Kelvin W. Chan

Rapid drug metabolite profiling using fast liquid chromatography, automated multiple-stage mass spectrometry and receptor-binding

Rapid drug metabolite profiling can be achieved using fast chromatographic separation and fast mass spectrometric scanning without compromising the separation efficiency. Fast chromatographic separations of drug and its metabolites can be achieved by eluting from a short narrow-bore guard cartridge column (20 x 2 mm I.D., 3 microns BDS Hypersil C8) at flow-rate of 1.0 ml/min and with a gradient volume greater than 90 column volumes. The need for chromatographic separation is important for automated data dependent multiple-stage mass spectrometry (MSn) experimentation. The total analysis time of 8 min permits profiling of metabolites in a 96-well plate in 13 h. The narrow chromatographic peaks resulting from the high flow-rate require the use of a mass spectrometer capable of fast scan speed due to the need to perform multiple MS experiments within the same chromatographic analysis. A method has been developed for screening potentially biologically active in vitro microsomal metabolites by affinity binding with a receptor. After separation by centrifugal ultrafiltration, the bound ligands are released and characterized by LC-MS. In vitro microsomal metabolites of tamoxifen, raloxifene and adatanserin were screened for potential biological activity using this method. The in vitro metabolites of tamoxifen captured by the receptor include N-demethyltamoxifen and three species of hydroxytamoxifen; these data are consistent with those from a conventional binding study and bioassay. In addition, both hydroxyraloxifene and dihydroxyraloxifene are also recognized by the receptor. The specificity of the molecular recognition process is illustrated by the absence of binding with control microsomal incubate and with adatanserin and its metabolites. Therefore, active metabolites can be rapidly profiled by fast LC, automated MSn, and receptor binding. This information can be obtained quickly and can add value to the drug discovery process.

High Performance Liquid Chromatographic Isolation and Spectroscopic Characterization of Three Major Metabolites from the Plasma of Rats Receiving Rapamycin (Sirolimus) Orally

Abstract Three major metabolites of rapamycin (M2, M3, and M5) were isolated from pooled plasma of orally dosed rats. Metabolites were extracted from the plasma with ethyl acetate/methanol prior to isolation by HPLC using a Supelcosil SPLC-18, 5μm, 10 × 250 mm column. The mobile phase was a methanol/ammonium acetate linear gradient system. The isolated metabolites were characterized by negative ion FAB MS, ion-spray MS and ion-spray MS/MS. Metabolite M2 is oxygenated in the southern portion of rapamycin and the macrolide ring is opened. M3 is a structural isomer of rapamycin where the lactone ring is opened. M5 is O-demethylated on the C41 methoxy moiety and the macrolide ring is intact.

High Performance Liquid Chromatographic Isolation, Spectroscopic Characterization, and Immunosuppressive Activities of Two Rapamycin Degradation Products

Abstract A high performance liquid chromatographic method has been developed for the isolation of two degradation products of rapamycin which is currently under development as an immunosuppressive agent. Prior to isolation, the drug was incubated at 37°C in rat bile or ammonium acetate (pH 8.0). The isolation was achieved by a Supelco, PLC-18 21.2 × 250 mm, 18 μm column using methanol/ammonium acetate gradient mobile phase. After evaporation of methanol, the remaining eluates were lyophilized. The isolated degradation products were characterized by negative ion fast atom bombardment mass spectrometry (FAB MS) and proton nuclear magnetic resonance spectroscopy (1H NMR). Degradation product A was found to be a macrolide ring-opened hydrolysis product of rapamycin where the C25 ester bond had been hydrolyzed. Degradation product B was determined to be a ring-opened isomer of rapamycin. B had less than 4% of the potency of rapamycin in a thymocyte proliferation assay, while A had minimal activity at concentra...

Base catalyzed degradations of rapamycin

Abstract The base catalyzed degradation of rapamycin was re-examined and a sequence of steps involving β-elimination, retro-aldol cleavage and benzilic acid rearrangement occur leading to several new products.

