Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2021 Dec 15.
Published in final edited form as: Toxicology. 2020 Nov 4;446:152626. doi: 10.1016/j.tox.2020.152626

Novel pyridinium oximes enhance 24-hour survivability against a lethal dose of nerve agent surrogate in adult female rats

Jason M Garcia 1, Edward C Meek 1, Janice E Chambers 1
PMCID: PMC7723323  NIHMSID: NIHMS1643944  PMID: 33159982

Abstract

Our laboratory has developed novel substituted phenoxyalkyl pyridinium oximes (US Patent 9,227,937) designed to more efficiently penetrate the central nervous system to enhance survivability and attenuate seizure-like signs and neuropathology. Previous studies with male Sprague-Dawley rats indicated that survivability was enhanced against the nerve agent (sarin) surrogate, 4-nitrophenyl isopropyl methylphosphonate (NIMP). In this study, female adult Sprague-Dawley rats, tested specifically in diestrus, were challenged subcutaneously with lethal concentrations of NIMP (0.6 mg/kg). After development of seizure-like behavior and other signs of cholinergic toxicity, human equivalent dosages of atropine (0.65 mg/kg) and one of four oximes (2-PAM, or novel oxime 15, 20, or 55; 0.146 mmol/kg) or Multisol vehicle was administered alone or in binary oxime combinations intramuscularly. Animals were closely monitored for signs of cholinergic toxicity and 24 h survivability. Percentages of animals surviving the 24 h NIMP challenge dose were 35% for 2-PAM and 55%, 70%, and 25% for novel oximes 15, 20, and 55, respectively. Improvements in survival were also observed over 2-PAM alone with binary combinations of 2-PAM and either oxime 15 or oxime 20. Additionally, administration of novel oximes decreased the duration of seizure-like behavior as compared to 2-PAM suggesting that these oximes better penetrate the blood-brain barrier to mitigate central nervous system hypercholinergic activity. Efficacies were similar between females and previously reported males. These data indicate that the novel pyridinium oximes enhance survivability against lethal OP toxicity as compared to 2-PAM in adult female rats.

Keywords: nerve agent surrogate, neuroprotection, oxime, nitrophenyl isopropyl methylphosphonate (NIMP), brain penetration, seizure-like behavior cessation

1. Introduction

Nerve agents are organophosphate (OP) compounds specifically designed for chemical warfare; however, they were initially developed as insecticides. These OP compounds can be easily absorbed through skin contact or via inhalation and, together with their potent anticholinesterase ability, they were deemed too dangerous for agricultural use, but were ideal for weaponization. The first group of nerve agents developed in the 1930s by Germany were the G-series agents including GB, otherwise known as sarin (Johnson et al., 2015).

The mechanism of action for the acute toxicity of OPs lies in their ability to phosphorylate the serine hydroxyl moiety within the active site of esterases. In a normal functioning nervous system, the action and degradation of the neurotransmitter acetylcholine (ACh) is extremely quick, occurring within a few milliseconds. Disruption of acetylcholinesterase (AChE) by an inhibitor such as an OP or N-methyl carbamate allows the neurotransmitter to accumulate and excessively prolong the stimulatory signal with no immediate remedy for termination (Kobayashi and Suzuki, 2010). A range of sympathetic and parasympathetic nervous system signs in mammals can occur including salivation, lacrimation, urination, and defecation, in a syndrome termed ‘SLUD’, as well as muscle spasms and tremors (Taylor, 1990). Overstimulation of the central nervous system from increased levels of ACh within the hippocampus of the brain activates muscarinic and nicotinic receptors which stimulate the release of the excitatory neurotransmitter glutamate inducing convulsions or seizure activity (Kozhemyakin et al., 2010; Shih and McDonough, 1997; Todorovic et al., 2012). Ultimately, the cause of death in most cases of acute OP toxicity is respiratory failure resulting from paralysis of muscles important for inspiration (Costa, 2008). Common therapy includes the administration of the muscarinic receptor antagonist atropine and an oxime AChE reactivator.

