DON, CONV and DONV—III. Pharmacologic and toxicologic studies

Cooney, David A.; Jayaram, Hiremagalur N.; Milman, Harry A.; Homan, Elton R.; Pittillo, Robert F.; Geran, R. I.; Ryan, Joan; Rosenbluth, Richard

doi:10.1016/0006-2952(76)90190-8

Cited by 18 publications

(8 citation statements)

References 11 publications

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“…DON inhibits glutaminase through covalent modification, resulting in cellular glutamate depletion (8,19). IEC-18 cells were pretreated with varying concentrations of DON for 1 h before glutamine was added to the incubation medium to the final concentration of 4 mM for two more hours, followed by 30 min heat shock and 2 h recovery.…”

Section: Don a Glutaminase Inhibitor Decreases Glutamine's Effect Tmentioning

confidence: 99%

Heat induction of heat shock protein 25 requires cellular glutamine in intestinal epithelial cells

Phanvijhitsiri¹,

Musch²,

Ropeleski³

et al. 2006

American Journal of Physiology-Cell Physiology

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Glutamine is considered a nonessential amino acid; however, it becomes conditionally essential during critical illness when consumption exceeds production. Glutamine may modulate the heat shock/stress response, an important adaptive cellular response for survival. Glutamine increases heat induction of heat shock protein (Hsp) 25 in both intestinal epithelial cells (IEC-18) and mesenchymal NIH/3T3 cells, an effect that is neither glucose nor serum dependent. Neither arginine, histidine, proline, leucine, asparagine, nor tyrosine acts as physiological substitutes for glutamine for heat induction of Hsp25. The lack of effect of these amino acids was not caused by deficient transport, although some amino acids, including glutamate (a major direct metabolite of glutamine), were transported poorly by IEC-18 cells. Glutamate uptake could be augmented in a concentration- and time-dependent manner by increasing either media concentration and/or duration of exposure. Under these conditions, glutamate promoted heat induction of Hsp25, albeit not as efficiently as glutamine. Further evidence for the role of glutamine conversion to glutamate was obtained with the glutaminase inhibitor 6-diazo-5-oxo-l-norleucine (DON), which inhibited the effect of glutamine on heat-induced Hsp25. DON inhibited phosphate-dependent glutaminase by 75% after 3 h, decreasing cell glutamate. Increased glutamine/glutamate conversion to glutathione was not involved, since the glutathione synthesis inhibitor, buthionine sulfoximine, did not block glutamine's effect on heat induction of Hsp25. A large drop in ATP levels did not appear to account for the diminished Hsp25 induction during glutamine deficiency. In summary, glutamine is an important amino acid, and its requirement for heat-induced Hsp25 supports a role for glutamine supplementation to optimize cellular responses to pathophysiological stress.

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Section: Don a Glutaminase Inhibitor Decreases Glutamine's Effect Tmentioning

confidence: 99%

Heat induction of heat shock protein 25 requires cellular glutamine in intestinal epithelial cells

Phanvijhitsiri¹,

Musch²,

Ropeleski³

et al. 2006

American Journal of Physiology-Cell Physiology

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“…Thereafter, the DON-treated mice developed parasitemias similar to those of the controls. The strong trypanocidal effect of DON is rather surprising, considering that the half-life of DON is only 5-8 min in the mouse bloodstream (17).…”

Section: Treatment Of Trypanosome-infected Mice With Donmentioning

confidence: 99%

Trypanosoma brucei CTP synthetase: A target for the treatment of African sleeping sickness

