CA1222699A - Pharmaceutical preparation for digestive ulcer - Google Patents
Pharmaceutical preparation for digestive ulcerInfo
- Publication number
- CA1222699A CA1222699A CA000455193A CA455193A CA1222699A CA 1222699 A CA1222699 A CA 1222699A CA 000455193 A CA000455193 A CA 000455193A CA 455193 A CA455193 A CA 455193A CA 1222699 A CA1222699 A CA 1222699A
- Authority
- CA
- Canada
- Prior art keywords
- nucleic acid
- ulcer
- acid
- bcg
- pharmaceutical preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
Abstract
ABSTRACT
DNAs, the salts thereof, the mixtures of DNA and RNA in which the DNA is mostly contained, and the mixtures of the salt of DNA and the salt of RNA in which the salt of DNA is mostly contained are used as active ingredients for pharmaceutical preparations for digestive ulcer, for example, DNA and RNA being obtained from BCG.
DNAs, the salts thereof, the mixtures of DNA and RNA in which the DNA is mostly contained, and the mixtures of the salt of DNA and the salt of RNA in which the salt of DNA is mostly contained are used as active ingredients for pharmaceutical preparations for digestive ulcer, for example, DNA and RNA being obtained from BCG.
Description
PHARMACEUTICAL PREPARATION FOR DIGESTIVE ULCER
Detailed Description of the Invention:
~ The present invention relates to a pharma-ceutical preparation for digestive ulcer containing as active ingredient deoxyribonucleic acid or salts thereof.
Patients of digestive ulcer are increasing these days as a result of enhanced mental stress caused by complication of social life. Much ~f the mechanism and pathophysiology of digestive ulcer is ye,t unknown, and no decisive therapeutic method for this disease has yet established.
Digestive ulcer is at present treated princi-pally by a pharmacotnerapeutical means, wherein antacids, antipeptic agents, or anticholinergic agents are used against digestive tract-attacking factors represented by gastric acid and pepsin.
On the other hand, it is assumed that in a living organism, the onset of digestive ulcer may be accompanied by the exercise of an arti-ulcer therapeutical mechanism, which is considered as one of the protective abilities endowed with living organisms. The idea of this protective ability of living organisms is now attracting interests, and some approaches have been made to encourage this r~ 1!;
~Z~9~
ability/ thereby accelerating the therapy of ulcer.
Extracts from vegetable or animal tissues and synthetic compounds have been found in line with this approach. In recent years, expectations have been enhanced particularly for the medicaments in this field.
As pharmaceutical preparations are usually employed in combination in the therapy of digestive ulcer, the turn up of such medicament is desired that has different characteristics in action and physical properties from those conventionally employed.
Moreover, there is a problem of side-effects when medicaments are administered for a prolonged period to prevent the recurrence of disgestive ulcer, which has a high recurrence ratio.
Much of the pharmacological effects of nucleic acid which is used in the present inventi.on is un-known, and almost nothing has been tried regarding its medical application. The present inventors have been interested in the medical application of nu~leic acid, and as the result of intensive studies have confirmed that nucleic acid, particularly deoxyribonucleic acid, shows a remarkable host-media-ted antitumor e~ect, as well as excellent ~L2Z~9~
safety, and filed a patent application (see Japanese Patent Laid-Open No. 139096/1982).
- In the process of the present study, the inventors examined the intracorporeal distribution of nucleic acid administered to a living organism~
It was notedly observed that nucleic acid was distributed in relatively high concentration in the digestive tracts centering around stomach. As the result of intensive studies regarding the pharma-cological effects of nucleic acid on digestive tracts, the inventors have completed the pr~sent invention based on the finding that nucleic acid remarkably accelerates the cure of digestive ulcer.
It has never been reported heretofore that nucleic acid shows the above-mentioned effect.
~ccordingly, the present invention relates to a pharmaceutical preparation for digestive ulcer containing as active ingredient nucleic acid or salts thereof, more particularly, to a pharma-ceutical preparation for digestive ulcer containing as active ingredient nucleic acid or salts thereof obtained from BCG.
The present invention further relates to a pharmaceutical preparation for digestive ulcer containing as active ingredient deo~yribonucleic - ~22269g acid or salts thereof, more particularly to a pharmaceutical preparation for digestive ulcer containing as active ingredient deoxyribonucleic acid or salts thereof obtained from BCG.
The present invention further relates to a pharmaceutical preparation for digestive ulcer containing as active ingredient a mixture of deoxyribonucleic acid and ribonucleic acid princi-pally comprising deoxyribonucleic acid or salts thereof, more particularly to a pharmaceutical pre-paration for digestive ulcer containing as active ingredient a mixture of deoxyribonucleic acid and ribonucleic acid principally comprising deoxyribo-nucleic acid or salts thereof obtained from BCG.
The nucleic acid to be used in the present invention includes those prepared from known calf thymus, salmon testes r microorganisms, or other natural products, among which those which have ~een already applied to food or medicaments are preferred from the viewpoint of safety. So-called artificially prepared nucleic acids, which are obtained by chemical or biochemical method, can also be employed.
They are represented by deoxyribonucleic acid, ribonucleic acid, mixtures of themr or fractions containing them. It is advisable that the ` ;l~Z2~99 pharmacological and physicochemical properties of each nucleic acid are consistent with the object of ~he present invention and the amount of impurities that may bring about fever or other side effects contained in said nucleic acid is as small as possible~ These nucleic acids may further be suh~
jected to heating, alkaline treatment, or other physicochemical treatments, or nuclease treatment or other biochemical treatments to be more suited as matexial for pharmaceutical preparation. Heat treatment is particularly preferable because of the easiness of operation. The object of these treat-ments is to improve the effect of nucleic acid as the active ingredient of the pharmaceutical prepara-tion of the present invention, facilitate the preparation, and further to hei~hten the solubility of said nucleic acid when it is administered.
