KR101424514B1 - Memantine as a therapeutic treatment of fetal valproate syndrome - Google Patents

Abstract

The present invention relates to memantine, a substance for treating social deficit symptoms, and more particularly, to a treatment for psychiatric diseases, wherein the NMDA receptor antagonist memantine effectively treats the symptoms of sociability deficiency and convulsive sensitivity increase in congenital valproic acid exposure syndrome Can be used.

Description

Use of memantine as a therapeutic agent for congenital valproic acid exposure syndrome {Mamantine as a therapeutic treatment of fetal valproate syndrome}

The present invention relates to memantine as a novel therapeutic substance for congenital valproic acid exposure syndrome, and more particularly, to a method for preventing and treating psychotic symptoms caused by exposure to valproic acid during pregnancy by the NMDA receptor antagonist memantine It can be used for the treatment of the deficiency of social function, which is a symptom of congenital valproic acid exposure syndrome.

Valproic acid is a widely used drug to alleviate epilepsy, migraine and bipolar symptoms. However, exposure to valproic acid in the early pregnancy (up to 14 weeks of gestation) induces skeletal abnormalities, cognitive impairment and behavioral abnormalities in the fetus, and these symptoms are associated with fetal valproate syndrome, Quot; This side effect of valproic acid has been suggested in the early 1980s (Dalens et al., J Pediatr, 97, 332-333, 1980; Gomez, J Pediatr, 98, 508-509, 1981; Bjerkedal et al., Lancet, Exposure to valproic acid during pregnancy in the 1990s (Ornoy, Reprod Toxicol, 28, 1-10, 2009), 2, 1096, 1982; Robert et al., Rev. Neurol (Langer et al., Fetal Diagn Ther, 9, 155-158, 1994; Moore et al., J Med Genet, 37, 489-497, 2000). In the 1990s, congenital valproic acid exposure syndrome was associated with autism (Christianson et al., Dev Med Child Neurol, 36, 361-369, 1994). Recently, epilepsy including valproic acid It has been reported that drugs can cause autism when the fetus is exposed during pregnancy (Guven et al., J Matern Fetal Neonatal Med, 19, 115-117, 2006).

In order to elucidate the mechanisms of various adverse effects of valproic acid on human fetuses, a method of using an animal model has been proposed (Wegner and Nau, Reprod Toxicol, 5, 465-471, 1991; Wegner and Nau, Neurology, 24, 1992, Amdt et al., Int J Dev Neurosci, 23, 189-199, 2005). As in humans, valproic acid induced neural tube defects and skeletal system abnormalities in pregnant rats, including rats (Briner and Lieske, Teratology, 52, 306-311, 1995; Lindhout and Schmidt, Lancet, 1, 1392-1393 Moore et al., J Med Genet, 37, 489-497, 2000). In addition, in rats similar to those with congenital valproic acid exposure syndrome, Schneider and Przewlocki, Neuropsychopharmacology, 30, 80-89, 2005), repetitive behavior (Schneider et al., Psychoneuroendocrinology , 33, 728-740, 2008), and epileptic symptoms (Kim et al., Toxicol Lett, 201, 137-142, 2011). Based on these reports, it has been widely used as an animal model for studying congenital valproic acid exposure syndromes (Narita et al., Pediatr Res 52, 576-579 , 2002, Ingram et al., Neurotoxicol Teratol, 22, 319-324, 2000).

Congenital valproic acid exposure syndromes have molecular biologic features based on these behavioral characteristics and typically have an imbalance between excitatory / inhibitory signaling in the brain. Such a signal imbalance in the brain has been identified as a major cause of sociability deficiency symptoms (Rubenstein and Merzenich, Genes Brain Behav, 2, 255-267, 2003; Dani et al., Proc Natl Acad Sci USA, 102, 12560-12565, 2005). An excitatory / inhibitory signaling imbalance was observed in the model of exposed rabbits during pregnancy as well as in human patients. (Fukuchi et al., Neurosci Res, 65, 35-43, 2009), decreased expression of GABA-producing enzyme Glutamic acid decarboxylase (GAD) Neurosci Res, 65, 35-43, 2009), reduction of brain inhibitory neurons (Gogolla et al., Journal of neurodevelopmental disorders, 1, 172-181, 2009) have been reported. In particular, the expression of NR2A and NR2B constituting the NMDA receptor and the expression of α-CaMKII mediating the NMDA receptor signaling have been reported, and thus synaptic organs have been reported (Rinaldi et al., Proc Natl Acad Sci USA , 104, 13501-13506, 2007).

