JPS6256134B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the therapeutic application of gangliosids to disorders of nerve impulse transmission in the central nervous system and peripheral nervous system in humans, and to a method for extracting gangliosids from mammalian nervous tissue. Gangliosides contain glycolipids in their molecules in addition to the amino alcohol sphingosine, fatty acids (mainly stearic acid, palmitic acid, arachidic acid, and behenic acid), and simple sugars (glucose and galactose). It has 4 to 4 N-acetylneuramine (sialic acid) residues. Ganglioside is by Billand Steiner
-Landsteiner and Leveuene and confirmed by Klenk in 1942. The method proposed by Svennerholm is the most commonly used nomenclature for gangliosids today. The method is to
The abbreviation G is followed by M, D, T and Q for each di-, tri- and tetrasialylated ganglioside. A letter followed by a number nomenclature is also used to distinguish compounds with the same sialic acid content but different chromatographic behavior. This nomenclature is also used in the specification of the present invention. The term ganglioside is also used to include glycosyl-ceramides containing sialic acid, but not sialyllactosylceramides (ematosides). It is usually included in the broader glycosphingolipids. According to the present invention, the active ingredient gangliosides used in the treatment include mixtures of mono-, di-, tri- and tetrasialylated gangliosides extracted from the nervous tissue of mammals, or more purified gangliosides containing smaller amounts of gangliosides. is used. According to the present invention, when gangliosides are used for treatment, an appropriately prepared aqueous buffer is injected intramuscularly or intravenously. Examples of injections include liquid vials, lyophilized vials, and intravenous injection vials. According to the present invention, gangliosids are extracted from mammalian neural tissue, for example, by the method shown in the Examples below. EXAMPLE 100 kg of bovine brain without membranes is washed with physiological saline and mechanically homogenized in 100 ml of 0.1 M phosphate buffer at pH 6.8. To this, add 1200 g of tetrahydrofuran while stirring vigorously. Continue stirring for 5 hours and separate the liquid phase from the solids using Celite under pressure. Add ethyl ether 400 to the liquid phase while stirring and continue stirring for 60 minutes. Discard the upper layer and concentrate the lower layer (approximately 80°C) under reduced pressure at a temperature below 40°C. The concentrated residue is dissolved in 1 part water/ethanol mixture (volume ratio=1:1). This solution was filtered through celite and DEAE-cellulose column (30 g DEAE-cellulose per 1 kg fresh brain).
Put it on. The DEAE-cellulose column is pre-equilibrated with a water/ethanol mixture. First, elute with 5 volumes of a water/ethanol mixture at a rate of approximately 50 ml/min. Then PH4.6
Elute with 5 volumes of 50% ethanol solution in 0.1 M acetate buffer. The first eluate is discarded and 20 g of barium hydroxide and 4 volumes of cold ethanol are added to the second eluate. The solution is stirred gently for 18 hours at 0°C. The white precipitate produced is separated by centrifugation. Dissolve this precipitate in 500ml of water,
Add HCl to adjust pH to 4.5. This solution is dialyzed and lyophilized. The yield of crude gangliosides at this stage is approximately 0.6 g per kg of fresh brain. This powder was dissolved in 20 parts (W/V) of chloroform/methanol (volume ratio = 2:1), the resulting solution was filtered through Celite, and 0.88% KCl solution in water was
Add and distribute volume with stirring. Separate the upper layer, dialyze and lyophilize. The final ganglioside yield is approximately 0.3 g/Kg of fresh brain. Before using the gangliosides obtained in the above manner for human treatment, they are processed to contain pyrogen, protein, and
The presence of anaphylactogens and histamine must be tested. Examples of formulations for novel therapeutic applications according to the invention include: Content per vial (made of neutral glass, volume 2 ml): - Gangliosides extracted from mammalian nervous tissue (calculated as N-acetyl-neuraminic acid)...X mg - Pyrogen-free sterile distilled water PH used
7.6 phosphate buffer (N/100)...2 ml where X varies depending on the titer of N-acetyl-neuraminic acid, which is 20-35% in the gangliosides used, or depending on the disease to be treated. is the amount of gangliosides, and is 5
The amount ranges from 100 mg to 100 mg. As an example of a typical dosage form, the formulation for a 2 ml ampoule is shown below. Ganglioside mixture (GM ₁ −GD _1a −GD _1b −GT
-GQ) ...10mg dibasic sodium phosphate, 12H ₂ O ...6mg monobasic sodium phosphate, 2H ₂ O ...0.5mg sodium chloride ...16mg pyrodiene-free double distilled water (with addition)
The ampoules thus obtained are usually injected intramuscularly or intravenously at 1 to 5 ampoules per day. According to the present invention, it is believed that gangliosides can be used for the treatment of all diseases caused by disturbances in the transmission of nerve impulses in the central and peripheral nervous systems; notable application aspects are listed below. Posterior medullary neuritis, motor sleep paralysis, trigeminal neuralgia,
Facial paralysis or Bell's palsy, Garcin hemibasilar syndrome, acute polyneuritis (Guillain-Barre syndrome), dorsal radiculitis, traumatic lesions of peripheral nerves, toxic lesions of peripheral nerves, diabetic and alcohol polyneuritis, paralysis of childbirth, paralysis of the sciatic nerve,
Motor neuron disease, amyotrophic lateral sclerosis, dorsal myelopathic muscular atrophy, progressive bulbar palsy, myasthenia gravis (Lambert-Eaton syndrome), muscular dystrophy, synaptic nerves in the central nervous system and peripheral nerves Modulation of transmission, disturbance of consciousness such as daze, concussion, cranial injury prognosis, cerebrovascular accident, thrombosis and embolism. The bipolar chemical composition of gangliosides and their localization on the cell membrane at nerve terminals
As a result of various studies, it is believed that this compound not only affects the chemical composition of nerve cells but also participates in the dynamic function of their receptors. Thus, gangliosids are considered to be extremely important in brain physiology;
This becomes even clearer in light of the fact that the amount of gangliosides in the cortex is known to be altered in many diseases of the central nervous system. In the present invention, extensive pharmacological and clinical experiments were conducted using gangliosides. The animal experiments conducted included protamine paralysis, prolonged barbiturate anesthesia, electric shock and metrazole convulsions, and the results of these tests were completely in line with the researchers' expectations. We also tested whether the brain ganglioside according to the present invention is effective in functional recovery obtained by regeneration or nerve replantation. It was tested whether gangliosids obtained from bovine brain cortex according to the present invention have a protective effect on human hearing loss. In contrast to the control group, subjects who were exposed to a narrow range of 2KHz sounds at 30-minute intervals showed no substantial shift in hearing thresholds. It was found that it has a protective effect against temporary hearing loss caused by such high and strong sounds. Furthermore, clinical experiments were conducted on patients suffering from various neurological diseases. This is because, considering the mechanism of action of the compounds according to the invention, they are effective against all pathological syndromes, the cause of which is known to be an altered stimulus due to inappropriate biochemical transmission or a change in the excitation threshold. This is because it was thought that there was. Thus, based on the pathological consideration that the origin of various different diseases lies in altered neurotransmission of nerve effectors in the central nervous system and peripheral nervous system, clinical trials of the compound according to the present invention were conducted. It was specifically tested on patients suffering from chronic alcoholism, Parkinson's disease, cerebrovascular abnormalities and demyelinating syndromes. A statistically significant difference in the therapeutic effect was observed compared to patients treated with conventional treatment methods. Clinical trials were conducted on a large number of patients suffering from various pathological conditions caused by alterations in neurotransmission in the central nervous system or peripheral nervous system, and the effects of the compound or its preparation according to the present invention were evaluated based on the subjective symptoms of the test subjects. It was evaluated by judgment and therapeutic coefficient based on several parameters. The toxicity of gangliosides is extremely low, and in rats (CFY strain), when subcutaneously injected,
LD ₅₀ is 8.0 g/Kg (body weight) or more, and for intravenous injection it is 2.0 g (1.6-2.1)/Kg (body weight).