Typically, adult female rats have been considered to be more sensitive to the lethal effects of a nerve agent such as sarin (24 hour LD50 = 67 μg/kg i.m.) than their male counterparts (24 hour LD50 = 88 μg/kg i.m.); however, this is not always the case (Pittel et al., 2018). Significantly higher 24 hour sarin LD50 (125 μg/kg s.c.) was observed in female rats while in the proestrus phase of the estrus cycle versus female rats in the estrus phase or those with ovaries surgically removed. Although not statistically significant, the LD50 of female rats in proestrus was higher than that of male rats (24 hour LD50 = 116 μg/kg s.c.) in the same experiment (Smith et al., 2015). In order to test new therapeutics using female animal models, it is incumbent on the researcher to know the stage of the estrus cycle because increased levels of sex hormones such as estrogen may affect the sensitivity to a given dose of OP compound and thereby confound results for testing therapeutic effectiveness (Wright et al., 2016).

One of the ways hypothesized to account for these observations is that estrogen is thought to have a protective effect on astrocytes which helps maintain the permeability characteristics of the blood-brain barrier (Pittel et al., 2018). Expression of inflammatory cytokines such as IL-6, TNFα, and IL-1β in response to OP exposure within the male brain increased in astrocytes, but not in astrocytes from female brains, suggesting that astrocytes in females are better protected from production of reactive oxygen species associated with inflammatory responses (Astiz et al., 2014). Additionally, increased concentrations of the monocyte chemoattractant cytokine MCP-1, found in the brains of female rats indicate that this higher concentration recruits monocytes from the peripheral circulation in order to better maintain the blood-brain barrier in females after sarin exposure as opposed to males. Overall, females demonstrated better protection against brain damage induced by sarin exposure but exhibited greater overall sensitivity (lower LD50) than their male counterparts when controlling for the estrus cycle (Pittel et al., 2018).

The currently approved oxime reactivator used in the United States for OP intoxication, pralidoxime (2-PAM), has significant limitations with its ability to penetrate the blood-brain barrier, and with broad spectrum capability against a variety of OP compounds. These reasons underscore the necessity of developing new and improved oximes as antidotes to attenuate acute OP toxicity. Our laboratory has developed novel substituted phenoxyalkyl pyridinium oximes designed to more effectively penetrate the blood-brain barrier (Chambers et al., 2013), and these novel oximes have shown functional evidence of efficacy in the brains of OP challenged rats through reduction of glial fibrillary acidic protein (GFAP, an indicator of gliosis and neural scarring) (Pringle et al., 2018) and preservation of neuronal architecture (Dail et al., 2019). Our previous studies investigating the survival efficacies of these novel oximes were performed on adult male rats (Chambers et al., 2016a). Typically oxime efficacy studies have been performed in male rats because of the potential confounding and inconsistencies that could arise because of experimental females being in different phases of the estrus cycle, as well as the inconvenience involved in characterizing the phase of the estrus cycle. If we are to further develop these novel oximes into drugs, experiments must be performed which take into account important biological variables such as sex. In addition, it is possible that a combination treatment of our novel oximes plus 2-PAM might be more efficacious than either one alone, and the efficacy of a combination treatment also needs to be evaluated in both sexes.

2. Materials and methods

2.1. Materials

2.1.1. Nerve agent surrogates

Several surrogate compounds for nerve agents were developed for research purposes (Ohta et al., 2006). The OP chosen for this work, 4-nitrophenyl isopropyl methylphosphonate (NIMP), is representative of the nerve agent sarin and was synthesized at Mississippi State University (Meek et al., 2012; Fig. 1). NIMP is less volatile than sarin but phosphorylates AChE with the same phosphoryl group making this compound highly relevant for studies of reactivators and significantly safer for research.

Figure 1.

Figure 1.

Open in a new tab

Structure of nitrophenyl isopropyl methylphosphonate (NIMP; sarin surrogate).

2.1.2. Novel oxime reactivators

Novel oximes 15, 20, and 55 were first synthesized by the late Dr. Howard Chambers at Mississippi State University (Chambers et al., 2015). These oximes, shown in Fig. 2, were later patented (US patent 9,277,937) and were recently synthesized by SRI International (Menlo Park, CA) for the present project.

Figure 2.

Figure 2.

Open in a new tab

Structures of novel substituted phenoxyalkyl pyridinium oximes. The R indicates the substitution on the phenoxy group and n indicates the number of methylene groups in the linker chain.

2.1.3. Animals

Adult female Sprague Dawley rats (Crl:CD(SD)BR) at postnatal day (PND) 70 were purchased from Envigo RMS, Inc. and housed in AAALAC accredited facilities at Mississippi State University. All animals were held in a temperature-controlled environment with a 12-hour dark-light cycle, used Envigo 70–90 Sani Chips laboratory grade bedding and had free access to Envigo Rodent Diet (18% protein laboratory rat chow) and tap water. Procedures performed for this research received prior approval by the Mississippi State University Animal Care and Use Committee.