These treatments, however, are not inevitable to accomplish the obiect of the present invention.
Method of the preparation of nucleic acid will now be described as Referential Examples.
Referential Example 1 Preparation of BCG-derived nucleic acid:
Mycobacterium boris BCG, ATCC 19015, was statically cultured on a meat extract ~lycerin ~2~3~
medium at 37C for 3 weeks, and the culture medium was centrifuged to obtain wet bacill.i. 3.3 kg of said wet bacilli was suspended in a 7-fold amount of a lO mM phosphate buffer solution (pH 7.0) and disrupted under ice-cooling wi~h a DYN0-MI~ (trade name) followed by cent.rifuge at 2~,000 x g for 20 minutes to obtain 21 ~ of cell extract.
63 g of streptomycin sulfate was added to said extract, mixed with suf~icient agitation and left to stand overnight at 4C, and the formed pre-cipitates were separated by centrifuge and sus-pended in a 10 mM phosphate buffer (pH 7.0) containing 0.5 M NaCQ.
This suspension was dialyzed against the same bu~fer and then against distilled water to obtain 8 Q of a suspension containing nucleic acid.
An equal amount of a 1.8% NaCQ was added to l Q of the suspension, mixed under stirring, and heated at 100C for 60 minutes. After the sus-pension was cooled, it was centrifuged at 10,000 x g for 20 minutes and the supernatant was separated, whereto NaCe was added so that the final concentra-tion was 0.4 M, and ag.itated, Cetyltrimethylammonium bromide (manufactured by Tokyo Kasei Kogyo) was iL2~2~99 ~ 7 --further added to the solution so that the final concentration was 0.2~ (w/v), mixed with sufficient agitation, and left to stand at room temperature for 30 minutes. The formed precipitates were collected by centrifuge and dissolved in 400 m~
of a l M NaC~.
~n equal amount of a chloroform-isoamyl alcohol (24:1) mixture was added to said solution, shaken, and centrifuged to separate the aqueous phase. After this operation was further repeated twice, a three fold amount of 99.5~ ethanol was added to the obtained aqueous phase, mixed under stirring, and left to stand overnight at 4C.
The formed precipitates were collected by centri-fuge, dissolved in distilled water, dialyzed against distilled water, and freeze-dried to obtain 1.04 g of BCG-derived nucleic acid. 'rhe thus obtained nucleic acid comprised 70~ of deoxyribonucleic acid, 28~ of ribonucleic acid, and trace amounts of other components. After deoxyribonucleic acid and ribonucleic acid had been fractionated (see Schneider, W. C. (1946) J. Biol.
Chem. 164, 747), the nucleic acids were subjected to quantitative determination. Deoxyribonucleic acid was determined by diphenylamine method (see ~ ~22~6~
Burton, Ko (1956) Biochem. ~. 62, 3, 5) with calf thymus deoxyribonucleic acid as a standard, and ribonucleic acid was de~ermined by orcinol method (see ~ejbaum, WO (1939) Hoppe-Seyler's Z. Physiol.
Chem. 258, 117) with yeast ribonucleic acid as a standard. (The determinations were performed in the same manner in the following examples.) Referential Example 2 Preparation of Bacillus-derived nucleic acid:
Bacillus subtilis, ATCC 6633, was cultured ~ . _ under shaking on a peptone medium at 37C for 6 hours and the culture medium was centrifuged to obtain wet bacilli. 98 g of the wet bacilli obtained was suspended in 100 mQ of a phosphate bu~fer (pH 7.0) and a Bacillus-derived nucleic acid solution was prepared in the same manner as in Referential Example 1. The solution obtained was neutralized with lN NaOH and freeze-dried to o~tain 158 mg of dried sample. 90 mg of it was disso~ved in 10 mQ of a 0.05 M acetate buffer (pH
4.5), whereto 200 U of ribonuclease T2 (manufactured by Sankyo) dissolved in 2 mQ of said buffer was added and incubated at 37C for 22 hours.
An equal amount of chloroform-isoamyl alcohol (24:1) mixture was added to the reaction liquid, -- ~222~9g shaken, and centrifuged to se.parate the aqueous layer. After this operation was repeated, the total amount of the aqueous layer was loaded on a (~ column (2.5 x 90 cm) of Sephadex G-100 (manufac-tured by Pharmacia Fine Chemicals, Inc.) previously equilibrated with a 0.5 M ammonium bicarbonate and eluted with said solution.
The fraction containing deoxyribonucleic acid, which had been eluted first, was obtained and dialyzed against distilled water.
The dialyPate was neutralized with 1 N NaOH
and freeze-dried to obtain 72 mg of a sodium salt of Bacillus-derived nucleic acid.
The nucleic acid thus obtained substantially comprised deoxyribonucleic acid.
Referential Example 3 The BCG-derived nucleic acid obtained in Reerential Example 1 was digested with ribonuclease (manufactured by Sigma Corp.) and further with pronase, and shaken with chloroform for deproteini-zation. The aqueous phase of this deproteinized nucleic acid was fraction-purified with a column of Sepharose CL 6B (manufactured by Pharmacia Fine Chemicals, Inc.) to be used in the Tests. The deoxyribonucleic acid content o the obtained ~~r~i~ mA~
`-` 12;~;~6~9 nucleic acid was not less than 98%.
Referential Example ~
- The BCG-derived nucleic acid obtained in Referential Example 1 was digested with deoxyribo-nuclease 1 (manufactured by Worthington Corp.) and shaken with chloroform for deproteinization. The aqueous phase of the deproteinized nucleic acid was used in the Tests after being subjected to fraction-purification with Sephadex G-50 (manu~
factured by Pharmacia Corp.) column. The ribo-nucleic acid content of the obtained nucleic acid was not less than 98%.
