregnant than in the pregnant rabbit. Moreover, lambda(z) was significantly lower in the non-pregnant than in the pregnant rabbit. However, AUC and CL showed no significant differences between the pregnant and non-pregnant rabbit. 3. When tissue concentrations were examined, it was found that in most of the tissues studied high concentrations of VER were found both in the pregnant and non-pregnant rabbit. Furthermore, VER concentrations in the uterus, heart, spleen and kidney were significantly higher in the non-pregnant than in the pregnant rabbit. 4. The results suggest that VER diffuses poorly through the placenta, given that VER blood concentrations were lower in blood foetuses than in maternal blood. Moreover, the concentrations of VER in the selected foetal tissues were either similar (brain and liver) or lower than those observed in maternal tissues.

Pharmacokinetics of verapamil in lactating rabbits. Prediction of verapamil distribution into rabbit milk.

In this work, we have studied the pharmacokinetics and milk penetration of verapamil following intravenous administration in lactating rabbits. Milk-to-serum drug concentration ratios (M/B(obs)) have been determined using area under the milk and serum concentration-time profiles, and the resulting values have then been compared with those obtained by theoretical classical diffusion milk transfer models that were described by Fleishaker et al. [J. Pharm. Sci. 76 (1987) 189.], Atkinson and Begg [Br. J. Clin. Pharmacol. 25 (1990) 495.], and Stebler and Guentert [Pharm. Res. 9 (1992) 1299.]. The pharmacokinetic profile of verapamil in lactating rabbits following endovenous administration is described in the form of a two-compartment model. Moreover, we detected an important milk transfer after endovenous administration of verapamil in lactating rabbits. M/B(obs) was near 15. The classical diffusional models mentioned were not able to predict this extensive transfer of verapamil into rabbit milk. However, when the classical Fleishaker equation was modified and a stepwise regression was carried out, we found that the M/B(obs) value could be predicted using the plasma and milk protein binding.

Comparative pharmacokinetics of enrofloxacin in mice, rats, rabbits, sheep, and cows.

OBJECTIVE: To compare pharmacokinetic variables of enrofloxacin (ENR) after IV administration in mice, rats, rabbits, sheep, and cows and to perform allometric analysis of ENR. ANIMALS: 47 mice, 5 rats, 5 rabbits, 5 sheep, and 5 cows. PROCEDURE: Serially obtained plasma samples were assayed for ENR concentration, using high-performance liquid chromatography. In vitro plasma protein binding was determined by ultrafiltration. Plasma ENR concentration versus time curves were fitted by use of nonlinear least-squared regression analysis. Pharmacokinetic variables were correlated further with body weight. RESULTS: In all species studied, the best fit was obtained for a two-compartment open model; ENR half-life ranged from 89 minutes in mice to 169 minutes in cows. Volume of distribution was large in all species studied, with values ranging from 10.5 L/kg in mice to 1.5 L/kg in sheep. Body clearance ranged from 68.1 ml/min/kg for mice to 4.6 ml/min/kg for sheep. Unbound ENR was found to be (mean +/- SD) 58+/-2, 50+/-6, 50+/-2, 31+/-2, and 40+/-3% in plasma of mice, rats, rabbits, sheep, and cows, respectively. The only pharmacokinetic variables that could be correlated with body weight were elimination half-life, clearance, and volume of distribution. Allometric exponents denoting proportionality of half-life, body clearance, and volume of distribution with body weight were 0.06, 0.82, and 0.90, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: An allometric approach could provide a suitable method for determining a scale for ENR pharmacokinetics among various mammalian species. This would faciliatate the administration of appropriate doses of ENR to all animals.

Determination of marbofloxacin in plasma samples by high-performance liquid chromatography using fluorescence detection.

