Inga Bjørnsdottir

Metabolism and Excretion of the Once-Daily Human Glucagon-Like Peptide-1 Analog Liraglutide in Healthy Male Subjects and Its In Vitro Degradation by Dipeptidyl Peptidase IV and Neutral Endopeptidase

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 [3H]liraglutide, a single subcutaneous dose of 0.75 mg/14.2 MBq was given to healthy males. The recovered radioactivity in blood, urine, and feces was measured, and metabolites were profiled. In addition, [3H]liraglutide and [3H]GLP-1(7–37) were incubated in vitro with dipeptidyl peptidase-IV (DPP-IV) and neutral endopeptidase (NEP) to compare the metabolite profiles and characterize the degradation products of liraglutide. The exposure of radioactivity in plasma (area under the concentration-time curve from 2 to 24 h) was represented by liraglutide (≥89%) and two minor metabolites (totaling ≤11%). Similarly to GLP-1, liraglutide was cleaved in vitro by DPP-IV in the Ala8-Glu9 position of the N terminus and degraded by NEP into several metabolites. The chromatographic retention time of DPP-IV-truncated liraglutide correlated well with the primary human plasma metabolite [GLP-1(9–37)], 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.

Selectivity enhancement in capillary electrophoresis using non-aqueous media

Abstract Efficiency and selectivity are two important parameters in separation science in order to facilitate resolution of solutes. High efficiencies are easily obtained using capillary electrophoresis. Selectivity can be improved using different additives like surfactants or cyclodextrins. Recently, it has been shown that also non-aqueous capillary electrophoresis provides large improvements in selectivity and without the use of the above-mentioned additives.

The analgesic effect of codeine as compared to imipramine in different human experimental pain models

&NA; 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.

Directly coupled HPLC-NMR and HPLC-MS approaches for the rapid characterisation of drug metabolites in urine: application to the human metabolism of naproxen

High resolution nuclear magnetic resonance (NMR) spectroscopy is a very powerful tool for the structural identification of xenobiotic metabolites in complex biological matrices such as plasma, urine and bile. However, these fluids are dominated by thousands of signals resulting from endogenous metabolites and it is advantageous when investigating drug metabolites in such matrices to simplify the spectra by including a separation step in the experiment by directly-coupling HPLC and NMR. Naproxen (6-methoxy-alpha-methyl-2-naphthyl acetic acid) is administered as the S-enantiomer and is metabolised in vivo to form its demethylated metabolite which is subsequently conjugated with beta-D-glucuronic acid as well as with sulfate. Naproxen is also metabolised by phase II metabolism directly to form a glycine conjugate as well as a glucuronic acid conjugate at the carboxyl group. In the present investigation, the metabolism of naproxen was investigated in urine samples with a very simple sample preparation using a combination of directly-coupled HPLC-1H NMR spectroscopy and HPLC-mass spectrometry (MS). A buffer system was developed which allows the same chromatographic method to be used for the HPLC-NMR as well as the HPLC-MS analysis. The combination of these methods is complementary in information content since the NMR spectra provide evidence to distinguish isomers such as the type of glucuronides formed, and the HPLC-MS data allow identification of molecules containing NMR-silent fragments such as occur in the sulfate ester.

Determination of opium alkaloids in crude opium using non-aqueous capillary electrophoresis

A method for the quantitative determination of the opium alkaloids morphine, codeine, thebaine, noscapine and papaverine in crude opium and in drug preparations based on non-aqueous capillary electrophoresis has been developed. The non-aqueous mode provides high separation selectivity and new possibilities for regulating the selectivity in capillary electrophoresis. The nature of the organic solvent, the acidity of the electrolytes as well as the temperature surrounding the capillary exhibit a major influence on the separation selectivity.

Assay of acetylsalicylic acid and three of its metabolites in human plasma and urine using non-aqueous capillary electrophoresis with reversed electroosmotic flow

The separation of acetylsalicylic acid and three of its metabolites--salicylic acid, salicyluric acid and gentisic acid--is demonstrated in a non-aqueous capillary electrophoresis system with reversed electroosmotic flow. Solvent mixtures of methanol and acetonitrile are used for the electrophoresis media and different electrolytes have been investigated. The flow is reversed by the addition of the polycation hexadimethrine bromide and thus a negative voltage is used. This system provides a fast and effective separation of the four analytes. The separation method was applied to the assay of acetylsalicylic acid and its major metabolites in plasma and urine and the limits of quantification for all of these compounds are about 5 microg ml(-1) in plasma and 25 microg ml(-1) in urine.

