Cytochrome P-450 2B6 is responsible for interindividual variability of propofol hydroxylation by human liver microsomes

BACKGROUND: Oxidation of propofol to 4-hydroxypropofol represents a significant pathway in the metabolism of this anesthetic agent in humans. The aim of this study was to identify the principal cytochrome P-450 (CYP) isoforms mediating this biotransformation. METHODS: Propofol hydroxylation activities and enzyme kinetics were determined using human liver microsomes and cDNA-expressed CYPs. CYP-specific marker activities and CYP2B6 protein content were also quantified in hepatic microsomes for correlational analyses. Finally, inhibitory antibodies were used to ascertain the relative contribution of CYPs to propofol hydroxylation by hepatic microsomes. RESULTS: Propofol hydroxylation by hepatic microsomes showed more than 19-fold variability and was most closely correlated to CYP2B6 protein content (r = 0.904), and the CYP2B6 marker activities, S-mephenytoin N-demethylation (r = 0.919) and bupropion hydroxylation (r = 0.854). High- and intermediate-activity livers demonstrated high-affinity enzyme kinetics (K(m) < 8 microm), whereas low-activity livers displayed low-affinity kinetics (K(m) > 80 microm). All of the CYPs evaluated were capable of hydroxylating propofol; however, CYP2B6 and CYP2C9 were most active. Kinetic analysis indicated that CYP2B6 is a high-affinity (K(m) = 10 +/- 2 microm; mean +/- SE of the estimate), high-capacity enzyme, whereas CYP2C9 is a low-affinity (K(m) = 41 +/- 8 microm), high-capacity enzyme. Furthermore, immunoinhibition showed a greater contribution of CYP2B6 (56 +/- 22% inhibition; mean +/- SD) compared with CYP2C isoforms (16 +/- 7% inhibition) to hepatic microsomal activity. CONCLUSIONS: Cytochrome P-450 2B6, and to a lesser extent CYP2C9, contribute to the oxidative metabolism of propofol. However, CYP2B6 is the principal determinant of interindividual variability in the hydroxylation of this drug by human liver microsomes.     

Propofol Stimulates Ciliary Motility via the Nitric Oxide-Cyclic GMP Pathway in Cultured Rat Tracheal Epithelial Cells

Background: Airway ciliary motility is impaired by inhaled anesthetics. Recent reports show that nitric oxide (NO) induces upregulation in ciliary beat frequency (CBF), and others report that propofol, an intravenous anesthetic, stimulates NO release; this raises the possibility that propofol increases CBF by stimulating the NO-cyclic guanosine monophosphate (cGMP) signal pathway. In this study, the authors investigated the effects of propofol on CBF and its relation with the NO-cGMP pathway using the pharmacologic blockers NG-monomethyl-l-arginine (l-NMMA), an NO synthase inhibitor; 1 H-[1,2,4]oxidazole[4,3-a]quinoxalin-1-one (ODQ), a soluble guanylyl cyclase inhibitor; and KT5823, a cGMP-dependent protein kinase inhibitor, in cultured rat tracheal epithelial cells.

Methods: Rat tracheal tissues were explanted and cultured for 3-5 days. Images of ciliated cells were videotaped using a phase-contrast microscope. Baseline CBF and CBF 25 min after exposure to propofol or blocker were measured using video analysis.

Results: Vehicle (0.1% dimethyl sulfoxide; n = 11) increased CBF by 0.2 +/- 1.7% (mean +/- SD) from baseline. Propofol stimulated CBF significantly (P < 0.01) and dose dependently (1 [mu]m, 2.0 +/- 1.9%, n = 6; 10 [mu]m, 8.2 +/- 6.7%, n = 9; 100 [mu]m, 14.0 +/- 4.7%, n = 10). Intralipid (0.05%), the clinical vehicle of propofol, did not affect CBF (-0.2 +/- 2.2%; n = 5). The enhancement of CBF with use of 100 [mu]m propofol was abolished (P < 0.01) by coadministration of 10 m[mu]m l-NMMA (2.4 +/- 3.6%; n = 5), 100 [mu]m ODQ (-0.3 +/- 2.2%; n = 6) or 30 [mu]m KT5823 (-0.1 +/- 4.1%; n = 8). l-NMMA, ODQ, or KT5823 alone did not change CBF.     

