Involvement of serotonin 5-HT3 receptors in the modulation of noradrenergic transmission by serotonin reuptake inhibitors: a microdialysis study in rat brain

Rationale

Selective serotonin reuptake inhibitors (SSRIs), in addition to being able to enhance serotonergic neurotransmission, are able to modulate other brain systems involved in depression.

Objectives

This study evaluates the neurochemical effect of the SSRI citalopram on brain noradrenergic activity and the serotonin receptor involved in this effect.

Methods

Dual-probe microdialysis in the locus coeruleus (LC) and prefrontal cortex (PFC) was performed in freely awake rats.

Results

Systemic citalopram (10 mg/kg, i.p.) increased noradrenaline (NA) in the LC (E _max?=?141?±?13 %) and simultaneously decreased NA in the PFC (E_max?=??46?±?7 %). In the local presence into the LC of the α₂-adrenoceptor antagonist RS79948 (1 μM), systemic citalopram increased NA in the LC (E_max?=?157?±?25 %) and PFC (E_max?=?175?±?24 %). Local citalopram (0.1–100 μM) into the LC induced NA increase in the LC (E_max?=?210?±?25 %) and decrease in the PFC (E_max?=??38?±?9 %). Local LC citalopram effect was abolished by LC presence of the 5-HT₃ receptor antagonist MDL72222 (1 μM) but not the 5-HT_1/2 receptor antagonist methiothepin (1 μM). Systemic citalopram in the LC presence of MDL72222 did not modify NA in the LC but increased NA in the PFC (E_max?=?158?±?26 %). Local citalopram into the PFC enhanced NA (E_max?=?376?±?18 %) in the area, which was prevented by MDL72222.

Conclusions

The SSRI citalopram modulates central noradrenergic neurotransmission by activation, through endogenous serotonin, of 5-HT₃ receptors expressed in the somatodendritic (LC) and terminal (PFC) areas, which subsequently promote an enhancement of local NA. Therefore, 5-HT₃ receptors and somatodendritic α₂-adrenoceptors in the LC play an important role in the global effect of SSRIs.     

In vivo potentiation of reboxetine and citalopram effect on extracellular noradrenaline in rat brain by α2-adrenoceptor antagonism

The therapeutic activity of noradrenaline reuptake inhibitors (NaRIs) and serotonin reuptake inhibitors (SSRIs) as antidepressant is based on their ability to increase monoamine concentrations in the synaptic cleft. α₂-Adrenoceptors inhibit noradrenaline (NA) release, which modulates antidepressant neurochemical activity. The present study assesses the influence of the addition of the selective α₂-adrenoceptor antagonist RS79948 to the NaRI reboxetine and the SSRI citalopram on brain extracellular NA. Dual-probe microdialysis technique in the locus coeruleus (LC) and prefrontal cortex (PFC) was performed in freely moving rats. Acute reboxetine (3 and 5 mg/kg i.p.) promoted a dose-dependent increase of NA in LC (164 ± 15%; 243 ± 24%) and PFC (140 ± 7%; 181 ± 30%). Acute citalopram (5 mg/kg i.p.) did not change NA in LC or PFC, but at 10 mg/kg i.p. increased NA in LC (144 ± 14%) and decreased it in PFC (− 42 ± 7%). An inactive dose of RS79948 (0.1 mg/kg i.p.) in rats pretreated with reboxetine (3 mg/kg i.p.) or citalopram (5 mg/kg i.p.) induced a significant enhancement of NA in LC (reboxetine: 462 ± 137%; citalopram: 142 ± 11%) and PFC (reboxetine: 281 ± 56%; citalopram: 130 ± 16%). The results indicate that co-administration of selective α₂-adrenoceptor antagonist drugs might improve the effects of NaRI or SSRI antidepressants by enhancing extracellular NA concentrations in the brain.     

