Intramuscular atenolol and levetiracetam reduces mortality in a rat model of paraoxon-induced status epilepticus

Abstract

Organophosphorus (OP) compounds are chemical threat agents and are irreversible inhibitors of the enzyme acetylcholinesterase that lead to a hypercholinergic response that could include status epilepticus (SE). SE particularly targets the heart and the brain and despite existing therapies it is still associated with significant mortality and morbidity. Here, we investigated the effect of intramuscular (i.m.) adjunct therapy consisting of atenolol (AT) and levetiracetam (LV) when administered after paraoxon (POX)-induced SE. The combination therapy was administered twice daily for 2, 7, or 14 days. POX exposure in rats produced rapid SE onset that was treated with atropine, pralidoxime chloride, and midazolam. Here, AT + LV therapy produced significant reductions in POX SE mortality assessed at 30 days post SE. AT + LV therapy exhibited muscle pathology inflammation scores that were not significantly different from saline-treated controls. Pharmacokinetic analyses revealed that the i.m. route achieved faster and stabler plasma therapeutic levels for both AT and LV under OP SE conditions compared with oral administrations. Our data provides evidence of the safety and efficacy of i.m. AT + LV therapy for reducing mortality following POX SE.

Graphical abstract

Organophosphorus (OP) compounds are irreversible inhibitors of the enzyme acetylcholinesterase that lead to a hypercholinergic response that includes status epilepticus (SE), which targets the heart and the brain and is associated with significant mortality and morbidity. Here, we investigated the effect of intramuscular adjunct therapy consisting of the β-adrenergic blocker atenolol and the antiepileptic drug levetiracetam when administered after SE induced by the OP compound paraoxon in a rat model.

Introduction:

Organophosphorus (OP) compounds are regarded as highly toxic compounds and include commonly used pesticides and nerve agents¹. Exposure to OP could occur occupationally, accidentally, intentionally, or through terrorism, and both military and civilian populations have been exposed to OP agents under these scenarios^2–6. Their relative ease of availability and high toxicity has resulted in increasing use of OP agents in recent geopolitical conflicts and has added to the urgency for developing effective countermeasures for OP exposures⁷.

OP compounds are potent inhibitors of the enzyme acetylcholinesterase (AChE) and consequently cause rapid accumulation of the neurotransmitter acetylcholine (ACh) at synapses. Dependent on the dose, symptoms of OP exposure rapidly progress from fluid secretions and muscle tremors into status epilepticus (SE), followed by respiratory depression, change in heart rate, and death that can occur within minutes following lethal OP exposure⁸. OP poisoning management follows the FDA-recommended 3-drug approach consisting of atropine, pralidoxime chloride (2-PAM), and midazolam, depending on the severity of symptoms^{9, 10}. It is important to stress here that not all OP exposures could lead to SE¹¹. Clinical data show that development of overt seizures is also dependent on the type of OP. Studies of OP pesticide poisonings have reported incidences of seizures ranging from 0 to 25%^12–15. Another factor that affects seizure presentation following OP poisoning is the age of the patient, with younger patients being 10-fold more susceptible to development of seizures than the adult population^16–18. However, when SE occurs after OP poisoning it is associated with a high mortality and morbidity and thus is an important area to address.

OP-induced SE causes mortality and morbidity in survivors of the initial OP intoxication. Rat models of OP exposures often produce SE and exhibit neuronal damage, benzodiazepine resistance, and neurological comorbidities that are seen in survivors of OP exposures^19–23. These rodent models have uncovered novel mechanisms for the development of comorbidities, including alterations in neuronal calcium (Ca²⁺) handling^{24, 25}, oxidative stress²⁶, neuroinflammation²⁷, cyclooxygenase signaling,²⁸ and the involvement of nitric oxide pathways²⁹. Current research approaches for countermeasures against OP exposures include development and optimization of the above outlined three-drug strategy and adjunct treatments for lowering OP-induced comorbidities^{7, 30}. In addition, there remains the need for effective therapeutics that could be administered outside of an acute care setting where treatment could be delayed following a mass exposure scenario³¹. Despite these important discoveries and advances, the issue of OP SE mortality has not been adequately addressed.

