Application of Magnetic Core-Shell Imprinted Nanoconjugates for the Analysis of Hordenine in Human Plasma-Preliminary Data on Pharmacokinetic Study after Oral Administration.

In this paper, we developed and validated a new analytical method to determine the pharmacokinetic profile of hordenine in plasma samples of human volunteers after oral administration of hordenine-rich dietary supplements. For this purpose, a magnetic molecularly imprinted sorbent was fabricated and characterized. The application of a magnetic susceptible material facilitates pretreatment step while working with a highly complex sample, reducing time and costs. An optimized, fast, and reliable separation step was combined with liquid chromatography tandem mass spectrometry, providing an analytical method for analysis of hordenine in human plasma after dietary supplement intake. The method was validated (lower limit of quantification of 0.05 µg/L), enabling the pharmacokinetic profile of hordenine to be determined. The highest concentration of hordenine was noted after 65 ± 14 min, reaching the value of 16.4 ± 7.8 µg/L. The average t1/2 was 54 ± 19 min. The apparent volume of distribution was 6000 ± 2600 L (66 ± 24 L/kg when adjusted for weight).

Absorption, Biokinetics, and Metabolism of the Dopamine D2 Receptor Agonist Hordenine (N,N-Dimethyltyramine) after Beer Consumption in Humans.

Hordenine, a natural constituent of germinated barley, is a biased agonist of the dopamine D2 receptor. This pilot study investigated the biokinetics of hordenine and its metabolites in four volunteers consuming beer equal to 0.075 mg hordenine/kg body weight. A new ultrahigh-performance liquid chromatography method coupled to electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) method determined maximum plasma concentrations of 12.0-17.3 nM free hordenine after 0-60 min. Hordenine phase-II metabolism was first dominated by sulfation, but later by glucuronidation. The elimination half-lives in plasma were 52.7-66.4 min for free hordenine and about 60/80 min longer for hordenine sulfate and hordenine glucuronide. Urinary excretion peaked 2-3.5 h after consumption and accumulated to 3.78 µmol within 24 h, corresponding to 9.9% of the ingested dose. The observed hordenine levels in plasma seem too low to provoke direct interaction with the dopamine D2 receptor related to food reward, but synergistic or additive effects with alcohol or N-methyltyramine may occur.

Therapeutic Effect of Controlled Release of Dual Growth Factor Using Heparin-Pluronic Hydrogel/Gelatin-Poly (Ethylene Glycol)-Tyramine Hydrogel System in a Rat Model of Cavernous Nerve Injury.

The number of cases of erectile dysfunction (ED) caused after radical prostatectomy (RP) prostate cancer treatment is increasing steadily. Although various studies have been conducted for treatment of post-RP ED, there is still a need for more effective methods. A dual growth factor incorporated heparin-pluronic/gelatin-poly(ethylene glycol)-tyramine (HP/GPT) hydrogel, which consists of a basic fibroblast growth factor (bFGF)-loaded HP hydrogel and nerve growth factor (NGF)-loaded GPT hydrogel, can control dose and rate of growth factor release. In this study, we demonstrated that dual growth factor incorporated HP/GPT hydrogel could further improve erectile function in a rat model of bilateral cavernous nerve injury (BCNI). We showed that erectile function was decreased after BCNI, but it was further improved by treatment with a dual growth factor incorporated HP/GPT hydrogel compared with groups treated with single growth factor in a rat model of cavernous nerve injury. Also, we observed an increase in cyclic guanosine monophosphate (cGMP) levels in the dual growth factor group when compared with the groups treated with single growth factor. This effect was associated with greater upregulation of nitric oxide synthase and endothelial nitric oxide synthase expression in the penile tissue of the group treated with dual growth factor incorporated HP/GPT than in the other experimental groups. Apoptosis in the penile tissue treated with the dual growth factor incorporated HP/GPT hydrogel was lower than those treated singly with either bFGF or NGF incorporated GPT hydrogel. Both α-smooth muscle actin and CD31 expression increased in the group treated with dual growth factor incorporated HP/GPT hydrogel when compared to in the other experimental groups. Altogether, our results proved that the sequential and continuous release of growth factors from dual growth factor incorporated HP/GPT hydrogel prevented fibrosis and nerve damage induced by BCNI in the corpus cavernosum, and promoted the recovery of erectile function. Dual growth factor incorporated HP/GPT hydrogel may be a potent clinical application for the treatment of post-RP ED and could potentially be used various biomedical application in tissue regnerative medicine.