Assessing the neurotoxic effects of palytoxin and ouabain,both Na+/K+-ATPase inhibitors,on the myelinated sciatic nerve fibres of the mouse: An ex vivo electrophysiological study

Palytoxin (PlTX) is a marine toxin originally isolated from the zoantharians of the genus Palythoa. It is considered to be one of the most lethal marine toxins that block the Na⁺/K⁺-ATPase. This study was designed to investigate the acute effects of PlTX and ouabain, also an Na⁺/K⁺-ATPase blocker, on the mammalian peripheral nervous system using an ex vivo electrophysiological preparation: the isolated mouse sciatic nerve. Amplitude of the evoked nerve compound action potential (nCAP) was used to measure the proper functioning of the sciatic nerve fibres. The half-vitality time of the nerve fibres (the time required to inhibit the nCAP to 50% of its initial value: IT₅₀) incubated in normal saline was 24.5 ± 0.40 h (n = 5). Nerves incubated continuously in 50.0, 10.0, 1.0, 0.5, 0.250 and 0.125 nM of PlTX had an IT₅₀ of 0.06 ± 0.00, 0.51 ± 0.00, 2.1 ± 0.10, 8.9 ± 0.30, 15.1 ± 0.30 h, and 19.5 ± 0.20 h, respectively (n = 5, 3, 4, 4, 10). PlTX was extremely toxic to the sciatic nerve fibres, with a minimum effective concentration (mEC) of 0.125 nM (n = 5) and inhibitory concentration to 50% (IC₅₀) of 0.32 ± 0.08 nM (incubation time 24 h). Ouabain was far less toxic, with a mEC of 250.0 μM (n = 5) and IC₅₀ of 370.0 ± 18.00 μM (incubation 24.5 h). Finally, when the two compounds were combined – e.g. pre-incubation of the nerve fibre in 250.0 μM ouabain for 1 h and then exposure to 1.0 nM PlTX – ouabain offered minor a neuroprotection of 9.1–17.6% against PlTX-induced neurotoxicity. Higher concentrations of ouabain (500.0 μM) offered no protection. The mouse sciatic nerve preparation is a simple and low-cost bioassay that can be used to assess and quantify the neurotoxic effects of standard PlTX or PlTX-like compounds, since it appears to have the same sensitivity as the haemolysis of erythrocytes assay – the standard ex vivo test for PlTX toxicity.     

Characterization of noradrenaline release in the locus coeruleus of freely moving awake rats by in vivo microdialysis

Rationale The origin and regulation of noradrenaline (NA) in the locus coeruleus (LC) is unknown.Objectives The neurochemical features of NA overflow (nerve impulse dependence, neurotransmitter synthesis, vesicle storage, reuptake, ₂-adrenoceptor-mediated regulation) were characterized in the LC.Methods Brain microdialysis was performed in awake rats. Dialysates were analyzed for NA.Results NA in the LC decreased via local infusion of Ca²⁺-free medium (–42±5%) or the sodium channel blocker tetrodotoxine (TTX) (–47±8%) but increased (333±40%) via KCl-induced depolarization. The tyrosine hydroxylase (TH) inhibitor -methyl-p-tyrosine (250 mg kg^–1, i.p.) and the vesicle depletory drug reserpine (5 mg kg^–1, i.p.) decreased NA. Therefore, extracellular NA in the LC satisfies the criteria for an impulse flow-dependent vesicular exocytosis of neuronal origin. Local perfusion of the ₂-adrenoceptor agonist clonidine (0.1–100 M) decreased NA (E_max=–79±5%) in the LC, whereas the opposite effect (E_max=268±53%) was observed with the _2A-adrenoceptor antagonist BRL44408 (0.1–100 M). This suggests a tonic modulation of NA release through local _2A-adrenoceptors. The selective NA reuptake inhibitor desipramine (DMI) (0.1–100 M) administered into the LC increased NA in the LC (E_max=223±40%) and simultaneously decreased NA in the cingulate cortex, confirming the modulation exerted by NA in the LC on firing activity of noradrenergic cells and on the subsequent NA release in noradrenergic terminals.Conclusion Synaptic processes underlying NA release in the LC are similar to those in noradrenergic terminal areas. NA in the LC could represent local somatodendritic release, but also the presence of neurotransmitter release from collateral axon terminals.     