Research using traditional chemoconvulsant models of SE have indicated involvement of increased sympathetic activity following SE³². Such an autonomic overload has been implicated in altering cardiac electrophysiology and myocardial damage following SE³³. Prophylactic treatment with a beta (β)-adrenergic blocker was demonstrated to be cardioprotective during kainite-induced SE³⁴. Subsequently, coadministration of atenolol along with diazepam following induction of kainic acid SE was shown to reduce the risk of aconitine-induced arrhythmias³⁵. Experimentally, β-adrenergic blockade has proved to be a significant intervention for preventing cardiac dysfunction following SE³⁶. Clinically, alterations in cardiac function have an important role in determining SE outcome, and cardiac decompensation is a leading factor in predicting mortality in SE patients³⁷. Consequently, there are reports of improved cardiac function following atenolol (AT) administration in ischemic patients^{38, 39}. However, it remains to be seen whether β-adrenergic blockade administered following a delay of treatment to mimic the treatment scenario in a mass casualty OP exposure event could confer cardioprotection and aid in lowering high OP SE mortality.

The antiepileptic drug levetiracetam (LEV) is a synaptic vesicle (SV) protein 2A inhibitor⁴⁰ and is reported to inhibit excitatory glutamate transmission in the brain⁴¹ and also inhibit synaptic glutamate machinery in rats with seizures⁴². Studies have also shown that LEV inhibits Ca²⁺-induced Ca²⁺-release (CICR) mechanisms in neurons^{43, 44}. Using a rat model of OP paraoxon (POX) intoxication²¹, we have shown development of neuronal injury, a hippocampal neuronal Ca²⁺ plateau, and occurrence of mood and memory dysfunction in rats that survive POX SE²⁴. The sustained Ca²⁺ increases in these rats had their origins in CICR mechanisms such that CICR inhibitors lowered Ca²⁺ levels and afforded significant neuroprotection when administered 60 min after POX SE^{24, 25, 45}. Interestingly, LEV, when combined with the anticholinergic drug procyclidine along with the oxime HI-6, significantly lowered immediate mortality and neuropathology when administered within 24 min of soman exposure^{46, 47}. In addition, imbalances in autonomic cardiovascular function in epilepsy patients were reported,^48,49 and LV has been successfully used to manage cardiac manifestations in epilepsy⁵⁰. Given these effects, LV could also play a role in modulating the central control of sympathetic function.

In this study, we tested the hypothesis that combined adjunct treatment with AT and LV would target both the cardiac and brain vulnerabilities and significantly lower the mortality associated with lethal OP SE. Given the necessity for ease of administration during a mass casualty scenario, we also tested the safety and availability of AT + LV administration following intramuscular (i.m.) treatment. Our studies show that i.m. AT + LV produced a significant reduction in the POX SE mortality, exhibited favorable pharmacokinetic profiles, and was associated with minimal muscle damage at the injection sites.

Materials and methods

Drugs and chemicals

All chemicals were obtained from Millipore-Sigma (St. Louis, MO) unless otherwise noted. POX is a yellow oily liquid. To prepare a working solution, the desired quantity of POX was removed using a Hamilton syringe and added to a glass bottle containing ice-cold phosphate buffered saline (1× PBS). The bottle was gently vortexed till the POX oil droplets were thoroughly mixed in PBS and no oily layer was visible on the PBS surface. The bottle was kept on ice, and syringes were drawn and kept on ice until the time of subcutaneous (s.c.) injections. Time on ice (between diluting in saline to injections) was never more than 10 min. Atenolol, atropine sulfate, and pralidoxime chloride (2-PAM) were dissolved in saline (0.9% NaCl) and sterile filtered. Midazolam and levetiracetam in injectable form were obtained from the VCU Health System Pharmacy. All the drugs were prepared fresh on the day of experiments.

Animals

All animal use procedures were in strict accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and approved by Virginia Commonwealth University’s Institutional Animal Care and Use Committee. Male Sprague-Dawley rats were obtained from Envigo (Indianapolis, IN) at 9 weeks of age. Animals were housed two per cage at 20–22 °C with a 12 h light: 12 h dark cycle (lights on 06:00–18:00 h) and free access to food and water.

Electrode implantation and seizure monitoring

Rats were stereotaxically implanted with three skull surface electrode screws attached to Teflon-insulated stainless steel MedWire® (Plastics One, Roanoke, VA, USA.) under general anesthesia with isoflurane/O₂ (5% induction; 2.5% maintenance). Electrode screws were positioned through burr holes above the right and left frontal and motor cortices (AP, ±3 mm and ML, ±3 mm from bregma); the third surface electrode screw was positioned over the cerebellum to serve as reference, and two additional (non-electrode) skull screws were inserted for structural support. The electrode screws were seated to contact, but not penetrate the dura mater. Female amphenol terminal pins connected to the electrode wire were seated into an electrode pedestal (Plastics One, Roanoke, VA) and this assembly was secured to the skull with Cerebond™ adhesive (Plastics One, Roanoke, VA). Rats were allowed 1 week of recovery time before the start of the experiment. Wire leads were securely connected into the threaded electrode pedestal on the rat and then connected to an electrical swivel commutator (Plastics One, Roanoke, VA) to allow for free movement of the rat while maintaining continuity of EEG signals. EEG signals were amplified using a Grass model 8–10D (Grass Technologies, West Warwick, RI) and digitized using a Powerlab 16/30 data acquisition system (AD Instruments, Colorado Springs, CO). Evaluation of digitally acquired EEG was performed with Labchart (AD Instruments, Colorado Springs, CO)