2.2. Methods

2.2.1. Estrus Cycle Stage Determination

The estrus cycle stage of each female rat was determined via a modified procedure from Cora et al. (2015) prior to advancing to the dosing protocol to ensure that each animal was in the diestrus phase of its estrus cycle. A syringe was loaded with 100 μL of 0.9% saline, and gently placed into the vaginal orifice, with flushing performed 2–3 times. Lavage fluid was placed on a glass slide and allowed to dry. Slides were stained in a three-step process utilizing Mercedes Medical QUIK-DIP™ hematological stain by dipping 5 times for 1–2 seconds in 80% methyl alcohol, xanthene dye (1 g/L), and thiazine dye (1.1 g/L) allowing the excess fluid to drain from the slide onto absorbent paper between each reagent. The slides were then read by standard light microscopy using 10x magnification to assess cell density and 40x magnification to identify cells.

2.2.2. Treatment Protocol

The experimental paradigm used for the adult female rats closely followed the same design used for adult males from previous studies (Chambers et al., 2016a). Initial testing was performed to ensure the correct dosage of NIMP (0.6 mg/kg) to achieve LD99 results after 24 hours with only the administration of 0.65 mg/kg atropine free base (Sigma Chemical Co., St. Louis, MO) in saline which was the same treatment paradigm as that used previously for the adult male rats (Chambers et al., 2016a). Each rat in an experimental cohort was challenged with NIMP subcutaneously (s.c.) except for one animal, which received only vehicle (Multisol, a biocompatible solvent consisting of 48.5% water, 40% propylene glycol, 10% ethanol, and 1.5% benzyl alcohol). Atropine in saline and oximes were administered intramuscularly (i.m.) at 25–30 minutes post challenge or immediately after onset of seizure-like signs (whichever came first). One animal received only atropine (0.65 mg/kg, i.m.) as an OP control, while the remaining animals received atropine plus 1 of 4 oximes in Multisol (0.146 mmol/kg, i.m.): 2-PAM, oxime 15, oxime 20, or oxime 55. This dosage of oxime is the human molar equivalent dosage for 3 auto-injectors of countermeasures, and these three novel oximes are currently the most promising in the platform. Further, additional rats received NIMP, atropine and binary combinations of oximes following the above paradigm; in the case of these binary combinations, rats received the same dosage (0.146 mmol/kg) of each oxime. To promote reproducibility, rats for all treatment groups came from multiple shipments. Rats which survived the 24-hour survivability challenge were humanely euthanized utilizing CO2 for anesthesia followed by decapitation. Animals which died during the 8-hour surveillance period and those that survived 24 hours had tissues harvested (brain and skeletal muscle) which were snap frozen in liquid nitrogen and stored at −80°C until they could be assayed. Whole blood was collected, separated into serum by centrifugation and kept frozen at −80°C until assay.

2.2.3. Documentation of seizure-like behavior

Animals were closely monitored following challenge and were assessed for signs of cholinergic toxicity following a modified Racine scale for scoring seizures (Luttjohann et al., 2009). These signs were scored as follows: (0) normal behavior; (1) oro-alimentary movements (chewing); (2) anterior limb clonus and rearing; (3) loss of balance (falling) and shaking; (4) tonic convulsions; (5) death. Stage 3 was considered seizure-like behavior, and return to stage 2 was recorded as the time to seizure cessation during the first 8 hour observation period. The observer was blinded as to which treatment each animal received. Animals were observed closely for the first 8 hours following treatment to assess and score signs of toxicity, and scored one last time at the 24-hour mark to terminate the in vivo portion of the experiment. Animals which did not exhibit seizure-like behavior after being challenged with a lethal dose of OP were removed from the study. Only 6 animals out of 100 were removed from the study and replaced due to possible dosing errors when administering NIMP.

