DE3424781A1 - Use of L-carnosine for tumour treatment - Google Patents

Abstract

The invention relates to the use of L-carnosine or its physiologically tolerated salts for the treatment of tumours.

Description

The development of drugs with anti-tumor effects

currently rests on two main considerations. One is the concept that cancer by preventing the active biosynthesis of nucleic acids in tumor tissue can be inhibited.

Bleomycin (Nippon Kayaku), Mytomycin C (MMC) (Kyowa Hakko Kogyo) and 5-FU (Kyowa Hakko Kogyo) are examples of medicinal substances made on this basis developed in Japan. The other concept is a non-specific active one Immunization therapy or drug therapy that accelerates immunity, whereby use is made of the host's immunity. Pycivanyl (OK-432) (Chugai Pharmaceutical), Maruyama Vaccine (SSM) (Zeria Pharmaceutical) and Krestine (PSK) (Kureha Chemical, Sankyo) belong to the second category.

However, all therapies with these drugs have a disadvantage, which prevents the full achievement of the clinical purpose. In the former case side effects are inevitable because the one that prevents nucleic acid synthesis Effect is not limited to cancer cells and therefore nucleic acid synthesis is also prevented in other tissues.

In immunity-accelerating drug therapy, that is Immune response not specific for cancer cells. Because the immunity accelerating Drugs are non-specific and active immuno-antigenic, which leads to administration of immunity-accelerating drugs only within physiological ones Threshold to an immune response and reactions above the threshold cannot be expected will. Since cancer cells multiply above the threshold of the defense effect, must them by increasing the defense potential to more than the usual physiological Worth and maintaining the balance of immunity and regeneration controlled will. So far it has been an essential aspect in medicine that spontaneous healing cannot be funded.

It was found that L-carhosine is a granulation accelerator Has an effect. Fundamental and clinical studies have now shown that spontaneous healing can be promoted by L-carnosine, so that the growth of tumors is interrupted.

The invention is based on the object of an effective medicament to create treatment for various types of tumors.

This task is accomplished through the use of L-Carnosine or one physiologically acceptable salt thereof dissolved for the treatment of tumors.

L-carnosine (B-alanyl-L-histidine) is made up of L-histidine and B-alanine existing dipeptide found in Liebig's meat extract from Gulewitsch in 1900 has been discovered. It occurs in large quantities in the skeletal muscles of various mammals on. Its physiological importance and pharmaceutical suitability have been recognized by numerous Research groups examined. So far, however, hardly any noteworthy conclusions have been drawn will.

In Japanese Patent Application 122986/1983, not prepublished it is described that with homocarnosine (L-histidinyl-y-aminobutyric acid) the systems from experimental cancer DDY-Sarcoma 180 (Sarcoma 180 is a from Komiyama, Department of Cancer and Pathology, Institute of Kitazato) and by BALB / C-METH-A (METH-A is one held at the Central Laboratory of Daiichi Pharmaceutical, the Hokkaido University belonging tribe) can be treated.

In view of the fact that these two strains are cancerous strains with positive proliferation ability to represent by immunity-accelerating Drugs can hardly be kept under control, it turns out that this new loading action very effective for cancer control in humans can be. The results show that homocarnosine is effective both on the same as well as acting on different systems of the same kind.

It has now been found that L-carnosine, which is in the side chain its structural formula has one less carbon atom than homocarnosine Has anti-tumor effects like homocarnosine.

L-Carnosine is a tasteless and odorless white crystalline powder, which is easily soluble in water, with a melting point of 250 ° C (decomp.); [α] D20 = 20.0 ° (H2O). It has the following chemical structural formula: The pH of its aqueous solution is 8.0 to 8.5.

L-carnosine is found primarily in the skeletal muscles of various mammals in an amount of 0.1 to 0.3%. It is made from carnal food into human Body and is a source of essential supplies Amino acid, L-histidine. It is also biosynthesized from L-histidine and B-alanine. The L-carnosine so absorbed into the body is made with the help of carnosinase broken down into L-histidine and B-alanine, which serve as nutrients. Simultaneously part of it is re-synthesized to L-carnosine. There is an intermediate stage in the biosynthesis of L-carnosine, known as ß-alanyl-1-methylhistidine (anserine) is.

