Degradation study of the investigational anticancer drug clanfenur.

Clanfenur belongs to a new group of substituted benzoylphenylureas. The drug shows both in vitro and in vivo antitumour activity. To assess its chemical stability, a study was carried out in which the effect of pH, temperature, ionic strength and buffer concentration on the reaction rate constant k(obs) were examined. A stability-indicating reversed-phase high performance liquid chromatography (RP-HPLC) system was used. The pH-log k(obs) degradation profile, obtained at 70 degrees C, shows that clanfenur has its maximum stability in the pH region 4-5. At pH 7, half-lives were calculated by extrapolation of the Arrhenius plot; at 4 degrees C the half-life was calculated to be 141 years and at 25 degrees C 9. 5 years. The activation energy was calculated to be 114 kJ/mol. In acidic, neutral, and alkaline media, the ionic strength has no effect on the degradation. The buffer concentration of citrate, phosphate, borate, and carbonate did not affect the value of k(obs). An RP-HPLC chromatogram of degraded clanfenur shows the presence of four degradation products, three of which were identified by LC-ESI-MS as p-chloroaniline, p-chlorophenylurea and 2-fluoro-6-dimethylaminobenzamide.

Qualitative and quantitative aspects of the degradation of several tripeptides derived from the antitumour peptide antagonist [Arg(6), D-Trp(7,9), MePhe(8)] substance P[6-11].

The tripeptides Arg-Trp-Phe, Arg-Trp-Phe-NH2, Phe-Trp-Arg and Phe-Trp-Arg-NH2 were subjected to a degradation study to get a more detailed insight into the degradation processes of the antitumor hexapeptide antagonist [Arg(6), D-Trp(7,9), MePhe(8)] substance P¿6-11¿ which was investigated in earlier research. Degradation kinetics as well as identities of degradation products of the tripeptides emerging in alkaline and acidic media were studied. The amidated forms (Arg-Trp-Phe-NH2, Phe-Trp-Arg-NH2) appear to be less stable than the carboxylic forms (Arg-Trp-Phe, Phe-Trp-Arg). Deamidation of the amide C-terminus, racemization of the Phe and Arg residues, ornithine formation, hydrolysis of the peptide backbone and diketopiperazine formation with elimination of the N-terminal fragments were the major degradative processes. Comparing these reactions with the reactions of antagonist [Arg(6), D-Trp(7,9), MePhe(8)] substance P¿6-11¿ it appeared that racemization of Phe and Arg, hydrolysis of the peptide backbone and diketopiperazine formation did not occur in detectable amounts in the hexapeptide. probably due to lower reaction rates of these reactions compared to the overall degradation rate of antagonist [Arg(6), D-Trp(7,9) MePhe(8)] substance P¿6-11¿.

Reduction of Cys36-Cys42 and Cys64-Cys74 disulfide bonds in recombinant human granulocyte colony stimulating factor.

The Cys36-Cys42 and Cys64-Cys74 disulfide bonds in recombinant methionyl human granulocyte colony-stimulating factor were reduced to sulfhydryls with dithiothreitol or mercury. Both reduction reactions are dependent on the pH. The reduction reaction with dithiothreitol increased in rate with increasing pH; between pH 7-9 and above pH 10.5 this increase was less than in other regions. These observations are explained by repulsive forces between dithiothreitol and regions in granulocyte colony-stimulating factor which intensify in these pH-regions. The hydroxyl catalysis causes the overall increase in k(obs) in the pH-region studied. The reduction of the disulfides with mercury is, as could be expected from the Nernst equation for disulfide reduction, also pH dependent: the half-wave potential decreases with increasing pH as predicted by theory.

Oxidation of recombinant methionyl human granulocyte colony stimulating factor.

The oxidation of methionine residues in recombinant methionyl human granulocyte colony stimulating factor with hydrogen peroxide has been investigated. Kinetic data of the oxidation were obtained by using reversed phase-high performance liquid chromatography. The stability-indicating capability of this system was confirmed with micellar electrokinetic capillary chromatography. In the pH range 1.9-7.5, the kobs value for the oxidation process is constant. Above pH 7.5, kobs tends to increase with increasing pH. In the pH range 1.9-11.8, four oxidation products were detected in RP-HPLC. Mass spectrometric analysis revealed that one mono-, one di- and two trioxidation products were formed. Using the cyanogen bromide cleavage method the nature of the oxidation products was determined. The mono-oxidation product is the protein with Met121 oxidized, while the dioxidation product has oxidized Met121 and Met126 residues. The trioxidation products are the proteins with Met121, Met126 and Met137 or Met0, Met121 and Met126 oxidized.

Isolation and structural confirmation of N-desmethyl topotecan, a metabolite of topotecan.

A sensitive high-performance liquid chromatography (HPLC) method for the determination of topotecan and total levels of topotecan (lactone plus its ring-opened hydroxycarboxylate form) was developed by the authors and used in several pharmacokinetics studies. During the analysis of plasma and urine samples collected in those studies, an additional peak eluting just after topotecan was observed. Approximately 100 ng of this potential metabolite was isolated from human urine using a solid-phase extraction procedure and purification by HPLC. Analysis of the isolated material by HPLC showed it to be approximately 95% pure. Mass spectrometry data along with the HPLC retention data and fluorescence data (in comparison with synthetic reference standard) are consistent with the metabolite's being N-desmethyl topotecan. The maximal concentrations of metabolite detected in human plasma and urine were relatively low. When topotecan was given as a 30-min infusion at 1.0 mg/m2 daily for 5 days every 3 weeks, the maximal plasma metabolite concentration (lactone plus the ring-opened hydroxycarboxylate form) was about 0.7% (n = 4) of the maximal total topotecan concentration. The average amount of metabolite excreted in urine during the treatment was 1-4% (n = 20) of the delivered dose.

Structural identification of the degradation products of the antitumor peptide antagonist [Arg6,D-Trp7,9,MePhe8]substance P (6-11).

The basic hexapeptide antagonist [Arg6,D-Trp7,9,MePhe8]-substance P (6-11) was degraded in acid and alkaline media. In acid solution, only one degradation product is found whereas in alkaline solution at least six products are formed. These compounds were analytically characterized and structurally identified by reversed-phase high-performance liquid chromatography, capillary electrophoresis, liquid chromatography/mass spectrometry, fast atom bombardment tandem mass spectrometry, optical rotation analysis, and chiral gas chromatography. The product formed in acidic solution is the terminally deamidated antagonist [Arg6,D-Trp7,9,MePhe8]substance P (6-11); this product was also found in alkaline degradation mixtures. Other important degradation products originate from racemization of the amino acid residue L-Met, formation of ornithine from Arg, and the oxidation of Met to its sulfoxide form.