Inhibition of intestinal motility by the putative BK<Subscript>Ca</Subscript> channel opener LDD175

LDD175 (4-chloro-7-trifluoromethyl-10H-benzo[4,5]furo[3,2-b]indole-1-carboxylic acid) is a benzofuroindole compound characterized previously as a potent opener of the large conductance calcium activated (BK_Ca) channels. Activators of the BK_Ca channels are potential therapies for smooth muscle hyperactivity disorders. The present study investigates the influence of LDD175 on the mechanical activity of the ileum smooth muscle. LDD175 inhibited spontaneous contractions of the ileum in a concentration-dependent manner (pEC₅₀=5.9 ± 0.1) (E _max=96 ± 1.0% at 100 μM, n=3). It also remarkably inhibited contractions due to acetylcholine (ACh) (pEC₅₀=5.3 ± 0.1)(E _max=97.7 ± 2.3%, n=6) and electrical field stimulation (EFS) (pEC₅₀=5.5 ± 0.1) (E _max=83.3 ± 6.0%, n=6). In strips precontracted by 20 mM KCl, LDD175 significantly reduced the contractions yielding a pEC₅₀ of 6.1 ± 0.1 and E _max of 96.6 ± 0.9%, (n=6). In 60 mM KCl, a concentration-dependent inhibition was observed with respective pEC₅₀ and E _max values of 4.1 ± 0.1 and 50.8 ± 5.0% (n=3). BK_Ca channel blockers iberiotoxin (IbTX) and tetraethylammonium chloride (TEA, 1 mM) attenuated the relaxative effect of LDD175 but not barium chloride (BaCl₂), and glibenclamide (K_IR and K_ATP channel blockers, respectively). These data demonstrate the antispasmodic activity of LDD175 attributable to the potentiation of the BK_Ca channels.     

Inhibition of T-cell activation and proliferation by mycophenolic acid in patients awaiting liver transplantation: PK/PD relationships

Mycophenolic acid (MPA) plasma concentrations were reported to be associated with a decrease in T-cell proliferation, and in both IL-2 α-chain (CD25) and transferin receptor (CD71) expression. The aim of this study was to confirm, quantify and model these PK/PD relationships.Full profiles of MPA plasma concentrations, T-cell proliferation, intracytoplasmic IL-2 and TNF-α expression, and both CD71 and CD25 expression were collected over the 12 h after dosing in 10 patients on the waiting list for liver transplantation. Data were analyzed using NONMEM^®.Both CD25 and CD71 expression and T cell proliferation clearly decreased (median of decrease from baseline 62%, 68% and 94%, respectively) with increasing MPA concentrations, in contrast to IL-2 and TNF-α expression. The CD25 and CD71 baseline expression (E₀) and maximum effect (E_max) were correlated with the E₀ and E_max values of T-cell proliferation (r² = 0.509 and r² = 0.622, respectively). The CD25, CD71 expression and T-cell proliferation profiles were adequately fitted using a sigmoid inhibitory E_max model. Low estimated values (≤2 mg/L) for 50% inhibitory MPA concentrations were obtained. This study confirmed a transient MPA concentration-dependent decrease in T-cells expressing CD25 and CD71 and a strong reduction of T-cell proliferation and showed that CD25 and CD71 expression was correlated with T-cell proliferation.     

Characterization of cannabinoid receptors coupled to vasorelaxation by endothelium-derived hyperpolarizing factor

We have recently proposed that an endogenous cannabinoid may be an endothelium-derived hyperpolarizing factor (EDHF), and we have now characterized the cannabinoid receptors mediating these responses. EDHF-mediated vasorelaxations to carbachol (ED₅₀=3.26±0.57 nmol; the maximum relaxation, R _max=87.0±2.5%) were opposed by the selective cannabinoid CB₁ antagonist, LY320135: at 2 μM ED₅₀ for carbachol was 10.4±2.6 nmol and R _max was 66.9±6.2%, at 10 μM ED₅₀ was 15.9±4.0 nmol and R _max was 34.0±4.3%. However, these responses were unaffected by another putative CB₁ ligand, AM630 (10 μM), or a CB₂ selective antagonist, SR144528 (100 nM–1 μM). None of the antagonists influenced vasorelaxation to either the potassium channel activator levcromakalim or sodium nitroprusside. Coupled to our previous observation that the CB₁ receptor antagonist SR141716A opposes EDHF-mediated relaxation, the present observations point to the involvement of a cannabinoid receptor, which may be CB₁ or CB₁-like, in EDHF-mediated vasorelaxation. Received: 6 July 1998 / Accepted: 2 October 1998     

