Kavalactone pharmacophores for major cellular drug targets.

A number of studies have identified differential kavalactone activity against a variety of molecular targets, including P-glycoprotein (Pgp), platelet monoamine oxidase (MAO-B), transcription factor binding domains, pregnane X (PXR) and GABA receptors, and cytochrome P450 and cyclo-oxygenase (COX) enzymes. The molecular structure of the kavalactones possesses a pharmacophore for several of these targets. In most cases, conformational stability is more significant than the substituents present. The analysis of these pharmacophores provides important insights for future medicinal chemistry-based approaches to kavalactone-type drugs.

Kavalactones Yangonin and Methysticin induce apoptosis in human hepatocytes (HepG2) in vitro.

While cases of severe kava hepatotoxicity have been reported, studies examining the toxicity of individual kavalactones are limited. The present study examined the in vitro hepatotoxicity of kavain, methysticin and yangonin on human hepatocytes (HepG2) and the possible mechanism(s) involved. Cytotoxicity was assessed using lactate dehydrogenase (LDH) and ethidium bromide (EB) assays. The mode of cell death was analysed with acridine orange/ethidium bromide dual staining with fluorescence microscopy. Glutathione oxidation was measured using the ortho-phthalaldehyde (OPT) fluorescence assay. Kavain had minimal cytotoxicity, methysticin showed moderate concentration-dependent toxicity and yangonin displayed marked toxicity with ~ 40% reduction in viability in the EB assay. Acridine orange/ethidium bromide staining showed the predominant mode of cell death was apoptosis rather than necrosis. No significant changes were observed in glutathione levels, excluding this as the primary mechanism of cell death in this model. Further studies may elucidate the precise apoptotic pathways responsible and whether toxic kavalactone metabolites are involved.

Role of ethanol in kava hepatotoxicity.

Kava is known for its recreational, ceremonial and medicinal use in the Pacific. The aqueous non-alcoholic drink of kava rhizome produces intoxicating, relaxing and soothing effects. While kava's medicinal effects receive worldwide recognition, kava-containing products came under scrutiny after over 100 reports of spontaneous adverse hepatic effects. Many mechanisms have been postulated to explain the unexpected toxicity, one being pharmacokinetic interactions between kavalactones and co-administered drugs involving cytochrome P450 enzyme system. Alcohol is often co-injested in kava hepatotoxicity cases. This review evaluates the possible hepatotoxicity mechanisms involving alcohol and kava.

Kavalactones fail to inhibit alcohol dehydrogenase in vitro.

In recent years, Kava kava (Piper methysticum, Forst. f., Piperaceae), a folkloric beverage and popular herbal remedy, has been implicated in a number of liver failure cases. Many hypotheses as to the mechanism of its hepatotoxicity, for example interactions with other co-ingested medication, have been postulated. This present study investigated whether pharmacokinetic interactions between kava constituents and alcohol via alcohol dehydrogenase (ADH) inhibition by individual kavalactones might explain its claimed hepatotoxic effects. Four kavalactones, (+/-)-kavain, methysticin, yangonin and desmethoxyyangonin, fail to inhibit ADH in vitro at 1, 10 or 100 microM concentrations.

Serum cholinesterase inhibition by omeprazole and lansoprazole.

Omeprazole inhibited human and rat serum cholinesterase by approximately 5 to 60% over the 0.5 to 50 mg/L (1.4-140 microM) concentration range. In contrast lansoprazole only produced 20-30% inhibition at the highest concentration of 10 mg/L (29 microM). Thus omeprazole but not lansoprazole is likely to potentiate the effect of succinylcholine at human clinical concentrations by inhibiting its hydrolysis in vivo by serum cholinesterases.

Onset and offset pharmacodynamics of propofol.

Propofol whole blood and plasma concentrations at offset of hypnosis in eighteen patients were inversely related to patient age and body fat. The relationship between propofol concentrations and body fat is derived from the relationship between age and body fat and age was the single independent predictor of concentrations at offset of propofol hypnosis.