Experimental design

Initially we used n = 6 rats (10 weeks of age) to demonstrate induction of SE following POX (4 mg/kg, s.c.) and immediate prophylactic treatment with atropine (0.5 mg/kg, i.m.) along with 2-PAM (25 mg/kg, i.m.), and termination of ensuing SE using midazolam (1.78 mg/kg, i.m.). These rat doses were selected using human equivalents calculated using a human-to-rat dose translation equation^{51, 52}. Upon confirmation that this dose of POX induces SE and successfully responds to midazolam treatment, in the second part, separate cohorts of rats were administered POX. Only those rats that underwent 1 h of SE and received midazolam were considered for this study. Such rats were randomized into four groups: those receiving either saline (SAL) or AT + LV therapy for the three treatment durations (2, 7, or 14 days). This design was repeated two more times. At the end of these three runs, each AT-LV group cumulatively had n = 15 rats and n = 21 rats receiving SAL (see Table S1 (online only) for sample size/group). These rats were followed for 30 days and the mortality rate was calculated. The data was then pooled together for mortality assessments.

For the pharmacokinetic studies, a separate cohort of rats were ordered, and blood was collected and stored in duplicate EDTA microcuvettes following SAL and AT + LV treatment under control and OP SE conditions following p.o. and i.m. routes of administration (see Table S1 for sample size/group). Blood samples were analyzed for drug levels at each of the 10 timepoints as described below.

For muscle pathology studies, to conserve animal numbers, surviving rats from these groups were euthanized and bilateral quadriceps isolated at various time points (1 day, 2 months, and 4 months; see Table S1 for sample size/group) and assessed for inflammation as described below.

Adjunct drug therapy

Rats surviving 1 h of POX (4 mg/kg, s.c.) SE were randomly assigned to receive either saline or pharmacological countermeasure therapy. A drug regimen consisting of AT (5 mg/kg, i.m.) and LV (50 mg/kg, i.m.) was instituted. The dose of AT was selected based on previous rat studies that demonstrated efficacy of this dose to reduce cardiac pathology and abnormal electrophysiology following chemoconvulsant-induced SE³⁴. The dose of LV was selected based on our previous studies that demonstrated effectiveness of this dose in lowering POX SE–associated hippocampal Ca²⁺ elevations²¹. This dose selection also based on a human-to-rat dose translation equation^{51, 52}. For i.m. administration, AT and LV were administered in the quadriceps muscle using a 28-gauge needle. The injection volume was 0.1 mL. Injection sites were alternated between the lateral and contralateral sides for the twice daily dosing protocol. Manifestations of distress and discomfort (such as limping, licking the injection site, and other ambulation-related issues) were subjectively graded as: none, mild, moderate, and severe. Mild discomfort was noted around days 6 and 7 of i.m. injections that quickly resolved without any intervention. No ambulatory deficits were observed in these rats. Rats in the 14-day treatment group exhibited moderate discomfort immediately after i.m. injection around day 9 onwards. However, rats resumed normal activity shortly thereafter and no ambulatory deficits were noted. Muscle pathology was noted as described below.

The first dose combination was administered along with midazolam at the end of 1 h SE. The second dose was administered 8 h after SE. After this, the drugs (AT, LV) were administered twice daily 12 h apart for 2 days or 7 days or 14 days post SE. The control group was given i.m. saline injections for the same duration and frequency. Figure 1 provides a schematic representation of the POX SE and drug treatment protocol.

Figure 1.

Timeline for induction of POX SE and pharmacological interventions after SE At time 0, male Sprague-Dawley rats were injected with the OP agent POX. One minute later atropine sulfate and 2-PAM were injected i.m. to prevent immediate death and allow progression into SE (~10 min). From the first appearance of Stage 4 SE, rats were allowed to seize for 1 h, at which point midazolam was injected to stop the SE. Surviving rats were injected with saline or test countermeasures (AT + LV) for 2 days, 7 days, or 14 days. Mortality was assessed by counting deaths up to a 30-day period following the termination of POX SE.

