Pharmacokinetics of Three Novel Pyridinium Aldoxime Acetylcholinesterase Reactivators in Female Rats

Abstract

A platform of novel lipophilic substituted phenoxyalkyl pyridinium oximes was invented to reactivate organophosphate-inhibited acetylcholinesterase. This platform has provided superior efficacy in rats to the current standard of care, 2-PAM, for survival of lethal doses of nerve agent surrogates as well as evidence of brain penetration and neuroprotection. The pharmacokinetics of three of these novel oximes in female rats was studied for comparison to previous data in male rats. Compared to the published half-life of 2-PAM (less than 2 hours), the lead novel oxime, Oxime 20, displayed a plasma half-life of about 5 hours in both sexes of rats following intramuscular administration. Very few sex differences in pharmacokinetic parameters were apparent. Oxime 20 displayed an increase in brain concentration to plasma concentration over the initial 2 hours following intramuscular administration in male rats, with a plateau at 1 hour; there were no differences in brain concentrations between the sexes at 2 hours. Hepatic metabolism of Oxime 20 was higher in rat microsomes than in human microsomes. The relatively long plasma half-life is likely an important factor in both the enhanced survival and the neuroprotection previously observed for Oxime 20. The metabolism data suggest that the clearance of Oxime 20 could be slower in humans than was observed in rats, which might allow less frequent administration than 2-PAM for therapy of organophosphate acute toxicity. Therefore, the pharmacokinetic data combined with our earlier efficacy data suggest that Oxime 20 has potential as a superior therapeutic for nerve agent poisoning.

Introduction

Organophosphate (OP) anticholinesterase compounds comprise a class of some of the deadliest synthetic toxicants. As such, exposures to OPs have been a consistent concern since their initial development in the late 1930s (Holstege et al., 1997; Howes, 2020; Koller et al., 1997; Stone, 2019). OPs evoke deleterious effects in the nervous systems of mammals through stimulation of the cholinergic system inducing a cholinergic crisis, central excitotoxicity resulting in seizures and long-term cognitive aberrations in survivors, or death from respiratory failure (Holstege et al., 1997).

The relative ease of synthesis and access to chemical precursors for OP-based chemical warfare agents (CWAs), such as sarin and VX, facilitated their stockpiling by a number of nations post World War II until the implementation of the Chemical Weapons Convention of 1997 (Tucker, 2007). Despite the mandated destruction imposed by the convention, OP-based CWAs have seen use across the world for pacification of civilian populations in the 1980s by Iraq and in 2013 by Syria, in the Tokyo subway terrorist attacks by the Aum Shinrikyo Cult in the mid-1990s (Sugiyama et al., 2020), and in the 2018 and 2020 attempted assassinations of former Russian spies and politicians (Howes, 2020; Stone, 2019). These events highlight a need for a more effective therapy of OP poisoning for both civilian and military populations.

The current standard of care for OP poisoning involves therapeutic cocktails composed of an oxime acetylcholinesterase (AChE) reactivator [pralidoxime (2-PAM) in the US], the muscarinic receptor antagonist atropine, and frequently an anticonvulsant (diazepam) to treat the major effects of OP poisoning. However, 2-PAM is limited in efficacy due to rapid clearance from plasma (T_1/2 ~0.5 hr to 2 hr) (Ellin et al., 1974; Green et al., 1986; Josselson and Sidell, 1978), different levels of reactivation of AChE inhibited by various OPs (Bajgar, 2010; Mercey et al., 2012), lack of appreciable entry into the central nervous system (CNS) (Mercey et al., 2012), and inadequate protection from permanent brain damage (Doctor and Saxena, 2005). Research is being conducted by several groups on reactivators that have structures that could allow brain penetration and therefore provide CNS protection (Sit et al., 2011; Zorbaz et al., 2020). However, a need still remains to develop an improved reactivator.

An ideal AChE reactivator would exhibit a high degree of druggability, enhanced blood-brain barrier (BBB) permeability, and broad-spectrum activity against various OP agents. Increasing the bioavailability of the oxime within the CNS is likely to reactivate inhibited brain AChE, potentially preventing or attenuating OP-induced seizures as well as the resultant neuropathology. Our laboratories have invented and tested a series of substituted phenoxyalkyl pyridinium oxime AChE reactivators (US Patent 9,227,937) (Chambers et al., 2013). Research resulted in down-selection to three lead candidates, Oximes 15, 20 and 55 (Figure 1) that have shown 1) greater lipophilicity than 2-PAM (Chambers et al., 2016a; Kitagawa et al., 2019), 2) extended biological half-lives compared to 2-PAM in male rats (Dail et al., 2019), and 3) neuroprotective effects in the brain following OP poisoning in male rats, whereas 2-PAM did not (Dail et al., 2019). In the present report, we provide pharmacokinetic data on these three oximes in female rats to provide a sex-based comparison to our previous data in male rats (Dail et al., 2019). In addition, the levels of these oximes in the brain of rats (both sexes) were determined, as well as initial information about the metabolism of these oximes by liver microsomes from rats of both sexes and from humans (pooled sample of both sexes). Based on the pharmacokinetic data presented here and previously reported efficacy data (Chambers et al., 2016b; Garcia et al., 2020), we have now selected Oxime 20 as our lead compound and Oxime 15 as our alternate compound for further development.

Figure 1.