As understood from the results of the Tests shown below, the acceleration of the cure of ulcer is considered to be one of the pharmacological e~fects of nucleic acid. Moreover, as demonstrated in Tests 1 and 3, the effect o~ acceleraiing the cure of ulcer of nucleic acid is mostly attributable to the effect o~ deox~ribonucleic acid, because less remarkable ef~ects are shown when only ribo-nucleic acid is administered than when only deoxyribonucleic acid is administered. It is noted, however, that the incorporation of an appropriate amount of ribonucleic acid with deoxyribonucleic acid exeri~-s a slightly greater effect than the use ~ ~L2~Z699 of either one of them.
The pharmaceutical preparation of the present invention shows a remarkable effect in curing the acetic acid ulcer of rats, which is considered as a morphologically similar model of human ulcer.
Moreover, it is understood that the effective dose of the preparation ranges widely.
The pharmaceutical preparation for digestive ulcer of the present invention have extremely low acute toxicity and are safe in antigenicity. It has been con~irmed as the result of the Tests that pyrogenicity, pain, prophlogistic properties, or other troubles are so slight as to be neglected in ordinary application as medicament.
As described above, since the pharmaceutical preparation of the present invention acts also tumoricidally, they are suitable for the cure of the case of digestive ulcer, for example, gastric ulcer which is proceeding to precancer or further to gastric cancer.
The pharmaceutical preparation for digestive ulcer of the present invention can be applied by itself or in combination with ordinarily employed additives or excipients. Dose, method, and route of administration are selected according to the case. Generally, it is advisable to administer 0.001 to 100 mg of it in one dose, at one- to seven-day intervals. The route of administration can be selected from intradermal, hypodermic, intramuscular, intravenous, oral, or direct administration to the seat of the disease.
The present invention will be more readily understood by the following Examples and the versatility of the present invention is further demonstrated by the results of the Tests shown below.
Example 1 Liquid preparations:
100 mg of the BCG-derived nucleic acid obtained in ~eferen lal Example 1 was dissolved in 100 mQ of phosphate buffered saline (manufactured by Nissui Seiyaku Co.) and filtered under sterile conditions using a Nuclepore filter (0.2 ~m; manuactured by Nuclepore Corp.). 1.5 m~ portions of the obtained filtrate were poured into vial bottles under sterile condi-tions to prepare the liquid preparation of the present invention.
Example 2 Lyophilized preparations:
100 mg of the BCG-derived nucleic acid was dissolved in 100 m~ of distilled water for injec-tlon, whereto 5 g of mannitol was added and tr~i~ marK
~ 12~2699 ~ 13 -dissolved, and filtered under sterile conditions using a Nuclepore filter (0.2 ~m). After l mQ
portions of the obtained filtrate were poured into vial bottles under sterile conditions, the solutlon was freeze-dried to obtain lyophilized preparation of the present invention.
Test 1 ,.
Effect of nucleic acid on acetic acid ulcer:
Acetic acid ulcer which was the rat chronic ulcer model, was prepared in the stomachs of male Wistar rats according to the method of Okabe et al. (Okabe, S. et al. Amer. J. Dig. Dis. Vol. 16, 277 (1971~) to examine the effect of a variety of nucleic acids. The nucleic acids dissolved in a physiological saline were hypodermically administered to the backs o~ the rats 6 times eve~y other day since the day following the opera-tion. The stomachs were delivered on the day following the final administration (the 12th day ater the operation) and lightly ~ixed with ~ormalin, and the maior and minor diameters of the ulcerated area were measured. The ulcer index and curative ratio were calculated rom the following formulae:
Ulcer index (mm2) = major diameter (mm) x minor diameter (mm) 12~;~699 Curative ratio (%) ulcer index of the ulcer index of the animals _ (control animals administered with the drug) ~ x 100 ulcer index of the control animals The results are summarized in Table 1.
Table 1 Amount of Number of Ulcer index Curative Sampleadministration 2 ratio , animals (average~SE: mm ) ln one dose ~g) (~
Control ~a) - 8 19.4+3.3 E. coli- 200 7 14.0+3.5 28 derived DNA (b) Bacillus- 200 7 12.9~2.6 33 derived NA (c) Salmon testes- 200 7 15.8+3.9 19 derived DNA (d) Calf thymus-200 7 17.1+3.1 12 Bakers yeast- 200 7 16.5~3.8 15 derived RNA (f) -(a) Administered with a physiological saline solution alone (b) The nucleic acid prepared from E. Coli K-12 according to the method of Marmur (see J~
Marmur, J. Mol. Biol. Vol. 3, 208 (1961)) was employed 1;~2~2699 (c) The Bacillus-derived nucleic acid obtained in Referential Example 2 was employed (d)- Salmon testes DNA (Type III, manufactured by Sigma Corp.) was employed (e) Calf thymus DNA (Type I, manufactured by 5igma Corp.) was employed (f) Bakers yeast RNA (Type III, manufactured by Sigma Corp.) was employed Test 2A
Effect of BCG-derived nucleic acid on acetic acid ulcer (Part l) The effect of the BCG-derived nucleic acid obtained in Referential Example 1 was examined ln the same manner as in Test 1. The results are summarized in Table 2.
Table 2 . . _ Amount of Wumber of U~cer index Curative administration 2 ratio in one dose ~g) animal- ~average+SE: mm )
Detailed Description of the Invention:
~ The present invention relates to a pharma-ceutical preparation for digestive ulcer containing as active ingredient deoxyribonucleic acid or salts thereof.