2.2.4. Cholinesterase Assay

Cholinesterase activities in harvested tissues were assayed utilizing a modified form of the Ellman discontinuous spectrophotometric cholinesterase assay (Ellman et al., 1961). Rat brain or skeletal muscle was homogenized in 50 mM Tris HCl buffer (pH 7.4 at 25°C) for final concentrations (FC) of 1 or 5 mg/mL wet weight equivalent in 2 mL total assay volume, respectively. Acetylthiocholine was used as a substrate (FC, 1 mM) and color was developed by the addition of 250 μL of a 4:1 mixture of 0.024 M 5,5’-dithio bis(2-nitrobenzoic acid) and 5% sodium dodecyl sulfate. Samples were read at 412 nm and background absorbance was corrected with eserine sulfate (FC, 0.01 mM) blanks. In addition to AChE activity, butyrylcholinesterase (BChE) activity was assayed in serum utilizing butyrylthiocholine (FC, 0.01 mM) as a substrate similar to the protocol outlined above scaled to a 1 mL total assay volume. The method of Lowry et al. (1951) was used to quantify protein content of each tissue sample using bovine serum albumin as the standard.

2.2.5. Statistical Methods

The 24-hour survival values and odds ratios were calculated using 2-PAM as the referent with PROC LOGISTIC in SAS for Windows 9.4 (SAS Institute, Inc., Cary, NC.). Statistical significance was determined using an alpha level of 0.1 (instead of the more common 0.05) in order to reduce the number of animals needed for the challenge protocol while still allowing for statistical power. Differences in mean time of cessation of seizure-like behavior were determined using PROC LIFETEST in SAS for Windows 9.4. Data were censored if the animal died before the end of the 8-hour observational period or if seizure-like behavior continued past this window. The log-rank test statistic was used to assess significance and Dunnett’s adjustment was used for multiple comparisons of the novel oximes compared to 2-PAM. Differences in mean cholinesterase activities from among oxime treatments were determined by ANOVA using PROC GLM in SAS for Windows 9.4 with TUKEY adjustment, and an alpha level of 0.05 was used to determine statistical significance.

3. Results

Determination of the rat estrus cycle was performed prior to initiation of the challenge protocol. Each phase of the estrus cycle was identified by examining the vaginal cytology of each rat as described above. After the female rats were determined to be in the diestrus phase of the estrus cycle, the experiment proceeded to the challenge protocol ensuring that enough animals were available to represent each oxime in every experimental group of rats. Of those animals challenged with NIMP receiving atropine only as therapy, none survived. Most died shortly after being challenged with NIMP during the 8-hour observational period with the average time to death being about 1 hour.

In this study, adult female rats treated with novel oximes showed improvement over 2-PAM in 24-hour survivability (Table 1). The percentages of animals challenged with NIMP surviving 24 hours and treated with the single novel oximes 15, 20, and 55 were 55%, 70%, and 25%, respectively, compared to 2-PAM at 35%. Novel oxime 20 performed the best and was significantly different from 2-PAM (p = 0.03) improving 24-hour survivability with an odds ratio of 4.3 compared to 2-PAM. Although improvement in 24-hour survival with oxime 15 was not statistically significant, the results are trending towards improvement over 2-PAM. Only oxime 55 did not increase 24-hour survivability as compared to 2-PAM when the animals were challenged with a lethal dose of NIMP.

Table 1.

Percent survival after 24 hours of adult (PND 70) female rats challenged at diestrus with 4-nitrophenyl isopropyl methylphosphonate (NIMP) (0.6 mg/kg s.c.) followed at 30 min by atropine (0.65 mg/kg i.m.) and oxime (0.146 mmol/kg i.m.) or Multisol vehicle control. Odds ratios were calculated for survival against 2-PAM.

Oxime Surv./Trted. %Surv. Odds Ratio
None 0/20 0
2-PAM 7/20 35
Oxime 15 11/20 55 2.3
Oxime 20 14/20 70 4.3*
Oxime 55 5/20 25 0.6
Open in a new tab
*

= statistical significance (p < 0.1) vs 2-PAM

Time to seizure cessation was evaluated for all four oximes against NIMP for rats surviving the 8-hour observational period. None of the animals treated with 2-PAM ceased seizure-like behavior during this observational period while all the novel oximes provided a decreased duration of seizure-like behavior. Novel oxime 20 performed the best against NIMP significantly reducing (p < 0.05) seizure-like behavior by 1.6 hours on average as compared to 2-PAM (Fig. 3). This reduction in seizure-like behavior aligned with the increased survival percentages seen with the novel oximes.

Figure 3.

Figure 3.

Open in a new tab

Kaplan-Meier analysis of time to cessation of seizure-like behavior. Cessation of seizure-like behavior during the first 8 h following NIMP administration (0.6 mg/kg, s.c.) in rats treated with atropine (0.65 mg/kg, i.m.) and oximes (0.146 mmol/kg, i.m.). Statistical analysis results of novel oximes compared to 2-PAM alone: Oxime 15, p = 0.661; Oxime 20, p = 0.004; Oxime 55, p = 0.468.

