CA1315717C - Nucleic acid derivatives - Google Patents
Nucleic acid derivativesInfo
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- CA1315717C CA1315717C CA000526797A CA526797A CA1315717C CA 1315717 C CA1315717 C CA 1315717C CA 000526797 A CA000526797 A CA 000526797A CA 526797 A CA526797 A CA 526797A CA 1315717 C CA1315717 C CA 1315717C
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
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Abstract
ABSTRACT
Novel nucleic acid derivatives wherein the purine or pyrimidine ring in the nucleic acid polymer is substituted with at least one SH group, or said derivatives contain a di-sulphide bond, or both. These derivatives have certain phys-iological activity and are useful in pharmaceutical prepara-tions.
Novel nucleic acid derivatives wherein the purine or pyrimidine ring in the nucleic acid polymer is substituted with at least one SH group, or said derivatives contain a di-sulphide bond, or both. These derivatives have certain phys-iological activity and are useful in pharmaceutical prepara-tions.
Description
NUCLEIC ACID DERIVATIVES
The present invention relates to nucleic acid deriva-tives useful as pharmaceuticals.
It is known in the art that nucleic acid is composed of sugars such as ribose bonded to purine or pyrimidine rings and they are arranged in chains via phosphoric acid. Among nucleic acids, RNA (ribonucleotide polymer) is a high molec-ular compound in chain structure in which ribose is contained as a sugar and sugar moieties are combined through diester bonds of phosphoric acid as follows:
f NIH2 HO-P--O ~
c~ `I
HO-P-O
~ 'H2 O R~
/ HN ~ r__ HO-P=O ~ ~I
oc~2ol \1~ o l l 11 /O R ~ NH
¦ ~N
Ribonucleic acid ~ 'H20 2 (R = OH, R' = H); /
Deoxyribonucleic acid \
(R = H, R' = CH3) ~ - 2 - 1 31 57 I7 The double chain is in spiral form in its stearic struc-ture by a combination of purine or pyrimidine ring moieties of the base (e.g. inosine, cytidine, uridine, etc.) which con-stitutes nucleic acid. Nucleic acids having double strands exhibit useful physiological ac-tivity and, accordingly, many studies have been done for them.
Among them, natural RNA derived from virus and syn-thetic double stranded RNA such as polyinosiric acid-polycy-tidylic acid derivatives (hereinafter abbreviated as "poly I:poly C"), polyadenylic acid-polyuridylic acid derivatives, etc. have been studied by many (e.g. Declark, et al: Texas Reports on Biology and Medicine, 41, 77, 1982).
N
HO--P--OH ( (5'-Cytidylic acid) 1H ~O~
~H' O OH OH
Jl l ~ N ~ N
HO-P-OH C
(5'-Inosinic acid) 1H ~ ~
OH OH
As such, many RNA nucleic acid derivatives have been synthesized and their physiological activities have been s-tudied.
The poly I:poly C is a substance having significant activity. Its usefulness has been evaluated and studies have been conducted therefore. A compound in which only a few parts `` 1 3 1 57 1 7 of cytidine in -the poly I:poly C is changed -to uridine (i.e.
mismatched RNA) has been studied because of its physiological activity similar to that of the poly I:poly C (cf. Japanese Laid Open 50/082226).
Studies for polycytidylic acid have also been conducted and there is a report (British Patent Application No. 2,038, 628) stating that, when a mercapto group (-SH) is introduced to an extent of 50~ or more in place of -NH2 groups of the pyrimidine ring of polycytidylic acid, the physiological ac-tivity increases.
The conventional physiologically active substances described above can be expected to exhibit useful effects through their toxicity as seen in the poly I:poly C (see Declark, et al: Infect. Immuni., 6, 344, 1972) It would therefore be desirable if the toxicity could be reduced and the activity increased.
We have found that RNA can form stabilized derivatives of high confirmation by certain covalence bond. When the physiological activity was measured, it has been found that they exhibit far stronger activity as compared with the con-ventional physiologically active substances with very low toxicity of the present invention.
Description of the Drawings Fig. 1 shows ultraviolet absorption spectra of sul-phurized polycytidylic acid obtained in Example 1. The ordi-nate and abscissa are absorption and wavelength (nm), respec-tively.
Fig. 2 shows ultraviolet absorption spectra of nucleic acid derivative obtained from Example 2. This is a UV pat-terns of reduction by sodium thiosulphate. The ordinate and abscissa are absorbancy and wavelength (nm), respectively.
Fig. 3 shows elution patterns by high performance li-quid chromatography of nucleic acid derivative of the present invention obtained from Example 3. The abscissa and ordinate are elution time (minute) eluted amount, respectively. Each arrow indicates the eluting posi-tion of size marker.
Fig. 4 shows fusion curve of the nucleic acid deriva--~I- 1315717 tive of the present invention in which the molecular size distribution is 50 to 2,000 residue numbers. The abscissa and ordinate are temperature (C) and relative absorption, respectively.
Nucleic acid derivatives oE the present invention can be synthesized from nucleic acid base to which -SH groups can be introduced. Specific examples of the present inven-tion nucleic acid derivatives are those having a complemen-tary double spiral structure of a chain compound (called "poly C" in this specification) which is a cytidylic acid polymer and another chain compound (called "poly I" in this specification) which is an inosinic acid polymer. Though said compound exhibits the structure similar to the already given poly I:poly C, the nucleic acid derivatives of the present invention still exhibits characteristics because of the following reasons.
The nucleic acid derivatives of the present invention having cross-linkage tS-S bond) have a covalent bond.
The sulphur atoms in the nucleic acid derivatives of the present invention can be introduced, before or after the polymer preparation, by utilizing an enzyma-tic reaction. The introduction is conducted by substituting nucleic acid base with -SH groups, such that an -NH2 group in a pyrimidine ring of cytidylic acid is substituted with an -SH group. The sub-stituent residue is called 4-thiouridylic acid.
i FIN
ll o ~ N
5'-4-Thiouridylic acid 111 k ~
OH OH
The introduced -SH is further oxidized by a suitable method as given later so that S-S bond bridge can be formed.
Derivatives having bo-th SH and S-S groups in a mole-`` - 5 1 31 571 ~
cule can be prepared either by a partial oxidation OL a com-pound in which -SH is introduced or by a partial reduc-tion of a compound in which S-S is introduced.
The above nucleic acid polymer (poly C~ is single stranded. The single strand nucleic acid polymer after for-mation of the SH substitution or the S-S bond bridge can be associated with a complementary single strand nucleic acid polymer, by a suitable method such as described below to form multiple strands. Conversely, it is also possible that the firstly-sulphurized single strand nucleic acid polymer is as-sociated with the complementary nucleic acid polymer to form multiple strands followed by oxidation to give an S-S bond bridge. The nucleic acid derivatives thus prepared are also part of the present invention. Thus, all nucleic acid poly-mers are included in the present invention so far as they contain nucleic acid bases -to which at least one -SH group is introduced. For example, the compounds obtained by di-sulphidation of nucleic acid polymers containing 4-thiouridine, 2-thiouridine, 2-thioguanosine, 6-mercaptopurine, 8-mercap-to-purine~ etc. are examples of the present invention~
The nucleic acid derivatives of the present invention can be cleaved at their phosphoric acid side chain to give lower molecular compounds and such lower molecular ones are also included in the present invention. Accordingly, in this sense, there is a difference between the present invention and the conventional poly I:poly C.
As a result of the characteristic feature of the nu-cleic acid derivatives of the present invention, i.e. sub-stitution with SH, formation of cross-linking by S-S bond and formation of low molecular substances by a cleavage of the phosphoric acid side chain, it is for the first time possible to obtain the effect of the present invention that (1) physio-logical activity is increased, and (2) safety can be increased because of lower toxicity.
The nucleic acid derivatives of the present invention are useful for their strong action as in-terferon-inducers.
In addition, they are us~ful for their TNF-productive ` - 6 - 1 3 1 ~7 1 7 ability, interleukin 1 productive ability, in-terleukin 2 pro-ductive ability, macrophage activating ability, activating ability on NK cell, inhibiting action for proliferation of tumor cells, inhibition action for proliferation of tumor in cancer-bearing mice, inhibition action for proliferation in nude mice bearing cancer of human tumor cells, preventing ac-tion for metastasis of tumor cells to lung, and the like.
Nucleic acid derivatives of the present invention have lower toxicity and hence are far more safe as compared with interferon-inducers such as conventional poly I:poly C. Ac-cordingly~ -the compounds of the present invention are useful as antiviral agents and antitumor agents.
The term "base pair" (abbreviated as "bp") frequently used to indicate the molecular size of nucleic acid is used to indicate the molecular size by the numbers of bases in the nucleic acid (i.e. 10 bp means the double strand polymer hav-ing ten bases) in each complementary strand. Since nucleic acid polymers other than double stranded polymers are also referred to in the present specification, the term "residue numbers" in place of bp will be used. Thus "10 residue num-bers" means the nucleic acid polymer having 10 bases in a strand.
The nucleic acid derivatives of the present invention contain substances with various kinds of molecular sizes and it is generally preferred that the size is not less than 50 residue numbers e.g., 50 to 10,000 residue numbers. The size may be even far larger, e.g. 200,000 residue numbers and it is believed that the molecular size has some influence on the physiological activity.
In giving numbers oi sulphur in the nucleic acid de-rivatives of the present invention, the degree of sulphuri-zation (n) is used in this specification. Cytidylic acid changes to ~-thiouridylic acid by substituting the -NH2 group in the pyrimidine ring with -SH group and the "n" indicates the numbers of cytidylic acid existent to one ~-thiouridylic acid. The nucleic acid derivatives of the present invention contain substances with many kinds of "n" and it is preferred ~ 7 ~ 131571, that said n is not less than 6. When n is less than -that, the physiological activity decreases. When n is 6 or more, and it may be as many as 39, n has little influence on the physiological activity so long as it is at least 6.
In manufacturing the nucleic acid derivatives of the present invention, various methods can be used. The poly C
and the like which are starting materials of the present in-vention, derivatives can be easily obtained. The poly C
may be easily sulphurized by, for example, the reaction with sulphurizing agents such as hydrogen sulphide. As a result of said reaction, certain numbers of cytidylic acid in the poly C can be changed to 4-thiouridylic acid. When the re-action conditions such as reaction temperature and reaction time are varied, poly C with the desired "n" values can be prepared.
The sulphurized poly C can be associated with poly I
which is easily obtained by known methods. The sulphurized poly C:poly I obtained as such can be introduced to the nu-cleic acid derivatives of the present invention by a disul-phide production reaction such as, for example, oxidation with an iodine reagent.
The nucleic acid derivatives of the present invention can be obtained almost quantitatively by the above methods and the overall yield is around 90%.
In the case of synthesis of other nucleic acids such as poly A:poly U having a disulphide bridge, the polymer is first prepared by an enzyme reaction using a nucleic acid base containing sulphur atom therein. Conven-tional methods in the manufacture of heteropolymers can be used.
Typical examples will be that, when 36mM of uridine-5'~diphosphate (UDP) and 7mM of 4-thiouridine-5'-diphosphate (4-SDH UDP) are made to react at 37C for 5 hours in 150mM
of Tris buffer (pH 8.2) using 0.5 unit/ml of polynucleotide phosphorylase (PNPase, type 15, PL Biochemical), a hetero-polymer in which one 4-thiouridine is contained for 13 uri-dine residue is obtained in around 50% yield. When 2-thio-uridine-5'-diphosphate is used in place of 4-thiouridine-5'-- 8 - l 31 5717 diphosphate, a poly U containing 2-thiouridine is obtained.