Patients of digestive ulcer are increasing these days as a result of enhanced mental stress caused by complication of social life. Much ~f the mechanism and pathophysiology of digestive ulcer is ye,t unknown, and no decisive therapeutic method for this disease has yet established.
Digestive ulcer is at present treated princi-pally by a pharmacotnerapeutical means, wherein antacids, antipeptic agents, or anticholinergic agents are used against digestive tract-attacking factors represented by gastric acid and pepsin.
On the other hand, it is assumed that in a living organism, the onset of digestive ulcer may be accompanied by the exercise of an arti-ulcer therapeutical mechanism, which is considered as one of the protective abilities endowed with living organisms. The idea of this protective ability of living organisms is now attracting interests, and some approaches have been made to encourage this r~ 1!;
~Z~9~
ability/ thereby accelerating the therapy of ulcer.
Extracts from vegetable or animal tissues and synthetic compounds have been found in line with this approach. In recent years, expectations have been enhanced particularly for the medicaments in this field.
As pharmaceutical preparations are usually employed in combination in the therapy of digestive ulcer, the turn up of such medicament is desired that has different characteristics in action and physical properties from those conventionally employed.
Moreover, there is a problem of side-effects when medicaments are administered for a prolonged period to prevent the recurrence of disgestive ulcer, which has a high recurrence ratio.
Much of the pharmacological effects of nucleic acid which is used in the present inventi.on is un-known, and almost nothing has been tried regarding its medical application. The present inventors have been interested in the medical application of nu~leic acid, and as the result of intensive studies have confirmed that nucleic acid, particularly deoxyribonucleic acid, shows a remarkable host-media-ted antitumor e~ect, as well as excellent ~L2Z~9~
safety, and filed a patent application (see Japanese Patent Laid-Open No. 139096/1982).
- In the process of the present study, the inventors examined the intracorporeal distribution of nucleic acid administered to a living organism~
It was notedly observed that nucleic acid was distributed in relatively high concentration in the digestive tracts centering around stomach. As the result of intensive studies regarding the pharma-cological effects of nucleic acid on digestive tracts, the inventors have completed the pr~sent invention based on the finding that nucleic acid remarkably accelerates the cure of digestive ulcer.
It has never been reported heretofore that nucleic acid shows the above-mentioned effect.
~ccordingly, the present invention relates to a pharmaceutical preparation for digestive ulcer containing as active ingredient nucleic acid or salts thereof, more particularly, to a pharma-ceutical preparation for digestive ulcer containing as active ingredient nucleic acid or salts thereof obtained from BCG.
The present invention further relates to a pharmaceutical preparation for digestive ulcer containing as active ingredient deo~yribonucleic - ~22269g acid or salts thereof, more particularly to a pharmaceutical preparation for digestive ulcer containing as active ingredient deoxyribonucleic acid or salts thereof obtained from BCG.
The present invention further relates to a pharmaceutical preparation for digestive ulcer containing as active ingredient a mixture of deoxyribonucleic acid and ribonucleic acid princi-pally comprising deoxyribonucleic acid or salts thereof, more particularly to a pharmaceutical pre-paration for digestive ulcer containing as active ingredient a mixture of deoxyribonucleic acid and ribonucleic acid principally comprising deoxyribo-nucleic acid or salts thereof obtained from BCG.
The nucleic acid to be used in the present invention includes those prepared from known calf thymus, salmon testes r microorganisms, or other natural products, among which those which have ~een already applied to food or medicaments are preferred from the viewpoint of safety. So-called artificially prepared nucleic acids, which are obtained by chemical or biochemical method, can also be employed.
They are represented by deoxyribonucleic acid, ribonucleic acid, mixtures of themr or fractions containing them. It is advisable that the ` ;l~Z2~99 pharmacological and physicochemical properties of each nucleic acid are consistent with the object of ~he present invention and the amount of impurities that may bring about fever or other side effects contained in said nucleic acid is as small as possible~ These nucleic acids may further be suh~
jected to heating, alkaline treatment, or other physicochemical treatments, or nuclease treatment or other biochemical treatments to be more suited as matexial for pharmaceutical preparation. Heat treatment is particularly preferable because of the easiness of operation. The object of these treat-ments is to improve the effect of nucleic acid as the active ingredient of the pharmaceutical prepara-tion of the present invention, facilitate the preparation, and further to hei~hten the solubility of said nucleic acid when it is administered.
These treatments, however, are not inevitable to accomplish the obiect of the present invention.
Method of the preparation of nucleic acid will now be described as Referential Examples.
Referential Example 1 Preparation of BCG-derived nucleic acid:
Mycobacterium boris BCG, ATCC 19015, was statically cultured on a meat extract ~lycerin ~2~3~
medium at 37C for 3 weeks, and the culture medium was centrifuged to obtain wet bacill.i. 3.3 kg of said wet bacilli was suspended in a 7-fold amount of a lO mM phosphate buffer solution (pH 7.0) and disrupted under ice-cooling wi~h a DYN0-MI~ (trade name) followed by cent.rifuge at 2~,000 x g for 20 minutes to obtain 21 ~ of cell extract.
63 g of streptomycin sulfate was added to said extract, mixed with suf~icient agitation and left to stand overnight at 4C, and the formed pre-cipitates were separated by centrifuge and sus-pended in a 10 mM phosphate buffer (pH 7.0) containing 0.5 M NaCQ.
This suspension was dialyzed against the same bu~fer and then against distilled water to obtain 8 Q of a suspension containing nucleic acid.