The binary combinations of a novel oxime plus 2-PAM in female rats yielded a significantly improved survival for all three novel oximes (Table 2). The binary combinations of novel oximes yielded significantly improved survival for only oximes 15 + 55 and a trend toward improved survival with oximes 15 + 20, but not for oximes 20 + 55. For easy comparison of the two sexes, also presented in Table 2, are the survival results for the binary combinations in male rats, with the combinations of novel oximes with 2-PAM previously reported (Chambers and Meek, 2020) and the combinations of novel oximes new data. The previously reported combination data from male rats showed higher survival by all three novel oximes + 2-PAM than 2-PAM alone with the improvements from oximes 15 and 20 being statistically significant. With the new data from binary combinations of novel oximes in male rats, the survival of the combinations of oximes 15 + 20 and 15 + 55 were significantly higher than 2-PAM but the combination of 20 + 55 was not different from 2-PAM, similar to what was observed in females.

Table 2.

Percent survival after 24 hours of adult (PND 70) male and female (at diestrus) rats challenged with 4-nitrophenyl isopropyl methylphosphonate (NIMP) (0.6 mg/kg s.c.) followed at 30 min by atropine (0.65 mg/kg i.m.) and combinations of oxime (0.146 mmol/kg i.m. each) or Multisol vehicle control. Odds ratios were calculated for survival against 2-PAM.

Male
 Female
Oxime Surv./Trted. %Surv. Odds Ratio Surv./Trted. %Surv. Odds Ratio
None 0/20 0 0/20 0
2-PAM 10/25 40 8/20 40
15 + 20 13/15 87 9.8* 9/15 60 2.8
15 + 55 12/15 80 6.0* 10/15 67 3.7*
20 + 55 7/15 47 1.3 7/15 47 2.1
15 + 2-PAM 10/15 67 3.0* 10/15 67 3.7*
20 + 2-PAM 13/15 87 9.8* 10/15 67 3.7*
55 + 2-PAM 8/15 53 1.7 11/15 73 5.1*
Open in a new tab
*

= statistical significance (p < 0.1) vs 2-PAM. (Male data on combinations of novel oximes and 2-PAM previously reported in Chambers and Meek, 2020).

The cholinesterase activity of brain, skeletal muscle, and serum was assessed for those adult female rats surviving 24 hours from the single oxime trials (Table 3). Activities from all OP + oxime treatment groups were inhibited compared to controls. In most of the assayed tissues, there was no difference in mean activity among the oxime treatment groups. There was one statistical difference (p < 0.05) among the oxime treatment groups, mean serum activity of AChE and BChE was higher in the oxime 15 treated group as compared to 2-PAM. Correspondingly, mean inhibition percentages were lower while recovery of enzymatic activity was higher for animals treated with oxime 15 as compared to 2-PAM (data not shown).

Table 3.

Specific activities expressed as nmoles min−1 mg protein−1, means ± standard deviation in rats surviving 24 hours after treatment with 4-nitrophenyl isopropyl methylphosphonate (NIMP) (0.6 mg/kg s.c.) followed at 30 min by atropine (0.65 mg/kg i.m.) and oxime (0.146 mmol/kg i.m.). The control animals were not treated with OP, only with Multisol vehicle and atropine (0.65 mg/kg i.m.), and are referenced here for comparison against OP treated animals.

Serum
Oxime n Brain AChE BChE SKM
Control 12 96.2 ± 12.0* 31.6 ± 5.3* 11.9 ± 1.8* 12.8 ± 1.7*
2-PAM 7 17.7 ± 1.5 13.6 ± 4.4 5.3 ± 2.1 4.1 ± 0.7
Oxime 15 11 15.1 ± 3.5 21.9 ± 7.3# 9.0 ± 3.1# 5.2 ± 1.5
Oxime 20 14 18.6 ± 11.4 17.3 ± 5.9 6.8 ± 3.0 5.1 ± 1.4
Oxime 55 5 14.3 ± 1.3 16.7 ± 2.1 7.0 ± 1.1 4.3 ± 1.7
Open in a new tab
*

= statistical significance from all oxime groups (p < 0.05);

#

= statistical significance from 2-PAM group. AChE = acetylcholinesterase; BChE = butyrylcholinesterase; SKM = skeletal muscle

