Y. Zhong et al.
Regulatory Toxicology and Pharmacology 123 (2021) 104927
cardiorespiratory arrest (Kerns and Kirk, 2006; Kirk and Stenhouse,
1953).
2.2. Test and control items
Various antidotes have been developed for acute cyanide poisoning,
acting to directly or indirectly sequester cyanide to prevent its binding
with cytochrome oxidase a3, and/or neutralize the compound so it can
be eliminated safely. For decades cyanide antidote kits have been uti-
lized in the United States (Chen and Rose, 1952; Shepherd and Velez,
2008), and their use has been associated with a decrease in death rates
from acute cyanide poisoning (Barillo, 2009). Amyl nitrite has been
historically used off-label via inhalation to treat cyanide poisoning but is
currently not approved by the FDA for this indication (Petrikovics et al.,
2015). Amyl nitrite works as an antidote for cyanide by oxidising the
central iron atom of hemoglobin from the ferrous (Fe2+) to the ferric
(Fe3+) state, producing methemoglobin (metHb) (Butler and Feelisch,
2008; Lavon and Bentur, 2010). Cyanide has a high binding affinity to
metHb, and results in the formation of the stable and non-toxic cyan-
methemoglobin complex (Chen and Rose, 1952; Chen et al., 1933). This
limits cyanide bonding to cytochrome oxidase a3 and allows aerobic
cellular respiration to resume. Extracellular cyanide can be eliminated
via metabolism in the liver as thiocyanate, a nontoxic compound that is
excreted in the urine (Kirk and Stenhouse, 1953; Megarbane et al.,
2003). Another antidotal mechanism of nitrites is attributed to vaso-
dilatory activity of nitric oxide (NO) and their ability to improve pe-
ripheral circulation and oxygen supply to vital organs (2011). Organic
nitrate, such as isosorbide dinitrate (ISDN), has also shown a beneficial
effect in the treatment of cyanide poisoning in rabbit and swine models
(Lavon et al., 2017, 2020).
The synthesis of isoamyl nitrite is based on synthesis of alkyl nitrites
as previously described (Noyes, 1936). Briefly, nitrous acid is generated
from an aqueous mixture of sodium nitrite and sulfuric acid in the
presence of isoamyl alcohol. The resulting isoamyl nitrite is mixed with
Epoxidized Linseed Oil (ELSO) to reduce the risk of chemical degrada-
tion. SIAN was manufactured in accordance with current Good
Manufacturing Practices (cGMP) by Southwest Research Institute
(SwRI), San Antonio, TX. SIAN was supplied in 2 mL ampoules filled to
approximately 1.2 mL and stored at 2–8 ◦C, protected from light, and
with no further formulation preparation. Ampoule contents (one
ampoule per animal) were transferred to a labeled glass vial right before
dosing using a glass transfer pipette, and the SIAN was drawn immedi-
ately using the delivery device.
Delivery Device: The intranasal (IN) spray delivery devices used to
deliver SIAN were produced and supplied by Southwest Research
Institute and consisted of a nozzle tip (modified from an off-the-shelf IN
atomization device to fit onto a variable volume syringe) and a modified
gas-tight glass syringe. Delivery devices were prepared under the flow
hood cabinet.
Potassium cyanide (KCN) (manufactured by Sigma-Aldrich) was
stored at room temperature. The KCN vehicle was 0.9% sodium chloride
for injection USP (saline) (Baxter Healthcare Corporation, Deerfield, IL)
purchased commercially and stored at room temperature. The KCN so-
lution was freshly prepared on the day of dosing by dissolving of KCN in
the appropriate volume of saline to make a 1 mg/mL (target) solution.