Memantine is a noncompetitive NMDA receptor inhibitor and is a drug developed to treat Alzheimer's dementia by acting on the glutamatergic system. Memantine binds to NMDA receptors with a higher affinity than Mg2 + ions and prevents the Ca2 + influx from the NMDA receptors in the extrasynaptic region that causes neuronal cytotoxicity (Chen et al., J Neurosci, 12, 4427-4436, 1992 ; Robinson and Keating, Drugs, 66, 1515-1534, 2006; Kornhuber et al., Eur J Pharmacol, 166, 589-590, 1989; Chen and Lipton, J Physiol, 499 Rogawski and Wenk, CNS Drug Rev, 9, 275-308, 2003).

WO 2005/06790B describes a method of treating mild to moderate Alzheimer's disease (AD) comprising administering an effective amount of memantine or a pharmaceutically acceptable salt thereof to a subject in need of such treatment. This method relates to a naive subject and a group of subjects who had been treated with other pharmacological compounds authorized for the treatment of AD, but who stopped AChEI treatment 1 day before the start of memantine administration.

As another related patent, WO 2006/009769 describes a pharmaceutically acceptable polymeric matrix carrier capable of prolonging the release rate of memantine from about 4 hours to about 24 hours after administration of the formulation.

Disclosure of Invention Technical Problem [8] The present invention has been made in view of the above-mentioned problems, and it is an object of the present invention to provide a new congenital valproic acid exposure treatment material.

Another object of the present invention is to provide a composition for treating a new social deficit symptom.

Another object of the present invention is to provide a composition for treating new epileptic symptoms.

In order to achieve the above object, the present invention provides a composition for treating congenital valproic acid exposure syndrome comprising memantine as an active ingredient.

The present invention also provides a composition for treating a symptom of sociability deficiency by valproic acid comprising memantine as an active ingredient.

The present invention also provides a composition for treating seizures by valproic acid comprising memantine as an active ingredient.

The present invention also relates to a method for the treatment and prophylaxis of cancer, comprising the steps of: a) injecting valproic acid into a pregnant rat, b) growing a female rat exposed to valproic acid during pregnancy, c) injecting memantine into the female rat; And d) performing a social interaction test, an open-field locomotor test, and an electroshock seizure threshold after the memantine injection, The present invention provides a method for analyzing the therapeutic effect of behavioral symptoms caused by

In one embodiment of the present invention, the rats of step a) are preferably 12 days of gestation,

In another embodiment of the present invention, the valproic acid of step a) is preferably injected subcutaneously at a dose of 400 mg / kg, but not limited thereto,

In another embodiment of the present invention, the rats of step b) are preferably but not limited to be born for up to four weeks,

In one embodiment of the present invention, the step c) is preferably performed by intraperitoneally injecting memantine 30 mg / kg to the grown rat, but is not limited thereto.

In the present invention, "memantine" means 1-amino-3,5-dimethyladamantane hydrochloride. The name for memantine in the United States is Namenda, in Germany it is Akatinol and Auxura, and in the rest of the European Union it is Ebixa. Memantine is the subject of U.S. Patent Nos. 4,122,193, 4,273,774 and 5,061,703.

Various salts of memantine may be used. The term "salt" may include acid addition salts or addition salts of free bases. Examples of acids which can be used to form pharmaceutically acceptable acid addition salts include salts derived from non-toxic inorganic acids such as nitric acid, phosphoric acid, sulfuric acid or hydrobromic acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, and aliphatic mono- and di Salts derived from non-toxic organic acids such as carboxylic acids, phenyl-substituted alkanoic acids, hydroxyl alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids and acetic acid, maleic acid, succinic acid or citric acid, . Non-limiting examples of such salts include, but are not limited to, napadisylate, besylate, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, But are not limited to, chloride, bromide, iodide, acetate, trifluoroacetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, Benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, methanesulfonate and the like. Also included are salts of amino acids such as arginate and the like, as well as gluconates and galacturonates (see Berge SM et al., "Pharmaceutical Salts: J. of Pharma. Sci., 1977; 66: 1) .