Assessment of mortality

Mortality assessment was carried by calculating the percent of the animals that died in each of the treatment groups in the 30-day observation period after the termination of POX SE and was identified from our human study on mortality and clinical presentations of SE⁵³. Similar population-based studies reported that death rates peak within 30 days after SE^{54, 55}. Acute mortality was measured during SE and every 3 h for the first 24 h after SE. Chronic mortality was evaluated every 24 h until 30 days post-SE.

Pharmacokinetic analyses

For the pharmacokinetics study, a fresh cohort of rats was used. Rats were dosed once with AT + LV and blood samples were obtained in duplicate from 3 unique rats at each of the 10 time points for the two routes of administration (i.m. and p.o.) following saline or POX administration. Following anesthesia, rats were euthanized and blood samples were collected through the cardiac ventricles at 0 and 30 min and at 1, 2, 3, 4, 6, 8, 12, and 24 h following drug administration. The blood was placed in an EDTA microcuvette and frozen at −80 °C until analyzed. Drug levels were measured in whole blood using liquid chromatography tandem mass spectrometry (LC-MS/MS). The linear range was 1–2000 ng/mL for AT and 5–10,000 ng/mL for LV.

Sample preparation

50 μL of rat whole blood was analyzed for AT and LV levels using a protein precipitation extraction. Briefly, each aliquot was transferred to a 1.5 mL microcentrifuge tube and 500 μL of extraction solvent consisting of 70% of methanol and 30% of water was added following addition of 50 μL of internal standard solution (AT day 7 and LV day 6 at a concentration of 500 ng/mL). Sample tubes were vortexed for 1 min and sonicated for 30 min. Samples were then centrifuged for 10 min at 13.2 × g and transferred to an ambler vial. AT and LV were analyzed using LC-MS/MS with multiple reaction monitoring (MRM) transitions on an AB Sciex 6500+ Qtrap (Sciex, USA) with LC-20-AD (Shimadzu, USA) UPLC system. Chromatographic separation was performed on a Zorbax Eclipse Plus C18 Column (2.1 × 100 mm, I.D. 3.5 μm, Agilent Technologies, USA). The column temperature was ambient; the flow rate was 1.0 mL/min, and the injection volume was 1 μL. The mobile phase A consisted of 0.2% formic acid in water and mobile phase B in methanol. Isocratic conditions were used at 95% acetonitrile and 5% 0.2% formic acid in reagent water. Data were analyzed with Analyst 1.6 Quantitation Wizard in accordance to the Bioanalytical Laboratories Standard Operating Procedures.

Assessment of muscle pathology

To conserve animal number, we used survivors from POX SE mortality groups for muscle pathology. To evaluate the safety of i.m. drug administrations, muscle specimens were obtained at 1 day, 2 months, and 4 months after the last injection of the 7-day treatment regimen. These time points were selected to assess whether our combination treatment regimen caused any muscle injury at the site of injection either acutely, subchronically, or chronically, respectively. Rats were euthanized and a wide surgical exposure of the muscles was performed, and the quadriceps muscles from each injection area were dissected free and placed in 10% neutral buffered formalin. These muscle samples were embedded in paraffin and 5-micron thick sections were obtained and stained with H & E or trichrome for histopathology. A standard four-point inflammation scale was used for scoring⁵⁶ (Table 1) and the score for each site was an average of the score for each of the three tissue blocks.

Table 1.

Inflammation scale for assessment of injection site muscle pathology

Score	Fibrosis	Infiltrates
0 = not present	0	None present
1 = minimal	<1 mm	Few, widely scattered infiltrates
2 = mild	1 to 1.9 mm	Scattered infiltrates, minimal small clusters
3 = moderate	2 to 3.9 mm	Loosely packed fields, many small clusters
4 = marked	>4 mm	Dense infiltrates, often packed fields

Data analysis

Data analysis and generation of graphs was accomplished using SigmaPlot 14.0 software. The mortality and muscle pathology data are expressed as mean ± SEM. The significance of the differences in the outcomes following drug treatment compared to saline controls was tested using a one-way analysis of variance (ANOVA) followed by a post-hoc Tukey test with significance set at P < 0.05. For mortality data, we also applied Fisher’s exact test to determine the significance of comparisons among all the groups. Model-independent methods were used to estimate the pharmacokinetic parameters of AT and LV in rat blood samples. The area under the curve (AUC) was determined utilizing the parameters programmed in Microsoft Excel. AUC from time zero to the last sampling time was calculated using the linear trapezoidal rule. The AUC from the last sampling time was extrapolated to infinity by dividing the last measured plasma concentration by the terminal elimination rate constant. C_max and t_max were obtained directly from the concentration–time curves. The AUC data are expressed as mean ± SD and compared using an ANOVA followed by a post-hoc Tukey test with a significance set at P < 0.05.