Structure of substituted phenoxyalkyl pyridinium oxime AChE reactivators MSU 15, 20 and 55.

Materials and Methods

Chemicals

Mesylate salts of the oximes were custom synthesized to >97% purity by SRI International (Menlo Park, CA) utilizing methods originally described by the late Dr. Howard Chambers at Mississippi State University (Chambers et al., 2016a). 12-[[(cyclohexylamino)carbonyl]amino]-dodecanoic acid (CUDA) was purchased from Cayman Chemical Company (Ann Arbor, MI). Nicotinamide adenine dinucleotide phosphate tetrasodium salt (NADPH) and Trizma base were purchased from Sigma-Aldrich (St. Louis, MO). Magnesium chloride hexahydrate and all solvents (Optima® LC/MS grade) were purchased from ThermoFisher Scientific (Waltham, MA).

Animal Treatments

Adult Sprague Dawley-derived female rats 8–9 weeks of age, 200–225 g, were obtained from Envigo and maintained within Mississippi State University AAALAC-accredited facilities in accordance with an approved IACUC protocol. Prior to shipment these female rats had been surgically implanted with jugular vein catheters containing either a dual port for intravenous (IV) oxime administration and blood sampling or singular port for blood sampling after intramuscular (IM) oxime administration. IM administration is likely the most practical initial route of oxime administration in a mass casualty situation, whereas IV administration would have utility in a subsequent hospital situation. All procedures performed on the female rats were the same as those used previously with male rats (Dail et al., 2019). All animals were humanely euthanized following sample collection.

Oxime Administration and Blood Sampling

The molecular weights of the novel oximes were very similar to one another: 436.52, 472.55 and 478.65 for Oximes 15, 20 and 55, respectively. Therefore the molarities administered for IM and IV were also similar among the compounds. Each oxime was dissolved aseptically in a biologically compatible vehicle to yield test doses of 5 mg oxime/kg body weight for IV administration and 50 mg oxime/kg body weight for IM administration (11.5, 10.6 and 10.4 µmoles/kg administered IV and 115, 106 and 104 µmoles/kg administered IM for Oximes 15, 20 and 55, respectively). The vehicle for IV injection was 3% N-methylpyrrolidone/45% polyethylene glycol 300/12% ethanol/40% sterile water, while the vehicle for IM injection was 1.5% benzyl alcohol/10% ethanol/40% propylene glycol/48.5% sterile water (multisol). Multisol is the vehicle that we have previously used for our in vivo experiments with these oximes administered IM. Blood samples (300 µL each) were collected from the jugular vein catheters at 5, 15, 30, and 45 min, and 1, 2, 4, 12, and 24 h post-dose into K₃EDTA coated tubes. Blood sampling was not performed past 24 hours because the time frame of value for oxime reactivators in survival is a very few hours since potent OP’s can be lethal very quickly. Three animals per route were used, each given a single dose. Plasma was prepared by centrifuging whole blood at 3 × 10³ g at 4C and was stored at −80C until extraction.

Plasma extraction

Plasma extraction procedures followed the protocol conducted earlier by SRI International in male rats with slight modifications (Dail et al., 2019). In general, plasma (20 µL) from treated rats was extracted at room temperature against a solution (110 µL) of 0.1% formic acid in acetonitrile fortified with 500 ng/mL of a quantifying internal standard (qIS); for samples containing Oximes 15 and 55, Oxime 20 served as the qIS and for samples containing Oxime 20, Oxime 15 served as the qIS. Extracted samples were clarified by centrifugation at 16 × 10³ g at 4C for 10 min, the supernatant was then transferred to autosampler vials, supplemented with 50 nM CUDA (final concentration) to serve as a type II instrument internal standard (iIS), and analyzed via UPLC-MS, as described below. Recovery experiments were performed by carrying blank plasma through the extraction process and then “adding back” the oxime being extracted at a known concentration and comparing the measured concentration to the nominal concentration as described earlier (Ross and Filipov, 2006); percent recoveries were found to be 75%, 72%, and 81% for Oximes 15, 20, and 55, respectively.

Brain Extraction Protocol

Whole brains were harvested from m ale rats (9–10 weeks of age, 250–300g) administered Oxime 15, 20 or 55 in multisol IM (50 mg/kg) at 30 min and Oximes 15 and 20 at 2 hr following administration. In addition, Oxime 20 (the lead compound) was also sampled at 5, 15, 45 and 60 min. No samples were obtained after 2 hours because OP-induced seizures occur within this 2-hr time frame, and the utility of a brain-penetrating oxime to provide neuroprotection would need to be during this short time interval. Three female rats (9–10 weeks of age, 225–275 g) were also treated with 50 mg/kg Oxime 20 IM and sampled at 2 hr. Animals were euthanized by CO₂ asphyxiation and the brains were quickly removed, rinsed with chilled 0.9% saline, snap frozen in liquid nitrogen and stored at −80C until homogenization and extraction. The rinsing of brain tissue is an accepted methodology (Sit et al., 2018); rinsing would not create a concentration gradient that perfusion could create between the brain and the perfusate where the oxime might diffuse out of the brain into the perfusate.

