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Abstract

In vivo antidotal efficacy of new bis- quaternary 2-(hydroxyimino)-N-(pyridin-3yl) acetamide derivatives (HNK series), to counter multiples of lethal doses of nerve agent sarin (GB) and reactivation of acetylcholinesterase (AChE), was evaluated in Swiss albino mice. [Protection index PI; median lethal dose (LD₅₀) of sarin with treatment/LD₅₀ of sarin] was estimated, using 0.05, 0.10, and 0.20 LD₅₀ as treatment doses of all the oximes with atropine against sarin poisoning. Dose-dependent time course study was conducted at 0.2, 0.4 and 0.8 LD₅₀ dose of sarin for estimating maximum AChE inhibition. At optimized time (15 min), in vivo enzyme half inhibition concentration (IC₅₀) was calculated. AChE reactivation efficacy of HNK series and pralidoxime (2-PAM) were determined by plotting shift of log IC₅₀ doses. HNK-102 with atropine showed three fold higher PI compared to 2-PAM. In vivo IC₅₀ of sarin for brain and serum AChE was found to be 0.87 LD₅₀ (139.2 µg/kg) and 0.48 LD₅₀ (77.23 µg/kg), respectively. Treatment with HNK-102 and HNK-111 (equal to their 0.20LD₅₀) significantly reactivated sarin-intoxicated AChE (p < 0.05) at 2× IC₅₀ dose of sarin, compared to 2-PAM. The study revealed that HNK-102 oxime was three times more potent as antidote, for acute sarin poisoning compared to 2-PAM in vivo.

Keywords

Sarin GB oximes HNK oximes AChE nerve agents

Introduction

Organophosphorus (OP) nerve agents (NAs) are the deadly poisonous chemical warfare agents (CWAs) and are being used during different military conflicts and terrorist attacks.¹ Recently, sarin attack in Syria (14th September 2013) made an alarming headline worldwide which destroyed the lives of hundreds of innocent civilians.² In this scenario, one should not deny the use of this kind of CWAs in future on mass scale, for the destruction of lives of civilian or armed forces. Therefore, development of effective medical countermeasures against OP CWAs poisoning is the thrust research area, at present time.

The acute toxicity of OP compounds is due to irreversible inhibition of an enzyme acetylcholinesterase (AChE) which hydrolyzes a key neurotransmitter acetylcholine (ACh).³ The underlying mechanism of toxicity involves irreversible inhibition of enzyme AChE leading to overstimulation of the cholinergic receptor in the synapse resulting in the breakdown of neuromuscular function and finally death of the victim takes place. This phosphorylated enzyme may further undergo dealkylation, resulting in a stable OP-AChE conjugate which virtually dose not undergo spontaneous hydrolytic reactivation. Although spontaneous reactivation of phosphorylated AChE within the body requires days to months (depending upon the nature of NAs) but the early treatment with oxime plays a vital role in AChE reactivation.⁴

Following the acute OP poisoning, the therapeutic regimen available includes anti-muscarinic drug (atropine) as competitive antagonist to ACh at muscarinic receptor, an oxime (AChE reactivator) and an anticonvulsant drug (diazepam) to control seizures and convulsion induced by NAs.^4
–6 At this stage, the use of oxime therapy becomes indispensible to counteract the early peripheral effects of anti-ChE intoxication.⁴ Most of the auto-injectors aid for self and buddy includes one of the oximes like pralidoxime (2-PAM), obidoxime, N,N′-Trimethylene bis (pyridinium-4-aldoxime) dichloride (TMB-4), or 1-(2- hydroxy-iminomethyl 1- pyridinio) 3- (4- carbamoyl-1-pyridino)-2-oxapropane dichloride (HI-6) alone or in combination with atropine.^7,8 But none of the reported single oxime antidote could provide an uniform protection efficacy against all kind of OP NAs. In addition, self-toxicity of oxime is also one of the major limitations in the development of oxime antidotes.⁸ In order to fill this void of potential oxime, the new series of amide conjugated oximes, namely bis-(2-(hydroxyimino)-N-(pyridin-3-yl)acetamide), were evaluated in vivo. It has been reported that these oximes (HNK-102, HNK-106, and HNK-111) significantly reactivated AChE and also offered appreciably more protection compared to that of 2-PAM in terms of survival against di-isopropylphosphorofluridate (DFP) in Swiss mice.⁹ In continuation, in vivo efficacy of these new oximes was evaluated against acute sarin (GB) poisoning in Swiss albino male mice.

