Plant macrofossil evidence for an early onset of the Holocene summer thermal maximum in northernmost Europe.

Holocene summer temperature reconstructions from northern Europe based on sedimentary pollen records suggest an onset of peak summer warmth around 9,000 years ago. However, pollen-based temperature reconstructions are largely driven by changes in the proportions of tree taxa, and thus the early-Holocene warming signal may be delayed due to the geographical disequilibrium between climate and tree populations. Here we show that quantitative summer-temperature estimates in northern Europe based on macrofossils of aquatic plants are in many cases ca. 2 °C warmer in the early Holocene (11,700-7,500 years ago) than reconstructions based on pollen data. When the lag in potential tree establishment becomes imperceptible in the mid-Holocene (7,500 years ago), the reconstructed temperatures converge at all study sites. We demonstrate that aquatic plant macrofossil records can provide additional and informative insights into early-Holocene temperature evolution in northernmost Europe and suggest further validation of early post-glacial climate development based on multi-proxy data syntheses.

Bioavailability of detomidine administered sublingually to horses as an oromucosal gel.

The objective of the study was to determine the absorption, bioavailability and sedative effect of detomidine administered to horses as an oromucosal gel compared to intravenous and intramuscular administration of detomidine injectable solution. The study was open and randomized, with three sequences crossover design. Nine healthy horses were given 40 µg/kg detomidine intravenously, intramuscularly or administered under the tongue with a 7-day wash-out period between treatments. Blood samples were collected before and after drug administration for the measurement of detomidine concentrations in serum. The effects of the route of administration on heart rate and rhythm were evaluated and the depth of sedation assessed. Mean (±SD) bioavailability of detomidine was 22% (±5.3%) after sublingual administration and 38.2% (±7.9%) after intramuscular administration. The sedative effects correlated with detomidine concentrations regardless of the route of administration. We conclude that less detomidine is absorbed when given sublingually than when given intramuscularly, because part of it does not reach the circulation. Sublingual administration of detomidine oromucosal gel at 40 µg/kg produces safe sedation in horses. Slow absorption leads to fewer and less pronounced adverse effects than the more rapid absorption after intramuscular injection.

CYP2B6 and CYP2C19 as the major enzymes responsible for the metabolism of selegiline, a drug used in the treatment of Parkinson's disease, as revealed from experiments with recombinant enzymes.

In view of conflicting data in the literature regarding the enzyme(s) responsible for metabolism of selegiline, a drug used in the treatment of Parkinson's disease, investigations were carried out in vitro using the human cytochrome P450 enzymes CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 recombinantly expressed in yeast to elucidate the enzyme specificity in selegiline metabolism. In the yeast microsomes used, desmethylselegiline and levomethamphetamine were formed from selegiline at significant rates. The highest contribution to the hepatic clearance of selegiline was calculated to be exerted by CYP2B6 (124 l/h) CYP2C19 (82 l/h), whereas CYP3A4 (27 l/h) and CYP1A2 (21 l/h) were of less importance. Antibodies against CYP2B6 inhibited metabolism of selegiline in microsomes containing CYP2B6 but not in microsomes without significant amounts of the enzyme. In contrast to previous reports, we could not find any role for CYP2D6 in the metabolism of selegiline. The data strongly indicate that the high extent of interindividual variation seen in vivo for selegiline clearance is caused by the metabolism of the compound by the highly polymorphic CYP2B6 and CYP2C19.

High-performance liquid chromatographic method combining radiochemical and ultraviolet detection for determination of low activities of uridine 5'-diphosphate-glucuronosyltransferase.