Hofer

Steverding

Chabes

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

109

102

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The drugs in clinical use against African sleeping sickness are toxic, costly, or inefficient. We show that Trypanosoma brucei, which causes this disease, has very low levels of CTP, which are due to a limited capacity for de novo synthesis and the lack of salvage pathways. The CTP synthetase inhibitors 6-diazo-5-oxo-L-norleucine (DON) and ␣-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin) reduced the parasite CTP levels even further and inhibited trypanosome proliferation in vitro and in T. bruceiinfected mice. In mammalian cells, DON mainly inhibits de novo purine biosynthesis, a pathway lacking in trypanosomes. We could rescue DON-treated human and mouse fibroblasts by the addition of the purine base hypoxanthine to the growth medium. For treatment of sleeping sickness, we propose the use of CTP synthetase inhibitors alone or in combination with appropriate nucleosides or bases. U p to 500,000 people are estimated to suffer from African sleeping sickness (1, 2), a fatal disease caused by the protozoan parasites Trypanosoma brucei gambiense or Trypanosoma brucei rhodesiense. These pathogens are transmitted by tsetse flies to their mammalian hosts, where they first establish an infection in the blood and lymph. In the second stage of the disease, the parasites invade the central nervous system. Most drugs used to treat sleeping sickness do not cross the bloodbrain barrier; as soon as the trypanosomes have entered the central nervous system, only eflornithine and arsenicals such as melarsoprol are effective. However, the arsenicals are highly toxic, whereas eflornithine is ineffective against T. b. rhodesiense (3) and is very expensive.Purine metabolism has been extensively studied in trypanosomes (4), and it is well established that they lack the ability to form purines de novo. They therefore absolutely depend on preformed purine bases or nucleosides for survival. Any purine source is fine, because they have all of the enzymes needed to interconvert IMP, AMP, and GMP. Much less attention has been paid to the synthesis of pyrimidines in trypanosomes (4); so far, no study of the pathways for CTP synthesis has been published.We have previously reported that cultured bloodstream T. brucei have a very small CTP pool in comparison with other NTP pools and the CTP pools of other cell types (5). In this paper, we demonstrate that the low CTP pools are a consequence of slow synthesis by the parasite CTP synthetase. We suggest that this enzyme is an excellent target for chemotherapy, because we found that the trypanosomes, unlike mammalian cells, cannot compensate for the inhibition of CTP synthetase by the salvage of cytidine. The rest of our work is focused on the use of two irreversible glutamine analog CTP synthetase inhibitors, 6-diazo-5-oxo-L-norleucine (DON) and ␣-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin). These drugs have been used extensively in clinical trials against various cancers, and at least acivicin is known to penetrate the blood-brain barrier (6). We found that both drug...

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“…The flow dialysis method of Colowick and Womack (16) was used to investigate binding of DON to plasma protein.…”

Section: Pharmacologic Studiesmentioning

confidence: 99%

Phase I study and clinical pharmacology of 6-diazo-5-oxo-L-norleucine (DON)

et al. 1985

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The toxicity of the glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON) administered as a 24 hour infusion has been evaluated. Studies of the clinical pharmacology of the drug have also been performed in 3 patients. The limiting toxicity of the drug was acute nausea, vomiting and diarrhea that was dose dependent in its severity and duration. The maximum tolerated dose was 600 mg/m2 over 24 hours. The other major toxicity was thrombocytopenia that was maximal 7-10 days after the completion of the infusion. The drug does not exhibit renal, hepatic or central nervous system toxicity. DON achieves steady state levels during these infusions and is eliminated by first order kinetics when the infusion is completed (t1/2 alpha = 1.81 h). The principal route of excretion is renal. A starting dose of 400 mg/m2 would be acceptable for Phase II studies of this drug administered on this schedule.

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DON, CONV and DONV—III. Pharmacologic and toxicologic studies

Cited by 18 publications

References 11 publications

Heat induction of heat shock protein 25 requires cellular glutamine in intestinal epithelial cells

Heat induction of heat shock protein 25 requires cellular glutamine in intestinal epithelial cells

Trypanosoma brucei CTP synthetase: A target for the treatment of African sleeping sickness

Phase I study and clinical pharmacology of 6-diazo-5-oxo-L-norleucine (DON)

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