Aims To identify the principal human cytochrome P450 (CYP) enzyme(s) responsible for the human in vitro biotransformation of repaglinide. Previous experiments have identified CYP3A4 as being mainly responsible for the in vitro metabolism of repaglinide, but the results of clinical investigations have suggested that more than one enzyme may be involved in repaglinide biotransformation. Methods [ 14 C]-Repaglinide was incubated with recombinant CYP and with human liver microsomes (HLM) from individual donors in the presence of inhibitory antibodies specific for individual CYP enzymes. Metabolites, measured by highperformance liquid chromatography (HPLC) with on-line radiochemical detection, were identified by liquid chromatography-mass spectrophotometry (LC-MS) and LC-MS coupled on-line to a nuclear magnetic resonance spectrometer (LC-MS-NMR). Results CYP3A4 and CYP2C8 were found to be responsible for the conversion of repaglinide into its two primary metabolites, M4 (resulting from hydroxylation on the piperidine ring system) and M1 (an aromatic amine). Specific inhibitory monoclonal antibodies against CYP3A4 and CYP2C8 significantly inhibited ( > 71%) formation of M4 and M1 in HLM. In a panel of HLM from 12 individual donors formation of M4 and M1 varied from approximately 160-880 pmol min -1 mg -1 protein and from 100-1110 pmol min -1 mg -1 protein, respectively. The major metabolite generated by CYP2C8 was found to be M4. The rate of formation of this metabolite in HLM correlated significantly with paclitaxel 6 a -hydroxylation ( r s = 0.80; P = 0.0029). Two other minor metabolites were also detected. One of them was M1 and the other was repaglinide hydroxylated on the isopropyl moiety (M0-OH). The rate of formation of M4 in CYP2C8 Supersomes TM was 2.5 pmol min -1 pmol -1 CYP enzyme and only about 0.1 pmol min -1 pmol -1 CYP enzyme in CYP3A4 Supersomes TM . The major metabolite generated by CYP3A4 was M1. The rate of formation of this metabolite in HLM correlated significantly with testosterone 6 b -hydroxylation ( r s = 0.90; P = 0.0002). Three other metabolites were identified, namely, M0-OH, M2 (a dicarboxylic acid formed by oxidative opening of the piperidine ring) and M5. The rate of M1 formation in CYP3A4 Supersomes TM was 1.6 pmol min -1 pmol -1 CYP enzyme but in CYP2C8 Supersomes TM it was only approximately 0.4 pmol min -1 pmol -1 CYP enzyme. Conclusions The results confirm an important role for both CYP3A4 and CYP2C8 in the human in vitro biotransformation of repaglinide. This dual CYP biotransformation may have consequences for the clinical pharmacokinetics and drug-drug interactions involving repaglinide if one CYP pathway has sufficient capacity to compensate if the other is inhibited. T. B. Bidstrup et al .306

ABSTRACT:Liraglutide is a novel once-daily human glucagon-like peptide (GLP)-1 analog in clinical use for the treatment of type 2 diabetes. To study metabolism and excretion of [ , and some of the NEP degradation products eluted very close to both plasma metabolites. Three minor metabolites totaling 6 and 5% of the administered radioactivity were excreted in urine and feces, respectively, but no liraglutide was detected. In conclusion, liraglutide is metabolized in vitro by DPP-IV and NEP in a manner similar to that of native GLP-1, although at a much slower rate. The metabolite profiles suggest that both DPP-IV and NEP are also involved in the in vivo degradation of liraglutide. The lack of intact liraglutide excreted in urine and feces and the low levels of metabolites in plasma indicate that liraglutide is completely degraded within the body.

Semaglutide is a human glucagon-like peptide-1 analogue in clinical development for the treatment of type 2 diabetes. The absorption, metabolism and excretion of a single 0.5mg/450μCi [16.7MBq] subcutaneous dose of [H]-radiolabelled semaglutide was investigated in healthy human subjects and compared with data from nonclinical studies. Radioactivity in blood, plasma, urine and faeces was determined in humans, rats and monkeys; radioactivity in expired air was determined in humans and rats. Metabolites in plasma, urine and faeces were quantified following profiling and radiodetection. The blood-to-plasma ratio and pharmacokinetics of both radiolabelled semaglutide-related material and of semaglutide (in humans only) were assessed. Intact semaglutide was the primary component circulating in plasma for humans and both nonclinical species, accounting for 69-83% of the total amount of semaglutide-related material, and was metabolised prior to excretion. Recovery of excreted radioactivity was 75.1% in humans, 72.1% in rats and 58.2% in monkeys. Urine and faeces were shown to be important routes of excretion, with urine as the primary route in both humans and animals. Semaglutide was metabolised through proteolytic cleavage of the peptide backbone and sequential beta-oxidation of the fatty acid sidechain, and metabolism was not confined to specific organs. Intact semaglutide in urine accounted for 3.1% of the administered dose in humans and less than 1% in rats; it was not detected in urine in monkeys. The metabolite profiles of semaglutide in humans appear to be similar to the profiles from the nonclinical species investigated.

The hypoalgesic effect of single oral doses of 100 mg imipramine and 125 mg codeine was evaluated in a randomised, placebo-controlled, double-blind, 3-way cross-over experiment including 18 healthy volunteers. Pain tests were performed before and 90, 180, 270, 360 and 450 min after medication. The tests included determination of pain tolerance thresholds to pressure, pain detection/tolerance thresholds to single electrical sural nerve stimulation and pain summation at tolerance threshold to repetitive electrical sural nerve stimulation (temporal summation) and pain experienced during the cold pressor test, rated as peak pain intensity, pain average intensity and discomfort. Compared to placebo, imipramine significantly increased pressure pain tolerance threshold (P = 0.03) and increased pain tolerance threshold (P = 0.05) and pain summation threshold (P = 0.03), but not pain detection threshold to electrical stimulation. Imipramine did not cause significant changes in pain perception during the cold pressor test. Codeine significantly increased pressure pain tolerance threshold (P = 0.02), pain detection (P = 0.04) and pain tolerance threshold (P = 0.01) and pain summation threshold (P = 0.02) to electrical stimulation. In addition, codeine reduced the pain experienced during the cold pressor test (P = 0.04-0.003). It is concluded that both imipramine and codeine inhibit temporal pain summation, whereas only codeine reduces cold pressor pain. Pain summation may be a key mechanism in neuropathic pain. Imipramine has a documented effect on such pain conditions on temporal summation. The present study showed that codeine also inhibits temporal summation, which is in line with the clinical observations indicating that opioids relieve neuropathic pain.

The on-line separation and structure elucidation of naphthodianthrones, flavonoids, and other constituents of an extract from Hypericum perforatum L. using high performance liquid chromatography (HPLC) coupled on-line with ultraviolet-visible, nuclear magnetic resonance (NMR), and mass spectrometry (MS) is described. A conventional reversed-phase HPLC system using ammonium acetate as the buffer substance in the eluent was used, and proton NMR spectra were obtained on a 500 MHz NMR instrument. The MS and MS/MS analyses were performed using negative electrospray ionization. In the present study, all of the major known constituents in extracts from Hypericum perforatum L. were identified, and two new substances which had not previously been reported as constituents of extracts of Hypericum perforatum L. were identified and their structures elucidated.