Analysis of harvested tissues from 24-hour survivors challenged with a lethal OP dose show that there was largely no difference in mean inhibition or reactivation of cholinesterase activity between the oxime treatment groups with the one exception being the performance of oxime 15 in NIMP-inhibited serum. In the absence of any additional trends in cholinesterase activity from the other tissues, it is difficult to ascertain if this statistically significant result bears any physiological significance. The lack of any appreciable differences is most likely due to the experimental design of this research which used high concentrations of OP and relatively quick administration of oxime (within 25–30 min of dosing with OP). These high concentrations of circulating OP would be available to re-inhibit cholinesterase enzyme molecules reactivated by oxime. Previous studies have been performed which were designed to prevent this phenomenon of re-inhibition by using a lower, sub-lethal dosage of OP and waiting about 1 hour until the peak AChE inhibition in the brain before oxime administration (Chambers et al., 2013). Using this earlier paradigm, oxime 20 demonstrated about 25% reactivation of NIMP-inhibited rat brain AChE with little to no reactivation by 2-PAM which would be more consistent with the 24-hour survivability data seen in this research (Chambers et al., 2016b).

4. Discussion

This study was designed to parallel previous studies done in this laboratory which tested the efficacy of the novel oximes in adult male rats (Chambers et al., 2016a). Compared to adult male rats, their female counterparts in this study displayed very similar 24-hour survivability improvements when treated with the novel oximes as compared to 2-PAM. These similarities were evident even with the fact that the same lethal dose of NIMP (0.6 mg/kg, s.c.) was used with both sexes. Comparing oxime-mediated survival efficacy against a lethal dosage of the sarin surrogate, NIMP, the percentages of adult male rats surviving 24 hours and treated with novel oximes 15, 20, and 55 were 60%, 55%, and 30%, respectively, compared to 2-PAM at 40% (Chambers et al., 2016a; Chambers and Meek, 2020), and the female results reported here were 55%, 70% and 25% for the novel oximes, respectively, and with 2-PAM 35%. Clearly, oximes 15 and 20 provided improved survival in both sexes while oxime 55 alone was ineffective.

The binary combinations of each of the three novel oximes with 2-PAM showed a similar improvement of survival efficacy with both sexes. The binary combinations of two novel oximes showed improved efficacy for two of the three combinations in male rats (80 or 87% survival compared to 40% for 2-PAM alone) but all three binary combinations of novel oximes showed only small improvements in survival in female rats. Similar trends were observed in male rats. The rank ordering of the three novel oximes was somewhat different between males and females, probably in part because, for humane reasons, the number of replications was reduced to 15 instead of the 20 used for the single oxime groups, providing less granularity in the data on the binary mixtures than the single oximes and only a difference of one rat surviving between the groups. The total data set for both males and females is sufficient, especially the results with the single oximes, to indicate that oximes 20 and 15 are more promising than oxime 55. Therefore there may be potential of a combination therapy with one of our novel oximes with 2-PAM. We are well aware of the fact that the combination paradigm delivered twice the molar dose of the 2-PAM alone paradigm, so clearly more experimentation would be needed before a combination therapy could be seriously considered.

The data on cessation of seizure-like behavior reported here for female rats was very similar to that reported earlier for male rats (Chambers et al., 2016a) in which oxime 20 showed the greatest potential for dampening seizure-like behavior and oxime 15 displayed some potential, while oxime 55 demonstrated the least efficacy.

The AChE data obtained from the 24 hour survivors showed substantial inhibition still present in brain, serum and muscle, which probably resulted from re-inhibition of AChE by the very high amount of NIMP administered to yield an LD99 paradigm. The reactivation elicited by the oximes would have been most effective early when the circulating levels of oxime were highest; the Cmax observed in pharmacokinetic studies of the novel oximes occurred at 5 min following IM administration (the earliest time point monitored) (Dail et al., 2019). However, the same study also showed an extended half life of the novel oximes (plasma half lives of 2.5, 5.3 and 13.8 hr for oximes 15, 20 and 55, respectively), compared to the reported half life of IM 2-PAM 74–77min (Meridian Medical Technologies, 2016). These longer plasma residence times suggest an extended reactivation period compared to 2-PAM, which is likely to contribute to the shortened time to cessation of seizure-like behavior observed compared to 2-PAM. The animals treated with the novel oximes were not observed to develop repeated seizures during the 8 hour observation period and the 24 hour survivors did not display seizure-like behavior.