The fact that L-carnosine is a harmless, food-like substance Substance that, after its ingestion by carnosinase, is found in various organs is present, is degraded, represents a decisive difference to the plural of the drugs on the market that are broken down in the liver and thus burden the liver function.

The following statements concern the toxicity of L-carnosine.

Acute Toxicity of L-Carnosine L-Carnosine is peritoneal and oral to test its acute toxicity 5 hours after administration to a group of 10 Administered to mice in different doses. The value LD50 is determined according to the procedure calculated by Van der Waerden on the basis of the mice which died after 72 hours.

In the experiment, L-carnosine is mixed with a physiological saline solution diluted so that its administration concentration is 0.1 to 0.3 ml / 10 g.

About 30 minutes after the peritoneal administration of 15,000 mg / kg (LD100) L-Carnosine occurs a decrease in spontaneous movement that occurs from irregular and decreased breathing. Frontal or outbreak reflexes will however not observed. Intermittent lifting of the tail and convulsions will occur observed in half of the test animals. If the symptoms continue to increase, roll over the animals repeat themselves horizontally and with touch stimulation the reflex becomes hyperreactive with subsequent cramp, which ultimately leads to death as a result of entasia. Half of the test animals were after 90 minutes, 80 % died after 2 hours and all test animals died after 5 hours. Oral administration of 15,000 mg / kg L-carnosine showed no noticeable effect in this regard, one However, mouse out of 10 died after 12 hours.

Table I LD50 of L-Carnosine Dosage LD50 with 95% reliability (mg / kg) Peritoneal 9.087 (8.320-9.925) Oral> 14.930 (minimum lethal dose) The The table above is based on the acute toxicity test of L-carnosine on males dd strain mice (75 hour value). It can be stated that L-carnosine is a has very low toxicity. The harmlessness of L-carnosine is already known. It has been used to treat anorexia or poor appetite for about 10 years by Lisa Ltd.

made in Spain. The effective amount of L-carnosine is transplanted cancer 1 mg / mouse or 50 mg / kg.

Since this corresponds to 1/181 of 9.087 mg / kg of the acute toxicity LD50, the harmlessness of L-carnosine can be seen.

A method for the production of L-carnosine is for example in Journal of Biological Chemistry, 108, 753 (1935). L-carnosine will be by converting carbobenzoxy-ß-alanine with phosphorus pentachloride into Chloride, esterification of the chloride to the methyl ester using methanol, Conversion to the azide with hydrazoic acid, coupling of the azide with L-histidine methyl ester and finally cleavage of the carbobenzoxy radical obtained by catalytic reduction.

Physiologically compatible salts of L-carnosine are also suitable for the purposes of the invention. Salts of the carboxyl group are used as salts of L-carnosine as well as addition salts with physiologically acceptable acids on the amino radical in question. Salts of the carboxyl group with the amino radical are also suitable.

The salts of the carboxyl group include metal salts such as sodium, Potassium, calcium, magnesium, zinc, aluminum, ammonium and substituted ammonium salts, such as trialkylamine salts, e.g. the triethylamine salt. The salts of the amino group include Salts with inorganic or organic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, Acetic acid, propionic acid, lactic acid, tartaric acid, citric acid, succinic acid, Maleic acid, benzenesulfonic acid and toluenesulfonic acid. These salts can according to known Process can be produced, for example by reacting L-carnosine in free Form with the desired acid or base in stoichiometric amount.

The following is an example of the anti-tumor effect of L-carnosine an experiment explained.

Antitumor effects of L-carnosine and OK-432 against sarcoma in the Mouse test animals: five-week-old male DDY mice. Tumor cells: Sarcoma 180 (Komiyama strain, Department of Cancer and Pathology, Institute of Kitazato).

Transplant: -4 5 x 10 4 cells are subcutaneously in the middle Scapular area transplanted (1 hour after cells separated from tumor became).

