NL8700016A - NUCLEIC ACID DERIVATIVES, PROCESS FOR THE PREPARATION OF MEDICINES CONTAINING SUCH DERIVATIVES, AND SUCH MEDICINES. - Google Patents

Description

NL 34005-dJ / cs * - -

Nucleic acid derivatives, process for the preparation of such derivatives containing drugs, as well as such drugs.

The present invention relates to nucleic acid derivatives, which by their physiological activity are suitable as pharmaceuticals.

A nucleic acid is composed of sugars, such as ribose, which are bound to a purination or pyrimidine ring and which are chained via phosphoric acid. Formula 1 of the formula sheet shows, for example, part of a nucleic acid, this part representing a ribonucleic acid in case R = OH and R '= H and a deoxyribonucleic acid in case R = H and R' = CH ^.

Among the nucleic acids, RNA (ribonucleotide polymer) is a high molecular chain compound having ribose when the sugar and the different sugar parts are linked by a diester bond of phosphoric acid.

Nucleic acids can be arranged in a double chain, which in its steric structure has a spiral shape created by a combination of purine or pyrimidine base units (eg, inosine, cytidine, uridine, etc.). Double-stranded nucleic acids exhibit suitable physiological activity and accordingly many studies have been conducted on them.

Among other things, studies have been conducted on single-stranded natural RNA (eg, RNA derived from a virus) and on double-stranded synthetic RNA, such as polyinosinyl acid - polycytidyl acid derivatives (hereinafter abbreviated as "poly I-poly C"), polyadenylic acid polyuridyl acid derivatives, etc. (e.g., Declark et al .: Texas Reports on Biology and Medicine, 41, 77, 1982). Formulas 2 and 3 of the formula sheet show the structural formulas for 5 * -cytidy Izmir and 5'-inosinyl acid, respectively. In this regard, many RNA derivatives have been synthesized and their physiological activities studied.

The poly I-poly C is a substance with a high activity and its usefulness has been evaluated by 8700016> i - 2 - studies. A compound in which only a small portion of the cytidine in the poly I-poly C has been changed to uridine (ie, mismatched RNA) has been tested for its physiological activity similar to that of the poly I-poly C (Compare Japanese Patent Application Laid-Open 50/082226).

The studies on polycytidylic acid are still ongoing and a report has been published (British Patent Application No. 2,038,628) stating that the physiological activity increases when the N 1 group of a pyrimidine ring of polycytidyl acid is replaced ( to a degree of 50% or more) by a mercapto group (-SH).

The conventional physiologically active substances as described above can be expected to have useful effects, however their toxicity, as in the case of poly i-poly C, cannot be denied (Declard et al .: Infect. Immuni ., 6, 344, 1972) and there is therefore a need for some improvement in this. It is also desirable to increase their physiological activity.

Over the course of many years of studies by the present inventors, it has been coincidentally found that RNA can form stabilized derivatives with a higher conformation due to a given covalent bond. When the physiological activity thereof was measured, it was found to exhibit a much stronger activity than the conventional physiologically active substances, accompanied by a very low toxicity.

A characteristic feature of the present invention is that due to a combination of a higher conformation by means of a covalent bridge bond or by the substitution of part of the molecular structure of the nucleic acid by one or more SH group (s). of the nucleic acid. is. : obtained, which can occur in stable form.

The nucleic acid derivatives of the present invention can be synthesized from a nucleic acid base into which an SH group can be introduced. A specific example of a nucleic acid derivative according to the invention is that which has a complementary double spiral structure 870001® ............................... .............

^ - has a chain compound which is a cytidyl acid polymer (herein referred to as "poly C") and another chain compound which is an inosinyl acid polymer (hereinafter further referred to as "poly I"). Although this compound has a structure similar to the aforementioned poly I-poly C, the nucleic acid derivatives of the present invention nevertheless exhibit characteristic properties for the following reasons.

The nucleic acid derivatives of the present invention are derivatives which are partially substituted by one or more sulfur atoms (SH group), which are optionally cross-linked (S-S bond), or which have both of these substituents.

The sulfur atoms in the nucleic acid derivatives of the present invention can be introduced before or after the polymer preparation using an enzyme reaction. The introduction is carried out by substituting a nucleic acid base with an SH group, such that an N 2 group in a pyrimidine ring of cytidyl acid is replaced by an SH group. The residue obtained is called 4-thio-uridylic acid.

In formula 4 of the formula sheet the 51-4-thiouri-dicylic acid is shown.

The introduced SH group can be further oxidized by an appropriate method, as will be described later, so that the S-S bridge bond can be formed.

Derivatives with both SH and S-S groups in a molecule can be prepared either by partial oxidation of a compound into which SH groups have been introduced or by partially reducing a compound into which S-S groups have been introduced.