In rabbits (New Zealand), the LD ₅₀ for subcutaneous injection is 4.1 (6.31-2.45) g/Kg (body weight) and for intravenous injection is 1.1 (1.315-
0.921) g/Kg (body weight). The efficacy of gangliosides as a medicine has been briefly described above, and the individual test results will be detailed below. Biological and clinical studies of gangliosids A Acute toxicity 1 Acute toxicity in rats 1.1 Subcutaneous injection (a) Experiment 1 CFY rats weighing 112 to 154 g were given 20% W/V of a gangliosid mixture.
The aqueous solution was injected subcutaneously. Maximum dose is 40
ml/Kg weight. The control group received 40ml/Kg of water. (b) Experiment 2 CFY rats weighing 108 to 129 g were injected with a solution of ganglioside mixture at 1.5 and 5.0% W/V in 0.01M phosphate buffer at pH 7.5 at a rate of 40 ml/Kg body weight. Injected subcutaneously. The control group received buffer only. The toxicity signs and mortality were observed for 14 days. Dead rats were visually observed and damaged organs were examined. Results As a result of the preliminary experiment of Experiment 1, LD ₅₀ was 8.0.
It was found that it was more than g/Kg body weight. Therefore, we used 5 male and 5 male rats to spread the dosage. Post-administration observations included piloerection (this was also observed in the control group), arched back posture (hunting), abnormal gait (staggering and toe walking), decreased respiratory rate, and catalepsy. etc. In experiment 2, groups treated with 0.6 g and 2.0 g/Kg body weight of gangliosides (2 female
The mortality rate was 0 for 2 males and 2 males).
Three animals in the 2.0 g/Kg group showed piloerection (same in the control group) and increased respiration immediately after treatment. 2 days after observation, 2.0g/Kg
Hunting and waddling gait were observed in two females in the administration group, and hunting was observed in all animals. Visually, all animals completely recovered 7 days after administration. Weight gain was the same as the control group. Final necropsy results were normal. Conclusion Skin ulcers were observed in rats subcutaneously administered gangliosides at a rate of 8.0 g/Kg using a 20% aqueous solution. This was not observed in the group administered at a rate of 2.0 g/Kg using 5% buffer. At doses of 2.0 g/Kg or less, the mortality rate was 0. LD ₅₀ could not be found. 1.2 Intravenous Injection CFY rats weighing 109 to 156 g were injected intravenously with the ganglioside mixture.
Gangliosides were made into a 20% W/V aqueous solution (under sterile conditions) and injected through the caudal vein. The dose was 2.5 to 10 ml/Kg body weight (full scale test). The control group received water 10
ml/Kg was administered. Observation for mortality and signs of toxicity was made for 14 days. Dead rats were autopsied.
The damaged organs were examined. From the mortality rate, _LD50 was calculated by the Litchfield and Wilcoxon method (Litchfield JTand
Wilcoxon F. (41949), J.pharmc.Exp.
Ther. 96 99−113). Results As a result of preliminary experiments, it was found that LD ₅₀ was approximately 2.0 g/Kg body weight. Based on this,
In order to obtain a more accurate LD ₅₀ , an experiment was conducted using 5 rats of each sex. Symptoms observed after administration were piloerection (same in the control group), hunting, abnormal gait, drowsiness, and decreased respiratory rate. Weight gain was the same as the control group. The results of the autopsy after the test were within normal limits. Conclusion The LD ₅₀ for intravenous injection in rats with 95% confidence limits was 1.8 (1.6-2.1) g/Kg body weight. 2 Rabbits 2.1 Intravenous Injection Forty-six female New Zealand rabbits weighing 1.5 to 2 Kg were injected intravenously (scalar doses) of the gangliosid mixture at a rate of 2 and 0.5 g/Kg. Gangliosides are 4
Sodium chloride was added at the rate of g/
It was used as a 30% solution in 0.01 phosphate buffer.
Observations were made for 7 days after administration. All animals were macroscopically necropsied. Results Catalepsy and drowsiness were exhibited at high doses. At doses of 2 and 1.5 g/Kg all died within 4 days. Conclusion LD ₅₀ is 1.1 (1.315−0.921) g/Kg (P
= 0.05). 2.2 Subcutaneous injection 8g/Kg to 2g/Kg to 27 New Zealand rabbits weighing 1.55 to 1.9Kg
A mixture of gangliosides was injected subcutaneously. Gangliosides were prepared at 20% in 0.01M phosphate buffer with 4g/ml of sodium chloride.
Administered as a solution. The animals were observed for 7 days after administration, and dead animals were macroscopically necropsied. Results: Drowsiness was observed, especially at high doses.
Gross autopsy revealed parenchymal organ abnormalities. That is, gel-like fluid was observed in the subcutaneous tissue, which accumulated in the abdominal cavity. 8g/Kg
The mortality rate is 100%, all after 50 days of administration.
Died in time. Conclusion LD ₅₀ is 4.1 (6.31−2.45) g/Kg (P=
0.05). B Subacute toxicity (4 weeks) 1 Rat (subcutaneous injection) The experiment was conducted as follows. Animal: Charles River CD rat. 25 females and 25 males, 28 days old. Weight 70-80g. The animals were divided into 4 groups with 5 males and 5 females in each group. Gangliosids: fauna groups 2, 3 and 4
150, 250 and 500 mg/Kg/group respectively.
Administered at a daily rate. The gangliosid is PH
7.5 in 0.01M phosphate buffer and administered. Between administration machines: Daily subcutaneous injection for 28 days. Breeding conditions: Placed in a breeding room with a temperature of 21±2℃ and a humidity of 50±5% for 12 hours (8 p.m. to 8 p.m.
12 hours) and darkness for 12 hours.
Water and feed were provided ad libitum. Observations: Observe signs, feed consumption, and weight changes. Group 4 was killed on the 15th day after administration, and the remaining groups were killed at the end of the period and macroscopically necropsied. Results 500 mg/Kg/day group: Subcutaneous thickening, crust formation, hair loss and swelling at the injection site. Reduced food consumption in male and female rats. Reduced weight gain rate in males. Macroscopic examination 15 days later revealed subcutaneous thickening and edema at the injection site.
