DE3700164C2 - - Google Patents

Description

The invention relates to nucleic acid derivatives with physiological Efficacy according to the above Claims for use as a medicament.

Nucleic acid is composed of such sugars as ribose, bound to a purine ring or pyrimidine ring and are arranged in chains via phosphoric acid.

Among the nucleic acids, RNA (ribonucleotide polymer) is a high molecular weight compound in chain structure in which ribose is contained as sugar and the sugar units via a Diesterbindung the phosphoric acid are combined.

The double chain has in its steric structure by the combination of purine or pyrimidine ring units of the base (such as Inosine, cytidine, uridine, etc.), which forms the nucleic acid, a spiral shape. Double-stranded nucleic acids have one very useful physiological efficacy, and they are intense been examined.

Of these, are natural (eg, virus-derived RNA) and synthetic double-stranded RNA, such as polyinosinic acid Polycytidylic acid derivatives (hereinafter abbreviated as "poly-I- Poly-C "), polyadenylic acid polyuridylic acid derivatives etc., has been thoroughly investigated (eg, De Clercq et al .: Texas Reports on Biology and Medicine, 41, 77, 1982).  

Many RNA nucleic acid derivatives are synthesized as such and their physiological activities have been studied.

The poly-I-poly-C is a substance with high efficiency, and their applicability has been evaluated based on research Service. A connection in which only a small Part of the cytidine in the poly-I-poly-C converted to uridine (for example, mismatched RNA) has been studied because of its physiological efficacy similar to that of poly-I-poly-C (See, Japanese Patent Publication 50/082226).

Studies on polycytidic acid have also been made and there is a report (British Patent Application No. 2038628) which states that when a mercapto group (-SH) replaces the -NH ₂ group of the pyrimidine ring Polycytidylic acid is introduced, the physiological activity increases. A control of the chain length is not provided here.

From the work "Modification of Cytosine Moieties of Nucleic Acids with Hydrogen Sulfides "by K. Miura and T. Ueda (Chem. Pharm. Bull. 28, 1980, pp. 3415-18) it is also known To sulfide nucleic acids in the cytidine moiety. But here too bridging and chain length control are not discussed or just mentioned.

The conventional physiologically active substances as above are expected to have beneficial effects despite their toxicity, as seen with poly-I-poly-C, which can not be denied (De Clercq et al .: Infect. Immuni., 6, 344, 1972), and improvements are needed here make. Furthermore, it is desirable that to increase physiological efficacy.

In the course of many years of investigations of the inventor is found have been that, despite some coincidence, RNA by a certain covalent linkage stabilized derivatives with a Conformation of higher order can form. If her physiological Effectiveness is measured, they are found to be have a higher efficacy with very low toxicity as the conventional physiologically active substances, which led to the present invention.  

The invention therefore nucleic acid derivatives, which consist of a Nucleic acid polymers in which a purine and / or pyrimidine rings, the contained in these polymers, substituted by sulfur atoms are converted by oxidation into disulfide bridges.

The nucleic acid derivatives according to the invention can be prepared from nucleic bases into which -SH groups are introduced can be. A specific example of the invention Nucleic acid derivatives is one that has a complementary having a double spiral structure of a chain connection (so-called "poly-C" in the present specification), which a cytidylic acid polymer, and another chain compound (referred to in the present specification as "poly-I"), which is an inosinic acid polymer. Although this connection a Having a structure similar to that of the above-mentioned poly-1 Poly-C resembles have the nucleic acid derivatives of the invention special properties for the following reasons.

Nucleic acid derivatives according to the present invention are those with partial crosslinking (-S-H bond), wherein a sulfur atom on 6-39 in the poly-C-containing cytidic acid comes.

The sulfur atoms in the nucleic acid derivatives of the invention may be introduced before or after the preparation of the polymer by the use of an enzyme reaction. The introduction is carried out by substituting -SH groups for the nucleic base, for example by substituting an -SH group on a -NH ₂ group in a pyrimidine ring of cytidine acid. The substituted radical is called 4-thiouridylic acid.

5'-4-Thiouridylsäure

The nucleic acid derivatives according to the invention can be used on their Phosphoric acid side chain to lower molecular weight compounds split and those of lower molecular weight also belong to the invention. Thus exists in this sense a difference to the conventional poly-I-poly-C.