ANALYSIS OF THE ANTAGONISM BY PRAZOSIN OF NORADRENALINE AND PHENYLEPHRINE INDUCED CONTRACTIONS OF THE RAT ANOCOCCYGEUS MUSCLE*

• 1 Blockade of α-adrenoreceptors was analyzed with prazosin in the rat anococcygeus muscle contracted with noradrenaline (NA) and phenylephrine (PHE).
• 2 Prazosin (1.1 times 10^–8– 8.8 times 10^–7M) competitively blocked phenylephrine-induced contractions (pA₂= 8.93 ± 0.04, slope = 0.85 ± 0.07) whilst its antagonism of NA was non-competitive (pA₂= 8.45 ± 0.04, slope = 0.46 ± 0.03).
• 3 At higher concentrations, (1.76 times 10^–5– 5.28 times 10^–6M) prazosin competitively antagonized NA-induced contractions (pA₂= 7.58 ± 0.11, slope = 0.93 ± 0.09).
• 4 Phentolamine also reduced the effect of NA competitively (pA₂= 8.11 ± 0.09, slope = 0.82 ± 0.26).
• 5 It is concluded that low concentrations of prazosin selectively blocked an α₁-adrenoreceptor component of NA-mediated response whilst higher concentrations of prazosin non-selectively blocked both α₁- and α₂-adrenoreceptors.

     

The prototypic pharmacogenetic drug debrisoquine is a substrate of the genetically polymorphic organic cation transporter OCT1

Debrisoquine is a probe drug for in vivo phenotyping of human CYP2D6 metabolic activity. However, debrisoquine is positively charged under physiological conditions and it is unclear how it enters the hepatocytes to undergo CYP2D6 metabolism. We analysed whether debrisoquine is a substrate of the hepatic organic cation transporter OCT1 and whether drug–drug interactions at OCT1, or polymorphisms in OCT1 gene, affect debrisoquine uptake.Debrisoquine showed low carrier-independent membrane permeability (P_e of 0.01 × 10^?6 cm/s in artificial PAMPA membranes) and strongly inhibited the uptake of the model OCT1 substrate MPP⁺ (IC₅₀ of 6.2 ± 0.8 μM). Debrisoquine uptake was significantly increased in HEK293 cells overexpressing OCT1 compared to control cells. The OCT1-mediated uptake of debrisoquine followed Michaelis–Menten kinetics (K_M of 5.9 ± 1.5 μM and V_max of 41.9 ± 4.5 pmol/min/mg protein) and was inhibited by known OCT1 inhibitors and by commonly used drugs. OCT1-mediated debrisoquine uptake was reduced or missing in cells expressing loss-of-function OCT1 isoforms. Deletion of Met420 or substitution of Arg61Cys or Gly401Ser reduced V_max by 48, 63 and 91%, respectively, but did not affect the K_M. The OCT1 isoforms carrying Cys88Arg or Gly465Arg substitutions completely lacked OCT1-mediated debrisoquine uptake.In conclusion, debrisoquine is a substrate of OCT1 and has the potential to be used as a phenotyping marker for OCT1 activity. Moreover, variations in debrisoquine metabolic phenotypes and their associations with diseases may be due not only to genetic variations CYP2D6, but also in OCT1.     

Evidence of competitive inhibition of methotrexate absorption by leucovorin calcium in rat small intestine

The effect of leucovorin calcium on the intestinal absorption of methotrexate in rat small intestine was investigated using an in situ rat gut technique. First, the kinetic absorption in situ parameters for methotrexate in solution were obtained: V_m=21.54 (±2.22) μM/h; K_m=10.51 (±1.08) μM; k_a=0.26 (±0.03) h⁻¹ and AIC=−188.63. The inhibitory effect of leucovorin calcium in methotrexate intestinal absorption has been investigated by perfusing of 10 μM methotrexate isotonic solutions containing increasing concentrations of leucovorin calcium (10–500 μM), and the remaining concentrations of both compounds were measured. A competitive inhibition of methotrexate absorption was detected: the apparent absorption rate constant of the drug decreased as the initial leucovorin calcium concentration increased. Higher leucovorin calcium concentrations, however, did not completely abolish the absorption of the drug (at 500 μM of leucovorin calcium, only 84% inhibition was observed). Apparent parameters characterizing the absorption of leucovorin calcium in the presence of methotrexate 10 μM were: V_mi=14.70 (±1.74) μM; K_mi=9.43 (±1.59) μM; k_ai=0.28 (±0.02) h⁻¹; AIC=−191.53). We can concluded that methotrexate and leucovorin calcium compete for the same intestinal carrier system. This means that since leucovorin calcium, because of its ready conversion to other tetrahydrofolic derivatives (McEvoy, 1996. AHFS Drug Information, Bethesda, MD, pp. 751–758), is administered together with methotrexate in order to prevent the hematopoietic and reticuloendothelial toxic effects of folic acid antagonists, using high leucovorin calcium concentrations, when the urine excretion is decreased, could prevent intestinal drug reabsorption and the drug could then be excreted in the feces, thereby decreasing the risk of poisoning.     