Once a nucleic acid polymer containing -SH groups is prepared as such, the following derivatives can be obtained by the same operation as in the case of sulphurized poly I:
poly C described in the Examples. More specifically, both sulphurized polyuridylic acid and equimolar polyadenylic acid are dissolved in neutral aqueous solution and subjected to an annealing for a complex forma-tion. Thus, after water-dissolving in an incubator, the temperature is gradually raised to 85C, then heated for 10 minutes, and allowed to stand at room temperature.
The complex thus obtained is oxidized with the same condition as in the disulphidation of poly I:poly C, i.e. ox-idized with lN iodine solution to afford poly A:poly U deri-vative having a disulphide bridge. The yield after the an-nealing is about 80% and the overall yield is about 40~.
The disulphide complex is well dialyzed to an aqueous solution and lyophilyzed to give white and fibrous solid.
The nucleic acid derivatives of the present invention give a certain clear melting curve as described later and, accordingly, it is apparent that they exhibit stable funda-mental structures.
With reference to the 50~ melting temperature (Tm value), it is 59.0C in the poly I:poly C under a neutral condition in the presence of 0.1M sodium ion while, in the cases of SH substituted nucleic acid derivative containing 4-thiouridine at the rate of n = 20 and S-S substituted nu-cleic acid derivative thereof, the values are 59.5C and 59.3C. They are stabilized. In the case of OFI substituted nucleic acid derivative containing uridine at the rate of n = 20 the same as above, -the value is as low as 53.1C.
Thus the introduction of sulphur atoms is more effec-tive in stabilizing the high molecular structure of nucleic acid as compared with other substituted derivatives such as those substituted with NH2 or with OH.
Then the resistance against hydrolysis by RNAase A
(an enzyme which decomposes RNA) was compared in terms of 13~5717 the time ratio until the decomposition arrives at 50%. Thus, under certain enzymatic reaction conditions, poly I:poly C
was 50 minutes while those of ~-thiouridine (SH group) and its nucleic acid derivative (n = 20; containing S-S) are 60 minutes and, by contrast, those of nucleic acid derivatives ~n = 20) containing uridine (OH group) were as short as 20 minutes. It is therefore apparent that sulphur atoms exhi-bit substituting effect even in biochemical stability.
When the nucleic acid derivatives of the present in-vention are administered as pharmaceuticals to humans andanimals, -they are given per se or as a pharmaceutical compo-sition containing, for example, 0.1 to 99.5% (more prefer-ably, 0.5 to 90~) of active ingredient in combination with a pharmaceutically acceptable carrier.
As to carriers, one or more liquid, solid or semisol-id diluent, filler and other auxiliary agents for pharmaceu-tical preparations may be used. It is desired that the phar-maceutical compositions are administered in unit dosage form.
Nucleic acid derivatives of the present invention may be gi-ven orally, parenterally, topically or rectally. They are of course given by forms suitable for each administration route. For example, they are administered in tablet or cap-sule form, by injection, inhalation, eye lotion, ointment, suppository, etc. Parenteral and topical administration is especially preferred.
It is desired that the dose in treating malignant tu-mors is regulated by considering a number of factors such as the status of the patient (e.g. age, body weight, etc.), ad-ministration route, and type and degree of condition to be treated. Generally, 0.05 to 1,000 mg per administration is an effective amount for adults, and preferably, 0.5 to 50 mg by intravenous infusion. In some cases, a lower does is suf-ficient and, in some other cases, a higher dose or more doses may be necessary. The administration may be one to several times a day or with an intermission of one to several days.
The dosage for the treatment of benign tumors or for diseases caused by viruses is preferably regulated by consid ering the type and degree of the disease, administration route, status of the patient, etc. and, in general, it is common that the dose is the same to about one fiftie-th of the dose des-crihed above for malignant tumors.
Oral administration can be effected utilizing solid and liquid dosage unit forms such as powders, tablets, capsules, granules and the like.
Powders are prepared by comminuting the compound to a suitable fine size and mixing with a similarly comminuted phar-maceutical carrier such as an edible carbohydrate as, for ex-ample, starch or mannitol. Flavoring, preservative, dispers-ing and coloring agents can also be present.
Capsules are made by preparing a powder mixture as des-cribed above and filling formed gelatin sheaths. Glidants and lubricants such as colloidal silica, talc, magnesium stearate, calcium stearate or solid polyethylene glycol can be added to the powder mixture before the filling operation. A disinte-grating or solubilizing agent such as agar-agar, calcium car-bonate or sodium carbonate can also be added to improve the availability of the medicament when the capsule is ingested.
Tablets are formulated, for example, by preparing a powder mixture, granulating or slugging, adding a lubricant and disintegrant and pressing into tablets. A powder mixture is prepared by mixing the compound, suitably comminuted, with a diluent or base as described above, and optionally, with a binder as carboxymethyl cellulose, an alginage, gelatin, or polyvinyl pyrrolidone, a solution retardant such as paraffin, a resorption accelerator such as a quaternary salt and/or an absorption agent such as bentonite, kaolin or dicalcium phos-phate. The powder mixture can be granulated by wetting with a binder such as syrup, starch paste, acadia mucilage or sol-utions of cellulosic or polymeric materials and forcing through a screen. As an alternative to granulating, the powder mixture can be run through the tablet machine and the resulting imper-fectly formed slugs broken into granules. The granules can be lubricated to prevent sticking to the tablet forming dies by means of the addition of stearic acid, a stearate salt, talc or 1 3 1 ~7 1 7 mineral oil. The lubricated mixture is then compressed into tablets. The compounds and pharmaceutically acceptable acid addition salts of the present invention can also be combined with free flowing inert carriers and compressed into tablets directly without going through the granulating or slugging steps. A clear or opaque protective coating consisting of a sealing coat of shellac, a coating of sugar or polymeric ma-terial and a polish coating of wax can be provided. Dyestuffs can be added to these coatings to distinguish different unit dosages.
Oral fluids such as solutions, syrups and elixirs can be prepared in dosage unit form so that a given quantity con-tains a predetermined amount of the compound. Syrups can be prepared by dissolving the compound in a suitably flavored aqueous solution, while elixirs are prepared through the use of a non-toxic alcoholic vehicle. Suspensions can be formu-lated by dispersing the compound in a non-toxic vehicle. Sol-ubilizers and emulsifiers such as ethoxylated isostearyl al-cohols and polyoxyethylene sorbitol esters, preservatives, flavor additives such as peppermint oil or saccarin, and the like can also be added.
Where appropriate, dosage unit formulations for oral administration can be microencapsulated. The formulation can also be prepared to prolong or sustain the release as for ex-ample by coating or embedding particulate material in poly-mers, wax or the like.
Parenteral administration can be effected utilizing liquid dosage unit forms such as sterline solutions and sus-pensions intended for subcutaneous, intramuscular or intra-venous injection. These are prepared by suspending or dis-solving a measured amount of the compound in a non-toxic li-quid vehicle suitable for in~ection such as aqueous or oleag-inous medium and sterilizing the suspension or solution. Al-ternatively, a measured amount of the compound is placed in a vial and the vial and its contents are sterilized and sealed.
An accompanying vial or vehicle can be provided for mixing prior to administration. Non-toxic salts and salt solutions can be added to render the injection isotonic. S-tabilizers, ~ . :
i!
preservatives and emulsifiers can also be added.
Rectal administration can be effected utilizing sup-positories in which the compound is admixed with low-melting, water-soluble or insoluble solids such as polyethylene glycol, cocoa butter, higher esters as for example flavored aqueous solution, while elixirs are prepared through myristyl palmi-tate or mixtures thereof.
The physiological activity of the nucleic acid deriv-atives of the present invention thus includes:
(1) Interferon-inducing activity:
With reference to sulphurized poly C:poly I deriva-tives which is one of the nucleic acid derivatives of the present invention, its action as in-terferon-inducer was de-termined by an antiviral activity assay method. The sulphur-ized poly C:poly I derivative used as sample I for the testis an SH substituted derivative while sample II is a deriva-tive having an S-S bond. In both n has a value of 13 and residue numbers in its molecule size of 50 to 2,000.
The lymphocyte (106 cells per ml) obtained from human peripheral blood was treated for 2 hours with the sample (10 micrograms/ml) in a culture liquid (RPMI 1640; 20% FCS). The reacted culture liquid was removed, the cells were again floated in a fresh culture liquid (RPMI 1640; 20~ FCS), incu-bated for 20 hours, and the supernatant liquid was subjected to a usual measuring method for interferon-antivirus activity using sindbi's virus and FL cell (cf. Rinsho Kensa, 28, 1726, 1984). The result is given in the following table. The titer of interferon was measured by a dilution concentration using a CPEI50 (Cytopathogenic Effect Inhibition 50) method. Sample II is the nucleic acid derivative having an S-S bond con-tain-ing 4-thiouridine at the rate of n = 20 while sample I was the SH substituted derivative. Conventional poly I:poly C
was used as a control sample.
Concentrations (micrograms/ml) Test Sample I 100 >6400 ~6400 ~6400 Test Sample II 100 >6400 >6400 ~6400 Control Sample 100 800 1600 ~6400 (Interferon titer: units/ml) ` - 13 - 1 31 ~ 7 1 7 It is apparent that the nucleic acid derivative of the present invention exhibits strong action as an interferon-inducer.
Among the sulphurized poly C:poly I derivatives of nu-cleic acid derivatives of the presen-t invention obtained in the example given later, one with the n-value of 20 and the molecular size of 50 to 300 residue number and another with the n-value of 20 and the molecular size of 200 to 2,000 res-idue were used as test samples and subjec-ted to the same phy-siological activity tests. Similar results were obtained.A similar result was also obtained on testing a nucleic acid derivative having an n-value of 20 and molecular size of not less -than 20,000 residue numbers before the low-moleculariza-tion.
(2) TNF (Tumor Necrosis Factor)-inducing ability:
Germinal vesicle macrophage from rabbits pretreated with BCG at 10 to 14 days before was taken, regulated to 2 x 106 per milliliter in a 10% FCS-added RP~I 1640 medium, one milliliter of i-t was placed on a plastic dish, and cul-tured in a carbon dioxide gase incuba-tor (5% CO2) in the presence or absence of the sample at 37C.
The supernatant liquid of cell culture after 2 or 8 hours was subjected to the TNF activity test. The result is given below. The TNF activity values were determined by measuring the cell hindrance activity to L~ cell after 72 hours using the dye in-take method and the dilution at 50%
cell hindrance was indicated as a titer. The fact that said cell hindrance was due to the TNF was confirmed by, using a monoclonal antibody to rabbit TNF, its activity neutralized.
30TNF Activity Neutralization by (2 hrs) (8 hrs) anti-TNF Antibody .
Control LPS 32 >64 +
Test Sample I 32 >64 -~
Test Sample II 32 >64 +
Control Sample 32 >64 +
- 14 - 1 31 5,~ 1 7 LPS was used at the concentration of 1 microgram/ml and the test and control samples were at the same concentra~
tions of 10 micrograms/ml. The test and control samples used were the same as those in (1). It is apparent that the TNF-inducing activity of the nucleic acid derivative of the present invention is strong.
The present invention relates to nucleic acid deriva-tives useful as pharmaceuticals.