Enzyme-mediated tissue adhesive hydrogels for meniscus repair.

Meniscus tissues have limited regenerative capacity once damaged. The treatment options for the meniscus tissue regeneration have been limited to arthroscopic meniscectomy or surgical interventions. The injectable hydrogels based system would provide an alternative to the conventional meniscus therapy by providing a minimally invasive treatment alternative. Here we utilized enzyme-based approaches to fabricate tissue adhesive hydrogels for meniscus repair. Tyramine (TA) conjugated hyaluronic acid (TA-HA) and gelatin are susceptible to tyrosinase (TYR)-mediated crosslinking in vitro and in vivo. Importantly, mechanical properties and degradation kinetics are modulated by the TA substitution and TYR concentrations. In addition, TYR -mediated crosslinking displayed tissue-adhesive properties. Furthermore, fibrochondrocyte-laden and TYR-crosslinked hydrogels demonstrated strong biocompatibility and resulted in enhancement of cartilage-specific gene expression and matrix synthesis. Overall, this represents a potential application of enzyme-mediated crosslinking hydrogels for meniscus tissue engineering.

Blood-brain barrier specific permeability assay reveals N-methylated tyramine derivatives in standardised leaf extracts and herbal products of Ginkgo biloba.

The linkage between the central nervous system availability and neuropharmacological activity of the constituents of Ginkgo biloba L. extracts (GBE) is still incomplete. In this study, the in vitro blood-brain barrier (BBB) permeability profile of the standardised GBE was investigated by the parallel artificial membrane permeability assay (PAMPA). Biomarkers, such as terpene trilactones, flavonoid aglycones and ginkgotoxin exerted moderate or good BBB-permeability potential (BBB+), while glycosides and biflavones were predicted as unable to pass the BBB. N-methyltyramine (NMT) and N,N-dimethyltyramine or hordenine (Hor) were identified among BBB+ compounds, while subsequent direct HRMS analysis revealed tyramine (Tyr) and N,N,N-trimethyltyramine or candicine (Can) in GBE as trace constituents. Distribution of Tyr, NMT, Hor and Can was determined by a validated ion-exchange mechanism-based liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS) method in G. biloba samples, such as herbal drugs and dietary supplements. The total content of the four tyramine derivatives in various GBEs ranged from 7.3 up to 6357µg/g dry extract with NMT and Hor as most abundant ones. Considering the pharmacological activities and the revealed fluctuation in the concentration of the analysed adrenergic protoalkaloids, the presented rapid LC-ESI-MS method is proposed for monitoring of the levels of Tyr, NMT, Hor and Can in G. biloba products.

Bioavailability of Alfrutamide and Caffedymine and Their P-Selectin Suppression and Platelet-Leukocyte Aggregation Mechanisms in Mice.

BACKGROUND: Alfrutamide and caffedymine are phenolic amides found in plants, including garlic and cocoa. However, the bioavailability of alfrutamide and caffedymine and their effects on cardiovascular diseases (CVDs), particularly via effects on P-selectin expression(PSE) and platelet-leukocyte aggregation (PLA), are unknown. OBJECTIVE: The objective of this study was to investigate the bioavailability of alfrutamide and caffedymine and their effects on PSE and PLA, which are frequently involved in the progression of CVDs. METHODS: Cyclooxygenase (COX) I and COX-II activities and cAMP were determined by using COX and cAMP kits. Bioavailability was determined by HPLC analysis of plasma samples from Swiss Webster mice orally administered alfrutamide and caffedymine (10 µg each). PSE and PLA were also measured by flow cytometry using blood samples from the same mice. RESULTS: At 0.05 µmol/L, alfrutamide and caffedymine inhibited COX-I and COX-II by 20-40% (P < 0.05) and 16-33% (P < 0.05), respectively, compared with the control. At 0.1 µmol/L, the 2 compounds also inhibited platelet PSE by 28% (P < 0.05) and 35% (P < 0.05), respectively, compared with the control. The ß2-adrenoceptor antagonists ICI118551 and butoxamine partially suppressed the inhibition of PSE by caffedymine, suggesting that ß2 receptors are involved in inhibition by caffedymine but not by alfrutamide. At the same concentration (0.1 µmol/L), however, these 2 compounds inhibited PLA by 24-32% (P < 0.05) compared with the control. In addition, mice administered caffedymine and alfrutamide orally (10 µg/35 g body weight) exhibited maximum concentrations >0.6 µmol/L and significant inhibition of PSE by 23-34% (P < 0.05) and PLA by 20-27% (P < 0.05) compared with control mice. CONCLUSIONS: These data show the adequate bioavailability of alfrutamide and caffedymine and their different mechanisms of suppressing PSE and PLA: alfrutamide exerts its effects only via COX inhibition, whereas caffedymine works through both COX inhibition and cAMP amplification.