A novel reversed-phase high-performance liquid chromatographic method was developed to measure UDP-glucuronosyltransferase (UGT) activity. Radiochemical and UV detection were combined in this UDP-[(14)C]glucuronic acid-utilizing method which was especially aimed at determination of low activities typical of N-glucuronidation of various amines and heterocycles. 4-Nitrophenol and levomedetomidine were used as substrates to validate this method, and applicability was tested with commonly used model substrates of N-glucuronidation, 4-aminobiphenyl and amitriptyline, and several 4-arylalkyl-1H-imidazole compounds. Detection limits were very low, 0.5-10 pmol, corresponding to UGT activities from 0.04 to 0.8 pmol/min/mg protein depending on UV absorbance of the glucuronide conjugate. The sensitivity was 10- to 100-fold compared with earlier HPLC assays using radiochemical detection. This method enabled quantitation without a reference glucuronide, and its high sensitivity allows for characterization of N-glucuronidation kinetics of various substrates. Using this method, human liver microsomal UGT activity was determined for a series of 4-arylalkyl-1H-imidazoles. Of these compounds, levomedetomidine was glucuronidated at the highest rate, 1.69 nmol/min/mg protein, using a 500 microM substrate concentration. In comparison, activities for the commonly used UGT substrates, 4-nitrophenol, 4-aminobiphenyl, and amitriptyline were 18.80, 3.23, and 0.23 nmol/min/mg protein, respectively.

Selegiline metabolism and cytochrome P450 enzymes: in vitro study in human liver microsomes.

Although being a drug therapeutically used for a long time, the enzymatic metabolism of selegiline has not been adequately studied. In the current work we have studied the cytochrome P450 (CYP)-catalyzed oxidative metabolism of selegiline to desmethylselegiline and 1-methamphetamine and the effects of selegiline, desmethylselegiline and 1-methamphetamine on hepatic CYP enzymes in human liver microsomes in vitro. The apparent Km values for desmethylselegiline and 1-methamphetamine formation were on an average 149 microM and 293 microM, and the apparent Vmax values, 243 pmol/min./mg and 1351 pmol/min./mg, respectively. Furafylline and ketoconazole, the known reference inhibitors for CYP1A2 and CYP3A4, respectively, inhibited the formation of desmethylselegiline with Ki value of 1.7 microM and 15 microM. Ketoconazole inhibited also the formation of 1-methamphetamine with Ki of 18 microM. Fluvoxamine, an inhibitor of CYP1A2, CYP2C19 and CYP3A4, inhibited the formation of desmethylselegiline and 1-methamphetamine with Ki values of 9 and 25 microM, respectively. On the basis of these results we suggest that CYP1A2 and CYP3A4 contribute to the formation of desmethylselegiline and that CYP3A4 participates in the formation of 1-methamphetamine. In studies with CYP-specific model activities, both selegiline and desmethylselegiline inhibited the CYP2C19-mediated S-mephenytoin 4'-hydroxylation with average IC50 values of 21 microM and 26 microM, respectively. The Ki for selegiline was determined to be around 7 microM. Selegiline inhibited CYP1A2-mediated ethoxyresorufin O-deethylation with a Ki value of 76 microM. Inhibitory potencies of selegiline, desmethylselegiline and 1-methamphetamine towards other CYP-model activities were much lower. On this basis, selegiline and desmethylselegiline were shown to have a relatively high affinity for CYP2C19, but no evidence about selegiline metabolism by CYP2C19 was obtained.

Comparison of the effects of acute and subchronic administration of atipamezole on reaction to novelty and active avoidance learning in rats.