It is known in the art that nucleic acid is composed of sugars such as ribose bonded to purine or pyrimidine rings and they are arranged in chains via phosphoric acid. Among nucleic acids, RNA (ribonucleotide polymer) is a high molec-ular compound in chain structure in which ribose is contained as a sugar and sugar moieties are combined through diester bonds of phosphoric acid as follows:
f NIH2 HO-P--O ~
c~ `I
HO-P-O
~ 'H2 O R~
/ HN ~ r__ HO-P=O ~ ~I
oc~2ol \1~ o l l 11 /O R ~ NH
¦ ~N
Ribonucleic acid ~ 'H20 2 (R = OH, R' = H); /
Deoxyribonucleic acid \
(R = H, R' = CH3) ~ - 2 - 1 31 57 I7 The double chain is in spiral form in its stearic struc-ture by a combination of purine or pyrimidine ring moieties of the base (e.g. inosine, cytidine, uridine, etc.) which con-stitutes nucleic acid. Nucleic acids having double strands exhibit useful physiological ac-tivity and, accordingly, many studies have been done for them.
Among them, natural RNA derived from virus and syn-thetic double stranded RNA such as polyinosiric acid-polycy-tidylic acid derivatives (hereinafter abbreviated as "poly I:poly C"), polyadenylic acid-polyuridylic acid derivatives, etc. have been studied by many (e.g. Declark, et al: Texas Reports on Biology and Medicine, 41, 77, 1982).
N
HO--P--OH ( (5'-Cytidylic acid) 1H ~O~
~H' O OH OH
Jl l ~ N ~ N
HO-P-OH C
(5'-Inosinic acid) 1H ~ ~
OH OH
As such, many RNA nucleic acid derivatives have been synthesized and their physiological activities have been s-tudied.
The poly I:poly C is a substance having significant activity. Its usefulness has been evaluated and studies have been conducted therefore. A compound in which only a few parts `` 1 3 1 57 1 7 of cytidine in -the poly I:poly C is changed -to uridine (i.e.
mismatched RNA) has been studied because of its physiological activity similar to that of the poly I:poly C (cf. Japanese Laid Open 50/082226).
Studies for polycytidylic acid have also been conducted and there is a report (British Patent Application No. 2,038, 628) stating that, when a mercapto group (-SH) is introduced to an extent of 50~ or more in place of -NH2 groups of the pyrimidine ring of polycytidylic acid, the physiological ac-tivity increases.
The conventional physiologically active substances described above can be expected to exhibit useful effects through their toxicity as seen in the poly I:poly C (see Declark, et al: Infect. Immuni., 6, 344, 1972) It would therefore be desirable if the toxicity could be reduced and the activity increased.
We have found that RNA can form stabilized derivatives of high confirmation by certain covalence bond. When the physiological activity was measured, it has been found that they exhibit far stronger activity as compared with the con-ventional physiologically active substances with very low toxicity of the present invention.
Description of the Drawings Fig. 1 shows ultraviolet absorption spectra of sul-phurized polycytidylic acid obtained in Example 1. The ordi-nate and abscissa are absorption and wavelength (nm), respec-tively.
Fig. 2 shows ultraviolet absorption spectra of nucleic acid derivative obtained from Example 2. This is a UV pat-terns of reduction by sodium thiosulphate. The ordinate and abscissa are absorbancy and wavelength (nm), respectively.
Fig. 3 shows elution patterns by high performance li-quid chromatography of nucleic acid derivative of the present invention obtained from Example 3. The abscissa and ordinate are elution time (minute) eluted amount, respectively. Each arrow indicates the eluting posi-tion of size marker.
Fig. 4 shows fusion curve of the nucleic acid deriva--~I- 1315717 tive of the present invention in which the molecular size distribution is 50 to 2,000 residue numbers. The abscissa and ordinate are temperature (C) and relative absorption, respectively.
Nucleic acid derivatives oE the present invention can be synthesized from nucleic acid base to which -SH groups can be introduced. Specific examples of the present inven-tion nucleic acid derivatives are those having a complemen-tary double spiral structure of a chain compound (called "poly C" in this specification) which is a cytidylic acid polymer and another chain compound (called "poly I" in this specification) which is an inosinic acid polymer. Though said compound exhibits the structure similar to the already given poly I:poly C, the nucleic acid derivatives of the present invention still exhibits characteristics because of the following reasons.
The nucleic acid derivatives of the present invention having cross-linkage tS-S bond) have a covalent bond.
The sulphur atoms in the nucleic acid derivatives of the present invention can be introduced, before or after the polymer preparation, by utilizing an enzyma-tic reaction. The introduction is conducted by substituting nucleic acid base with -SH groups, such that an -NH2 group in a pyrimidine ring of cytidylic acid is substituted with an -SH group. The sub-stituent residue is called 4-thiouridylic acid.
i FIN
ll o ~ N
5'-4-Thiouridylic acid 111 k ~
OH OH
The introduced -SH is further oxidized by a suitable method as given later so that S-S bond bridge can be formed.
Derivatives having bo-th SH and S-S groups in a mole-`` - 5 1 31 571 ~
cule can be prepared either by a partial oxidation OL a com-pound in which -SH is introduced or by a partial reduc-tion of a compound in which S-S is introduced.
The above nucleic acid polymer (poly C~ is single stranded. The single strand nucleic acid polymer after for-mation of the SH substitution or the S-S bond bridge can be associated with a complementary single strand nucleic acid polymer, by a suitable method such as described below to form multiple strands. Conversely, it is also possible that the firstly-sulphurized single strand nucleic acid polymer is as-sociated with the complementary nucleic acid polymer to form multiple strands followed by oxidation to give an S-S bond bridge. The nucleic acid derivatives thus prepared are also part of the present invention. Thus, all nucleic acid poly-mers are included in the present invention so far as they contain nucleic acid bases -to which at least one -SH group is introduced. For example, the compounds obtained by di-sulphidation of nucleic acid polymers containing 4-thiouridine, 2-thiouridine, 2-thioguanosine, 6-mercaptopurine, 8-mercap-to-purine~ etc. are examples of the present invention~
The nucleic acid derivatives of the present invention can be cleaved at their phosphoric acid side chain to give lower molecular compounds and such lower molecular ones are also included in the present invention. Accordingly, in this sense, there is a difference between the present invention and the conventional poly I:poly C.
As a result of the characteristic feature of the nu-cleic acid derivatives of the present invention, i.e. sub-stitution with SH, formation of cross-linking by S-S bond and formation of low molecular substances by a cleavage of the phosphoric acid side chain, it is for the first time possible to obtain the effect of the present invention that (1) physio-logical activity is increased, and (2) safety can be increased because of lower toxicity.
The nucleic acid derivatives of the present invention are useful for their strong action as in-terferon-inducers.
In addition, they are us~ful for their TNF-productive ` - 6 - 1 3 1 ~7 1 7 ability, interleukin 1 productive ability, in-terleukin 2 pro-ductive ability, macrophage activating ability, activating ability on NK cell, inhibiting action for proliferation of tumor cells, inhibition action for proliferation of tumor in cancer-bearing mice, inhibition action for proliferation in nude mice bearing cancer of human tumor cells, preventing ac-tion for metastasis of tumor cells to lung, and the like.
Nucleic acid derivatives of the present invention have lower toxicity and hence are far more safe as compared with interferon-inducers such as conventional poly I:poly C. Ac-cordingly~ -the compounds of the present invention are useful as antiviral agents and antitumor agents.
The term "base pair" (abbreviated as "bp") frequently used to indicate the molecular size of nucleic acid is used to indicate the molecular size by the numbers of bases in the nucleic acid (i.e. 10 bp means the double strand polymer hav-ing ten bases) in each complementary strand. Since nucleic acid polymers other than double stranded polymers are also referred to in the present specification, the term "residue numbers" in place of bp will be used. Thus "10 residue num-bers" means the nucleic acid polymer having 10 bases in a strand.
The nucleic acid derivatives of the present invention contain substances with various kinds of molecular sizes and it is generally preferred that the size is not less than 50 residue numbers e.g., 50 to 10,000 residue numbers. The size may be even far larger, e.g. 200,000 residue numbers and it is believed that the molecular size has some influence on the physiological activity.
In giving numbers oi sulphur in the nucleic acid de-rivatives of the present invention, the degree of sulphuri-zation (n) is used in this specification. Cytidylic acid changes to ~-thiouridylic acid by substituting the -NH2 group in the pyrimidine ring with -SH group and the "n" indicates the numbers of cytidylic acid existent to one ~-thiouridylic acid. The nucleic acid derivatives of the present invention contain substances with many kinds of "n" and it is preferred ~ 7 ~ 131571, that said n is not less than 6. When n is less than -that, the physiological activity decreases. When n is 6 or more, and it may be as many as 39, n has little influence on the physiological activity so long as it is at least 6.
In manufacturing the nucleic acid derivatives of the present invention, various methods can be used. The poly C
and the like which are starting materials of the present in-vention, derivatives can be easily obtained. The poly C
may be easily sulphurized by, for example, the reaction with sulphurizing agents such as hydrogen sulphide. As a result of said reaction, certain numbers of cytidylic acid in the poly C can be changed to 4-thiouridylic acid. When the re-action conditions such as reaction temperature and reaction time are varied, poly C with the desired "n" values can be prepared.
The sulphurized poly C can be associated with poly I
which is easily obtained by known methods. The sulphurized poly C:poly I obtained as such can be introduced to the nu-cleic acid derivatives of the present invention by a disul-phide production reaction such as, for example, oxidation with an iodine reagent.
The nucleic acid derivatives of the present invention can be obtained almost quantitatively by the above methods and the overall yield is around 90%.
In the case of synthesis of other nucleic acids such as poly A:poly U having a disulphide bridge, the polymer is first prepared by an enzyme reaction using a nucleic acid base containing sulphur atom therein. Conven-tional methods in the manufacture of heteropolymers can be used.
Typical examples will be that, when 36mM of uridine-5'~diphosphate (UDP) and 7mM of 4-thiouridine-5'-diphosphate (4-SDH UDP) are made to react at 37C for 5 hours in 150mM
of Tris buffer (pH 8.2) using 0.5 unit/ml of polynucleotide phosphorylase (PNPase, type 15, PL Biochemical), a hetero-polymer in which one 4-thiouridine is contained for 13 uri-dine residue is obtained in around 50% yield. When 2-thio-uridine-5'-diphosphate is used in place of 4-thiouridine-5'-- 8 - l 31 5717 diphosphate, a poly U containing 2-thiouridine is obtained.
Once a nucleic acid polymer containing -SH groups is prepared as such, the following derivatives can be obtained by the same operation as in the case of sulphurized poly I:
poly C described in the Examples. More specifically, both sulphurized polyuridylic acid and equimolar polyadenylic acid are dissolved in neutral aqueous solution and subjected to an annealing for a complex forma-tion. Thus, after water-dissolving in an incubator, the temperature is gradually raised to 85C, then heated for 10 minutes, and allowed to stand at room temperature.
The complex thus obtained is oxidized with the same condition as in the disulphidation of poly I:poly C, i.e. ox-idized with lN iodine solution to afford poly A:poly U deri-vative having a disulphide bridge. The yield after the an-nealing is about 80% and the overall yield is about 40~.
The disulphide complex is well dialyzed to an aqueous solution and lyophilyzed to give white and fibrous solid.
The nucleic acid derivatives of the present invention give a certain clear melting curve as described later and, accordingly, it is apparent that they exhibit stable funda-mental structures.
With reference to the 50~ melting temperature (Tm value), it is 59.0C in the poly I:poly C under a neutral condition in the presence of 0.1M sodium ion while, in the cases of SH substituted nucleic acid derivative containing 4-thiouridine at the rate of n = 20 and S-S substituted nu-cleic acid derivative thereof, the values are 59.5C and 59.3C. They are stabilized. In the case of OFI substituted nucleic acid derivative containing uridine at the rate of n = 20 the same as above, -the value is as low as 53.1C.