Enlarged subfluid lymph glands in 1 male and 2 females, 2 males
Paralytic enlargement was observed in the animal. 250 mg/Kg/day administration group: Subcutaneous thickening, swelling, and hair loss at the injection site. Decreased feed consumption (decreased utilization efficiency) and decreased weight gain in both sexes. Visual inspection revealed subcutaneous thickening (4 males, 5 females) and edema (2 males, 2 females) at the injection site.
observed), crust formation (1 male), loss of hair (1 male, 1 female), and enlarged axillary and inguinal lymph glands (5 males, 4 females). 150 mg/Kg/day group: Subcutaneous thickening and hair loss at the injection site. Decreased feed intake in females and decreased utilization efficiency in males. Reduced rate of weight gain (both sexes). Macroscopic examination revealed subcutaneous thickening (4 males, 5 females), edema (1 male), and hair loss (1 female) at the injection site. Enlarged inguinal and/or axillary lymph glands (3 males,
2 females). Conclusion From the above results, 250mg/Kg/
The daily dose was found to be too high. 150mg/Kg/judging from the effect on weight gain (approximately 6 and 10% reduction in males and females, respectively)
Days were found to be suitable as a high dose for long-term administration tests. 2 Dog (intramuscular injection) The experiment was conducted as follows. Animal: Beagle dogs (3 males, 5 females) Administration method: Intramuscular injection Administration period: Within 4 weeks Dose: 160mg/Kg/using 20% aqueous solution
40 mg/Kg/day using 1.5% phosphate buffer solution, 6 mg/Kg/day using 5% phosphate buffer solution, and 20 mg/Kg/day using 5% phosphate buffer solution. Feed: In the morning, give 400g of new feed after dosing and measure the remaining weight from the previous day. Give about 200ml of milk every day except on weekends. Give water ad libitum. Preparation of injection solution: A 20% aqueous solution of gangliosides is adjusted to pH 7.4 with 1N HCl. A 0.01M phosphate buffer (PH7.5) is prepared by mixing a 0.01M potassium dihydrogen orthophosphate solution and a 0.01M disodium hydrogen orthophosphate solution. Add sodium chloride to this buffer solution at a rate of 4 g/ml. 1.5 to 5 using this buffer
% gangliosid solution. Clinical Observation Observation of any signs. Weigh yourself at least once a week. Measurement of unconsumed feed. Whether or not milk is refused. Tests at the end of the period After the end of the administration period, the animals were sacrificed, tissue was observed, and the brain, liver, prostate, uterus, pituitary gland, spleen, kidneys, heart, pancreas, thyroid, lungs, thymus, adrenal glands, and gonads were removed. The weight was measured. These tissues and the aorta (aortic arch and abdominal aorta), trachea, lymph nodes (cervical and mesenteric lymph nodes), gallbladder, stomach (corpus and antrum), duodenum, jejunum, ileum, colon (ascending and descending), skin Preparations were taken from the mammary gland, osseous muscle, bone marrow, sciatic nerve, eye with optic nerve, and muscle at the injection site and placed in fixative. Results and Conclusions The animals in the 160 mg/Kg/day administration group were sacrificed on the 10th day because the pain caused by the administration was severe. As a result of post-mortem autopsy, a significant local reaction was observed at the injection site. Pain was also observed in the 6 mg/Kg/day administration group, but it was slight. There were also no signs of local reactions. Edema of the fat layer and/or pallor of the muscles were also observed at the injection site. Doses of 3, 6 and 12 mg/Kg/day were found to be suitable for long-term administration. Although the minimum lethal dose for intramuscular injection is 160 mg/Kg/day or more, it was found that doses higher than this are not practically preferred. C. Long-term toxicity (26 weeks) 1 Rat (subcutaneous injection) The experiment was conducted as follows. Animals: 200 Charles River rats (100 each sex), 28 days old, weighing 75-85 g. Each group consisted of 20 males and 20 females. Dose: 1st group is control, 2nd group is 6mg/Kg/
30mg/Kg/day for group 3 and 150mg/kg/day for group 4.
mg/Kg/day. Administration method and duration: Subcutaneous injection, daily for 26 weeks. Rearing conditions: Same as for _B1 subacute toxicity Gangliosids: 0.01M phosphate buffer (NaCl
Administer as a solution. Observations: signs, mortality, feed intake, weight changes,
Water intake, ophthalmoscopy, laboratory tests ((a) urine analysis: pH, specific gravity, protein reducing substances, glucose, ketones, bile pigments, urobilinogen and hemoglobin; (b) blood analysis: packed red blood cell volume, hemoglobin red blood cell count,
MCHV±MCV, WBC total white blood cell count. Differential calculation: neurotrophiles, lymphocytes, eosinophils, basophils, monocytes platelet count test. (c) Blood chemistry: serum urea, plasma glucose, serum total protein, serum albumin, phosphatase, pyruvate transaminase, serum sodium, serum potassium, serum chloride, serum calcium, serum inorganic phosphorus, serum creatinine and serum cholesterol. ) Passive cutaneous anaphylaxis test During 26 weeks, 2 ml of blood is taken from the orbital sinus of all surviving rats under light ether anesthesia. All rats in the 150 mg/Kg/day treatment group underwent a passive skin anaphylaxis test. Final Tests Gross Pathology and Organ Weight Distribution After 26 weeks, all surviving animals were sacrificed and tissues were observed macroscopically. The following organs were dissected, fat removed, and weighed: adrenal glands, pituitary glands;
brain, spleen, heart, testicles, kidneys, thyroid, liver, uterus and ovaries. Specimens were taken from the following tissues (including others that were macroscopically abnormal) and preserved in 10% formalin buffer (except for the eyes, which were preserved in Davinson's fixative): adrenal gland, liver, seminal sac, aorta. , lung, bone nucleus muscle,
Bladder, lymph nodes (cervical and mesenteric), skin (normal and injection site), bone marrow, brain (medulla, cerebellum and cortex), mammary glands, spleen, central colon, stomach (glandular and non-glandular), cecum, esophagus, Duodenum, ovaries, testicles, eyes, pancreas, thymus, heart, pituitary gland, thyroid, ileum, prostate, tongue, jejunum, salivary glands, trachea, kidneys, sciatic nerve and uterus. Samples for macroscopic examination were embedded in paraffin wax, sliced into 5μ slices, and all sections were stained with hematoxylin and eosin. Kidney sections were stained with periodic acid Schiff (for basal membranes). Frozen liver and kidney sections were fixed in formalin, cut into 12μ pieces on a cryostat, and fat stained with Oil Red O. Prepare fresh frozen liver sections;
Stained with PAS to demonstrate glycogen. Macroscopic evaluation Histological examination was performed on the following. 1. Abnormal tissue in animals that died 2. Injection site in all animals in the high dose group and 10 males and 10 females in the control group killed after 26 weeks 3. Site of injection in all animals killed after 26 weeks Statistical method Analysis of variance followed by Student's t-test The significance of between-group differences was evaluated. Results The findings of each treatment group compared with the control group are as follows. 150 mg/Kg/day administration group: 1. Thickening of the skin at the injection site, 2. Decreased weight gain rate in both sexes,
In part, there was a decrease in feed intake, a decrease in feed utilization efficiency, a decrease in serum albumin, total protein and A/G ratio in 3-25 and 6-week-old females, and lumbar hypertrophy in rats of both sexes killed after the end of the 4th period. Enlargement of inguinal and axillary lymph glands, increased weight of spleens of male and female rats killed after the 5th period (however, no morphological changes were observed as a result of histological examination);