L-DOPA pharmacokinetics in the MPTP-lesioned macaque model of Parkinson's disease

ObjectiveThe 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned non-human primate is widely used to model Parkinson's disease (PD) and to evaluate the efficacy of new therapies. However, some doubts have been raised about the translatability of findings in the MPTP-lesioned monkey, because the doses of l-3,4-dihydroxyphenylalanine (l-DOPA) required to alleviate parkinsonism and elicit dyskinesia are high, on a mg/kg basis, when compared to clinical practice. Thus, in the MPTP-lesioned macaque, doses ranging from 20 to 40 mg/kg might be used, while in the clinic single l-DOPA administrations ranging from 100 to 200 mg are more typical. However, bioavailability of drugs varies between species and it is unknown how plasma l-DOPA levels providing therapeutic benefit in the non-human primate compare to those having similar actions in PD patients.MethodsWe administered acute challenges of l-DOPA 30 mg/kg orally to MPTP-lesioned macaques with established dyskinesia, and determined plasma, brain and cerebrospinal fluid (CSF) levels of l-DOPA using high-performance liquid chromatography–mass spectrometry/mass spectrometry.ResultsThe maximal plasma concentration of l-DOPA (C_max) was 18.2 ± 3.8 nmol/ml and was achieved 1.6 ± 0.3 h after administration (t_max). Half-life was 58.8 ± 22.7 min. l-DOPA levels in the caudate nucleus at peak behavioural effect were 3.3 ± 0.7 μg/g tissue protein while they were 1.5 ± 0.1 nmol/ml in the CSF.ConclusionsAlthough therapeutically-active doses of l-DOPA administered to the MPTP-lesioned macaque are higher on a mg/kg basis than those administered in clinical settings, they lead to l-DOPA C_max similar to those achieved with 200 mg l-DOPA in clinic. l-DOPA t_max and half-life are also similar to those reported in human.     

Non-genomic effects of catecholestrogens in the in vitro rat uterine contraction

• 1.1. The effects of catecholestrogens 2-hydroxyestradiol (2-OH E₂, 0.6–30 μM), 4-hydroxy-estradiol (4-OH E₂, 1–30 μM) and 2-methoxyestradiol (2-MeO E₂, 0.6–30 μM) on rat uterine contraction induced by KCI (60 mM), have been assayed.
• 2.2. All drugs assayed relaxed the tonic-contraction induced by KCI in a concentration-dependent way. The EC₅₀s were: 4.4 ± 0.5, 4.2 ± 0.3 and 8.5 ± 0.7 μM for 2-MeO E₂, 2-OH E₂ and 4-OH E₂, respectively. This relaxing effect was counteracted by CaCl₂ (1–10 mM) but not by the calcium channel agonist Bay k 8644 (1 nM-1 μM).
• 3.3. The effect of 2-MeO E₂ is not modified by propranolol (1 μM), cycloheximide (35 μM), actinomycin D (4 μM), α-difluoromethyl-ornithine (10 μM) or genistein (10 μM). Not did cycloheximide (35 μM) or actinomycin D (4 μM) modify the relaxing effect of 2-OH E₂ and 4-OH E₂. Propranolol (1 μM) significantly increased the effect of 4-OH E₂ but not the effect of 2-OH E₂.
• 4.4. Our results suggest that the relaxing effect of catecholestrogens in the rat uterus is a non-genomic effect and could be related to inhibition of extracellular calcium entry.