Synthesis, HPLC measurement and bioavailability of the phenolic amide amkamide.

Amkamide is a phenolic amide whose analogues were recently reported to have potent mitochondria protective activity, although their bioavailability is still unknown. Therefore, in this study, amkamide was synthesized and confirmed by nuclear magnetic resonance, and a high-performance liquid chromatography (HPLC) method was developed for analyzing the amides in biological samples. HPLC separation was performed on a Nova-Pak C18 column using a gradient condition and a coulometric electrochemical detector. The HPLC method was able to produce excellent and reproducible separations of amkamide from other amides (oretamide, becatamide, enferamide and veskamide). All five amides yielded outstanding peak resolutions with detection limits as low as 100 fmol. Therefore, the bioavailability of amkamide together with becatamide was determined in a mouse model. Their plasma concentrations were measured following two oral administrations (2 and 4 mg per 30 g body weight) using the HPLC method. As expected, relatively high amounts of amkamide and becatamide (51-58 µM) were detected at the time after administration when the maximum concentration was reached (approximately 20 and 25 min) at the high dose. The data indicate that HPLC methods with amide standards may be utilized to measure amkamide and its analogues in biological samples with excellent resolution and detection limits.

Human liver enzymes responsible for metabolic elimination of tyramine; a vasopressor agent from daily food.

Dietary tyramine is associated with hypertensive crises because of its ability to induce the release of catecholamines. The roles of monoamine oxidase (MAO); flavin-containing monooxygenase (FMO); and cytochrome P450 2D6 (CYP2D6) were studied in terms of the enzymatic elimination of tyramine in vitro at a substrate concentration of 1.0 µM; which is relevant to in vivo serum concentrations. Tyramine elimination by human liver supernatant fractions was decreased by ˜70% in the absence of NADPH. Pargyline; an MAO inhibitor; decreased tyramine elimination rates by ˜30%. Among recombinant P450 and FMO enzymes; CYP2D6 had a high activity in terms of tyramine elimination. Tyramine elimination rates were inhibited by quinidine and significantly correlated with bufuralol 1'-hydroxylation activities (a CYP2D6 marker). Liver microsomes genotyped for CYP2D6*10/*10 and CYP2D6*4/*4 showed low and undetectable activities; respectively; compared with the wild-type CYP2D6*1/*1. The present results suggest that tyramine is eliminated mainly by polymorphic CYP2D6. Tyramine toxicity resulting from differences in individual metabolic elimination is thus genetically determined.

Serotonin-transporter mediated efflux: a pharmacological analysis of amphetamines and non-amphetamines.

The physiological function of neurotransmitter transporter proteins like the serotonin transporter (SERT) is reuptake of neurotransmitter that terminates synaptic serotoninergic transmission. SERT can operate in reverse direction and be induced by SERT substrates including 5-HT, tyramine and the positively charged methyl-phenylpyridinium (MPP(+)), as well as the amphetamine derivatives para-chloroamphetamine (pCA) and methylene-dioxy-methamphetamine (MDMA). These substrates also induce inwardly directed sodium currents that are predominantly carried by sodium ions. Efflux via SERT depends on this sodium flux that is believed to be a prerequisite for outward transport. However, in recent studies, it has been suggested that substrates may be distinct in their properties to induce efflux. Therefore, the aim of the present study was a pharmacological characterization of different SERT substrates in uptake experiments, their abilities to induce transporter-mediated efflux and currents. In conclusion, the rank order of affinities in uptake and electrophysiological experiments correlate well, while the potencies of the amphetamine derivatives for the induction of efflux are clearly higher than those of the other substrates. These discrepancies can be only explained by mechanisms that can be induced by amphetamines. Therefore, based on our pharmacological observations, we conclude that amphetamines distinctly differ from non-amphetamine SERT substrates.