Administration of the drug: 0.1 ml of physiological saline solution is placed subcutaneously on the back, about 2 cm from the location of the graft, or subcutaneously on the intraperitoneum, 15 times starting 48 hours after the transplant and then at intervals of 48 Hours at each Comparison animal administered. To the laboratory animals are placed subcutaneously on the back, about 2 cm from the location of the graft in the same way as above 1 mg L-carnosine (when given alone), or 0.2 mg, 1 mg and 2 mg L-carnosine (when used together with OK-432), dissolved in 0.1 ml physiological Saline solution, administered. The OK-432 is supplied in an amount of 0.5 KE in 0.1 ml physiological saline solution to the test animals in the same way in both cases as given above.

The results are summarized in Table II. Tabel II Effect of the sole use of L-carnosine and the combined use of L-carnosine and OK-432 on Sarcoma 180 treatment mean over- (1) number of ver (2) death rate,% regression, (3) tumor size, (4) lifespan, animals ended after 60 days mm² days (after 60 days) (20 days) comparison 29.2 12/12 100 545.8 L-carnosine 1 mg 26.4 7/10 70 3 280.1 OK-432 0.5KE 29.8 10/12 83 2 427.3 Ok-432 0.5KE 32.6 10/10 100 423.4 + L-carnosine 0.2 mg OK-432 0.5KE 29.7 6/13 46 7 * 177.8 + carnosine 1 mg OK-432 0.5 KE + L-carnosine 2 mg 29.5 8/11 73 3 330.0 Remarks Re Table II: (1) The mean survival time is the total number of days of life of all test animals divided by the total number of test animals. Since the falls for which regression occurred were excluded from the calculation of the mean survival time are, the effectiveness of the drug can be determined by the fact that the mean survival is related to regression cases.

(2) The number of dead animals is the number of dead animals / number of the test animals.

(3) "Regression" means that the tumor cells were successfully transplanted and clearly grew into a tumor of macroscopic size. Then, however, continued Retraction and eventually molting and the spot could no longer be distinguished will. The cases of regression in the table refer to transplants Tumors identified by autopsy and histological examination 60 days after transplant could no longer be distinguished from normal tissue.

The transplanted tumor cells grew in two out of seven cases did not approach a tumor, but disappeared, like microscopic observation showed from the transplanted area.

(4) The tumor size is as larger diameter x smaller diameter specified in mm2.

In Figures 1 and 2, the duration of the experiment is on the horizontal Axis (1 to 60 days) versus number of surviving animals on the vertical axis applied in%.

Discussion of Experimental Results: Table II and Figures 1 and 2 show that L-carnosine has a marked anti-tumor effect on transplanted Possesses cancer when administered alone. With the gift of L-carnosine in conjunction with OK-432 (a common drug used to boost immunity) shows an even stronger anti-tumor effect. This combination is more effective than the single administration of either L-carnosine or OK-432, with this effectiveness in particular becomes evident in cases of regression. In addition, the effectiveness is also due to the Tumor size recognizable. The Sarcoma 180 strain used is one of the most powerful proliferative strains kept in Japan. The in this attempt at Inhibition shown to such a vigorous tumor strain proves the anti-tumor effect of L-carnosine.

Following the explanation above is the use of L-carnosine itself or the combined use of L-carnosine and another immunity-accelerating agent Medicines, such as OK-432, are effective against cancer of various organs and against malignant ones Tumors such as malignant neoplasms of the gastrointestinal tract, rectum, the Breast, uterus, oral cavity, esophagus, bile, biliary tract (cholangioma), of the pancreas, kidneys (nephromas), prostate, lungs, brain, liver, of the tongue, as well as malignant strumata, thymus tumors, epitheliomata and sarcomata.

The anti-tumor drug of the invention can be used in more usual Way for oral administration or non-oral administration, for example as an injection liquid, Powder, granules, tablets, capsules, in form soluble in the intestine, as an ointment, suppository or pastille can be prepared. The drug can be used individually or in combination given with other active substances, the condition of the patient the type of tumor and symptoms are observed. The clinical dosage for adults is due to of the basic experiments 0.5 to 3.0 g orally per day. The daily dose is preferably divided given at the appropriate time interval.