Results

Induction and termination of POX SE

One minute after POX (4 mg/kg, s.c.) exposure, rats (n = 6) implanted with EEG electrodes were injected with atropine sulfate (0.5 mg/kg, i.m.) and 2-PAM (25 mg/kg, i.m.). Rats displayed hypercholinergic symptoms, including Racine 4–5 seizures within 7–10 minutes, that evolved into SE. The tonic-clonic activity was paralleled by rhythmic epileptiform activity with a spike frequency in 7–10 Hz. Rats were treated with midazolam (1.78 mg/kg, i.m.) at 1 h following the onset of SE (Fig. 2A). A single midazolam injection successfully stopped EEG epileptiform discharges, and SE-like activity stayed suppressed over the next 48-h period (Fig. 2B).

Figure 2.

Induction and termination of POX SE. (A) Representative continuous EEG recording from a rat before, during, and after POX administration. Baseline EEG activity was noted before POX exposure (4 mg/kg, s.c.). One minute later, rats were treated with atropine sulfate (0.5 mg/kg, i.m.) and 2-PAM (25 mg/kg. i.m.). SE activity is characterized by high frequency spiking minutes after POX administration. SE was robust, intense and did not wax and wane for the entire 1 h. SE was stopped at 1 h following administration of midazolam (MDZ, 1.78 mg/kg, i.m.). (B) SE remained suppressed and did not reoccur over the next 48 h period following MDZ. Each trace represents a continuous 30-min EEG recording obtained during the morning period at the respective time point. Traces are representative of six animals.

Time course for mortality following POX SE

We conducted a retrospective analysis of our previously acquired POX SE data to establish a time course of mortality following termination of POX SE. In these studies, a total of 50 rats (male, Sprague-Dawley rats, 10 weeks of age, 290–320 g) had received POX (4 mg/kg, s.c.), 45 rats had survived 1 h of SE and received midazolam treatment (1.78 mg/kg, i.m.). We calculated the number of deaths at various time points leading up to 30 days after the termination of POX SE. As shown in Figure 2, 3/45 rats (6.6%) succumbed to post-SE complications on Day 1. The number of deaths on subsequent days of observations were as follows: Day2: 5 rats (11.1%); Day3: 4 rats (8.8%); Day5: 3 rats (6.6%); and Day7: 1 rat (2.2%). The mortality rate dramatically tapered off following this period and no further deaths occurred beyond the two-week period. The cumulative POX SE mortality was 16/45 (35.5%), with most of the deaths occurring within the first week after POX SE (Fig. 3).

Figure 3.

Time-course for mortality following termination of POX SE. Mortality over a continuous 30-day period following termination of POX SE was retrospectively calculated (n = 50). Of the 46 rats that survived 1 h of POX SE and received midazolam, 4/46 rats succumbed to post-SE complications on Day 1. Additional new deaths on subsequent days after POX SE were as follows: Day2: 5; Day3: 3; Day5: 2; and on Day7: 1 rat was dead. The mortality rate dramatically tapered off around the second week and no further deaths occurred beyond the two-week period. The cumulative mortality (15/46) during this period was around 35%, with most of the deaths occurring within the first week after POX SE.

Mortality after i.m. treatment with AT + LV in the POX SE model

Based on the above studies, we investigated whether a 2-day, 7-day, or 14-day treatment regimen consisting of twice daily AT (5 mg/kg, i.m.) + LV (50 mg/kg, i.m.) could improve the POX SE mortality outcome. Figure 3 demonstrates that the 2-day AT+LV treatment reduced the 30-day POX SE mortality from approximately 35% down to 19 ± 4%. The 7-day and 14-day AT + LV treatment regimens also reduced POX SE mortality to 11 ± 3% and 13 ± 3%, respectively. One-way ANOVA revealed that there were significant differences between the three durations of AT + LV and SAL treatment. Post-hoc analysis revealed that the 2-day, 7-day, and 14-day AT + LV treatments were significantly different than SAL but not significantly different from each other (n = 45, one-way ANOVA, *P < 0.05, Fig. 4).

Figure 4.

Effect of various durations of AT + LV therapy on POX SE–induced mortality. Following termination of POX SE, AT + LV therapy was instituted for 2 days (black bar), 7 days (red bar), or 14 days (blue bar). Mortality was assessed once daily over the subsequent 30-day period following the termination of POX SE. The data shows average SE mortality for saline conditions across the three treatment groups. All the three treatment durations for AT + LV caused significant reductions in the POX SE mortality compared with saline treatment, but the three treatment durations were not different from each other (n = 45, one-way ANOVA, Tukey test, and chi-square test of independence, *P < 0.05).