Interaction of organic cations with a newly identified plasma membrane monoamine transporter.

Many endogenous compounds and xenobiotics are organic cations that rely on polyspecific organic cation transporters (OCTs) to traverse cell membranes. We recently cloned a novel human plasma membrane monoamine transporter (PMAT) that belongs to the equillibrative nucleoside transporter (ENT) family. We have reported previously that, unlike other ENTs, PMAT (also known as ENT4) is a Na+-independent and membrane potential-sensitive transporter that transports monoamine neurotransmitters and the neurotoxin 1-methyl-4-phenylpyridinium (MPP+). These compounds are the known substrates for OCTs, which raises the possibility that PMAT functions as a polyspecific transporter like the OCTs. In the present study, we analyzed the interaction of PMAT with a series of structurally diverse organic cations using MDCK cells stably expressing human PMAT. Our study showed that PMAT interacts with many organic cations that have heterogeneous chemical structures. PMAT transports classic OCT substrates, such as tetraethylammonium, guanidine, and histamine. Prototype OCT inhibitors, including cimetidine, and type II cations (e.g., quinidine, quinine, verapamil, and rhodamine123) are also PMAT inhibitors. An analysis of molecular structures and apparent binding affinities revealed that charge and hydrophobicity are the principal determinants for transporter-substrate/inhibitor interaction. A planar aromatic mass seems to be important for high affinity interaction. trans-Stimulation and efflux studies demonstrate that PMAT is able to mediate bidirectional transport. These functional properties of PMAT are strikingly similar to those of the OCTs. We therefore conclude that PMAT can function as a polyspecific organic cation transporter, which may play a role in organic cation transport in vivo.

A new low-dose formulation of selegiline: clinical efficacy, patient preference and selectivity for MAO-B inhibition.

