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A Novel Paradigm A Novel Paradigm for Assessing Efficacies of Potential Antidotes against Neurotoxfor Assessing Efficacies of Potential Antidotes against Neurotoxins in Mice.ins in Mice.

Daune L. Crankshaw, David J. W. Goon, Jacquie E. Briggs, David DeLong, Michael Kuskowski, Steven E. Patterson, and Herbert T. Nagasawa

METHODSMETHODSThe recovery test paradigm is as follows: Mice are treated with a 4.8 mg/kg dose of sodium cyanide intraperitoneally, which induces a knock-down state with recovery periods of approximately one h. Before each day of experimentation, three or more animals are treated with cyanide at this dose to verify that the recovery times are close to one h. Each treatment requires 8 animals.

The mouse moves his head and starts to perambulate about 60 min post-CN (or less, when the mouse also received an antidote). At thispoint, the mouse is placed on top of a 10x10 cm fine mesh wire screen which is subsequently inverted. The time from cyanide administration to the time the mouse successfully rights itself from the inverted screen (see inset below) is taken as the endpoint and recorded.

Premature testing of an animal without full neuromuscular coordination will artificially prolong the recovery period. Critical signs that the animal is ready to be tested include bodily appearance, both eyes open and bright, head up and moving, steady on feet, un-hunched back and certain movements about the cage, e.g., definite exploration of environment with alert, quick ambulation and social interaction. Recognition of these signs are easily gained by experience.

INTRODUCTIONINTRODUCTION RESULTSRESULTS

AcknowledgmentsAcknowledgmentsStartup funding was provided by The Center for Drug Design, Academic Health Center, University of Minnesota. The project was subsequently funded by the National Institutes of �Health CounterACT Program through the National Institute of Neurological Disorders and Stroke (Award #U01NS058087-01). The Department of Veterans Affairs Medical Center, Minneapolis, MN provided small animal barrier facilities and additional medicinal chemistry laboratories.

CounterACT has identified cyanide as a ‘Higher Priority Chemical Threat’ with a need for enhancement of diagnostic and treatment response capabilities during an emergency, especially antidotes that provide post-cyanide brain protection. Since IACUC guidelines strongly discourage LD 50 determinations, it was necessary to devise a new test paradigm that allowed for maximal survival of cyanide-treated animals with greatly reduced numbers of animals. A prime target for cyanide is the brain, resulting in rapid disruption of muscular coordination. The end point of this paradigm is recovery of neuromuscular coordination, i.e., the righting reflex (Koplowitz et al., 1989; Baskin et al., 1999). An effective antidote reduces the neurobiological recovery time. The relationship between recovery of righting reflex, % survival, and cyanide dose was established to test antidote efficacy (Crankshaw et al., Toxicology Letters. 2007, 175:111-117).

SUMMARY & CONCLUSIONSSUMMARY & CONCLUSIONSOur data has clearly established the relationship between the neuromuscular recovery of righting reflex and the LD50 slopes, both slopes shifting in tandem to the right, as demonstrated in Graph A and B. The LD50 values obtained in these studies for cyanide alone and cyanide with antidote protection, 6.1 and 40.0 mg/kg are consistent with those of other investigators.The efficacy of prototype drug #2 and 3-MPDT delivered orally before cyanide suggests a prophylactic use which is not a pharmacological option for the currently available antidotes.

The power of the recovery paradigm is demonstrate with our post-cyanide data. Prototype drug #2 and 3-MPDT are efficacious when delivered i.p. 40 min after cyanide. In contrast, the currently available antidotes are ineffective 10 to 20 min after a cyanide insult.

These graphs show the relationship between (i) time required for recovery of righting reflex (neuromuscular coordination) and (ii) survival as the dose of cyanide is increased. The left y-axis shows the time in min required for the recovery of the righting reflex, while the right y-axis indicates % survival.