High-Performance Liquid Chromatographic (HPLC) and HPLC-Mass Spectrometric (MS) Analysis of the Degradation of the Luteinizing Hormone-Releasing Hormone (LH-RH) Antagonist RS-26306 in Aqueous Solution

The kinetics of the degradation of an LH-RH antagonist, RS-26306,1, in aqueous solution from pH 1 to pH 11 were studied by reverse-phase HPLC. The pH–rate profiles at 50, 60, and 80°C were U-shaped with the rate law of kobs = kHaH + kw + kOHaOH. The predicted 25°C shelf life at the pH of maximum stability, pH ∼5, is greater than 10 years. The products from the degradation were analyzed by HPLC-MS using thermospray ionization. Below pH 3, the primary product, 2, forms from the acid-catalyzed deamidation of the C-terminal amide. Above pH 7, epimerization of the individual amino acids is the principal reaction. Between pH 4 and pH 6, intramolecular serine-catalyzed peptide hydrolysis becomes important, yielding a tripeptide, 3, and a heptapeptide, 4. At the pH of maximum stability all three pathways for degradation are observed.

Acid catalyzed functionalization of rapamycin

Abstract Rapamycin rapidly undergoes demethoxylation at C-7 in the presence of Lewis acids (BF 3 ·Et 2 O, SnCl 4 etc.) to give a highly stabilized carbocation. This intermediate gives a tetraene or is trapped by nucleophiles to give functionalized trienes. Several examples of the substitution reaction and elaboration of the reaction scheme are reported.

Metabolic Disposition of 14C-Bromfenac in Healthy Male Volunteers

The metabolic disposition of 14C‐bromfenac, an orally active, potent, nonsteroidal, nonnarcotic, analgesic agent was investigated in six healthy male subjects after a single oral 50‐mg dose. The absorption of radioactivity was rapid, producing a mean maximum plasma concentration (Cmax) of 4.9 ± 1.8 μgṁequiv/mL, which was reached 1.0 ± 0.5 hours after administration. Unchanged drug was the major component found in plasma, and no major metabolites were detected in the plasma. Total radioactivity recovered over a 4‐day period from four of the six subjects averaged 82.5% and 13.2% of the dose in the urine and feces, respectively. Excretion into urine was rapid; most of the radioactivity was excreted during the first 8 hours. Five radioactive chromatographic peaks, a cyclic amide and four polar metabolites, were detected in 0‐ to 24‐hour urine samples. Similarity of metabolite profiles between humans and cynomolgus monkeys permitted use of this animal model to generate samples after a high dose for structure elucidation. Liquid chromatography/mass spectrometry (LC/MS) analysis of monkey urine samples indicated that the four polar metabolites were two pairs of diastereoisomeric glucuronides whose molecular weight differed by two daltons. Enzyme hydrolysis, cochromatography, and LC/MS experiments resulted in the identification of a hydroxylated cyclic amide as one of the aglycones, which formed a pair of diastereoisomeric glucuronides after conjugation. Data also suggested that a dihydroxycyclic amide formed by the reduction of the ketone group that joins the phenyl rings formed the second pair of diastereoisomeric glucuronides. Further, incubation of various reference standards in control (blank) urine and buffer with and without creatinine indicated that the hydroxy cyclic amide released from enzyme hydrolysis can undergo ex vivo transformations to a condensation product between creatinine and an α‐keto acid derivative of the hydroxy cyclic amide that is formed by oxidation and ring opening. Further experiments with a dihydroxylated cyclic amide after reduction of the keto function indicated that it too can form a creatinine conjugate.

High Performance Liquid Chromatographic Isolation and Spectroscopic Characterization of Metabolites from the Bile of Rats Receiving Rapamycin (Sirolimus) Intravenously

Abstract Ten major metabolites of rapamycin (M2, M3, M8, M9, M10, M11, M13, M14, M15, and M16) were isolated from pooled bile of intravenously dosed rats. Metabolites were extracted from the bile with ethyl acetate prior to isolation by HPLC using a Supelcosil SPLC-18, μm, 10 × 250 mm column. The mobile phase was a methanol/ammonium acetate linear gradient system. The isolated metabolites were characterized by negative ion FAB MS, ion-spray MS and ion-spray MS/MS analyses. Metabolite M2 is oxygenated in the southern portion of rapamycin and the macrolide ring is opened. M3 is a structural isomer of rapamycin where the lactone ring is opened. M10 is oxygenated in the southern portion of rapamycin and the macrolide ring is intact. M13 is a monohydroxylation and demethylation metabolite and both biotransformations occurred at the southern portion. M8, M9, and M11 are monohydroxylation and demethylation metabolites. M14 and M15 are di-hydroxylation metabolites. M16 is mainly a dihydroxylation metabolite. † Th...