Thus the introduction of sulphur atoms is more effec-tive in stabilizing the high molecular structure of nucleic acid as compared with other substituted derivatives such as those substituted with NH2 or with OH.
Then the resistance against hydrolysis by RNAase A
(an enzyme which decomposes RNA) was compared in terms of 13~5717 the time ratio until the decomposition arrives at 50%. Thus, under certain enzymatic reaction conditions, poly I:poly C
was 50 minutes while those of ~-thiouridine (SH group) and its nucleic acid derivative (n = 20; containing S-S) are 60 minutes and, by contrast, those of nucleic acid derivatives ~n = 20) containing uridine (OH group) were as short as 20 minutes. It is therefore apparent that sulphur atoms exhi-bit substituting effect even in biochemical stability.
When the nucleic acid derivatives of the present in-vention are administered as pharmaceuticals to humans andanimals, -they are given per se or as a pharmaceutical compo-sition containing, for example, 0.1 to 99.5% (more prefer-ably, 0.5 to 90~) of active ingredient in combination with a pharmaceutically acceptable carrier.
As to carriers, one or more liquid, solid or semisol-id diluent, filler and other auxiliary agents for pharmaceu-tical preparations may be used. It is desired that the phar-maceutical compositions are administered in unit dosage form.
Nucleic acid derivatives of the present invention may be gi-ven orally, parenterally, topically or rectally. They are of course given by forms suitable for each administration route. For example, they are administered in tablet or cap-sule form, by injection, inhalation, eye lotion, ointment, suppository, etc. Parenteral and topical administration is especially preferred.
It is desired that the dose in treating malignant tu-mors is regulated by considering a number of factors such as the status of the patient (e.g. age, body weight, etc.), ad-ministration route, and type and degree of condition to be treated. Generally, 0.05 to 1,000 mg per administration is an effective amount for adults, and preferably, 0.5 to 50 mg by intravenous infusion. In some cases, a lower does is suf-ficient and, in some other cases, a higher dose or more doses may be necessary. The administration may be one to several times a day or with an intermission of one to several days.
The dosage for the treatment of benign tumors or for diseases caused by viruses is preferably regulated by consid ering the type and degree of the disease, administration route, status of the patient, etc. and, in general, it is common that the dose is the same to about one fiftie-th of the dose des-crihed above for malignant tumors.
Oral administration can be effected utilizing solid and liquid dosage unit forms such as powders, tablets, capsules, granules and the like.
Powders are prepared by comminuting the compound to a suitable fine size and mixing with a similarly comminuted phar-maceutical carrier such as an edible carbohydrate as, for ex-ample, starch or mannitol. Flavoring, preservative, dispers-ing and coloring agents can also be present.
Capsules are made by preparing a powder mixture as des-cribed above and filling formed gelatin sheaths. Glidants and lubricants such as colloidal silica, talc, magnesium stearate, calcium stearate or solid polyethylene glycol can be added to the powder mixture before the filling operation. A disinte-grating or solubilizing agent such as agar-agar, calcium car-bonate or sodium carbonate can also be added to improve the availability of the medicament when the capsule is ingested.
Tablets are formulated, for example, by preparing a powder mixture, granulating or slugging, adding a lubricant and disintegrant and pressing into tablets. A powder mixture is prepared by mixing the compound, suitably comminuted, with a diluent or base as described above, and optionally, with a binder as carboxymethyl cellulose, an alginage, gelatin, or polyvinyl pyrrolidone, a solution retardant such as paraffin, a resorption accelerator such as a quaternary salt and/or an absorption agent such as bentonite, kaolin or dicalcium phos-phate. The powder mixture can be granulated by wetting with a binder such as syrup, starch paste, acadia mucilage or sol-utions of cellulosic or polymeric materials and forcing through a screen. As an alternative to granulating, the powder mixture can be run through the tablet machine and the resulting imper-fectly formed slugs broken into granules. The granules can be lubricated to prevent sticking to the tablet forming dies by means of the addition of stearic acid, a stearate salt, talc or 1 3 1 ~7 1 7 mineral oil. The lubricated mixture is then compressed into tablets. The compounds and pharmaceutically acceptable acid addition salts of the present invention can also be combined with free flowing inert carriers and compressed into tablets directly without going through the granulating or slugging steps. A clear or opaque protective coating consisting of a sealing coat of shellac, a coating of sugar or polymeric ma-terial and a polish coating of wax can be provided. Dyestuffs can be added to these coatings to distinguish different unit dosages.
Oral fluids such as solutions, syrups and elixirs can be prepared in dosage unit form so that a given quantity con-tains a predetermined amount of the compound. Syrups can be prepared by dissolving the compound in a suitably flavored aqueous solution, while elixirs are prepared through the use of a non-toxic alcoholic vehicle. Suspensions can be formu-lated by dispersing the compound in a non-toxic vehicle. Sol-ubilizers and emulsifiers such as ethoxylated isostearyl al-cohols and polyoxyethylene sorbitol esters, preservatives, flavor additives such as peppermint oil or saccarin, and the like can also be added.
Where appropriate, dosage unit formulations for oral administration can be microencapsulated. The formulation can also be prepared to prolong or sustain the release as for ex-ample by coating or embedding particulate material in poly-mers, wax or the like.
Parenteral administration can be effected utilizing liquid dosage unit forms such as sterline solutions and sus-pensions intended for subcutaneous, intramuscular or intra-venous injection. These are prepared by suspending or dis-solving a measured amount of the compound in a non-toxic li-quid vehicle suitable for in~ection such as aqueous or oleag-inous medium and sterilizing the suspension or solution. Al-ternatively, a measured amount of the compound is placed in a vial and the vial and its contents are sterilized and sealed.
An accompanying vial or vehicle can be provided for mixing prior to administration. Non-toxic salts and salt solutions can be added to render the injection isotonic. S-tabilizers, ~ . :
i!
preservatives and emulsifiers can also be added.
Rectal administration can be effected utilizing sup-positories in which the compound is admixed with low-melting, water-soluble or insoluble solids such as polyethylene glycol, cocoa butter, higher esters as for example flavored aqueous solution, while elixirs are prepared through myristyl palmi-tate or mixtures thereof.
The physiological activity of the nucleic acid deriv-atives of the present invention thus includes:
(1) Interferon-inducing activity:
With reference to sulphurized poly C:poly I deriva-tives which is one of the nucleic acid derivatives of the present invention, its action as in-terferon-inducer was de-termined by an antiviral activity assay method. The sulphur-ized poly C:poly I derivative used as sample I for the testis an SH substituted derivative while sample II is a deriva-tive having an S-S bond. In both n has a value of 13 and residue numbers in its molecule size of 50 to 2,000.
The lymphocyte (106 cells per ml) obtained from human peripheral blood was treated for 2 hours with the sample (10 micrograms/ml) in a culture liquid (RPMI 1640; 20% FCS). The reacted culture liquid was removed, the cells were again floated in a fresh culture liquid (RPMI 1640; 20~ FCS), incu-bated for 20 hours, and the supernatant liquid was subjected to a usual measuring method for interferon-antivirus activity using sindbi's virus and FL cell (cf. Rinsho Kensa, 28, 1726, 1984). The result is given in the following table. The titer of interferon was measured by a dilution concentration using a CPEI50 (Cytopathogenic Effect Inhibition 50) method. Sample II is the nucleic acid derivative having an S-S bond con-tain-ing 4-thiouridine at the rate of n = 20 while sample I was the SH substituted derivative. Conventional poly I:poly C
was used as a control sample.
Concentrations (micrograms/ml) Test Sample I 100 >6400 ~6400 ~6400 Test Sample II 100 >6400 >6400 ~6400 Control Sample 100 800 1600 ~6400 (Interferon titer: units/ml) ` - 13 - 1 31 ~ 7 1 7 It is apparent that the nucleic acid derivative of the present invention exhibits strong action as an interferon-inducer.
Among the sulphurized poly C:poly I derivatives of nu-cleic acid derivatives of the presen-t invention obtained in the example given later, one with the n-value of 20 and the molecular size of 50 to 300 residue number and another with the n-value of 20 and the molecular size of 200 to 2,000 res-idue were used as test samples and subjec-ted to the same phy-siological activity tests. Similar results were obtained.A similar result was also obtained on testing a nucleic acid derivative having an n-value of 20 and molecular size of not less -than 20,000 residue numbers before the low-moleculariza-tion.
(2) TNF (Tumor Necrosis Factor)-inducing ability:
Germinal vesicle macrophage from rabbits pretreated with BCG at 10 to 14 days before was taken, regulated to 2 x 106 per milliliter in a 10% FCS-added RP~I 1640 medium, one milliliter of i-t was placed on a plastic dish, and cul-tured in a carbon dioxide gase incuba-tor (5% CO2) in the presence or absence of the sample at 37C.
The supernatant liquid of cell culture after 2 or 8 hours was subjected to the TNF activity test. The result is given below. The TNF activity values were determined by measuring the cell hindrance activity to L~ cell after 72 hours using the dye in-take method and the dilution at 50%
cell hindrance was indicated as a titer. The fact that said cell hindrance was due to the TNF was confirmed by, using a monoclonal antibody to rabbit TNF, its activity neutralized.
30TNF Activity Neutralization by (2 hrs) (8 hrs) anti-TNF Antibody .
Control LPS 32 >64 +
Test Sample I 32 >64 -~
Test Sample II 32 >64 +
Control Sample 32 >64 +
- 14 - 1 31 5,~ 1 7 LPS was used at the concentration of 1 microgram/ml and the test and control samples were at the same concentra~
tions of 10 micrograms/ml. The test and control samples used were the same as those in (1). It is apparent that the TNF-inducing activity of the nucleic acid derivative of the present invention is strong.
(3) The TNF-inducing ac'civity in vivo:
Meth-A tumor (2 x 105) was transplanted behind the abdomen skin of BALB/c mice (6 to 8 weeks age; male), drugs were given on 2nd and 12th days after transplantation, blood was taken at one hour after the second administration, and the TNF activity in the serum was measured. The necrosis phenomenon at the cancer-carrying part occurred thereafter was also observed. The result is given below. The values in the table are the same titer as used in the above (2).
Treatments TNF Activity Necrosis (lst) (2nd) in Serum BCG LPS 192 observed BCG Test Sample I 48 observed BCG Test Sample II 50 observed BCG Control Sample 24 observed Test Sample I Test Sample I 12 observed Test Sample II Test Sample II 24 observea -Both test and control samples were the same as those in the above (1). It is apparent that the TNF producing abil-ity in vivo of the nucleic acid derivative of the present in-vention is s-trong.
Meth-A tumor (2 x 105) was transplanted behind the abdomen skin of BALB/c mice (6 to 8 weeks age; male), drugs were given on 2nd and 12th days after transplantation, blood was taken at one hour after the second administration, and the TNF activity in the serum was measured. The necrosis phenomenon at the cancer-carrying part occurred thereafter was also observed. The result is given below. The values in the table are the same titer as used in the above (2).
Treatments TNF Activity Necrosis (lst) (2nd) in Serum BCG LPS 192 observed BCG Test Sample I 48 observed BCG Test Sample II 50 observed BCG Control Sample 24 observed Test Sample I Test Sample I 12 observed Test Sample II Test Sample II 24 observea -Both test and control samples were the same as those in the above (1). It is apparent that the TNF producing abil-ity in vivo of the nucleic acid derivative of the present in-vention is s-trong.