The effects of an alpha2-adrenoceptor antagonist, atipamezole, on exploratory behaviour in a novel environment, spontaneous motor activity and active avoidance learning were studied after acute injection and continuous infusion (0.1 mg/kg h) for 24 h and 6-9 days in rats. The effects of atipamezole on biogenic amines and their main metabolites in brain were studied after an acute injection (0.3 mg/kg s.c.) and continuous infusion (0.1 mg/kg h) for 24 h and 10 days. The level of central alpha2-adrenoceptor antagonism and the drug concentration in blood and in the brain were measured after continuous infusion for 24 h and 10 days. In behavioural tests, atipamezole had no effect on spontaneous motor activity at any of the doses studied. However, after both acute administration and continuous 24-h infusion, atipamezole decreased exploratory behaviour in a staircase test, but no longer after 6 days of continuous infusion. Acute administration of atipamezole impaired performance in active avoidance learning tests causing a learned helplessness-like behaviour. When the training was started after 7 days of continuous infusion, atipamezole significantly improved active avoidance learning. There was a significant increase in the metabolite of noradrenaline (NA), 3-methoxy-4-hydroxyphenylethyleneglycol sulphate (MHPG-SO4), after 24 h but not any longer after 10 days of continuous atipamezole infusion, although the extent of central alpha2-adrenoceptor antagonism was unchanged and the atipamezole concentration present in brain was even elevated at 10 days compared to levels after 24-h infusion. In conclusion, these results reveal that acute and subchronic atipamezole treatments have different and even opposite effects on behaviour in novel, stressful situations. After acute treatment, atipamezole potentiates reaction to novelty and stress, causing a decrease in exploratory activity and impairment in shock avoidance learning. After subchronic treatment, there was no longer any effect on exploratory behaviour and, in fact, there was an improvement in the learning of a mildly stressful active avoidance test. The changes in behaviour occurred in parallel with attenuation in the MHPG-SO4-increasing effect, thus the suppressed behaviour in the present test conditions after acute atipamezole injection is associated with a major increase in central NA release. The results support the role of alpha2-adrenoceptors and noradrenergic system in reactions both to novelty and stress and have possible implications in cognitive functions as well as in depression.

Effects of immobilization with medetomidine and reversal with atipamezole on blood chemistry of semi-domesticated reindeer (Rangifer tarandus tarandus L.) in autumn and late winter.

Blood chemistry was studied in 8 adult female reindeer, of which 5 were pregnant. Half of them received only medetomidine (150 micrograms/kg i.m.) and half of them medetomidine and atipamezole (750 micrograms/kg) in March. Three weeks later the drug regimens were reversed. The same procedure was carried out during the next September and October. Seasonal differences in pretreatment values could be seen in serum urea, phosphorous, and cholesterol, with the highest concentrations during the autumn; and creatinine, ASAT, ALAT, and CK values, which were higher in the non-pregnant reindeer in late winter. Their low-protein and low-energy diet during the winter explains most of the differences. Increased enzyme activities in serum indicate decreased membrane stability of certain organs in late winter, possibly due to nutritional deficiencies. Treatment effects could be seen in several parameters. The increase in blood glucose and decrease in serum FFA were most probably due to alpha 2-adrenoceptor activation, which inhibits insulin release and lipolysis. These effects were partly or totally inhibited after treatment with the antagonist atipamezole. The earlier increase in serum CK and ASAT activities in those receiving atipamezole can be explained by increased tissue perfusion due to atipamezole itself and the fact that these animals stood up and began to move much earlier than did those which received medetomidine only. A significant decrease in serum Na+, K+, Cl-, Pi, cholesterol, total Ca, and total protein concentration observed during the first 10 to 40 min of the medetomidine sedation could be explained by possible haemodilution and diuresis. More effective metabolism of medetomidine in autumn could explain the shorter recovery times of reindeer receiving only medetomidine and most of the differences in treatment effects between the seasons: faster increase in protein and cholesterol concentrations after the decrease, and the antagonistic effect of atipamezole on glucose and Pi changes in autumn. Based on these results, medetomidine seems to be a good sedation agent for reindeer both in autumn and in late winter; the effects of medetomidine can be rather effectively antagonized by atipamezole.

Systemic absorption and systemic effects of ocularly administered dexmedetomidine in rabbits.

Dexmedetomidine is a selective alpha 2-adrenoceptor agonist which has previously been shown to reduce the ocular pressure of normotensive rabbits as well as those with pressures artificially elevated by laser irradiation. In this study instillation of an equivalent hypotensive dose (12.5 micrograms) did not cause changes in heart rate, blood pressure, blood glucose or plasma catecholamine content even though dexmedetomidine could be detected in plasma. However, this dose given intravenously (i.v.) was also without effect. Higher ocular doses resulted in equivalent bradycardia and changes in blood glucose levels as when the dose was given i.v. These two parameters proved to be most sensitive indicators of systemic alpha 2-agonism, blood pressure did not change and plasma catecholamine levels were too low to be reliably assayed. It is concluded that when hypotensive doses of dexmedetomidine are instilled into the eye, intraocular concentrations are sufficiently high to exert pharmacological effects. As it is absorbed into the general circulation, it is diluted such that its systemic effects are minimal.