6. Subcutaneous infiltration of mononuclear cells at the injection site. 30mg/Kg/day administration group: No deaths, feed intake was the same as the control group, weight gain rate was the same as the control group,
Subcutaneous infiltration of mononuclear cells (some with a foamy appearance) was observed at the injection site in 11 out of 20 females. 6mg/Kg/day administration group: No deaths, feed intake was the same as the control group, weight gain rate was the same as the control group,
No changes due to ganglioside administration were observed. Water intake Increased water intake in both sexes in the 150 mg/Kg/day treatment group from 6 to 12 weeks compared to the control group. However, the difference from the control group was not statistically significant, and no particular effect on urination was observed. Laboratory Tests Urine Analysis: At week 25, there were no differences between groups that could be attributed to medication. Blood analysis: At week 25, there were no differences between groups that appeared to be due to medication. Blood chemistry: There were no differences between groups that could be attributed to medication. Conclusion At the injection site, (1) there was subcutaneous infiltration of mononuclear cells with or without foamy cytoplasm in most of the 150 mg/Kg/day group and some in the 30 mg/Kg/day group; (2) 150mg/
Foamy mononuclear cell nests were observed in some lymph glands of the Kg/day administration group. No abnormalities different from those in the control group due to ganglioside administration were observed in the examined tissues. Passive cutaneous anaphylaxis test All sera collected from rats in the 150 mg/Kg/day group that survived for 26 weeks showed no PCA reaction. 2 Dog (intramuscular injection) The experiment was conducted as follows. Animals: 48 beagles (divided into 4 groups, 6 males and 6 females), 5-6 months old and weighing 7.4-12.4 kg. Dosage: Inject intramuscularly by making a solution of the ganglioside mixture (0.01M phosphate buffer (NaCl added)). Once a day for 7 days/week. The first group is the control, the second group is 3 mg/Kg/day, and the third group is 6 mg/Kg/day.
mg/Kg/day, group 4 12mg/Kg/day. The total administration period was one year (52 weeks), but some of the animals were killed after 26 weeks. Breeding conditions: Feed 400g of fresh dry pellet feed per day (in the morning), and measure the remaining amount from the previous day. Food was given 1 hour after dosing. I gave him 200ml of milk every day except on weekends (but only for the first 26 laps). Water was available ad libitum. Observations: clinical findings, mortality, body weight, feed intake,
Water intake, ophthalmoscopy, laboratory tests (blood tests, biochemical tests, urine analysis) Passive cutaneous anaphylaxis test (PCA) Whole blood from dogs in the high dose group for 18 to 26 weeks
10ml of blood was collected. The blood is left to clot for 2 to 4 hours at room temperature and then overnight at 4°C. Separate the serum, aliquot it into small portions, and store it frozen at -20°C until antibody analysis. Next, the IgE antibody titer is measured. Three susceptible animals were used for each serum sample, and if at least two of the three showed a positive (+) blue reaction, it was considered positive. Results (26 weeks after treatment) Death: none, clinical findings: restless and cries during administration. This is the same for the control group, but 12
Significant in the mg/Kg/day administration group, Body weight: No adverse effect on body weight considered to be due to administration, Feed intake: No effect, Ophthalmoscopy: No effect, PCA test: 12mg/Kg/day administration group As a result of testing, it was found that gangliosides were not sensitized. Laboratory tests Blood analysis: There was some slight disturbance in the white blood cell count, but all were within normal limits. Biochemical analysis: Serum cholesterol concentration, including control group, is considered to be at the upper limit of normal
Some cases exceeded 212mg%. Urine analysis: All were within normal limits. Postmortem macroscopic observations: The only finding that appears to be related to ganglioside administration is reddening of the dermis over the injection site muscle. Organ weights: were within normal range. Histological examination: Histopathological changes were observed only at the injection site. Conclusion: No changes that could be attributed to gangliosides were observed in the tissues examined. D Antigenic potency test 1 week for female guinea pigs (Dunkin-Hartley)
Ganglioside mixtures were injected subcutaneously for 4 weeks to test for antigenic potency. Eight guinea pigs weighing 350-400 g were used as one group, except for the second group (10 animals), and 1 ml/Kg of the sample containing gangliosides in phosphate buffer solution was injected subcutaneously: the first group had only the vehicle; Group ovalbumin 100mg/Kg;
Groups 3, 4, 5 and 6 received 1, 10, 100 and 100 mg/Kg of ganglioside mixture, respectively.
administered. The egg albumin administration group was administered only twice in the 1st week and the 3rd week, while the other groups were administered at weekly intervals for 4 weeks. Antigenicity was determined by (1) measurement of guinea pig IgE/IgG _1b antibody titer, (2) measurement of histamine release from isolated lungs after antibody administration, and (3) measurement of bronchoconstriction response in anesthesia-sensitized guinea pigs by antibody administration. I evaluated it. Results Passive cutaneous anaphylaxis: At any dose, ganglioside mixtures
No IgE/IgG _1b antibody titers were raised. Histamine release from removed lungs: Histamine was not released. In vivo administration: In vivo administration of gangliosides had little or no effect on bronchial resistance in 7 of 8 guinea pigs in the 100 mg/Kg dose group. Conclusion Gangliosids are not antigens for guinea pigs. E Fetotoxicity 1 Embryo malformation test 1.1 Rat (subcutaneous injection) Sexually mature rats (SPF, CrL: COBS
CD (SD) BR, Charles River, weight
155-200g) during the 6th and 15th day of pregnancy;
Gangliosides were injected subcutaneously at doses of 0, 50, 100, and 200 mg/Kg/day to examine the effects of gangliosides on pregnancy. Mothers were sacrificed 20 days after pregnancy, parity values were measured, and fetal bone and visceral abnormalities were examined. Gangliosides were used after being dissolved in 0.01M phosphate buffer. Observation After 20 days of pregnancy, the surviving mothers were examined for congenital abnormalities and gross pathological changes in maternal organs. For the ovaries and uterus, (a) number of corpus luteum, (b) distribution of live fetuses, (c) distribution of dead embryos/fetuses, (d) parity group value (from which average fetal weight is calculated), ( e) Fetal abnormalities were investigated. Embryo/fetal death was divided into early stage (finally, only the placenta was observed) and late stage (finally, placenta and embryonic remains were observed). Surviving fetuses were visually observed, weighed, and examined for bone core and visceral abnormalities. Results Local reactions (skin thickening at the injection site,
Eschar formation) was observed. There were no significant systemic reactions, and the piloerection appears to be due to administration difficulties. Pale limbs and slow movements in the 200 mg/Kg administration group appear to be indirect effects of local reactions. In the 200 mg/Kg administration group, feed intake decreased and maternal weight gain decreased.