The left half of the brain was thawed on ice, weighed, rinsed briefly with chilled 0.9% saline, and homogenized in 50 mM Tris-HCl buffer (pH 7.7 at 25C) to a concentration of 0.25 g tissue/mL buffer. Because of the lipophilicities of these novel oximes, brain tissue processing drew from protocols originally developed for lipid and brain extractions (Bligh and Dyer, 1959; Folch et al., 1957). The homogenate (150 µL) was extracted with 975 µL of a solution of (2:1 v/v) methanol:chloroform with 0.1% formic acid fortified with 500 ng/mL of the appropriate qIS. The sample was centrifuged, and the supernatant was collected and dried under a stream of nitrogen with gentle heating. The dried residue was reconstituted to 0.3 mL in a solution of (3:1 v/v) methanol:water supplemented with 0.33% formic acid and iIS CUDA (500 nM). The reconstituted solution was passed through a Costar Spin-X 0.22 µm cellulose acetate centrifugal filter, collected, and then analyzed via UPLC-MS (method described below). Brain homogenates from each animal were extracted in triplicate (technical replicates) and the average value ± the standard deviation reported for n=3 animals. Quality control samples were also prepared in brain homogenates at a concentration of 30 ng/mL and were extracted as described above. Recovery experiments were performed similarly to plasma recovery using brain tissue from naïve male and female rats. Percent recoveries were 33%, 63%, and 40% for Oximes 15, 20, and 55, respectively.

Microsomal Preparations

Livers were collected from three separate rats per sex, rinsed with 0.9% saline and homogenized with a Teflon-glass tissue grinder in 50 mM Tris HCl buffer with 250 mM sucrose (pH 7.4 at 25C) at 0.5 g liver/mL. Microsomal preparations were the 100,000 g (1 hr) pellets of 17,000 g (30 min) supernatants (Forsyth and Chambers, 1989). Protein concentrations, as quantified by the method of Lowry et al. (1951), for female derived microsomes ranged from 9 to 11 mg/mL, and for male derived microsomes ranged from 14 to 18 mg/mL. Human liver microsomes were purchased from Xenotech (Kansas City, KS) and used as received (mixed sex pool of 200 donors containing 20 mg of protein/mL).

Rat Microsomal Metabolism Studies.

Stock concentrations (20 mM) of oximes were prepared in a 1:1 solution of absolute ethanol: 50 mM Tris-HCl (pH 7.4 at 25C) and subsequently diluted in 50 mM Tris-HCl containing 3 mM MgCl•6 H₂O (pH adjusted to 7.4 at 37C) to a 10X working concentration of 50 µM. Oximes 15 and 20 were sampled at times 0, 5, 10, 15, 20, and 30 min and Oxime 55 was sampled at time 0 and 30 min.

For rat metabolism studies, 50 µL of the resuspended microsomal preparation (0.5 g wet weight equivalent of tissue/mL) was added to a glass test tube followed by the addition of 50 µL of the 50 µM oxime solution and vortexed. Next, 400 µL of either a control buffer, 50 mM Tris-HCl containing 3 mM MgCl•6 H₂O prewarmed to 37C (pH adjusted to 7.4 at 37C), or reaction buffer, 50 mM Tris-HCl containing 3 mM MgCl•6 H₂O and 1.25 mM NADPH prewarmed to 37C (pH adjusted to 7.4 at 37C), was added to the test tube. Final reaction assay conditions were 5 µM oxime, 0.05 g wet weight equivalent of liver/mL, and 1 mM NADPH. Microsomal protein concentrations in the reactions were 1.6 and 1.0 mg protein/mL for males and females, respectively. The test tubes were vortexed to mix components and oxygenate the reaction mix, and were then incubated at 37C with shaking. Aliquots (200 µL) of the reaction mixture were removed and the reactions were stopped by the addition of an equal volume of acetonitrile containing 0.1% formic acid and 500 ng/mL of the appropriate qIS. Samples were centrifuged at 16,000 g at 4C for 10min; the supernatants were analyzed via UPLC-MS. Because the chemical nature of the metabolites was unknown, oxime metabolism was quantified as the % loss of the parent compound over 30 min.

For human liver metabolism the following adjustments were made: microsomal protein concentration was adjusted to reflect a concentration between the concentrations of male and female rats (1.4 mg protein/mL), reaction sample volume was reduced to 100 µL, reaction sampling times were 0, 15, and 30 min, control samples (-NADPH) were sampled at time 0 min and 30 min, and oxime 55 was omitted.

UPLC/MS Analysis

Analysis of the three oximes was performed with a UPLC-MS system configured with a Waters Acquity UPLC (a binary pump, autosampler, and column oven) connected to a TSQ Quantum Max Access mass spectrometer (Thermo Fisher Scientific, Waltham, MA). Separation of analytes was achieved on a Waters Acquity UPLC^® BEH C-18 column (2.1 × 100mm, 1.7µm) equipped with a Waters Acquity UPLC^® BEH C-18 VanGuard™ pre-column (2.1 × 5mm, 1.7µm) set at a flow rate of 0.2 mL/min. Ten µL of the extract was injected and chromatographed using a binary mobile phase that consisted of component A: (95:5 v/v) water:acetonitrile + 0.1% formic acid and component B: acetonitrile + 0.1% formic acid utilizing the following gradient: 0 – 1 min, 95% A : 5% B; 1 – 8 min, 20% A : 80% B; 8 – 8.5 min, 100% B; 8.5 – 10 min, 100% B; 10 – 11 min, 95% A : 5% B; 11 – 14 min, 95% A : 5% B. The column oven and autosampler were maintained at 50C and 4C, respectively. Selected reaction monitoring (SRM) was used to detect and quantify target analytes in the positive ion mode. SRM transitions for oxime 15, 20, 55, and CUDA were m/z 341.204>163.107, 377.208>255.106, 383.294>219.147 and 341.289>216.193, respectively. Data were processed using Thermo Xcalibur 2.2 SP1.48 software.