A simple and sensitive HPLC method has been developed for the determination of marbofloxacin (MAR) in plasma. Sample preparations were carried out by adding phosphate buffer (pH 7.4, 0.1 M), followed by extraction with trichloromethane. MAR and the internal standard, enrofloxacin (ENR), were separated on a reversed-phase column and eluted with aqueous solution-acetonitrile (80:20). The fluorescence of the column effluent was monitored at lambda(ex) = 338 and lambda(em) = 425 nm. The retention times were 2.20 and 3.30 min for MAR and ENR, respectively. The method was shown to be linear from 15 to 1500 ng/ml (r2 = 0.999). The detection limit was 15 ng/ml. Mean recovery was determined as 90% by the analysis of plasma standards containing 150, 750, and 1500 ng/ml. Inter- and intra-assay precisions were 3.3% and 2.7%, respectively.

Simultaneous determination of enrofloxacin and its primary metabolite, ciprofloxacin, in plasma by HPLC with fluorescence detection.

A simple and sensitive HPLC method has been developed for the simultaneous determination of enrofloxacin (ENR) and ciprofloxacin (CIP) in plasma. Plasma sample preparation was carried out by adding phosphate buffer (pH 7.4, 0.1 M), followed by extraction with trichloromethane. ENR, CIP and the internal standard, sarafloxacin (SAR), were separated on a reversed-phase column, and eluted with aqueous acetonitrile (80:20). The fluorescence of the column effluent was monitorized at lambda(ex) 338 and lambda(em) 425 nm. The retention times were 2.28, 3.30 and 4.40 min for CIP, ENR and SAR, respectively. The detection limit for the two compounds was 10 ng/mL. Standard curves were linearly related to concentration in the range from 1 to 1500 ng/mL. The recovery was 93% for ENR and 75% for CIP.

Determination of orbifloxacin in rabbit plasma by high-performance liquid chromatography with fluorescence detection.

A simple and sensitive high-performance liquid chromatography method is developed for the determination of orbifloxacin (ORB) in rabbit plasma. Sample preparations are carried out by adding phosphate buffer (pH 7.4, 0.1 M) and extracting with trichloromethane. ORB and the internal standard, norfloxacin (NOR), are separated on a reversed-phase column using an aqueous phosphate buffer-acetonitrile (80:20, v/v) mobile phase. The concentrations of ORB and NOR eluting from the column with retention times of 2.16 and 3.09 min, respectively, are monitored by fluorescence detection at 338 (excitation) and 425 nm (emission). The method is shown to be linear from 4 to 1500 ng/mL (regression coefficient r2 = 0.999). The quantitation and detection limits are 4 and 9 ng/mL, respectively. Mean recovery is determined as 92% by the analysis of plasma standards containing 150, 750, and 1500 ng/mL. Inter- and intra-assay precisions were 4 and 3%, respectively.

A common method for the determination of several calcium channel blockers using an HPLC system with ultraviolet detection.

We report a common HPLC method for the single or simultaneous determination of four calcium channel blockers (CCB), namely diltiazem (DTZ), verapamil (VER), nifedipine (NIF) and nitrendipine (NIT) and their active metabolites demetildiltiazem and deacetildiltiazem (MA and M1), norverapamil (NOR), and dehydronifedipine (DHN). DHN was first synthesised in our laboratory and different pH values of the mobil phase were subsequently prepared and tested for chromatographic separation. The detection system and the environmental light conditions were optimised. The best separations of all analytes were obtained using a C(18) column and a mobile phase of methanol, 0.04 M ammonium acetate, acetonitrile and triethylamine (2:2:1:0.04 v/v). Quantitation was performed using imipramine (IMI) as the internal standard. For DTZ and its metabolites (M1 and MA), the wavelength chosen was 237 nm; for VER and its metabolite NOR, it was 210 nm; and, finally for NIF and its metabolite DHN and NIT it was 216 nm. When a simultaneous analysis was carried out the wavelength was of 230 nm. The optimum pH were 7.90 and 7.10 when the separation of NIT and DTZ or VER and NIF were carried out, respectively, and 7.90 when a simultaneous separation was carried out. The detection limit of the assay was less than 8 ng ml(-1) for all compounds, with coefficients of variation less than 7% (for inter- and intra-day) over the concentration range of 1-1000 ng ml(-1). The retention times were less than 11 min. When NIF or NIT were studied, it was necessary to use a sodium vapour lamp in order to avoid the photodegradation which takes place under daylight conditions.