Identification of detomidine carboxylic acid as the major urinary metabolite of detomidine in the horse.

Horse urine was investigated for metabolites by chromatography and mass spectrometry following the oral administration of the large animal analgesic sedative detomidine to two stallions and intravenous administration of [3H]-detomidine to a mare. Detomidine carboxylic acid and hydroxydetomidine glucuronic acid conjugate were identified in the urine after the oral doses. In addition, traces of free hydroxydetomidine were observed. About half of the radioactivity of [3H]-detomidine was excreted in the urine in 12 h after the i.v. dose (80 micrograms/kg). Most of the excretion occurred between 5 and 12 h in contrast to urine output which was highest 2-5 h after the dosing. The major radioactive metabolite in the urine was detomidine carboxylic acid. It comprised more than two thirds of the total metabolites in all the urine fractions collected. Its excretion profile was similar to that of total radioactivity. Hydroxydetomidine glucuronide was also excreted. It contributed 10-20% of the total metabolites in the urine. The free aglycone was only seen in the samples collected during the peak urine flow. A minor metabolite was tentatively characterized as the glucuronide of N-hydroxydetomidine.

Tissue-specificity of hydroxylation and N-methylation of arylalkylimidazoles.

Hydroxylation and N-methylation of three potent alpha 2-adrenoceptor agonists detomidine, medetomidine and dexmedetomidine by rat brain, kidney, liver and lung were studied in vitro. NADPH-dependent hydroxylation of the 3'-methyl group was catalyzed mainly by the microsomal fraction of liver. Monooxygenation by the other tissues was negligible. The hepatic monooxygenase reaction was characterized by high affinity (Km 5.5-9.8 microM) and low capacity (Vmax 29-66 pmol/min. per mg protein). Statistically significant (P less than 0.05) differences between the Km and Vmax values of medetomidine and dexmedetomidine were observed suggesting stereoselective oxidation of the drug molecule. N-Methylation of the arylalkylimidazoles occurred mainly in the cytosolic fraction of the kidney. With S-adenosylmethionine as the methyl donor, the transferase activities in other tissues were less than 10% of the specific activity of the renal enzyme (about 7 pmol/min. per mg protein for medetomidine). Even the renal enzyme showed a relatively low capacity (Km 250-300 microM and Vmax 15-33 pmol/min. per mg protein). Different tissue distributions of the arylalkylimidazole N-methyltransferase and histamine N-methyltransferase suggest that they are two distinct enzyme species. Based on these results, aliphatic hydroxylation is the major (metabolic) elimination mechanism of the arylalkylimidazole drugs, and liver is the principal eliminating organ. The importance of N-methylation is diminished by its low capacity and probable regeneration of the parent drug by demethylation.

Pharmacological effects and pharmacokinetics of atipamezole, a novel alpha 2-adrenoceptor antagonist--a randomized, double-blind cross-over study in healthy male volunteers.

1. Single doses (10, 30 and 100 mg) of atipamezole (MPV-1248), a new potent and selective imidazole-type alpha 2-adrenoceptor antagonist, and saline placebo were administered as 20 min intravenous infusions to six healthy male volunteers in a randomized double-blind, cross-over phase I study. Later, 100 mg atipamezole was given orally to the same subjects in an open fashion. 2. The i.v. doses resulted in linearly dose-related concentrations of atipamezole in plasma. Pharmacokinetic calculations revealed an elimination half-life of 1.7-2.0 h, an apparent volume of distribution of 3.0-3.5 l kg-1 and a total plasma clearance of 1.1-1.5 l h-1 kg-1. No atipamezole could be detected in plasma after oral dosing. 3. Subjective drug effects were seen mainly after the largest i.v. dose and included increased alertness and nervousness, coldness and sweating of hands and feet, tremor and shivering, motor restlessness, and increased salivation. Salivation was also quantitated using dental cotton rolls, with dose-related increases produced by the i.v. doses. 4. The 100 mg i.v. dose increased plasma noradrenaline concentrations on average by 484 +/- 269 (s.d.)%, and also elevated both systolic and diastolic blood pressure (mean increases 17 +/- 7/14 +/- 2 mm Hg). The 30 mg dose had minor and the 10 mg dose no effects on these variables. Adrenaline and cyclic AMP levels in plasma were increased only after the largest dose. No drug effects were observed after oral dosing. 4. Plasma C-peptide and blood glucose levels were not markedly influenced by the drug, and cortisol secretion was not stimulated. 5. The observed effects are compatible with the presumed alpha 2-adrenoceptor antagonistic action of atipamezole and are in general concordance with the reported results of other alpha 2-adrenoceptor antagonists (yohimbine and idazoxan). 6. Although not orally active, atipamezole may prove to be a useful agent in studies of alpha 2-adrenoceptor function in man.