The analytical method was developed based on FDA guidance for bioanalytical method validation. A calibration curve was generated with known concentrations of oxime in plasma or brain tissue homogenate from naïve animals and calibrants were subjected to the same processing protocols as the unknown samples. Blank samples of tissue containing no added analyte or internal standard were processed in parallel in each run and assessed for any interfering isobaric peaks present near peaks of interest. Quantification was performed by extracting a series of calibrators (50 – 10,000 ng/mL, plasma; 10 – 500 ng/mL of brain homogenate; 100 nM – 50 µM, liver microsomal metabolism) in parallel with the test samples, then generating a calibration curve by fitting a 1/x weighted quadratic regression for plasma and brain samples using the peak area ratio (PAR) of the analyte of interest to the qIS versus the calibrator concentration, whereas for liver metabolism a 1/x weighted linear regression was used. The concentration of the oxime in the treated sample was determined by solving the quadratic regression using its respective PAR from the LC/MS chromatogram. The lower limits of oxime quantitation were 50 ng/ml for plasma and 40 ng/g for brain.

Pharmacokinetic Analysis

A non-compartmental model for calculations was used. Plasma oxime concentrations were input into Phoenix WinNonLin (version 8.3.1) to calculate time to maximum plasma concentration (T_max), maximum plasma concentration (C_max), terminal elimination half-life (T_1/2), area under the curve to the last time point for the plasma concentration curve (AUC_last), area under the curve for the plasma concentration curve extrapolated to infinity (AUC_inf), mean residence time to the last time point (MRT_last), and the mean residence time extrapolated to infinity (MRT_∞). Doses were entered as mg of oxime/kg of body weight. Because this version of WinNonLin was an upgraded version compared to that used in the earlier male study (Dail et al., 2019), the male plasma data were reanalyzed to ensure valid male to female comparisons.

Statistical Analysis

Sample sets were analyzed for outliers using Grubb’s test for single outliers. For analysis of sex differences, test groups were compared using an unpaired Student’s t-test with a p-value of 0.05.

Results

Plasma Oxime Concentrations

Data are presented as weights and not molarities because of the preference of the US Food and Drug Administration; as noted above, the molecular weights of all three oximes were similar to one another. Data from IM administration indicated that the T_1/2 in both sexes followed the trend of Oxime 55 > Oxime 20 > Oxime 15, while the opposite trend was found in the C_max for both sexes Oxime 15 > Oxime 20 > Oxime 55 (Table 1); there were no statistical differences between the sexes. T_max’s were rapid for Oximes 20 and 55 with both reaching T_max at the first sampling time and taking about 1.6 times longer to reach T_max in females than males for Oxime 15 (Table 1). Oxime 20 was found to have about 2 times the AUC_last and AUC_∞ for females compared to males, although this difference was not statistically significant. Oximes 15 and 55 had similar AUC_last and AUC_∞ for both females and males. For Oximes 15 and 20, the mean values for MRT_last and MRT_∞ did not differ between males and females, whereas for Oxime 55 the mean MRT_last was about 1.2 times higher in females than males and the mean MRT_∞ was about 1.6 times higher in females than in males. Oxime 20 had about 1.7 times higher V_d in males than females, whereas the V_d’s for oximes 15 and 55 were similar in both sexes. Finally, the Cl for each of the three oximes was similar between the sexes following IM administration. The AUC_∞’s for all three oximes were higher in females than males with statistical differences for both Oximes 15 and 20. The Cl for Oxime 15 was significantly higher in males than females, and there was no significant difference between the sexes for Oximes 20 and 55. The V_d’s were found to be similar in both sexes for Oximes 15 and 55, while the V_d for Oxime 20 was found to be statistically different between the sexes. The MRT_last and MRT_∞ for Oxime 55 were the only values to differ between the sexes. The plasma concentration time course values following IM administration are shown in Figure 2.

Table 1.

Pharmacokinetic data for the 50 mg/kg intramuscular dose of Oximes 15, 20 and 55 in female and male rats. Data are expressed as the mean ± the standard deviation (n=3). Male data were previously reported in (Dail et al., 2019) and reprocessed in Phoenix WinNonLin (version 8.3.1).