(4) Inducing activity of Interleukin 1:
Normal human heparin-added peripheral blood was sepa-rated from mononuclear particles by specific gravity centri-fugation method using Ficoll-Hypaque (Farmacia, Ficoll-paque, trademark), the numbers were adjusted to 5 x 106/ml in a 10%
FCS-added RPMI medium, placed in a plastic dish, incubated at 37C for one hour, and the cohesive cells were used as an interleukin 1 producing source.
The sample to be tested was added to the 5 x 105/ml of single particle, incubated at 37C for 24 hours, and the in-\
- 15 - 1 31 5 7 l7 terleukin 1 activity in the supernatant liquid was determined by measuring a H-thymidine in-take method into a PHA-stimu-lated mice thymus cells using a proliferation ability of the thymus cells as a target. The result is given in the follow-ing table. The control was physiological saline solution.The concentration of interleukin was 1.25 units/ml and those of test samples I and II and of control sample were in 100 ~g/ml.
The test samples I and II and control sample used were the same as those used in the above (1).
In-Take Amount Samples Used(DPM(x 104)) Control 3370 Test Sample I 11384 Test Sample II 11560 Control Sample 8397 Interleukin 1 12517 It is apparent that the nucleic acid derivatives of the present invention exhibit strong interleukin 1 inducing activ-ity.
Normal human heparin-added peripheral blood was sepa-rated from mononuclear particles by specific gravity centri-fugation method using Ficoll-Hypaque (Farmacia, Ficoll-paque, trademark), the numbers were adjusted to 5 x 106/ml in a 10%
FCS-added RPMI medium, placed in a plastic dish, incubated at 37C for one hour, and the cohesive cells were used as an interleukin 1 producing source.
The sample to be tested was added to the 5 x 105/ml of single particle, incubated at 37C for 24 hours, and the in-\
- 15 - 1 31 5 7 l7 terleukin 1 activity in the supernatant liquid was determined by measuring a H-thymidine in-take method into a PHA-stimu-lated mice thymus cells using a proliferation ability of the thymus cells as a target. The result is given in the follow-ing table. The control was physiological saline solution.The concentration of interleukin was 1.25 units/ml and those of test samples I and II and of control sample were in 100 ~g/ml.
The test samples I and II and control sample used were the same as those used in the above (1).
In-Take Amount Samples Used(DPM(x 104)) Control 3370 Test Sample I 11384 Test Sample II 11560 Control Sample 8397 Interleukin 1 12517 It is apparent that the nucleic acid derivatives of the present invention exhibit strong interleukin 1 inducing activ-ity.
(5) Inducing activity of Interleukin 2:
Lymph particles ob-tained from spleen cells of BALB/c mice were used as a source for production of interleukin 2.
To 5 x 106/ml of lymph particles was added the sample to be listed, the mixture was incubated at 37C for 24 to 48 hours, and the interleukin 2 activity in the supernatant li-quid was determined by a ~I-thymidine incorporation method using CTLL-2 or NK-7 which is cell strain proliferating de-pending upon interleukin 2 using the proliferation ability as a target. The results are given in the following table.
The control is physiological saline solution. The interleu-kin 2 was in a concentration of 5 units/ml and both test sam-ples I and II and control sample were in the same concentra-tion of 100 ~g/ml. The test samples I and II and control sam-ple used were the same as those in (1).
Incorporated Amount Samples Used(DPM(x 103)) Control 230 Test Sample I 3247 Test Sample II 3320 Control Sample 653 Interleukin 2 3778 It is apparent that the nucleic acid derivatives of the present invention exhibit strong in-terleukin 2 inducing activity.
Lymph particles ob-tained from spleen cells of BALB/c mice were used as a source for production of interleukin 2.
To 5 x 106/ml of lymph particles was added the sample to be listed, the mixture was incubated at 37C for 24 to 48 hours, and the interleukin 2 activity in the supernatant li-quid was determined by a ~I-thymidine incorporation method using CTLL-2 or NK-7 which is cell strain proliferating de-pending upon interleukin 2 using the proliferation ability as a target. The results are given in the following table.
The control is physiological saline solution. The interleu-kin 2 was in a concentration of 5 units/ml and both test sam-ples I and II and control sample were in the same concentra-tion of 100 ~g/ml. The test samples I and II and control sam-ple used were the same as those in (1).
Incorporated Amount Samples Used(DPM(x 103)) Control 230 Test Sample I 3247 Test Sample II 3320 Control Sample 653 Interleukin 2 3778 It is apparent that the nucleic acid derivatives of the present invention exhibit strong in-terleukin 2 inducing activity.
(6) Macrophage activation:
Samples were administered to BALB/c mice (7 to 10 weeks age; male) intraperitoneally, cells exuded from the peritoneum at 3 to 5 days after administration were col-lected, plastic-cohesive cells (mainly macrophage) were sep-arated as effector cells, and the macrophage activation was investigated by a 3H-thymidine isolation method using Meth-A
tumor cell as a target (the ~/T ratio being 15-20:1). The results are given in the following table. The control was 0.2 ml of physiological saline solution. Each 50 micrograms/
mouse of test samples I and II or control sample was admin-istered. The test samples I and II and control sample used were the same as those in (1).
% Cytotoxicity was calculated by:
(experimental value) - ( background 3 - x 100 (100% H release) - ( background Samples Used% Cytotoxicity Control 0.5 Test Sample I 10.3 Test Sample II 10.8 Control Sample 5.7 It is apparent that the nucleic acid deriva-tives of the present invention exhibit strong macrophage activation.
Samples were administered to BALB/c mice (7 to 10 weeks age; male) intraperitoneally, cells exuded from the peritoneum at 3 to 5 days after administration were col-lected, plastic-cohesive cells (mainly macrophage) were sep-arated as effector cells, and the macrophage activation was investigated by a 3H-thymidine isolation method using Meth-A
tumor cell as a target (the ~/T ratio being 15-20:1). The results are given in the following table. The control was 0.2 ml of physiological saline solution. Each 50 micrograms/
mouse of test samples I and II or control sample was admin-istered. The test samples I and II and control sample used were the same as those in (1).
% Cytotoxicity was calculated by:
(experimental value) - ( background 3 - x 100 (100% H release) - ( background Samples Used% Cytotoxicity Control 0.5 Test Sample I 10.3 Test Sample II 10.8 Control Sample 5.7 It is apparent that the nucleic acid deriva-tives of the present invention exhibit strong macrophage activation.
(7) NK cell activation:
The activation of NK cells in human peripheral blood was determined by measuring hindrance activity using K562 as a target cell by an isolation method of lCr. The result is given below. The control used was physiological saline solu-tion. The test samples I and II and control sample were the same as those in the above (1).
Samples (micrograms/ml)Melting % (% Cr Isolation) Control 30 Test Samples I ( 10) 47 ( 30) 55 (100) 57 (300) 50 (500) 30 Table continued Samples (micrograms/ml) Melting % (% Cr Isolation) Test Samples II ( 10) 51 ( 30) 59 5(100) 63 (300) 60 (500) 45 Control Samples ( 10) 52 ( 30) 60 10(100) 65 (300) 62 (500) 52 It is apparent that the nucleic acid derivatives of the present invention exhibit NK cell activation.
The activation of NK cells in human peripheral blood was determined by measuring hindrance activity using K562 as a target cell by an isolation method of lCr. The result is given below. The control used was physiological saline solu-tion. The test samples I and II and control sample were the same as those in the above (1).
Samples (micrograms/ml)Melting % (% Cr Isolation) Control 30 Test Samples I ( 10) 47 ( 30) 55 (100) 57 (300) 50 (500) 30 Table continued Samples (micrograms/ml) Melting % (% Cr Isolation) Test Samples II ( 10) 51 ( 30) 59 5(100) 63 (300) 60 (500) 45 Control Samples ( 10) 52 ( 30) 60 10(100) 65 (300) 62 (500) 52 It is apparent that the nucleic acid derivatives of the present invention exhibit NK cell activation.
(8) Inhibitory action for proliferation of tumor cells:
Inhibitory action for proliferation of cell line -tumor cells was measured. Cells (3 x 104/ml) was treated for 48 hours in a culture liquid containing 10% FCS together with samples (10 micrograms/ml) and 100 micrograms/ml) and then incuba-ted for 48 hours with tritium-labelled thymidine. The inhibition % was given by an incorporated amount of -thymidine to a control which was not treated with the sample. The results are given below. The -test samples I and II and control sample were the same as those in the above (1).
25Inhibition % Control Samples Test Sample I Test Sample IImicrograms/ml Cells 100 10 100 10 100 10 NAMALWA65.6 55.7 61.2 46.4 56.9 37.4 RAJl 68~3 64.6 67.8 61.5 74.7 69.4 L929 36.9 35.7 36.8 34.7 32.7 20.5 RAMOS 40.8 42.2 45.5 40.5 40.1 52.4 It is apparent that the nucleic acid derivatives of the present invention exhibit inhibitory action for proliferation of tumor cells.
Inhibitory action for proliferation of cell line -tumor cells was measured. Cells (3 x 104/ml) was treated for 48 hours in a culture liquid containing 10% FCS together with samples (10 micrograms/ml) and 100 micrograms/ml) and then incuba-ted for 48 hours with tritium-labelled thymidine. The inhibition % was given by an incorporated amount of -thymidine to a control which was not treated with the sample. The results are given below. The -test samples I and II and control sample were the same as those in the above (1).
25Inhibition % Control Samples Test Sample I Test Sample IImicrograms/ml Cells 100 10 100 10 100 10 NAMALWA65.6 55.7 61.2 46.4 56.9 37.4 RAJl 68~3 64.6 67.8 61.5 74.7 69.4 L929 36.9 35.7 36.8 34.7 32.7 20.5 RAMOS 40.8 42.2 45.5 40.5 40.1 52.4 It is apparent that the nucleic acid derivatives of the present invention exhibit inhibitory action for proliferation of tumor cells.
(9) Inhibitory action for proliferation of tumor in cancer-carrying mice:
The inhibitory action for tumor proliferation was mea-sured by the following method in Meth-A cancer-carrying mice which is the transplanted same type cancer. Meth-A cell (3 x 105/0.1 ml) was suspended in physiological saline solution, hypodermically injected into BALB/c mice (5 weeks age; male) - 1~ - 1 3 1 57 I 7 and, after 2 days, started in administration of the drug three times a week for two weeks. On the second day after final ad-ministration, tumor cells were extracted and the weight was measured. The results are given below. The test samples I and 5 II and control sample were the same as those in the above (1).
Nos.
Samples of (micrograms/mouse) Mice Mean ~ S.E.Inhibition Test Sample I ( 10) 7 2.09 + 0.15 3 10( 30) 7 1.52 + 0.2830 (100) 5 0.96 + 0.4356 Test Sample II ( 10) 7 2.10 + 0.27 ( 30) 7 1.62 + 0.25 (100) 7 0.97 + 0.30 15Control Sample ( 10) 7 2.12 + 0.27 2 ( 30) 8 1.38 ~ 0.3836 (100) 8 1.08 + 0.2750 It is apparent that the nucleic acid derivatives of the present invention inhibit the proliferation of tumor cells in cancer-bearing mice.
(10) Proliferation inhibiting action in nude mice bear-ing cancer from human tumor cells:
Each 2.5 x 106 of cell strain HeLa S3 derived from human uterus neck and cell strain Hep-2 derived from throat cancer cell were transplanted under the skin of abdomen and, on the 7th to 10th day after the transplantation, living cohesion of tumor was confirmed. Then 100 ~g/mouse of test sample (intra-venously) and 25 mg/kg of 5-FU (in-traperitoneally) were admin-istered twice a week for four weeks. Tumor was extracted on the fourth week after the administration and the weight was measured. The results are given in the following table~ The test samples used were the same as that in the above (1).