Material and methods

Reagents

Sarin (O-isopropylmethylphosphonoflouridate; GB), 1,1′-(ethane-1,2-diyl)bis(3-(2-(hydroxyl-imino)acetamido)pyridinium)dibromide (HNK-102), 1,1′-(hexane-1,6-diyl)bis(3-(2-(hydroxyl-imino) acetamido)pyridinium)dibromide (HNK-106), and 1,1′-(1,4-phenylenebis(methylene)) bis(3-(2-(hydroxyimino) acetamido)pyridinium)dibromide (HNK-111; Figure 1) were synthesized in this establishment with > 98% purity (gas chromatography (GC) and nuclear magnetic resonance spectroscopy (NMR)).¹⁰ Acetylthiocholiniodide (ASChI), 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB), atropine sulfate, sodium chloride, and propylene glycol were procured from Sigma Chemicals Co. (St. Louis, Missouri, USA) and 2-PAM or pralidoxime chloride (I.P.) from Kwality Pharma, India.

Figure 1.

Bis-quaternary 2-(hydroxyimino)-N-(pyridin-3-yl) acetamide derivatives.

All the oximes and sarin were diluted in freshly prepared mixture solution of normal saline (sodium chloride 0.9% in distilled water) and propylene glycol in a ratio of 9:1 v/v, respectively, and used in the entire study as solvent for all injections.¹¹

Animals

Randomly outbred Swiss albino male mice (Animal House, DRDE, Gwalior, India) weighing 25–30 g were used for the study. Four mice were housed in each cage and dust-free steam-autoclaved paddy husk was used as bedding material. The cages were maintained in environmentally controlled room (25 ± 2°C, relative humidity 40–60%). For all the experiment, maximum four mice per dose were used. The replace, reduce and refine (3Rs) of animal’s ethics were strictly followed. The study was approved by Institutional Animal Ethics Committee, a statutory committee constituted by Committee for Purpose of Control and Supervision of Experiments on Animals, Animal Welfare Cell, Ministry of Environment, Forests and Climatic change, Government of India.

Sample preparation

The animals were anesthetized with anesthetic ether I.P. (Narsans Pharma, India). Blood from orbital plexus was collected in sterilized tubes by puncturing the orbital plexus gently and was allowed to clot at 37°C. The blood samples were centrifuged for 10 min at 513g, and 100 µL serum was collected and stored at −80°C until use. Parallel to it, the whole brain of anesthetized animals was dissected out quickly and stored at −80°C until use. At the time of assay, the whole brain tissue was thawed, diluted 1:10 (w/v) in 0.25 M sucrose solution, and homogenized using vertical homogenizer (REMI Motors, India). However, after every 20 s of homogenization, the homogenizer was deepen for 10 s into crushed ice for cooling. The homogenates were twice centrifuged (Model 3–18K, Sigma® Laborzentrifugen, Germany) at 8500g at 4°C for 10 min. The supernatant was decanted and the pellet was diluted in 0.35 M sucrose solution for assay.

For AChE estimation, 2.6 mL phosphate buffer (pH 8.0), 100 µL of DTNB (prepared in phosphate buffer pH 7.0), and 20 µL of test sample (either homogenized brain or serum) were added together. The reaction mixture was then incubated for 3 min at 37°C and then 20 µL of ASChI (prepared in phosphate buffer pH 7.0) was added. The blank was taken by adding the phosphate buffer in the place of substrate. The enzyme activity was read in kinetic mode (UV VIS Spectrophotometer Specord® 200Analytik Jena AG, Germany) at 412 nm up to 4 min. Processed blood and tissue sample were analyzed using modified Ellman method.¹² AChE activity was expressed as micromoles of ASChI hydrolyzed per minute per gram of brain tissue and micromoles of ASChI hydrolyzed per minute per 10 µL of serum.