Oxime	Sex	Tmax (hr.)	T1/2 (hr.)	Cmax (ng/mL)	AUClast (ng·hr/mL)	AUC∞ (ng·hr/mL)	Vd (mL/kg)	CI (mL/hr./kg)	MRTlast (hr.)	MRT∞ (hr.)
15	Female	0.8	3.0 ± 0.9	2977 ± 811	9618 ± 3251	11553 ± 591	18820 ± 6829	4290 ± 221	2.4 ± 0.6	3.9 ± 1.0
	Malea	0.50	2.5 ± 0.4	2925 ± 983	9072 ± 3257	9416 ± 66*	19249 ± 3146	5311 ± 37*	2.8 ± 0.3	3.2 ± 0.5
20	Female	0.083	5.4 ± 2.0	2385 ± 793	11748 ± 4619	12880 ± 5266	30563 ± 3248	4242 ± 1459	5.4 ± 2.0	7.2 ± 2.0
	Male	0.083	5.2 ± 0.6	2547 ± 640	6689 ± 945	7006 ± 1094*	52709 ± 2391*	7105 ± 1122	5.4 ± 0.7	6.5 ± 1.0
55	Female	0.083	19.3 ± 5.0	1040 ± 372	5913 ± 2420	10821 ± 5083	152162 ± 79033	5190 ± 1914	10.6 ± 0.8	28.7 ± 3.0
	Male	0.083	13.7 ± 0.3	1481 ± 654	5672 ± 1015	7729 ± 1344	128527 ± 22803	6527 ± 1203	8.8 ± 0.3*	18.1 ± 0.1*

^aOxime 15 male IM data n=2.

^bT_1/2 = ln 2/k_el, k_el is the terminal elimination rate constant (units: hr⁻¹).

^*Statistical difference between female and male (P≤0.05).

Figure 2.

Plasma concentrations for each oxime following a single 50-mg/kg IM dose of Oximes 15, 20 or 55 to female (A) or male (B) rats. [Male data originally reported in (Dail et al., 2019) were reprocessed in Phoenix WinNonLin (version 8.3.1)]. Each point represents the mean ± the standard deviation of n = 3 animals except for Oxime 15 in males, n = 2.

Following IV administration of oximes, the trends in T_1/2, C_max, and T_max were the same for both sexes (Table 2) but not when the routes of administration (IM vs IV) were compared. Oxime 55 showed the longest T_1/2, followed by Oxime 15 and Oxime 20 (Table 2). Males had a longer T_1/2 for Oxime 15 (~1.7 times longer) and Oxime 55 (~2.2 times longer) than females (Table 2). The half-lives were 1 hour or less for all three oximes. C_max values for all three oximes, like the T_1/2 data, followed the same trend between sexes; however, females had higher concentrations (not statistically different) of all three oximes; the trend was Oxime 55 > Oxime 15 > Oxime 20 (Table 2). Of the three compounds, Oxime 15 had the largest difference in C_max between males and females, with females having 4.9 times more oxime found. Oxime 55, on the other hand, had the smallest difference between males and females; females treated with Oxime 55 had 1.7 times greater C_max. The female C_max for Oxime 20 was found to be 3.6 times greater than males (Table 2). As expected T_max for all three oximes was the same (0.083 hr) after the IV dose, which was the earliest time point measured (Table 2). T_1/2 values for Oximes 15 and 55 were found to vary significantly between males and females, whereas animals treated with Oxime 20 did not. Unlike the T_1/2 data, statistically significant variations between the sexes were not found for C_max values. The same T_max value, 0.083 hr, was found for all oximes in both sexes. The plasma concentration time course values following IV administration are shown in Figure 3

Table 2.

Pharmacokinetic data for the 5 mg/kg intravenous dose of oximes 15, 20 and 55 in female and male rats. Data are expressed as the mean ± the standard deviation (n=3 animals).

Oxime	Sex	Tmax (hr.)	T1/2 (hr.)	Cmax (ng/mL)	AUClast (ng·hr/mL)	AUC∞ (ng·hr/mL)	Vd (mL/kg)	CI (mL/hr./kg)	MRTlast (hr.)	MRT∞ (hr.)
15	Female	0.083	0.15 ± 0.051	2935 ± 3670	457 ± 542	480 ± 528	3518 ±3227	13917 ± 10561	0.09 ± 0.02	0.15 ±0.10
	Male	0.083	0.25 ± 0.030*	600 ± 181	171 ± 50	181 ± 50	10438 ± 3407	29107 ± 7663 *	0.12 ± 0.01	0.17 ± 0.02
20	Female	0.083	0.13 ± 0.035	1852 ± 1831	312 ± 309	324 ± 314	2795 ± 1501	16346 10473	0.09 ± 0.01	0.11 ± 0.01
	Male	0.083	0.12 ± 0.010	511 ± 122	142 ± 41	146 ± 41	5904 ± 1054	34847 ± 9054	0.11 ± 0.03	0.13 ± 0.03
55	Female	0.083	0.45 ± 0.28	3108 ± 2676	622 ± 519	691 ± 542	6017 ± 5623	11493 ± 13762	0.20 ± 0.10	0.33 ± 0.15
	Male	0.083	1.0 ± 0.15*	1870 ± 1020	658 ± 349	699 ± 353	11755 ± 4360	8329 ± 3578	0.17 ± 0.10	0.39 ± 0.15

Figure 3.

Plasma concentrations for each oxime following a single 5 mg/kg IV dose of Oximes 15, 20 or 55 to female or male rats. A: plasma concentration curves for female rats; B: plasma concentration curves for male rats. [Male data reprocessed in Phoenix WinNonLin (version 8.3.1), originally reported in (Dail et al., 2019)]. Each point represents the mean ± the standard deviation of n = 3 except for Oxime 15 in males, n = 2.

Brain Oxime Concentrations.