We also analyzed our results using a Fisher’s exact test. A chi-square test of independence was performed to examine the relation between SAL and various durations AT + LV treatment on POX SE mortality percentages. The relation between these variables was significant. For the 2-day AT + LV treatment: χ² (2, 3) = 6.4, P = 0 .0400. For the 7-day AT + LV treatment: χ² (2, 3) = 6.5, P = 0.0381, and for the 14-day AT + LV treatment: χ² (2, 3) = 6.8, P = 0.0326. Thus, all three durations of AT+LV treatment were more likely to reduce POX SE mortality than SAL treatment. We then examined if there were differences between AT + LV treatment durations on POX SE mortality. A chi-square test of independence showed that there was no significant effect of the 2-day versus the 7-day AT + LV treatment on POX SE mortality: χ² (2, 3) = 0.26, P = 0.874. Similarly, POX SE mortality percentages did not differ between the 7-day versus the 14-day AT + LV treatment: χ² (2, 3) = 0.01, P = 0.995. Thus, chi-square analysis confirmed the results from the one-way ANOVA that all three AT + LV treatment durations (2 days, 7 days, and 14 days) significantly lowered the POX SE mortality compared with SAL-treatment, but they were not significantly different from each other. Although the 2-day and 7-day/14-day protocols are not statistically significant, the 2-day treatment seems a little bit less effective.

Pharmacokinetic studies after i.m. and p.o. treatment with AT + LV

Blood levels of AT and LV were determined in control and SE rats at various time intervals following i.m. and p.o. dosing. Table 2 provides data on the pharmacokinetics for AT and LV treatment in control rats while Table 3 summarizes these data for SE rats following i.m. and p.o. administration. As shown in Figure 5, higher concentration levels for both AT and LV were rapidly achieved following i.m. administration under SE conditions. One-way ANOVA analysis revealed that for time points ranging between 30–180 min, the i.m. route provided significantly higher drug levels compared with the p.o. route (n = 3 rats/timepoint, one-way ANOVA, post-hoc Tukey test, *P < 0.05, **P < 0.01). Mean value calculations for AT and LV plasma levels in control and OP SE rats are provided in Tables S2 and S3 (online only).

Table 2.

Summary of intramuscular and oral pharmacokinetics for levetiracetam and atenolol in control rats

Levetiracetam (LV)
	AUCtrap [ng/mL*min]	AUC∞ [ng/mL*min]	tmax [min]	t1/2 [min]	Cmax [ng/mL]
i.m.	7762428.75	8208483.94	30.00	181.87	28050.00
p.o.	9970850.00	10586510.38	60.00	251.03	29533.33
Atenolol (AT)
	AUCtrap [ng/mL*min]	AUC∞ [ng/mL*min]	tmax [min]	t1/2 [min]	Cmax [ng/mL]
i.m.	191031.85	454526.74	30.00	107.44	2135.00
p.o.	59415.00	239377.77	120.00	74.73	238.25

Levetiracetam (LV, 50 mg/kg) and atenolol (AT, 5 mg/kg) were administered to control rats separately via intramuscular (i.m.) or oral (p.o.) route. Animals were sacrificed at various time points after injection and blood collected for analysis. Descriptive pharmacokinetic parameters are: C_max, maximal plasma concentration, where plasma concentration is in ng/mL; t_max, time to reach C_max; t_½, terminal half-life; AUC_trap, area under the plasma concentration time curve 0→∞ via trapezoidal method; AUC_∞, area under the plasma concentration time curve extrapolated to infinity.

Table 3.

Summary of intramuscular and oral pharmacokinetics for levetiracetam and atenolol in OP SE rats

Levetiracetam (LV)
	AUCtrap [ng/mL*min]	AUC∞ [ng/mL*min]	tmax [min]	t1/2 [min]	Cmax [ng/mL]
i.m.	3853050	3853050	60.00	96.97	21975
p.o.	1193785	1193785	180.00	76.64	3626
Atenolol (AT)
	AUCtrap [ng/mL*min]	AUC∞ [ng/mL*min]	tmax [min]	t1/2 [min]	Cmax [ng/mL]
i.m.	1131797	1131797	30.00	104.97	8125
p.o.	299531	299531	360.00	181.10	616

Levetiracetam (LV, 50 mg/kg) and atenolol (AT, 5 mg/kg) were administered to OP-SE rats separately via intramuscular (i.m.) or oral (p.o.) route. Animals were sacrificed at various time points after injection and blood collected for analysis. Descriptive pharmacokinetic parameters are C_max, maximal plasma concentration, where plasma concentration is in ng/mL; t_max, time to reach C_max; t_½, terminal half-life; AUC_trap, area under the plasma concentration time curve 0→∞ via trapezoidal method; AUC_∞, area under the plasma concentration time curve extrapolated to infinity.

