Pharmacological Characteristics Of Benthiactzine's Antagonism On Cholinergic Receptors And Its Antidotal Effects Against Cholinesterase Inhibitors Poisoning

Cholinesterase inhibitors (ChEI) include nerve agents and the majority of organophosphorus pesticides. The main underlying mechanism of ChEI intoxication are due to irreversible inhibition of acetylcholinesterase (AChE) by these compounds and subsequent increase in the concentration of the neurotransmitter acetylcholine(ACh) and hyperstimulation of cholinergic system at central and peripheral sites. In the periphery, acetylcholine accumulation leads to persistent muscarinic receptor stimulation that triggers a syndrome including miosis, profuse secretions, hypotension, bradycardia, emesis, bronchoconstriction, diarrhea, and urination. It also leads to overstimulation followed by desensitization of nicotinic receptors, causing severe skeletal muscle fasciculations, weakness, dyspnea, and paralysis. Central nervous system-related effects include anxiety, restlessness, confusion, tremors, seizures, cardiorespiratory paralysis, and coma. The emergency medical treatment of ChEI poisoning represents a major public-health concern worldwide. Currently, WHO-recommended standard therapeutic strategies to decrease ChEI poisoning consist of administration of combination of atropine, oxime and diazepam with other supportive measures when necessary. However, the fatality rate of severe acute organophosphorus pesticides poisoning (AOPP) remains at more than 20% even after application of standard atropine-centered treatment. The limitations of these strategies have been well recognized now. First, there are controversial for the use of cholinesterase reactivators. Second, the clinical practices have shown the limitations of atropine in reducing mortality and morbidity associated with ChEI poisoning. In addition, atropine often causes serious toxic reactions and may even lead to death through its potent antimuscarinic effect. Third, the high death rate may not be attributed to the insufficient muscarinic antagonism but to the lack of nicotinic antagonism. Because it was well documented that the antagonist of nicotinic receptors not only can block the nicotinic syndrome, but also can decrease the toxicity mediated by desensitized neuronal nicotinic acetylcholine receptors though facilitating mAChRs functions. Thus, there is an urgent need for the development of a more effective and safer antidote with both antimuscarinic and antinicotinic receptors to counteract ChEI poisoning.Presently, it is well recognized that loss of the gut mucosa barrier function can result in endotoxemia or pyemia, and cause a sustained inflammatory response syndrome. Gut barrier function has been implicated as a potential source of multiple organ dysfunction syndrome (MODS). The maintenance of gut mucosa barrier function is crucial to the development and prognosis of severe diseases. From clinical observations, one reason accounted for the high mortality of AOPP patients is due to MODS or multiple system organ failure (MSOF). Thus, it is very meaningful to investigate the changes of gut mucosa barrier function during ChEI poisoning and to develop an anticholinergic agent which can protect against the damage of gut mucosa barrier function induced by ChEI intoxication.Benthiactzine, which has both antimuscarinic and antinicotinic effects, was our newly developed ChEI poisoning antidote. Previously, we have shown that: (1) Benthiactzine is absorbed swift and more prone to penetrate blood-brain barrier, and it is mainly distributed in lung, gland, brain, heart, et al. (2) Benthiactzine is weaker than atropine in binding to mAChRs expressed in various tissues or to five different mAChRs subtypes in a specific [3H]QNB-binding assay, while afforded much better protection than atropine against the most lethal ChEI, sarin, soman, tabun, or VX, in a mouse model. (3) Benthiactzine has potent protective effects against circulatory or respiratory failure. Interestingly, benthiactzine can inhibit the increase of serum endotoxin content in mouse during VX poisoning. This suggests that benthiactzine can protect against injury of the gut barrier function induced by ChEI intoxication. However, subsequent study demonstrated that benthiactzine has the lowest affinity to gastrointestinal tract mucosa, which is much weaker than that of atropine. Thus, we hypothesize that the treatment of benthiactzine may not evoke enteroparalysis and aggravate the injury of gut mucosa