Since L-carnosine is easily soluble in water, there are no difficulties in the preparation of isotonic aqueous solutions containing 3, 5 or 10% under aseptic conditions. The solutions prepared in this way are under inert gas filled into ampoules and injected using ordinary syringes. It can also freeze-dried L-carnosine, which is packaged in ampoules under aseptic conditions or vial located for injections. To do this, before 3, 5 or 10 percent isotonic solutions are prepared after use. In the form of Powder, granules, tablets or capsules for oral administration can be L-Carnosine in usual Way using binders such as syrup, gum arabic, gelatin, sorbitol, Gum tragacanth or polyvinylpyrrolidone, carriers such as lactose, corn starch, Calcium phosphate, sorbitol or glycine, lubricants such as magnesium stearate, tallow, polyethylene glycol, Hydroxypropylmethyl cellulose or silicon dioxide, disintegrants such as potato starch, and wetting agents such as sodium lauryl sulfate.

Tablets can be coated in a known manner. Ointments in a known way by mixing finely powdered L-carnosine in an amount, which leads to the desired concentration in the finished salts, with an ointment base, such as bleached beeswax, whale wax, anhydrous lanolin, white petrolatum, higher alcohols, macrogols or plastic base (ointment base based on hydrocarbon gel, manufactured by Taisho Pharmaceutical). Hydrophilic ointments, obtained water-absorbent ointments or mixtures thereof. If desired, can Oils, such as sesame oil, peanut oil or olive oil, resinous substances, glycerine, propylene glycol, Wetting agents, germicides, fungicides or antioxidants can be added. The thus obtained Mixture is kneaded to produce L-carnosine ointment of uniform quality.

Suppositories are made in much the same way as ointments. For example, by adding anti- septic acting Substances and L-Carnosine into a melted suppository base, careful Mixing and pouring the mixture into a mold, as well as removing it from the mold solidification can be obtained.

Below are various preparations in which L-carnosine is used as the main active ingredient.

Preparation example 1 (solution for injection) Under aseptic conditions is synthetic L-carnosine in aqueous isotonic solutions with a concentration of 3, 5 or 10% prepared, which is filled into ampoules for injection.

Preparation example 2 (granules) Granules of the following composition are obtained manufactured: component L-carnosine 0.2 lactose 0.34 corn starch 0.45 hydroxypropylmethylcellulose 0.01 granules 1.00 Preparation example 3 (ointment) Using synthetic L-carnosine and a hydrocarbon gel-based ointment base is one. Ointment made with a content of 5%.

Component L-carnosine 5.0 ointment base 95.0 ointment 100.0 Preparation example 4 (suppository) Using synthetic L-carnosine and Hosco S-55 (suppository base by Maruishi Pharmaceutical) suppositories are made.

Ingredient L-Ca rnosine 0.2 p-Oxyethylbenzoate 0.00085 Hosco S-55 1.5 (per suppository) L-carnosine and p-Oxyäthylbenzoat sieved through a sieve with a mesh size vcrt C, 075 mm and gradually added to the suppository base melted at 500C. Included a uniform mixture is obtained, which is poured into a mold at 380C and cured in the refrigerator after cooling to room temperature. The thus obtained Product will. removed from the mold and wrapped in paraffin paper.

The life-prolonging effect, the regression effect of L-carnosine and the synergistic effect of L-carnosine with the potentiator OK-432, was first detected in transplanted cancer cells from Sarcoma 180. in DDY mice. The healing effect of L-carnosine was then also increased by giving it in the form of a suppository shown in three cases of squamous cervical cell carcinoma. The three cases are below listed as clinical examples of therapy with L-carnosine.

The suppositories used in the treatment contain 200 mg L-carnosine per suppository (1.7 g).

The cytological findings are given according to the following classification: Great I: absence of atypical or abnormal cells Class II: atypical cytology, but no indication of malice.

Class III: Cytological evidence of malignancy, which, however, is not are beyond doubt (class III is further subdivided into class IIIa and class IIIb. IIIa is close to benignMeit, while IIIb approaches malignancy.) Class IV: Suspected severe malignancy.

Class V: Definitely malignant.

