Åse Ripel

The extent of postmortem drug redistribution in a rat model.

The aim of this study was to investigate the postmortem redistribution of several drugs in a rat model and to examine if any of the pharmacological properties was related to the extent of this phenomenon. One of the following drugs: phenobarbital (phenobarbitone), acetaminophen (paracetamol), carbamazepine, codeine, verapamil, amphetamine, mianserin, trimeprazine (alimemazine) or chloroquine was administered together with nortriptyline orally to rats 90 min prior to sacrifice. Heart blood was sampled immediately before sacrifice and after 2 h postmortem, as it has previously been shown that this is sufficient time for postmortem concentration changes to occur in heart blood. Blood was also sampled from the clamped abdominal inferior vena cava (representing peripheral blood) and tissue samples were taken from lungs, myocardium, liver, kidney, thigh muscle, forebrain, and vitreous humor together with a specimen from the minced carcass. Drugs were analyzed by high performance liquid or gas chromatography. For phenobarbital, acetaminophen and carbamazepine the postmortem to antemortem blood drug concentration ratios were close to 1.0 and tissue concentrations were low. The postmortem to antemortem heart blood drug concentration ratio for chloroquine (6.9 +/- 1.5) was higher than for nortriptyline (3.5 +/- 0.3), and the remaining drugs (codeine, verapamil, amphetamine, mianserin, and trimeprazine) showed ratios of the same magnitude as nortriptyline. The postmortem to antemortem blood drug concentration ratios for both heart blood and blood from the vena cava and also the lung to antemortem blood drug concentration ratio were closely related to the apparent volume of distribution for the drugs studied (p < 0.001). Accordingly, an apparent volume of distribution of more than 3-4 L/kg is a good predictor that a drug is liable to undergo postmortem redistribution with significant increments in blood levels. The postmortem drug concentration in blood from vena cava was closely related to the antemortem blood level, confirming that among the postmortem samples, the peripheral blood sample was the most representative for the antemortem blood concentration.

Morphine-6-Glucuronide might Mediate the Prolonged Opioid Effect of Morphine in Acute Renal Failure

1 A 43-year-old male developed acute kidney failure due to ethylene glycol poisoning. He was treated with bicarbonate to combat metabolic acidosis, ethanol as an antimetabolite and haemodialysis to remove the glycol and its toxic metabolites. He was kept on a respirator and sedated with morphine. Peritoneal dialysis was given for 36 d. Following sedation with morphine for 11 d, the patient was given naloxone and then extubated. The antidote had to be continued for 14 d to prevent respiratory depression, until kidney function improved. 2 Only morphine-6-glucuronide (M-6-G) was found in the plasma and CSF at concentrations which might explain the opioid effects observed in the patient during the days after the cessation of morphine treatment. The ratio of the area under the concentration-time curve (AUC) of morphine-3-glucuronide (M-3-G) to M-6-G was 2:1. The elimination half-lives of M-3-G and M-6-G were 55 and 82 h, respectively. The clearance data indicate that most of the glucuronides were eliminated by peritoneal dialysis during renal failure. 3 The data suggest that M-6-G exerts opioid effects and is retained in acute kidney failure. Morphine should therefore not be used preferentially as a sedative/analgesic in pronounced kidney failure.

Pharmacokinetic differences of morphine and morphine-glucuronides are reflected in locomotor activity

The main metabolites of morphine, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G), have been considered to participate in some of the effects of morphine. There is limited knowledge of the pharmacokinetics and dynamics of morphine and the main metabolites in mice, but mice are widely used to study both the analgesic effects and the psychomotor effects of morphine. The present study aimed to explore pharmacokinetic differences between morphine and morphine-glucuronides in mice after different routes of administration, and to investigate how possible differences were reflected in locomotor activity, a measure of psychostimulant properties. Mice were given morphine, M3G or M6G by different routes of administration. Serum concentrations versus time curves, pharmacokinetic parameters and locomotor activity were determined. Intraperitoneal administration of morphine reduced the bioavailability compared to intravenous and subcutaneous administration, but not so for morphine-glucuronides. The two morphine-glucuronides had similar pharmacokinetics, but morphine demonstrated higher volume of distribution and clearance than morphine-glucuronides. The present results demonstrated no locomotor effect of M3G, but a serum concentration effect relationship for morphine and M6G. When serum concentrations and effect changes were followed over time, there was some right hand shifts with respect to locomotor activity, especially during the declining phase of the concentration curve and particularly for M6G.