Three studies were performed using a fast dissolving formulation of selegiline hydrochloride designed for buccal absorption "Zydis Selegiline". The aim of the first study was to compare the therapeutic efficacy of Zydis Selegiline (1.25 mg or 10 mg) with conventional selegiline hydrochloride tablets "conventional selegiline tablets" (10 mg) in patients with Parkinson's disease (PD) who were previously treated with conventional selegiline tablets as an adjunct to levodopa/dopamine agonist therapy. Patients were observed for 4 weeks to ensure that they were stable. Stable patients (n=197) were then randomised to continue with conventional selegiline tablets 10 mg (n=68), or to treatment with Zydis Selegiline 1.25 mg (n=64) or Zydis Selegiline 10 mg (n=62) for 12 weeks in this randomised, parallel group study. A further aim was to establish the acceptability of Zydis Selegiline compared with conventional selegiline tablets. Patient preference for Zydis Selegiline was also evaluated in a second study, a single-dose, randomised, two-way crossover study conducted in patients with PD (n=148). Patients were stratified by the presence or absence of swallowing and salivation problems and were randomised to either Zydis Selegiline 5 mg or a placebo fast-dissolving formulation. In a third study, the degree of potentiation of the tyramine pressor effect following Zydis Selegiline was compared with that following conventional selegiline tablets in healthy volunteers. A total of 24 healthy volunteers were randomised to receive Zydis Selegiline 1.25 mg or conventional selegiline tablets 10 mg for 14-16 days in an open-label, randomised parallel group study. Both Zydis Selegiline (1.25 mg and 10 mg) treatments were shown to be therapeutically equivalent to conventional selegiline tablets 10 mg based on comparison of mean total Unified Parkinson's Disease Rating Scale (UPDRS) scores. Therapeutic equivalence was defined a priori as the 90% confidence interval (CI) for the difference in total UPDRS scores between groups to lie entirely within the range +/-5. The difference (90% CI) in mean adjusted total UPDRS between Zydis Selegiline 1.25 mg and conventional selegiline tablets 10 mg was -2.50 (-4.84, -0.17), and for Zydis Selegiline 10 mg and conventional selegiline tablets 10 mg, 0.04 (-2.30, 2.38). For the motor subscores of the UPDRS, differences between adjusted means (90% CI) compared with the conventional selegiline tablets group were: Zydis Selegiline 1.25 mg, -2.14 (-3.94, -0.33) and Zydis Selegiline 10 mg, -0.90 (-2.70, +0.91). Patients who switched from conventional selegiline tablets to Zydis Selegiline 1.25 mg showed a slight improvement in UPDRS scores following 12 weeks of treatment (standard error of difference 1.039; p=0.01). In the single-dose crossover study, most (61%) patients liked Zydis Selegiline 5 mg; a significantly greater proportion than the null hypothesis of 50% (p<0.002). However, only 62 patients (46%) indicated that they liked the taste of Zydis Selegiline. Nevertheless, the proportion of patients who preferred Zydis Selegiline (65%) to their usual medication was significantly greater than the null hypothesis of 50% (p<0.001). Similar findings were demonstrated in the 12-week study where a higher proportion of patients who received up to 3 months of treatment indicated a preference for either Zydis Selegiline 1.25 mg (90%) or Zydis Selegiline 10 mg (86%) over conventional selegiline tablets 10 mg. More than 90% of patients found Zydis Selegiline easy to take, with 61% rating it as extremely easy. Most (81%) patients taking Zydis Selegiline 1.25 mg liked the taste compared with 45% taking Zydis Selegiline 5 mg (in the previous study). Zydis Selegiline did not potentiate the tyramine effect: a pressor effect was elicited after 400 mg tyramine both before and after 14 days of treatment with Zydis Selegiline 1.25 mg. In contrast, after 14 days treatment with conventional selegiline tablets 10 mg, the threshold dose required to elicit the tyramine pressor response was significantly (p<0.0001) reduced from 400 mg to 200 mg. In summary, Zydis Selegiline at doses of 1.25 mg and 10 mg was therapeutically equivalent to conventional selegiline tablets 10 mg. The Zydis Selegiline formulation was well-liked by all patients, with most preferring Zydis Selegiline 1.25 mg to their usual selegiline tablet. Furthermore, Zydis Selegiline was well tolerated and, unlike conventional selegiline tablets, appeared to retain specificity for inhibition of monoamine oxidase type B (MAO-B), since it did not potentiate the pressor response to tyramine.

Duloxetine increases serotonin and norepinephrine availability in healthy subjects: a double-blind, controlled study.

Evidence suggests that compounds that increase the synaptic availability of more than one neurotransmitter have greater efficacy in the treatment of depression than single-acting drugs. Preclinical studies indicate that duloxetine acts to inhibit serotonin (5-HT) and norepinephrine (NE) transporters. The ability of duloxetine to alter 5-HT and NE reuptake was tested in 12 healthy male subjects. Placebo, desipramine 50 mg b.i.d., and duloxetine (80 mg q.d. or 60 mg b.i.d.) were compared in a randomized, double-blind, three-period crossover study in 12 healthy male subjects. Whole-blood 5-HT, urinary excretion of NE and major metabolites, and TYR PD30 (IV tyramine pressor dose needed to increase systolic blood pressure by 30 mmHg) were measured at steady state. Vital signs were measured periodically. Duloxetine affected 5-HT reuptake, with whole-blood 5-HT depletion vs placebo (80 mg q.d.: p=0.07; 60 mg b.i.d.: p=0.02; combined regimens: p=0.01). Cardiovascular changes reflecting increased sympathetic tone were observed with both duloxetine and desipramine, and both treatments significantly decreased whole body NE turnover (p<0.01). Duloxetine and desipramine were associated with similar mean increases in fractional extraneuronal NE concentration, although these changes did not reach statistical significance. TYR PD30 increased significantly with desipramine dosing (p<0.01). In conclusion, whole-blood measurements confirm that duloxetine inhibits platelet 5-HT uptake in vivo. Urinary and cardiovascular measurements suggest that duloxetine has an effect on NE synthesis and turnover, indicative of NE reuptake inhibition. The lack of a detectable impact of duloxetine on TYR PD30 suggests that this may not be the most sensitive indirect measure of NE reuptake when assessing dual reuptake inhibitors.