(HeLa S3) Inhibition Sample Weight (g) + S.E. Rate (~) 35Control 3.85 + 0.23 --Test Sample I 1.57 + 0.19 59 Test Sample II 1.55 + 0.20 60 5-FU 2.06 + 0.35 47 (Hep-2) Inhibition Sample Weight (g) + S.E.Rate (%) Control 0.88 + 0.07 --Test Sample I 0.38 + 0.07 57 Test Sample II 0.35 + 0.05 60 5-FU 0.56 + 0.12 30 It is apparent that the nucleic acid derivatives of the present invention exhibit strong inhibitory action for proliferation.
The inhibitory action for tumor proliferation was mea-sured by the following method in Meth-A cancer-carrying mice which is the transplanted same type cancer. Meth-A cell (3 x 105/0.1 ml) was suspended in physiological saline solution, hypodermically injected into BALB/c mice (5 weeks age; male) - 1~ - 1 3 1 57 I 7 and, after 2 days, started in administration of the drug three times a week for two weeks. On the second day after final ad-ministration, tumor cells were extracted and the weight was measured. The results are given below. The test samples I and 5 II and control sample were the same as those in the above (1).
Nos.
Samples of (micrograms/mouse) Mice Mean ~ S.E.Inhibition Test Sample I ( 10) 7 2.09 + 0.15 3 10( 30) 7 1.52 + 0.2830 (100) 5 0.96 + 0.4356 Test Sample II ( 10) 7 2.10 + 0.27 ( 30) 7 1.62 + 0.25 (100) 7 0.97 + 0.30 15Control Sample ( 10) 7 2.12 + 0.27 2 ( 30) 8 1.38 ~ 0.3836 (100) 8 1.08 + 0.2750 It is apparent that the nucleic acid derivatives of the present invention inhibit the proliferation of tumor cells in cancer-bearing mice.
(10) Proliferation inhibiting action in nude mice bear-ing cancer from human tumor cells:
Each 2.5 x 106 of cell strain HeLa S3 derived from human uterus neck and cell strain Hep-2 derived from throat cancer cell were transplanted under the skin of abdomen and, on the 7th to 10th day after the transplantation, living cohesion of tumor was confirmed. Then 100 ~g/mouse of test sample (intra-venously) and 25 mg/kg of 5-FU (in-traperitoneally) were admin-istered twice a week for four weeks. Tumor was extracted on the fourth week after the administration and the weight was measured. The results are given in the following table~ The test samples used were the same as that in the above (1).
(HeLa S3) Inhibition Sample Weight (g) + S.E. Rate (~) 35Control 3.85 + 0.23 --Test Sample I 1.57 + 0.19 59 Test Sample II 1.55 + 0.20 60 5-FU 2.06 + 0.35 47 (Hep-2) Inhibition Sample Weight (g) + S.E.Rate (%) Control 0.88 + 0.07 --Test Sample I 0.38 + 0.07 57 Test Sample II 0.35 + 0.05 60 5-FU 0.56 + 0.12 30 It is apparent that the nucleic acid derivatives of the present invention exhibit strong inhibitory action for proliferation.
(11) Inhibitory action against metastasis of tumor cells to lung:
To C57BL/6 mice (5 weeks age; male) was transplanted to intravenous vein, 2 x 10 B16F10 cells which are transplant-able melanoma of the same type and the numbers of nodes movedto lung (numbers of colonies) on the second week after the transplantation were counted. The samples were administered intravenously at 24 hours prior to the transplantation of sl6F10 melanoma. Numbers of the cases were 9. The results are given below. The test samples I and II and control sam-ple used were the same as those in the above ~1).
Numbers of Nodes Sample (micrograms/mouse)moved to Lung -~ S.E.
Control 112 + 17 25Test Sample I ( 1) 33 + 6*
( 10) 21 + 10*
(100) 4 + 2*
Test Sample II ( 1) 21 + 6*
( 10) 15 + ~*
30(100) 4 + 1*
Control Sample ( 1) 36 + 16*
( 10) 3 + 0.7*
(100) 7 + 3*
It is apparent that the nucleic acid derivatives of the present invention exhibit inhibitory action against metastasis of tumor cells to lung.
Safety of the Nucleic Acid Derivatives of the Presen-t Invention:
Safety of the nucleic acid derivatives of the present invention will be illustrated below. Sulphurized poly C:poly I
derivative which one of the nucleic acid derivatives of the - 20 - 13~571~
present invention was taken and its cytotoxic effect to bone marrow main cells was examined. The sulphurized poly C:poly I
derivative used had an n-value of 20 and molecular size of 150 to 2,000 residues.
The sample was injected intravenously to mice (S weeks age; male; each group comprises 5 mice) at the does of 100 micrograms/mouse and, after 24 hours, bone marrow cells of them were collected. The cells were fixed and dyed with Gi-emsa. Cells of the smeared~sample were observed wi-th a mi-croscope and the degree of appearance of reticulocytes was counted by %. Known poly I:poly C was used as a control.
Physiological saline solution was administered to the control.
The results are given in the following table:
Erythrocyte Cells (%) Control 40 Test Sample I 38 Test Sample II 39 Control Sample 11 This is clear evidence that the nucleic acid deriva-tives of the present invention exhibit high safety.
The pyrogenic effect of the nucleic acid derivatives of the present invention were examined for pyrogenic effect.
Injection of the poly I:poly C in vivo has been known to be pyrogenic. As a result of a pyrogen test to rabbits, it has been found that the poly I:poly C gives 1.45C of body temperature rise in average. On the contrary, the nucleic acid derivatives of the present invention (both S~ and S-S
substances in the above cytotoxic experiment) show only 0.25C
rise in body temperature on average and the result of the py-rogenic test was negative. In those experiments, three rab-bits per one group were used and a solution of the sample (0.2 microgram/kg) in 10 ml of physiological saline solution was intravenously injected beneath the ear of rabbits and the body temperature at 4 hours after the injection was observed.
This is further evidence -that the nucleic acid deriv-atives of the present invention exhibit very high safety.
The results of acute toxicity tests of the nucleic 13157i7 acid derivatives of the present invention is set forth below.
With reference to normal ddY mice, the dose of the nucleic acid derivatives of the present invention is restricted by the upper limit of the solubility of the drug given and the LD50 value was not less than 394 mg/kg. The e~fect was judged by whether the mouse was dead or alive at one week after the intravenous injection. The similar test using other mice strains (C57BL6 and BALB/c) also gave the result that the nu-cleic acid derivatives (the same as above) of the present in-vention show much lower toxicity than known poly I:poly C.
This toxicity data is further evidence of the safetyof the nucleic acid derivatives of the present invention.
Route of Test Sam- Test Sam- Control Strain Administration ple I LD50 ple II Sample ddY intravenously >39~ mg/kg >394 mg/kg 132 mg/kg The following non-limitative examples more particularly illustrate the present invention.
Exarnple 1 Synthesis of Sulphurized Polycytidylic Acid Poly C (0.5 g) was dissolved in a mixed solvent of 4 ml of water and 2 ml of pyridine, the solution was placed in a 30 ml stainless steel reaction tube together with S ml of li-quid hydrogen sulphide, and made to react at 37C for 6 hours.
The pressure in the sealed tube during the reaction was 20 to 22 kg/cm2.
After the reaction, the reaction tube was cooled to 0C or lower to decrease the pressure sufficiently and the reaction tube was opened. An excess of unreacted hydrogen sulphide was removed, the sulphurized poly C solution was transferred to a 50 ml round flask, and unreacted hydrogen sulphide was removed in vacuo. The resulting solution was dialyzed three times against Tris buffer (pH 7.5) containing lO liters of 50mM sodium chloride and the resulting trans-parent liquid was further lyophilyzed to give 0.47 g of white fibrous substance.
The ultraviolet absorption spectra of the resulting ~- ~* Trade Mark - 22 ~ 1 3 1 5 7 i 7 substance were measured in a neutral aqueous solu-tion to give the result of Fig. 1. It is known that the maximum absorp-tion wavelength oE cytidylic acid is 271 nm and that the ab-sorption wavelength of 4-thiouridylic acid in which -NH2 group at 4-position of pyrimidine ring of cytidylic acid was substituted with an -SH group is shown at 330 nm.
Out of the ratio of heights of peaks in Fig. 1, it has been confirmed that there is one 4--thiouridylic acid to 13 cytidylic acid.
In the same manner, -the reaction temperatures and re~
action time of the stainless tube were varied and sulphurized polycytidylic acids having the degrees of sulphurization as given in the following table were obtained. Here the degree of sulphurization is given by "n" and said "n" means the num-15 bers of cytidylic acid to one 4-thiouridylic acid.
Reaction Temperature Reaction Time n 45C 12 hours 6 6 + 1 37 6 13 + 2 37 4 26 ~ 2 37 2.5 39 + 2 -Example 2 Manufacture of Double Strand Nucleic Acid Derivatives Poly I and sulphurized poly C obtained hereinabove (1) were dissolved, in the same moles, in a Tris buffer contain-ing 50mM of sodium chloride to make the concentration of 10 to 20 mg/ml and, in the water bath, the temperature was grad-ually raised from the room temperature to 68C. The mixture was maintained at 68C for about 10 minutes, allowed to stand until it became to room temperature, and s-tored at 4C.
The resulting solution is lyophilized to give 1.2 g of a nucleic acid derivative having SH groups in whi-te solid~
Then, to this was added about 10 times volume (by molar ratio) of lN iodine solution (a mixture of 1/3 mole of iodine and 2/3 mole of sodium iodide). The mixture was well mixed to make it uniform and allowed to stand at 0C for 1 hour.
The reaction solution was well dialyzed against water until `" - 23 - 1 3~ ~7 17 the yellow color of iodine vanished.
The solution thus obtained was lyophilyzed to give 0.98 g of white solid.
The fact that the resulting solid contains an S-S link-age was confirmed as follows. Thus, 0.1 g of this solid wasdissolved in lO ml of Tris buffer (pH 7.5) containing 0.03M
of sodium sulphite, stored at room temperature for 0 to 7.5 hours, and the ultraviolet absorption spectra at each time were observed. The result is given in Fig. 2. It is known that the S-S bond are generally reversible and that the S-S
bond cleaves by reduction giving an S-H group. With an elapse of time, a shoulder peak of 310 nm which is an absorption wave-length due to the S-S bond decreased while an absorption at 330 nm which is due to -SH substance (4-thiouridine) increased and, after 7.5 hours, the degree of absorption at 330 nm be-came to be identical with that of the sulphurized poly C be-fore the oxidation. It is -therefore apparent that the S-S
bond was quantitatively reduced to -SH group.
Example 3 Cross-Linking Structure and Biological Activity Among the nucleic acid derivatives into which sulphur atoms are introduced, synthetic methods and physiological ac-tivities of the test sample I (having all-reduced type SH
groups) and the test sample II (having all-oxidized type S-S
groups) were described as hereinabove. The cross-linking substances of the nucleic acid derivatives described here are the compounds in which a part of sulphur atoms introduced into the same molecule exhibits a disulphide bond between or in the molecules.
For example, the cross-linking derivatives having 80%
of SH groups and 20% of S-S groups in the same molecule can form a multistrand cross-linking structure in 20% of the total part. In other words, in the case of poly C with l,000 bp for example, it has a cross-linking structure at ten parts.
Any nucleic acid derivative having various cross-linking num-bers may be easily prepared by a partial oxidation of the SH
substance under a mild condition or by a par-tial reduction of - 2~ - 1 3 1 5 7 1 7 the S-S substance under a mild condition. The synthetic con-dition is the same as that described in the examples.