An equal amount of a 1.8% NaCQ was added to l Q of the suspension, mixed under stirring, and heated at 100C for 60 minutes. After the sus-pension was cooled, it was centrifuged at 10,000 x g for 20 minutes and the supernatant was separated, whereto NaCe was added so that the final concentra-tion was 0.4 M, and ag.itated, Cetyltrimethylammonium bromide (manufactured by Tokyo Kasei Kogyo) was iL2~2~99 ~ 7 --further added to the solution so that the final concentration was 0.2~ (w/v), mixed with sufficient agitation, and left to stand at room temperature for 30 minutes. The formed precipitates were collected by centrifuge and dissolved in 400 m~
of a l M NaC~.
~n equal amount of a chloroform-isoamyl alcohol (24:1) mixture was added to said solution, shaken, and centrifuged to separate the aqueous phase. After this operation was further repeated twice, a three fold amount of 99.5~ ethanol was added to the obtained aqueous phase, mixed under stirring, and left to stand overnight at 4C.
The formed precipitates were collected by centri-fuge, dissolved in distilled water, dialyzed against distilled water, and freeze-dried to obtain 1.04 g of BCG-derived nucleic acid. 'rhe thus obtained nucleic acid comprised 70~ of deoxyribonucleic acid, 28~ of ribonucleic acid, and trace amounts of other components. After deoxyribonucleic acid and ribonucleic acid had been fractionated (see Schneider, W. C. (1946) J. Biol.
Chem. 164, 747), the nucleic acids were subjected to quantitative determination. Deoxyribonucleic acid was determined by diphenylamine method (see ~ ~22~6~
Burton, Ko (1956) Biochem. ~. 62, 3, 5) with calf thymus deoxyribonucleic acid as a standard, and ribonucleic acid was de~ermined by orcinol method (see ~ejbaum, WO (1939) Hoppe-Seyler's Z. Physiol.
Chem. 258, 117) with yeast ribonucleic acid as a standard. (The determinations were performed in the same manner in the following examples.) Referential Example 2 Preparation of Bacillus-derived nucleic acid:
Bacillus subtilis, ATCC 6633, was cultured ~ . _ under shaking on a peptone medium at 37C for 6 hours and the culture medium was centrifuged to obtain wet bacilli. 98 g of the wet bacilli obtained was suspended in 100 mQ of a phosphate bu~fer (pH 7.0) and a Bacillus-derived nucleic acid solution was prepared in the same manner as in Referential Example 1. The solution obtained was neutralized with lN NaOH and freeze-dried to o~tain 158 mg of dried sample. 90 mg of it was disso~ved in 10 mQ of a 0.05 M acetate buffer (pH
4.5), whereto 200 U of ribonuclease T2 (manufactured by Sankyo) dissolved in 2 mQ of said buffer was added and incubated at 37C for 22 hours.
An equal amount of chloroform-isoamyl alcohol (24:1) mixture was added to the reaction liquid, -- ~222~9g shaken, and centrifuged to se.parate the aqueous layer. After this operation was repeated, the total amount of the aqueous layer was loaded on a (~ column (2.5 x 90 cm) of Sephadex G-100 (manufac-tured by Pharmacia Fine Chemicals, Inc.) previously equilibrated with a 0.5 M ammonium bicarbonate and eluted with said solution.
The fraction containing deoxyribonucleic acid, which had been eluted first, was obtained and dialyzed against distilled water.
The dialyPate was neutralized with 1 N NaOH
and freeze-dried to obtain 72 mg of a sodium salt of Bacillus-derived nucleic acid.
The nucleic acid thus obtained substantially comprised deoxyribonucleic acid.
Referential Example 3 The BCG-derived nucleic acid obtained in Reerential Example 1 was digested with ribonuclease (manufactured by Sigma Corp.) and further with pronase, and shaken with chloroform for deproteini-zation. The aqueous phase of this deproteinized nucleic acid was fraction-purified with a column of Sepharose CL 6B (manufactured by Pharmacia Fine Chemicals, Inc.) to be used in the Tests. The deoxyribonucleic acid content o the obtained ~~r~i~ mA~
`-` 12;~;~6~9 nucleic acid was not less than 98%.
Referential Example ~
- The BCG-derived nucleic acid obtained in Referential Example 1 was digested with deoxyribo-nuclease 1 (manufactured by Worthington Corp.) and shaken with chloroform for deproteinization. The aqueous phase of the deproteinized nucleic acid was used in the Tests after being subjected to fraction-purification with Sephadex G-50 (manu~
factured by Pharmacia Corp.) column. The ribo-nucleic acid content of the obtained nucleic acid was not less than 98%.
As understood from the results of the Tests shown below, the acceleration of the cure of ulcer is considered to be one of the pharmacological e~fects of nucleic acid. Moreover, as demonstrated in Tests 1 and 3, the effect o~ acceleraiing the cure of ulcer of nucleic acid is mostly attributable to the effect o~ deox~ribonucleic acid, because less remarkable ef~ects are shown when only ribo-nucleic acid is administered than when only deoxyribonucleic acid is administered. It is noted, however, that the incorporation of an appropriate amount of ribonucleic acid with deoxyribonucleic acid exeri~-s a slightly greater effect than the use ~ ~L2~Z699 of either one of them.
The pharmaceutical preparation of the present invention shows a remarkable effect in curing the acetic acid ulcer of rats, which is considered as a morphologically similar model of human ulcer.
Moreover, it is understood that the effective dose of the preparation ranges widely.
The pharmaceutical preparation for digestive ulcer of the present invention have extremely low acute toxicity and are safe in antigenicity. It has been con~irmed as the result of the Tests that pyrogenicity, pain, prophlogistic properties, or other troubles are so slight as to be neglected in ordinary application as medicament.
As described above, since the pharmaceutical preparation of the present invention acts also tumoricidally, they are suitable for the cure of the case of digestive ulcer, for example, gastric ulcer which is proceeding to precancer or further to gastric cancer.