Experimental procedure

Determination of median lethal dose and protection index

Sarin was injected subcutaneously, at dorsoproximal site of thorax, in the mice. LD₅₀ of sarin was determined following “moving average” method¹³ and expressed as micrograms per kilogram of body weight. Briefly, for LD₅₀ determination, scales of doses were taken in logarithm (increase or decrease by 0.301log) with four animals for each dose, for example, 200 µg/kg dose = 2.301log, 400 µg/kg dose = 2.602, and so on. The LD₅₀ value was then calculated using the formula: log m = log D + df, where m is the LD₅₀, D is the lowest dose, d is the score or number of mice died out of four, and f is the table value against the score from Gad and Weil.¹³ The obtained values were compared with the reported data of sarin by Clement.¹⁴ Determination of protection index (PI) was based on the formula, PI = LD₅₀ of sarin with treatment/LD₅₀ of sarin. In other words, PI indicated that the given treatment protected against how many fold LD₅₀ of sarin. Atropine (10 mg/kg) was injected through i.p. route as common treatment with all oximes. Animals were then treated with oximes within 20 s of post-sarin exposure and evaluated for protection efficacy against acute sarin poisoning. Three treatment doses of HNK-102, HNK-106 and HNK-111 equal to their 0.05, 0.10, and 0.20 of their LD₅₀ (LD₅₀ HNK-102 = 282.8 mg/kg, HNK-106 = 35.35 mg/kg, and HNK-111 = 35.35 mg/kg, intramuscular) were used. However, one dose of 2-PAM (30 mg/kg)^12,15 was used. All oximes were injected intramuscularly. Volume of all the injections was kept between 0.10 and 0.20 mL. PI of each oxime with or without atropine treatment was also determined against sarin poisoning following the same procedure. The animals were observed for mortality up to 24 h. The higher treatment dose which offered best protection (equal to 0.20 LD₅₀ of oxime) was selected for in vivo determination of AChE enzyme inhibition/reactivation studies. Total 232 mice were used in the protection studies.

In vivo determination of time-dependent peak (maximum) AChE inhibition

Sarin was injected subcutaneously at 32, 64, and 128 µg/kg (subcutaneous). Atropine (10 mg/kg, i.p.) was injected 5 min prior to sarin exposure (128 µg/kg, i.p.) to ensure the chances of their survival until being killed. Serum and whole brain were collected at 3.75, 7.5, 15, 30, 60, 120, 240, and 960 min intervals post-sarin exposure. Linear regression of AChE enzyme inhibition induced by sarin was observed at 15 min post exposure. This time period of (15 min) sarin post exposure was used in further AChE enzyme inhibition/reactivation studies.

In vivo determination of half inhibition concentration dose of sarin and its shift for AChE activity

Half inhibition concentration (IC₅₀) value was determined in samples collected from the mice injected with 0.1, 0.2, 0.4, 0.8, and 1.6 LD₅₀ of sarin. IC₅₀ value was then calculated using regression line equation: Y = mx + C. Whole brain and serum samples were collected 15 min postexposure and AChE enzyme inhibition was estimated accordingly. Using log values of IC₅₀ (0.4, 0.8, and 1.6 LD_50, corresponding to 64, 128, and 256 µg/kg, respectively) as determined above, AChE enzyme reactivation was studied using 0.20 LD₅₀ as treatment dose of HNK series of oximes and 30 mg/kg (i.m.) dose of 2-PAM in vivo. The animals of positive control group were injected with sarin and atropine, in the same volume of solvent as used for treated groups.

Statistical analysis

Results are expressed as mean ± standard error of mean. Data were analyzed by one-way analysis of variance followed by Dunnett’s test and Student’s t test. The value of p < 0.05 was considered significant.