Molecular Mechanisms of the Inhibitory Effects of Propofol and Thiamylal on Sarcolemmal Adenosine Triphosphate-sensitive Potassium Channels

Background: Both propofol and thiamylal inhibit adenosine triphosphate-sensitive potassium (KATP) channels. In the current study, the authors investigated the effects of these anesthetics on the activity of recombinant sarcolemmal KATP channels encoded by inwardly rectifying potassium channel (Kir6.1 or Kir6.2) genes and sulfonylurea receptor (SUR1, SUR2A, or SUR2B) genes.

Methods: The authors used inside-out patch clamp configurations to investigate the effects of propofol and thiamylal on the activity of recombinant KATP channels using COS-7 cells transfected with various types of KATP channel subunits.

Results: Propofol inhibited the activities of the SUR1/Kir6.2 (EC50 = 77 [mu]m), SUR2A/Kir6.2 (EC50 = 72 [mu]m), and SUR2B/Kir6.2 (EC50 = 71 [mu]m) channels but had no significant effects on the SUR2B/Kir6.1 channels. Propofol inhibited the truncated isoform of Kir6.2 (Kir6.2[DELTA]C36) channels (EC50 = 78 [mu]m) that can form functional KATP channels in the absence of SUR molecules. Furthermore, the authors identified two distinct mutations R31E (arginine residue at position 31 to glutamic acid) and K185Q (lysine residue at position 185 to glutamine) of the Kir6.2[DELTA]C36 channel that significantly reduce the inhibition of propofol. In contrast, thiamylal inhibited the SUR1/Kir6.2 (EC50 = 541 [mu]m), SUR2A/Kir6.2 (EC50 = 248 [mu]m), SUR2B/Kir6.2 (EC50 = 183 [mu]m), SUR2B/Kir6.1 (EC50 = 170 [mu]m), and Kir6.2[DELTA]C36 channels (EC50 = 719 [mu]m). None of the mutants significantly affects the sensitivity of thiamylal.     

Physostigmine Reverses Propofol-induced Unconsciousness and Attenuation of the Auditory Steady State Response and Bispectral Index in Human Volunteers

Background: It is postulated that alteration of central cholinergic transmission plays an important role in the mechanism by which anesthetics produce unconsciousness. The authors investigated the effect of altering central cholinergic transmission, by physostigmine and scopolamine, on unconsciousness produced by propofol.

Methods: Propofol was administered to American Society of Anesthesiologists physical status 1 (n = 17) volunteers with use of a computer-controlled infusion pump at increasing concentrations until unconsciousness resulted (inability to respond to verbal commands, abolition of spontaneous movement). Central nervous system function was assessed by use of the Auditory Steady State Response (ASSR) and Bispectral Index (BIS) analysis of electrooculogram. During continuous administration of propofol, reversal of unconsciousness produced by physostigmine (28 [mu]g/kg) and block of this reversal by scopolamine (8.6 [mu]g/kg) were evaluated.

Results: Propofol produced unconsciousness at a plasma concentration of 3.2 +/- 0.8 (+/- SD) [mu]g/ml (n = 17). Unconsciousness was associated with reductions in ASSR (0.10 +/- 0.08 [mu]V [awake baseline 0.32 +/- 0.18 [mu]V], P < 0.001) and BIS (55.7 +/- 8.8 [awake baseline 92.4 +/- 3.9], P < 0.001). Physostigmine restored consciousness in 9 of 11 subjects, with concomitant increases in ASSR (0.38 +/- 0.17 [mu]V, P < 0.01) and BIS (75.3 +/- 8.3, P < 0.001). In all subjects (n = 6) scopolamine blocked the physostigmine-induced reversal of unconsciousness and the increase of the ASSR and BIS (ASSR and BIS during propofol-induced unconsciousness: 0.09 +/- 0.09 [mu]V and 58.2 +/- 7.5, respectively; ASSR and BIS after physostigmine administration: 0.08 +/- 0.06 [mu]V and 56.8 +/- 6.7, respectively, NS).     

A Brief Regional Ischemic-reperfusion Enhances Propofol-induced Depression in Left Ventricular Function of in situ Rat Hearts

Background: Propofol is short-acting intravenous general anesthetics that reduces cardiovascular hemodynamics. The effects of propofol on intrinsic myocardial contractility, however, remain debatable. The aim of the current study was to test the hypothesis that inhibitory effects of propofol on left ventricular (LV) contractility and mechanical work capability of in situ ejecting rat hearts are attenuated after a brief regional ischemia and reperfusion.