Rocuronium plasma concentrations during three phases of liver transplantation: relationship with early postoperative graft liver function.

BACKGROUND: Previous studies have suggested that neuromuscular blocking agents might be used to assess liver function during liver transplantation. This study examines changes in rocuronium plasma concentration during liver transplantation, to assess graft function. METHODS: A constant-rate infusion of rocuronium was administered to 17 adult patients undergoing liver transplantation. Blood samples were taken at 30-min intervals throughout the procedure, which was divided into three phases: paleo-, an-, and neohepatic. Assay of plasma concentrations of rocuronium was by a gas chromatographic-mass spectrometry technique. Postoperative liver function was followed for up to five days by measuring plasma aminotransferases. RESULTS: In 14 of the 15 patients who survived the transplantation procedure, there was a 7-50% decrease in rocuronium concentration during the neohepatic phase compared with the anhepatic phase. In contrast, rocuronium concentrations increased in the two patients who died after surgery, one as a result of primary non-function and one from massive bleeding. In one patient who survived there was no change in rocuronium concentration. The increase in plasma rocuronium concentration during the neohepatic phase in the two patients who died was consistent with high levels of plasma aminotransferases. CONCLUSIONS: Comparison of changes in plasma rocuronium concentration during the neohepatic phase with early postoperative liver function tests suggests the potential use of rocuronium as a pharmacokinetic probe for predicting liver function during liver transplantation. Further study of rocuronium's potential as an intraoperative pharmacodynamic probe of liver function by measuring neuromuscular paralysis is suggested.

Gas chromatographic-mass spectrometric assay for rocuronium with potential for quantifying its metabolite, 17-desacetylrocuronium, in human plasma.

A rapid, sensitive and selective method has been developed for the quantification of plasma concentrations of neuromuscular blocking drug, rocuronium, using gas chromatography with mass spectrometric detection. 3-Desacetylvecuronium served as the internal standard. The method involved iodide ion pair formation and a single-step liquid-liquid extraction with dicholoromethane. This method also permits simultaneous determination of its putative metabolite, 17-desacetylrocuronium, although the high detection limit for the metabolite limits the practical application of this method in pharmacokinetic study of the metabolite. The extraction efficiency was approximately 75% for rocuronium and approximately 50% for 17-desacetylrocuronium. The limit of quantification was 26 ng/ml for rocuronium and 870 ng/ml for its metabolite. The assay was used successfully in a patient undergoing liver transplantation and receiving rocuronium as a constant rate infusion and in a patient undergoing general elective surgery receiving the drug as an intravenous bolus. This assay is a time-saving alternative to published gas or liquid chromatographic methods for assaying rocuronium.

Application of a high-performance liquid chromatographic assay for the neuromuscular blocker gallamine to analysis of rat plasma, muscle and microdialysate samples.

A reliable high-performance liquid chromatographic method has been validated for determination of gallamine in rat plasma, muscle tissue and microdialysate samples. A C18 reversed-phase column with mobile phase of methanol and water containing 12.5 mM tetrabutyl ammonium (TBA) hydrogen sulphate (22:78, v/v) was used. The flow-rate was 1 ml/min with UV detection at 229 nm. Sample preparation involved protein precipitation with acetonitrile for plasma and muscle tissue homogenate samples. Microdialysate samples were injected into the HPLC system without any sample preparation. Intra-day and inter-day accuracy and precision of the assay were <13%. The limit of quantification was 1 microg/ml for plasma, 1.6 microg/g for muscle tissue and 0.5 microg/ml for microdialysate samples. The assay was applied successfully to analysis of samples obtained from a pharmacokinetic study in rats using the microdialysis technique.

Potential use of pharmacological markers to quantitatively assess liver function during liver transplantation surgery.