Results

Signs of toxicity and lethality

Gross clinical signs and symptoms were observed in the mice following 1.0 LD₅₀ (160 µg/kg) dose of sarin exposure. With sarin exposure, 5 of 16 animals died (31%), when used for determination of LD₅₀. The mice treated with 1.0 LD₅₀ dose of sarin showed bout of convulsions, tremors, seizures, and muscle fasciculation and were culminated in death within 5 min. In survived animals, the symptoms gradually disappeared within 15–20 min; however, severe lethargy persisted for 2–6 h. Treatment with atropine, with or without oxime, could not prevent sarin-induced aforesaid clinical signs of toxicity. The animals did not show noticeable signs of toxicity when exposed to 128 µg/kg of sarin or below.

PI of the oximes in vivo

The animals were poisoned with multiples of LD₅₀ of sarin and treated with atropine and oximes. Treatment with 2-PAM, HNK-102, HNK-106, and HNK-111 with atropine showed PI of 2.49, 6.03, 3.26, and 4.2 (fold increase in LD₅₀), respectively, against sarin poisoning. The protection profile was comparable with 0.05 LD₅₀ and 0.10 LD₅₀ treatment doses of HNK-102, HNK-106, and HNK-111oximes. Among the used oximes (2-PAM, HNK-102, HNK-106, and HNK-111), HNK-102 offered significant increase in protection following dose–response relationship. Table 1 and Figure 2 depict the summary of protection showed by the oximes.

Table 1.

In vivo protection offered by atropine and oximes (2-PAM, HNK-102, HNK-106, and HNK-111) against sarin poisoning in Swiss albino mice.

S. No.	Treatment		LD₅₀ ^b sarin (µg/kg, s.c.) (n = number of mice)	No. of experiments	PI
S. No.	Atropine (mg/kg, i.p.)	Oxime (mg/kg, i.m.)	LD₅₀ ^b sarin (µg/kg, s.c.) (n = number of mice)	No. of experiments	PI
Column	2	3	4	5	6
1	—	—	160.0 (97.2–259.1) (n = 8)	1	1
2	10.00	2-PAM (30.00)	400.0 ± 58.5 (153.9–551.3) (n = 32)	4	2.5
3	10.00	HNK-102 (14.14)	300.0 ± 100 (132.9–1602) (n = 16)	2	1.87
4	10.00	HNK-102 (28.28)	393.1 ± 110.5 (308.7–822.7) (n = 16)	2	2.45
5	10.00	HNK-102 (56.56)	965.6 ± 95.6 (199.6–3000)^a (n = 32)	4	6.03
6	10.00	HNK-106 (1.76)	267.3 ± 15.4 (154.3–411.3) (n = 16)	2	1.67
7	10.00	HNK-106 (3.53)	358.6 ± 41.2 (117.2–877.8) (n = 16)	2	2.24
8	10.00	HNK-106 (7.00)	524.1 ± 41.4 (165.4–1011.5)^a (n = 32)	4	3.27
9	10.00	HNK-111 (1.76)	325.9 ± 74.0 (83.9–1072.0) (n = 16)	2	2.03
10	10.00	HNK-111 (3.53)	437.8 ± 37.8 (278.5–689.7) (n = 16)	2	2.73
11	10.00	HNK-111 (7.00)	682.8 ± 165.7 (166.8–968.7) (n = 32)	4	4.26

PI: protection index; LD₅₀: median lethal dose; i.p.: intraperitoneal; i.m.: intramuscular; s.c.: subcutaneous.

^a p < 0.01 compared to respective lowest treatment doses of the oxime. Total 232 mice were used.

^bLD₅₀ determined following the moving average method of Gad and Weil.¹³ Three increasing treatment doses of HNK-102, HNK-106, and HNK-111 are corresponding to their 0.05, 0.10, and 0.20 LD₅₀. PI = LD₅₀ of sarin with treatment/LD₅₀ of sarin. Values in column 4 are (i) mean ± standard error of mean of number of experiments shown in column 5; (ii) in parenthesis are 95% confidence limits; and (iii) n or number of mice used.

Figure 2.

Dose–response of the protection offered in terms of PI by HNK-102, HNK-106, and HNK-111 at 0.05, 0.10, and 0.20 of their respective LD₅₀ doses. PI = LD₅₀ of sarin with treatment/LD₅₀ of sarin. LD₅₀ of HNK-102 = 282.8 mg/kg, LD₅₀ of HNK-106 = 35.35 mg/kg, and LD₅₀ of HNK-111 = 35.00 mg/kg. Values are in mean ± standard error of mean with four animals per group. *p < 0.01 compared to 2-PAM, HNK-106, and HNK-111. PI: protection index; LD₅₀: median lethal dose.

