KR0176321B1 - Antidote for organophosphonate compounds poisoning - Google Patents

Abstract

The present invention provides an antidote for the prevention of lethal or brain damage by organophosphate agents consisting of NMDA receptor antagonists. The antidote of the present invention may be used alone or in combination with an existing antidote. Addiction by the organophosphate preparation can be effectively detoxified by the antidote of the present invention.

Description

Organophosphate poisoning antidote

Figure 1 depicts the major pharmacological regulatory sites of the N-methyl-D-aspartate (NMDA) receptor complex.

2 is an optical micrograph showing brain damage by organophosphate (CD) (A: low magnification, B: high magnification).

Figure 3 is an optical micrograph showing the protective effect of the antidote of the present invention against brain damage by organophosphate (CD) (A: procyclidine (30mg / kg), B: diazepam (10m g / kg)) .

The present invention relates to an antidote capable of effectively detoxifying an organophosphate agent poisoning consisting of an N-methyl-D-aspartate (NMDA) receptor antagonist.

Anticholinesterases, such as organophosphates, have been widely used as insecticides due to their strong toxicity. It has been developed and used as a CB chemical agent known as isopropyl derivative.

Toxic expression during organophosphate poisoning is associated with excessive acetylcholine (ACh) at cholinergic nerve synapses and neuroeffector junctions following inhibition of the enzyme cholinesterases (ChEs). Due to accumulation, it is divided into muscarinic signs, nicotinic signs, and central effects.As a result, the main cause of death is bronchial contraction due to repression of the respiratory center. It is known to be respiratory failure due to broncho-constriction and broncho-spasm, hypersecretion and respiratory paralysis (see Dunn, MA and Sidell, FR (1989). JAMA 262; 649: Richett, DJ et al. (1987) Mil. Med. 152; 35: and Dretchen, KL et al. (1986). Toxicol. Appl. Pharmacol. 83; 584.

The detoxification of organophosphate poisoning has been composed of the combined administration of atropine (At), an antimuscarinic agent, as a symptomatic agent, and oximes, a reactivator of an enzyme inhibited as a causative agent. However, the combined administration of these two drugs alone does not provide satisfactory detoxification effects on various organophosphates with different properties. In other words, in the case of organophosphate preparations that are rapidly chemically aged during the enzyme inhibition process, such as CD, the reactivation effect of oximes cannot be expected (cf. Dunn, MA, quoted above: Berry, WK and Davies, DR ( 1970) Biochem. Pharmacol. 19; 927: and Berman, HA and Decker, MM (1986). J. Biol. Chem. 261; 10646. In view of this, carbamates such as pyridostigmine (PS) have been developed as a preventive premeasure that can improve survival rate (see Berry, WK's). Cited above: Koester, R. (1946) J. Pharmacol. Exp. Ther. 88; 39: and Gordon, JJ, et al. (1978). Toxicol. Appl. Pharmacol. 43; 207. However, even if they survive the complex prescription, they may have behavioral disorders, mental concentration disorders, schizophrenia, depression, insomnia, memory loss, neurological hypersensitivity, heart failure, and visual acuity due to sequelae of brain injury (cf. Metcalf). , DR and Hmlmes, JH (1969) Ann. NY Acad. Sci. 160; 357. Recently, attention has been focused on efforts to resolve brain damage in terms of detoxification and prevention (cf. McLeod, CG, et al. (1985) Fund.Appl.Toxicol. 5; S10: Tryphones, L. and Clement, JG (1995) .Toxicol.Pathol. 23; 393: and McDonough, JH, Jr. et al. (1989). Appl.Toxicol. 13; 256).

Brain damage caused by organophosphate preparations, etc., is followed by severe spasms, or epileptic seizures, which are effectively prevented by the administration of γ-aminobutanoic acid (GABA) receptor agonists (see McDonough). , JH, Jr. et al., Supra. Recently, excitatory amino acid (EAA) receptors have been found to be a major neurotransmitter of seizure attacks, and drugs that act on EAA receptors have been extensively studied (see Meldrum, B. and Garthwalte, J. (1990)). TiPS 11; 379). EAA (glutamate, aspartate) receptors are classified into three ionotrophic subtypes and one metabotropic receptor, depending on the selectivity of the agonist for each receptor. NMDA receptors, quisqualate / AMPA receptors, kainate receptors, and metabotropic receptors that detect quisqualate / ibothenates (see Choi, DW (1988)). Neuron 1 623: Makowiec, RL et al. (1991) Neuroscience 42; 671: and Albin, R. et. Al. (1992) Neuroscience 46; 35). The major spasm attacks of these receptors are known to be caused by activation of NMDA receptors.