Feed intake reduction in 100mg/Kg group and 50 or
The reduction in body weight gain in the 100 mg/Kg treatment group was not different from the control group. There were no deaths and pregnancy rates did not differ between groups. Conclusion The following factors were not affected by gangliosids: (a) congenital group values;
Post-implantation losses, mean fetal weight, (b) embryonic and fetal development (in terms of severe malformations), nuclear and visceral abnormalities and bone variants. 1.2 Rabbit (subcutaneous injection) Animal: The following experiment was conducted using sexually mature New Zealand rabbits.
Feed and water intake was ad libitum. Pre-test: 24 to determine optimal dosage
The experiment was conducted using rabbits (4 groups of 6 rabbits per group). The sample was dissolved in 0.01M phosphate buffer containing sodium chloride at a rate of 4g/0, 50, 100 and 200mg/
It was injected subcutaneously at a rate of Kg/day. This treatment continued from the first day of pregnancy until day 13. Malformation test: 0 from 6th day to 18th day of pregnancy,
It was injected subcutaneously at a rate of 10, 20 and 40 mg/Kg. The gangliosid mixture consists of 4
Contains sodium chloride in the proportion of g/
It was used after being dissolved in 0.01 phosphate buffer (PH7.5). Observations All rabbits were sacrificed on day 29 of gestation, the litters were examined by gross necropsy, and individual fetuses were examined internally and externally for bone core and visceral malformations. Autopsies were performed on the parents to examine congenital organ malformations and gross pathological changes. In particular, the ovaries and uterus were examined for (a) the number of corpus luteum, (b) the distribution of viable fetuses, (c) the distribution of dead embryos or fetuses, (d) individual fetal weights, and (e) fetal abnormalities. Ta. Results: No systemic symptoms or weight gain were observed due to ganglioside administration. Pregnancy and mortality rates were also unaffected. Conclusion Ganglioside administration had no effect on the following factors. (a) Parity values, post-implantation losses, mean fetal weight, (b) embryonic and fetal development (in terms of severe malformations), nuclear and visceral abnormalities and bone variants. 2. Intrapartum and postpartum studies 2.1 Rats (subcutaneous injection) 80 sexually mature SPF rats (Crl: COBS CD (SD) BR. Charles River) were incubated at a temperature of 20 ± 4°C and a relative humidity of 50 ± 5%.
It was bred in Animals were given free access to food and water, and artificial irradiation was performed from 8 a.m. to 8 p.m. To investigate the effects of gangliosides on the growth of rats before and after parturition, we conducted a study of 0,
Gangliosides were administered subcutaneously at 25, 50, and 100 mg/Kg/day. The 80 female rats were divided into 4 groups of 20 rats each. The ganglioside mixture was administered as a solution in 0.01M phosphate buffer. Observation: Parents and offspring were killed 21 days after delivery, and abnormalities were observed. From that observation, (a) the number of congenital groups;
(b) Same birth group and average baby rat body weight, (c)
The mortality rate of young rats was calculated. Results: No systemic effects were observed. 100
Thickening of the skin at the injection site (sometimes accompanied by crust formation and hair loss) was observed on the 5th day of administration in the mg/Kg dose group. 100
In the mg/Kg administration group, there was a decrease in body weight gain at the beginning of administration, and this tendency was also seen in the 50 mg/Kg administration group, but the difference from the control group was not significant. There was no effect on gestational age. Conclusions: There was no effect on litter values, cumulative pup mortality, or mean pup weight (from birth to weaning). Although a dose-related decrease in litter weight was observed on days 8 and 12 of calving, there was no significant difference between the groups and this is not considered to be biologically important. Abnormalities and malformations were occasionally observed during post-mortem examination of young rats, but were not due to ganglioside administration. F. Mutagenesis test The following experiment was conducted to test whether gangliosides have a mutagenic effect. 1 Salmonella typhimurium
Ames test using metabolically active and non-metabolically active TA1535, TA1537, TA1538,
As a result of testing the TA98 and TA100 strains at a dose of 10 mg/medium, no mutagenic effect was observed for any of the strains. 2 In vitro tests on mammalian cells: Growth of Syrian baby hamster kidney cells on soft agar. The ganglioside mixture was found not to be carcinogenic based on the criteria used in cell transformation assays. 3 DNA repair test: Induction of DNA repair synthesis in E. coli W3110/polA ⁺ and p3478/polA ^- . No changes were observed in cellular DNA. Gangliosids did not exhibit toxicity to any of the above E. coli bacteria. 4 Saccharomyces
cerevisiae): D5 strain with and without metabolic activity. Results with negative and positive controls were as expected, showing low levels of spontaneous mitotic recombination induced by ethyl methanesulfonate. Ganglioside mixtures have a D5 value of 2 hours after incubation without metabolic activity or 18 hours after incubation with metabolic activity.
showed no toxicity. This test compound did not exhibit dose-dependent recombinant activity up to a saturated solution. This indicates that the test compound has no activity in the beer yeast D5 assay, regardless of whether it has metabolic activity or not. Overall Activity Conclusion Ganglioside mixtures exhibit no mutagenic effects even at their highest concentrations, regardless of whether they are metabolically active. G. Pharmacological experiments In vivo experiments using muscle function measurements showed that gangliosides promote the regeneration and recovery of nerve impulse transmission in autonomic and motor nerves whose innervation has been removed by amputation or cold degeneration. (CECCARELLI)
B.ET COLL., Adv.Exp.Med.Biol. 71
275.1976 and ibid. , 83 283.1977). This experiment also revealed that ganglioside action increases the sensitivity of nerve endings to adrenergic and corinergic stimuli. Based on these experiments, Carmignoto et al. showed in experiments using rats that gangliosides rapidly restore the release mechanism of nerve transmitters and that nerve endings rapidly develop (CARMINOTO G .ET COLL.,
Int.Conf.on Biol.Memb., Crans−Sur−Sierre
1979-Communication). In an extensive study of muscle nerve replantation, Gorio et al. performed intracellular recordings using appropriate electrophysiology techniques (GORIO A. ETCOLL,
Proceeding of International Symposium on
Cholinergic Mechanisms.Florence11−
15March1980.Plenum Press−in Press). This study included histochemical, histological, and morphological studies designed to examine the effects of gangliosids on the differentiation of axons into terminals that can form muscle nerve terminals. Electrophysiological data obtained using rat EDL muscles deinnervated by crushing showed that synapses in ganglioside-treated rats matured faster than in untreated control animals, indicating that the postsynaptic membrane was double-innervated. It was found that the rate was high. Albizzati et al. also examined muscle enzyme activity parameters indicative of nerve regeneration after treatment with gangliosides, using a neurodegeneration and nerve regeneration model (ALBIZZATI
MGET COLL.Peripheral Neuropathies.
Dev.Neurol 1 Ed.by CANAL N.et al.