Methods: The authors obtained steady-state LV pressure-volume loops and intermittently obtained LV end-systolic pressure-volume relation and evaluated effects of propofol on LV function by end-systolic pressure (ESPmLVV), systolic pressure-volume area (PVAmLVV) at midrange LV volume (mLVV).

Results: Propofol (5.2 +/- 0.3~11.1 +/- 3.7 [mu]g[middle dot]ml-1) significantly decreased ESP0.08 to 78 +/- 12%~64 +/- 13% of prepropofol and PVA0.08 to 76 +/- 13%~63 +/- 16% of prepropofol in normal hearts, whereas propofol at a lower concentration (4.1 +/- 1.0 [mu]g/ml) did not. Although brief ischemic-reperfusion per se did not affect LV function, propofol after that, even at a lower concentration (4.1 +/- 1.0 [mu]g/ml), significantly decreased ESP0.08 to 70 +/- 27% of prepropofol and PVA0.08 to 68 +/- 33% of prepropofol. Pretreatment with a protein kinase C (PKC) inhibitor, bisindolylmaleimide reduced the propofol (4.1 +/- 1.0 [mu]g/ml)-induced greater decreases in ESP0.08 and PVA0.08 after brief ischemic-reperfusion to 94 +/- 33% and 92 +/- 39% of prepropofol. In the propofol-infused hearts after brief ischemic-reperfusion, protein kinase C-[epsilon] translocation to the nucleus-myofibril fraction was found.     

Myocardial and Coronary Effects of Propofol in Rabbits with Compensated Cardiac Hypertrophy

Background: Myocardial effects of propofol have been previously investigated but most studies have been performed in healthy hearts. This study compared the cardiac effects of propofol on isolated normal and hypertrophic rabbits hearts.

Methods: The effects of propofol (10-1,000 [mu]m) on myocardial contractility, relaxation, coronary flow and oxygen consumption were investigated in hearts from rabbits with pressure overload-induced left ventricular hypertrophy (LVH group, n = 20) after aortic abdominal banding and from sham-operated control rabbits (SHAM group, n = 10), using an isolated and erythrocyte-perfused heart model. In addition, to assess the myocardial and coronary effects of propofol in more severe LVH, hearts with a degree of hypertrophy greater than 140% were selected (severe LVH group, n = 7).

Results: The cardiac hypertrophy model induced significant left ventricular hypertrophy (136 +/- 21%, P < 0.05). The pressure-volume relation showed normal systolic function but an altered diastolic compliance in hypertrophic hearts. Propofol only decreased myocardial contractility and relaxation at supratherapeutic concentrations (>= 300 [mu]m) in SHAM and LVH groups. The decrease in myocardial performances was not significantly different in SHAM and LVH groups. Propofol induced a significant increase in coronary blood flow which was not significantly different between groups. In severe LVH group, the degree of hypertrophy reached to 157 +/- 23%. Similarly, the effects of concentrations of propofol were not significantly different from the SHAM group.     

Low-dose Propofol-induced Amnesia Is Not due to a Failure of Encoding: Left Inferior Prefrontal Cortex Is Still Active

Background: Propofol may produce amnesia by affecting encoding. The hypothesis that propofol weakens encoding was tested by measuring regional cerebral blood flow during verbal encoding.

Methods: Seventeen volunteer participants (12 men; aged 30.4 +/- 6.5 yr) had regional cerebral blood flow measured using H2O15 positron emission tomography during complex and simple encoding tasks (deep vs. shallow level of processing) to identify a region of interest in the left inferior prefrontal cortex (LIPFC). The effect of either propofol (n = 6, 0.9 [mu]g/ml target concentration), placebo with a divided attention task (n = 5), or thiopental at sedative doses (n = 6, 3 [mu]g/ml) on regional cerebral blood flow activation in the LIPFC was tested. The divided attention task was expected to decrease activation in the LIPFC.

Results: Propofol did not impair encoding performance or reaction times, but impaired recognition memory of deeply encoded words 4 h later (median recognition of 35% [interquartile range, 17-54%] of words presented during propofol vs. 65% [38-91%] before drug; P < 0.05). Statistical parametric mapping analysis identified a region of interest of 6.6 cm2 in the LIPFC (T = 7.44, P = 0.014). Regional cerebral blood flow response to deep encoding was present in this region of interest in each group before drug (T > 4.41, P < 0.04). During drug infusion, only the propofol group continued to have borderline significant activation in this region (T = 4.00, P = 0.063).     