In vivo determination of sarin-induced inhibition of brain AChE

Control AChE activities in brain and serum are shown in Figures 3 and 4. Various doses of sarin, that is, 0.2, 0.4, and 0.8 LD₅₀ corresponding to 32, 64, and 128 µg/kg, respectively, showed brain AChE inhibition at different time points (Figure 3). Hence, following dose–time response curve. At 15 min post-sarin exposure, 18.2%, 19.9%, and 56.1% AChE inhibition was observed at 32, 64, and 128 µg/kg doses, respectively, thus following the linear trend. Significant inhibition of brain AChE was observed at 15 min, thus optimized for IC₅₀ determination.

Figure 3.

Effect of sarin on brain acetylcholinesterase using 32, 64, and 128 μg/kg (s.c.) doses at various time points. Each bar represents mean ± standard error of mean of four experiments. s.c.: subcutaneous.

Figure 4.

Effect of sarin on serum acetylcholinesterase using 32, 64, and 128 μg/kg (s.c.) doses at various time points. Each bar represents mean ± standard error of mean of four experiments. s.c.: subcutaneous.

In vivo determination of sarin-induced inhibition of serum AChE

Sarin induced inhibition of serum AChE at 32, 64, and 128 µg/kg dose, using different time points is depicted in Figure 4. No significant inhibition pattern of serum AChE was observed in dose–time response study.

Determination of IC₅₀

Dose-dependent AChE inhibition at 15 min (optimized time) post-sarin exposure is shown in Figures 5 and 6. IC₅₀ value for brain AChE was calculated as 139.2 µg/kg, correspond to 0.87 LD₅₀ of sarin. Similarly for serum AChE, IC₅₀ was calculated 77.23 µg/kg, correspond to 0.482 LD₅₀ of sarin.

Figure 5.

Effect of sarin on brain acetylcholinesterase at various doses, 15 min postexposure. Each point represents mean ± standard error of mean of four experiments.

Figure 6.

Effect of sarin on serum acetylcholinesterase at various doses, 15 min postexposure. Each point represents mean ± standard error of mean of four experiments.

In vivo reactivation of phosphorylated brain AChE

Percent activity of brain AChE was estimated at 15 min post-sarin exposure with 2-PAM and HNK analogs at 64, 128 (ca. IC₅₀ dose), and 256 µg/kg of sarin. Significant AChE enzyme reactivation (70% and 78%) from HNK-102 and HNK-111, respectively, at 64 µg/kg dose of sarin was observed. Approximately, 12.5% (p < 0.05) AChE reactivation was shown by HNK-111 at 256 µg/kg of sarin, as shown in Figure 7 .

Figure 7.

Reactivation inhibited brain acetylcholinesterase by oximes 15 min post-sarin exposure. Doses: sarin, 64, 128, and 256 μg/kg; oximes: HNK-102, HNK-106, and HNK-111 at 0.20 LD₅₀ of their respected doses; and 2-PAM, 30 mg/kg. Each bar represents mean ± standard error of mean of four experiments. *p < 0.05 compared to sarin; ^# p < 0.01 compared to sarin + 2-PAM. 2-PAM: pralidoxime; LD₅₀: median lethal dose.

In vivo reactivation of phosphorylated serum AChE

Similarly, serum AChE activity in percentage was estimated at 64, 128, and 256 µg/kg doses of sarin with 2-PAM and HNK analogs. Oximes showed reactivation at all the three doses, 15 min post-sarin exposure. HNK-102 at 128 µg/kg (double of serum IC₅₀ dose) showed ca. 45% (p < 0.001) significant reactivation in comparison to 2-PAM in vivo. The results are shown in Figure 8.

Figure 8.