Case I patient: # 56 years old Pathological: squamous cervical cell c arzinom tissue diagnosis cytoscopy: uterocervical canal: class V portiovaginalis Uteri: Class V Therapy: The treatments are carried out according to the following two plans: 1) Treatments as an outpatient.

A suppository containing L-carnosine is administered once a day or every other day in the vagina near the cervical canal and portio vaginalis uteri inserted.

2) 100 rads. Cobalt 60 Fccus irradiation on an irradiation field of 12 cm² from the front and back once a day or every other day.

Course of therapy: 1st day: On the 1st day of treatment (28 days after the first diagnosis) is with the irradiation with cobalt 60 from the front and back began. A suppository is introduced.

Day 3: On the first day of diagnosis, there will be a moderate amount of bleeding observed from the genital organ, however, 2 days after the administration of Suppositories stops.

Day 45: No vaginal bleeding at all.

local erosion is noted, but no abnormality of the Appendix.

Cytoscopy: Uterocervic Canal: Class II Portio vaginalis Uteri: Class II The focus irradiation with cobalt 60 is 4650 rads up to this point in time. and 20 suppositories had been taken. The total administration of L-carnosine is 4000 mg.

63rd day: The irradiation with cobalt 60 is completed.

Later a Ralstrors therapy (insertion of a piece of radium into the endocervix) at the National Cancer Center Research Institute should, it was decided to continue the treatment with suppositories.

Day 69: Cytoscopy: Uterocervical canal: Class II portio vaginalis Uteri: Klasee IIIa total amount of L-carnosine administered: 6200 mg (31 x) 83rd day: The diagnosis at the National Cancer Center-Research Institute says that the results therapy are satisfactory and that a decision will be made in two weeks whether Ralstron TheraFie is required. It will be the continuation of sole treatment decided with suppositories.

98th day: cytoscopy: uterocervical canal: class I portio vaginalis Uteri: Class I The patient's condition is considered cured; it will however Continuation of therapy nit 1 suppository per day decided.

Total amount of L-carnosine administered: 9400 mg (47 x) 114th day: Tue Diagnosis at the National Cancer Center Research Institute shows further improvement of symptoms since diagnosis on the 83rd day. Ralstron Therapy is not needed for deems.

136th day: cytoscopy: uterocervical canal: class I portio vaginalis Uteri: Class I After that, weekly diagnosis shows excellent cytoscopic results. The patient is cured. Administration of 1 suppository per day per observation however, will continue.

176, day: cytoscopy: lower ocervicyl canal: class I portio vaginalis Uteri: Class I Total amount of L-carnosine administered: 25,000 mg (125 x) Since the diseased area recovered very well, monitoring will continue, however Without histological tissue examination, the decision is made to avoid tissue injuries from specimen collection to avoid. The patient is instructed to continue the weekly exam and to take a suppository when diagnosing.

403rd day: 305 days have been cured since the patient was judged past. The cytoscopic results were continuous in class I. The patient is instructed to have weekly examination and ingestion of a suppository to continue with the diagnosis. The observation continues.

Total intake of L-carnosine until healing: 9400 mg (47 x) irradiation with cobalt 60. 4650 wheel.

It has been 26 months since the healing and the tissue exams always resulted in class I. The observation is continued.

Case II patient: 8 47 years of age Pathological squamous cervical cell carcinoma Tissue diagnosis: cytoscopy: uterocervical canal: class V portiovaginalis uteri: Class V treatment: The treatment is carried out as an outpatient. L-carnosine is given at an amount of 200 mg x 2 per day or every other day. Additionally is irradiated with cobalt 60 as in case 1.

Course of therapy: 1st day: start of treatment with suppositories for 3 days after diagnosis.

Day 9: Beginning of irradiation with cobalt 60 Day 20: Total amount administered L-carnosine: 6,400 mg (16 x) cytoscopy: uterocervical canal: class V Portiovaginalis uteri: Class V The apparition is "in a malignant state".

Day 46: Cytoscopy: Uterocervical canal: Class V vaginal port Uteri: Class V Pathological Tissue Diagnosis: There will be many collapsing cells found, but there are still a noteworthy number of unchanged malignancies Cells left.