Propofol Modulates γ-Aminobutyric Acid-mediated Inhibitory Neurotransmission to Cardiac Vagal Neurons in the Nucleus Ambiguus

Background: Although it is well recognized that anesthetics modulate the central control of cardiorespiratory homeostasis, the cellular mechanisms by which anesthetics alter cardiac parasympathetic activity are poorly understood. One common site of action of anesthetics is inhibitory neurotransmission. This study investigates the effect of propofol on [gamma]-aminobutyric acid-mediated (GABAergic) and glycinergic neurotransmission to cardiac parasympathetic neurons.

Methods: Cardiac parasympathetic neurons were identified in vitro by the presence of a retrograde fluorescent tracer, and spontaneous GABAergic and glycinergic synaptic currents were examined using whole cell patch clamp techniques.

Results: Propofol at concentrations of 1.0 [mu]m and greater significantly (P < 0.05) increased the duration and decay time of spontaneous GABAergic inhibitory postsynaptic currents. To determine whether the action of propofol was at presynaptic or postsynaptic sites, tetrodotoxin was applied to isolate miniature inhibitory postsynaptic currents. Propofol at concentrations of 1.0 [mu]m and greater significantly (P < 0.05) prolonged the decay time and duration of miniature inhibitory postsynaptic currents, indicating that propofol directly alters GABAergic neurotransmission at a postsynaptic site. Propofol at high concentrations (>=50 [mu]m) also inhibited the frequency of both GABAergic inhibitory postsynaptic currents and miniature inhibitory postsynaptic currents. Propofol at concentrations up to 50 [mu]m had no effect on glycinergic neurotransmission.     

Effect of Propofol on Carotid Body Chemosensitivity and Cholinergic Chemotransduction

Background: Propofol decreases the acute hypoxic ventilatory response in humans and depresses in vivo carotid body chemosensitivity. The mechanisms behind this impaired oxygen sensing and signaling are not understood. Cholinergic transmission is involved in oxygen signaling, and because general anesthetics such as propofol have affinity to neuronal nicotinic acetylcholine receptors, the authors hypothesized that propofol depresses carotid body chemosensitivity and cholinergic signaling.

Methods: An isolated rabbit carotid body preparation was used. Chemoreceptor activity was recorded from the whole carotid sinus nerve. The effect of propofol on carotid body chemosensitivity was tested at three different degrees of Po2 reduction. Nicotine-induced chemoreceptor response was evaluated using bolus doses of nicotine given before and after propofol 10-500 [mu]m. The contribution of the [gamma]-aminobutyric acid A receptor complex was tested by addition of [gamma]-aminobutyric acid A receptor antagonists.

Results: Propofol reduced carotid body chemosensitivity; the magnitude of depression was dependent on the reduction in Po2. Furthermore, propofol caused a concentration-dependent (10-500 [mu]m) depression of nicotine-induced chemoreceptor response, with a 50% inhibitory concentration (propofol) of 40 [mu]m. Bicuculline in combination with propofol did not have any additional effect, whereas addition of picrotoxin gave a slightly more pronounced inhibition.     

Respiratory Sites of Action of Propofol: Absence of Depression of Peripheral Chemoreflex Loop by Low-dose Propofol

Background: Propofol has a depressant effect on metabolic ventilatory control, causing depression of the ventilatory response to acute isocapnic hypoxia, a response mediated via the peripheral chemoreflex loop. In this study, the authors examined the effect of sedative concentrations of propofol on the dynamic ventilatory response to carbon dioxide to obtain information about the respiratory sites of action of propofol.

Methods: In 10 healthy volunteers, the end-tidal carbon dioxide concentration was varied according to a multifrequency binary sequence that involved 13 steps into and 13 steps out of hypercapnia (total duration, 1,408 s). In each subject, two control studies, two studies at a plasma target propofol concentration of 0.75 [mu]g/ml (Plow), and two studies at a target propofol concentration of 1.5 [mu]g/ml (Phigh) were performed. The ventilatory responses were separated into a fast peripheral component and a slow central component, characterized by a time constant, carbon dioxide sensitivity, and apneic threshold. Values are mean +/- SD.