Graph A shows the optimum dose for antidote screening was 4.8 mg NaCN/kg body weight, with a resultant 68 min average time required for the recovery of the righting reflex (shown at the arrow). The survival rate at this optimum dose was 99%. The linear regression of the survival slope for doses ranging from 4.8 to 7.0 mg/kg is Y= 244.943-30.982 * X; R2=0.915, while the simple regression analysis recovery of of the cyanide dose-response slope was Y= -46.638 + 24.688 X; R2=0.832. The estimated LD50 is at a NaCN dose of 6.1 mg/kg (shown at asterisk). Values reflect means, n = 8/group, except control.I.e., 0 cyanide, n = 20.

Graph B differs in that a standard dose of nitrite/thiosulfate (1.45 and 6.32 mmol/kg), respectively was administered as a protective antidote given 30 min pre-cyanide. The linear regression of the survival slope for doses ranging from 34.2 to 43.8 mg/kg is Y= 411.56-9 X; R2=0.929. The estimated LD50 for NaCN was 40.0 mg/kg (shown at asterisk). The simple regression analysis recovery of of the cyanide dose-response slope was Y= -9.4 + 3.957 X; R2=0.923. Values reflect means, n = 4/group, except the 34.2 and 39.0 mg/kg doses, n= 8.

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The effect of current antidotes administered pre- and post-cyanide. Recovery time of the righting reflex in min. The current antidotes, hydroxocobalamin (HC), at 0.22 mmol/kg and nitrite plus thiosulfate (N/T) at 1.45 and 6.32 mmol/kg, respectively, were administered i.p. at the times indicated before and after cyanide (NaCN, 4.8 mg/kg, I.p.). The current antidotes at 60, 30, and 5 min pre- and 1 min post-cyanide were all significantly different from the control, (p<0.0001). However, at the 5, 10, and 20 min post-cyanide, the antidotal efficacies were drastically compromised. Not only was efficacy reduced, there was a significant increase in recovery time with nitrite plus thiosulfate given 20 min post-cyanide (p<0.0001).

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The effect of prodrug#2 (0.29 mmol/kg and 3-MPDT (0.58 mmol/kg) administered 5, 10, 20, 30, and 40 min after 4.8 mg/kg NaCNgiven i.p. Also the effect of prodrug #2 and 3-MPDT given orally at 1.45 mmol/kg 30 min pre-cyanide. Recovery of righting reflex shown in min. These drugs were all significantly different from control (p<0.0001).

Dose effect of PD#2 in mmol/kg at 30 min before cyanide and 10 minutes after cyanide. The PD#2 was administered orally by gavage pre-CN and i.p. post-CN. Recovery times of righting reflex are in minutes. There was a significant dose effect for both pre- and post-cyanide, (F(3,37)=84.652, p<0.0001) and F(4,56)=77.219, p<0.0001), respectively.

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Prodrug #2These compounds are sulfhydryl-protected

derivatives of 3-mercaptopyruvate, a substrate for the predominately cytosolic 3-mercaptopyruvate cyanide sulfurtransferase (MST). The specific activity ratio of MST to rhodanese is much higher in heart, lung, and cerebellum, important areas targeted by cyanide.

This model was used to evaluate cyanide toxicity using current cyanide antidotes: nitrite-thiosulfate (USA) and hydroxocobalamin(Europe & USA) in addition to novel antidotes designed and synthesized in our laboratories. These compounds are sulfhydryl-protected derivatives (prodrugs) of 3-mercaptopyruvate (3-MP) derivates which, in a cellular environment, release this natural substrate for the predominantly cytosolic enzyme, 3-mercaptopyruvate: cyanide sulfurtransferase (Nagasawa et al., J. Med. Chem. 2007, Dec 27; 50(26):6462-4).

Abstract # 228Poster # 443

3-MPDT

S

SHO

EtO2C

CO2Et

OHS

SHO

NaO2C

CO2Na

OH