As a consequence of the characteristic feature of the nucleic acid derivatives according to the invention, d. H. the substitution with -SH, and the Formation of lower molecular substances by splitting at the Phosphorsäureseitenkette, it is possible for the first time, the effect according to the invention, that (1) the physiological Effectiveness increases and (2) safety is improved can.

The physiological activity of the nucleic acid derivatives according to The invention is extremely advantageous for pharmaceuticals. As will be explained below, have the nucleic acid derivatives according to the invention a strong activity as interferon inducers. This effect is just one of many physiological Effects of the nucleic acid derivatives of the invention. What the physiological effects of the nucleic acid derivatives As regards the invention, the following may be mentioned numerous effects are called. These include the Abilities to form TNF, interleukin 1 and interleukin 2, to activate the macrophages, to activate NK cells, to inhibit the proliferation of tumor cells that  To inhibit tumor proliferation in cancerous mice, carrying the proliferation in human tumor cells to inhibit nude mice, the formation of metastases of Prevent tumor cells in the lungs, etc.

Nucleic acid derivatives according to the invention have a lot greater safety than interferon inducers, such as conventional ones Poly-I-poly-C. Consequently, the compounds according to the invention useful antiviral and antitumor agents.

The term "base pair" (abbreviated BP) used to indicate the Molecular size of the nucleic acid is often used the molecular size by the number of bases in the nucleic acid in each complementary strand (i.e., "10 BP" means that the double-stranded polymer has 10 bases). There too other nucleic acid polymers than double-stranded polymers according to the invention will be used instead of "BP" here Expression "remaining number" used. For example, means "Residual number 10" indicates that the nucleic acid polymer has 10 bases in having a strand.

Nucleic acid derivatives according to the invention contain substances with a variety of molecular sizes, and it's common to prefer that the remaining number is not less than 50, d. h., That the remaining number is 50 to 10,000. The Size can be even bigger, d. H. 200,000 are too to tolerate, and it has been shown that the molecule size a small influence on the physiological effectiveness according to the invention has.

In order to be able to indicate the number of sulfur atoms in the nucleic acid derivatives according to the invention, the term "sulfurization (s)" is used in the present specification. The cytidylic acid is converted to 4-thiouridic acid by substitution of the -NH ₂ group in the pyrimidine ring with the -SH group, and "n" is the number of cytidylic acids on a 4-thiouridylic acid. The nucleic acid derivatives of the invention contain substances having a plurality of "n", and it is preferable that n = 6 or more. When n is smaller, it has been found that the physiological efficacy decreases. If n = 6 or more, there is no particular restriction, and n may even be 39. There is only a small influence of n on the physiological activity according to the invention.

For the preparation of the nucleic acid derivatives according to the invention Numerous methods can be used. The poly-C and the same substances, the starting materials for the invention Derivatives are readily available. The poly-C can be replaced by, for example, reaction with sulfurizing agents, like hydrogen sulfide, easily sulfurized. As a result This reaction may involve a certain number of cytidic acid residues in the poly-C are converted into 4-thiouridylic acid. If the Reaction conditions, such as reaction temperature and reaction time, Poly-C can be modified with the desired n- Values are produced.

The nucleic acid derivatives according to the invention can be applied to the obtained above methods almost quantitatively and it has been shown that the overall yield is about 90%.  

For example, when 36 mmol uridine-5'-diphosphate (UDP) and 7 mmol 4-thiouridine-5'-diphosphate (4-SDH UDP) at 37 ° C for 5 hours in 150 mmol Tris buffer (pH 8.2) using 0.5 unit / ml polynucleotide phosphorylase (PNPase, type 15, PL Biochemical), a heteropolymer obtained, in which a 4-thiouridine is contained on 13 uridine residues, in a yield of about 50%. When 2-thiouridine 5'-diphosphate instead of 4-thiouridine diphosphate is used 2-thiouridine-containing poly-U obtained.

Once an -SH group-containing nucleic acid polymer is used once is prepared, the following derivatives can be made to the same Be obtained as it is in sulfurized poly-I-poly-C the case is as described in the examples. In particular Both the sulfurized polyuridylic acid and the equimolar Amount of polyadenylic acid dissolved in neutral aqueous solution and heated to complex formation. Then, after dissolving in water in an incubator, the temperature gradually increased to 85 ° C, then heated for 10 minutes and finally at Room temperature is allowed to stand.  

The nucleic acid derivatives of the invention give a particular clear melting curve, as explained below becomes, and it becomes clear that they are stable basic structures respectively.

