Effects of fadolmidine, an α2 -adrenoceptor agonist, as an adjuvant to spinal bupivacaine on antinociception and motor function in rats and dogs.

α2 -Adrenoceptor agonists such as clonidine and dexmedetomidine are used as adjuvants to local anesthetics in regional anesthesia. Fadolmidine is an α2 -adrenoceptor agonist developed especially as a spinal analgesic. The current studies investigate the effects of intrathecally administered fadolmidine with a local anesthetic, bupivacaine, on antinociception and motor block in conscious rats and dogs. The antinociceptive effects of intrathecal fadolmidine and bupivacaine alone or in combination were tested in the rat tail-flick and the dog's skin twitch models. The durations of motor block in rats and in dogs were also assessed. In addition, the effects on sedation, mean arterial blood pressure, heart rate, respiratory rate and body temperature were evaluated in telemetrized dogs. Concentrations of fadolmidine in plasma and spinal cord were determined after intrathecal and intravenous administration in rats. Co-administration of intrathecal fadolmidine with bupivacaine increased the magnitude and duration of the antinociceptive effects and prolonged motor block without hypotension. The interaction of the antinociceptive effect was synergistic in its nature in rats. Concentration of fadolmidine in plasma was very low after intrathecal dosing. Taken together, these studies show that fadolmidine as an adjuvant to intrathecal bupivacaine provides enhanced sensory-motor block and enables a reduction of the doses of both drugs. The results indicate that co-administration of fadolmidine with intrathecal bupivacaine was able to achieve an enhanced antinociceptive effect without hypotension and could thus represent a suitable combination for spinal anesthesia.

Regio- and stereospecific N-glucuronidation of medetomidine: the differences between UDP glucuronosyltransferase (UGT) 1A4 and UGT2B10 account for the complex kinetics of human liver microsomes.

Medetomidine is a chiral imidazole derivate whose dextroenantiomer is pharmacologically active. The major metabolic pathway of dexmedetomidine [(+)-4-(S)-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole] in humans is N-glucuronidation at the imidazolate nitrogens. We have purified the N3- and N1-glucuronides of dexmedetomidine, termed DG1 and DG2, respectively, according to their elution order in liquid chromatography and determined their structure by 1H nuclear magnetic resonance (NMR). Studying medetomidine glucuronidation by human liver microsomes (HLMs) and recombinant UDP glucuronosyltransferase (UGT) 1A4 indicated that another human UGT plays a major role in these activities. We now demonstrate that this enzyme is UGT2B10. HLMs catalyzed DG1 and DG2 formation, at a ratio of 3:1, with two-enzyme kinetics that contain both a high-affinity component, K(m1) values of 6.6 and 8.7 microM, and a low-affinity component, K(m2) values > 1 mM. The DG1/DG2 ratio in the case of UGT2B10 was lower, 1.4:1, whereas the substrate affinity for both reactions was high, K(m) values of 11 and 16 microM. UGT1A4 produced mainly DG1 (DG1/DG2 ratio of 6.6:1) at low substrate affinities, K(m) values above 0.6 mM, but superior expression-normalized V(max) values. Levomedetomidine [(-)-4-(R)-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole] glucuronidation by HLMs yielded mostly the N3-glucuronide (LG1, structure determined by NMR), with monophasic kinetics and a K(m) value of 14 microM. The activity of UGT1A4 toward levomedetomide was low and generated both LG1 and LG2, whereas UGT2B10 exhibited relatively high activity and sharp regioselectivity, yielding only LG1, with a K(m) value of 7.4 microM. The results highlight the contribution of UGT2B10 to medetomidine glucuronidation and its potential importance for other N-glucuronidation reactions within the human liver.

Comparative studies on the cytochrome p450-associated metabolism and interaction potential of selegiline between human liver-derived in vitro systems.

Selegiline was used as a model compound in a project aimed at comparing, evaluating, and integrating different in vitro approaches for the prediction of cytochrome p450 (p450)-catalyzed hepatic drug metabolism in humans (EUROCYP). Metabolic predictions were generated using homology modeling, cDNA-expressed p450 enzymes, human liver microsomes, primary cultured human hepatocytes, and precision-cut human liver slices. All of the in vitro systems correctly indicated the formation of two dealkylated metabolites, desmethylselegiline and methamphetamine. The metabolic instability of selegiline was demonstrated by all of the in vitro systems studied. Estimates of clearance varied from 16 l/h to 223 l/h. With the exception of one approach, all systems underpredicted the in vivo clearance in humans (236 l/h). Despite this, all approaches successfully classified selegiline as a high clearance compound. Homology modeling suggested the participation of CYP2B6 in the demethylation of selegiline and of CYP2D6 in the depropargylation of the drug. Studies with recombinant expressed enzymes and with human hepatic microsomal fraction supported the involvement of CYP2B6 but not of CYP2D6. These techniques also suggested the involvement of CYP1A2, CYP2C8, and CYP2C19 in the biotransformation of selegiline. In vitro, CYP2B6 was the most active form of p450 involved in selegiline metabolism. Metabolism by several enzymes operating in parallel implies a low interaction potential for the drug. None of the techniques alone was able to predict all aspects of the metabolic and kinetic behavior of selegiline in vivo. However, when used as an integrated package, all significant characteristics were predictable.

N-Glucuronidation of some 4-arylalkyl-1H-imidazoles by rat, dog, and human liver microsomes.

N-Glucuronidation in vitro of six 4-arylalkyl-1H-imidazoles (both enantiomers of medetomidine, detomidine, atipamezole, and two other closely related compounds) by rat, dog, and human liver microsomes and by four expressed human UDP-glucuronosyltransferase isoenzymes was studied. Human liver microsomes formed N-glucuronides of 4-arylalkyl-1H-imidazoles with high activity, with apparent V(max) values ranging from 0.59 to 1.89 nmol/min/mg of protein. In comparison, apparent V(max) values for two model compounds forming the N-glucuronides 4-aminobiphenyl and amitriptyline were 5.07 and 0.56 nmol/min/mg of protein, respectively. Atipamezole showed an exceptionally low apparent K(m) value of 4.0 microM and a high specificity constant (V(max)/K(m)) of 256 compared with 4-aminobiphenyl (K(m), 265 microM; V(max)/K(m), 19) and amitriptyline (K(m), 728 microM; V(max)/K(m), 0.8). N-Glucuronidation of medetomidine was highly enantioselective in human liver microsomes; levomedetomidine exhibited a 60-fold V(max)/K(m) value compared with dexmedetomidine. Furthermore, two isomeric imidazole N-glucuronides were formed from dexmedetomidine, but only one was formed from levomedetomidine. Dog liver microsomes formed N-glucuronides of 4-arylalkyl-1H-imidazoles at a low rate and affinity, with apparent V(max) values ranging from 0.29 to 0.73 nmol/min/mg of protein and apparent K(m) values from 279 to 1640 microM. Rat liver microsomes glucuronidated these compounds at a barely detectable rate. Four expressed human UDP-glucuronosyltransferase isoenzymes (UGT1A3, UGT1A4, UGT1A6, and UGT1A9) were studied for 4-arylalkyl-1H-imidazole-conjugating activity. Only UGT1A4 glucuronidated these compounds at an activity of about 5% of that measured for 4-aminobiphenyl. The observed activity of UGT1A4 does not explain the high efficiency of glucuronidation of 4-arylalkyl-1H-imidazoles in human liver microsomes.