Anticonvulsants for preventing brain damage include GABA receptor agonists, NMDA receptor antagonists, barbiturates, calcium iontophoresis agents, and cholinergic nerve blockers (cf. McDonough's quote: Domino, EF ( Neurotoxicology 8; 113: Coudray-Lucas, C. et al. (1984) .Acta Pharmacol, Toxicol. 5; 153: Shih, T.-M. et al. (1991). Neurosci.Behav. 15; 349: McDonough, JH and Shin, T.-M. (1993) Neurosci. Behav. Rev. 17; 203. In developed countries, GABA receptor agonists are used to alleviate cramps caused by organophosphates, but problems such as severe sleep effects are emerging. In addition, barbiturates have a strong anesthetic effect, calcium ion inflow blockers and cholinergic nerve blockers are ineffective. On the other hand, GABA receptor agonists have also recently been shown to exhibit strong antagonistic effects on NMDA receptors (McDonough, JH and Shih, T.-M. (1995). Neurosci. Behav. Rev. 51; printing) The fact is that NMDA receptor antagonists can alleviate cramps caused by organophosphates. Accordingly, the present inventors further studied the NMDA receptor antagonists in this regard, it was confirmed that it is possible to effectively prevent brain damage while increasing the survival rate without any other problem when organophosphate poisoning.

It is therefore an object of the present invention to provide an antidote for the prevention of lethal or brain damage by organophosphate preparations consisting of NMDA receptor antagonists.

The NMDA receptor antagonist of the present invention exhibits a synergistic effect on organophosphate agents, in particular CD and CB poisoning, and anti-brain damage.

The NMDA receptor antagonist of the present invention is selected from procyclidine (PC), diazepam (DZ), and lorazepam (LZ). Procycletridin 5-30mg / kg, diazepam 3.5-10 mg / kg, Lorazepam is preferably used in an intramuscular dose of 1-2 mg / kg. As described above, setting the dose of each antidote suggests a dose that exhibits a minimal detoxifying effect to a dose that exhibits sufficient detoxifying effect without any side effects of the drug itself.

In addition, the NMDA receptor antagonist of the present invention is a conventional antidote, for example, a carbamate drug that is a prophylactic agent such as pyridostigmine, a muscarinic nerve blocker such as atropine, which is a symptomatic agent, and a palm (2-proli) Can be used in combination with at least one of the enzyme reactivators such as venom, PAM).

Hereinafter, the antidote for organophosphate poisoning of the present invention will be described in more detail.

1) Selection of Organophosphate Preparation

Among the highly toxic organophosphate preparations, CD (LD50 approximately 75 µg / kg) in which chemical aging occurs rapidly and CB (LD50 approximately 140 µg / kg) in which chemical aging occurs relatively slowly were selected.

2) Selection of NMDA Receptor Antagonists

NMDA receptor complexes are regulated by several sites, as shown in FIG. Many drugs have been reported as antagonists acting on these sites, such as MK-801, mecamylamine, ketamine, beactyzine, pentobarbital (McDonough, JH and Shih, T. -M. (1995) Neurosci.Behav. Rev. 51; printing), mecamylamine and benathigin were severely toxic and discontinued, ketamine and pentobarbital were anesthetics, and MK-801 was applied clinically. There is no bar. Accordingly, in the present invention, procyclidine used for Parkinson's disease and diazepam and lorazepam used in antispasmodic diseases were selected. Procyclidine binds to the ion channel domain, or the pencyclidine (PCP) binding site, blocks EAA neurotransmission, and at the same time has an antimuscarinic effect, an atropine-like effect. Known (see Olney, JW et al. (1991). Science 254; 1515). The mechanisms of NMDA receptor antagonistic effects of diazepam and lorazepam are not yet clear, but are thought to be related to nitric oxide (NO) produced by NMDA receptor activation (Hanbauer, I. et al. (1992) NeuroReport 3; 409.

3) Evaluation of Detoxification Effect on Organophosphate Addiction

The detoxification efficacy of the NMDA receptor antagonist of the present invention was evaluated by the synergistic effect of survival after organophosphate poisoning, that is, the drug's protection ratio (PR). The drug's defense rate is expressed as the ratio of LD50 after drug administration to the median lethal dose (LD50) of organophosphate preparation.

4) Evaluation of protective effect against brain injury by organophosphate preparation

The protective effect of brain damage on the antidote of organophosphates was assessed as an indicator of brain cell death after organophosphate poisoning. In other words, histopathological observation of the death and degeneration of the hippocampus pyramidal cells after organophosphate poisoning was evaluated to evaluate the protective effect of the drug.

Hereinafter, the present invention will be described with reference to Examples. The present invention is not intended to limit the scope of the present invention.