The half-value melting temperature (Tm value) is at poly I Poly-C under neutral conditions in the presence of 0.1 M Sodium ions 59.0 ° C, while the value in the case of SH- substituted nucleic acid derivative containing 4-thiouridine in one Contains amount of n = 20, 59.5 ° C, and consequently are they the same or even more stabilized? In the case of OH substituted nucleic acid derivative containing uridine in an amount of n = 20, as above, the value is 53.1 ° C low.

Thus, the introduction of sulfur atoms has a more effective stabilizing effect on the high molecular structure of the nucleic acid compared to other substituted derivatives such as those substituted with NH ₂ or with OH.

Then the resistance to hydrolysis by RNAase A became (an enzyme that degrades RNA) based on the time ratio, where the decomposition reaches 50%. So this was Value under certain enzymatic reaction conditions for  Poly-I-poly-C at 50 minutes while that of 4-thiouridine (SH groups) and its nucleic acid derivative (n = 20; S-S) was 60 minutes while, in contrast, that of Nucleic acid derivatives (n = 20) containing uridine OH groups, was very short with 20 minutes. It becomes clear that the sulfur atoms have a substitution effect even at biochemical stability.

When the nucleic acid derivatives according to the invention as a medicament administered to living things including humans be, the active ingredient concentration is 0.1 to 99.5% (preferably 0.5 to 90%) in a pharmaceutically acceptable and non-toxic and inert carrier.

As carriers, one or more liquid, solid or semi-liquid diluents, fillers and other auxiliaries be used for pharmaceutical preparations. It is desirable that the pharmaceutical preparations in Shapes are given with dosable units. nucleic acid derivatives According to the invention, perorally, into the tissue, administered locally or rectally. You can of course in Shapes are given for each route of administration are suitable. For example, they are considered orally administrable Form, injection, inhalation, eye lotion, ointment, Suppositories, etc. given. Particularly advantageous is the Administration into the tissue or locally.

It is desirable that the dose be used as a cure malignant tumors taking into account the status of the patient (such as age, body weight, etc.), route of administration and type and Extent of the disease is regulated. In general, be 0.05 to 1000 mg at once as effective amount of nucleic acid  given to adults according to the invention, and preferably 0.5 to 50 mg of an intravenous infusion is usual. In some cases a lower dose is sufficient while in some other cases higher doses are required could be. It is also possible that the dose at once given or on several doses a day or with an interruption from one to several days.

The doses when used as a cure for benign Tumors or viral diseases are preferably under Consideration of the nature and extent of the disease, the route of administration, the status of the patient, etc., and in general it is common for the dose to be just as large down to 1/50 of the dose as in the above malignant Tumors is.

The physiological activity of the nucleic acid derivatives according to The invention will be described below.

(1) Interferon-inducing action

With reference to sulfurized poly-C-poly-I derivative, the is one of the nucleic acid derivatives according to the invention its effect as an interferon inductor by a method for Investigation of antiviral efficacy. The sulfurized Poly-C-poly-I derivative which is S-H substituted has been reported as Sample I in the test; it has an n-value of 20 and a remainder in their molecular size from 50 to 2000.

The lymphocytes (10 ⁶ cells per ml) obtained from human peripheral blood were treated with the sample (10 μg / ml) in a culture fluid (RPMI 1640, 20% FCS) for 2 hours. The reacted culture fluid was removed, the cells were floated in fresh culture fluid (RPMI 1640, 20% FCS), incubated for 20 hours and the supernatant fluid was analyzed using Sindbi virus and FL cells (see Rinsho Kensa, 28, 1726 , 1984) a standard measurement method for interferon antiviral efficacy. The results are summarized in the following table. The titer of interferon was measured by a dilution concentration using a CPEI ₅₀ method (50% inhibition of cytopathogenic effect). Sample I is an SH-substituted nucleic acid derivative containing 4-thiouridine in an amount of n = 20. Conventional poly-I-poly-C was used as a comparative sample.

It is clear that the nucleic acid derivative according to the present Invention a higher effect than interferon structure Has.

Among the sulfurized poly-C-poly-I derivatives of the nucleic acid derivatives according to the invention, according to the following Example given was one with the  n value of 20 and the molecular size with the number of residues 50 to 300 and another with the n value of 20 and a molecular size with Residual number from 200 to 2000 taken as test samples and the same subjected to physiological efficacy tests, wherein comparable results were obtained. This result was also obtained when the nucleic acid derivative (n-value 20 and molecular size with residual number of not less than 20,000) studied before conversion to lower molecular weight substances has been.