The pharmaceutical preparation for digestive ulcer of the present invention can be applied by itself or in combination with ordinarily employed additives or excipients. Dose, method, and route of administration are selected according to the case. Generally, it is advisable to administer 0.001 to 100 mg of it in one dose, at one- to seven-day intervals. The route of administration can be selected from intradermal, hypodermic, intramuscular, intravenous, oral, or direct administration to the seat of the disease.
The present invention will be more readily understood by the following Examples and the versatility of the present invention is further demonstrated by the results of the Tests shown below.
Example 1 Liquid preparations:
100 mg of the BCG-derived nucleic acid obtained in ~eferen lal Example 1 was dissolved in 100 mQ of phosphate buffered saline (manufactured by Nissui Seiyaku Co.) and filtered under sterile conditions using a Nuclepore filter (0.2 ~m; manuactured by Nuclepore Corp.). 1.5 m~ portions of the obtained filtrate were poured into vial bottles under sterile condi-tions to prepare the liquid preparation of the present invention.
Example 2 Lyophilized preparations:
100 mg of the BCG-derived nucleic acid was dissolved in 100 m~ of distilled water for injec-tlon, whereto 5 g of mannitol was added and tr~i~ marK
~ 12~2699 ~ 13 -dissolved, and filtered under sterile conditions using a Nuclepore filter (0.2 ~m). After l mQ
portions of the obtained filtrate were poured into vial bottles under sterile conditions, the solutlon was freeze-dried to obtain lyophilized preparation of the present invention.
Test 1 ,.
Effect of nucleic acid on acetic acid ulcer:
Acetic acid ulcer which was the rat chronic ulcer model, was prepared in the stomachs of male Wistar rats according to the method of Okabe et al. (Okabe, S. et al. Amer. J. Dig. Dis. Vol. 16, 277 (1971~) to examine the effect of a variety of nucleic acids. The nucleic acids dissolved in a physiological saline were hypodermically administered to the backs o~ the rats 6 times eve~y other day since the day following the opera-tion. The stomachs were delivered on the day following the final administration (the 12th day ater the operation) and lightly ~ixed with ~ormalin, and the maior and minor diameters of the ulcerated area were measured. The ulcer index and curative ratio were calculated rom the following formulae:
Ulcer index (mm2) = major diameter (mm) x minor diameter (mm) 12~;~699 Curative ratio (%) ulcer index of the ulcer index of the animals _ (control animals administered with the drug) ~ x 100 ulcer index of the control animals The results are summarized in Table 1.
Table 1 Amount of Number of Ulcer index Curative Sampleadministration 2 ratio , animals (average~SE: mm ) ln one dose ~g) (~
Control ~a) - 8 19.4+3.3 E. coli- 200 7 14.0+3.5 28 derived DNA (b) Bacillus- 200 7 12.9~2.6 33 derived NA (c) Salmon testes- 200 7 15.8+3.9 19 derived DNA (d) Calf thymus-200 7 17.1+3.1 12 Bakers yeast- 200 7 16.5~3.8 15 derived RNA (f) -(a) Administered with a physiological saline solution alone (b) The nucleic acid prepared from E. Coli K-12 according to the method of Marmur (see J~
Marmur, J. Mol. Biol. Vol. 3, 208 (1961)) was employed 1;~2~2699 (c) The Bacillus-derived nucleic acid obtained in Referential Example 2 was employed (d)- Salmon testes DNA (Type III, manufactured by Sigma Corp.) was employed (e) Calf thymus DNA (Type I, manufactured by 5igma Corp.) was employed (f) Bakers yeast RNA (Type III, manufactured by Sigma Corp.) was employed Test 2A
Effect of BCG-derived nucleic acid on acetic acid ulcer (Part l) The effect of the BCG-derived nucleic acid obtained in Referential Example 1 was examined ln the same manner as in Test 1. The results are summarized in Table 2.
Table 2 . . _ Amount of Wumber of U~cer index Curative administration 2 ratio in one dose ~g) animal- ~average+SE: mm )
2~ .
0 11 15.7+1.7 11 9.3+1.6* 41 200 10 9.0~1.9* 43 2,000 12 9.4+2.4** 40 _ * p< 0.02, ~* p< 0,0 ~ l~Z~699 Test 2B
Effect of BCG-derived nucleic acid on acetic acid ulcer (Part 2) The effect of the BCG-derived nucleic acid obtained in Referential Example 1 and the deoxyribo-nucleic acid and ribonucleic acid separated from said BCG-derived nucleic acid were tested in the same manner as in Test 1. The results are summarized in Table 3.
Table 3 Amount of Curative . . Number of Ulcer index ~i Sampleadminlstratlon animals (average+SE: mm2) ra~
in one dose (~g) (~) Control (a) - 12 15.4+1.8 BCG-derived 100 12 8.9+1.3** 42 NA
DNA (b) 100 12 10.6+1.4** 31 BCG-derived 100 12 13.2+2.5 14 RNA (c) **P<0.05 (a) administered with 5~ mannitol ~b) obtained in Re~erential Example 3 (c) obtained in Reerential Example 4 Test 3A
Acute toxicity test o BCG-derived nucleic acid:
lZ2269~
The BCG-derived nucleic acid obtained in Referential Example 1 dissolved in a physiological saline solution was intravenously administered at a dose level of 1 g per kg of the body weight to male ddY mice which were 5 weeks of age and divided into groups of 10 animals each (weighing 23 g on the average). During the observation period of one week after the administration, no hindrance of weight incxease nor death were observed. It is understood from the result that the 50~ lathal dose LD50 of the present substance in intravenous a~ministratlon is not lower than 1 g/kg.