Biotransformation of medetomidine in the rat.

1. The metabolites of a novel alpha 2-adrenoceptor agonist, medetomidine, in rat urine after subcutaneous administration at two dose levels (80 micrograms/kg or 5 mg/kg), and after incubation with rat liver fractions, were characterized by h.p.l.c., 1H-n.m.r and mass spectrometry. 2. Hydroxylation of a methyl substituent was the main biotransformation in vitro. Hydroxylation occurred at a rate sufficient for high metabolic clearance. 3. The major urinary metabolites were the glucuronide of hydroxymedetomidine (about 35% of urinary metabolites) and medetomidine carboxylic acid (about 40%). 4. Medetomidine unchanged represented about 1% or 10% of the urinary excretion products, dependent on dose. 5. A metabolic pathway consisting of hydroxylation with subsequent glucuronidation, or further oxidation to carboxylic acid, is suggested.

Single-dose pharmacokinetics of detomidine in the horse and cow.

The pharmacokinetics of detomidine, a novel analgesic sedative, was studied in the major target species after high (80 micrograms/kg) i.v. and i.m. doses. In addition, drug residues in some organs were determined. Concentrations were measured using a sensitive, detomidine-specific radio-immunoassay method. Rapid absorption following i.m. dosing occurred. Absorption half-lives were 0.15 h (horse) and 0.08 h (cattle). The mean peak concentration in the horse (51.3 ng/ml) was achieved in 0.5 h and in the cow (65.8 ng/ml) in 0.26 h. The areas under the concentration curve after i.m. dosing were 66% (horse) and 85% (cow) of the corresponding i.v. values. Distribution was rapid with half-lives of 0.15 h (horse, i.v.) and 0.24 h (cow, i.v.). The apparent volume of distribution was higher after the i.m. dosing (horse 1.56 l/kg, cow 1.89 l/kg) than after i.v. dosing (horse 0.74 l/kg, cow 0.73 l/kg). Elimination half-lives were 1.19 h (horse) and 1.32 h (cow) for the i.v. dose and 1.78 h (horse) and 2.56 h (cow) for the i.m. dose. Total clearances ranged from 6.7 (horse, i.v.) to 12.3 (cow, i.m.) ml/min/kg. Renal clearances were less than 1% of the total clearances showing negligible excretion of the drug in urine and suggesting elimination by metabolism. A cross-reacting metabolite in urine corresponded to less than 1.5% of the detomidine dose's immunoreactivity. High-dose detomidine increased urine flow significantly. Excretion of detomidine in milk in cattle was extremely low. No detectable amounts were present 23 h after dosing.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics and metabolism of selegiline.

Selegiline is readily absorbed from the gastrointestinal tract. It is distributed rapidly into the tissues, including the brain. It is the L-form of selegiline that is an active MAO-B inhibitor, the D-(+)-form being 25 times less active. Selegiline is metabolised into L-(-)-desmethylselegiline (DES), L-(-)-amphetamine (A) and L-(-)-methamphetamine (MA), mainly in the liver. We measured the steady state concentrations of the metabolites in the serum and cerebrospinal fluid (CSF) of patients with Parkinson's or Alzheimer's diseases who were on continuous selegiline therapy. The mean concentrations in serum and CSF were similar, and were not affected by the addition of levodopa. The mean concentrations of patients with Alzheimer's or Parkinson's disease were 6.5 +/- 2.5 ng/ml for A, 14.7 +/- 6.5 ng/ml for MA and 0.9 +/- 0.7 ng/ml for DES. The metabolites of selegiline were excreted in urine, and the recovery as metabolites was 87%. Due to the stereospecificity and the low CSF concentrations of the (-)amphetamine metabolites during the therapy with 10 mg selegiline, these metabolites do not seem to contribute significantly to the clinical efficacy of selegiline.