barrier function.PartⅠPharmacological characteristics of benthiactzine's antagonism on cholinergic receptorsThe antagonism of benthiactzine on central and peripheral cholinergic receptor activities were evaluated and compared with the effects of atropine. The in vivo experiments were all performed in a mouse model. The methods are list below: (1)The central tremor and peripheral salivation caused by arecoline, the peripheral salivation caused by pilocarpine, and the isolated guinea pig ileum construction induced by acetylcholine or oxotremorine were carried out to assess the effects of benthiactzine on central and peripheral muscarinic receptors. (2)The convulsions produced by Nicotine and the isolated guinea pig ileum construction induced by Nic were used to assess the effects of benthiactzine on central and peripheral nicotinic receptors. (3)The mixed tremor evoked by nicotine combined with pilocarpine was also used to assess the antagonism of benthiactzine on cholinergic receptors. (4) A poison resistance (PR) value was introduced to evaluate the antilethal effect of benthiactzine and atropine in organophosphorus pesticide DDVP poisoning mice. In addition, effects of benthiactizine on brain or plasma ChE activities in rat were carried out by the micro DTNB method.1.1 The antagonism of benthiactzine on central muscarinic receptorsThe ED50±L95 value of benthiactzine antagonizing to central tremor caused by arecoline (8mg/kg, sc) in mice was 0.31±0.06mg/kg, which was 3.3 times stronger than that of atropine (1.33±0.17mg/kg, P<0.05).1.2 The antagonism of benthiactzine on peripheral muscarinic receptorsThe ED50±L95 value of benthiactzine (ip 15min) antagonizing to peripheral salivation caused by arecoline (8mg/kg, sc) in mice was 0.38±0.09mg/kg, which was 1.2 times weaker than that of atropine (0.17±0.03mg/kg, p<0.05). The ED50±L95 value of benthiactzine antagonizing to peripheral salivation caused by pilocarpine (40mg/kg, sc) in mice was 5.57±1.30mg/kg, which was 11 times weaker than that of atropine (0.46±0.18mg/kg, p<0.01). In isolated guinea pig ileum preparations, benthiactzine inhibits ACh-induced or Oxo-induced contractions in dose-dependent manner as well as atropine. There was no significant difference between the IC50 values of the two compounds.1.3 The antagonism of benthiactzine on central nicotinic receptorsThe ED50±L95 value of benthiactzine antagonizing to central convulsions induced by nicotine (1.0mg/kg, speed tail iv) in mice was 2.87±0.76mg/kg; under the same condition, the percentage of antagonizing convulsion of atropine was below 20% even its dose was high at 60mg/kg.1.4 The antagonism of benthiactzine on peripheral nicotinic receptorsIn isolated guinea pig ileum preparations, the IC50±L95 value of benthiactzine inhibiting the nicotine-induced contractions was 1.1±0.18μM, which was 13 times stronger than that of atropine (13.84±1.68μM, P<0.05). Benthiactzine was found to antagonize nicotine-induced contractions due to excitation of ganglionic nicotinic receptors.1.5 The antagonism of benthiactzine on mixed tremor evoked by nicotine combined with pilocarpine.The ED50±L95 value of benthiactzine antagonizing to mixed tremor caused by pilocarpine (50 mg/kg, ip) combined with nicotine (2.5mg/kg, ip) in mice was 2.64±0.35mg/kg, which was approximate 1 times stronger than that of atropine (4.95±0.65mg/kg, P<0.05). PartⅡEffects of benthiactzine on ChE activitiesBenthiactzine has no effects on brain or plasma ChE activities in rat at the concentrations of 10-9 M to 10-5M in vitro.PartⅢThe antidotal effects of benthiactzine against ChEI poisoning3.1 The antidotal effect of benthiactzine against DDVP poisoning in miceThe PR value was calculated as (DDVP LD50 in presence of antidote)/(DDVP LD50 in absence of antidote). Administration of benthiactzine at a dose of 10 mg/kg raised the PR value of DDVP to 17.84, whereas atropine at the same dose increased it only to 1.81, showing a weaker effect of atropine compared with benthiactzine. The ED50±L95 value of benthiactzine antagonizing to the lethal effect caused by DDVP (1.2LD, sc) was 2.87±0.40mg/kg, which was much lower than that of atropine (6.87±3.92mg/kg, P<0.05). These results suggest that benthiactzine possesses a more potent effect than atropine in counteracting ChEI poisoning.3.2 Protective effects of benthiactzine on the injury of gut mucosa barrier function induced by VX poisoningThe objective of this secssion is to investigate the functional, morphological changes of the gut barrier during