Results: Plasma propofol concentrations were approximately 0.5 [mu]g/ml for Plow and approximately 1.3 mg/ml for Phigh. Propofol reduced the central carbon dioxide sensitivity from 1.5 +/- 0.4 to 1.2 +/- 0.3 (Plow;P < 0.01 vs. control) and 0.9 +/- 0.1 l [middle dot] min-1 [middle dot] mmHg-1 (Phigh;P < 0.001 vs. control). The peripheral carbon dioxide sensitivity remained unaffected by propofol (control, 0.5 +/- 0.3; Plow, 0.5 +/- 0.2; Phigh, 0.5 +/- 0.2 l [middle dot] min-1 [middle dot] mmHg-1). The apneic threshold was reduced from 36.3 +/- 2.7 (control) to 35.0 +/- 2.1 (Plow;P < 0.01 vs. control) and to 34.6 +/- 1.9 mmHg (Phigh;P < 0.01 vs. control).     

Propofol Potentiates Phenylephrine-induced Contraction via Cyclooxygenase Inhibition in Pulmonary Artery Smooth Muscle

Background: The authors previously demonstrated in vivo that the pulmonary vasoconstrictor response to the [alpha] agonist phenylephrine is potentiated during propofol anesthesia compared with the conscious state. The current in vitro study tested the hypothesis that propofol potentiates phenylephrine-induced contraction by inhibiting the synthesis and/or activity of vasodilator metabolites of the cyclooxygenase pathway.

Methods: Canine pulmonary arterial rings were suspended for isometric tension recording. Intracellular calcium concentration ([Ca2+]i) was measured in pulmonary arterial strips loaded with acetoxylmethyl ester of fura-2. After phenylephrine-induced contraction, propofol (10-7 to 10-4 m) was administered in the presence or absence of the cyclooxygenase inhibitor ibuprofen (10-5 m). The effects of propofol on the arachidonic acid and prostacyclin relaxation-response curves were assessed. The amount of 6-keto prostaglandin F1[alpha](stable metabolite of prostacyclin) released from pulmonary vascular smooth muscle in response to phenylephrine was measured with enzyme immunoassay in the presence or absence of propofol and ibuprofen.

Results: Propofol potentiated phenylephrine-induced contraction in pulmonary arterial rings in a concentration-dependent and endothelium-independent manner. In endothelium-denuded strips, propofol (10-4 m) increased tension by 53 +/- 11%, and increased [Ca2+]i by 56 +/- 9%. Ibuprofen also potentiated phenylephrine-induced contraction but abolished the propofol-induced increases in tension and [Ca2+]i. Propofol had no effect on the relaxation response to prostacyclin, whereas propofol and ibuprofen attenuated the relaxation response to arachidonic acid to a similar extent. Phenylephrine markedly increased 6-keto prostaglandin F1[alpha] production, and this effect was virtually abolished by propofol and ibuprofen.     

Propofol Neuroprotection in Cerebral Ischemia and Its Effects on Low-molecular-weight Antioxidants and Skilled Motor Tasks

Background: Propofol is neuroprotective when administered immediately after stroke. The therapeutic window, duration of administration, and antioxidant mechanisms of propofol in neuroprotection are not known. The effects of propofol after stroke were examined in the conscious animal. The authors have previously shown that light propofol anesthesia (25 mg [middle dot] kg-1 [middle dot] h-1) for a period of 4 h, even if delayed 1 h after the onset of ischemia, decreases infarct volume 3 days after the stroke.

Methods: Cerebral ischemia was induced in awake Wistar rats by a local intracerebral injection of the potent vasoconstrictor, endothelin (6 pmol in 3 [mu]l) into the striatum. Propofol treatment after ischemia was delayed up to 4 h, and the infusion period shortened from 4 h to 1 h. Infarct volume was assessed 3 or 21 days after the stroke. Neurologic outcome was evaluated on days 14-21 after ischemia. Tissue ascorbate and glutathione concentrations were evaluated at 4 h and 3 days after ischemia.

Results: Infarct volumes were reduced 3 days after ischemia when propofol treatment (25 mg [middle dot] kg-1 [middle dot] h-1) was delayed for 2 h (0.5 +/- 0.3 mm3) but not 4 h (2.0 +/- 0.9 mm3), compared with intralipid controls (2.4 +/- 0.7 mm3). The propofol infusion period of 3 h but not 1 h reduced infarct volume. Propofol treatment did not reduce infarct volume 21 days after the stroke, although motor function improvements (Montoya staircase test) were observed 14-21 days after the stroke. Propofol neuroprotection was independent of tissue ascorbate and glutathione concentrations.     

