Jørgen Olsen

In vivo proof of concept of oral insulin delivery based on a co-administration strategy with the cell-penetrating peptide penetratin.

Oral delivery of insulin is blocked by low intestinal absorption caused by the poor permeability of insulin across cellular membranes and the susceptibility to enzymatic degradation in the gastrointestinal tract. Cell-penetrating peptides (CPPs) have been investigated for a number of years as oral absorption enhancers for hydrophilic macromolecules. Penetratin, a cationic and amphipathic CPP, effectively enhances insulin absorption and we were able to alleviate the enzymatic barrier by using the enzymatic resistant D-form of penetratin. In this study, mice were dosed orally with a physical mixture of insulin and penetratin. Blood glucose concentrations were measured and a pharmacological availability (PA) of 18.2% was achieved in mice dosed with insulin and D-penetratin. Following the promising data, we investigated the degradation parameters of insulin and penetratin in rat intestinal fluid. As expected, L-penetratin was degraded rapidly whereas D-penetratin had a halflife of 67±7min in 10-fold diluted gastrointestinal fluid. Insulin degradation was slowed by the presence of penetratin in intestinal fluid. The half-life of insulin increased from 24.9±4.5min to 55.6±14min and 90.5±11.8min in the presence of L- and D-penetratin respectively. In conclusion, both Land D-penetratin acted as oral absorption enhancers at select CPP concentrations for insulin and the current study is the first solid evidence of pharmacological activity of oral insulin delivery systems based on non-covalent intermolecular interactions with penetratin.

Separation, purification and identification of the major selenium metabolite from human urine by multi-dimensional HPLC-ICP-MS and APCI-MS

When humans are supplied with selenium-containing nutritional preparations, one of the selenium-containing metabolites in urine increases relatively more than the other selenium metabolites. The purpose of this study was to identify this major selenium metabolite. Urine samples from six male volunteers were collected and analysed by ion-pair chromatography with ICP-MS detection for this major selenium metabolite. Samples containing the metabolite were pooled and solid phase extracted to remove ionic substances. The extracted pool was purified and preconcentrated twice by preparative reversed-phase chromatography. The fractions containing the selenium metabolite were collected and further purified by size exclusion chromatography. It was not possible to ionize the selenium metabolite by electrospray ionization mass spectrometry, ESI-MS. Instead, atmospheric pressure chemical ionization, APCI, was applied. The m/z of the selenium metabolite was 300 for the 80Se isotope. MS/MS experiments indicated that the metabolite was a selenosugar, and it is proposed that the selenium metabolite is a Se-methyl-N-acetylselenohexosamine.

Electrochemical oxidation of troglitazone: identification and characterization of the major reactive metabolite in liver microsomes.

Troglitazone (TGZ) was developed for the treatment of type 2 diabetes but was withdrawn from the market due to hepatotoxicity. The formation of reactive metabolites has been associated with the observed hepatotoxicity. Such reactive metabolites have been proposed to be formed via three different mechanisms. One of the proposed mechanisms involves the oxidation of the chromane moiety of TGZ to a reactive o-quinone methide. The two other mechanisms involve metabolic activation of the thiazolidinedione moiety of TGZ. In the present study, it is shown that electrochemical oxidations can be used to generate a reactive metabolite of TGZ, which can be trapped by GSH or N-acetylcysteine. From incubations of TGZ with rat and human liver microsomes in the presence of either GSH or N-acetylcysteine, it was shown that similar conjugates were formed in vitro as formed from electrochemical oxidations of TGZ. One- and two-dimensional NMR studies of the troglitazone- S-( N-acetyl)cysteine conjugate revealed that N-acetylcysteine was attached to a benzylic carbon in the chromane moiety, showing that the conjugate was formed via a reaction between the o-quinone methide of TGZ and N-acetylcysteine. From electrochemical oxidations of rosiglitazone, pioglitazone, and ciglitazone in the presence of GSH, no GSH conjugates could be identified. These three compounds all contain a thiazolidinedione moiety. In conclusion, it has been shown that the primary reactive metabolite of TGZ formed from electrochemical oxidation was the o-quinone methide, and this metabolite was similar to what was observed to be the primary reaction product in human and rat liver microsomes.

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.

Combination of LC-ICP-MS, LC-MS and NMR for investigation of the oxidative degradation of selenomethionine.

Selenomethionine (SeMet) was oxidized by heating an acidic solution with hydrogen peroxide. Samples were taken before and during the oxidation process. The oxidation products were separated by cation exchange chromatography followed by ICP-MS detection to identify the selenium containing compounds as well as electrospray ionization MS detection to determine the masses of the degradation products. Furthermore, the samples were analyzed by (77)Se-NMR. The first appearing degradation product was selenomethionine selenoxide, which was converted via the deaminated selenoxide to methane seleninic acid and selenite.

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.

Identification of coenzyme A-related tolmetin metabolites in rats: relationship with reactive drug metabolites.