Increased Locomotor Activity Induced by Heroin in Mice: Pharmacokinetic Demonstration of Heroin Acting as a Prodrug for the Mediator 6-Monoacetylmorphine in Vivo

We investigated the relative importance of heroin and its metabolites in eliciting a behavioral response in mice by studying the relationship between concentrations of heroin, 6-monoacetylmorphine (6MAM), and morphine in brain tissue and the effects on locomotor activity. Low doses (subcutaneous) of heroin (≤5 μmol/kg) or 6MAM (≤15 μmol/kg) made the mice run significantly more than mice given equimolar doses of morphine. There were no differences in the response between heroin and 6MAM, although we observed a shift to the left of the dose-response curve for the maximal response of heroin. The behavioral responses were abolished by pretreatment with 1 mg/kg naltrexone. Heroin was detected in brain tissue after injection, but the levels were low and its presence too short-lived to be responsible for the behavioral response observed. The concentration of 6MAM in brain tissue increased shortly after administration of both heroin and 6MAM and the concentration changes during the first hour roughly reflected the changes in locomotor activity. Both the maximal and the total concentration of 6MAM were higher after administration of heroin than after administration of 6MAM itself. The morphine concentration increased slowly after injection and could not explain the immediate behavioral response. In summary, the locomotor activity response after injection of heroin was mediated by 6MAM, which increased shortly after administration. Heroin acted as an effective prodrug. The concentration of morphine was too low to stimulate the immediate response observed but might have an effect on the later part of the heroin-induced behavioral response curve.

Different biotransformation of morphine in isolated liver cells from guinea pig and rat.

The biotransformation of morphine was characterized in freshly isolated parenchymal and non-parenchymal liver cells from rats and guinea pigs in suspension culture to establish an in vitro model for morphine metabolism. Liver cells were prepared by a collagenase perfusion technique, and separated by differential centrifugation. Morphine metabolism was investigated at different concentrations (1, 5, 100 and 200 microM). Samples were taken repeatedly during 2-4 hr of incubation, and subsequently analysed on a HPLC system employing both UV and electrochemical detection. In suspensions of hepatocytes from both animal species morphine-3-glucuronide (M3G) was the major metabolite of morphine, and was formed at comparable rates at all concentrations examined. Guinea pig hepatocytes in addition produced considerable quantities of morphine-6-glucuronide (M6G), whereas this metabolite was detected only intracellularly in minor quantities in rat hepatocytes. The ratio between the two morphine glucuronides (M3G/M6G) in suspensions of guinea pig hepatocytes was approximately 4:1. N-Demethylation of morphine was more pronounced per mg cell protein in rat hepatocytes compared to guinea pig cells. Metabolic activity towards morphine was not detected in non-parenchymal cells of the two species. The morphine glucuronidation pattern found in guinea pig hepatocytes resembles to a greater extent than that found in rat hepatocytes the pattern found in in vivo studies of humans. It was concluded that isolated guinea pig parenchymal cells appeared to be a promising in vitro system for studies of morphine glucuronidation, and to observe metabolism in general.

Levels of heroin and its metabolites in blood and brain extracellular fluid after i.v. heroin administration to freely moving rats.

Heroin, with low affinity for μ‐opioid receptors, has been considered to act as a prodrug. In order to study the pharmacokinetics of heroin and its active metabolites after i.v. administration, we gave a bolus injection of heroin to rats and measured the concentration of heroin and its metabolites in blood and brain extracellular fluid (ECF).

Postmortem Amitriptyline Pharmacokinetics in Pigs after Oral and Intravenous Routes of Administration

In this study we have evaluated the postmortem pharmacokinetics of amitriptyline (Ami) and metabolites in pigs after oral and intravenous administration, and the results are compared with previous studies in rats and humans. In addition a meticulous investigation of blood and tissue concentrations after postmortem intravenous infusion of Ami was undertaken. Of a total of 9 over-night fasted pigs, 3 were given 25 mg/Kg Ami orally, and another 3 pigs received an intravenous infusion lasting 1 h of 3.3 mg/Kg Ami prior to death. The final 3 pigs were sacrificed and then given the intravenous infusion after death. After approximately 5 h at room temperature, all carcasses were subsequently stored at 4-5 degrees C. Postmortem blood samples were collected at 0.25, 1, 2, 4, 8, 24, 48, and 96 h through an indwelling intracardial needle. Postmortem examination with blood and tissue sampling was performed 96 h after death. Analysis was carried out by high performance liquid chromatography with ultraviolet detection. Postmortem blood samples from the heart of the orally dosed animals revealed large and variable concentration increases of 99(30-243)% for Ami and 96(52-429)% for the main metabolite 10-OH-Ami at 96 h. In the intravenously infused live pigs heart blood Ami increased by 55(33-69)% and 10-OH-Ami increased by 232(76-240)%. Blood from the atria had significantly higher Ami concentrations than blood from both ventricles in the animals dosed while alive, and the drug concentration in femoral blood was higher than in heart blood (p < 0.01). In the orally dosed pigs the left lobe of the liver had significantly higher Ami levels than the right lobe. Tissue/blood Ami concentration ratios were generally lower than previously reported in rats and approximating the levels reported in humans. The animals infused intravenously after death demonstrated high drug levels in blood samples from central vessels, heart, lungs as well as cerebrospinal fluid and vitreous humour. This implies that the presence of a lethal concentration of a drug in just one sample of heart blood can prove worthless in a case where agonal drug infusion may have occurred.