The aforementioned nucleic acid polymer (poly C) has a single strand. This single-stranded nucleic acid polymer can be combined with a complementary single-stranded nucleic acid polymer after the SH substitution or the formation of an SS-bridge bond, using an appropriate method, as provided later herein, to form a multi-strand nucleic acid. On the other hand, it is also possible that the first-sulfurized single-stranded nucleic acid poly-40 is more combined with the complementary nucleic acid-8700016 '? * - 4 - polymer to form the multi-strand nucleic acid, followed by oxidation to yield the S-S bridge bond.

The nucleic acid derivatives thus prepared are also among the nucleic acid derivatives which have physiological activity and are the subject of the present invention. Therefore, the present invention includes all nucleic acid polymers, insofar as they contain nucleic acid bases into which SH groups have been introduced. For example, the present invention includes compounds obtained by disulfiding 10 nucleic acid polymers containing 4-thiouridine, 2-thiouridine, 2-thioguanosine, 6-mercaptopurine, 8-mercaptopurine, etc.

The nucleic acid derivatives of the present invention can be cleaved in their phosphoric acid side chain to yield lower molecular weight compounds and such lower molecular weight compounds are also included in the present invention. As a result, there is a difference in this respect from the conventional poly I-poly C.

Due to the characteristics of the 20 nucleic acid derivatives of the present invention, ie substitution by SH, cross-linking by an SS bond, and formation of low molecular weight substances by a cleavage of the phosphoric acid side chain, it is possible for the first time obtain the following effects: (1) the physiological activity can be increased and (2) the safety can be increased, ie the toxicity is reduced.

Due to the physiological activity of the nucleic acid derivatives of the present invention, they are very suitable as pharmaceuticals. As will be described later, the nucleic acid derivatives of the present invention show strong activity as interferon inducers. This action is only one of several physiological actions of the nucleic acid derivatives of the present invention. As physiological actions of the nucleic acid derivatives of the present invention, the following, which will be described later, may be mentioned: TNF-inducing ability, interleukin 1-inducing ability, interleukin 2-inducing ability, macrophage activating ability, activating power 8700013 ^ .......... one, - 5 - * a on the NK cell, inhibitory effect on the proliferation of tumor cells / inhibitory effect on the proliferation of tumors in cancer-bearing mice, inhibitory effect on the proliferation in nude mice suffering from cancer of human tumor-5 cells, the preventive effect on the metastasis of tumor cells to the lung, and so on.

The nucleic acid derivatives of the present invention are much safer compared to interferon inducers, such as conventional poly I-poly C. Thus, the compounds of the present invention are suitable as anti-viral and anti-tumor agents.

The term "base pair" (abbreviated as "bp, f) is often used herein to indicate the molecular size of the nucleic acid, that is, to indicate the molecular size by the number of bases in the nucleic acid (i.e., 10 bp means that the double-stranded polymer has ten bases in each complementary strand) in each complementary strand. Since the present invention also relates to nucleic acid polymers other than double-stranded polymers, the term uresido number "will be used instead of bp. For example," 10 residue numbers "means that the nucleic acid polymer has 10 bases in a strand.

The nucleic acid derivatives of the present invention include substances of various kinds of molecular sizes, and it is usually preferred that the size is not less than 50 residue numbers, for example, 50-10,000 residue numbers. The size may even be much larger, for example residue numbers of 200,000 will also suffice, and the molecular size is known to have little influence on the physiological activity of the present invention.

To indicate the number of sulfur atoms in the nucleic acid derivatives of the present invention, the sulfurization degree (s) is used in this description. The cytidylic acid changes to 4-thiouridylic acid by substituting the group 35 in the pyrimidine ring for an SH group, and "n" represents the number of cytidylic acid units present relative to one 4-thiouridylic acid unit. The nucleic acid derivatives of the present invention include substances with many values for "n" and it is preferred that 8700016 1 - 6 - n is not less than 6. It has been confirmed that the physiological activity decreases when n is less than this value. When n is 6 or greater, there will be no further limitation and the value may be as high as 39 and the value of n has little influence on the physiological activity of the subject derivatives.

Various methods can be used for the preparation of the nucleic acid derivatives of the present invention. The poly C and the like, which are starting materials for the present derivatives, are readily available. The poly C can be easily sulfurized by, for example, the reaction with a sulfurizing agent such as hydrogen sulfide. As a result of this reaction, a certain number of cytidyl acids in the poly C can be changed to 4-thiouri-15-dyl acid. When the reaction conditions, such as reaction temperature and reaction time, are varied, the poly C of the desired n value can be prepared.

The sulfurized poly C can be combined with poly I, which is readily available using known methods. The sulfurized poly C-poly I thus obtained can be converted to nucleic acid derivatives of the present invention by a disulfide formation reaction, such as, for example, by oxidation with iodine reagent.

By the above methods, the nucleic acid derivatives of the present invention can be obtained almost quantitatively and the total yield has been found to be about 90%.

In the case of the synthesis of other nucleic acids such as poly A-poly U with disulfide bridges, the polymer is first prepared by an enzyme reaction using a nucleic acid base containing a sulfur atom. Conventional methods for preparing heteropolymers can be used.