Test 3B
Acute toxicity test of BCG-derived deoxyribonucleic acid:
The acute toxicity of-the BCG-derived deoxyribo-nucleic acid employed in Test 2B was examined in the same manner as in Test 3A. It is understood rom the result of the test that the S0~ lethal dose LD50 of the present substance in intravenous admin-istxation is not lower than 1 kg/kg.
Test 4A
___ Antigenicity test of BCG-derived nucleic acid:
The BCG-derived nucleic acid obtained in Referential Example 1 dissolved in a physiological -` 122~;9~
saline solution was intradermally administered at a dose level of 1 mg per animal to female Hartley guinea pigs which had been divided into groups of 6 animals each (weighing 350 g on the average) 6 times (3 times a week) for sensitization. Two weeks after the final sensitization, the same BCG-derived nucleic acid dissolved in a physiological saline solution was intravenously administered at a dose level of 10 mg or 2 mg per kg of the body weight. It was understood from the result of the examination of the antigenicity of the present substance by observing the guinea pigs' behaviour around challenge that no anaphylaxis shock was induced at all at the above dose level.
Test_4B
Antigenicity test of BCG-derived deoxyribonucleic acid:
The antigenicity of the BCG-derived deoxyribo-nucleic acid employed in Test 2B was tested in the same manner as in Test 4A. As the result of the test, it was found that the present substance induced no anaphylactic shock.
0 11 15.7+1.7 11 9.3+1.6* 41 200 10 9.0~1.9* 43 2,000 12 9.4+2.4** 40 _ * p< 0.02, ~* p< 0,0 ~ l~Z~699 Test 2B
Effect of BCG-derived nucleic acid on acetic acid ulcer (Part 2) The effect of the BCG-derived nucleic acid obtained in Referential Example 1 and the deoxyribo-nucleic acid and ribonucleic acid separated from said BCG-derived nucleic acid were tested in the same manner as in Test 1. The results are summarized in Table 3.
Table 3 Amount of Curative . . Number of Ulcer index ~i Sampleadminlstratlon animals (average+SE: mm2) ra~
in one dose (~g) (~) Control (a) - 12 15.4+1.8 BCG-derived 100 12 8.9+1.3** 42 NA
DNA (b) 100 12 10.6+1.4** 31 BCG-derived 100 12 13.2+2.5 14 RNA (c) **P<0.05 (a) administered with 5~ mannitol ~b) obtained in Re~erential Example 3 (c) obtained in Reerential Example 4 Test 3A
Acute toxicity test o BCG-derived nucleic acid:
lZ2269~
The BCG-derived nucleic acid obtained in Referential Example 1 dissolved in a physiological saline solution was intravenously administered at a dose level of 1 g per kg of the body weight to male ddY mice which were 5 weeks of age and divided into groups of 10 animals each (weighing 23 g on the average). During the observation period of one week after the administration, no hindrance of weight incxease nor death were observed. It is understood from the result that the 50~ lathal dose LD50 of the present substance in intravenous a~ministratlon is not lower than 1 g/kg.
Test 3B
Acute toxicity test of BCG-derived deoxyribonucleic acid:
The acute toxicity of-the BCG-derived deoxyribo-nucleic acid employed in Test 2B was examined in the same manner as in Test 3A. It is understood rom the result of the test that the S0~ lethal dose LD50 of the present substance in intravenous admin-istxation is not lower than 1 kg/kg.
Test 4A
___ Antigenicity test of BCG-derived nucleic acid:
The BCG-derived nucleic acid obtained in Referential Example 1 dissolved in a physiological -` 122~;9~
saline solution was intradermally administered at a dose level of 1 mg per animal to female Hartley guinea pigs which had been divided into groups of 6 animals each (weighing 350 g on the average) 6 times (3 times a week) for sensitization. Two weeks after the final sensitization, the same BCG-derived nucleic acid dissolved in a physiological saline solution was intravenously administered at a dose level of 10 mg or 2 mg per kg of the body weight. It was understood from the result of the examination of the antigenicity of the present substance by observing the guinea pigs' behaviour around challenge that no anaphylaxis shock was induced at all at the above dose level.
Test_4B
Antigenicity test of BCG-derived deoxyribonucleic acid:
The antigenicity of the BCG-derived deoxyribo-nucleic acid employed in Test 2B was tested in the same manner as in Test 4A. As the result of the test, it was found that the present substance induced no anaphylactic shock.
Claims (4)
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A pharmaceutical preparation comprising as an active ingredient deoxyribonucleic acid or a pharmaceutically accept-able salt thereof mixed with a pharmaceutically acceptable diluent or carrier, the preparation being adapted for the treatment of digestive ulcers.
2. A pharmaceutical preparation comprising as an active ingredient a mixture of a major amount of deoxyribonucleic acid and a minor amount of ribonucleic acid or pharmaceutically acceptable salts thereof mixed with a pharmaceutically accept-able diluent or carrier, the preparation being adapted for the treatment of digestive ulcers.