Reactivation inhibited serum acetylcholinesterase by oximes 15 min post-sarin exposure. Doses: sarin, 64, 128, and 256 μg/kg; oximes: HNK-102, HNK-106, and HNK-111 at 0.20 LD₅₀ of their respected doses; and 2-PAM, 30 mg/kg. Each bar represents mean ± standard error of mean of four experiments. *p < 0.001 compared to sarin; ^# p < 0.001 compared to sarin + 2-PAM. 2-PAM: pralidoxime; LD₅₀: median lethal dose.

Discussion

Many efforts are being made for the development of a broad spectrum oxime, effective against various OP poisoning. Standard treatment for OP poisoning involves the administration of atropine and oxime to counteract excess of ACh and reactivation of AChE at the synapse, but the efficacy of an oxime as a therapeutic drug for various OP compounds is still debatable. Since, more than five decades, the oxime 2-PAM has been used as standard antidote but the inability of 2-PAM to cross the blood brain barrier and its poor efficacy against structurally different OP compounds poisoning has urge the need for the development of better, efficacious AChE reactivator in comparison to 2-PAM.

Previously, we reported the efficacy of these bis-quaternary oximes, that is, HNK analogs in terms of survival and enzyme reactivation against DFP poisoning in vivo.⁹ The present study unveils antidotal efficacy of the oximes against NA sarin and its comparison with 2-PAM in vivo. Earlier, acute effect of sarin was assessed measuring the inhibition of AChE in specific brain regions and plasma BChE inhibition as marker of the toxicity.¹⁶ However, the present study reports dose- and time-dependent sarin-induced AChE inhibition in brain and serum samples of Swiss mice (in vivo).

LD₅₀ values of sarin and the new oximes were determined by “moving average method” described by Gad and Weil,¹³ which supposed to be more accurate, highly reproducible with use of two to six more animals. At least four experiments for each treatment dose⁹ were performed for confidence building and to ensure reproducibility of the results. The s.c. route of administration of sarin was selected so as to slightly delay appearance of toxic effects, so as to closely mimic actual condition. Administration of atropine and oximes was done following i.p. and i.m. routes, respectively, for quick and uniform drug absorption.¹⁷ Using previously reported data of LD₅₀ of effective oximes (HNK-102, HNK-106, and HNK-111), PI was determined and compared with antidote 2-PAM (dose 30 mg/kg, LD₅₀ 180 mg/kg, i.p),¹⁵ considering it as standard oxime antidote. As reported, the treatment doses of oximes in combination with atropine showed ninefold protection (synergistic effect) against DFP poisoning in vivo.⁹ Also in the present study, similar beneficial synergistic antidotal effects were observed against sarin poisoning, when the new oximes used in combination with atropine. HNK-102 showed about three times more protection in comparison to 2-PAM in vivo. In earlier studies, it was indicated that higher doses of oximes showed marked increase in protection ratio against sarin toxicity.¹⁸ In light of the report, ¹⁸ three treatment doses of HNK oximes, corresponding to their 0.05, 0.10, and 0.20LD₅₀, were used and a dose-dependent protection was observed. At the higher treatment dose (0.20 LD₅₀), all the three HNK oximes offered their maximum and better protection compared to that of 2-PAM. HNK-102 showed about three times more protection as antidote compared to 2-PAM. Based on PI, the noted results (Figure 2) can be arranged in the order: HNK-102 > HNK-106 = HNK-111 > 2-PAM. Interesting to note that evaluation of antidotal efficacy of oximes, on the basis of better PI, found to be a promising approach for in vivo studies.

Further, to study AChE enzyme reactivation, the oximes were tested against acute poisoning of sarin and compared to 2-PAM in vivo. Earlier, the effect of sarin on brain and plasma cholinesterases via dose-dependent time course study has shown differential effect of the drug on the rodents.¹⁶ Likewise, we determined the sarin-induced AChE inhibition in whole brain and serum using various log doses and time points. The rationale behind choosing the Swiss mice as an experimental animal model, as it contains the highest level of carboxylesterase enzyme, that is, AChE in brain and blood, and thus can be used as an effective animal model to assess the effectiveness of oximes against OP toxicity.¹⁹ When sarin was injected at 128 µg/kg dose ca. 50% inhibition in whole brain was observed at 7.5–15 min postexposure. While at same dose and time point, ca. 53% AChE inhibition was seen in serum which persisted up to 16 h. As reported elsewhere, aging of sarin-exposed AChE takes place within 3–4 h¹⁹ and AChE inhibition follows dose–response curve.¹⁶ Hence, the present findings are in agreement with the earlier reported data that the inhibitory effect of sarin at the lower doses (64 µg/kg) is less compared to higher doses such as 128 and 256 µg/kg (Figure 3).