Early functioning of the transplanted liver is of crucial importance to the recipient. This function may be assessed by measuring the disposition of substances that are mainly eliminated via the liver. None of the agents currently used is ideal for this purpose. Measurement of mono-ethyleneglycinexylidide (MEGX) formation from lignocaine is useful and has been widely used in liver transplantation to assess liver graft function. MEGX formation can be affected by the use of drugs that influence liver perfusion or interfere with the CYP450 enzyme system. Indocyanine green clearance is a convenient method but both blood flow and hepatocellular function affect the test results. Tests of caffeine clearance, galactose elimination capacity and antipyrine clearance all require time-consuming, technically cumbersome and expensive serial blood sampling. The aminopyrine breath test is non-invasive, but gastric emptying and the patient's physical state affect results. The potential hazard of exposure to radioactive compounds limits the wide clinical use of both aminopyrine and erythromycin breath tests. Monitoring the rate of recovery from neuromuscular blockade induced by vecuronium and rocuronium can provide valuable information on liver function.

Neuromuscular paralysis as a pharmacodynamic probe to assess organ function during liver transplantation.

Potential for assessing liver function during liver transplantation surgery by monitoring muscle paralysis from nondepolarizing neuromuscular blockers that are hepatically cleared is critically assessed. Rocuronium is strongly favored as a promising pharmacodynamic probe for predicting allograft liver function because it is predominantly eliminated via the liver and its putative metabolites are not active. Prolongation of recovery from rocuronium paralysis is closely correlated with allograft liver function postoperatively. Vecuronium, pancuronium, and perhaps pipecuronium may also prove to be useful probes, but the two former blockers have active metabolites. Further prospective studies are necessary with more precise measurement of neuromuscular function to confirm the predictive value of this method. Alterations in neuromuscular blocker plasma concentrations that are correlated with changes in liver function and either the dose required or the intensity or duration of paralysis needs to be demonstrated for this technique to be clinically useful.

Assessment of in vitro and in vivo recovery of gallamine using microdialysis.

The application of microdialysis technique for the investigation of pharmacokinetics and pharmacodynamics of drugs requires careful assessment of probe performance to ensure validity of the data obtained using this technique. The aim of this study was to establish and validate the microdialysis technique for investigation of the pharmacokinetics and pharmacodynamics of the neuromuscular blocker, gallamine. In vitro recovery of gallamine from the microdialysis probe when different perfusion flow rates were employed was evaluated leading to selection of a flow rate of 2 microl/min with 15-min sampling intervals for the subsequent studies. In vitro recovery of gallamine from the microdialysis probe was independent of concentration, stable over an 8-h period and reproducible. Comparable in vitro recoveries were obtained by different established approaches including recovery estimation by gain, loss and the zero-net flux (ZNF) method. Recovery by loss was used to study the in vivo recovery of gallamine from rat muscle tissue. The in vivo recovery was stable over a 5.5-h sampling period. In vitro performance of the probe subsequent to the in vivo study remained stable supporting reusage of the probe. These data highlight the importance of a systematic examination of microdialysis probe validation.

Effect of haemorrhage on plasma propofol concentrations in a patient undergoing orthotopic liver transplantation.

We report a patient who underwent repeat orthotopic liver transplantation complicated by intraoperative blood loss of more than 59 litre. During liver transplantation, a constant rate propofol infusion was given and plasma samples were obtained throughout the procedure for propofol assay. Before the anhepatic phase of the procedure, plasma propofol concentrations reached a plateau at 1 microgram ml-1, and these concentrations were consistent with those predicted using a pharmacokinetic model. During the anhepatic phase of surgery, serum propofol concentrations increased progressively and exceeded those predicted by the pharmacokinetic model. Large intravascular fluid shifts associated with blood loss and subsequent blood replacement influenced plasma propofol concentrations observed in this patient.

