Disposition and metabolism of rifapentine, a rifamycin antibiotic, in mice, rats, and monkeys.

Rifapentine is a cyclopentyl derivative of rifampin under development for the treatment of Mycobacterium tuberculosis and Mycobacterium avium complex infections. These studies were designed to investigate the disposition and biotransformation of single iv and oral doses of 14C-rifapentine in mice, bile duct-cannulated and uncannulated rats, and monkeys. Mass balance studies included 14C analyses of urine, feces, bile, cage wash, carcasses, and cage air collected for up to 120 hr postdose. Separation of radioactive compounds extracted from urine, bile, and feces was conducted using high-performance liquid chromatography and radioisotope detection. The mass spectra of selected chromatographic peaks were obtained. Disposition results were similar for all three species. Less than 5% of the radioactive dose of 14C-rifapentine was recovered in urine, indicating that renal excretion is a minor route of elimination in these species. The major route of elimination of radioactivity was into the feces, where more than 75% of the radioactivity was recovered. Biliary excretion was the major route of elimination of radioactivity in bile duct-cannulated rats dosed either po or IV. Radiochromatograms were similar for fecal samples from animals dosed by IV or orally. Ten regions of radioactivity were observed in mouse and rat fecal sample radiochromatograms, and seven regions of radioactivity were observed in monkey fecal sample radiochromatograms. The most abundant compound identified in feces was usually intact rifapentine (27%-41% of dose in mouse, 3%-35% of dose in rat, and 17%-29% of dose in monkey). Other peaks identified or characterized in feces based on liquid chromatography/ultraviolet/14C and/or liquid chromatography/mass spectrometry methods included 25-desacetyl-rifapentine, 3-formyl-25-desacetyl-rifapentine, and 3-formyl-rifapentine. The compounds rifapentine, 25-desacetyl-rifapentine, and 3-formyl-rifapentine were present in rat bile samples. These studies show that the metabolism and disposition of rifapentine in mice, rats, and monkeys were similar.

Disposition and metabolism of 14C-rifapentine in healthy volunteers.

Rifapentine is a long-acting cyclopentyl-derivative of rifampin. This study was designed to investigate the mass balance and biotransformation of 14C-rifapentine in humans. Four healthy male volunteers received a single 600-mg oral dose of 14C-rifapentine in a hydroalcoholic solution. Whole blood, urine, and fecal samples were collected before and at frequent intervals after drug administration. Amount of radioactivity recovered in urine and feces was assessed for up to 18 days postdose. Metabolite characterization in urine and feces was conducted using high-performance liquid chromatography with radiometric detection and liquid chromatography/mass spectroscopy. The total recovery of radioactive dose was 86.8%, with the majority of the radioactive dose recovered in feces (70.2%). Urine was a minor pathway for excretion (16.6% of the dose recovered). More than 90% of the excreted radioactivity was profiled as 14C chromatographic peaks and 50% was structurally characterized. These characterized compounds found in feces and urine were rifapentine, 25-desacetyl-rifapentine, 3-formyl-rifapentine, and 3-formyl-25-desacetyl-rifapentine. The 25-desacetyl metabolite, formed by esterase enzymes found in blood, liver, and other tissues, was the most abundant compound in feces and urine, contributing 22% to the profiled radioactivity in feces and 54% in urine. The 3-formyl derivatives of rifapentine and 25-desacetyl-rifapentine, formed by nonenzymatic hydrolysis, were also prominent in feces and, to a lesser extent, in urine. In contrast to the feces and urine, rifapentine and 25-desacetyl-rifapentine accounted for essentially all of the plasma radioactivity (99% of the 14C area under the concentration-time curve), indicating that 25-desacetyl-rifapentine is the primary metabolite in plasma. It appears, therefore, that the nonenzymatic hydrolysis of rifapentine to 3-formyl byproducts occurs primarily in the gut and the acidic environment of the urine.

Quantification of a dual angiotensin I-converting enzyme-neutral endopeptidase inhibitor and the active thiol metabolite in dog plasma by high-performance liquid chromatography with ultraviolet absorbance detection.

MDL 100,240 ([4S-[4 alpha,7 alpha(R*), 12b beta]]-7-[[2- (acetylthio)-1-oxo-3-phenylpropyl]amino]-1,2,3,4,6,7,8,12b-octahyd ro-6-oxo- pyrido[2,1-a][2]benzazepine-4-carboxylic acid, I) is the thioacetyl prodrug of the active thiol, MDL 100,173 (II), a dual inhibitor of angiotensin-I converting enzyme (ACE) and neutral endopeptidase (NEP). A drug which simultaneously inhibits both ACE and NEP may provide a unique therapy for hypertension and congestive heart failure. Methods based on high-performance liquid chromatography with UV absorbance detection at 200 nm were developed to support preclinical pharmacokinetic investigations. One method is used to measure unchanged I and free II, while the second method is used to quantify the total level of the thiol II after the plasma is incubated with the disulfide reducing agent, dithiothreitol. By either method, the analytes are quantified over the range of 25-1000 ng/ml with good accuracy and precision. The overall extraction efficiencies of unchanged I and free II in dog plasma were 79% and 86%, respectively, while the extraction efficiency of total II averaged 75%. Described in this report are the results obtained in validating the assay methods for measuring the compounds in plasma. Pharmacokinetic data are presented which were obtained by applying these methods to plasma collected from dogs dosed with I.