Having established the relationship between LD₅₀ of DFP and its AChE inhibition (IC₅₀),⁹ AChE enzyme IC₅₀ of sarin in brain and serum was determined at the optimized 15 min postexposure. Calculated IC₅₀ value of sarin for whole brain is 139.2 µg/kg, corresponds to 0.87 LD₅₀. Similarly, in serum, IC₅₀ value is 77.23 µg/kg, corresponding to 0.48 LD₅₀ of sarin. Thus, difference of about two times was noted between LD₅₀ and the dose for AChE inhibition by 50% (IC₅₀) in serum. Whereas, in whole brain, approximately 0.87 LD₅₀ (139.2 µg/kg) causes ca. 50% AChE inhibition. The plausible explanation to this, in brain highest AChE enzyme, is present followed by serum, muscles, and intestine.^20,21 In other words, it may be stated that about double the dose of sarin was required to inhibit 50% AChE in brain compared to serum. Also, it is a well-known fact that sarin (as non-charged agents) penetrates rapidly via blood brain barrier, and its IC₅₀ values are significantly greater in brain compare to RBCs, plasma, or whole blood.²² Our in vivo findings are in agreement with these observations. But herein, our results also showed that it causes only 50% AChE inhibition in brain at its LD₅₀ dose where it is toxic enough to cause lethality in exposed animals. Henceforth, these results are supporting the theory which says that OP/NAs along with AChE may be involved in other additional targets also.²³ These may be the involvement of muscaranic receptors in brain (higher affinity), neuropathy target esterase, and acylpeptide hydrolases (less pronounced in inhibition)^24,25 which are also majorly sensitive to OPCs toxicity. These receptors in brain along with other higher affinity proteins may decrease the free concentration of sarin to interact with AChE enzyme. Hence, this atypical sensitivity of AChE inhibition in brain and serum clearly indicates that the lethality due to sarin toxicity may not be solely attributed by AChE inhibition but may be involving other non-AChE targets as well.²⁶

Three treatment doses of oximes corresponding to their 0.05, 0.10, and 0.20 LD₅₀ values were considered practically applicable and safe via i.m. route for these experimental studies. The present study depicts the IC₅₀ shift curve using higher treatment (0.20 LD₅₀) dose of oximes against sarin toxicity, in comparison to well established and clinically used 30 mg/kg dose of 2-PAM which is 0.1666 of its LD₅₀ intramuscularly.

Careful analysis of the present results showed that HNK-102 and HNK-111 were capable of reactivating AChE at all the selected doses, that is, 64, 128, and 256 µg/kg of sarin. Additionally, HNK-111 showed significant reactivation (p < 0.05) at 1.6 LD₅₀ 256 µg/kg of sarin up to 4.5% cholinesterase reactivation. It is known that, 2-PAM-reactivated cholinesterase in blood and peripheral tissues intoxicated by sarin (GB) and VX.²⁷ However, the present results showed that the new oximes, namely HNK-102 and HNK-111, were able to reactivate sarin-intoxicated brain AChE significantly (p < 0.01) at lower dose only (Figure 7). The rank order for in vivo AChE reactivation by oximes in whole brain may be arranged in the order: HNK-111 > HNK-102 > 2-PAM > HNK-106, not in agreement with rank order profile of PI (HNK-102 > HNK-106 = HNK-111 > 2-PAM). Based on these findings, it is stated that there is no direct correlation between inhibition of brain AChE and lethality.

