Characterization of cisplatin degradation as affected by pH and light.

The stability of cisplatin in 0.9% sodium chloride solution was studied. Cisplatin was reconstituted with sterile water for injection to achieve a concentration of approximately 1 mg/mL in 0.9% sodium chloride solution. The solutions were placed in flexible polyvinyl chloride containers, clear and amber glass flasks, and plastic syringes and stored at 22-25 degrees C in the dark or exposed to measured amounts of light and assayed periodically for up to 96 days. The initial pH of some of the solutions was adjusted. The stability of cisplatin in the solutions was determined by measuring the concentrations of cisplatin, trichloroammineplatinate(II) (TCAP), and trans-dichlorodiammineplatinum(II) (transplatin) with high-performance liquid chromatography and by measuring solution pH. The effect of adding ethylene oxide and other materials was also determined. TCAP was identified as the predominant degradation product of cisplatin in all the solutions. The rate of degradation was dependent on pH: In the dark, about 0.04% and 0.21% of the cisplatin degraded to TCAP per week at pH 4.3 and 6.3, respectively. The presence of ethylene oxide accelerated cisplatin degradation. Degradation also occurred during exposure to short-wavelength (350-490-nm) visible light. Storage in amber glass flasks offered more protection than storage in clear flasks. Isomerization of cisplatin to transplatin was not observed. The major degradation product of cisplatin was trichloroammineplatinate(II). Solution pH was the predominant factor affecting cisplatin stability.

Identification of the amino acid bound to the labile adduct formed during inactivation of monoamine oxidase by 1-phenylcyclopropylamine.

Three reactions are carried out on the reversible adduct formed when 1-phenylcyclopropylamine (1-PCPA) inactivates monoamine oxidase (MAO) in order to determine the identity of the amino acid involved in reversible adduct formation. Raney nickel treatment yields trans-beta-methyl[14C]styrene, the compound that would result from carbon-sulfur bond reduction of a (3-hydroxy-3-phenyl-propyl)cysteine adduct. A 5,5'-dithiobis(2-nitrobenzoic acid) assay for cysteine residues indicates that upon reversible inactivation of MAO by 1-PCPA, one cysteine is lost. The third reaction involves sodium periodate and hydrogen peroxide oxidation, but no definitive result is obtained. The first two reactions provide evidence that the amino acid residue involved in reversible adduct formation is a cysteine.

1-Phenylcyclobutylamine, the first in a new class of monoamine oxidase inactivators. Further evidence for a radical intermediate.

1-Phenylcyclobutylamine (PCBA) is shown to be both a substrate and a time-dependent irreversible inactivator of monoamine oxidase (MAO). Inactivation results in attachment to the flavin cofactor. For every molecule of PCBA leading to inactivation, 325 molecules are converted to product. The first metabolite formed is identified as 2-phenyl-1-pyrroline; then after a lag time, 3-benzoylpropanal and 3-benzoylpropionic acid are generated. The 3-benzoylpropanal is a product of MAO-catalyzed oxidation of 2-phenyl-1-pyrroline (presumably, of its hydrolysis product, gamma-aminobutyrophenone). The aldehyde is nonenzymatically oxidized by nascent hydrogen peroxide to the carboxylic acid. These results are consistent with a one-electron oxidation of PCBA to the amine radical cation followed by homolytic cyclobutane ring cleavage. The resulting radical can partition between cyclization (an intramolecular radical trap) to the 2-phenylpyrrolinyl radical and attachment to the flavin. The cyclic radical can be further oxidized by one electron to 2-phenyl-1-pyrroline. PCBA represents the first in the cyclobutylamine class of MAO inactivators and strongly supports involvement of a radical mechanism for MAO-catalyzed amine oxidations.

1-Benzylcyclopropylamine and 1-(phenylcyclopropyl)methylamine: an inactivator and a substrate of monoamine oxidase.

1-Benzylcyclopropylamine (1) and 1-(phenylcyclopropyl)methylamine (2), cyclopropane analogues of phenethylamine, were tested as inactivators for monoamine oxidase (MAO). Compound 1 is a potent competitive reversible inhibitor of the oxidation of benzylamine and also is a mechanism-based inactivator. It requires 2.3 equiv of 1 to inactivate 1 equiv of MAO. The excess equivalents of 1 are converted into benzyl vinyl ketone. A one-electron mechanism of inactivation is proposed. Compound 2 is a substrate for MAO and is converted into 1-phenylcyclopropanecarboxaldehyde without inactivation of the enzyme. Mechanistic consequences are discussed as a result of this observation.

Mechanism of inactivation of monoamine oxidase by 1-phenylcyclopropylamine.

1-Phenylcyclopropylamine (1-PCPA) is shown to be a mechanism-based inactivator of mitochondrial monoamine oxidase (MAO). The strained cyclopropyl ring is important to inactivation since alpha,alpha-dimethylbenzylamine, the acyclic analogue of 1-PCPA, is neither an inactivator nor a substrate of MAO. Two different pathways occur during inactivation by 1-PCPA, both believed to be derived from a common intermediate. One pathway leads to irreversible inactivation of the enzyme and a 1:1 stoichiometry of radioactivity to the active site when 1-[phenyl-14C]PCPA is used as the inactivator; the other pathway results in a covalent reversible adduct. Three organic reactions are carried out on the irreversibly labeled enzyme in order to determine the structure of the active site adduct. Sodium boro[3H]hydride reduction results in the incorporation of 0.73 equiv of tritium, suggesting a carbonyl functionality. Baeyer-Villiger oxidation followed by saponification gives 0.8 equiv of phenol, indicating the presence of a phenyl ketone. Treatment of the labeled enzyme with hydroxide produces acrylophenone, as would be expected from the retro-Michael reaction of beta-X-propiophenone. The identity of X is determined in two ways. The optical spectrum of the flavin cofactor is reduced during inactivation; no reoxidation occurs upon denaturation. Pronase treatment of the radioactively labeled enzyme produces fragments that contain both the radioactivity and the flavin. The X group, therefore, is the flavin. The results of two tests designed to differentiate N5 from C4a attachment to the flavin suggest an N5 adduct. In addition to formation of this stable covalent adduct, another pathway occurs 7 times as often. This alternate reaction of 1-[phenyl-14C]PCPA with MAO produces 7 equiv of [14C]acrylophenone during the course of irreversible inactivation and is believed to arise from formation of the same type of adduct as described above except that X is something other than the N5-flavin (Y). Upon denaturation of this labeled enzyme, the flavin is completely oxidized when most of the radioactivity is still bound to the enzyme. This indicates that Y is not a C4a-flavin adduct and suggests attachment to an active site amino acid residue. More facile elimination of Y from this beta-substituted propiophenone adduct would give acrylophenone on the time scale of the inactivation. Treatment of the reversible adduct with sodium borohydride prior to denaturation prevents release of radioactivity.(ABSTRACT TRUNCATED AT 400 WORDS)