Mechanisms Underlying the Inhibitory Effect of Propofol on the Contraction of Canine Airway Smooth Muscle

Background: Propofol has been shown to produce relaxation of preconstricted airway smooth muscle. Although the inhibition of calcium mobilization is supposed to be the major mechanism of action, the whole picture of the mechanisms is not completely clear.

Methods: Contractile response was performed using canine tracheal rings. The effects of propofol on carbachol-induced mobilization of intracellular Ca2+ and phosphoinositide hydrolysis were measured using cultured canine tracheal smooth muscle cells by monitoring fura-2 signal and assessing the accumulation of [3H]-inositol phosphates. To detect the effect of propofol on muscarinic receptor density and affinity, [3H]N-methyl-scopolamine was used as a radioligand for receptor binding assay.

Results: Pretreatment with propofol shifts the concentration-response curves of carbachol-induced smooth muscle contraction to the right in a concentration-dependent manner without changing the maximal response. Propofol not only decreased the release of Ca2+ from internal stores but also inhibited the calcium influx induced by carbachol. In addition, carbachol-induced inositol phosphate accumulation was attenuated by propofol; the inhibitory pattern was similar to the contractile response. Moreover, propofol did not alter the density of muscarinic receptors. The dissociation constant value was not altered by pretreatment with 100 [mu]M propofol but was significantly increased by 300 [mu]M (propofol, 952 +/- 229 pM; control, 588 +/- 98 pM; P < 0.05).     

Propofol Suppresses Macrophage Functions and Modulates Mitochondrial Membrane Potential and Cellular Adenosine Triphosphate Synthesis

Background: Propofol is an intravenous anesthetic agent that may impair host defense system. The aim of this study was to evaluate the effects of propofol on macrophage functions and its possible mechanism.

Methods: Mouse macrophage-like Raw 264.7 cells were exposed to propofol, at 3, 30 (a clinically relevant concentration), and 300 [mu]m. Cell viability, lactate dehydrogenase, and cell cycle were analyzed to determine the cellular toxicity of propofol to macrophages. After administration of propofol, chemotactic, phagocytic, and oxidative ability and interferon-[gamma] mRNA production were carried out to validate the potential effects of propofol on macrophage functions. Mitochondrial membrane potential and cellular adenosine triphosphate levels were also analyzed to evaluate the role of mitochondria in propofol-induced macrophage dysfunction.

Results: Exposure of macrophages to 3 and 30 [mu]m propofol did not affect cell viability. When the administered concentration reached 300 [mu]m, propofol would increase lactate dehydrogenase release, cause arrest of cell cycle in G1/S phase, and lead to cell death. In the 1-h-treated macrophages, propofol significantly reduced macrophage functions of chemotactic and oxidative ability in a concentration-dependent manner. However, the suppressive effects were partially or completely reversed after 6 and 24 h. Propofol could reduce phagocytic activities of macrophages in concentration- and time-dependent manners. Exposure of macrophages to lipopolysaccharide induced the mRNA of interferon-[gamma], but the induction was significantly blocked by propofol. Propofol concentration-dependently decreased the membrane potential of macrophage mitochondria, but the effects were descended with time. The levels of cellular adenosine triphosphate in macrophages were also reduced by propofol.     

Comparative Pharmacokinetics and Pharmacodynamics of the New Propofol Prodrug GPI 15715 and Propofol Emulsion

Background: GPI 15715 is a new water-soluble prodrug that is hydrolyzed to release propofol. The objectives of this crossover study in volunteers were to investigate the pharmacokinetics and pharmacodynamics of GPI 15715 in comparison with propofol emulsion.

Methods: In two separate sessions, nine healthy male volunteers (19-35 yr, 70-86 kg) received GPI 15715 and propofol emulsion as a target controlled infusion over 60 min. In the first 20 min, the propofol target concentration increased linearly to 5 [mu]g/ml. Subsequently, the targets were reduced to 3 [mu]g/ml and 1.5 [mu]g/ml for 20 min each. The plasma concentrations of GPI 15715 and propofol were measured from arterial and venous blood samples up to 24 h and pharmacokinetics were analyzed. The pharmacodynamic effect was measured by the median frequency of the power spectrum of the electroencephalogram, and a sigmoid model with effect compartment was fitted to the data.