Oxime 15 concentrations in male brains at 30 min were 735.5 ± 703.5 ng/g brain (n=4). However, three of the values were very close and the fourth was much higher; omitting that very high value yielded 390.3 ±165.8 ng/g brain. The value for 2 hr was 742 ±151 ng/g brain (n = 3). Omitting this very high value at 30 min, which does not match the other three, shows an increase in concentration from 30 min to 2 hr. The values for brains, ng/g brain, at 2 hr between males (742 ±151) and females (611 ± 78) were not significantly different. The higher variability with the brain data than the plasma data is probably due to the difficulty of efficient extraction of these lipophilic compounds from a high lipid tissue such as brain.

For Oxime 20 in male rats, there was also an increase in brain concentrations over the 2 hr time course (Figure 4). Brains from female rats sampled at 2 hr displayed similar concentrations (126 ± 18 ng/g) to the males (134 ± 54 ng/g), and there was no significant difference between the sexes.

Figure 4.

Male rat brain concentration data after a single IM 50 mg/kg dose of Oxime 20. Open circles (o) represent the values for each individual animal whereas the x represents the mean value for all the replicates at that time point. Error bars about the mean represent the standard deviation. For times 5 min, 45 min, and 1 hr, n = 2; time 2 hr, n = 3; and time 15 min and 30 min, n = 4.

Three male and three female rats were treated with 50 mg/kg Oxime 55, their brain tissue was extracted, and analyzed at 2 hr post-dose. The levels of oxime were detectable; however, they were below the limits of quantitation (40 ng/g).

As can be seen in Figure 5, the brain to plasma concentration of Oxime 20 increased with time over the first hour, with all points except the initial sampling time of 5 min being above 0.04, indicating brain penetration. There was no significant difference between males and females in this brain to plasma ratio at 2 hours.

Figure 5.

Evidence for brain penetration of Oxime 20 following its IM administration (50 mg/kg). (A) For male rats, the [Oxime 20]brain (ng/g brain)/[Oxime 20]plasma (ng/mL plasma) ratio at each time point increases as a function of time and is >0.04 (indicated by horizontal dashed line) except at the first sampling time. (B) [Oxime 20]brain (ng/g brain)/[Oxime 20]plasma (ng/mL plasma) ratio for male and female rats at 2 hours after administration. Data in (A) represents the mean ratio at each time point, and data in (B) represents the mean ± SD (n=3 rats per time point). ns, not significant (Student’s t-test).

Rat Liver Metabolism of Oximes.

Oximes 20 and 55 were metabolized in an NADPH-dependent manner more extensively than Oxime 15 (Table 3). Some slight variations between the sexes were noted. For example, Oxime 55 was metabolized more rapidly in females, whereas Oxime 20 was metabolized slightly faster in males. However, no statistical differences in the rates of metabolism were noted between the sexes for any of the oximes (Table 3).

Table 3.

NADPH-dependent metabolism of Oximes 15, 20 and 55 by rat liver microsomes of both sexes as assessed by substrate disappearance in 30 min. Data are expressed as the mean of three biological replicates ± the standard deviation.

Oxime	Sex	NADPH	T0 (nmol/g liver)	T30 (nmol/g liver)	% Substrate Loss
15	Female	−	11.7 ± 2.8	10.4 ± 3.5	11
		+	10.2 ± 1.8	7.8 ± 0.7a,b	24
	Male	−	12.5 ± 2.4	11.3 ± 2.8	12
		+	10.9 ± 2.5	9.1 ± 1.7	17
20	Female	−	8.9 ± 1.3	8.8 ± 1.2	1
		+	8.8 ± 1.0	2.1 ± 0.9a,b	76
	Male	−	11.1 ± 4.1	12.8 ± 1.7	−15
		+	13.8 ± 3.3	3.0 ± 2.1a,b	78
55	Female	−	10.2 ± 1.0	9.0 ± 1.1	12
		+	10.5 ± 1.2	1.9 ± 0.2a,b	82
	Male	−	20.5 ± 9.9	17.7 ± 4.3	14
		+	14.6 ± 5.1	4.7 ± 1.8a,b	68

^ap<0.05 relative to T₀ (nmol/g liver) with supplemented NADPH;

^bp<0.05 relative to T₃₀ (nmol/g liver) without supplemented NADPH.

Human Liver Metabolism of Oximes.

In contrast to the trends found in rat liver microsomes, human liver microsomes metabolized Oxime 15 in an NADPH-dependent manner nearly two times more effectively than Oxime 20 (Table 4). Moreover, human liver microsomes were found to metabolize Oxime 20 about 4 times more slowly than rat liver microsomes. Because Oxime 55 was not being considered for further development, its metabolism by human microsomes was not studied.

Table 4.

NADPH-dependent metabolism of Oximes 15 and 20 by pooled human liver microsomes as assessed by substrate disappearance in 30 min. Microsomal metabolism was assayed in duplicate on two different days and the data were pooled together. Data are expressed as the mean ± the standard deviation.

Oxime	NADPH	T0 (nmol/mg protein)	T30 (nmol/mg protein)	% Substrate Loss
15	−	4.9 ± 0.3	4.8 ± 0.2	2
	+	4.6 ± 0.5	3.1 ± 0.2	33
20	−	6.0 ± 0.5	6.4 ± 0.5	−7
	+	6.5 ± 0.4	5.3 ± 0.1	18

Discussion

The pharmacokinetic parameters determined in this study following IM administration of the novel oximes in female rats were generally similar to those previously reported in male rats. Oximes 20 and 15 reached plasma C_max’s greater than 1.2 µg/mL in 30 min or less following IM administration, with plasma half-lives greater than 2 hr (Dail et al., 2019). There was less than a 170 ng/mL difference between the female and male C_max’s for each of the oximes. There were no statistically significant differences observed in both T_1/2 and T_max between the sexes. Therefore, we do not expect sex differences in pharmacokinetics from these oximes in future in vivo tests following this route of exposure.