It is noteworthy that efficacy of oximes in reactivating sarin-intoxicated AChE enzyme was markedly different in brain and serum. Although, all the new oximes including 2-PAM significantly reactivated serum AChE but HNK-102 outshine among all other reported oximes. It is well understood that oximes due to proximity effect²⁷ have better chance to act on intoxicated serum cholinesterase enzyme than in brain, hence up to ca. 50% reactivation at lower dose, that is, 64 µg/kg and ca. 20% AChE reactivation in serum at higher doses (128 and 256 µg/kg) were shown by the oximes, 15 min post-sarin exposure. It is interesting to note that HNK-102 showed significant AChE reactivation in serum (p < 0.001) at 128 µg/kg dose of sarin (ca. 27% AChE reactivation) which is double the IC₅₀ dose 77.23 µg/kg) of serum, compared to brain AChE (Figure 8). As HNK series of oximes (HNK-102, 106 and 111) are ionic in nature like 2-PAM, therefore, it is obvious to expect that these oximes could not cross the blood–brain barrier and could not produce the protection inside the brain region.²⁸ But one of the in vivo study indicated that certain amount of unmetabolized carbonyl derivatives of 2-PAM entered some anatomical areas of the brain as in Paraoxon (diethyl 4-nitrophenyl phosphate)-poisoned rats, 2-PAM showed significant reactivation inside the cerebral cortex.^29,30 Since HNK-102 showed protection inside the brain (in case of sarin poisoning); therefore, HNK-102 might have entered in the brain region. This observation can be supported by the structural difference between the HNK analogs and simple oximes (2-PAM). HNK series of oximes are the bis(pyridinium) amide-conjugated oximes. This kind of structural feature, up to certain extent, mimics peptide bonds (amide), which might have facilitated a little more to cross the BBB compared to that of 2-PAM. Thus, HNK-102 has shown the better protection inside the brain region than 2-PAM. Studies with other toxic NAs may also unveil some interesting finding on antidotal efficacy of these new oximes.

All the three oximes offered better reactivation in serum. But higher activity in serum cannot be an indicator for increase in survival rate at the lethal doses of sarin.³¹ Also measuring only the levels of serum AChE, the severity of OP poisoning can never be validated.^29,32 In light of the discussed points, it is not justified to predict the better reactivation efficacy of the oximes only on the grounds of serum cholinesterase enzyme activity. Moreover, dissimilar rate of AChE reactivation in various species including primates and guinea pigs having none or low levels of blood carboxylesterases, while rat and mice, having highest carboxylesterases level, showed that OP compounds differently inhibit the cholinesterase enzyme in different species.^33
–35

Conclusion

HNK series of oximes showed better antidotal efficacy against sarin poisoning in Swiss mice as evidenced by (i) threefold more protection (in terms of survival) shown by HNK-102, in comparison to 2-PAM; (ii) reactivation of sarin (64 µg/kg) inhibited brain AChE by 17% (HNK-111) and 25% (HNK-102) compared to 2-PAM; and (iii) reactivation of sarin (256 µg/kg) inhibited serum cholinesterase by 20%.

Footnotes

Acknowledgments

The authors thank Dr Lokendra Singh, Scientist ‘H’ & OS, the Director, Defence Research and Development Establishment, Jhansi Road, Gwalior, India, for his encouragement and providing necessary facilities. They also thank Ms Pooja Phatak, JRF, for her help during the study.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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In vivo protection studies of bis -quaternary 2-(hydroxyimino)- N -(pyridin-3-yl) acetamide derivatives against sarin poisoning in mice

Abstract

Keywords

Introduction

Material and methods

Reagents

Animals

Sample preparation

Experimental procedure

Determination of median lethal dose and protection index

In vivo determination of time-dependent peak (maximum) AChE inhibition

In vivo determination of half inhibition concentration dose of sarin and its shift for AChE activity

Statistical analysis

Results

Signs of toxicity and lethality

PI of the oximes in vivo

In vivo determination of sarin-induced inhibition of brain AChE

In vivo determination of sarin-induced inhibition of serum AChE

Determination of IC50

In vivo reactivation of phosphorylated brain AChE

In vivo reactivation of phosphorylated serum AChE

Discussion

Conclusion

Footnotes

Acknowledgments

Declaration of Conflicting Interests

Funding

References

Determination of IC₅₀