(2) TNF capability (tumor necrosis factor induction)

Rabbit microbial macrophages were pretreated with BCG 10 to 14 days prior to collection and were adjusted to 2 x 10 ⁶ per milliliter in a 10% FCS-shifted RPMI 1640 medium. 1 ml of it was placed on a plastic dish and cultured in a carbon dioxide gas incubator (5% CO ₂ ) in the presence or absence of the sample at 37 ° C.

The supernatant of cell culture became 2 or 8 hours subjected to the TNF activity test. The result is listed below. The TNF activity levels were determined by measuring cell inhibitory activity on LM cells 72 hours using the dye uptake method, and the dilution at 50% cell inhibition was titers specified. The fact that cell inhibition is due to TNF was, was using a monoclonal antibody on rabbit TNF through its activity neutralization approved.  

LPS was used at a concentration of 1 μg / ml, and the test and control samples were at the same concentrations of 10 μg / ml. The test and comparison samples were the same as in (1). It becomes clear that the TNF-inducing activity of the nucleic acid derivatives is high according to the invention.

(3) TNF-inducing activity in vivo

Meth A tumor (2 × 10 ⁵ ) was transplanted under the abdominal skin of BALB / c mice (age 6 to 8 weeks, male). The drugs were given on the 2nd and 12th day after the transplant. Blood was withdrawn 1 hour after the second dose, and serum TNF activity was measured. The necrosis phenomenon on the cancerous part which appeared afterwards was also observed. The result is shown below. The values in the table were the same as those used under (2) above.

Both the test and control samples were the same as used under (1) above. It is obvious that the TNF-producing ability of the nucleic acid derivatives according to the Invention in vivo is large.

(4) Efficacy for inducing interleukin 1

Normal human heparin-displaced peripheral blood was separated from mononuclear cells by a weight centrifugation method using Ficoll-Paque®, the numbers were adjusted to 5 × 10 ⁶ / ml in a 10% FCS-added RPMI medium, placed in a plastic dish. Incubated for 1 hour at 37 ° C, and the cohesive cells were used as interleukin 1 generating source.

The drug was added to the 5 x 10 ⁵ / ml of individual particles, incubated for 24 hours at 37 ° C, and the interleukin-1 activity in the supernatant was measured by measuring by the ³ H-thymidine uptake method in PHA stimulated mouse thymic cells using the proliferation ability of the thymus cells as a target. The results are given in the table below. As a comparison, physiological saline was used. The concentration of the interleukin was 1.25 units / ml, and those of the test sample I and the control sample were 100 μg / ml. Test Sample I and the comparative sample used were the same as in (1).

Used samples Recorded quantity (DPM (× 10⁴)) comparison 3 370 Test sample I 11,384 comparison sample 8 397 Interleukin 1 12 517

It will be appreciated that the nucleic acid derivatives of the invention had a large interleukin 1 inducing effect.

(5) Efficacy for inducing interleukin 2

Lymphocytes obtained from spleen cells of BALB / c mice were used as a source for the production of interleukin 2.

To 5 x 10 ⁶ / ml lymphoid particles, the drug was added, the mixture was incubated at 37 ° C for 24 to 48 hours, and the interleukin 2 activity in the supernatant was determined by the ³ H-thymidine uptake method using CTLL-2 or NK-7, which are cell-proliferating in response to interleukin 2, using proliferative capacity as the target. The result is given in the following table. As a comparison, physiological saline was used. The interleukin 2 was present at a concentration of 5 units / ml and both test samples I and II and the control had the same concentration of 100 μ / ml. The test and control samples used were the same as in (1).

rehearse Amount received (DPM (× 10³)) comparison 230 Test sample I 3247 comparison sample 653 Interleukin 2 3778

It will be apparent that the nucleic acid derivatives of the invention have a strong interleukin 2 inducing activity.  

(6) macrophage activation

BALB / c mice (age 7 to 10 weeks, male) were given intraperitoneally the samples, cells secreted from the peritoneum 3 to 5 days after administration were collected, and the plastic-cohesive cells (mainly macrophages (seizure cells)) were separated as effector cells and seizure cell activation was assayed by ³ H-thymidine isolation method using target Meth A tumor cells (at an E / T ratio of 15-20: 1). The result is given in the following table. As a comparison, 0.2 ml of physiological saline was used. 50 μg / mouse each of test sample I and the control were administered. The test and control samples used were the same as in (1).