Administration of a crystalloid fluid preload does not prevent the decrease in arterial blood pressure after induction of anaesthesia with propofol and fentanyl.

Anaesthesia was induced in 58 women (ASA I or II) undergoing elective gynaecological procedures, using propofol 2.5 mg kg-1 and fentanyl 1.5 micrograms kg-1. Patients were allocated to receive 20 ml kg-1 of crystalloid fluid preload over 20 min or to receive no fluids before induction of anaesthesia. A significant decrease in systolic arterial pressure (< 75% of baseline value) occurred in both the fluid-loaded and the control groups, and was similar in both groups. Administration of a fluid preload did not attenuate the decrease in systolic arterial pressure after induction of anaesthesia with propofol and fentanyl.

A novel substance associated with gallamine-induced myoclonus.

If gallamine or d-tubocurarine gains access to the central nervous system it produces a myoclonus, a synchronized jerking of many skeletal muscles. Each jerk is accompanied by a slow wave in the inferior olive. The jerking continues for 24 h or more after the gallamine or d-tubocurarine can no longer be detected in the CSF. We report here that a novel substance appears in the CSF and persists for a long period of time, possibly as long as the twitching. This substance is not corticotrophin-releasing factor (CRF) nor does CRF or harmaline (a substance causing a tremor by an action on the inferior olive) lead to the appearance of the novel substance. At present the nature of this substance is not known.

Droperidol elimination after cardiopulmonary bypass surgery.

A high-dose (0.75 to 2.8 mg/kg) pharmacokinetic study of droperidol was undertaken in patients during the recovery phase after cardiac surgery involving hypothermic cardiopulmonary bypass (CPB). The elimination half-life of droperidol in these patients, determined from concentration-time data obtained after CPB, was significantly prolonged relative to previously reported mean values in younger surgical patients not undergoing CPB and receiving lower doses of the drug (0.05-0.20 mg/kg). On stratification of the patients by droperidol dose, there was an inverse correlation between the size of the dose and the elimination half-life of droperidol: mean half-life decreased as mean dose increased. This difference in elimination half-life was not related to the duration of the CPB procedure, or the total anesthetic time, both of which were not significantly different between the patient groups receiving the three different doses of droperidol. The magnitude or duration of hypothermia after CPB did not differ between the three patient groups. The differences in half-lives are more likely due to the clinical condition of the patients, such that the patients who received the higher doses of droperidol were also judged clinically to be less ill and thus eliminated droperidol more efficiently. This hypothesis, however, could not be supported due to the small number of patients studied. The results obtained in this study indicate that droperidol elimination is significantly prolonged after high-dose administration to elderly patients undergoing hypothermic CPB procedures during cardiac surgery.

Acute in vitro neuromuscular effects of carbamazepine and carbamazepine-10,11-epoxide.

We examined the acute neuromuscular effects of the anticonvulsant, carbamazepine, and its major metabolite, carbamazepine-10,11-epoxide, using the in vitro rat phrenic nerve-hemidiaphragm muscle preparation. Carbamazepine produced 8.8% +/- 2.2% (n = 12) neuromuscular paralysis as its concentration was increased from 1 to 50 microg/mL (4.2-210 microM). In contrast, carbamazepine-10,11-epoxide produced maximum paralysis of 65% +/- 8% (n = 10) in the concentration range 1-100 microg/mL (4-400 microM) and the concentration required to produce half this paralysis was 36 +/- 7 microg/mL (144 +/- 28 microM). Carbamazepine 10 microg/mL (42 microM) shifted the response-concentration curve for both a depolarizing (succinylcholine) and a nondepolarizing (atracurium) neuromuscular blocker, reducing their concentrations required for 50% paralysis by approximately 30%. In contrast, the metabolite, which was a more potent neuromuscular blocker by itself, failed to alter either succinylcholine or atracurium effect. These results concur with previous clinical reports where anticonvulsants have acutely reduced neuromuscular blocker dose requirements.