Tyramine pharmacokinetics and reduced bioavailability with food.

Tyramine challenge studies have demonstrated that it requires approximately twice the amount of tyramine administered with a meal compared to administration after a fast to elicit the same effect, suggesting a reduction in bioavailability of tyramine when administered with food. The pharmacokinetics of tyramine when administered in a fasted versus a fed state were studied. A single 200-mg dose of tyramine was administered orally to healthy subjects both after an overnight fast and during a meal. Systemic exposure to tyramine was reduced by 53% (p < 0.05), and the maximum concentration of tyramine was reduced by 72% (p < 0.05) when the dose was administered during a meal. Tyramine maximum serum concentration was observed between 20 minutes and 1 hour when the dose was administered after an overnight fast and appeared to be delayed and/or prolonged by administration during a meal. Tyramine oral clearance was 135 +/- 55.4 L/min, maximum observed serum concentration was 37.7 +/- 26.01 ng/mL, and tyramine elimination half-life was 0.533 (range: 0.330-0.668) hours after administration to fasted subjects. Tyramine bioavailability was significantly reduced when administered with a meal compared to after a fast. The results suggest that larger amounts of dietary tyramine will be required to induce a pressor response equivalent to that following encapsulated tyramine administered in the fasted state.

Acute limb ischemia does not facilitate but inhibits norepinephrine release from muscle sympathetic nerve endings in anesthetized rabbit.

Although myocardial ischemia is associated with regional cardiac sympathetic nerve deterioration, it remains unknown whether acute hindlimb ischemia impairs muscle sympathetic nerve function. In the study presented here we implanted dialysis probes in the adductor muscle of anesthetized rabbits and measured dialysate norepinephrine levels as an index of muscle sympathetic nerve activity. Acute hindlimb ischemia was induced by injection of microspheres and occlusion of the common iliac artery. Dialysate norepinephrine levels decreased from 19.3 +/- 3.5 pg/ml at control to 9.4 +/- 3.7 pg/ml at 30 min of ischemia and further to 1.7 +/- 0.2 pg/ml at 75 min of ischemia. During acute hindlimb ischemia, baroreflex (bilateral carotid occlusion) and high potassium level-induced norepinephrine response was inhibited, but tyramine-induced norepinephrine response was preserved. In conclusion, acute hindlimb ischemia caused decreases in dialysate norepinephrine levels. This reduction may be mediated by an impairment of axonal conduction and/or of norepinephrine releasing function at skeletal muscle sympathetic nerve endings.

Linezolid, a novel oxazolidinone antibiotic: assessment of monoamine oxidase inhibition using pressor response to oral tyramine.

The primary objective of this study was to compare the effects of oral linezolid with moclobemide and placebo on the pressor response to oral tyramine. Secondary objectives were to determine possible mechanisms of the effect based on changes in the pharmacokinetics of tyramine and to evaluate alternative methods for quantifying the pressor effect. Subjects received linezolid (625 mg bid orally), moclobemide (150 mg tid orally), or placebo for up to 7 days. Using the oral tyramine dose producing a >30 mmHg increase in systolic blood pressure (SBP) (PD>30), a positive pressor response was defined as a PD>30 index (pretreatment/treatment ratio of PD>30) of > or = 2. There were 8/10, 11/11, and 1/10 responders with linezolid, moclobemide, and placebo, respectively. Responses returned to baseline within 2 days of drug discontinuation. The ratio of mean greatest SBP and heart rate at the time of greatest SBP (GSBP/HR) increased linearly with tyramine dose both pretreatment and during treatment with linezolid and moclobemide. During treatment, responses to tyramine when subjects took linezolid or moclobemide were significantly different from placebo. Both drugs significantly decreased tyramine oral clearance compared with placebo. Urinary excretion of catecholamines and metabolites was consistent with MAOI activity of the drugs, but results were variable. The MAOI activity of linezolid is similar to that of moclobemide, a drug used clinically without food restrictions. Restrictions to normal dietary intake of tyramine-containing foods are not warranted when taking linezolid.

Assessment of the serotonin and norepinephrine reuptake blocking properties of duloxetine in healthy subjects.