The acid addition salts of the basic compounds are prepared by bringing the free base form into contact with a sufficient amount of the desired acid in the customary manner to produce the salt. The free base form can be regenerated by contacting the salt form with a base and separating the free base in the conventional manner. The free base forms are somewhat different from their respective salt forms in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free base for the purposes of the present invention.

Pharmaceutically acceptable base addition salts are formed using metals or amines such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N, N'-dibenzylethylenediamine, chloropropane, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine and procaine.

The base addition salt of the acidic compound is prepared by contacting the free acid with a sufficient amount of the desired base in a conventional manner to form the salt. The free acid form can be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner.

The phrase "pharmaceutically acceptable ", as used in connection with the composition of the present invention, is intended to include those that are physiologically acceptable when administered to a mammal (e.g., a human) and that do not generally produce undesirable reactions The molecular body of the composition and other components. Preferably, the term "pharmaceutically acceptable " as used herein is intended to encompass a mammal, particularly a human, approved by a federal or state agency for use in humans, or a US pharmacopeia or other generally recognized Means the one listed in the pharmacopoeia.

The term "carrier" as applied to the pharmaceutical composition of the present invention means a diluent, excipient or vehicle to be administered with an active compound (e.g., memantine). Such pharmaceutical carriers may be sterile liquids such as water, saline, aqueous dextrose solutions, aqueous glycerol solutions, and oils comprising petroleum, animal, vegetable, or synthetic origin oils such as peanut oil, soybean oil, mineral oil, . However, because memantine is very soluble, an aqueous solution is preferred. Suitable pharmaceutical carriers are described in " Remington ' s Pharmaceutical Sciences "by E. W. Martin, 18th Edition. Particularly preferred in the present invention is a carrier which is suitable for immediate release, i. E. Release of most or all of the active ingredient over a short period of time, such as not longer than 60 minutes, and which allows rapid absorption of the drug.

As used herein, the term "subject in need of treatment" means a mammal.

In connection with the method of the present invention, there is also provided a pharmaceutical composition comprising a therapeutically effective amount of memantine. The compositions of the present invention may further comprise a carrier or excipient, all of which are pharmaceutically acceptable. The composition may be formulated for administration once a day, or twice a day.

Memantine (NAMENDA (TM)) is available commercially as hydrochloride salt as a 5 or 10 mg film-coated tablet. However, according to the present invention, the dosage form of memantine may be a solid, semi-solid or liquid preparation according to the following.

Memantine may be administered orally, topically, parenterally, or mucosally (e. G., By oral, by inhalation, or rectally, in unit dosage forms containing conventional, non-toxic, pharmaceutically acceptable carriers, ≪ / RTI > It is usually preferred to use oral routes. In a preferred embodiment by administration to a pediatric individual, memantine is formulated in a directional liquid, for example, peppermint direction. Memantine may be administered orally in the form of a capsule, tablet or the like, or in a semi-solid or liquid formulation (Remington's Pharmaceutical Sciences, Mack 5 Publishing Co., Easton, Pa.). Orally administered medicaments may also be administered in the form of (but not limited to) time-controlled release vehicles, including, but not limited to, diffusion-regulating systems, osmotic devices, dissolution-regulating matrices and caustic / .

When orally administered in the form of tablets or capsules, memantine may be formulated with binders (e.g., pregelatinised corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); Fillers (e.g., lactose, sucrose, glucose, mannitol, sorbitol and other reducing and non-reducing saccharides, microcrystalline cellulose, calcium sulfate or calcium hydrogen phosphate); Lubricants (e. G., Magnesium stearate, talc or silica, stearic acid, sodium stearyl fumarate, glyceryl behenate, calcium stearate and the like); Disintegrants (e. G., Potato starch or sodium starch glycolate); (E.g., acacia, tragacanth or alginate), buffer salts, carboxymethylcellulose, polyethylene glycols, waxes (e.g., sodium lauryl sulfate), colorants and perfumes, gelatin, sweeteners, , ≪ / RTI > and the like.