Pharmacokinetics of medetomidine.

The pharmacokinetics of medetomidine administered as a single dose (80 micrograms/kg) were studied in rat, dog and cat with the tritium labelled drug. The results showed a rapid distribution, after an s.c. dose, of medetomidine radioactivity into rat tissues including the brains. In plasma/serum a distribution phase with a half-life of only a few minutes was observed. Peak concentration after i.m. administration (dog & cat) was seen within 0.5 h in complete accordance with the rapid onset of clinical effects. The apparent volumes of distribution ranged from 2.8 (dog, i.v.) to 3.5 l/kg (cat, i.m.) and clearances from 27.5 (dog, i.m.) to 33.4 ml/min kg (dog, i.v.). Elimination of medetomidine from plasma/serum occurred with half-lives ranging from 0.97 to 1.60 h. Differences between dosing routes were small. Elimination of radioactivity from rat brain tissue followed approximately the same time course as elimination from plasma suggesting that termination of clinical effects is controlled by removal of drug from CNS. Excretion of radioactivity was from 28.6 to 74.7% of the dose in three days. In each species most of the activity was excreted in urine. Fecal excretion was significant only in the rat. No measurable levels of the parent drug were found in excreta. Instead a hydroxylated product(s) and (their) conjugates (except in the cat) were present in urine. Other metabolites were not identified. It was concluded that elimination occurs mainly by biotransformation in the liver.

Metabolism of detomidine in the rat. I. Comparison of 3H-labelled metabolites formed in vitro and in vivo.

The biotransformation of detomidine, a new alpha 2-adrenoceptor agonist, was studied using rat as the model animal. In vivo metabolism of the tritiated drug was compared to in vitro incubations with liver homogenates and intact, isolated hepatocytes. Metabolites were analysed by HPLC with radioactivity detection. The metabolic patterns in all systems were closely related. HPLC of urine gave twelve radioactive peaks. Tritiated water and unchanged 3H-detomidine were minor components. The two major peaks were tentatively identified as hydroxylated detomidine (14%) and its O-glucuronide (43%). Sulphate conjugates were not found. Isolated hepatocytes converted detomidine to the same two major products; the relative amount of the glucuronide increased with incubation time. In liver post-mitochondrial supernatant, hydroxylation was the dominant reaction, and the hydroxylated product comprised 74% of the total metabolites with non-induced and 50% with phenobarbital-induced liver. The major biotransformation in rat was thus concluded to be hydroxylation by the liver monooxygenases followed by glucuronic acid conjugation. The maximal rate of oxidation or the enzymatic capacity of a whole liver was estimated to be at least 100 nmol/min allowing for a high hepatic extraction ratio for detomidine. Together with the effective excretion of the glucuronide, this reaction sequence alone could account for the rapid elimination of the drug.

Metabolism of detomidine in the rat. II. Characterisation of metabolites in urine.

In order to investigate the biotransformation of a new alpha 2-adrenoceptor agonist, detomidine, metabolites were isolated from rat urine by solid phase extraction and purified by TLC. The isolated compounds were structurally analysed by 1H-NMR, MS and GC-MS as such or as their methyl and/or silyl derivatives. In addition to detomidine, which was found in trace amounts, four major metabolites were identified: hydroxymethyldetomidine, the corresponding O-glucuronide, detomidine carboxylic acid, and detomidine mercapturate. Together the identified components make up about 80% of urinary detomidine derived compounds. On the basis of these findings a major biotransformation pathway could be suggested. The reaction sequence is initiated by a hydroxylation. Subsequent glucuronidation, glutathione conjugation or secondary oxidation divide the route into three branches each producing one of the other three identified metabolites.