Elsevier/North Holland (Amsterdam) 1978
year, p453). Caccia et al. also used electrophysiological parameters to confirm the effects of exogenous gangliosides on peripheral nerve trunk regeneration. EDL denervated by cold degeneration
In studies of neurological functional recovery, these authors demonstrate the stimulatory effect of gangliosids extracted from the brain cortex of bovines. That is, they found that gangliosides increased the excitability of survival motor units and stimulated the recovery of neuron trophic control (CACCIAM.R.ET
COLL., Muscle Nerve 2 382, 1979). Furthermore, there is an interesting report on the influence of gangliosids on the regeneration of sensory nerves in the rat tail that have been denervated by amputation (NORIDO F.ET COLL., Submitted to
Experientia1980). Savattieri et al. found that when nerve transmission does not reach the denervated muscle,
We succeeded in showing how exogenous gangliosides act on other factors that change (SAVATTIERI G.ET COLL., XXI
Congress of SIN Catania 1979; Poster).
They looked at amino acid exchange between blood and muscle and increases in acid proteases throughout the animals. The results showed that the former parameter was not affected by gangliosides, but the increase in acid proteases was significantly reduced. Wojcik et al. obtained interesting results on the influence of exogenous gangliosides on the regeneration of the central collinergic pathway after medial-ventral injury of the septum using biochemical and histochemical methods (WOJCIK M.ET COLL. ,Submitted to
Neuroscience1980). Many experiments have demonstrated the action of gangliosides in experimental polyneuritis of various causes (toxic and metabolic).
Aporti et al. showed through in vivo experiments that the conduction velocity of peripheral nerves previously denatured with toxic substances, such as trioltocresyl phosphate, nitrofurantoin, and alcohol, was normalized by the administration of gangliosides (Aporti et al. F.ET COLL.Med.Lav. 68
296.1977). Eulgheroni et al. demonstrated electrophysiologically that exogenous gangliosides have a protective effect on neuropathy induced by carbon sulfide (BULGHER ONIC.ET
COLL., Peripheral neuropathies, Dev.
Neurol. 1 Ed.by Canal N.et al.Elsevier/
North Holland (Amsterdam) 1978.p487). Maroni et al. also evaluated the effects of gangliosides in animals with CS ₂ -induced polyneuritis (MARONI M.ET COLL., Int.
Congr.of Neurotoxicology.Varese 1979−
Communication−). They found earlier nerve fiber recovery and reduced morphological features of the injury in treated animals compared to untreated animals. Piccoli et al. have illustrated the effect of gangliosides in preventing degeneration of isoenzyme LDH activity in the sciatic nerve and retina in alloxan-induced diabetic neuropathy (PICOOLI
F.ET COLL., Ricerca Sientifica ed.
Educazione Permanente.Suppl. 9 71.Univ.
Milano 1978). Norido and Canella studied experimental diabetic neuropathy and treated genetically diabetic mice with gangliosides (daily doses of 1 and 10
mg/Kg for 30 days). As a result, we were able to demonstrate that motor nerve conduction velocity (MNCV) was significantly increased in treated animals compared to untreated animals. Although the effects of gangliosides have not yet been fully elucidated, it is thought that they act on myelin sheaths based on the conduction velocity data (NORIDO F.ET
COLL., Report Fidia Res.Labs). It has long been well known that neural transmission is damaged in demyelinating conditions, and this issue is still being clarified. Other experimental models showing distinct activities of gangliosids in relation to neurotransmission phenomena include
There is hearing loss due to physical damage (noise) or ototoxic drugs such as etbacrynic acid, kanamycin, ouabain, and streptomycin. Such experiments measure various electrophysiological parameters such as auditory nerve action potentials, cochlear microphone potentials and auditory nerve evoked potentials (APOR TI F.ET
COLL., Inner ear biology/biol ogie de
I´Oreille interne.Ed.by Port mann M et al
Inserm (Paris), 68 , p371, 1977).
(MOLINARI G.ET COLL., XIII Congr.Int.
d´Audiologie, Firenz 1976−Communication
−) APORTI F.ET COLL., 15th Workshop
on Inner Ear Biology, Seefeld.Innsbruck
1978−Communication−). H. Clinical experiments A. Central nervous system Lipparini et al. have made an interesting report on acute cerebrovascular disorders (LIPPARINI R.
ET COLL., Min.Med. 66 3774, 1975). Ganglioside activity was investigated in 107 patients with acute CNS vasculopathy. The basic parameters investigated were state of consciousness and motor deficits. Patients received intramuscular injections of 20 mg ganglioside per day for 20-30 days. When measuring primarily the time to functional recovery, patients treated with gangliosides had a shorter recovery time. That is, the time reduction was less than 50% compared to patients treated with neuromodulators and anti-edema agents alone. Miceli et al. investigated the effects of gangliosides in presenile dementia in a double-blind study (MICELI G.ET COLL., Acta Psychiat.
Scand. 55 102, 1977). Neurological examinations were performed on all patients in treated and untreated groups before and after dosing. And compared the two.
Patients treated with gangliosides (20 per day)
mg, 30 days, intramuscular injection) showed statistically significant improvement compared to patients treated with placebo.
The tests conducted were short-term memory, long-term memory,
and the Raven test. Ferromilone et al. conducted a controlled study on 28 patients with neuroses, pyramidal and extrapyramidal syndromes, multiple sclerosis, myelopathy, and general disorders of the brain. In this study, the parameters for improvement in running, reflexes, muscle strength, sensitivity, tremor, and mental function were as follows:
Therapeutic activity was observed after administration for 10 to 30 days. Mazzoni conducted a study on 35 patients with demyelinating syndromes and vascular disorders (MAZZONI S., Gazz.Med.Ital. 135
393, 1976). Thirty untreated patients served as controls. Patients treated with 20 mg of ganglioside per day for 10 to 36 days showed significant improvement in tremor, asthenia, mild motor and sleep symptoms compared to the control group. B. Peripheral Nervous System Treatment of patients with diseases of the peripheral nervous system (PNS) with gangliosides has been shown to protect and restore motor and sensory nerve transmission, and also to improve neuromuscular junctions and functional nerve transmission. A direct effect was seen in restoring transmission. All these effects are conducive to faster and more complete functional recovery. Negrin investigated the therapeutic activity of gangliosides in a controlled study of the clinical and EMG evolution of 24 patients with facial paralysis "a frigore." The course of disability in these patients was evaluated using the anti-inflammatory agent cortisone and vitamins.
Patients with the same symptoms treated with B ₁ , B ₆ and B ₁₂
Compared to 40 people. Patients treated with gangliosides were further divided into groups according to whether their facial nerve damage was neuropractic or axonal. In the former case, 10 mg of ganglioside per day was administered for 3 weeks, and in the latter case, the same dose was administered for a period of at least 4 weeks and up to 10 months. More distinctive results were seen in this latter group. That is, early and long-term administration of ganglioside substantially reduced the risk of permanent, quantitatively and qualitatively severe facial paralysis (limited clinical evidence for continuous hemifacial spasm). and EMG recovery was observed). The cure rate for the ganglioside group was 84%, compared to 34% for the control group.
It was hot. D´Agostini administered gangliosides to 30 patients with peripheral neuritis due to chronic alcoholism.