The tablets may be coated by methods well known in the art. The core may also be coated with a concentrated sugar solution, which may contain, for example, acacia rubber, gelatin, talc, titanium dioxide, and the like. Alternatively, the tablets may be coated with polymers known to those skilled in the art, wherein the polymer is dissolved in a readily volatile organic solvent or mixture of organic solvents. In a preferred embodiment, memantine is formulated into immediate-release (IR) or modified-release (MR) tablets. The immediate-release solid dosage form allows release of most or all of the active ingredient over a short period of time, such as 60 minutes or less, and allows rapid absorption of the drug. Modified-release solid oral dosage forms similarly allow sustained release of the active ingredient over an extended period of time in an effort to maintain therapeutically effective plasma levels and / or alter other pharmacological properties of the active ingredient over prolonged intervals .

For the formulation of soft gelatin capsules, the active substance may be mixed with, for example, vegetable oils or poly-ethylene glycols.

Hard gelatin capsules are prepared by conventional means known to those skilled in the art for the purification of active agents using the abovementioned excipients such as lactose, saccharose, sorbitol, mannitol, starch (for example, potato starch, corn starch or amylopectin), cellulose, Component granules. In addition, a liquid or semisolid of the drug may be filled into the hard gelatin capsule.

The compositions of the present invention may also be incorporated into microspheres or microcapsules prepared from, for example, polyglycolic acid / lactic acid (PGLA) (see, for example, U.S. Patent Nos. 5,814,344; 5,100,669 and 4,849,222; 95/11010 and WO 93/07861). Biocompatible polymers useful for achieving controlled release of a drug include, for example, polylactic acid, polyglycolic acid, copolymers of polylactic acid and polyglycolic acid, polyepsilon caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, Polyhydroxypolypyrroles, polycyanoacrylates, and cross-linked or amphipathic block copolymers of hydrogels.

Since the active ingredient is highly soluble in aqueous media, the formulation of memantine in the form of a semi-solid or liquid is within the skill in the art. Typically, the active ingredient, memantine, may comprise from 0.1 to 99% by weight of the formulation, more particularly from 0.5 to 20% by weight in the case of injectable preparations and from 0.2 to 50% by weight in the case of preparations suitable for oral administration .

In certain embodiments of the present invention, memantine is formulated into an oral liquid preparation. Liquid preparations for oral administration may take the form of, for example, solutions, syrups, emulsions or suspensions, or they may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Formulations for oral administration may be suitably formulated to provide controlled or extended release of the active compound. Specific examples of oral time-controlled release pharmaceutical formulations are described in U.S. Patent No. 5,366,738.

For oral administration in liquid form, memantine is a non-toxic pharmaceutically acceptable inert carrier (e.g., ethanol, glycerol, water), a suspending agent (e.g., sorbitol syrup, a cellulose derivative or hydrogenated edible fats ), An emulsifying agent (e.g., lecithin or acacia), a non-aqueous vehicle (e.g., castor oil, oily esters, ethyl alcohol or fractionated vegetable oils), a preservative (such as methyl or propyl- Phosphate or sorbic acid) or the like. Stabilizers such as antioxidants (BHA, BHT, propyl gallate, sodium ascorbate, citric acid) may also be added to stabilize the dosage form. For example, the solution may contain from about 0.2 to about 20 weight percent memantine, with the remainder being a mixture of sugars and ethanol, water, glycerol, and propylene glycol. Optionally, these liquid preparations may contain coloring agents, fragrances, saccharin, and carboxymethyl-cellulose as a thickening agent or other excipients known to those skilled in the art.

In certain embodiments, a therapeutically effective amount of memantine is administered in an oral solution containing a preservative, a sweetener, a solubilizing agent, and a solvent. Oral solutions of the invention may comprise one or more buffering agents, flavoring agents or additional excipients. In a further preferred embodiment, peppermint or other flavoring is added to the oral liquid memantine preparation.

Preferably, the optimal therapeutically effective amount will depend upon a variety of factors, including the exact mode of administration in which the drug is being administered, the indication for which administration is indicated, the entities involved (e.g. weight, health, age, sex, etc.) and the preference of the physician or veterinarian It should be determined experimentally considering experience.