The data presented here demonstrate improved survivability with our novel oximes as compared to 2-PAM against the sarin surrogate, NIMP. Two of the novel oximes also demonstrated improvements when combined with 2-PAM. Reduced seizure-like behavior was seen in these female animals treated with novel oximes as compared to 2-PAM. These data provide additional support to the concept that our novel oximes can enter the brain. These data also indicate that the novel oximes are equally efficacious in promoting survival in both sexes.

Highlights.

  • Novel oxime AChE reactivators support survival of a sarin surrogate in female rats.

  • Novel oximes also shorten the time to cessation of seizure-like behavior from a sarin surrogate in female rats.

  • These female data are very similar to earlier male data, showing no sex differences in oxime efficacy.

  • This novel oxime platform continues to show promise as a countermeasure to OP anticholinesterases.

Acknowledgements

Research presented in this publication was supported by the National Institutes of Health under Award Numbers U01NS083430 and U01NS107127. The views expressed in this publication are those of the authors and do not reflect the official policy or position of the National Institutes of Health, United States Air Force, Department of Defense, or the U.S. Government.

The authors wish to thank W. Shane Bennett for assistance with the technical procedures and Dr. Robert Wills for his advice on the statistical methods, both at Mississippi State University.

Footnotes

Conflicts of interest

The platform of novel oximes described here are patented by Mississippi State University under US patent 9,277,937, and are licensed by Defender Pharmaceuticals, which did not have input into the design of these studies.