     

Effects of receptor density on Nociceptin/OrphaninFQ peptide receptor desensitisation: studies using the ecdysone inducible expression system

Pretreatment of the G-protein coupled nociceptin receptor (NOP) with nociceptin/orphaninFQ (N/OFQ) produces desensitisation. The influences of receptor expression and genomic effects are largely unknown. We have used an ecdysone-inducible NOP expression system in a CHO line (CHO_INDhNOP) to examine the effects of N/OFQ pretreatment upon receptor density, GTPγ[³⁵S] binding, cAMP formation and NOP-mRNA. CHO_INDhNOP induced with 5 and 10 μM PonasteroneA (PonA) for 20 h produced NOP densities (B _max) of 194 and 473 fmol. mg^-1 protein, respectively. This was accompanied by decreased NOP mRNA. The lower B _max is typical of the central nervous system. Pretreatment with 1 μM N/OFQ significantly (p < 0.05) reduced B _max at 5 and 10 μM PonA to 100 and 196 fmol. mg^-1 protein, respectively. There was no change in binding affinity. Along with the reduction in B _max, potency and efficacy for N/OFQ-stimulated GTPγ[³⁵S] binding were also reduced (5 μM PonA: pEC₅₀-control = 8.55 ± 0.06, pretreated = 7.88 ± 0.07; E _max-control = 3.52 ± 0.43, pretreated = 2.48 ± 0.10; 10 μM PonA: pEC₅₀-control = 8.41 ± 0.18, pretreated = 7.76 ± 0.03; E _max-control = 5.07 ± 0.17, pretreated = 3.38 ± 0.19). For inhibition of cAMP formation, there was a reduction in potency (5 μM PonA: pEC₅₀-control = 9.78 ± 0.08, pretreated = 8.92 ± 0.13; 10 μM PonA: pEC₅₀-control = 9.99 ± 0.07, pretreated = 9.04 ± 0.14), but there was no reduction in efficacy. In addition, there were 39 and 31% reductions in NOP mRNA at 5 and 10 μM PonA, respectively, but these measurements were made following concurrent N/OFQ challenge and PonA induction. In CHO_INDhNOP, we have shown a reduction in cell surface receptor numbers and a reduction in functional coupling after N/OFQ pretreatment. This was observed at pseudo-physiological and supraphysiological receptor densities. Moreover, we also report a reduction in NOP mRNA, but further studies are needed which include ‘pulsing’ PonA and desensitizing following wash-out.     

Mediation by 5-HT2 Receptors of 5-Hydroxytryptamine-Induced Contractions of Human Placental Vein

• 1.In isolated human placental chorionic vein segments, 5-hydroxytryptamine (5-HT; 10⁻⁸ to 5×10⁻⁵ M) elicited concentration-dependent contractions with EC₅₀=5.5 (5.2–5.7)×10⁻⁸ M) and E_max=93.1±7.3% of 75 mM KCl-induced contraction.
• 2.The agonist of 5-HT₂ receptors, α-methyl-5-hydroxytryptamine, and the selective agonist of 5-HT₁ receptors, N,N-dipropyl-5-carboxamidotryptamine and 5-carboxamidotryptamine, induced pronounced concentration-related contractions, which reached 71.1±6.0%, 53.0±5.0% and 75.0±7.8% at the highest dose tested, respectively. The agonist of 5-HT₃ receptor, 2-methyl-5-hydroxytryptamine, reached a maximum averaging 36.7±5.1% of the maximal response to KCl.
• 3.The 5-HT₁ and 5-HT₃ receptor antagonists, methiothepin and metoclopramide (10⁻⁷ to 10⁻⁶ M) did not alter the response to 5-HT. However, ketanserin (10⁻⁷ to 10⁻⁶ M), a 5-HT₂ receptor antagonist, induced significant inhibition of the concentration–response curve to 5-HT.
• 4.Contractile responses to 5-carboxamidotryptamine and 2-methyl-5-hydroxytryptamine were not affected by methiothepin and metoclopramide, respectively, whereas ketanserin significantly attenuated the contractile response to these agonists.
• 5.In conclusion, our study shows that 5-HT₂ receptors mediate contraction of the human placental vein with no obvious role for 5-HT₁-like, or 5-HT₃ receptors.