The percent cytotoxicity was calculated by:

rehearse % Cytotoxicity comparison 0.5 Test sample I 10.3 comparison sample 5.7

It will be apparent that the nucleic acid derivatives of the invention have strong macrophage activation.  

(7) NK cell activation

The activation of NK cells in human peripheral blood was determined by measuring the inhibitory activity using K562 as target cells by the isolation method of ⁵¹ Cr. The result is shown below. As a comparison, physiological saline was used. The test and control samples were the same as in (1).

Samples (μg / ml) Melt% (% ⁵¹Cr insulation) comparison 30 Test sample I @ (10) 47 (30) 55 (100) 57 (300) 50 (500) 30 comparison sample @ (10) 52 (30) 60 (100) 65 (300) 62 (500) 52

It will be apparent that the nucleic acid derivatives of the invention activate the NK cells.  

(8) Inhibitory effect on the proliferation of tumor cells

The inhibitory effect on the proliferation of cell line tumor cells was measured. Cells (3 × 10 ⁴ / ml) were treated for 48 hours in a culture broth containing 10% FCS together with samples (10 μg / ml and 100 μg / ml) and then incubated for 48 hours with tritiated thymidine. % Inhibition was given by the amount of thymidine consumed versus a control that was not treated with the sample. The result is shown below. The test and control samples were the same as in (1).

Percent inhibition

It will be apparent that the nucleic acid derivatives of the invention an inhibitory effect on the proliferation of Own tumor cells.  

(9) Inhibitory effect on the proliferation of tumor in cancer-bearing mice

The inhibitory effect on tumor proliferation was examined for the following method in Meth-A cancer infested mice, which is the same transplanted type of cancer. Meth A cells (3 × 10 ⁵ / 0.1 ml) were suspended in physiological saline, injected hypodermically into BALB / c mice (age 5 weeks, male), and after 2 days, the drug was administered three times weekly started over a period of two weeks. On the second day after the last dose, the tumor cells were extracted and their weight measured. The result is given below. The test and control samples were the same as in (1).

It will be apparent that the nucleic acid derivatives of the invention the proliferation of tumor cells in cancer patients Inhibit mice.  

(10) Proliferation inhibitory effect in nude, with Cancer of human tumor cells infested mice

2.5 × 10 ⁶ cells each of HeLa S ₃ strain originating from human cervix and Hep-2 strain originating from laryngeal cancer cells were transplanted under the abdominal skin, and at 7-10 days after the transplantation, the living one was transplanted Cohesion of the tumor confirmed. Then, 100 μg / mouse of the test sample (intravenously) and 25 mg / kg of 5-FU (intraperitoneal) were administered twice weekly for four weeks. The tumor was extracted in the fourth week after the administration and the weight was measured. The results are given in the following table. The test sample used was the same as in (1).

HeLa S₃

Hep-2

It will be apparent that the nucleic acid derivatives of the invention have a strong inhibitory effect on proliferation.

(11) Inhibitory activity against metastasis of Tumor cells in the lung

In C57BL / 6 mice (male, aged 5 weeks), 2 × 10 ⁵ B16F10 cells transplanted, which are transplantable melanomas of the same type, and the number of nodules that formed in the lung (number of colonies) were intravenously transplanted second week after the transplant was counted. The sample was given intravenously 24 hours before the transplantation of B16F10 melanoma. The number was 9. The result is given below. The test and control samples used were the same as in (1).

Sample (μg / mouse) Number of lungs migrated to the lungs ± standard error comparison 112 ± 17 Test sample I @ (1) 33 ± 6 * (10) 21 ± 10 * (100) 4 ± 2 * comparison sample @ (1) 36 ± 16 * (10) 3 ± 0.7 * (100) 7 ± 3 *

It will be apparent that the nucleic acid derivatives of the invention an inhibitory activity against metastasis of Own tumor cells in the lungs.  

Safety of the nucleic acid derivatives of the invention

The safety of the nucleic acid derivatives according to the invention is explained below. Sulfurized poly-C-poly-I derivative, the is one of the nucleic acid derivatives according to the invention taken, and its cytotoxic effect on bone marrow major cells was tested. The sulfurized poly-C-poly-I used Derivative had an n-value of 20 and a molecular size with a residual number of 150-2000.