Isolation and characterization of porcine somatotropin containing a succinimide residue in place of aspartate129.

Aspartate129 in porcine somatotropin was converted into a cyclic imide residue (succinimide) under acidic solution conditions. Reversed-phase high performance liquid chromatography was utilized to isolate and quantitate this altered species, which accounted for approximately 30% of the total protein. The molecular mass of this modified species was determined by electrospray mass spectrometry to be 18 Da less than normal porcine somatotropin, indicative of a loss of 1 H2O molecule. Tryptic peptide mapping demonstrated that the peptide composed of residues 126-133 was altered in this modified protein. Amino acid analysis, amino acid sequencing, mass spectrometry, and capillary zone electrophoresis were used to demonstrate that aspartate129 in this peptide had been converted into a succinimide residue. Further confirmation that this peptide contained a succinimide was obtained by hydrolyzing the modified peptide at pH 9.0, which yielded both the aspartate and isoaspartate peptides.

Determination of the disulfide bond pairings in bovine transforming growth factor-alpha.

A rapid method for determining the three disulfide bond pairings in bovine transforming growth factor-alpha (bTGF-alpha) was developed by digesting bTGF-alpha with thermolysin followed by separation of the generated peptides by reversed-phase HPLC. The disulfide-bonded peptides were identified by amino acid sequencing and fast atom bombardment mass spectrometry. The disulfide bond pairings in bTGF-alpha were determined to be homologous to those in the human and mouse TGF-alpha molecules. A species of low bioactivity isolated from the folding/oxidation mixture of chemically synthesized bTGF-alpha was demonstrated to contain two incorrect disulfide bonds. These results indicate that mispairing of disulfide bonds in bTGF-alpha significantly reduces the activity of this molecule.

Formation of isoaspartate 99 in bovine and porcine somatotropins.

Asparagine 99 in bovine (BST) and porcine somatotropins (PST) was converted to an isoaspartate residue during incubation at neutral or alkaline pH. Isoaspartate 99 BST or isoaspartate 99 PST was resolved from the normal somatotropin by reversed-phase high-performance liquid chromatography (HPLC). The altered peptide of residues 96-108 which contains isoaspartate 99 was detected by tryptic peptide mapping of the modified BST or PST. Amino acid sequencing, amino acid analysis, mass spectrometry, and co-elution with a chemically synthesized peptide containing isoaspartate 99 were used to demonstrate the existence of isoaspartate in the modified peptides. Peptide bond cleavage between Asn 99 and Ser 100 also occurred during incubation of BST and PST at neutral or alkaline pH. This chemically cleaved product was resolved on reversed-phase HPLC from both the isoaspartate 99 and normal somatotropin molecules.

Sulfation of a tyrosine residue in the plasmin-binding domain of alpha 2-antiplasmin.

Sulfation of human alpha 2-antiplasmin, the major plasma inhibitor of fibrinolysis, was examined using both protein isolated from human plasma and protein synthesized and biosynthetically labeled with [35S]sulfate by a human hepatoma-derived cell line. Linkage of sulfate to tyrosine was demonstrated by recovery of labeled tyrosine sulfate after base hydrolysis of sulfate-labeled alpha 2-antiplasmin. Analysis by reverse-phase high performance liquid chromatography of peptides released from alpha 2-antiplasmin by cleavage with trypsin or cyanogen bromide indicated that sulfate is linked to a single segment of the protein. A cyanogen bromide peptide corresponding to the sulfate-labeled peptide was prepared from alpha 2-antiplasmin isolated from human plasma. Consistent with the presence of tyrosine sulfate in this peptide, its chromatographic elution was altered by treatment with acid under conditions which release sulfate from a tyrosine residue. No peptide in the total digest of alpha 2-antiplasmin by cyanogen bromide eluted at the position of the peptide following desulfation, suggesting that all of the protein is in a sulfated form. The sequence of the sulfate-containing cyanogen bromide peptide as determined by sequential Edman degradation, amino acid composition, and fast atom-bombardment-mass spectrometry was: Glu-Glu-Asp-Tyr(SO4)-Pro-Gln-Phe-Gly-Ser-Pro-Lys-COOH. This peptide is a segment of the previously identified plasmin-binding domain of alpha 2-antiplasmin.

Determination of impurities in nucleoside 3'-phosphoramidites by fast atom bombardment mass spectrometry.

Negative-ion fast atom bombardment mass spectrometry is quite useful for the identification of products and by-products formed during the synthesis of nucleoside 3'-phosphoramidites. The data show that detritylation and oxidation are side reactions which occur during the synthesis of monomeric units used in the construction of oligodeoxyribonucleotides by the phosphite triester method.