Duloxetine is a dual inhibitor of norepinephrine (NE) and serotonin (5-HT) uptake. Initial trials conducted in depressed patients using regimens of 20 mg/day or less did not convincingly demonstrate its efficacy as an antidepressant. The aim of this study was to assess the effects of duloxetine on the 5-HT and NE reuptake processes in healthy human volunteers. Twenty-seven healthy young males without a history of psychiatric disorder were randomly assigned to four groups, each group receiving one of the following daily drug regimens: placebo, clomipramine (a potent 5-HT/NE reuptake blocker) 100 mg/day, duloxetine 20 mg/day, or duloxetine 60 mg/day. In order to assess the NE reuptake process, the pressor response to intravenous tyramine (4 and 6 mg) was measured. Determination of the whole blood 5-HT content was used to evaluate the 5-HT reuptake blockade. These measurements were performed at baseline and repeated after 7 and 14 days of drug intake. Both duloxetine, at doses of 20 to 60 mg/day, and clomipramine significantly interfered with the 5-HT reuptake process, as demonstrated by marked decreases in blood 5-HT concentrations. However, the same doses of duloxetine, unlike clomipramine, failed to impede the usual increase in blood pressure that follows a tyramine intravenous infusion, indicating that clomipramine but not duloxetine blocked NE reuptake. At doses tested in a population of healthy volunteers, duloxetine acted as a selective 5-HT reuptake inhibitor, having no clear effect on the NE reuptake process. Nevertheless, given that the highest dose of duloxetine increased supine systolic blood pressure, it is possible that it represents the threshold regimen for NE reuptake inhibition.

Increased low density lipoprotein degradation in aorta of irradiated mice is inhibited by preenrichment of low density lipoprotein with alpha-tocopherol.

We previously reported that upper thoracic exposure to ionizing radiation (IR) accelerates fatty streak formation in C57BL/6 mice and that such effects are inhibited by overexpression of the antioxidant enzyme CuZn-superoxide dismutase (SOD). Notably, IR-accelerated lesion formation is strictly dependent on a high fat diet (i.e., atherogenic lipoproteins) but does not involve alterations in circulating lipid or lipoprotein levels. We thus proposed that IR promotes changes in the artery wall that enhance the deposition of lipoprotein lipids. To address this hypothesis, we examined the effects of IR on aortic accumulation and degradation of low density lipoproteins (LDL). Ten-week-old C57BL/6 mice were exposed to a single (8-Gy) dose of (60)Co radiation to the upper thoracic area or were sham irradiated (controls) and were then placed on the high fat diet. Five days postexposure, the mice received either (125)I-labeled LDL ((125)I-LDL) (which was used to measure intact LDL) or (125)I-labeled tyramine cellobiose ((125)I-TC)-LDL (which was used to measure both intact and cell-degraded LDL) via tail vein injection. On the basis of trichloroacetic acid (TCA)-precipitable counts in retroorbital blood samples, > or =95% of donor LDL was cleared within 24 h and there were no differences in time-averaged plasma concentrations of the two forms of LDL among irradiated and control mice. Aortic values increased markedly within the first hour and thereafter exhibited a slow increase up to 24 h. There were no differences between irradiated and control mice at 1 h, when values primarily reflected LDL entry, but a divergence was observed thereafter. At 24 h, (125)I-TC-associated counts were 1.8-fold higher in irradiated mice (P = 0.10). In contrast, (125)I-LDL-associated counts were 30% lower in irradiated mice (P< 0.05), suggesting that most of the retained (125)I-TC was associated with LDL degradation products. Consistent with the proposed involvement of oxidative or redox-regulated events, IR-induced LDL degradation was lower in SOD-transgenic than wild-type mice (P<0.05). The importance of LDL oxidation was suggested by observations that IR-induced LDL degradation was significantly reduced by preenriching LDL with alpha-tocopherol. On the basis of these results, we propose that IR elicits SOD-inhibitable changes in the artery wall that enhance LDL oxidation and degradation leading to the deposition of LDL-borne lipids. These studies provide additional support for the role of oxidation in lipoprotein lipid deposition and atherogenesis and suggest that IR promotes an arterial environment that stimulates this process in vivo.

Uptake and degradation of radioactively labelled albumin microspheres as markers for Kupffer cell phagocytosis.