We conducted a controlled clinical trial of intramuscular injection of 10mg per day (D´AGOSTINI N. Relazione sulla).
clinica del preparato
“GL/5” Per USO intramucolare
deilaboratori FIDIA Farmaceutici di
Abano Terme). This treatment lasted for a period of at least 13 days and up to 32 days. The results were compared with a control group. The dose adopted was found to be adequate to show statistically significant therapeutic activity (Student's t-test). This drug is well tolerated;
There were no side effects. Mazzoni conducted a controlled trial in which 12 patients with alcoholic neuropathy were given 20 mg of ganglioside per day for up to 36 days (MAZZONI S.Gazz.Med.Ital. 135
559, 1976), the results were subjected to Student's t-test, and there was a statistically significant difference between the 10 control group (P<0.01) and the ambulation group (P<0.01). Mamoli et al. 37 patients with alcoholic polyneuritis who presented with sensory and motor deficits.
A controlled clinical trial was conducted to investigate the effects of gangliosides on humans (MAMOLI G.ET COLL., Ricerca
Scientifica ed Educazione Permanente,
Universita di Milano, Suppl. 9
139.1978). Gangliosides in 17 patients
20 mg/day was administered for 4 weeks. The remaining patients served as the control group. All patients underwent neurological and EMG examinations at the beginning and end of the study. Neurological examination showed that patients treated with ganglioside showed clear improvement in paresthesias and Achilles tendon reflexes compared to the control group (P<0.02). Ferromilcne et al. administered gangliosides at a dose of 10 mg per day for 11 to 30 days to 12 patients with polyneuritis and polyradiculoneuritis.
compared to a human control group (FERROMILCNE
F.ET COLL.Gazz.Med.Ital.135 559,
976). Statistical analysis using Student's t-test revealed that tremor (P=0.01), gait (P=0.01), and muscle strength (P=0.01) were significantly improved in the ganglioside treatment group. Azzoni conducted a controlled clinical trial to determine whether gangliosides were protective against vincristine-induced neuropathy in seven patients receiving vincristine for various malignancies. results
The Armitage serial analysis showed that ganglioside administered at a rate of 10 mg per day during vincristine treatment had a clear protective effect on the neuropathies caused by this anticancer drug, particularly loss of tendon reflexes and paresthesias. I found out that I can see it (AZZONI P.
Poiiclinico (Sed.Med.) 85 255 1978). Dantona et al. administered 20 mg of ganglioside per day for 20 days, then 10 mg per day to 40 patients with existing vincristine neuropathy, which is primarily characterized by paresthesias and limb weakness.
It was administered for 10 days. The therapeutic activity of ganglioside was 87%, with decreased paresthesia 4 days after administration (DANTONA A.ET COLL.,
Ricerca Scientifica ed Educazione
Parmanente, Universita di Milano, Supp.
9 155, 1978). Catizone administered gangliosides at 20 mg/day to 10 patients undergoing hemodialysis who had clinical and neuropathic neuropathy.
Administered for 30 days (CATIZONE L.ET COLL.
, Clin.Terap. 85 395, 1978). 10 who have the same symptoms and are being treated only with artificial kidneys
When compared with human patients, the gangliosid-treated group had fewer motor disorders (restless legs, spasms, muscle weakness) and sensory disorders (paresthesias,
Her pain (pain and burning legs) decreased significantly, and she began to feel noticeably happier. Overall improvement in subjective complaints and objective findings regarding motor and sensory disorders was 60% in the ganglioside group compared to only 12% in the untreated control group.
It was hot. Pozza et al. administered ganglioside 20 mg/day for 40 days to 19 insulin-dependent diabetic patients with existing peripheral neuropathy (POZZA G.ET COLL., Ricerca Scietifica
ed Educazione Permanete, Universita
Milano, Suppl 9 115, 1978). A complete electrophysiological examination and autonomic function tests were performed on all patients before and after treatment. There was a stable and statistically significant improvement in electrophysiological parameters, with a decrease in clinical complaints along with an improvement in peak motor conduction velocity (P<0.001).
This observed improvement was independent of changes in carbohydrate balance. Sandrini et al. described 11 patients with polyneuritis of various etiologies (alcoholic, diabetic, diphenylhydantoin-induced).
EMG research was carried out and the effects of gangliosides on nerve impulse transmission (20 mg/day, 30
(SANDRINI ET COLL.,
Ricerca Scientifica ed Educazione
Permanente, Universita di Milano,
Suppl. 9 129, 1978). The EMG parameters selected before and after treatment were the maximal motor conduction velocity in the lateral popliteal nerve and the reflex response (H-reflex) derived from the medical gastrocnemius muscle. Improvements were seen in these parameters, and at the same time, subjective symptoms were reduced. Leoni et al. administered gangliosid to 26 patients with herpes zoster in various locations and investigated the subjective symptoms (hypersensitivity, burning, pain) and objective findings (erythema, vesicle formation, edema, exudate). Observed the effect of gangliosides (LEONI A.ET COLL., Policlinico
(Sez.Med,) 85 428, 1978). Ganglioside was administered at 20 or 40 mg per day for 10 days, but clinically all symptoms disappeared in 4 cases.
The medication was discontinued within 10 days as it completely decreased within 10 days. Improvement in symptoms appeared 2 days after administration and peaked 4 to 6 days later. It turns out that the sooner you start treatment after contracting herpes zoster, the faster your symptoms will improve. Lonati Grillo conducted a controlled clinical trial on 33 pediatric patients (5 days to 16 months of age) with neonatal traumatic disorders of the peripheral nervous system (PNS).
V., Eur.Med.Phys. 13 1, 1977). Ganglioside 5 mg/day was administered in 20 day cycles with a 10 day washout period between cycles. Clinical improvement was assessed in terms of nutritional status, tonus, strength, and spontaneous and reflex motor activity. The treatment group showed significant overall improvement compared to the control group, with functional recovery lasting through the drug washout period and stable improvement when examined six months after the end of the study. Pavanini et al. conducted a controlled clinical trial administering ganglioside to 13 pediatric patients with paresis receiving exercise therapy (PAVANINI G.ET COLL., Eur. Med.
Phys.in.Press, 1980). The dose was 10 mg per day, administered three times in 15-20 day cycles, with a 45-day washout period in between each cycle. Compared to the control group, patients treated with gangliosides showed a clear recovery in motor activity, tonus and nutritional status. Additionally, the time required for functional recovery was shorter in the treatment group than in the control group. Saraceni administered gangliosides by iontophoresis to patients with traumatic peripheral neuropathy and investigated their therapeutic activity (SARACENI V.
ET COLL., Clin.Terap. 85 517, 1978).
This particular method of administration has been found to be effective as an adjunct to standard physical-motor therapy and to be effective in the majority of patients receiving electrical stimulation therapy. Improvement in this case was measured by objective EMG findings (fibrillation-to-intentional activity ratio, spontaneous activity during isometric contractions, peak motor conduction velocity, and antegrade sensory latency). Mingione et al. conducted a controlled clinical trial on 60 patients with altcred conduction of upper extremity nerves due to surgical nerve ablation (MINGIONE A.ET COLL.