     

Detection of lithospermate B in rat plasma at the nanogram level by LC/MS in multi reaction monitoring mode

Low bioavailability and high binding affinity to plasma proteins led to the difficulty for the quantitative detection of lithospermate B (LSB) in plasma. This study aimed to develop a protocol for detecting LSB in plasma. A method was employed to quantitatively detect LSB of 5–500 ng/mL by LC/MS spectrometry in multi reaction monitoring mode via monitoring two major fragments with m/z values of 519 and 321 in the MS2 spectrum. To set up an adequate extraction solution to release LSB captured by plasma proteins, recovery yields of LSB extracted from rat plasma acidified by formic acid or HCl in the presence or absence of EDTA and caffeic acid were detected and compared using the above quantitative method. High recovery yield (～90%) was achieved when LSB (5–500 ng/mL) mixed in rat plasma was acidified by HCl (5 M) in the presence of EDTA (0.5 M) and caffeic acid (400 μg/mL). The lower limit of detection and the lower limit of quantification for LSB in the spiked plasma were calculated to be 1.8 and 5.4 ng/mL, respectively. Good accuracy (within ±10%) and precision (less than 10%) of intra- and inter-day quality controlled samples were observed. Oral bioavailability of LSB in rat model was detected via this optimized extraction method, and the maximum plasma concentration (C_max) was found to be 1034.3 ± 510.5 μg/L at t_max around 10 min, and the area under the plasma concentration–time curve (AUC) was 1414.1 ± 851.2 μg·h/L.     

Clinical applicability of current pharmacokinetic models: Splanchnic elimination of 5-fluorouracil in cancer patients

What can be inferred from limited clinical data by using current models of hepatic elimination? We examined this question by analyzing previously published data on the steady-state uptake of the anticancer agent 5-fluorouracil (5-FU) in seven cancer patients in terms of the venous equilibration model, the undistributed and distributed forms of the sinusoidal perfusion model, and the convection-dispersion model. Because of appreciable extrasplanchnic removal of 5-FU, the value of the steady infusion rate was not used in our analysis. When the data from all patients were pooled by plotting the measured hepatic venous concentration against the measured hepatic arterial concentration, the high concentration data fell on a limiting straight line of slope 1, indicating that at high dose rates elimination of 5-FU in both the liver and gastrointestinal tract was close to saturation. The intercept of this line gave a model-independent estimate of V_max/Q= 48.0±11.6 (SD) μM for the pooled data set, where V_max is the maximum splanchnic elimination rate of 5-FU, and Q is the hepatic blood flow. The low concentration data points fell on a limiting straight line through the origin, from which model-dependent values of the Michaelis constant were determined. The venous equilibration model gave K _m=9.4μM,while the undistributed sinusoidal perfusion model gave K _m ^* =26,5μM. With these values of K _m,both models fit the pooled data equally well. These methods were applied to analyses of the five individual data sets which contained sufficiently high concentration data points. The resulting mean values were V_max/Q=41.0±5.1 (sem) μM,K _m=8.4±1.3μM and K _m ^* =23.2±3.2 μM. However, the splanchnic region is a highly heterogeneous organ system, for which an undistributed analysis provides no more than an upper bound on the Michaelis constant K _m ⁺ (K _m ⁺ ?K _m ^* ).A perfusion model distributed to represent total splanchnic elimination is developed in the Appendix. Using previous estimates of the degree of functional heterogeneity in the liver alone, this model yields K _m ⁺ values for individual patients which have a mean of 20.3±2.8 μM.     

