Identification of Platinum(II) Sulfide Complexes Suitable as Intramuscular Cyanide Countermeasures

Abstract

The development of rapidly acting cyanide countermeasures using intramuscular injection (IM) represents an unmet medical need to mitigate toxicant exposures in mass casualty settings. Previous work established that cisplatin and other platinum(II) or platinum(IV)-based agents effectively mitigate cyanide toxicity in zebrafish. Cyanide's in vivo reaction with platinum-containing materials was proposed to reduce the risk of acute toxicities. However, cyanide antidote activity depended on a formulation of platinum-chloride salts with dimethyl sulfoxide (DMSO) followed by dilution in phosphate-buffered saline (PBS). A working hypothesis to explain the DMSO requirement is that the formation of platinum-sulfoxide complexes activates the cyanide scavenging properties of platinum. Preparations of isolated NaPtCl₅-DMSO and Na (NH₃)₂PtCl-DMSO complexes in the absence of excess DMSO provided agents with enhanced reactivity toward cyanide in vitro and fully recapitulated in vivo cyanide rescue in zebrafish and mouse models. The enhancement of the cyanide scavenging effects of the DMSO ligand could be attributed to the activation of platinum(IV) and (II) with a sulfur ligand. Unfortunately, the efficacy of DMSO complexes was not robust when administered IM. Alternative Pt(II) materials containing sulfide and amine ligands in bidentate complexes show enhanced reactivity toward cyanide addition. The cyanide addition products yielded tetracyanoplatinate(II), translating to a stoichiometry of 1:4 Pt to each cyanide scavenger. These new agents demonstrate a robust and enhanced potency over the DMSO-containing complexes using IM administration in mouse and rabbit models of cyanide toxicity. Using the zebrafish model with these Pt(II) complexes, no acute cardiotoxicity was detected, and dose levels required to reach lethality exceeded 100 times the effective dose. Data are presented to support a general chemical design approach that can expand a new lead candidate series for developing next-generation cyanide countermeasures.

Figure 1

Starting materials and DMSO complexes under investigation as cyanide antidotes.

Scheme 1. Structural Representation of Pt Amine–Sulfide Containing Complexes Isolated and Pharmacologically Evaluated in this Study

Structures represent a major isomer assigned based upon heteronuclear NMR spectra consistent with prior studies on related materials.

Figure 2

Reaction between platinum and cyanide monitored by HPLC. (A) Complex 6 (blue trace) and the product with cyanide (red trace) were quantified using a Restek Ultra IBD. (B, C) Photodiode array (200–300nm) of 6 shown as the blue trace in panel A. (C) Photodiode array (200–300 nm) of Pt(CN)₄^2– having a strong charge transfer band at 260 nm consistent with the formation of Pt(CN)₄^2–. (D) Complex 6 was titrated with 1–10 molar equivalents of cyanide.

Figure 3

¹⁹⁵Pt NMR spectra of 6 (51 mM in 200 mM sodium phosphate, 10% D₂O, pH 7.5. at 291.5 K titrated by cyanide) (A) before addition of cyanide; (B) after addition of approximately 50 mM cyanide; and (C) after addition of 200 mM (final concentration) cyanide. The observed signals correspond to the reactant (PtMet₂), with Pt chemical shifts around −3650 ppm, and the final product [Pt(CN)₄^2–] with the Pt chemical shift at −4700 ppm.

Figure 4

Pt amine–sulfide complex 11 reaction with KCN was monitored by ¹H NMR at 298K. (A) 1 mM compound 11 alone in 50 mM sodium phosphate, pH 7.5, 10% D₂O; (B) with 1 mM KCN added; and (C) with 5 mM (final concentration) KCN added. Ligand (2-methylthioethylamine, or MTEA) can be easily identified by the characteristic sharp peaks at 2.69 and 2.62 ppm for the bound form, and 2.1 ppm for the free form. In the absence of KCN, MTEA is found to be fully bound to Pt. Addition of KCN leads to the quantitative release of MTEA, as the signal at 2.1 ppm becomes incrementally higher in (B and C). The complete disappearance of the bound ligand is observed in (C).

Figure 5

¹³C NMR of the reaction between 6 and cyanide in 100% rabbit serum. (A) Rabbit serum with 400 μM ¹³C–KCN and (B) 100 μM complex 6 was added. The asterisk-labeled signal at 117 ppm is generated from KCN, as it exists mainly as HCN at neutral pH. The signal at 125 ppm is assigned to Pt(¹³CN)₄^2–while satellites from ¹⁹⁵Pt coupling are not clearly observed presumably due to the low signal-to-noise ratio and line-broadening. Semiquantitation of Pt(¹³CN)₄^2–, based on the signal at 117 ppm (for 400 μM) suggests that its concentration is about 100 μM, consistent with the expectation of full scavenging of cyanide by compound 6.

Figure 6

Cardiotoxicity testing of Pt complexes in zebrafish. This assay was performed on zebrafish embryos in the presence of Pt complex agents and dofetilide as the positive control. Target concentrations were chosen from the zebrafish cyanide rescue efficacy doses. Analysis was performed using a multiple comparison test between the means of the ventricle and atrium. The differences between the two means were not significant for all of the doses administered. P < 0.00005 for the dofetilide control.

Figure 7

Complex 6 results in rapid recovery of oxy/deoxy hemoglobin. (A) Nonlethal rabbit cyanide exposure was performed with 100% oxygen ventilation for the first 30 min and infusion of sublethal cyanide over 70 min prior to injecting the drug intramuscularly. (B) Oxygenated (red) deoxygenated (blue) blood hemoglobin for rabbits treated intramuscularly with 3 after cyanide infusion shows the recovery of normal homeostatic levels. (C) Similarly, 6 was injected intramuscularly after the cyanide infusion showing a dramatic recovery of precyanide exposure levels. (D) Platinum blood plasma analysis determined by ICP-MS with AUC as the mean value of n = 3 replicates calculated for the first 60 min after treatment. The highest documented concentration (C_max) from a single injection paired with the time observed (T_max).