Electromyogr.clin.Neurophysiol. 19 353,
1979). The dose was 10 mg per day (later increased to 20 mg per day) and was administered intramuscularly. Dosing cycles were 30 days with a washout period of 10 to 15 days between cycles. Compared to the control group, the medicated group had an earlier recovery of sensorimotor activity and an earlier reduction in pain and paresthesias. Viva administered ganglioside 20 mg/day for 20 days to 56 patients with traumatic or compressive neuropathy of the 15th and 17th cranial nerves (VIVA E.ET COLL., Min. Stomatol. 27
177, 1978). Good results were obtained for patients with trigeminal hypoesthesia secondary to nerve block paresthesia, with clear improvement observed 10 days after administration. 20 or more in patients with systemic hypoglandectomy
Complete recovery of facial nerve function was observed within 30 days, and the characteristic signs (Bell's Negro's sign and platysma sign) disappeared. Patients with axonal rupture of nerve fibers due to deep wounds or neuroplastic lesions made a full recovery in 15 days. Marangolo et al. observed the effects of gangliosides on 84 patients with modulated neurotransmission caused by toxin-infected nerve damage, specifically involving cranial nerves. When the therapeutic activity was compared with that of patients treated with conventional methods, the ganglioside-treated group showed earlier recovery of neurological function, even in what would previously be considered chronic progressive disease. Castellazzo et al., myopic chorioretinal degeneration,
Ganglioside was administered to 74 patients with optic neuritis, optic atrophy, ophthalmoplegia, limbal defects, macular degeneration, and amblyopia (CASTELLAZZO
R.ET COLL., Min.Oftalmol. 20 93,
1978). The dosage is 10 mg per day, 10 or
Injected intramuscularly for 90 days. As a result of examining the degree of functional improvement in many parameters, it was found that recovery was accelerated compared to conventional methods.
A reduction in the duration of ophthalmoplegia was observed, especially in cases of moderate maralytic strabismus. Good results were also obtained in extrinsic ophthalmoplegia and chorioretinal modulation (particularly degenerative ones). Buonfiglio investigated the effect of gangliosides on cerebral syphilis, intoxication or chitosinosis and ocular muscle lesions with an etiology characterized by modulation of neurotransmission of sinile and postoperative variants (BUONFIGLIO R..Min.
Oftalmnl. 18 99, 1976). The dosage is 10- per day.
20mg in 30 day cycles with a 15 day washout period
-It was hot on the 25th. A total of 35 patients were treated with optic nerve paralysis, convergence insufficiency, postoperative motor insufficiency (after surgery for strabismus), toxic optic neuritis, and mild optic nerve atrophy associated with glaucoma or cataract. All of these diseases cause impaired sensory activity, loss of visual acuity, and visual field modulation (concentric stenosis and central scotoma). As a result of treatment, it was found that gangliosides are more effective when administered early in the disease. Patients who showed significant improvement in visual acuity (power) were examined several months after treatment and found that the improvement had not decreased. The mechanism of action of gangliosides suggests that improved vision (power) may represent a more rapid communication between viable retinal cells and corresponding brain structures. This hypothesis was confirmed by the results of the treatment of patients with optic neuritis due to mixed nicotine intoxication from tobacco and alcohol, who had decreased vision (power) and altered visual fields, using gangliosides. That is, in this case,
Significant improvements were seen in both weaving accuracy and perimetry findings. At a later date, this
Buonfiglio also administered ganglioside to 26 patients with extrinsic ophthalmoplegia (10 cases), postoperative muscle failure due to overcorrection (4 cases), amblyopia (2 cases), and convergence insufficiency (10 cases). R.,G.
Ital. Ortottica 5 1977). Gangliosides were injected intramuscularly at 10-20 mg per day. The treatment period was at least 20 days and up to 30 days. The results obtained were good, and gangliosids were found to be effective as an aid to visual acuity training. Feira investigated the effects of gangliosides on compimetric abnormalities in patients with chronic glaucoma (FEIRA C.ET COLL., Min Oftalmol. 20
127, 1978). This is based on the fact that such modulation reflects the difficulty in hypoxic retinal nerve fibers due to ischemia caused by disk compression. Ganglioside 20 in patients with chronic glaucoma who are stable with regard to intraocular pressure and visual field modulation.
mg/day for 30 days. As a result, the scotoma area was significantly reduced. Meduri et al. studied 50 patients with moderate to severe strabismic amblyopia (MEDURI
R.ET COLL., G.Ital.Ortottica 5 97,
1977). 21 patients received orthodontic
pleptic method) alone, while the remaining 29 patients were treated with gangliosides (10 per day) at the same time.
mg, intramuscular injection). Post-treatment psychophysical and bioelectrical changes were examined and both groups were compared. Analysis of psychophysical parameters showed that ganglioside therapy, which was used as an adjunct to orthodontic treatment, increased its therapeutic efficacy. Regarding bioelectrical parameters, (1) the electroretinogram, which was normal in both groups before treatment, showed no significant changes; (2) the optic nerve evoked, which before treatment, showed disease-specific modulations; The potential did not change in the control group, but
The group of patients treated with gangliosides showed a significant increase in the strength of deflection 3, both in the response under conditions of binocular vision and in the response in the amblyopic eye only. The results of these controlled clinical trials show that ganglioside therapy is more effective than conventional treatments (vitamin B complex, cortisone, and anti-inflammatory drugs), especially in severe cases and those requiring hospitalization. It turns out that it is. Effective here means early functional recovery, and in some cases it means recovery from previous chronic symptoms, but in any case, it means shortening the length of hospital stay. It has a rapid and clear effect on symptoms of the sensory cortex associated with peripheral neuropathy caused by infection, poisoning, trauma and mechanical factors.
No other drugs comparable to gangliosides are currently available. Bevilacqua investigated the effects of gangliosides on high-risk patients with otosclerosis undergoing stapedectomy (BEVILAQUA F.
In press Valsava, 1980). 15 patients were treated with ganglioside (10 mg/day,
(administered by intramuscular injection for over 30 days), 12 similar patients were treated with different drugs for the same period of time. Hearing thresholds were measured by audiometry before stapal resection and 10 and 30 days after surgery.
As a result, the postoperative course of patients treated with gangliosides was clearly better than that of the control group, and this is thought to be due to the protective effect of gangliosides on cochlear fluff cells and vascular striae.

Claims (1)

[Claims] 1. A tetrahydrofuran extract of animal nerve tissue homogenized in a phosphate buffer solution, with the ethyl ether eluate removed, is introduced into an ion exchange column, and the aqueous ethanol eluate is treated with barium hydroxide-ethanol to remove the precipitate. After filtering, it is made into an aqueous solution, and after dialysis, the hydrochloric acid acidic solution is dispersed in a mixed solvent of chloroform and methanol. After adding an aqueous potassium chloride solution to this, the upper layer is separated and dialyzed, and if desired, it is further placed in a silicic acid column. , a method for extracting a ganglioside mixture, characterized by elution with a methanol-chloroform mixed solvent.