1. It has recently been proposed that acyl coenzyme A thioesters (acyl-CoAs) of xenobiotic carboxylic acids are electrophilic, reactive metabolites that may react with proteins. 2. The primary objective was to investigate the reactivity of the tolmetin acyl coenzyme A thioester (Tol-CoA). The second objective was to identify and quantify tolmetin (Tol) metabolites in vivo that were formed via Tol-CoA, e.g. the glycine (Tol-Gly) and taurine (Tol-Tau) conjugates. This finding would be indicative of Tol-CoA formation and thus of other acyl-CoA-related reactions that might occur, e.g. covalent binding to proteins. 3. In order to study the chemical reactivity, Tol-CoA (0.5 mM) was incubated with glutathione (5 mM) in a 0.1 M phosphate buffer (pH 7.4) at 37°C. Tol-CoA reacted rapidly with glutathione in vitro to form the S -acyl glutathione conjugate at a rate of 14.9 ± 0.7 µ M min − 1 (mean ± SD, n = 3) from 0 to 10 min. Compared with acyl-CoAs of other xenobiotic carboxylic acids, naproxen and clofibric acid, the rate by which Tol-CoA reacted with glutathione was high. 4. Following administration of 3 H-Tol (100 mg kg − 1, 200 µ Ci kg − 1, p.o.) to male Sprague-Dawley rats, Tol-Tau and Tol-Gly were identified in urine by electrospray ionization MS-MS in both positive- and negative-ion modes. The conjugates were only formed at trace levels (< 0.5%). However, the presence of Tol-Tau and Tol-Gly showed the reactive Tol-CoA was formed in vivo.

Identification of the amino acids of human serum albumin involved in the reaction with the naproxen acyl coenzyme A thioester using liquid chromatography combined with fluorescence and mass spectrometric detection.

Xenobiotic carboxylic acids, that via their metabolites covalently modify proteins, have been associated with serious side effects in man. Such reactive metabolites may be acyl glucuronides or alternatively, the corresponding acyl-CoA thioesters. In this study, the reaction of a model xenobiotic acyl-CoA, the naproxen-CoA, with human serum albumin (HSA), was characterized by high-performance liquid chromatography employing fluorescence and mass spectrometric detection. One mM naproxen-CoA was incubated for 6h with HSA (0.45 mM) at 37 degrees C in a 0.1M phosphate buffer (pH 7.4). The tryptic digest of the reduced and alkylated protein was analyzed in order to identify the amino acids in the sequence that were covalently modified with naproxen. Fluorescent peptides, that represented naproxen-modified peptides, were characterized using HPLC-MS-MS and HPLC-MS in zoom scan mode, which provided information on the structure and the charge of the modified peptides. The naproxen-CoA reacted predominantly with lysine 199, lysine 541, and lysine 351, which was in agreement with the binding pattern that has previously been reported for the reactive acyl glucuronides and their reaction with HSA.

Metabolism of peptide YY 3–36 in Göttingen mini-pig and rhesus monkey

Peptide YY 3-36-amide (PYY3-36) is a peptide hormone, which is known to decrease appetite and food-intake by activation of the Y2 receptor. The current studies were designed to identify the metabolites of PYY3-36 in mini-pig and rhesus monkey. Plasma samples were analyzed by high resolution LC-MS (and MS/MS) in order to unambiguously identify the metabolites of PYY3-36. In summary, the metabolism of PYY3-36 was similar in mini-pig and rhesus monkey. Several metabolites were identified and PYY3-34 was identified at the highest levels in plasma. In addition, mini-pigs were also dosed with PYY1-36-amide, PYY3-35, PYY3-34 and [N-methyl 34Q]-PYY3-36-amide in order to investigate the mechanisms by which PYY was metabolized. PYY3-35 was rapidly converted to PYY3-34 whereas dosing of PYY3-34 to mini-pigs only showed circulating degradation products at low levels, i.e., PYY3-34 was metabolically more stable than PYY3-36 and PYY3-35. [N-methyl 34Q]-PYY3-36-amide was hypothesized to be stable toward cleavage between 34Q and 35R and after i.v. administration to mini-pigs, one major cleavage product was identified as [N-methyl 34Q]-PYY3-35. Overall, this showed that cleavage between 35R and 36Y was possible as well as between 34Q and 35R (as shown for PYY3-35), which indicated that metabolism of PYY3-36 to PYY3-34 may be a two-step process. PYY1-36 was also dosed to mini-pigs, which showed that PYY1-36 was metabolized in the C-terminal as PYY3-36. The overall degradation pattern of PYY1-36 was more complex due to the simultaneous enzymatic degradation in the N-terminal to form PYY2-34/36 and PYY3-34/36. In vitro incubations with heparin stabilized plasma showed that PYY3-36 was degraded with a half-life of 175 min, whereas incubations with PYY3-35 (half-life of 6 min) showed a rapid formation of PYY3-34. In conclusion, the present studies showed that PYY3-36 underwent enzymatic degradation in the C-terminal part and that the major circulating metabolite was PYY3-34. Furthermore, it may be a sequential two-step process leading to the formation of PYY3-35 and subsequently the metabolically more stable PYY3-34.