Impact of aminophylline on the pharmacodynamics of propofol in beagle dogs

This study aimed to characterize pharmacodynamic interaction between propofol and aminophylline. Nine beagle dogs were randomly allocated at the propofol rates of 0.75 (group A), 1.00 (group B), and 1.25 (group C) mg/kg/min. During period 1, propofol only was infused, while during period 2, aminophylline only, at the rate of 0.69 (group A), 1.37 (group B), and 2.62 (group C) mg/kg/h. During periods 3–5, the two drugs were co-administered. The aminophylline infusion rate was 0.69 (period 3), 1.37 (period 4), and 2.62 (period 5) mg/kg/h. The aminophylline was infused from 0 to 30 h, and the propofol was infused at 24 h for 20 min. Blood samples and electroencephalograms were obtained at preset intervals. In the linear regression between log-transformed doses of aminophylline and AUC _inf , the slope was 0.6976 (95 % CI 0.5242–0.8710). Pharmacokinetics of aminophylline was best described by a one-compartment, with enzyme auto-induction, model. Pharmacokinetics and pharmacodynamics of propofol were best described by a three-compartment model and a sigmoid E _max model, respectively. Pharmacodynamic parameter estimates of propofol were: k _e0 = 0.805/min, E ₀ = 0.76, E _max  = 0.398, Ce _{50 na}  = 2.38 μg/mL (without aminophylline-exposure), Ce _{50 wa}  = 4.49 μg/mL (with aminophylline-exposure), and γ = 2.21. Propofol becomes less potent when exposed to aminophylline. Pharmacodynamic antagonistic interaction of aminophylline with propofol sedation, may occur, not in a dose-dependent manner, but in an all-or-none response.     

Systemic absorption of ocular pilocarpine is modified by polymer matrices

Systemic absorption of ocularly applied pilocarpine (1.2 mg) was studied after administration in aqueous solution, in hydroxypropylcellulose (HPC) matrix, and in a matrix of n-butyl half-ester of poly(methyl vinyl ether/maleic anhydride) (PVM/MA). In vitro release of pilocarpine from the HPC-matrix deviated slightly and positively from the diffusional square root of time dependence. The rate of drug release was independent of the phosphate buffer concentration of the dissolution medium with an initial pH of 7.4; the rate of release was 10.91 ± 0.59% min^?0.5 in 1.3 mM buffer and 9.91 ±0.37% min^?0.5 in 66.7 mM buffer. A matrix of n-butyl half-ester of PVM/MA released pilocarpine according to zero-order kinetics. The rate of drug release was 0.22 ± 0.02% min^?1 in 1.3 mM phosphate buffer and 0.95 ± 0.06% min^?1 in 66.7 mM phosphate buffer. From the 2% aqueous solution, pilocarpine was absorbed efficiently into the plasma (t_max = 3.6 ± 0.9 min, c_max = 0.384 ± 0.024 μg/ml). Pharmacokinetic analysis of data for drug absorption revealed that the conjunctiva of the eye was the most important site for systemic absorption of pilocarpine. Both the HPC matrix (t_max = 35.0 ± 7.9 min, c_max = 0.256 ± 0.022 μg/ml) and the matrix of n-butyl half-ester of PVM/MA (t_max = 204 ± 17.5 min, c_max = 0.112 ± 0.014 μg/ml) delayed and decreased the peak concentrations of pilocarpine in general circulation. (AUC_{0–6 h}/AUC_{0–6 h,i,v.}) values were 0.72 ± 0.08, 0.67 ± 0.16, and 0.41 ± 0.05 for the aqueous solution, HPC matrix, and n-butyl half-ester of PVM/MA matrix, respectively. During the in vivo study, HPC matrices dissolved in 7–12 min in the tear fluid. n-Butyl half-ester of PVM/MA neither dissolved totally nor released all the drug from the matrix in the tear fluid during 8 h. Besides improving ocular drug absorption, as shown in earlier studies, the pilocarpine concentrations in systemic circulation can be decreased by administering the drug in polymer matrices.     

Identification and characterization of diarylimidazoles as hybrid inhibitors of butyrylcholinesterase and amyloid beta fibril formation

In this contribution, a chemical collection of aromatic compounds was screened for inhibition on butyrylcholinesterase (BChE)’s hydrolase activity using Ellman’s reaction. A set of diarylimidazoles was identified as highly selective inhibitors of BChE hydrolase activity and amyloid β (Aβ) fibril formation. New derivatives were synthesized resulting in several additional hits, from which the most active was 6c, 4-(3-ethylthiophenyl)-2-(3-thienyl)-1H-imidazole, an uncompetitive inhibitor of BChE hydrolase activity (IC₅₀ BChE = 0.10 μM; K_i = 0.073 ± 0.011 μM) acting also on Aβ fibril formation (IC₅₀ = 5.8 μM). With the aid of structure–activity relationship (SAR) studies, chemical motifs influencing the BChE inhibitory activity of these imidazoles were proposed. These bifunctional inhibitors represent good tools in basic studies of BChE and/or promising lead molecules for AD therapy.