Figure 5.

Blood levels of AT and LV following p.o. and i.m. administration in POX SE rats. Comparison of the pharmacokinetic profile of (A) AT (5 mg/kg, i.m.) and (B) LV (50 mg/kg, i.m.) with p.o. and i.m. administration. Levels of AT and LV were analyzed using LC-MS/ MS of blood specimens collected from POX SE rats at time points ranging from 0 min to 1440 min (24 h). Each data point represents the mean ± SD of concentration values (ng/mL) from 3 rats. *P < 0.05, **P < 0.01. Tukey post-hoc analysis was used to compare concentrations.

Muscle safety studies after i.m. treatment with AT + LV

As shown in Figure 6, the AT + LV pathological score at 1-day post injection in control rats was not different from the saline control. At 2 and 4 months after injection, the muscle pathology scores for the saline control were also not different from the AT and LV tissue (n = 5, Student’s t-test, P = 0.37). At 1 day, 2 months, or 4 months after injection, no differences were found in SE rats between the pathology inflammation scores for POX + saline and the POX + (AT-LV) group (n = 5, Student’s t-test, P = 0.217). Interestingly, significant differences were observed in inflammation scores at 1 day after injection between saline + saline and POX + saline group and between SAL + (AT-LV) and POX + (AT-LV) groups (n = 5/group, Student’s t-test, #*P < 0.05).

Figure 6.

Acute and chronic muscle pathology following 7 days i.m. therapy. Data present the mean muscle damage score ± SEM for the 7-day therapy protocol when evaluated 1 day, 2 months, and 4 months after injections in the following groups: saline + saline control (white), saline + (AT-LV) (grey), POX + saline control (black), and POX + (AT-LV) (red). In control rats, the saline muscle pathology score at 1 day after injection was 1 ± 0.2 and the AT + LV score was 1.3 ± 0.3. The saline and AT + LV muscle pathology scores at 2 months were 0.66 ± 0.08 and 0.7 ± 0.04, respectively. At 4 months after injection the muscle pathology scores were 0.58 ± 0.04 for saline control and 0.60 ± 0.04 for the AT + LV muscles. AT + LV muscle pathology scores were not different from the saline control. In SE rats, at 1 day after the last injection of the 7-day regimen, the POX + saline control value for mean inflammation score was 2.06 ± 0.37 and the POX + (AT-LV) score was 2.4 ±0.2. The POX + saline and POX + (AT-LV) pathology inflammation scores at 2 months were 0.54 ± 0.04 and 0.62 ± 0.08, respectively. At 4 months after injection the muscle pathology scores were 0.49 ± 0.07 for the POX + saline control and 0.66 ± 0.11 for the POX + (AT-LV) muscles. While, no differences were found between the pathology inflammation scores for POX + saline and the POX + (AT-LV) group (n = 5/group, Student’s t-test, P < 0.05), significant differences were observed in mean inflammation scores acutely at 1 day after 7-day therapy between saline + saline and POX + saline group (n = 5/group, Student’s t-test, *P < 0.05). Similarly, the POX + (AT-LV) treated group exhibited higher muscle inflammation scores compared with the SAL + (AT-LV) group (n = 5/group, Student’s t-test, #P < 0.05).

Discussion

This study assessed whether targeting the cardiac and brain vulnerabilities following the termination of OP SE could afford reductions in mortality following lethal POX intoxication. Our results show that institution of a treatment regimen consisting of i.m.-administered AT and LV during the first-week after POX SE significantly lowered the mortality rates. Our studies further showed that the i.m. route exhibited favorable pharmacokinetic parameters and produced rapid increases in blood drug levels compared with p.o. administration. In addition, the i.m. treatment protocol did not produce overt and prolonged muscle pathology at injection sites, as indicated by lower, comparable inflammation scores between the treatment groups.

Dependent on the dose of OP and availability of emergency medical procedures, OP exposure could lead to SE. If SE is not controlled promptly, significant mortality and morbidity could occur. Indeed, several investigators are exploring mechanisms for development of morbidities following OP SE and therapies for effective treatment of such chronic symptoms^{27, 29, 57, 58}. As far as mortality data is concerned, mortality in animal models of SE induced by chemoconvulsants⁵⁹, OP pesticides²¹, nerve agents⁶⁰ and their surrogates²² approaches 20–30%. These findings were confirmed in our study and further showed that most of the death occurred during the first seven-day period following termination of POX SE.