Chemical reactivity of the naproxen acyl glucuronide and the naproxen coenzyme A thioester towards bionucleophiles

Drugs may be metabolised to reactive electrophilic species that spontaneously react with proteins. The presence of such drug-protein adducts has been associated with drug toxicity. In this study, the reactivity of the major metabolite of naproxen--the 1-beta-O-glucuronide (Nap-GlcU)--was compared to the corresponding naproxen coenzyme A (Nap-CoA) thioester. The reactivity of the two metabolites was assessed in vitro in a phosphate buffer (pH 7.4; 0.1 M) at 37 degrees C towards the model bionucleophiles glutathione and human serum albumin (HSA). The reaction between the electrophilic species (Nap-GlcU and Nap-CoA) and glutathione forming the Nap-glutathione conjugate was monitored using LC-MS-MS and LC-UV, respectively. It was shown that Nap-CoA resulted in an approximate 100-fold higher formation of Nap-glutathione conjugate than Nap-GlcU. The presence of Nap-CoA also resulted in acylated HSA with a rate and a yield that was significantly higher than reported for Nap-GlcU. In summary, the data suggest that CoA metabolites may be more reactive species than acyl glucuronides that previously have been associated with severe drug related side effects in vivo.

Fast separation of 16 seizure drug substances using non-aqueous capillary electrophoresis

A fast and simple method for separation of 16 seizure drug substances using capillary electrophoresis in a non-aqueous separation medium is described. The separation medium consists of a mixture of acetonitrile, methanol and glycerol with ammonium acetate/acetic acid as the electrolyte. The analytes are detected by UV detection at 214 nm. Injection from the detection end (8.5 cm to detector) combined with the usage of a short capillary (32.5 cm total length) makes it possible to separate all 16 amines within 2 min. The choice of solvents, electrolytes and viscosity increasing additives are discussed with special emphasis to their influence on the separation selectivity.

Studies on the Metabolism of Tolmetin to the Chemically Reactive Acyl-Coenzyme A Thioester Intermediate in Rats

Carboxylic acids may be metabolized to acyl glucuronides and acyl-coenzyme A thioesters (acyl-CoAs), which are reactive metabolites capable of reacting with proteins in vivo. In this study, the metabolic activation of tolmetin (Tol) to reactive metabolites and the subsequent formation of Tol-protein adducts in the liver were studied in rats. Two hours after dose administration (100 mg/kg i.p.), tolmetin acyl-CoA (Tol-CoA) was identified by liquid chromatography-tandem mass spectrometry in liver homogenates. Similarly, the acyl-CoA-dependent metabolites tolmetin-taurine conjugate (Tol-Tau) and tolmetin-acyl carnitine ester (Tol-Car) were identified in rat livers. In a rat bile study (100 mg/kg i.p.), the S-acyl glutathione thioester conjugate was identified, providing further evidence of the formation of reactive metabolites such as Tol-CoA or Tol-acyl glucuronide (Tol-O-G), capable of acylating nucleophilic functional groups. Three rats were treated with clofibric acid (150 mg/kg/day i.p. for 7 days) before dose administration of Tol. This resulted in an increase in covalent binding to liver proteins from 0.9 nmol/g liver in control rats to 4.2 nmol/g liver in clofibric acid-treated rats. Similarly, levels of Tol-CoA increased from 0.6 nmol/g to 4.4 nmol/g liver after pretreatment with clofibric acid, whereas the formation of Tol-O-G and Tol-Tau was unaffected by clofibric acid treatment. However, Tol-Car levels increased from 0.08 to 0.64 nmol/g after clofibric acid treatment. Collectively, these results confirm that Tol-CoA is formed in vivo in the rat and that this metabolite can have important consequences in terms of covalent binding to liver proteins.

Electrospray ionization mass spectrometric method for the determination of cannabinoid precursors: N-acylethanolamine phospholipids (NAPEs)†

N-Acylethanolamine phospholipids (NAPEs) serve as endogenous precursors of N-acylethanolamines (NAEs), e.g. N-arachidonoylethanolamine (anandamide) and N-palmitoylethanolamine that are endogenous ligands of cannabinoid receptors. Under physiological conditions, NAPE is found in very low concentrations in mammalian tissue (3-12 nmol g(-1)). However, pathophysiological conditions may increase the endogenous NAPE levels, which again may cause an increase in endocannabinoid concentrations. This paper presents a simple and selective method for the determination of NAPE standards using negative electrospray ionization mass spectrometry (ESI-MS). The procedure provides complete positioning of all acyl and alkenyl groups contained in each NAPE species. The calibration curve for standard NAPE was linear over the range 100 fmol-50 pmol (0.1-50 ng) per injection. The lower limit of detection (signal-to-noise ratio of 3) was 100 fmol, implying that this method is superior to previous methods for the determination of NAPE. These results suggest that this ESI-MS method can be used to identify and quantify NAPE species in mammalian tissues and provide information on the corresponding NAEs to be released from the endogenous NAPE pool.