There are some associated risks with nitrite administration in the
context of cyanide exposure. Nitrite-induced methemoglobinemia is a
side effect and can reduce oxygen carrying capacity of hemoglobin
(Cooling, 2014). Additionally, efficacy studies of amyl nitrite in animal
models of cyanide toxicity provide limited information regarding the
antidotal effects of amyl nitrite on respiratory and hemodynamic pa-
rameters, as well as dose- or concentration-response correlations after
cyanide exposure. These studies were mostly performed on dogs and
mice (Chen and Rose, 1952; Jandorf and Bodansky, 1946; Klimmek and
Krettek, 1988; Vick and Froehlich, 1985, 1991). Nonhuman primates
(NHPs) are one of the most commonly used preclinical species due to
their genetic, cardiovascular and metabolic similarities to humans;
however, only a few studies on cyanide antidote testing have used NHPs
as a model. The NHP model allows for telemetered respiratory and
cardiovascular monitoring, and has been successfully employed and
validated in a number of studies (Authier et al., 2007; Benardeau et al.,
2000; Gauvin et al., 2006; Hassimoto and Harada, 2003).
The solution was filtered through a 0.22 μm filter into a non-pyrogenic
sterile container. The solution was kept at room temperature prior to
administration. Samples of the KCN solution were analyzed by poten-
tiometric titration at initiation and completion of the dosing period and
the concentrations were within acceptance criteria (1.02 and 0.97 mg/
mL, respectively).
2.3. General animal handling
A total of 22 (11 males/11 females) rhesus monkey, aged 2.0–5.3
years, and weighing 3.4–5.4 kg, were included in the study. The animal
room environment was maintained at a temperature of 21 ± 3 ◦C with a
relative humidity of 50 ± 20%, a light dark cycle of 12 h light/12 h dark,
and 10–15 air changes per hour. Temperature and relative humidity
were monitored continuously.
NHPs were fed a standard certified commercial chow (ENVIGO
Teklad Certified Hi-Fiber Primate Diet #7195C in form of cookies) twice
daily. Treats or fruits/vegetables were provided as part of the animal
enrichment program. Water, which had been exposed to ultraviolet light
and purified by reverse osmosis, was provided to the animals ad libitum.
The current study aimed to assess the pharmacodynamic response of
telemeterized NHPs (Macaca mulatta) to cyanide exposure and efficacy
of the administration of subsequent antidotal stabilized isoamyl nitrite
(SIAN) therapy. This strategy allowed for an in-depth characterization of
respiratory and cardiovascular changes in NHPs upon cyanide chal-
lenge, and the efficacy of SIAN therapy on survival and recovery.
2.4. Surgical instrumentation
All animals underwent surgical instrumentation for cardiovascular
monitoring of the arterial blood pressure, left ventricular pressure,
respiration, electrocardiogram, body temperature and locomotor
activity.
2. Materials and methods
2.1. Ethics statement
General anesthesia and surgical preparation. The animals were fasted
overnight prior to the surgical procedures. The animals were sedated
using a mixture of Ketamine (9.09 mg/kg IM) and Acepromazine (0.9
mg/kg IM) followed by intubation. Lidocaine spray (10% w/w) was
administered onto the glottis prior to intubation as needed. An
ophthalmic ointment was applied to both eyes to prevent drying of the
cornea prior and after anesthesia. Anesthesia was maintained with iso-
flurane by inhalation with an oxygen flow at approximately 200 mL/kg/
min or as needed, and animals were mechanically ventilated, as needed,
at a rate of 8–20 breaths/minute with inspiratory pressure of 18–25
cmH2O. Intravenous fluid therapy was given throughout the anesthesia
using Sterile Lactated Ringer’s solution at a rate of 10 mL/kg/hr.
All experimental procedures were performed in accordance with
Institutional Animal Care and Use Committee (IACUC) and the Canadian
Council on Animal Care (CCAC) guidelines for use of experimental an-
imals. The procedures were also approved by the Office of Laboratory
Animal Welfare/Vertebrate Animal Section (OLAW/VAS). The Charles
River Laval (formally known as Citoxlab North America) facility is
AAALAC accredited and all protocols, including humane euthanasia
criteria were reviewed and approved by the IACUC prior to conduct. The
study was approved by the Office of Laboratory Animal Welfare
(OLAW). All procedures were conducted as per Standard Operating
Procedures (SOPs) in place.
2