SE causes cardiac irritability and damage during this time of vulnerability that can result in sudden death by triggering lethal cardiac arrhythmias or cardiac failure^61–72. Studies have indicated chronic increased synaptic activity in the brain^{73, 74} and sympathetic stimulation of the heart and chronic systemic catecholamine release^{32, 37, 62, 75} that can last for almost a week after SE has stopped. This can lead to tachycardia followed by stress related ST-T wave changes on the EKG^{65, 67, 69, 70, 76–78}. Catecholamine released directly from nerve terminals into the myocardium leads to cardiac damage and contraction band necrosis⁷⁹. There is also evidence that LV can decrease this increased synaptic activity^{41, 42, 44, 80, 81} and that AT can stabilize the cardiac irritability after SE^{32, 34, 36, 82}. Indeed, β-adrenergic blockade is reported to decrease SE-induced cardiac pathology and subsequent arrhythmias^{32, 34–36}. In addition, LV can stabilize synaptic activity and decrease neurotransmitter release in the brain^{33,42, 44, 81}, decrease cardiac manifestations of epilepsy⁵⁰, and minimize neuronal damage following experimental SE^{24, 25, 45}. In line with these studies, our data here indicate that a combination therapy consisting of the β-blocker AT and the synaptic vesicle inhibitor LV significantly decreased the mortality rate when administered during the first seven-day period following termination of POX SE. The effect of this combination on cardiac electrophysiology and myocardial pathology are currently being studied in rat models of OP SE in our laboratory.

During a mass casualty scenario, particularly where an OP agent is involved, there could be delays in first responders arriving and treating everyone in a timely fashion. Moreover, outside of an acute care setting a treatment that could be administered quickly is of paramount importance^{30, 83}. With these considerations, our AT + LV treatment was administered one hour after the development of POX SE using a repeated i.m. route of administration. In a real-world scenario, following hospital admission, other routes of drug administration would become available and could be preferred over i.m. administrations. In this study, as a proof of concept, we continued with i.m. regimen to first identify the optimal dose and duration for our treatment combination. Administering the first couple of doses as i.m. to mimic pre-hospital setting interventions and then switching the subsequent dosing to other routes in order to determine the impact on drug availability is something that could be examined in future studies. We conducted studies to compare the pharmacokinetics for AT + LV following p.o. and i.m. administration under both control and SE conditions. Our studies indicated that i.m. route produced greater drug plasma levels much faster than the p.o. route for both AT and LV. The C_max for both AT and LV showed that the i.m. administration was superior to the p.o. route in SE animals. These data (also see supplementary information) showed that i.m. was preferable given that p.o. administration had a lower absorption rate, longer t_max, and high variability. Taken together, these studies indicated that the i.m. route of administration of AT and LV could be used for OP SE treatment.

We also conducted safety studies to investigate the effects of repeated i.m. administration by conducting muscle histology at the injection site⁵⁶. Our studies showed that i.m. treatment regimen with AT + LV was well tolerated by rats and did not produce any overt inflammation or other pathology at the injection sites either immediately or months after the end of the injection period. Although the 2-day and 7-day/14-day protocols are not statistically significant, a 2-day treatment seems a little bit less effective. Further studies aiming at determining if 2 days are really less effective than 7 days or if treatment may be terminated after only 2 days are needed. While there may not be a need for 7-day i.m. administration, and exposed patients could be switched to p.o. routes once admitted to the hospital, there is a possibility that i.m. route could still be preferred in a forward-care or resource-limited setting. These studies establish the safety of such administrations.

In conclusion, our work provides evidence that i.m. AT + LV treatment can significantly reduce mortality from OP SE. The data from this study demonstrate that the i.m. protocol can achieve stable therapeutic blood levels and faster kinetics in comparison with p.o. administration under SE conditions and that the i.m. route is more effective and preferred over the p.o. route of administration. Finally, we showed that the i.m. administration of AT + LV for 7 days did not cause significant muscle pathology acutely or chronically after injection in POX SE animals. Thus, adjunct treatment with AT + LV could provide an effective countermeasure for improving survival outcomes after OP intoxication.

Supplementary Material

Supinfo S1

Table S1. Summary of sample sizes for POX SE outcomes following AT-LV treatment.

Table S2. Mean value calculations for atenolol and levetiracetam plasma levels in naive rats.

Table S3. Mean value calculations for atenolol and levetiracetam plasma levels in OP SE rats.