3. A pharmaceutical preparation as claimed in claim 1, wherein the deoxyribonucleic acid is BCG derived.
4. A pharmaceutical preparation as claimed in claim 2, wherein the mixture of deoxyribonucleic acid and ribonucleic acid is BCG derived.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP58094141A JPS59219235A (en) | 1983-05-30 | 1983-05-30 | Drug for peptic ulcer |
| JP58-094141 | 1983-05-30 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1222699A true CA1222699A (en) | 1987-06-09 |
Family
ID=14102101
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000455193A Expired CA1222699A (en) | 1983-05-30 | 1984-05-25 | Pharmaceutical preparation for digestive ulcer |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US4657896A (en) |
| JP (1) | JPS59219235A (en) |
| CA (1) | CA1222699A (en) |
| DE (1) | DE3418820A1 (en) |
| FR (1) | FR2547500B1 (en) |
| GB (1) | GB2140689B (en) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6020322A (en) * | 1993-11-09 | 2000-02-01 | Pro-Neuron, Inc. | Acyl deoxyribonucleoside derivatives and uses thereof |
| ATE142221T1 (en) * | 1987-10-28 | 1996-09-15 | Pro Neuron Inc | ACYL DEOXYRIBONUCLEOSIDE DERIVATIVES AND USES THEREOF |
| US5246708A (en) * | 1987-10-28 | 1993-09-21 | Pro-Neuron, Inc. | Methods for promoting wound healing with deoxyribonucleosides |
| US6919320B1 (en) | 1987-10-28 | 2005-07-19 | Wellstat Therapeutics Corporation | Pharmaceutical compositions containing deoxyribonucleosides for wound healing |
| US6348451B1 (en) | 1987-10-28 | 2002-02-19 | Pro-Neuron, Inc. | Acyl deoxyribonucleoside derivatives and uses thereof |
| US6743782B1 (en) * | 1987-10-28 | 2004-06-01 | Wellstat Therapeutics Corporation | Acyl deoxyribonucleoside derivatives and uses thereof |
| GB2216416B (en) * | 1988-03-11 | 1992-06-24 | Sandoz Ltd | Nucleobase source for the stimulation of the immune system |
| ZA892928B (en) * | 1988-04-25 | 1991-01-30 | Pro Neuron Inc | Pharmaceutical compositions containing deoxyribonucleosides for wound healing |
| US7169765B1 (en) | 1988-10-27 | 2007-01-30 | Wellstat Therapeutics Corporation | Acyl deoxyribonucleoside derivatives and uses thereof |
| US5081997A (en) † | 1989-03-09 | 1992-01-21 | Vance Products Incorporated | Echogenic devices, material and method |
| DK0502180T3 (en) * | 1990-09-21 | 1997-09-22 | Amgen Inc | Enzymatic synthesis of oligonucleotides |
| DE19516773A1 (en) * | 1995-03-11 | 1996-09-12 | Eftag Entstaubung Foerdertech | Use of DNA to prepare medicaments |
| KR102247169B1 (en) * | 2014-01-02 | 2021-05-03 | 주식회사 파마리서치프로덕트 | Composition comprising mixture of DNA fragments separated from fish's testis for the prevention or treatment of peptic ulcer |
Family Cites Families (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR4696M (en) * | 1965-06-23 | 1966-12-26 | ||
| US3534017A (en) * | 1967-03-14 | 1970-10-13 | Kyowa Hakko Kogyo Kk | Process for the preparation of nucleoside-5'-diphosphates and triphosphates and mono- and oligo-nucleotidyl-nucleoside-5'-diphosphates and triphosphates |
| US3626054A (en) * | 1967-10-25 | 1971-12-07 | Bard Hamilton Co Inc | Lupus erythematosus skin test |
| NL7003690A (en) * | 1969-03-17 | 1970-09-21 | ||
| BE757653A (en) * | 1969-10-21 | 1971-04-16 | Ugine Kuhlmann | NEW DRUGS DERIVED FROM NUCLEIC ACIDS AND METHODS FOR THEIR PREPARATION |
| DE2154279A1 (en) * | 1970-11-03 | 1972-05-25 | Crinos Industria Farmaco | Medicines for the fibrinolytic system |
| FR2201879B2 (en) * | 1972-05-19 | 1976-01-23 | Tixier Georges Fr | |
| GB1440626A (en) * | 1973-05-02 | 1976-06-23 | Farmaceutici Italia | |
| JPS5221570B2 (en) * | 1973-08-30 | 1977-06-11 | ||
| US4062949A (en) * | 1974-04-30 | 1977-12-13 | Eisai Co., Ltd. | Abrin composition of reduced toxicity |
| FR2353293A1 (en) * | 1976-06-03 | 1977-12-30 | Beljanski Mirko | POLYRIBONUCLEOTIDES WITH AN ACTIVITY IN THE GENESIS OF LEUCOCYTES AND BLOOD PLATELETS |
| DK413681A (en) * | 1980-09-18 | 1982-03-19 | Wellcome Found | SYNTHETIC DNA AND PROCEDURES FOR PRODUCING THEREOF |
| JPS57139096A (en) * | 1981-02-21 | 1982-08-27 | Mitsui Toatsu Chem Inc | Heat denaturing deoxyribonucleic acid md-011 having antitumor activity and antitumor agent |
| US4579941A (en) * | 1982-08-13 | 1986-04-01 | Mitsuitoatsu Chemicals, Inc. | Thermally-denatured deoxyribonucleic acid MD-011 and antitumor agent |
-
1983
- 1983-05-30 JP JP58094141A patent/JPS59219235A/en active Granted
-
1984
- 1984-05-17 US US06/611,160 patent/US4657896A/en not_active Expired - Fee Related
- 1984-05-21 DE DE19843418820 patent/DE3418820A1/en active Granted
- 1984-05-25 CA CA000455193A patent/CA1222699A/en not_active Expired
- 1984-05-28 FR FR8408312A patent/FR2547500B1/en not_active Expired
- 1984-05-29 GB GB08413576A patent/GB2140689B/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| GB2140689A (en) | 1984-12-05 |
| FR2547500A1 (en) | 1984-12-21 |
| US4657896A (en) | 1987-04-14 |
| FR2547500B1 (en) | 1988-01-08 |
| GB8413576D0 (en) | 1984-07-04 |
| JPS59219235A (en) | 1984-12-10 |
| DE3418820A1 (en) | 1984-12-13 |
| JPH0427964B2 (en) | 1992-05-13 |
| GB2140689B (en) | 1986-12-17 |
| DE3418820C2 (en) | 1987-12-17 |
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