Rats were injected with liposomes containing iodixanol (CTP10 Injection; 100 mg iodine per kg body weight) followed by a second injection of 125I-tyramine-cellobiose-albumin microspheres. The amounts of phagocytosed and degraded labelled albumin in liver were measured. A reduced uptake and degradation of albumin microspheres was observed when the labelled microspheres were injected 2 h or 24 h after the liposomes compared with that obtained in control animals receiving saline. No effect on the uptake and degradation of labelled microspheres was observed when the time lag between the injection of liposomes and labelled microspheres was 1 week. The data show that the uptake and degradation of 125I-tyramine-cellobiose-albumin microspheres can be used as indicators of Kupffer cell phagocytotic function following drug uptake by these cells.

Analysis and prediction of absorption profile including hepatic first-pass metabolism of N-methyltyramine, a potent stimulant of gastrin release present in beer, after oral ingestion in rats by gastrointestinal-transit-absorption model.

The prediction method for the plasma concentration-time profile of N-methyltyramine (NMT), a potent stimulant of gastrin release present in beer after oral ingestion in rats was examined using the previously developed Gastrointestinal (GI)-Transit-Absorption Model, with the addition of a process of hepatic first-pass metabolism. Phenol red was used as a nonabsorbable marker for estimation of the GI transit rate constant for eight segments in the GI tract. The first order absorption rate constant for each segment was estimated by means of a conventional in situ closed loop method. The results of in situ absorption experiments showed that NMT is well absorbed in the small intestine, especially in the duodenum and jejunum. Using the GI-Transit-Absorption Model, it was demonstrated that more than 90% of orally ingested NMT is absorbed in the small intestine, and that the substantial absorption site for NMT in vivo is the lower jejunum and the ileum. However, the observed bioavailability was only 39.0%. The in vitro metabolism study clarified that NMT is metabolized in the liver, but not in the small-intestinal mucosa. With the hepatic intrinsic clearance value (2.0 liters/h) calculated from the rate of metabolism in vitro, the hepatic availability was estimated to be 0.510 on the basis of a well stirred model, which was validated by two other methods to calculate the hepatic availability of NMT. The plasma concentration-time curve and bioavailability of NMT after oral ingestion were well predicted by the GI-Transit-Absorption Model with the hepatic first-pass metabolism process.

Catecholamine handling in the porcine heart: a microdialysis approach.

Experimental findings suggest a pronounced concentration gradient of norepinephrine (NE) between the intravascular and interstitial compartments of the heart, compatible with an active neuronal reuptake (U1) and/or an endothelial barrier. Using the microdialysis technique in eight anesthetized pigs, we investigated this NE gradient, both under baseline conditions and during increments in either systemic or myocardial interstitial fluid (MIF) NE concentration. At steady state, baseline MIF NE (0.9 +/- 0.1 nmol/l) was higher than arterial NE (0.3 +/- 0.1 nmol/l) but was not different from coronary venous NE (1.5 +/- 0.3 nmol/l). Local U1 inhibition raised MIF NE concentration to 6.5 +/- 0.9 nmol/l. During intravenous NE infusions (0.6 and 1.8 nmol. kg(-1). min(-1)), the fractional removal of NE by the myocardium was 79 +/- 4% to 69 +/- 3%, depending on the infusion rate. Despite this extensive removal, the quotient of changes in MIF and arterial concentration (DeltaMIF/DeltaA ratio) for NE were only 0.10 +/- 0.02 for the lower infusion rate and 0.11 +/- 0.01 for the higher infusion rate, whereas U1 blockade caused the DeltaMIF/DeltaA ratio to rise to 0.21 +/- 0.03 and 0.36 +/- 0.05, respectively. From the differences in DeltaMIF/DeltaA ratios with and without U1 inhibition, we calculated that 67 +/- 5% of MIF NE is removed by U1. Intracoronary infusion of tyramine (154 nmol. kg(-1). min(-1)) caused a 15-fold increase in MIF NE concentration. This pronounced increase was paralleled by a comparable increase of NE in the coronary vein. We conclude that U1 and extraneuronal uptake, and not an endothelial barrier, are the principal mechanisms underlying the concentration gradient of NE between the interstitial and intravascular compartments in the porcine heart.