Protection by the heavy metal chelator N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN) against the lethal action of botulinum neurotoxin A and B

Abstract

The ability of N,N,N',N'-tetrakis (2-pyridylmethyl)-ethyenediamine (TPEN) to protect against botulinum neurotoxin (BoNT) A and B was examined in vivo in mice. To determine the protective efficacy of TPEN, mice were injected i.p. with TPEN as a single bolus or as multiple injections 30 min before and 0, 2, 4 and 6 hr following i.v. challenges with BoNT-A or -B. TPEN treatment did not alter the 24 hr lethality of BoNT but did produce a significant delay in the time to death. For a moderate dose of serotype A (20 LD50), five divided doses of TPEN prolonged the time to death from 7.8 +/- 0.4 hr to 9.9 +/- 0.5 hr. For serotype B, examined under comparable conditions, the prolongation of the time to death was from 6.1 +/- 0.2 hr to 9.4 +/- 0.6 hr. The range of TPEN doses that could be examined in vivo was limited by its acute toxicity. Although low doses of TPEN (< or = 10 mg/kg) were well tolerated, higher doses (> or = 30 mg/kg) led to ataxia, loss of coordination, convulsions and death in 20.3 min or less. In clonal NG108-15 cells, TPEN was found to produce cytotoxicity as revealed by increases in the secretion of the marker enzyme lactate dehydrogenase (LDH), and enhanced reactivity with the vital dye trypan blue. From LDH concentration-response data determined 24 hr after addition of TPEN, the threshold concentration for observing cytotoxicity was 10 microM and the IC50 was 19.8 microM. At the highest TPEN concentration tested (100 microM), cytotoxicity was detected 8 hr after TPEN addition and increased in severity over a 3 day period. The cytotoxicity in NG108-15 cells appears to be distinct from the rapid-onset toxicity observed in whole animals. These results suggest that TPEN may be of potential benefit in delaying the lethal actions of BoNT-A and -B, but its use is limited by its initial and delayed toxicity. Since the therapeutic and toxic actions of TPEN are both related to zinc chelation, the use of TPEN would need to be restricted to low doses as part of a combination therapy.