Declaration of interests

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

The oxime platform described here is patented by Mississippi State University and is licensed by Defender Pharmaceuticals.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Astiz M, Acaz-Fonseca A, and Garcia-Segura LM (2014). Sex differences and effects of estrogenic compounds on the expression of inflammatory molecules by astrocytes exposed to the insecticide dimethoate. Neurotox. Res 25: 271–285. doi 10.1007/s12640-013-9417-0 [DOI] [PubMed] [Google Scholar]
  2. Chambers JE, Chambers HW, Meek EC, Funck KE, Bhavaraju M, Gwaltney SR and Pringle RB 2015. Novel nucleophiles enhance the human serum paraoxonase 1 (PON1)-mediated detoxication of organophosphates. Toxicol. Sci 143(1):46–53. [DOI] [PubMed] [Google Scholar]
  3. Chambers JE, Chambers HW, Meek EC, and Pringle RB (2013). Testing of novel brain-penetrating oxime reactivators of acetylcholinesterase inhibited by nerve agent surrogates. Chemico-Biol. Interact 203:135–138. [DOI] [PubMed] [Google Scholar]
  4. Chambers JE, and Meek EC (2020). Novel centrally active oxime reactivators of acetylcholinesterase inhibited by surrogates of sarin and VX. Neurobiology of Disease. 133: 104487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chambers JE, Meek EC, Bennett JP, Bennett WS, Chambers HW, Leach CA, Pringle R, and Wills R (2016a). Novel Substituted phenoxyalkyl pyridinium oximes enhance survival and attenuate seizure-like behavior of rats receiving lethal levels of nerve agent surrogates. Toxicology. 339: 51–57. doi 10.1016/j.tox.2015.12.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chambers JE, Meek EC, and Chambers HW (2016b). Novel brain-penetrating oximes for reactivation of cholinesterase inhibited by sarin and VX surrogates. Ann. N.Y. Acad. Sci 1374: 52–58. doi 10.1111/nyas.13053 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cora CC, Kooistra L, and Travolos G (2015). Vaginal cytology of the laboratory rat and mouse: Review and criteria for the staging of the estrous cycle using stained vaginal smears. Toxicologic Pathol. 43: 776–793. doi 10.1177/0192623315570339 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Costa LG. (2008). Toxic Effects of Pesticides In: Klaassen CD, editor. Casarett’s and Doull’s Toxicology. McGraw Hill; 883–930. [Google Scholar]
  9. Dail MB, Leach CA, Meek EC, Olivier AK, Pringle RB, Green CE and Chambers JE 2019. Novel brain-penetrating oxime acetylcholinesterase reactivators attenuate organophosphate-induced neuropathology in the rat hippocampus. Toxicol. Sci 169:465–472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ellman GL, Courtney KD, Andres V Jr., and Featherstone RM (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol 7: 88–95. doi 10.1016/0006-2952(61)90145-9 [DOI] [PubMed] [Google Scholar]
  11. Johnson NH, Larson JC, and Meek EC (2015). Historical perspectives of chemical warfare agents In: Gupta RC, editor. Handbook of Toxicology of Chemical Warfare Agents, London, U.K.: Elsevier; 7–16 doi 10.1016/B978-0-12-800159-2.00002-6 [DOI] [Google Scholar]
  12. Kobayashi H and Suzuki T (2010). Acetycholinesterase and acetylcholine receptors: brain regional heterogeneity In: Satoh T and Gupta RC, editors. Anticholinesterase Pesticides. John Wiley & Sons, Inc.; 3–14. [Google Scholar]
  13. Kozhemyakin M, Rajasekaran K, & Kapur J (2010). Central cholinesterase inhibition enhances glutamatergic synaptic transmission. J. Neurophysiol 103(4): 1748–1757. doi 10.1152/jn.00949.2009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem 193: 265–275. [PubMed] [Google Scholar]
  15. Lüttjohann A, Fabene P, and van Luijelaar G (2009). A revised Racine’s scale for PTZ-induced seizures in rats. Physiology & Behavior. 98: 579–586. doi 10.1016/j.physbeh.2009.09.005 [DOI] [PubMed] [Google Scholar]
  16. MarvinSketch. (2017). Version 17.13.0, developed by ChemAxon, https://www.chemaxon.com/products/marvin/marvinsketch.
  17. Meek EC, Chambers HW, Coban A, Funck KE, Pringle RB, Ross MK and Chambers JE (2012). Synthesis and in vitro and in vivo inhibition potencies of highly relevant nerve agent surrogates. Toxicol Sci. 126(2): 525–533. doi 10.1093/toxsci/kfs013 [DOI] [PubMed] [Google Scholar]
  18. Meridian Medical Technologies, Inc. (2016). Pralidoxime chloride- pralidoxime chloride injection. [Accessed 25 September 2020] https://www.meridianmeds.com/sites/default/files/pralidoxime_chloride_uspi_2016.pdf
  19. Ohta H, Ohmori T, Suzuki S, Ikegaya H, Sakurada K and Takatori T (2006). New safe method for preparation of sarin-exposed human erythrocytes acetylcholinesterase using non-toxic and stable sarin analogue isopropyl p-nitrophenyl methylphosphonate and its application to evaluation of nerve agent antidotes. Pharmaceut. Res 23(12): 2827–2833. doi 10.1007/s11095-006-9123-1 [DOI] [PubMed] [Google Scholar]
  20. Pittel Z, Grauer E, Gez R, Shlomovich Y, Baranes S, and Chapman S (2018). Sex modulated effects of sarin exposure in rats: toxicity, hypothermia and inflammatory markers. Neurotoxicol. 66: 121–127. doi 10.1016/j.neuro.2018.04.002 [DOI] [PubMed] [Google Scholar]
  21. Pringle RB, Meek EC, Chambers HW and Chambers JE (2018). Neuroprotection from organophosphate-induced damage by novel phenoxyalkyl pyridinium oximes in rat brain. Toxicol. Sci 166: 420–427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Shih T, and McDonough JH Jr. (1997). Neurochemical Mechanisms in Soman-induced Seizures. J. Appl. Toxicol 17(4): 255–264 doi [DOI] [PubMed] [Google Scholar]
  23. Smith C, Wright L, Garcia G, Lee R, and Lumley L (2015). Hormone-dependence of sarin lethality in rats: Sex differences and stage of the estrous cycle. Toxicol. Appl. Pharmacol 287: 253–257. doi 10.1016/j.taap.2015.06.010 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Taylor P (1990). Anticholinesterase agents In: Pharmacological Basis of Therapeutics. (Gilman AG, Nies AS, Rall TW, and Taylor P, Eds.). Macmillan, New York: pp. 131–150. [Google Scholar]
  25. Todorovic MS, Cowan ML, Balint CA, Sun C, and Kapur J (2012). Characterization of status epilepticus induced by two organophosphates in rats. Epilepsy Res. 100: 268–276. doi 10.1016/j.eplepsyres.2012.04.014 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wright L, Lee R, Vincelli N, Whalley C, and Lumley L (2016). Comparison of the lethal effects of chemical warfare nerve agents across multiple ages. Toxicol. Let 241: 167–174 doi 10.1016/j.toxlet.2015.11.023 [DOI] [PubMed] [Google Scholar]

RESOURCES