The degradation of the antitumor agent gemcitabine hydrochloride in an acidic aqueous solution at pH 3.2 and identification of degradation products.

A study of the degradation kinetics of gemcitabine hydrochloride (2'-deoxy-2',2'-difluorocytidine) in aqueous solution at pH 3.2 was conducted. The degradation of gemcitabine followed pseudo first-order kinetics, and rate constants were determined at four different temperatures. These rates were used to construct an Arrhenius plot from which degradation rates at lower temperatures were extrapolated and activation energy calculated. Four major degradation products were identified. Only one of these degradation products, the uridine analogue of gemcitabine, was a known degradation product of gemcitabine and was identified by comparison with synthesized material. The other three degradation products were isolated and characterized by spectroscopic techniques. Two of these products were determined to be the diastereomeric 6-hydroxy-5, 6-dihydro-2'-deoxy-2',2'-difluorouridines, and the other product was determined to be O(6),5'-cyclo-5,6-dihydro-2'-deoxy-2', 2'-difluorouridine. The mechanisms of formation of these degradation products are discussed.

Liquid chromatography/nuclear magnetic resonance spectroscopy and liquid chromatography/mass spectrometry identification of novel metabolites of the multidrug resistance modulator LY335979 in rat bile and human liver microsomal incubations.

Compound LY335979 is a P-glycoprotein inhibitor currently entering phase I clinical trials for potential reversal of multidrug resistance to cancer chemotherapy. In early exploratory studies, LY335979 was found to be rapidly transformed in incubations with liver microsomes from rats, dogs, monkeys, and humans. Although the parent compound was completely metabolized, no prominent metabolite peaks were observed. One peak did appear early in the time course, but it did not increase over time. In another preliminary experiment, rats were treated iv with [3H]LY335979 (prepared for pharmacology studies), and urine and bile fractions were collected. Analysis of the urine by reverse-phase HPLC with UV and radioactivity detection revealed that almost all of the material eluted with the solvent front. More than half the radioactivity in bile was accounted for by two peaks eluting earlier than the parent compound (the rest eluted at the solvent front). With both bile and the incubations with microsomes, initial attempts to isolate metabolites were not successful. There was also evidence in both systems of products derived from cleavage of LY335979 (by both further metabolism and degradation). LC/NMR was thus used to analyze materials directly in their respective matrices. An N-oxide metabolite (LY389551) formed by oxidation of the quinoline nitrogen was identified in the microsomal incubations; in bile, three glucuronide metabolites were identified, all of which were conjugates of products formed by oxidation of the quinoline ring of LY335979. There have been few reports in the literature of LC/NMR analysis of bile, which is a more complex matrix than either urine or microsomal suspensions. However, the HPLC techniques developed in this work for the HPLC/UV and LC/MS analyses of LY335979 metabolites in the microsomal matrix and in bile proved readily adaptable for LC/NMR. Using a 500-MHz instrument, basic 1H NMR spectra could be obtained in 2-3 hr with approximately 100 ng of material in the LC/NMR microprobe. With approximately 1.5 microg of material injected onto the column, 1H-1H correlation spectroscopy spectra could be acquired overnight. Along with LC/MS data, the LC/NMR technique facilitated direct identification of a number of metabolites of LY335979 at a point at which their identification by traditional methods would not have been pursued.

Maillard reaction of lactose and fluoxetine hydrochloride, a secondary amine.