Limited capacity of neonatal rabbits to eliminate enrofloxacin and ciprofloxacin.

The pharmacokinetics of enrofloxacin (ENR) and ciprofloxacin (CIP) in newborn and young rabbits were studied. Rabbits of different ages (1-, 8-, 16-, and 30-day-old) were administered, by the intraperitoneal route (i.p.), a dose of 7.5 mg of either drug/kg. In 1-, 8-, and 16-day-old rabbits, blood samples were drawn by cardiac puncture, under light ether anaesthesia, at predetermined times after drug administration. In 30-day-old rabbits, serial blood samples were drawn through an arterial catheter. Plasma was immediately obtained and analysed using an HPLC method. ENR and CIP plasma protein binding was also determined. The plasma pharmacokinetic profiles of ENR and CIP obtained for 30-day-old rabbits agreed with those reported in the literature for healthy adult rabbits. Nevertheless, significant differences were observed for the body clearance, the slope of the terminal phase, the volume of distribution, and the area under the curve when compared with those for younger animals (1-, 8-, and 16-day-old rabbits), indicating a limited capacity of neonatal rabbits to eliminate ENR and CIP. No differences were found when we compared the calculated values for ENR or CIP plasma protein binding as a function of the postnatal age, indicating that development does not seem to alter the free fraction of these drugs in the rabbit. Taking into account that extensive placental and milk transfer has been reported for these drugs after administration to pregnant or nursing rabbits, a cautious, attitude regarding their use in these animals must be adopted.

Simultaneous determination of verapamil and norverapamil in biological samples by high-performance liquid chromatography using ultraviolet detection.

In this paper we develop an high-performance liquid chromatographic method with ultraviolet detection for the determination of verapamil and its primary metabolite norverapamil in biological samples. Both compounds, as well as the internal standard, imipramine, were extracted from alkalinised blood, with n-hexane-isobutyl alcohol, back-extracted into 0.01 M phosphoric acid and determined using a reversed-phase column and ultraviolet monitoring at 210 nm. The average coefficient of variation obtained over the concentration range of 1-1000 ng/ml is about 3%. The detection limit is below 5 ng/ml for both compounds, and extraction recoveries close to 80%. The method was applied to a pharmacokinetic study of the drug and its active metabolite and used to analyse blood samples from verapamil treated rabbits.

Enhanced diltiazem deacetylase activity in pre-term and full-term rabbits compared with adult rabbits.

The in vitro metabolic activity of the esterase responsible for the hydrolysis of diltiazem (DTZ) to its deacetylated metabolite (M1) was determined in an age-dependent fashion using the rabbit as an animal model. The presence of the enzyme in several tissues (liver, lung, small intestine, and brain) and in whole blood from pre-term and full-term fetuses, full-term newborns, yound and adult rabbits was examined. To this end, DTZ was spiked to 10,000-g tissue homogenates and whole blood to yield a final concentration of 1 microgram/ml. Serial samples were withdrawn from the incubation medium up to 240 min and assayed for DTZ and M1 concentration. In all tissues examined there was a net production of M1. Chemical breakdown and stability studies confirmed the metabolic origin of the M1 formed throughout the incubation. In pre-term fetuses (25 days of gestation) the brain was found to be the most active tissue in eliminating DTZ (brain > liver > lung > small intestine). This trend changed in young and adult rabbits (lung = brain > liver > small intestine). Although an important age-dependent DTZ deacetylase activity was observed in blood, it was not included in the comparison between organs because of the unequal composition of the incubation medium. In conclusion, results showed that fetuses and newborn rabbits have a similar, and in some instances higher, DTZ deacetylase activity to that in adults (p < 0.05). In vitro findings were further confirmed by in vivo experiments.

