Inhibition of glutathione synthesis as a chemotherapeutic strategy for trypanosomiasis.

With the expectation that trypanosomal glutathione (GSH) plays a major protective role against the endogenous oxidant stress that results form high intracellular levels of H2O2, we sought to deplete Trypanosoma brucei brucei of their GSH through inhibition of its biosynthesis. Administration of buthionine sulfoximine (BSO), a reversible inhibitor of gamma-glutamylcysteine synthetase, to parasitemic mice resulted in a progressive decrease in trypanosome GSH content, such that parasites isolated after 5 h or BSO treatment contained 50% of normal values. When BSO administration was continued for 18 h (intraperitoneal injection of 4 mmol/kg every 1.5 h), parasitemias temporarily cleared. When inhibitory plasma levels of BSO were maintained for about 27 h, two out of six infected mice were cured and the rest had significantly prolonged survival. These findings demonstrate the potential value of GSH depletion for the treatment of trypanosomiasis.

Pharmacokinetics of buthionine sulfoximine (NSC 326231) and its effect on melphalan-induced toxicity in mice.

Intravenous doses of buthionine sulfoximine (BSO, NSC 326231), an inhibitor of glutathione synthesis, were eliminated rapidly from mouse plasma in a biexponential manner. The initial phase of the plasma concentration versus time curve had a half-life of 4.9 min and accounted for 94% of the total area under the curve. The half-life of the terminal phase of the curve was 36.7 min and the area accounted for only 6% of the total area under the curve. Plasma clearance of BSO was 28.1 ml/min/kg and the steady state volume of distribution was 280 ml/kg. The oral bioavailability of BSO, based on plasma BSO levels, was extremely low. However, comparable glutathione depletion was apparent after i.v. and p.o. doses of BSO, suggesting a rapid tissue uptake and/or metabolism of BSO. Therefore, due to the rapid elimination of BSO from mouse plasma, plasma drug levels do not directly correlate with BSO-induced tissue glutathione depletion. Administration of multiple i.v. doses of BSO to male and female mice resulted in a marked 88% depletion of liver glutathione at doses of 400-1600 mg/kg/dose. Toxicity of i.v. administered BSO was limited to a transient depression of peripheral WBC levels in female mice given six doses of 1600 mg/kg. Multiple i.v. doses of BSO of up to 800 mg/kg/dose (every 4 h for a total of six doses) did not alter the toxicity of i.v. administered melphalan. However, multiple doses of 1600 mg/kg/dose of BSO did potentiate histopathological evidence of melphalan-induced bone marrow toxicity in 30% of the mice and, additionally, the combination of BSO and melphalan produced renal tubular necrosis in 80% of the male mice. The potentiation of melphalan induced toxicity did not appear to be related to GSH depletion, since: quantitatively similar amount of GSH depletion occurred at lower dose of BSO without any increase in melphalan toxicity.

Determination of L-buthionin (SR)-sulfoximine, gamma-glutamylcysteine synthetase inhibitor in rat plasma with HPLC after prelabeling with dansyl chloride.

L-(SR)-Buthionin sulfoximine (L-(SR)-BSO) is a potent and specific inhibitor of gamma-glutamylcysteine synthetase, which catalyzes the first reaction of glutathione biosynthesis. A selective, sensitive, and simple high-performance liquid chromatographic method was developed for the determination of L-(SR)-BSO in rat plasma. After the compound was labeled with dansyl chloride (Dns-Cl) under optimal conditions, it was separated in a Zolbax-ODS column with a mobile phase that consisted of 0.01M phosphate buffer, methanol, and acetonitrile (8:1:3, v/v). The compound was detected with a fluorescence detector at an excitation wavelength of 335 nm and an emission wavelength of 525 nm using a xenon lamp. The coefficients of variation (DV) from the interassay in the low and high concentrations (10 and 500 micrograms/mL of L-(SR)-BSO in rat plasma) were 2.5 and 4.8%, respectively. The CVs from the intra-assay in the low and high concentrations were 3.2 and 5.6%, respectively. The minimum concentration of L-(SR)-BSO that could be determined was 10 micrograms/mL when 100-microL serum samples were used. The detection limit was 50 ng per injection volume. This method enables pharmacokinetic and pharmacodynamic studies in rats.

Determination of L-buthionine-(S,R)-sulfoximine in plasma by high-performance liquid chromatography with o-phthalaldehyde derivatization and fluorometric detection.

The development of a high-performance liquid chromatography system for the analysis of L-buthionine-(S,R)-sulfoximine (BSO) in human plasma is described. o-Phthalaldehyde derivatization and fluorescence detection were used. The R- and S-BSO peaks were partially separated from each other and completely separated from the matrix components. The limit of detection for BSO was 2 micrograms/ml plasma.

High-performance liquid chromatographic determination of the S- and R-diastereoisomers of L-buthionine (SR)-sulfoximine in human plasma and urine.

A sensitive and reproducible HPLC procedure was developed for the simultaneous determination of the diastereoisomers of the synthetic amino acid L-buthionine-(SR)-sulfoximine (BSO) in human plasma and urine. Plasma samples were prepared for analysis by addition of internal standard (L-norleucine) followed by ultrafiltration using disposable centrifugal filtration units. Urine samples received internal standard followed by solid phase extraction using disposable C18 cartridges. All samples were derivatized with phenylisothiocyanate (PITC). The derivatized amino acids were separated by HPLC on an octyldecyl column (250 mm x 4.6 mm I.D., 5 microns particle size) using a mobile phase of sodium acetate-acetonitrile-triethylamine-ethylaminediaminetetraacetic acid. The column effluent was monitored at 254 nm and quantitation was performed using peak areas. The linear range for each diastereoisomer of L-(SR)-BSO was from 2 to 100 micrograms/ml in plasma and from 10 to 1000 micrograms/ml in urine. The method is reproducible, convenient and sensitive, illustrating its utility for application in pharmacokinetic studies.

Effects of methionine sulphoximine treatment on renal amino acid and ammonia metabolism in rats.

Renal glutamine metabolism in relation to ammoniagenesis has been extensively studied during chronic metabolic acidosis, when arterial glutamine levels are reduced. However, little is known about the effects of reduced glutamine delivery on renal glutamine and ammonia metabolism at physiological systemic pH values. Therefore, a model of decreased arterial glutamine concentrations at normal pH values was developed using methionine sulphoximine (MSO). Renal glutamine and ammonia metabolism was measured by determining fluxes and intracellular concentrations after an overnight fast in ether anaesthetized normal rats, MSO-treated rats and their pair-fed controls. Moreover, fluxes and intracellular concentrations of several other amino acids were determined concomitantly. After 2 and 4 days of MSO treatment, arterial glutamine concentrations were reduced to 55%, while arterial ammonia concentrations increased by 70%. Kidney glutamine uptake reduced, but systemic pH was unchanged. Fractional extraction of glutamine remained unchanged, suggesting that also in vivo net uptake of glutamine by the kidney at subnormal levels is related to arterial glutamine concentrations. As a result, at day 2 but not at day 4, the kidney reduced the net release of ammonia into the renal vein and thus reduced net renal ammonia addition to body ammonia pools. Therefore at day 2, the kidney seems to play an important role in adaptation to both hyperammonaemia and hypoglutaminaemia.