Example 1

Detoxification efficacy evaluation by single dose

As experimental animals, SD rats (weight 200-300g, age 6-7 weeks) were used after breeding and breeding under appropriate breeding conditions to adapt to the laboratory environment.

At the dosages shown in Table 1, procyclidine, diazepam and lorazepam were administered intramuscularly to experimental animals (about 30), respectively. At this time, procyclidine was dissolved in physiological saline, diazepam and lorazepam in 40% glycerin, and administered intramuscularly in an amount of 0.5 ml / kg. After 15 minutes, the organophosphate CD (10 mg) was dissolved in a small amount of ethanol (1 ml), and then diluted in physiological saline to various CD doses before the experiment, and subcutaneously administered in an amount of 1 ml / kg. After 24 hours, the survival rate was observed. LD50 was estimated based on the survival rate for each dose, and the magnification (defense ratio) of LD50 at the time of drug administration relative to LD50 when organophosphate alone was administered was determined. The results are shown in Table 1.

In Table 1, the detoxification effect of the antidote agent of the present invention on the organophosphate CD was good in order of procyclidine diazepam lorazepam, and the protection rate of about 2.6 times when 30 mg / kg of procyclidine was administered was shown. Indicated.

Example 2

Evaluation of detoxification efficacy by combination administration

In the same manner as in Example 1, the conventional antidote, pyridostigmine, atropine and palm were respectively administered to procyclidine and diazepam at the dosages shown in Table 2. At this time, pyridostigmine was dissolved in distilled water and orally administered in an amount of 1 ml / kg, and atropine and palm were dissolved in physiological saline and administered intramuscularly in an amount of 0.5 ml / kg. However, in the case of pyridostigmine, atropine and palm were immediately administered 60 minutes before organophosphate. Obtain the ERA at the time of drug administration and show the results in Table 2.

In Table 2, the detoxification effect on the CD administered with the conventional antidote in addition to the antidote PC and DZ of the present invention was higher than that of diazepam with procyclidine, and the combined protection rate of about 8.0-fold with PS + PC + At + PAM was combined. Indicated.

Example 3

Comparison of Detoxification Efficacy with Existing Antidote for CB

To compare the detoxification efficacy for organophosphate CB with conventional antidote, use CB instead of CD as organophosphate and use the types and dosages of the antidote shown in Table 3, respectively, according to the methods shown in Examples 1 and 2. Single dose and multiple doses with palm. Obtain the ERA at the time of drug administration and present the results in Table 3.

In Table 3, the detoxifying effect of the present invention on the organophosphate CB and the antidote of the present invention was better than that of procyclidine alone or in combination with palm, and the PC + RAM When administered in combination, the ERA was 60 times higher.

Example 4

Comparison of Detoxification Efficacy by Combination Administration with CD

In order to compare the detoxification efficacy of the conventional antidote against the organophosphate CD with the coadministration, the combination and the dosage of the antidote shown in Table 4 were administered according to the method shown in Example 2. The protection rate at the time of drug administration is calculated and the results are shown in Table 4.

In Table 4, the detoxification effect by the combined administration of the antidote procyclidine of the present invention to the organophosphate CD was superior to the combined administration of atropine and showed about 6-fold protection rate upon the combined administration of PS + PC + PAM.

Example 5

Evaluation of Protective Effect Against Brain Damage by Organophosphate Preparation

Procyclidine (30 mg / kg) and diazepam (10 mg / kg) were administered alone to the experimental animals according to the method set forth in Example 1, and the animals were anesthetized 24 hours after administration of the organophosphate CD and then heart Through 2 IU The saline solution containing heparin / ml and 4% paraformaldehyde brain were perfusion-fixed. The brain was extracted and fixed after 24 hours with 10% neutral formalin. The immobilized tissues were sliced to a thickness of 3 μm after embedding paraffin and stained with hematoxylin and eosin (HE). The stained tissue samples were observed under an optical microscope, and the brains with or without drug administration were examined. Protective efficacy was assessed by the degree of death degeneration of cells.

Observed optical micrographs are shown in FIGS. 2 and 3.

FIG. 2 shows brain damage caused by organophosphate CD. The nuclear concentration of the cerebral hippocampal vertebral cells is apparent, and the brain tissue around the cells is fear-denatured. 3 shows the protective effect of procyclidine (FIG. 3a) and diazepam (FIG. 3b) against brain injury caused by organophosphate CD. Do not see. Therefore, the protective effect of the antidote of the present invention is excellent.

Claims (2)

An antidote for preventing lethal or brain injury by organophosphate preparation, comprising a complex composition of procyclidine and palm. The antidote of claim 1, further comprising pyridostigmine.