The ratio of the SH group numbers and the S-S group numbers, i.e. the degree of cross-linking, can be calculated as a ratio of the values obtained by dividing the ultraviolet absorbances at 310 nm of S-S group and 330 nm of SH group, respectively, by the molecular absorption coefficients.
An alternative will be as follows. Thus, nucleic acid derivative is decomposed with an RNAase (e.g., ribonuclease Pl), the decomposed product is subjected to a high performance liquid chromatography (HPLC) of the reverse phase system, and 4-thiouridine (or 4-thiouridylic acid) and a bis substance formed by a disulphide bond thereof are separated, and their amounts are measured.
With reference to physiological activities, it was found that nucleic acid derivatives having any degree of cross-linking from 1 (totally S-S substance; e.g. test sam-ple II) to 0 (to-tally SH substance; e.g. test sample I) ex-hibits the similar activity and safety as -those of the afore-mentioned test samples I and II.
This fact suggests that, even if those nucleic acid derivatives may be partially oxidizable or reduceable, they can exhibit the same stable physiological activity as the original ones.
Example 4 Low-Molecularization The SH-containing nucleic acid derivative (1 g) ob-tained in the Example 2 was dissolved in 120 ml of wa-ter and 30 ml of 5M sodium chloride solution and 150 ml of forma-mide were added thereto. The mixture was vigorously stirred to give uniform solution. The reaction solution was heated at 80C for 8 hours, well dialyzed against water, and lyo-philyzed to give 0.95 g of white solid.
When the S-S bond-containing nucleic acid derivative obtained in the above is subjected to low molecularization the same as above, the same result is obtained.
This was subjected to a hiyh performance liquid chro-.i~V
- 25 ~ 1 31 571 7 matography by a gel Eiltration and the resulting pattern is given in Fig. 3. The condition applied was that TSK-gel G4000SW (0.65 x 60 cm) was used and, as an eluate, 50mM
Tris HCl buffer (pH 7.5) containing 0.5M sodium chloride was used.
Each arrow in the figure shows the elution position of each standard size marker ~unit of said size marker was bp) and, out of Fig. 3, it is apparent that this has a max-imum distribution at around 500 residue numbers and that it has a molecular size which distributes 150 to 1,000 residue numbers. The result well agreed with the value obtained from an electrophoresis using polyacrylamide gel or agarose gel. As already stated, the molecular size is indicated in this specification by the term "residue number: and, in Fig.
3, the "residue number" is in agreement with the "bp" as units.
When the reaction time and temperature of this low-molecularization were varied, the substances having the fol-lowing molecular sizes were obtained by the same manner.
Maximum Reaction ReactionDistributionDistribution Temp. Time (bp) (bp) - 0 hr. - 2000 80C 2~ 30 10 - 55 Among the nucleic acid derivatives obtained by the ab-ove example, those with 200 to 5,000 residue numbers of mol-ecular size distribution were taken and their fusing curveswere measured.
The sample I or II (0.7 OD/ml) in 10mM Tris buffer (pH
7.5) containing 0.1M sodium chloride was warmed, at the ris-ing rate of 2C/4 minutes, from 20C to 90C, the ultraviolet absorbancy at each tempera-ture was measured at the wavelength of 260 nm, and an increase of ultraviolet absorbancy by a hy-perchromicity was represented by a relative ra-tio giving -the state at 20C as a base. A Beckmann-DU-8B spectrophotometer - 2~ - 1 31 57 l7 was used for the measurement. The result is given in Fig.
4. Fusion of the nucleic acid derivatives was gradually ob-served during 35C to 55C and, a-t around 59C, a sudden fu-sion curve was obtained. At higher than 70C, the curve nearly arrived at a plateau and this means the disappearance of high dimension structure and spiral structure formation by hydrogen bond of the nucleic acid deriva-tives.
The 50% fusing temperatures for the samples I and II
were 59.5C and 59.3C, respec-tively. Out of the calcula-tion from the increasing/decreasing rate in ultraviolet ab-sorbancy at 260 nm between 90C and 25C, it has been con-firmed that the hyperchromicity (color darkening effect) for the samples I and II were 43.5gO and 42.4% respectively. This indicates that the nucleic acid deriva-tives are with very stable structures under physiological conditions.
To C57BL/6 mice (5 weeks age; male) was transplanted to intravenous vein, 2 x 10 B16F10 cells which are transplant-able melanoma of the same type and the numbers of nodes movedto lung (numbers of colonies) on the second week after the transplantation were counted. The samples were administered intravenously at 24 hours prior to the transplantation of sl6F10 melanoma. Numbers of the cases were 9. The results are given below. The test samples I and II and control sam-ple used were the same as those in the above ~1).
Numbers of Nodes Sample (micrograms/mouse)moved to Lung -~ S.E.
Control 112 + 17 25Test Sample I ( 1) 33 + 6*
( 10) 21 + 10*
(100) 4 + 2*
Test Sample II ( 1) 21 + 6*
( 10) 15 + ~*
30(100) 4 + 1*
Control Sample ( 1) 36 + 16*
( 10) 3 + 0.7*
(100) 7 + 3*
It is apparent that the nucleic acid derivatives of the present invention exhibit inhibitory action against metastasis of tumor cells to lung.
Safety of the Nucleic Acid Derivatives of the Presen-t Invention:
Safety of the nucleic acid derivatives of the present invention will be illustrated below. Sulphurized poly C:poly I
derivative which one of the nucleic acid derivatives of the - 20 - 13~571~
present invention was taken and its cytotoxic effect to bone marrow main cells was examined. The sulphurized poly C:poly I
derivative used had an n-value of 20 and molecular size of 150 to 2,000 residues.
The sample was injected intravenously to mice (S weeks age; male; each group comprises 5 mice) at the does of 100 micrograms/mouse and, after 24 hours, bone marrow cells of them were collected. The cells were fixed and dyed with Gi-emsa. Cells of the smeared~sample were observed wi-th a mi-croscope and the degree of appearance of reticulocytes was counted by %. Known poly I:poly C was used as a control.
Physiological saline solution was administered to the control.
The results are given in the following table:
Erythrocyte Cells (%) Control 40 Test Sample I 38 Test Sample II 39 Control Sample 11 This is clear evidence that the nucleic acid deriva-tives of the present invention exhibit high safety.
The pyrogenic effect of the nucleic acid derivatives of the present invention were examined for pyrogenic effect.
Injection of the poly I:poly C in vivo has been known to be pyrogenic. As a result of a pyrogen test to rabbits, it has been found that the poly I:poly C gives 1.45C of body temperature rise in average. On the contrary, the nucleic acid derivatives of the present invention (both S~ and S-S
substances in the above cytotoxic experiment) show only 0.25C
rise in body temperature on average and the result of the py-rogenic test was negative. In those experiments, three rab-bits per one group were used and a solution of the sample (0.2 microgram/kg) in 10 ml of physiological saline solution was intravenously injected beneath the ear of rabbits and the body temperature at 4 hours after the injection was observed.
This is further evidence -that the nucleic acid deriv-atives of the present invention exhibit very high safety.
The results of acute toxicity tests of the nucleic 13157i7 acid derivatives of the present invention is set forth below.
With reference to normal ddY mice, the dose of the nucleic acid derivatives of the present invention is restricted by the upper limit of the solubility of the drug given and the LD50 value was not less than 394 mg/kg. The e~fect was judged by whether the mouse was dead or alive at one week after the intravenous injection. The similar test using other mice strains (C57BL6 and BALB/c) also gave the result that the nu-cleic acid derivatives (the same as above) of the present in-vention show much lower toxicity than known poly I:poly C.
This toxicity data is further evidence of the safetyof the nucleic acid derivatives of the present invention.
Route of Test Sam- Test Sam- Control Strain Administration ple I LD50 ple II Sample ddY intravenously >39~ mg/kg >394 mg/kg 132 mg/kg The following non-limitative examples more particularly illustrate the present invention.
Exarnple 1 Synthesis of Sulphurized Polycytidylic Acid Poly C (0.5 g) was dissolved in a mixed solvent of 4 ml of water and 2 ml of pyridine, the solution was placed in a 30 ml stainless steel reaction tube together with S ml of li-quid hydrogen sulphide, and made to react at 37C for 6 hours.
The pressure in the sealed tube during the reaction was 20 to 22 kg/cm2.
After the reaction, the reaction tube was cooled to 0C or lower to decrease the pressure sufficiently and the reaction tube was opened. An excess of unreacted hydrogen sulphide was removed, the sulphurized poly C solution was transferred to a 50 ml round flask, and unreacted hydrogen sulphide was removed in vacuo. The resulting solution was dialyzed three times against Tris buffer (pH 7.5) containing lO liters of 50mM sodium chloride and the resulting trans-parent liquid was further lyophilyzed to give 0.47 g of white fibrous substance.
The ultraviolet absorption spectra of the resulting ~- ~* Trade Mark - 22 ~ 1 3 1 5 7 i 7 substance were measured in a neutral aqueous solu-tion to give the result of Fig. 1. It is known that the maximum absorp-tion wavelength oE cytidylic acid is 271 nm and that the ab-sorption wavelength of 4-thiouridylic acid in which -NH2 group at 4-position of pyrimidine ring of cytidylic acid was substituted with an -SH group is shown at 330 nm.
Out of the ratio of heights of peaks in Fig. 1, it has been confirmed that there is one 4--thiouridylic acid to 13 cytidylic acid.
In the same manner, -the reaction temperatures and re~
action time of the stainless tube were varied and sulphurized polycytidylic acids having the degrees of sulphurization as given in the following table were obtained. Here the degree of sulphurization is given by "n" and said "n" means the num-15 bers of cytidylic acid to one 4-thiouridylic acid.
Reaction Temperature Reaction Time n 45C 12 hours 6 6 + 1 37 6 13 + 2 37 4 26 ~ 2 37 2.5 39 + 2 -Example 2 Manufacture of Double Strand Nucleic Acid Derivatives Poly I and sulphurized poly C obtained hereinabove (1) were dissolved, in the same moles, in a Tris buffer contain-ing 50mM of sodium chloride to make the concentration of 10 to 20 mg/ml and, in the water bath, the temperature was grad-ually raised from the room temperature to 68C. The mixture was maintained at 68C for about 10 minutes, allowed to stand until it became to room temperature, and s-tored at 4C.
The resulting solution is lyophilized to give 1.2 g of a nucleic acid derivative having SH groups in whi-te solid~
Then, to this was added about 10 times volume (by molar ratio) of lN iodine solution (a mixture of 1/3 mole of iodine and 2/3 mole of sodium iodide). The mixture was well mixed to make it uniform and allowed to stand at 0C for 1 hour.
The reaction solution was well dialyzed against water until `" - 23 - 1 3~ ~7 17 the yellow color of iodine vanished.
The solution thus obtained was lyophilyzed to give 0.98 g of white solid.
The fact that the resulting solid contains an S-S link-age was confirmed as follows. Thus, 0.1 g of this solid wasdissolved in lO ml of Tris buffer (pH 7.5) containing 0.03M
of sodium sulphite, stored at room temperature for 0 to 7.5 hours, and the ultraviolet absorption spectra at each time were observed. The result is given in Fig. 2. It is known that the S-S bond are generally reversible and that the S-S
bond cleaves by reduction giving an S-H group. With an elapse of time, a shoulder peak of 310 nm which is an absorption wave-length due to the S-S bond decreased while an absorption at 330 nm which is due to -SH substance (4-thiouridine) increased and, after 7.5 hours, the degree of absorption at 330 nm be-came to be identical with that of the sulphurized poly C be-fore the oxidation. It is -therefore apparent that the S-S
bond was quantitatively reduced to -SH group.