Results: Compared with propofol emulsion, propofol from GPI 15715 showed a different disposition function and especially larger volumes of distribution. The propofol effect site concentration for half maximum effect was 2.0 +/- 0.5 [mu]g/ml for GPI 15715 and 3.0 +/- 0.7 [mu]g/ml for propofol emulsion (P < 0.05). Propofol from GPI 15715 did not show a hysteresis between plasma concentration and effect.     

Anesthetic Concentrations of Propofol Protect against Oxidative Stress in Primary Astrocyte Cultures: Comparison with Hypothermia

Background: The extracellular concentration of glutamate in the brain increases after oxidative damage. This increase may be caused, in part, by changes in glutamate transport by astrocytes. The authors hypothesized that propofol and hypothermia mitigate the effects on astrocytes of oxidative stress.

Methods: Primary cultures of rat cerebral astrocytes were subjected to oxidative stress by incubation with tert-butyl hydroperoxide for 30 min, followed by a 30-90-min washout period. The effects of prophylactic (simultaneous with tert-butyl hydroperoxide application) and delayed (administered 30 min after the oxidant) propofol or hypothermia were determined by measuring the uptake of glutamate as well as the release of preloaded d-aspartate (a nonmetabolizable analog of glutamate) and endogenous lactate dehydrogenase (a cytosolic marker).

Results: Delayed administration of an anesthetic concentration of propofol (1-3 [mu]m) prevented the inhibition of high-affinity glutamate uptake, stimulation of d-aspartate release, and increase in lactate dehydrogenase release caused by tert-butyl hydroperoxide (1 mm, 37[degrees]C). The protective effect of propofol (EC50 = 2 [mu]m) on glutamate uptake was 20-fold more potent than that of [alpha]-tocopherol (EC50 = 40 [mu]m). Prophylactic hypothermia (28 and 33[degrees]C) also protected astrocytes from tert-butyl hydroperoxide. Delayed hypothermia was not protective but did not compromise rescue by propofol.     

Effects of Subhypnotic Doses of Propofol on Gastric Emptying in Volunteers

Background: Drugs which accelerate gastric emptying (GE) decrease nausea and vomiting. This could contribute to the antiemetic potential of subhypnotic doses of propofol. On the contrary, subhypnotic doses of propofol used for sedation could decrease GE and thus favor regurgitation and pulmonary inhalation. Therefore, the aim of this study was to assess the effect of low-dose propofol infusion on GE.

Methods: On three separate occasions, 10 volunteers received either a propofol infusion at a rate set to achieve a target plasma concentration of 0.5 [mu]g/ml or equivalent volumes of 10% Intralipid(R) or 0.9% saline. GE for solids was measured by using the octanoic acid breath test. An acetaminophen absorption technique measured the GE rate for liquids. Blood samples were assayed for acetaminophen and propofol. Breath samples were analyzed for 13CO2 concentration by isotope-ratio mass spectrometry. Carbon dioxide production ([latin capital V with dot above]co2) was measured instead of calculated by indirect calorimetry. Sedation was evaluated by the Bispectral Index of the electroencephalogram.

Results: Propofol blood concentrations were 0.32 +/- 0.20 and 0.45 +/- 0.18 [mu]g/ml at 60 and 165 min, respectively. These concentrations were not sedative. Propofol or its solvent did not modify GE for solids or liquids. In all groups, differences in GE were obtained if measured [latin capital V with dot above]co2 was integrated in the formula instead of calculated [latin capital V with dot above]co2 (P < 0.002).     

Propofol Has Both Enhancing and Suppressing Effects on Human Platelet Aggregation In Vitro

Background: Volatile anesthetics are known to suppress platelet aggregation. In contrast, there is conflicting information regarding the effect of propofol on platelet function. The present study was designed to clarify the effects of propofol on platelet function and the mechanisms underlying these effects.

Methods: Propofol or an equivalent volume of 10% Intralipos (as a control) was added to test tubes 5 min before the induction of each reaction. Platelet aggregation induced by epinephrine, arachidonic acid (AA), prostaglandin G2 (PGG2), or STA2 (a thromboxane A2 [TXA2] analog) was measured using an eight-channel aggregometer. To determine type 1 cyclooxygenase activity, AA (0.5 mM) was added to an assay mixture containing type 1 cyclooxygenase, and the concentration of the final product, malonaldehyde, was measured by spectrophotometry. Epinephrine-, adenosine diphosphate-, AA-, and PGG2-induced TXA2 formation was measured using a commercially available radioimmunoassay kit. To estimate TXA2 receptor-binding affinity, 3H-S145, a specific TXA2 receptor antagonist, was added, and the radioactivity of receptor-bound 3H-S145 was determined using a liquid scintillation analyzer. Inositol 1,4,5-triphosphate formation was measured in STA2-stimulated platelets using a commercially available inositol 1,4,5-triphosphate assay kit.