The toxic and therapeutic efficacy of the compositions of the present invention can be determined by standard pharmaceutical procedures in laboratory animals, for example, LD50 (doses that kill up to 50% of the population) and ED50 (50% ). ≪ / RTI > The dose ratio between the therapeutic effect and the toxic effect is the therapeutic index, which can be expressed as a non-ED50 / LD50. Compositions exhibiting a large therapeutic index are preferred.

The daily dose of the active compound of the present invention suitable for human therapeutic treatment is about 0.01 to 10 mg per kg of body weight when orally administered and 0.001 to 10 mg per kg of body weight in the case of parenteral administration.

In the case of an adult, the appropriate daily dose of memantine is in the range of about 5 mg to about 100 mg / day, preferably about 20 to about 40 mg / day.

In the case of 4 to 14 year old pediatric subjects, memantine is preferably administered in an oral liquid dosage form at a dose of 0.5 mg / kg / day up to about 20 mg / day. It is highly recommended to adjust from a lower initial starting capacity to a maximum capacity over about the fourth week. In the case of liquid oral administration, memantine is dissolved in approximately half of the liquid equivalent of the dose. For example, 12.5 mg of memantine will be dissolved in 10 ml of liquid formulation for administration.

Hereinafter, the present invention will be described.

The present invention is based on the finding that Memantine, an NMDA receptor antagonist, will treat societal deficiency symptoms and epileptic syndromes, which are manifested by a person exposed to valproic acid during pregnancy, suggesting a therapeutic agent for socially deficient symptoms and laying the foundations for the target of psychiatric diseases .

The present inventors have completed the present invention by confirming that Memantine restores the socio-deficiency symptom and convulsive sensitivity increase to a normal level in a rat model of congenital valproic acid exposure syndrome.

The present invention provides that exposure to valproic acid at 12 days of gestation specifically induces neural tube defects (tail deformity), societal deficit symptoms, increased locomotor activity, and increased susceptibility to convulsions in rats.

Hereinafter, the present invention will be described in detail.

On the 12th day of gestation, the present inventors injected 400 mg / kg of sodium valproate (Sigma) subcutaneously with 280 g of SD female rats to prepare an animal model exposed to congenital valproic acid. Valproic acid is dissolved in physiological saline at a concentration of 100 mg / ml. The female of the rat is born 9 days after exposure to valproic acid (on the 21st day of pregnancy) and lives with the mother rat until 28 days after birth. 79% of the rats in the rats exhibited a caudate anomaly (Fig. 1). Memantine 30 mg / kg is intraperitoneally injected into a newborn 28 days old. Memantine is dissolved in physiological saline at a concentration of 3 mg / ml. After 30 minutes of administration of Memantine, 1) locomotor activity, 2) sociability, and 3) seizure threshold were measured. As a result, it was confirmed that the exercise activity which was increased by the exposure of valproic acid was decreased by administration of memantine (see FIG. 2). F (1, 28) = 1.494, P = 0.2318], but decreased by administration of Memantine [F (1, 28) 8.189, P = 0.0079]. In the post-hoc comparisons, the pedophysial vehicle group had more distance (P <0.05) than the control group (P <0.05) and the distance between the pedophylic memantine group and the valproic acid vehicle group was shorter ). There was a significant correlation between valproic acid exposure and Memantine dose [F (1, 28) = 5.712, P = 0.0238]. [F (1, 28) = 2.570, P = 0.1201], but decreased by administration of Memantine [F (1, 28) = 5.731, P = 0.0236 ]. In the post-hoc comparisons, the valproic acid vehicle group had more time (P <0.01) than the control group (P <0.01) and the valproic acid memantine group had less time (P <0.001) . There was a significant correlation between valproic acid exposure and Memantine administration [F (1, 28) = 8.010, P = 0.0497].

Exposure to valproic acid during pregnancy decreased the sociability of the rats. The time spent on the Stranger 1 side decreased compared to the control, and the time spent on the Empty side increased (see FIG. 3). The time spent on the Stranger 1 side did not change by exposure to valproic acid [F (1, 28) = 2.893, P = 0.1000] and was increased by administration of Memantine [F (1, 28) = 10.49, P = 0.0031 ]. Post-hoc comparisons showed that the valproic acid vehicle group remained on the Stranger 1 side for a shorter period of time than the control group (P <0.05) (P <0.001), respectively. There was a significant correlation between valproic acid exposure and Memantine administration [F (1, 28) = 6.159, P = 0.0193].