Simultaneous measurement of heroin and its metabolites in brain extracellular fluid by microdialysis and ultra performance liquid chromatography tandem mass spectrometry.

INTRODUCTION The pharmacokinetic profile and systemic bioavailability of a substance is often described by blood or total tissue concentrations. For centrally acting drugs, like opioids, the free fraction of active compound in brain extracellular fluid (ECF) is more likely to be correlated to the pharmacodynamic effects than the blood concentrations. Drugs of abuse, like heroin, are often administered intravenously as bolus injections, and the blood concentrations might change rapidly due to metabolism and distribution. The aim of our study was to establish a method to measure the free fraction of heroin and its metabolites in brain ECF, and follow their fast changes in concentration. METHODS Sprague-Dawley rats were injected intravenously with a bolus of heroin. Heroin and its main metabolites 6-monoacetylmorphine, morphine and morphine-3-glucuronide were measured simultaneously. Brain microdialysis was used for sampling and a method for quantification using ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed. Deuterated analogues for each analyte were included in the microdialysis perfusion solution as calibrators for recovery estimation. RESULTS A highly sensitive UPLC-MS/MS method allowed short sampling intervals, down to one minute, and the simultaneous detection of each analyte and its specific deuterated analogues, making possible the individual recovery calculation for each compound of interest. This method allowed us to determine the pharmacokinetic profiles of heroin and its metabolites in brain-ECF in the same animal after an intravenous injection of heroin. DISCUSSION Our method makes detecting concurrently the rapid changes in concentrations of heroin and its metabolites in brain ECF possible, despite the rapid metabolism of heroin. Recovery was measured specifically for each analyte in the same sample by carefully combining different deuterated analogues. This technique can be applied to pharmacokinetic studies where more than one compound of interest has to be monitored, and to study distribution of prodrugs or drugs with active metabolites.

Morphine‐3‐glucuronide inhibits morphine induced, but enhances morphine‐6‐glucuronide induced locomotor activity in mice

The main metabolite of morphine, morphine-3-glucuronide (M3G) has no opioid effects. Some studies have rather indicated that it antagonizes the antinociceptive and respiratory depressive effects of both morphine and the active metabolite morphine-6-glucuronide (M6G). We studied the possible influence of M3G on the psychostimulant properties of morphine and M6G measured by locomotor activity. Mice were given two injections, one with either 80, 240 or 500 micromol/kg M3G or saline followed by an injection of 20 or 30 micromol/kg morphine or M6G. M3G influenced the locomotor activity induced by both morphine and M6G, but in opposite directions. M3G reduced the morphine induced locomotor activity during the first hour following morphine injection in a concentration dependent manner. M3G pretreatment did not significantly influence brain concentrations of morphine indicating that the interaction was of a pharmacodynamic type. In contrast M3G pretreatment increased the M6G induced locomotor activity. M3G pretreatment increased serum and brain M6G concentrations to an extent indicating that this interaction was mainly of a pharmacokinetic type. In conclusion our results disclose complicated interactions between morphine and its two metabolites with respect to induction of locomotor activity and possibly also with respect to mechanisms related to drug reward.

Determination of dopamine concentrations in brain extracellular fluid using microdialysis with short sampling intervals, analyzed by ultra high performance liquid chromatography tandem mass spectrometry

INTRODUCTION An increase in striatal dopamine is considered essential for the rewarding and reinforcing effects of drugs of abuse. We have developed and validated an ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for the analysis of dopamine in rat brain extracellular fluid (ECF) sampled with microdialysis. The method was applied to monitor changes in dopamine concentrations over time after an intravenous bolus injection of heroin. METHODS Dopamine and dopamine-d3 were analyzed using a 2.1×100mm Aquity T3 column, 1.7μm particle size, with a formic acid and methanol gradient. The run time of the method was 2.5min including equilibration time. RESULTS The method had an LOQ of 0.15ng/mL, which equals 0.55pg on column. The calibration curves were linear in the tested area of 0.15 to 16ng/mL. Inter-assay coefficients of variation varied between 5-17%, with an accuracy expressed as bias of -10 to 5%. The intra-assay coefficients of variation varied between 9-15%, with an accuracy of -3-7%. DISCUSSION Heroin metabolism is very rapid. Sampling intervals of only 2min were thus required to obtain an adequate number of samples of dopamine analysis accompanying the concentration-time profile of opioids in the brain. Applying a flow of 2μL/min, 4μL of dialysate were sampled at 2min intervals, in 7μL internal standard. The injection volume onto the UPLC column was 10μL. Analyses of microdialysate samples from a rat given heroin i.v. showed that it was possible to measure baseline levels and rapid changes in dopamine concentrations with very short sampling periods.