Pharmacokinetics of fosfomycin in chickens after a single intravenous dose and tissue levels following chronic oral administration.

This paper describes the pharmacokinetics of fosfomycin following a single i.v. bolus dose (10 mg/kg of body weight) in broiler chickens. Serial blood samples were collected up to 5 h post-administration. Fosfomycin serum concentrations were determined by a microbiological method, using Proteus mirabilis as the test microorganism. The serum concentration versus time curves after i.v. administration followed a biexponential decline. The main pharmacokinetic variables were t1/2 lambda l = 23 min, t1/2 lambda n = 112 min, VDarea = 575 +/- 190 mL/kg and CLb = 3.12 +/- 0.44 mL.min-1.kg-1. Tissue levels of fosfomycin in kidney, liver, lung, muscle, heart, fat, gizzard and serum were also determined after oral chronic administration of the drug in drinking water (150 micrograms/mL). During the oral chronic administration period, high FOS concentrations in serum were maintained (mean 6.1 +/- 1.1 micrograms/mL), but a significant decline over time could be observed (P < 0.05). Fosfomycin was detected in all tissues except muscle, with mean concentrations ranging from 0.63 microgram/g in fat to 13.48 micrograms/g in kidney. Twenty-four hours after the treatment was finished, fosfomycin levels were below the assay detection limit in all tissues tested.

Deacetylation of diltiazem by several rabbit tissues.

PURPOSE: Diltiazem (DTZ) undergoes extensive metabolism yielding several metabolites, some of which retain a certain degree of pharmacological activity. N-demethylating activity has been detected mainly in the liver. Nevertheless, the organs involved in the formation of the deacetylated metabolite of DTZ (M1) have not been fully elucidated. In order to address this issue, we have carried out in vitro studies using the blood, lung, brain, small intestine, and liver as enzyme sources. METHODS: DTZ (1,000 ng/ml) was incubated in 10,000 x g supernatant homogenates of selected tissues or in whole blood for 240 minutes at 37 degrees C. Multiple samples were withdrawn, and DTZ and its metabolite M1 were assayed by HPLC. RESULTS: The apparent degradation rate constant of DTZ was in the rank order blood > lung > brain > liver > small intestine. This trend can also be observed for the AUC and for the percentage of DTZ metabolized. In all the tissue homogenates examined there was a net production of the deacetylated metabolite. The M1 metabolite was also detected in the blood (500 ng/ml after 240 minutes of incubation). CONCLUSIONS: The widespread distribution of the DTZ deacetylase activity described in this study suggests that extrahepatic metabolism of DTZ to M1 may play a relevant role in the overall pharmacokinetics of DTZ.

Pharmacokinetics of ciprofloxacin in sheep after single intravenous or intramuscular administration.

The disposition and urinary excretion of ciprofloxacin (CIP) following intravenous (IV) or intramuscular (IM) administration of 7.5 mg/kg body weight in sheep (n = 5) was studied. The intravenous plasma concentration curve was best described pharmacokinetically by a two-compartment open model, while the intramuscular administration data fitted better to a one-compartment open model. Mean elimination half-lives after IV and IM administration were 72 and 184 minutes, respectively. The absorption of intramuscularly administered CIP in sheep was fast: maximal plasma concentration (Cmax) was reached quickly (tmax 31.93 min) and attained values of 0.69 +/- 0.27 mg/l. The bioavailability was 49%. The urinary data showed a significant decrease in the elimination rate constant of CIP when CIP was administered intramuscularly. The other parameters calculated did not display differences between the two routes of administration. The results obtained suggest that when CIP was administered by the IM route in the assayed dose, it was able to maintain serum concentrations above the MIC of most common pathogens over an 8-hour period.