The sample was administered intravenously to mice (age 8 weeks, male; each group consisted of 5 mice) at a dose of 100 μg / Mouse injected, and after 24 hours were bone marrow cells taken from the animals. The cells were fixed and with Colored Giemsa. The cells of the swab specimen were submerged observed by the microscope, and the extent of occurrence of Reticulocytes were counted in%. Known poly-I-poly-C was used as a comparison. Physiological saline was given as a comparison. The result is in the following Table specified.

Erythrocyte cells (%) comparison 40 Test sample I 38 comparison sample 11

It will be apparent that the nucleic acid derivatives of the invention have a high level of security.  

The following is the pyrogenic effect of the nucleic acid derivatives explained according to the invention.

It is known that the injection of poly-I-poly-C in vivo pyrogen acts. Pyrogen tests with rabbits have shown Poly-I-Poly-C is an increase in body temperature Average of 1.45 ° C. In contrast, show the nucleic acid derivatives according to the invention (both the SH- as well as the S-S substances in the above cytotoxic Experiment) only an average increase in Body temperature of 0.25 ° C, so that the pyrogen test negative ran. In these experiments, three rabbits per Group used, and a solution of the sample (0.2 μg / kg) in 10 ml of physiological saline was intravenously below the ears of rabbits injected, and body temperature 4 hours after the injection was measured.

It turns out that the nucleic acid derivatives according to the invention have a very high level of security.

The results of the acute toxicity tests of the nucleic acid derivatives according to the invention are given below. In normal ddY mice, the dose of the nucleic acid derivatives of the invention is limited by the upper limit of the solubility of the drug, and the LD ₅₀ values were not less than 394 mg / kg. The effect was judged by the fact that the mouse was dead or alive one week after the intravenous injection. The same test using other mouse strains (C57BL6 and BALB / c) also concluded that the nucleic acid derivative (same as above) of the invention has lower toxicity than poly-I-poly-C.

It becomes clear that the safety of nucleic acid derivatives is very high according to the invention.

LD₅₀

The invention will be explained in more detail below with reference to examples, the production methods for nucleic acid derivatives relate to the invention.

Examples (1) Synthesis of Sulfurized Polycytidylic Acid (Starting Material)

0.5 g of poly-C was dissolved in a mixed solvent of 4 ml Dissolved water and 2 ml of pyridine, the solution was combined with Add 5 ml of liquid hydrogen sulfide to a 30 ml reaction tube made of stainless steel, and it was 6 hours long reacted at 37 ° C. The pressure was in the sealed Tube during the reaction 20 to 22 bar.

After the reaction, the reaction tube was at 0 ° C or below cooled to lower the pressure sufficiently, and the Reaction tube was opened. An excess of unreacted Hydrogen sulfide was removed, and the sulfurized poly-C Solution was transferred to a 50 ml round bottom flask and others  Unreacted hydrogen sulfide was removed in vacuo. The resulting solution was tris buffered three times dialyzed (pH 7.5) containing 10 l of 50 M sodium chloride, and the obtained transparent liquid was continued lyophilized, with 0.47 g of a white, fibrous substance containing one sulfur atom per 20 cycles of cytidic acid Contained units in poly-C.

The ultraviolet absorption spectra of the obtained substance were measured in a neutral aqueous solution, whereby the results plotted in Fig. 1 were obtained. It is known that the maximum absorption wavelength of cytidic acid is 271 nm and that the absorption wavelength of 4-thiouridic acid in which the -NH ₂ group is substituted at the 4-position of the pyrimidine ring of cytidic acid by the -SH group 330 nm is shown.

From the ratio of peak heights in Fig. 1, it was ascertained that a 4-thiouridylic acid is present on 13 cytidylic acids.

In the same way were reaction temperatures and reaction times in the stainless tube, and sulfurized polycytidylic acids with sulfurization levels, as in the following Table given were obtained. Here is the degree of sulfurization is indicated by "n", and this means "n" the number of cytidylic acids on a 4-thiouridylic acid.

(2) Preparation of Double-Stranded Nucleic Acid Derivatives: (Inventive Product)

Poly-I and sulfurized poly-C obtained as in (1) above, were prepared at the same molar ratios in a Tris Buffer containing 50 M sodium chloride, up to one Concentration of 10 to 20 mg / ml dissolved, and in one Water bath, the temperature was gradually from room temperature increased to 68 ° C. The mixture became about 10 minutes long kept at 68 ° C, then allowed to stand until room temperature was reached and stored at 4 ° C.