The time spent in the central compartment was increased by exposure to valproic acid [F (1, 28) = 36.96, P <0.0001] and decreased by administration of Memantine [F (1, 28) = 15.28 P = 0.0005]. Post-hoc comparisons showed that the valproic acid vehicle group had more time (P <0.001) than the control group (P <0.001), while the valproate memantine group remained less than the valproic acid vehicle group <0.001) and had more time (P <0.05) than the control group Memantine. There was a significant correlation between valproic acid exposure and Memantine administration [F (1, 28) = 5.789, P = 0.0230].

The time spent on the empty side was not affected by valproic acid exposure [F (1, 28) = 0.2978, P = 0.5896] or Memantine administration [F (1, 28) = 0.01921, P = 0.8908]. There was no significant correlation between valproic acid exposure and Memantine dose [F (1, 28) = 0.2211, P = 0.6416].

In the social preference test, the time spent on the Novel side decreased and the time spent on the Familiar side increased. These socially deficient symptoms were restored to the control level by Memantine treatment (see FIG. 3). The time spent on the Familiar side was increased by valproic acid exposure [F (1, 28) = 143.1, P <0.0001] and decreased by Memantine administration [F (1, 28) = 198.9, P <0.0001]. In the post-hoc comparisons, the valproic acid vehicle group had more time on the familial side than the control group (P <0.001), and the valproate memantine group had less time than the valproic acid vehicle group <0.001). Memantine administration significantly reduced the time spent on the familial side of control rats (P <0.001). There was a significant correlation between valproic acid exposure and Memantine administration [F (1, 28) = 6.159, P = 0.0193].

The time spent in the central compartment was increased by exposure to valproic acid [F (1, 28) = 11.09, P = 0.0024] and decreased by administration of Memantine [F (1, 28) = 40.50, P <0.0001]. In the post-hoc comparisons, the valproic acid vehicle group had more time (P <0.05) than the control group, and the valproic acid memantine group had less time than the valproic acid vehicle group (P <0.001 ). The time spent in the central compartment of control rats by memantine treatment was significantly decreased (P <0.01). There was no significant correlation between valproic acid exposure and Memantine dose [F (1, 28) = 0.8942, P = 0.3524].

The time spent on the novel side was decreased by valproic acid exposure [F (1, 28) = 35.68, P <0.0001] and was increased by administration of Memantine [F (1, 28) = 170.7, P <0.0001]. Post-hoc comparisons showed that the valproic acid vehicle group spent less time on the novel side than the control vehicle group (P <0.001) and the valproic acid memantine group had more time than the valproic acid vehicle group P <0.001). The time spent on the novel side of control rats was significantly increased by Memantine administration (P <0.01). The duration of stay on the novel side of valproic acid Memantine group was shorter than that of control Memantine group (P <0.01). There was no significant correlation between valproate exposure and Memantine administration [F (1, 28) = 0.9772, P = 0.3314].

 This social recovery effect can be easily confirmed through the Sociability Index (SI) and the Social Preference Index (SPI) (see FIG. 4). SI was significantly decreased by valproic acid exposure [F (1, 28) = 15.52, P = 0.0005] and was increased by administration of memantine [F (1, 28) = 13.79, P = 0.0009]. In the post-hoc comparisons, SI values in the valproic acid vehicle group were lower (P <0.05) than in the control group (P <0.05) . Serum SI levels were increased in the large arm (P <0.05) and in the memantine group (P <0.05). There was no significant correlation between valproic acid exposure and Memantine dose [F (1, 28) = 0.1021, P = 0.7517].

SPI was significantly decreased by valproic acid exposure [F (1, 28) = 83.78, P <0.0001] and was increased by administration of Memantine [F (1, 28) = 120.1, P <0.0001]. Post-hoc comparisons showed that the SPI values in the valproic acid vehicle group were lower than those in the control group (P <0.001), and the SPI values in the valproic acid-exposed group were increased by Memantine (P <0.01) . The SPI level in the major arm was increased (P <0.001) and the SPI level in the valproate arm was lower than the SPI level in the control arm (P <0.001). There was a significant correlation between valproic acid exposure and Memantine administration [F (1, 28) = 39.85, P <0.0001].