Day 56: Total amount of L-carnosine administered: 8,000 mg (20 x) focus irradiation with cobalt 60: 6000 rad Pathological diagnosis: Although many cells multiply had numerous others are destroyed and difficult to identify. They are obviously not cancer cells but inflamed cells.

Day 70: Cytoscopy: Uterocervical canal: Class I portio vaginalis Uteri: Class II Pathological diagnosis: Cancer cells cannot be detected.

Obviously, malignant and unproliferated flat epithelial cells appear established. Other cells are swollen with altered tissue.

The focus irradiation with cobalt 60 is 7350 at this point in time Wheel.

Total amount of L-carnosine administered: 18,800 mg (47 x).

The cancer is cured after 70 days.

Day 88: Cytoscopy: Uterocervical canal: Class I Potio vaginalis Uteri: Class II The patient shows clear improvement; further observation is however required.

21 months after healing shows cytoscopy of uterocervicylic canal and Portiovaginalis Uteri class I. There are no special features in the uterine appendix established.

Case III patient: 8 71 years of age Pathological squamous cervical cell carcinoma Tissue diagnosis: Cytoscopy: Uterocervical canal: Class V Portio vaginali s Uteri: Class V Therapy: treatment as an inpatient.

L-carnosine dosage: 200 mg / day.

Irradiation with cobalt 60 as in case I Course of therapy: 1st day: Start of treatment with L-carnosine and irradiation with cobalt 60 (42 days after the first diagnosis).

Day 28: Pathological diagnosis: Neither malignant symptoms nor cancer cells are detected. Since there are no malignant cells at all, will the patient is considered cured. The observation continues.

Total amount of L-carnosine administered: 5600 mg (28 x) focus irradiation with cobalt 60: 4200 rad.

Summary: The three cases can be summarized as follows: Case no. Total amount administered- Total irradiation days until the patient tes L-carnosine treatment with cobalt 60 is judged to be cured 1 9 400 / mg / 47 times 4650 rad 98 days II 18 800 mg / 47 times 7350 rad. 70 days III 5 600 mg / 28 times 4200 rad 28 days The therapy with 200 mg L-carnosine, administered daily or every other day as a suppository, gives a remarkable effect together with the irradiation with cobalt 60 squamous cervical cell carcinoma.

It cannot be determined with certainty whether the irradiation with cobalt 60 or the treatment with L-carnosine Main share in the therapeutic effects in the cases mentioned. General and scientific Experience makes it unlikely, however, that indirect irradiation of the abdomen is possible with cobalt 60 the observed effects on squamous cervical cell carcinoma in the specified cases. Irradiation with cobalt 60 inhibits proliferation of cancer cells, but at the same time reduces the vital immune immunity and the Reintegration functions.

It follows that cobalt 60 is not the same as in the cases mentioned observed healing of canker sores.

The administration of L-carnosine promotes the physiological defense effect by its active granulation accelerating effectiveness by removing these obstacles to be overcome.

L-carnosine has an anti-tumor effect and heals cancerous ulcers.

Irradiation with cobalt 60 can help.

The healings observed in the cases mentioned are therefore to the a large part of the cancer-inhibiting and the reactivating the physiological defense Effect of L-carnosine.

Since the anti-tumor effects of L-carnosine administered alone already has been proven by experiments with transplanted cancer cells, can the cures observed in the clinical cases mentioned are very likely to be attributed to the effect of L-carnosine.

In cancer, cells proliferate above the physiological threshold Defense effects. To cure cancer is therefore an artificial strengthening of the physiological Defense effects required. Therefore, the anti-tumor effect of L-carnosine is fundamental not limited to the treatment of squamous cervical cell carcinoma, but also applicable to cancers of any other organs.

Claims (4)

II Use of L-carnosine for tumor treatment "claims 1. Use of L-carnosine or a physiologically acceptable salt thereof for Treatment of tumors. 2. L-carnosine or a physiologically acceptable salt thereof for Use in the treatment of tumors. 3. Medicines for use in the treatment of tumors, marked through a content of L-carnosine or a physiologically acceptable salt thereof as an active ingredient. 4. Medicament according to claim 3, characterized by an additional Content of a substance that accelerates the build-up of immunity.