The resulting solution was lyophilized to give 1.2 g of nucleic acid derivative obtained with SH groups as a white solid has been.  

(4) formation of lower molecular substances

1 g of the obtained according to (2) -S-H-containing nucleic acid derivative was dissolved in 120 ml of water and 30 ml of 5 M sodium chloride solution and 150 ml of formamide were added. The mixture was vigorously stirred to obtain a uniform solution. The reaction solution was raised to 80 ° C for 8 hours  heated, dialysed well against water and lyophilized, taking 0.95 g of a white solid was obtained.

These were subjected to high performance liquid chromatography by gel filtration, and the obtained pattern is shown in FIG . In this case, the column material used was a material used essentially for hydrophilic silica gel for gel filtration chromatography or size separation chromatography, and 50 mmol Tris-HCl buffer (pH 7.5) containing 0.5 mol of sodium chloride as eluate.

Each arrow in the figure shows the elution position of each standard size mark (unit of magnitude mark is BP), and it can be seen from Fig. 3 that it has a maximum distribution at residue numbers of about 500 and that the molecular size distribution is between 150 and 1000 residue numbers. This result agrees well with the value obtained by electrophoresis using polyacrylamide gel or agarose gel. As explained above, in the present specification, the molecular size is indicated by the term "residual numbers", and in Fig. 3, "residual number" is expressed in terms of "BP" as units.

When reaction time and temperature at this conversion in lower molecular compounds are modified in the same way, compounds having the following molecular sizes receive:  

Among the nucleic acid derivatives obtained by the above Example were those with a molecular size distribution taken from 200 to 5000 remnants, and their melting curves were determined.

Sample I (0.70 D / ml) in 10 M Tris buffer (pH 7.5) containing 0.1 M sodium chloride became 90 ° at a temperature rise of 2 ° C / 4 minutes from 20 ° C C was heated, and the ultraviolet absorbances at each temperature were measured at the wavelength of 260 nm. The increase in ultraviolet extinction by hyperchrominance ws was represented by a relative ratio indicating the state at 20 ° C as a base. For this measurement, a Beckman DU-8B spectrophotometer was used. The result is plotted in FIG . The melting of the nucleic acid derivatives gradually proceeded from 35 to 55 ° C, and at about 59 ° C, a sudden melting curve was obtained. At above 70 ° C, the curve nearly reached a plateau, and this means the disappearance of a high-dimensional structure and helical structure formation by hydrogen bonding of the nucleic acid derivatives.

The half-value melting temperatures were for the sample I 59.5. From the calculation of the increase / decrease  Ratio at an ultraviolet absorbance of 260 nm between 90 ° C and 25 ° C it was ensured that the hyperchrominance (Color darkening effect) for Samples I and II 43.5% and 42.4% respectively. This indicates that the nucleic acid derivatives extremely under physiological conditions have stable structures.

(5) Brief explanation of the attached drawings

Fig. 1 shows the ultraviolet absorption spectra of sulfurized polycytidic acid used as the starting material obtained in (1) of Example. The ordinate and the abscissa show the absorption and the wavelength (nm), respectively.

Fig. 2 shows the elution patterns by high performance liquid chromatography of nucleic acid derivative according to the invention obtained according to (3) of the example. The abscissa and the ordinate show the Eulierzeit (min) or the eluted amount. Each arrow indicates the elution position of the size mark.

Fig. 3 shows the melting curve of the nucleic acid derivatives according to the invention in which the molecular size distribution is 50 to 2000 residual numbers. The abscissa and the ordinate indicate the temperature (0 ° C) and the relative absorption.

Claims (2)

A nucleic acid derivative wherein in a nucleic acid polymer, a portion of the purine or pyrimidine ring constituting one of its constituents is substituted by one or more sulfur atoms, characterized in that the nucleic acid polymer is a double-stranded ribonucleotide polymer composed of poly-I and poly-C, wherein an SH group introduced by substitution of an NH₂ group in the pyrimidine ring of cytidic acid is present on 6 to 39 cytosolic acid present in the poly-C. 2. nucleic acid derivative according to claim 1, characterized that the length of the polymer is 50 to 10,000, calculated on the number of base pairs.