 Exposure to valproic acid during pregnancy significantly increased susceptibility to spasm induced by electrical stimulation in rats. These symptoms recovered to the control level by Memantine treatment (see FIG. 5).

From the above results, the present inventors found that, when injected 400 mg / kg of valproic acid subcutaneously to the rats on the 12th day of gestation, the behavioral symptoms such as increase in locomotor activity, lack of sociability, And that the symptoms of the syndrome were successful.

Based on the above results, the present invention provides that memantine can be a therapeutic substance for congenital valproic acid exposure syndrome.

As can be seen from the present invention, the present invention confirms that the NMDA receptor antagonist, Memantine, is an effective substance for treating the behavioral abnormality manifested in the congenital valproic acid exposure syndrome, and it is a psychiatric disorder It can be used to present therapeutic targets and to develop biopharmaceuticals.

FIG. 1 is a diagram showing a case in which a tail deformity, which is a symptom of a neural tube defect, is induced in a rat by exposure to valproic acid during pregnancy. Tissue anomalies are seen by exposure on the 12th day of pregnancy and the rate is 79%. By 9.5 days of pregnancy, the number of births was greatly decreased due to reproductive toxicity, and some of the births did not form a tail.
FIG. 2 shows the results of measuring the locomotor activity in the control group and the volunteers exposed to valproic acid, showing that the increased locomotor activity by administration of memantine is reduced again to the control level. # P < 0.05, # P < control vehicle group. ** P < VPA vehicle group.
FIG. 3 is a graph showing the sociability of the control group and the volunteers exposed to valproic acid, showing that the absence of sociability and social preference in valproic acid-exposed group is restored to the control level by administration of memantine. # P < 0.05, ### P < control vehicle group. * P < 0.05, *** P < VPA vehicle group. beta P < 0.05, beta ' P < 0.01, beta ' control Memantine group. alpha ' P < 0.01, alpha ' P < control vehicle group.
FIG. 4 is a graph showing the social index and social preference index values obtained from the results of FIG. 3. FIG. 4 shows that the index value decreased by valproic acid was restored to the control level by administration of Memantine. # P &lt; 0.05, ### P &lt; control vehicle group. * P < 0.05, ** P < VPA vehicle group. ? P < 0.05,? '' P < 0.001 vs.? control Memantine group. alpha ' P &lt; 0.01, alpha &apos; P &lt; control vehicle group.
FIG. 5 is a graph showing the susceptibility to electrical stimulation induced by convulsions in the control group and in the volunteers exposed to valproic acid. It is shown that the increase in the convulsive sensitivity in the valproic acid-exposed group is restored to the control level by the administration of Memantine.

The present invention will now be described in more detail by way of non-limiting examples. It should be noted, however, that the following examples are illustrative of the present invention and are not to be construed as limiting the scope of the present invention.

Example  1: During pregnancy Valproic Mountain  Congenital due to exposure Valproic Mountain  Exposure syndrome Rat  Model induction

The pregnant rats are kept on the 5th day of pregnancy from the laboratory animal company, Korea BioLink, in a space maintaining constant temperature (22 ± 2 ℃) and constant humidity (55 ± 5%). On the 12th day of pregnancy, 280 g of SD female rats are subcutaneously injected with 400 mg / kg of valproate (Sigma) to make an animal model exposed to congenital valproic acid. Valproic acid is dissolved in physiological saline at a concentration of 100 mg / ml and administered with 1.12 ml of valproic acid solution per 280 g. The control group is administered with physiological saline. For each experimental animal group, the animals from eight pregnant rats were used for the experiment.

Example  2: 4 weeks In the rat Memantine  administration

Memantine 30 mg / kg is intraperitoneally injected into a newborn 28 days old. Memantine is dissolved in physiological saline at a concentration of 3 mg / ml. The average weight of a 28-day-old female is 70 g, and thus 0.7 ml of 3 mg / ml of Memantine is administered based on 70 g of a rat. The control group is administered with physiological saline. After 30 minutes of administration, conduct a behavioral experiment.

Example  3: Congenital Valproic Mountain  Exposure syndrome Rat  Measuring behavioral symptoms in a model

The rats exposed to valproic acid during pregnancy measure their behavioral abnormalities at 4 weeks of age.