IV administration of the oximes showed considerably shorter T_1/2’s than IM administration, as was expected because it is likely that the oximes formed a transient depot in the muscle and entered the blood stream from this depot over a short period of time. IV administration showed a greater variability between the sexes for T_1/2, T_max, and C_max. Whereas Oximes 15 and 55 displayed significant differences in T_1/2 values (both compounds were more rapidly cleared in females than males), Oxime 20 did not exhibit this sex difference. In addition, T_max and C_max values showed no difference between the sexes for any of the oximes. As shown in Figure 4, females showed a higher degree of variability among the biological replicates when compared to males, probably leading to differences for Oximes 15 and 55 in the T_1/2. However, since treatment of the female rats was not performed at a specific stage of the estrus cycle, metabolic efficiency and blood lipids and proteins could have been influenced by different levels of sex hormones. Because these novel oximes are quite lipophilic (Chambers et al., 2016a), their retention in the plasma could be influenced by potential reversible binding to proteins or lipoproteins in the blood to which they might bind during transport. In addition, the experiments on males and females were performed in different laboratories and using different personnel and sources of animals, so some differences between the sexes are not surprising. Regardless, the overall shapes of the curves indicate that the pharmacokinetic trends are similar between sexes. This suggests that if a sex-based difference in PK behavior following IV administration exists, it is probably negligible, and probably of less practical relevance since the anticipated route of administration is IM for initial mass casualty use. Finally, from the IM and IV plasma curves, the terminal elimination rate of Oxime 55 was slower than those of Oximes 15 and 20, suggesting that Oxime 55 is either more slowly metabolized, excreted, or more distributed into peripheral tissues than the other oximes. As the most lipophilic of the three oximes tested, it may have been bound to plasma proteins to a greater extent than the other two and therefore was retained in the blood longer.

In the event of an exposure to an OP, current therapy uses an oxime to reactivate inhibited AChE. The United States employs the oxime reactivator 2-PAM, whereas asoxime (HI-6) and obidoxime are approved for use in the European Union (EU). IM injections in rat models showed C_max values of 14.5 µg/mL for 40 mg/kg 2-PAM (Green et al., 1986), 0.9 µg/mL for 50 mg/kg asoxime (Cassel and Fosbraey, 1996), and 26 µg/mL for 50 mg/kg obidoxime. The pharmacokinetic data presented here and earlier (Dail et al., 2019) show that our oximes reach C_max values (2.9–3.0 and 2.4–2.5 µg/mL for Oximes 15 and 20, respectively) higher than those of asoxime, but lower than those for 2-PAM and obidoxime. These pharmacokinetic data taken in consideration with earlier reports from our group showing that our compounds increase survival in rats challenged with lethal levels of the highly relevant sarin surrogate NIMP in comparison to 2-PAM and that Oxime 20 attenuates signs of cholinergic toxicity more rapidly than 2-PAM (Garcia et al., 2020) suggest that continued development of Oxime 20 has merit. We understand, of course, that humans and rats differ in pharmacokinetics of drugs and that the current animal experiments may not reflect the half-lives and other parameters that would be observed in humans.

The oximes approved to treat OP exposure in the US and EU (2-PAM, asoxime, obidoxime) have relatively short plasma T_1/2s that are less than 90 min, 2-PAM at 8, 40 or 77 min (Houze et zl., 2010; Lorke and Petroianu, 2019; Jokanović, 2015), asoxime at 48 min (Cassel and Fosbraey, 1996), and obidoxime at 35 min (Alioth-Streichenberg et al., 1991). The administration guidelines for the use of these compounds vary on the initial dose to maintain therapeutic efficacy. For example, in adults the initial recommended doses of 2-PAM are 1 to 2 g either IM or IV whereas with asoxime and obidoxime the recommendations are 0.5 g and 0.25 g either IM or IV, respectively (EMEA / CPMP Guidance Document on the Use of Medicinal Products for the Treatment of Patients Exposed to Terrorist Attacks with Chemical Agents, 2003). The high initial doses and the recommended repeat administration of 2-PAM, asoxime and obidoxime are likely a result of their short plasma half-lives. By comparison, our compounds have much longer plasma half-lives, T_1/2 2.5–3.0 and 5.3–5.4 hr for Oximes 15 and 20, respectively, for IM administration. The presence of Oxime 20 in the blood for several hours probably contributed to the greater efficacy that it displayed compared to 2-PAM in promoting 24 hr survival of rats of both sexes to lethal levels of a sarin surrogate (Chambers et al., 2016b; Garcia et al., 2020). This longer half-life feasibly could result in dosing regimens that would require less compound and/or fewer administrations to provide the same level of therapeutic efficacy. If so, one of these novel oximes could result in better patient outcome post-OP challenge with less likelihood of adverse side effects or drug-drug interactions.