Example 3 Cross-Linking Structure and Biological Activity Among the nucleic acid derivatives into which sulphur atoms are introduced, synthetic methods and physiological ac-tivities of the test sample I (having all-reduced type SH
groups) and the test sample II (having all-oxidized type S-S
groups) were described as hereinabove. The cross-linking substances of the nucleic acid derivatives described here are the compounds in which a part of sulphur atoms introduced into the same molecule exhibits a disulphide bond between or in the molecules.
For example, the cross-linking derivatives having 80%
of SH groups and 20% of S-S groups in the same molecule can form a multistrand cross-linking structure in 20% of the total part. In other words, in the case of poly C with l,000 bp for example, it has a cross-linking structure at ten parts.
Any nucleic acid derivative having various cross-linking num-bers may be easily prepared by a partial oxidation of the SH
substance under a mild condition or by a par-tial reduction of - 2~ - 1 3 1 5 7 1 7 the S-S substance under a mild condition. The synthetic con-dition is the same as that described in the examples.
The ratio of the SH group numbers and the S-S group numbers, i.e. the degree of cross-linking, can be calculated as a ratio of the values obtained by dividing the ultraviolet absorbances at 310 nm of S-S group and 330 nm of SH group, respectively, by the molecular absorption coefficients.
An alternative will be as follows. Thus, nucleic acid derivative is decomposed with an RNAase (e.g., ribonuclease Pl), the decomposed product is subjected to a high performance liquid chromatography (HPLC) of the reverse phase system, and 4-thiouridine (or 4-thiouridylic acid) and a bis substance formed by a disulphide bond thereof are separated, and their amounts are measured.
With reference to physiological activities, it was found that nucleic acid derivatives having any degree of cross-linking from 1 (totally S-S substance; e.g. test sam-ple II) to 0 (to-tally SH substance; e.g. test sample I) ex-hibits the similar activity and safety as -those of the afore-mentioned test samples I and II.
This fact suggests that, even if those nucleic acid derivatives may be partially oxidizable or reduceable, they can exhibit the same stable physiological activity as the original ones.
Example 4 Low-Molecularization The SH-containing nucleic acid derivative (1 g) ob-tained in the Example 2 was dissolved in 120 ml of wa-ter and 30 ml of 5M sodium chloride solution and 150 ml of forma-mide were added thereto. The mixture was vigorously stirred to give uniform solution. The reaction solution was heated at 80C for 8 hours, well dialyzed against water, and lyo-philyzed to give 0.95 g of white solid.
When the S-S bond-containing nucleic acid derivative obtained in the above is subjected to low molecularization the same as above, the same result is obtained.
This was subjected to a hiyh performance liquid chro-.i~V
- 25 ~ 1 31 571 7 matography by a gel Eiltration and the resulting pattern is given in Fig. 3. The condition applied was that TSK-gel G4000SW (0.65 x 60 cm) was used and, as an eluate, 50mM
Tris HCl buffer (pH 7.5) containing 0.5M sodium chloride was used.
Each arrow in the figure shows the elution position of each standard size marker ~unit of said size marker was bp) and, out of Fig. 3, it is apparent that this has a max-imum distribution at around 500 residue numbers and that it has a molecular size which distributes 150 to 1,000 residue numbers. The result well agreed with the value obtained from an electrophoresis using polyacrylamide gel or agarose gel. As already stated, the molecular size is indicated in this specification by the term "residue number: and, in Fig.
3, the "residue number" is in agreement with the "bp" as units.
When the reaction time and temperature of this low-molecularization were varied, the substances having the fol-lowing molecular sizes were obtained by the same manner.
Maximum Reaction ReactionDistributionDistribution Temp. Time (bp) (bp) - 0 hr. - 2000 80C 2~ 30 10 - 55 Among the nucleic acid derivatives obtained by the ab-ove example, those with 200 to 5,000 residue numbers of mol-ecular size distribution were taken and their fusing curveswere measured.
The sample I or II (0.7 OD/ml) in 10mM Tris buffer (pH
7.5) containing 0.1M sodium chloride was warmed, at the ris-ing rate of 2C/4 minutes, from 20C to 90C, the ultraviolet absorbancy at each tempera-ture was measured at the wavelength of 260 nm, and an increase of ultraviolet absorbancy by a hy-perchromicity was represented by a relative ra-tio giving -the state at 20C as a base. A Beckmann-DU-8B spectrophotometer - 2~ - 1 31 57 l7 was used for the measurement. The result is given in Fig.
4. Fusion of the nucleic acid derivatives was gradually ob-served during 35C to 55C and, a-t around 59C, a sudden fu-sion curve was obtained. At higher than 70C, the curve nearly arrived at a plateau and this means the disappearance of high dimension structure and spiral structure formation by hydrogen bond of the nucleic acid deriva-tives.
The 50% fusing temperatures for the samples I and II
were 59.5C and 59.3C, respec-tively. Out of the calcula-tion from the increasing/decreasing rate in ultraviolet ab-sorbancy at 260 nm between 90C and 25C, it has been con-firmed that the hyperchromicity (color darkening effect) for the samples I and II were 43.5gO and 42.4% respectively. This indicates that the nucleic acid deriva-tives are with very stable structures under physiological conditions.
Claims (7)
1. A nucleic acid derivative of a nucleic acid polymer, said nucleic acid polymer being a single strand ribonucleotide polymer wherein the derivative is a poly C
containing one 4-thiouridylic acid moiety containing sulphur atoms, obtained as a result of substituting an -NH2 group in a pyrimidine ring of the cytidylic acid in the poly C of the nucleic acid polymer with an -SH group, for every 6 to 39 cytidylic acid moieties.
containing one 4-thiouridylic acid moiety containing sulphur atoms, obtained as a result of substituting an -NH2 group in a pyrimidine ring of the cytidylic acid in the poly C of the nucleic acid polymer with an -SH group, for every 6 to 39 cytidylic acid moieties.
2. A nucleic acid derivative as defined in Claim 1 further having at least one disulphide bond.
3. A nucleic acid derivative as defined in Claim 1 in which the length of the polymer is 50 to 5,000 as calculated by base numbers.
4. A nucleic acid derivative of a nucleic acid polymer, said nucleic acid polymer being a double strand ribonucleotide polymer, wherein the derivative is composed of poly I and poly C containing one 4-thiouridylic acid moiety containing sulphur atoms by substituting an -NH2 group in the pyrimidine ring of a cytidylic acid in the poly C of the nucleic acid polymer with an -SH group, for every 6 to 39 cytidylic acid moieties.
5. A nucleic acid derivative as defined in Claim 4 further having at least one disulphide bond.
6. A nucleic acid derivative as defined in Claim 4 in which the length of the polymer is 50 to 10,000 calculated as base pair numbers.
7. A nucleic acid derivative of a nucleic acid polymer wherein said derivative contains a disulphide bond, and further wherein said nucleic acid polymer is a single strand ribonucleotide polymer or a double strand ribonucleotide polymer, wherein said single strand ribonucleotide polymer is a poly C containing one 4-thiouridylic acid moiety containing sulphur atoms, obtained as a result of substituting an -NH2 group in a pyrimidine ring of the cytidylic acid in the poly C of the nucleic acid polymer with an -SH group, for every 6 to 39 cytidylic acid moieties, and said double strand ribonucleotide polymer is composed of poly I and poly C containing one 4-thiouridylic acid moiety containing sulphur atoms by substituting an -NH2 group in the pyrimidine ring of cytidylic acid in the poly C of the nucleic acid polymer with an -SH group, for every 6 to 39 cytidylic acid moieties.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP107686 | 1986-01-06 | ||
| JP61-001076 | 1986-01-06 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1315717C true CA1315717C (en) | 1993-04-06 |
Family
ID=11491410
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000526797A Expired - Fee Related CA1315717C (en) | 1986-01-06 | 1987-01-06 | Nucleic acid derivatives |
Country Status (11)
| Country | Link |
|---|---|
| JP (1) | JPS62252798A (en) |
| KR (1) | KR940004076B1 (en) |
| BE (1) | BE906157A (en) |
| CA (1) | CA1315717C (en) |
| CH (1) | CH671960A5 (en) |
| DE (1) | DE3700164A1 (en) |
| ES (1) | ES2004038A6 (en) |
| FR (1) | FR2596762B1 (en) |
| GB (1) | GB2185026B (en) |
| NL (1) | NL8700016A (en) |
| SE (1) | SE469594B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ATE473748T1 (en) * | 1998-05-25 | 2010-07-15 | Nippon Shinyaku Co Ltd | METHOD FOR PRODUCING A COMPOSITION COMPOSITION CONTAINING NUCLEIC ACID |
| JPWO2003023031A1 (en) * | 2001-09-04 | 2004-12-24 | 日本新薬株式会社 | Methods for diagnosing the effectiveness of nucleic acid therapy |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB906851A (en) * | 1960-05-03 | 1962-09-26 | Schwarz Bio Res Inc | Thiolation of polymers |
| DE2041735A1 (en) * | 1970-08-22 | 1972-02-24 | Merck Patent Gmbh | Thio-pyrimidine derivatives |
| GB1364987A (en) * | 1971-06-19 | 1974-08-29 | Merck Patent Gmbh | Biologically active two component complexes |
| GB2038628A (en) * | 1978-11-14 | 1980-07-30 | Searle & Co | Physiological compositions comprising poly(4-thiouridylic acid) or copolymers thereof and the use thereof as anti-viral agents or serum cholesterol lowering agents |
-
1986
- 1986-11-25 JP JP61280452A patent/JPS62252798A/en active Pending
- 1986-12-30 SE SE8605616A patent/SE469594B/en not_active IP Right Cessation
- 1986-12-31 BE BE0/217694A patent/BE906157A/en not_active IP Right Cessation
- 1986-12-31 FR FR8618433A patent/FR2596762B1/en not_active Expired - Fee Related
-
1987
- 1987-01-05 DE DE19873700164 patent/DE3700164A1/en active Granted
- 1987-01-05 ES ES8700012A patent/ES2004038A6/en not_active Expired
- 1987-01-06 CA CA000526797A patent/CA1315717C/en not_active Expired - Fee Related
- 1987-01-06 KR KR1019870000061A patent/KR940004076B1/en active IP Right Grant
- 1987-01-06 NL NL8700016A patent/NL8700016A/en not_active Application Discontinuation
- 1987-01-06 GB GB8700183A patent/GB2185026B/en not_active Expired - Fee Related
- 1987-01-06 CH CH23/87A patent/CH671960A5/de not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| GB2185026A (en) | 1987-07-08 |
| SE8605616L (en) | 1987-07-07 |
| KR940004076B1 (en) | 1994-05-11 |
| FR2596762A1 (en) | 1987-10-09 |
| KR870007198A (en) | 1987-08-17 |
| BE906157A (en) | 1987-07-01 |
| CH671960A5 (en) | 1989-10-13 |
| DE3700164C2 (en) | 1993-02-25 |
| DE3700164A1 (en) | 1987-07-09 |
| GB8700183D0 (en) | 1987-02-11 |
| SE8605616D0 (en) | 1986-12-30 |
| ES2004038A6 (en) | 1988-12-01 |
| NL8700016A (en) | 1987-08-03 |
| JPS62252798A (en) | 1987-11-04 |
| SE469594B (en) | 1993-08-02 |
| GB2185026B (en) | 1990-08-08 |
| FR2596762B1 (en) | 1993-10-08 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKLA | Lapsed | ||
| MKLA | Lapsed |
Effective date: 19951008 |