<3-1> In the rat  Measurement of locomotor activity Open - field locomotor test )

The measurement of locomotor activity in the rat model follows the following method (Park et al., Eur J Pharmacol, 574, 112-119, 2007); To observe the movement of the rats, a black plastic box with a length of 42 cm, a width of 42 cm and a height of 42 cm is used. The subject's rats are placed in a box and accustomed to the environment for 5 minutes and then measured for activity for 10 minutes. The total distance (cm) and the total time (sec) moved in the box are measured using a device and software to track movement based on the CCD camera (EthoVision 3.1, Noldus information Technology, the Netherlands). For each experimental group, 8 rats were used for the experiment.

<3-2> In the rat  Social measurement Social interaction test ; sociability  & social preference )

The measurement of sociality in the rat model follows the following method (Crawley, Ment Retard Dev Disabil Res Rev, 10, 248-258, 2004); In a transparent box structure divided into three compartments, a round cage made of wire mesh is placed in both compartments, and the experiment is carried out by placing the experimental rat in the middle compartment. Put a homozygous mouse in one cage and leave the other cage empty to compare and analyze the degree of interest in the homologous rat and the degree of interest in the new object (cage). Measurements are performed for 10 minutes using the EthoVision System (Sociability test). The Sociability Index (SI) is defined as the time spent in the Stranger 1 side of the experimental rat, divided by the time in the empty cage (Empty side) (Kim et al. Toxicol Lett., 201, 137-142, 2011).

The social preference test was conducted at the end of the sociability test and immediately after that, the new homologous mouse was added to the empty cage, and the experimental rat showed the degree of interest in the existing mouse and the degree of interest in the new mouse Respectively. Measurements are performed for 10 minutes using the EthoVision System. The social preference index (SPI) is defined as the value of the time spent on the novel side of the experimental rat divided by the time spent on the familial side of the mouse (Kim et al. Toxicol Lett., 201, 137-142, 2011). For each experimental group, 8 rats were used for the experiment.

<3-3> In the rat  Measuring spasticity sensitivity to electrical stimulation

In the rat model, convulsive sensitivity to electrical stimulation is measured by the following method (Park et al., Eur J Pharmacol, 574, 112-119, 2007); Electrical stimulation is given by a mechanism that produces a constant current, and the response by cramp stimulation is determined by whether or not the overt hind limb extension is present. The spiking stimulus starts at 50 mA, and when the spasticity reaction occurs, the next animal will lower 3 mA, and if no spasticity response, the next animal will be given 3 mA higher (Browning et al., Epilepsy Res, 6 , 1-11, 1990). The CC50 (convulsive current 50), which is the stimulation level at which 50% of the animals experience seizures in the experimental group, is calculated using the Litchfield-Wilcoxon II method (Litchfield and Wilcoxon, J Pharmacol Exp Ther, 96, 99-113, 1949). Twelve rats were used for each experiment group.

Example  4: Rat  Binary variance analysis of behavioral test results of model

Values obtained from behavioral experiments are expressed as mean ± standard deviation. Two-way ANOVA analysis revealed a correlation between valproic acid exposure, Memantine dose, or both. If statistically significant effects are found, the significance is tested through post-hoc comparisons using Bonferroni's post-tests method. Statistical analysis was performed using PASW Statistics (18.0; SPSS Inc, Chicago, IL, USA).

Claims (8)

delete delete delete a) injecting valproic acid on the 12th day of pregnancy in pregnant rats;
b) the step of rabbits exposed to valproic acid during the pregnancy being born;
c) injecting memantine to the grown rat; and
d) Performing a social interaction test, an open-field locomotor test, and an electroshock seizure threshold after the above menstrual injection
Methods for the analysis of behavioral symptom treatment effects caused by congenital valproic acid exposure. delete 5. The method according to claim 4, wherein the step (a) of step (a) is administered subcutaneously at a dose of 400 mg / kg. 5. The method according to claim 4, wherein the step (b) of the step (a) is a step of growing until 4 weeks of age. [Claim 5] The method according to claim 4, wherein step c) is performed by intraperitoneally injecting memantine 30 mg / kg to the grown rat.

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