Results: Propofol (40 [mu]M) enhanced, whereas 100 [mu]M suppressed, adenosine diphosphate- and epinephrine-induced secondary aggregation without affecting primary aggregation. Propofol (40 [mu]M) also enhanced, but 100 [mu]M suppressed, AA-induced aggregation. Propofol (100 [mu]M) enhanced PGG2- and STA2-induced aggregation. Propofol (100 [mu]M) suppressed AA-induced TXA2 formation but did not alter that induced by PGG2. Propofol (30-100 [mu]M) suppressed AA- induced malonaldehyde formation, indicating inhibition of type 1 cyclooxygenase activity. Propofol did not alter TXA2 receptor-binding affinity. Propofol (30 and 100 [mu]M) augmented inositol 1,4,5-triphosphate formation in STA2-stimulated platelets.     

Sympathetic Neural Activation Evoked by μ-Receptor Blockade in Patients Addicted to Opioids Is Abolished by Intravenous Clonidine

Background: [mu]-Opioid receptor blockade by naloxone administered for acute detoxification in patients addicted to opioids markedly increases catecholamine plasma concentrations, muscle sympathetic activity (MSA), and is associated with cardiovascular stimulation despite general anesthesia. The current authors tested the hypothesis that the [alpha]2-adrenoceptor agonist clonidine (1) attenuates increased MSA during [mu]-opioid receptor blockade for detoxification, and (2) prevents cardiovascular activation when given before detoxification.

Methods: Fourteen mono-opioid addicted patients received naloxone during propofol anesthesia. Clonidine (10 [mu]g kg-1 administered over 5 min + 5 [mu]g kg-1 h-1 intravenous) was infused either before (n = 6) or after (n = 6) naloxone administration. Two patients without immediate clonidine administration occurring after naloxone administration served as time controls. Muscle sympathetic activity (n = 8) in the peroneal nerve, catecholamine plasma concentrations (n = 14), arterial blood pressure, and heart rate were assessed in awake patients, during propofol anesthesia before and after [mu]-opioid receptor blockade, and after clonidine administration.

Results: [mu]-Receptor blockade markedly increased MSA from a low activity (burst frequency: from 2 burst/min +/- 1 to 24 +/- 8, means +/- SD). Similarly, norepinephrine (41 pg/ml +/- 37 to 321 +/- 134) and epinephrine plasma concentration (13 pg/ml +/- 6 to 627 +/- 146) significantly increased, and were associated with, increased arterial blood pressure and heart rate. Clonidine immediately abolished both increased MSA (P < 0.001) and catecholamine plasma concentrations (P < 0.001). When clonidine was given before [mu]-opioid receptor blockade, catecholamine plasma concentrations and hemodynamic variables did not change.     

Fentanyl inhibits metabolism of midazolam: competitive inhibition of CYP3A4 in vitro

Fentanyl decreases clearance of midazolam administered i.v., but the mechanism remains unclear. To elucidate this mechanism, we have investigated the effect of fentanyl on metabolism of midazolam using human hepatic microsomes and recombinant cytochrome P450 isoforms (n = 6). Midazolam was metabolized to l'-hydroxymidazolam (l'-OH MDZ) by human hepatic microsomes, with a Michaelis-Menten constant (K(m)) of 5.0 (SD 2.7) microgramsmol litre-1. Fentanyl competitively inhibited metabolism of midazolam in human hepatic microsomes, with an inhibition constant (Ki) of 26.8 (12.4) microgramsmol litre-1. Of the seven representative human hepatic P450 isoforms, CYP1A2, 2A6, 2C9, 2C19, 2D6, 2E1 and 3A4, only CYP3A4 catalysed hydroxylation of midazolam, with a K(m) of 3.6 (0.8) microgramsmol liter-1. Fentanyl competitively inhibited metabolism of midazolam to l'-OH MDZ by CYP3A4, with a Ki of 24.2 (6.8) microgramsmol litre-1, comparable with the Ki obtained in human hepatic microsomes. These findings indicate that fentanyl competitively inhibits metabolism of midazolam by CYP3A4.