Analysis of commercially available generic formulations of fluoxetine HCl revealed the presence of lactose as the most common excipient. We show that such formulations are inherently less stable than formulations with starch as the diluent due to the Maillard reaction between the drug, a secondary amine hydrochloride, and lactose. The Amadori rearrangement product was isolated and characterized; the characterization was aided by reduction with sodium borohydride and subsequent characterization of this reduced adduct. The lactose-fluoxetine HCl reaction was examined in aqueous ethanol and in the solid state, in which factors such as water content, lubricant concentration, and temperature were found to influence the degradation. N-Formylfluoxetine was identified as a major product of this Maillard reaction and it is proposed that N-formyl compounds be used as markers for this drug-excipient interaction since they are easy to prepare synthetically. Many characteristic volatile products of the Maillard reaction have been identified by GC/MS, including furaldehyde, maltol, and 2,3-dihydro-3,5-dihydroxy-6-methyl-4 H-pyran-4-one. Close similarity between the degradation products of simple mixtures and formulated generic products was found; however, at least one product decomposed at a rate nearly 10 times that predicted from the simple models. Maillard products have also been identified in unstressed capsules. The main conclusion is that drugs which are secondary amines (not just primary amines as sometimes reported) undergo the Maillard reaction with lactose under pharmaceutically relevant conditions. This finding should be considered during the selection of excipients and stability protocols for drugs which are secondary amines or their salts, just as it currently is for primary amines.

Characterization of impurities formed by interaction of duloxetine HCl with enteric polymers hydroxypropyl methylcellulose acetate succinate and hydroxypropyl methylcellulose phthalate.

Duloxetine hydrochloride ((S)-N-methyl-3-(1-naphthalenyloxy)-2-thiophenepropanamine hydrochloride) has been found to react with polymer degradation products or residual free acids present in the enteric polymers hydroxypropyl methylcellulose acetate succinate (HPMCAS) and hydroxypropyl methylcellulose phthalate (HPMCP) in dosage formulations to form succinamide and phthalamide impurities, respectively. The rate of formation of the impurities is accelerated by heat and humidity. The structures were deduced using molecular weights obtained from LC-MS experiments and confirmed by comparison of UV spectra, HPLC retention times, and electrospray mass spectra to independently synthesized material. It is proposed that polymer-bound succinic and phthalic substituents can be cleaved from the polymer, resulting in the formation of either the free acids or the anhydrides. It is postulated that the reaction is enabled by migration of either (1) the free acid or anhydride or (2) the parent drug through the formulation. The formation of these impurities was minimized by increasing the thickness of the physical barrier separating the enteric coating from the drug.

Degradation chemistry of gemcitabine hydrochloride, a new antitumor agent.

The anti-tumor agent gemcitabine hydrochloride, a beta-difluoronucleoside, is remarkably stable in the solid state. In 0.1 N HCI solution at 40 degrees C, deamination of gemcitabine occurs, yielding its uridine analogue. Approximately 86% of the initial gemcitabine remains after 4 weeks under these conditions. Cleavage of the N-glycosidic bond of gemcitabine or conversion to its alpha-anomer in 0.1 N HCI solution is not observed over a 4-week period. However, this work has shown that gemcitabine hydrochloride anomerizes in 0.1 N NaOH at 40 degrees C. Approximately 72% of the initial gemcitabine remains after 4 weeks under the basic conditions used. Uridine hydrolysis products are also formed under these conditions. The anormerization reaction, which is unusual under basic conditions, has been confirmed by characterization of the chromatographically isolated alpha-anomer by NMR and mass spectrometry. A mechanism involving an acyclic intermediate is proposed.

Acid stabilization of insulin.

The effect of pH on the conformational stability of insulin was studied. Surprisingly, the Gibbs free energy of unfolding increased approximately 30% by acidification. pH titration of insulin's conformational stability is described by a transition involving a single proton with an apparent pK(a) of 7.0. The acid stabilization of insulin's conformation was attributed to the protonation of histidine at position 5 on the B-chain (HB5) as determined by 1H-NMR of the histidines, selective amino acid alteration, and enthalpies of ionization. Further acidification (at least to pH 2) does not decrease the free energy of unfolding. A conformational change in the tertiary structure, as indicated by the near-UV circular dichroism spectrum, accompanies this change in stability. We propose that this acid stabilization of insulin is physiologically important in maintaining insulin stability in the acid environment of the secretory/storage granules of the beta-cell of the pancreatic islets of Langerhans.