Positional isomers of monopegylated interferon alpha-2a: isolation, characterization, and biological activity.

The success of recombinant interferon alpha in the clinic in part is limited by two properties of the protein: short serum half-life and immunogenicity. To improve these properties, interferon alpha-2a was conjugated with polyethylene glycol (PEG-5000). A homogeneous preparation of monopegylated interferon alpha-2a was subjected to vigorous analytical and activity characterization. A newly developed ampholyte-free chromatofocussing-like cation-exchange HPLC method utilizing a sulfopropyl resin was used to separate the monopegylated protein into 11 species. Peptide mapping, sequencing, and mass spectrometric analyses indicated that these species are positional isomers where each isomer represents a single polymer molecule conjugated to one specific lysine residue. No species with a modification at the amino terminus was observed. All 11 isomers show antiviral and antiproliferative activities in the same range as the parent monopegylated interferon alpha-2a.

A versatile plasmid expression vector for the production of biotinylated proteins by site-specific, enzymatic modification in Escherichia coli.

A versatile plasmid vector was designed to direct the synthesis of recombinant proteins in either one of two forms that will be biotinylated in Escherichia coli with high efficiency at a single, unique site. The protein of interest can be produced with a peptide substrate for E. coli biotin holoenzyme synthetase (BirA) joined directly to its N terminus, or alternatively, as a fusion to the C terminus of a maltose-binding protein domain (MalE) with the peptide substrate on its N terminus. To maximize the yield of biotinylated protein, the vector is designed to express the substrate in a coupled translation arrangement with the enzyme.

The metabolism of (-)-3-phenoxy-N-methylmorphinan in dogs.

The disposition of radioactive (-)-3-phenoxy-N-methyl[2-3H]morphinan in dogs after oral administration has been investigated. Unchanged drug was not found in bile, urine, or feces. Excretion of total radioactivity in feces ranged from 67 to 78% of an oral dose. Two unconjugated metabolites were isolated from feces and identified by NMR and GC/MS. Both were substituted on the phenoxy group; they were found to be the p-hydroxy (pOH-PMM) and the m-methoxy-p-hydroxy (mOCH3-pOH-PMM) metabolites. Further, levorphanol and norlevorphanol were identified in feces both as free and conjugated metabolites, as well as a small amount of levomethorphan. Urine contained mostly unknown metabolites and conjugated levorphanol and pOH-PMM. Although the glucuronide of mOCH3-pOH-PMM was the major metabolite in bile, smaller amounts of the glucuronide and sulfate conjugated of both levorphanol and pOH-PMM were also found. Estimates for the total urinary and fecal excretion (as percentages of the dose) by two dogs for the five known metabolites were as follows: levorphanol, 18.8-21.5%; pOH-PMM, 14.4-20.6%; mOCH3-pOH-PMM, 14.9%; norlevorphanol, 2.8-6.1%; levomethorphan, 0.5%. Two of these metabolites, pOH-PMM and levorphanol, are potent analgesics.

Metabolism of carprofen, a nonsteroid anti-inflammatory agent, in rats, dogs, and humans.

The metabolic disposition of 14C-labeled carprofen [(+/-)-6-chloro-alpha-methylcarbazole-2-acetic acid] was investigated in rats, dogs, and humans. Carprofen is eliminated predominantly by biotransformation in these three species. In dogs and rats, the direct conjugation of carprofen to form an ester glucuronide and oxidation to the C-7 and the C-8 phenols followed by their conjugation represent the major metabolic pathways. Small amounts of the alpha-hydroxy derivative also are formed by these species and are excreted free in the urine. In dogs, biliary secretion predominates, and 70% of an intravenous dose of carprofen is excreted in the feces while 8--15% of the dose is excreted in the urine. In rats, fecal excretion due to biliary secretion varies from 60 to 75%, and urinary excretion accounts for 20--30% of an intravenous dose. In humans direct conjugation of carprofen represents the only significant pathway of metabolism. Between 65 and 70% of the orally administered carprofen was found to be excreted as the ester glucuronide in the urine, and most of the remaining dose was estimated to be secreted as this metabolite in the bile. Due to enterohepatic circulation, only a fraction of the biliary metabolite was recovered in the feces in humans. Less than 5% of the dose was excreted in human urine as free, intact carprofen. In dogs and humans, plasma levels of carprofen and of total radioactivity exhibit a multiphasic decline. In the three human subjects studied, the terminal component declined with a 13--26 hr half-life; the terminal half-life was approximately 40 hr in dogs.