In vitro metabolism of opioid tetrapeptide agonists in various tissues and subcellular fractions from rats.

The metabolism of three mu-selective opioid tetrapeptide agonists, Tyr-D-Arg-Phe-Nva-NH(2) (TArPN), Tyr-D-Arg-Phe-Phe-NH(2) (TArPP), and Tyr-D-Ala-Phe-Phe-NH(2) (TAPP), was investigated in different rat tissues. High metabolic activity (<20% peptide remaining after 30 min) was found against the three peptides in the kidney homogenate and against TArPN in spleen homogenate. Low metabolic activity (>80% peptide remaining after 30 min) was found for all peptides in brain homogenate and plasma, and for TArPN and TArPP in blood. The other tissue homogenates, prepared from the small and large intestine, liver and lung, all exhibited intermediate metabolic activity (20-80% peptide remaining after 30 min) against the peptides. In all tissues investigated, the tetrapeptides were metabolized at the C-terminal amide by deamidation.A further in depth metabolic investigation was performed in subcellular fractions isolated from three tissues (small intestine, liver and kidney). In the liver, the deamidation was predominantly localized to the mitochondrial/lysosomal fraction, while hydrolysis at the N-terminal Tyr residue was the major metabolic pathway in the microsomal/brush-border membrane fraction from the kidney and small intestine.

Investigations of the in-vitro metabolism of three opioid tetrapeptides by pancreatic and intestinal enzymes.

The metabolism of three opioid tetrapeptides, Tyr-D-Arg-Phe-Nva-NH2, Tyr-D-Arg-Phe-Phe-NH2 and Tyr-D-Ala-Phe-Phe-NH2, was investigated in the presence of pure pancreatic enzymes (trypsin, chymotrypsin, elastase, carboxypeptidase A and carboxypeptidase B), as well as in the presence of pure carboxylesterase and aminopeptidase N. The cleavage patterns of the pure pancreatic enzymes were then compared with those found in rat and human jejunal fluid. Metabolism was also studied in homogenates from different intestinal regions (duodenum, jejunum, ileum and colon) and in enterocyte cytosol from rats. The effect of various protease inhibitors was investigated in the jejunal homogenate. The parent peptides were assayed by high-performance liquid chromatography and metabolites were identified by means of liquid chromatography-mass spectrometry. Of the pure enzymes, the quickest hydrolysis of the peptides was observed for the pancreatic enzymes chymotrypsin, trypsin and carboxypeptidase A. In most cases they formed the corresponding deamidated tetrapeptides (chymotrypsin and trypsin) or tripeptides with a missing C-terminal amino acid (carboxypeptidase A). Regional differences in intestinal metabolism rates were found for all three peptides (P < 0.001), with the highest rates observed in jejunal and/or colonic homogenates. The deamidated tetrapeptides were formed both in rat intestinal homogenates and in enterocyte cytosol. Metabolism in the jejunal homogenate was markedly inhibited by some serine and combined serine and cysteine protease inhibitors. In conclusion, the C-terminal amide of these tetrapeptides did not fully stabilise them against intestinal deamidase and carboxypeptidase activities. The significant hydrolysis of the peptides by pure chymotrypsin, trypsin and carboxypeptidase A showed that lumenal pancreatic proteases might be a clear metabolic obstacle in oral delivery even for small peptides such as these tetrapeptides.

Studies on sulfation of synthesized metabolites from the local anesthetics ropivacaine and lidocaine using human cloned sulfotransferases.

The metabolism of the local anesthetics lidocaine and ropivacaine (ropi) involves several steps in humans. Lidocaine is mainly hydrolyzed and hydroxylated to 4-OH-2,6-xylidine (4-OH-xyl). The metabolism of ropi, involving dealkylation and hydroxylation, gives rise to 3-OH-ropi, 4-OH-ropi, 3-OH-2'6'-pipecoloxylidide (3-OH-PPX), and 2-OH-methyl-ropi. Because the metabolites are hydroxylated, they are particularly prone to subsequent Phase II conjugation reactions such as sulfation and glucuronidation. This study focused on the in vitro sulfation of these metabolites as well as another suspected metabolite of ropi, 2-carboxyl-ropi. All the metabolites were synthesized for the subsequent enzymatic studies. Five cloned human sulfotransferases (STs) were used in this study, namely, the phenol-sulfating form of ST (P-PST-1), the monoamine-sulfating form of ST (M-PST), estrogen-ST (EST), ST1B2, and dehydroepiandrosterone-ST (DHEA-ST), all of which are expressed in human liver. The results demonstrate that all of the metabolites except 2-OH-methyl-ropi and 2-carboxyl-ropi can be sulfated. It was also found that all of the STs can conjugate the remaining hydroxylated metabolites except DHEA-ST. However, there are large differences in the capacity of the individual human ST isoforms to conjugate the different metabolites. P-PST-1 sulfates 3-OH-PPX, 3-OH-ropi, and 4-OH-xyl; M-PST and EST conjugate 3-OH-PPX, 3-OH-ropi, and 4-OH-ropi whereas ST1B2 sulfates only 4-OH-xyl. The most extensively sulfated ropi metabolite is 3-OH-PPX. In conclusion, all of the hydroxylated metabolites of lidocaine and ropi can be sulfated if the hydroxyl group is attached to the aromatic ring in the metabolites. The human ST enzymes that are considered to be responsible for the sulfation of these metabolites in vivo are P-PST-1, M-PST, EST, and ST1B2. These enzymes are also found in the liver; this is the most important tissue for the metabolism of ropi in humans, demonstrated by.