VX poisoning and the therapeutic effect of benthiactzine. Forty eight male Wistar rats were randomly divided into six groups (n=8, each): control group, VX poisoning (model) group, benthiactzine 1, 3, 9mg/kg groups, and atropine 9mg/kg groups. The above mentioned drugs were respectively given (ip) 5 min after VX poisoning at the dose of 13μg/kg (sc). The plasma concentration of D-lactate and the diamine oxidase(DAO)activity, which reflected the gut barrier function, were measured at 3 hours after VX poisoning. At the same time point, the specimens from jejunum and ileum were taken respectively. The morphological changes of the intestinal mucosa were determined by light microscope and electron microscope. 3.2.1 The changes of plasma D-lactate concentration after VX intoxication and the therapeutic effects of benthiactzine3 hours after VX poisoning, the plasma D-lactate concentration in model group (87.75±22.91μg/mL) was significantly increased compared with that of control group (29.07±6.55μg/mL, P < 0.01). This change could be decreased dose-dependently after benthiactzine administration at doses of 1, 3, 9mg/kg during the VX poisoning (64.23±24.88,56.94±10.37,45.29±11.14μg/mL). The plasma D-lactate content was significantly decreased compared with that of model group after benthiactzine treatment at doses of 3 and 9 mg/kg, respectively. (P<0.01) And the increase of D-lactate concentration could also be inhibited by atropine administration at the dose of 9 mg/kg. (P<0.01)3.2.2 The changes of plasma DAO activity after VX intoxication and the therapeutic effects of benthiactzine3 hours after VX poisoning, the DAO activity in model group (6.72±0.93 U/L) was significantly increased compared with that of control group. (2.99±0.43 U/L,P<0.01). This change could be decreased dose-dependently after benthiactzine administration at doses of 1, 3, 9mg/kg during the VX poisoning (5.26±0.81,4.11±1.11,3.17±0.68 U/L; P<0.05 or P<0.01). The increase of DAO activity could be totally reversed by benthiactzine at the dose of 9mg/kg as well as atropine treatment.3.2.3 Morphological changes of the gut mucosa barrier during VX poisoning and therapeutic effect of benthiactzineThe mucosal epithelial injury were obvious in small intestine at the 3 hours after VX poisoning by light microscope, including the mucosal thickness and the height of villi in jejunum and ileum diminished, interstitial edema, angiotelectasis. Under the electron microscope, the damaged mucosal epithelial cells with organelles and cell tight junction injury were observed. These pathological changes could be improved dose-dependently after benthiactzine administration at doses of 1, 3, 9mg/kg, and the changes could be totally reversed by benthiactzine at dose of 9mg/kg as well as atropine treatment. From these observations, conclusion could be drawn:1. The pharmacological characteristics of benthiactzine in antagonizing cholinergic receptors are entirely distinct from those of atropine, a commonly studied antidote against ChEI poisoning. Benthiactzine has different antagonism between central and peripheral muscarinic receptors. Benthiactzine exhibits stronger central antimuscarinic effects than atropine owing to its capability of going through blood-brain barrier rapidly. In contrast, benthiactzine has weaker peripheral antimuscarinic effects than atropine. This prevents benthiactzine having a number of side effects which are undesirable such as dry mouth and possibly enteroparalysis. The predominant feature of benthiactzine is that it has definite antagonism on central and peripheral ganglionic nicotinic receptors, while atropine has no effects on nicotinic receptors.2. Benthiactzine has much better antidotal effects than atropine during ChEI poisoning, which is contributed to its definite antagonism on muscarinic and nicotinic receptors but not reactivating acetylcholinesterase.3. The gut mucosa barrier function was seriously damaged during the cholinesterase inhibitors poisoning, including the injury of intestinal mucosal epithelial cells, the loss of cellular tight junction, and the increase of intestinal permeability. The functional and morphological changes of gut mucosa barrier could be improved after benthiactzine administration. As a novel anticholinergic drug, benthiactzine is able to ameliorate the injury of gut mucosa barrier function besides its potent effects in counteracting ChEI poisoning. Thus, it is meaningful to prevent or treat the development and progression of MODS induced by ChEI poisoning.