Throughout this study we chose to emphasize plasma pharmacokinetics of IM administration over IV because IM is the most likely practical route of antidote administration in mass casualty scenarios. The results from our experiments in this study and in our prior study with males showed that 1) our compounds were able to enter the blood quickly as shown by detection at our earliest sampling, 5 min, 2) reach comparable plasma concentrations to oximes currently utilized in OP treatment, and 3) our oximes have a much longer IM T_1/2.

One of the major unmet needs in current OP treatment is the ability to reactivate AChE within the central nervous system. To date, our group has been able to show functional in vivo evidence for brain penetration of Oxime 20 through the blood brain barrier by the earlier cessation of OP-induced seizure-like behavior and neuroprotection as evidenced immunohistochemically by the preservation of neurons (NeuN) and little evidence of glial scarring (GFAP); in addition these oximes show efficacy in increasing survival post-OP challenge in rats (Chambers et al., 2016b; Garcia et al., 2020; Pringle et al., 2018). These studies used the sarin surrogate nitrophenyl isopropyl methylphosphonate (NIMP) at a dosage that was lethal with only a single administration of atropine as a therapy. In this current study, we were able to develop a method of extraction of our compounds from brain tissue based on previously reported methods for lipophilic compounds (Bligh and Dyer, 1959; Folch et al., 1957). Method development for brain tissue was carefully chosen to account for the lipophilic nature of our compounds and the tissue itself to allow for accurate data collection. We were able to show that Oximes 15 and 20 are quantifiable in the brain, and our lead compound, Oxime 20, displayed a general increase in brain concentration over the first hour with an apparent plateau between 1 and 2 hours. While these data from brain are more variable than those from plasma, this variability is not unexpected because of the difficulty in extracting lipophilic compounds from a very high lipid-containing tissue such as brain. This difficulty might be the reason that we did not quantify Oxime 55, the most lipophilic of the three oximes, in the brain. These data are consistent with our previous studies on seizure cessation with NIMP, a highly relevant sarin surrogate (Chambers et al., 2016b; Garcia et al., 2020). These pharmacokinetic data represent our first information about actual accumulation of our lead oxime, Oxime 20, in the brain during the first 2 hours following IM administration. A molecule is deemed to be “brain penetrant” if its brain-to-plasma concentration ratio is >0.04 using nonperfused brain tissue, as cerebral blood volume approximates 4% of total brain volume (Shaffer et al., 2010). Except for the initial 5 minute sample, the brain to plasma ratios all exceed 0.04, providing evidence that Oxime 20 does accumulate in the brain, and provides additional support for our functional data that Oxime 20 does enter the brain and likely is providing therapy within the brain.

The comparison of pharmacokinetic data between male and female rats indicated very few significant differences. Part of the reason for the differences may have been the fact that the male data were generated by SRI International and the female data were generated by our group, with the source of the animals being different vendors. Nevertheless, there were few differences observed that indicate that males and females are processing our oximes differently and no differences in a dosing regimen would be needed.

As a final addition to this study, we examined the metabolism of these oximes in rat liver microsomes from both sexes and in human liver microsomes. Oxime 15 was the least metabolized by rat liver microsomes and Oxime 20 was metabolized most, whereas in human liver microsomes the trend was the opposite. The difference in the metabolism is likely from the differences in the hepatic expression of individual cytochrome P450 (CYP) isoforms in rats and humans. Because our oximes exhibited a relatively long T_1/2 in vivo, their pronounced metabolism by rat liver microsomes was an unexpected finding, although the compounds (particularly Oxime 20) were more stable in NADPH-fortified human liver microsomes than rat liver microsomes, which suggests that the AUC and/or T_1/2 might be even higher in humans than what we observed in rats. A previous report on the anaerobic metabolism of aryl aldoximes by phenobarbital-induced rat liver microsomes supplemented with NADPH demonstrated a unique CYP-catalyzed dehydration reaction that yielded the corresponding nitrile products of the aldoximes (Boucher et al., 1994). Further, the stereochemistry of the aldoxime C=N bond (i.e., the Z- and E-isomers) was an important determinant for this biotransformation pathway. However, the microsomal incubations in our study were not anaerobic, and several sites on our novel pyridinium aldoximes exist that could be attacked by a CYP-activated oxygen atom. More work will be necessary to characterize the CYP-catalyzed metabolites formed under aerobic conditions. One possibility is that the aldoxime moiety is enzymatically reduced in an NADPH-dependent way to give the aldimine, which is hydrolyzed to an aldehyde and then oxidized to the corresponding carboxylic acid (Heberling et al., 2006). At the time that this study was conducted, our laboratories did not have the capability to identify the nature of the metabolites, but identification of metabolites is under current investigation. However, the lack of identification of the metabolites does not detract from our ability to evaluate the compounds for oxime stability, safety and tolerability.

In summary, our novel oximes were administered to female rats and their pharmacokinetics, brain levels in male and female rats, and preliminary metabolic stability trends were determined. The results combined with our previously published data indicate that there is little to no difference between female and male rats in the pharmacokinetics, brain accumulation, and metabolism of these compounds. In addition, a species-dependent difference in the metabolism of the oximes was also observed, which may be necessary to consider for their further development in humans. Overall, the data reported here combined with the efficacy data obtained earlier indicate that our novel oximes, particularly our lead Oxime 20, have potential promise as therapeutics against OP poisoning and they warrant further development.