Typical examples are the following: when 36 mM

Uridine 5'-diphosphate (UDP) and 7 mM 4-thiouridine-5'-diphosphate (4-SDH UDP) are reacted at 37 ° C for 5 hours in 150 mM Tris buffer (pH 8.2) using 0.5 units / ml polynucleotide phosphorylase (PNPase, type 15, PL Biochemical), a heteropolymer is obtained in which S70 0 0 1 6 - 7 - * one 4-thiouridine is present per 13 uridine residues, in a yield of about 50 %. When 2-thiouridine-diphosphate is used instead of 2-thiouridine-51-diphosphate, poly (2-thiouridine) is obtained.

When an SH-containing nucleic acid polymer is prepared as such, the following derivatives can be obtained by the same operations as in the case of sulfurized poly I-poly C, as described in the examples. More specifically, both sulfurized polyuridylic acid and equimolar polyadenylic acid are dissolved in a neutral aqueous solution and subjected to a heat-cooling treatment to form a complex. Thus, after dissolving in water in an incubator, the temperature is gradually raised to 85 ° C, then. heated for 10 more minutes, then left to stand at room temperature.

The complex thus obtained is oxidized under the same conditions as in the disulfidation of poly I-poly C, ie oxidized with 1N iodine solution, to obtain a poly A-poly U derivative with a disulfide bridge. The yield after the heat-cooling treatment is about 80% and the total yield is about 40%.

The disulfide complex dialyses well in an aqueous solution and can be lyophilized to yield a white and fibrous solid.

It is not always clear what type of steric structure the nucleic acid derivatives of the present invention are due to their characteristic S-S bond.

The nucleic acid derivatives of the present invention give clear melting curves, as will be described later, and therefore, they are evident to have a stable fundamental structure.

The 50% melting temperature (Tm value) is 59.0 ° C

in the case of poly I-poly C under neutral conditions in the presence of 0.1M sodium ion, while in the cases of an SH-substituted nucleic acid derivative containing 4-thiouridine in an amount represented by n = 20 if the >. S-S-substituted nucleic acid acidic ivate thereof have values of 59.5 ° C and 59.3 ° C, respectively, which are therefore equally or more stabilized. In the case of OH-8700016 $ * - 8 - substituted nucleic acid derivative containing uridine in the same amount as above n = 2O, the value is as low as 53.1 ° C.

Therefore, the introduction of sulfur atoms 5 provides a more effective stabilization of the high molecular structure of a nucleic acid as compared to otherwise substituted derivatives, such as those substituted by NH2 or OH.

Subsequently, the resistance to hydrolysis by RNA-ase A (an enzyme that degrades RNA) was compared in terms of the time until degradation approaches 50%. Therefore, under given enzymatic reaction conditions for poly I-poly C, this value was 50 min, while that for 4-thiourvin (SH group) and its nucleic acid derivative (n = 20; containing SS 15) was 60 min and, on the other hand , those of nucleic acid derivatives (n = 20) containing uridine (OH group) were as short as 20 min. Thus, it is clear that sulfur atoms exhibit a substitute effect even in biochemical stability.

When the nucleic acid derivatives of the present invention are administered as pharmaceuticals, they are administered as such or as a pharmaceutical composition containing, for example, 0.1-99.5% (more preferably 0.5-90%) active ingredient in a pharmaceutically acceptable, non-toxic and inert carrier, to animals, including humans.

As carriers, one or more liquid, solid or semi-solid diluents, fillers and other auxiliaries for pharmaceutical preparations can be used. It is desirable that the pharmaceutical preparations be administered in unit dosage form. The nucleic acid derivatives of the present invention can be administered by mouth, tissue, rectum or applied locally.

They are of course administered in the form suitable for any route of administration. For example, they are administered in an oral administration form, as an injection, as an inhalation, as an eye lotion, as an ointment, as a suppository, and so on. It is especially preferred to administer in the tissue or locally.

It is desirable that the dose, as a remedy for a 87 0 0 0 f é - 9 - * * malignant tumor, be determined depending on the patient's condition (e.g., age, body weight, etc.), route of administration and type and the severity of the disease. Usually 0.05-1000 mg at a time is used as an effective amount of the nucleic acid of the present invention for adults and, preferably 0.5-50 mg is normal for an intravenous infusion. In certain cases a smaller dose is sufficient and in some cases a higher dose may be needed. It is also possible that the administration is carried out once or several times a day or with an interruption of one to several days.

A dose as a remedy for benign tumors or for diseases caused by viruses is preferably determined depending on the type and severity of the disease, route of administration, patient condition, etc., and generally the dose is about one-fiftieth of the dose for the aforementioned malignant tumor.

The physiological activity of the nucleic acid-20 derivatives of the present invention is further explained below.

(1) Interferon-inducing efficacy:

The sulfurized poly C-poly I derivative, which is one of the nucleic acid derivatives of the present invention, was determined to function as an interferon inducer using an antiviral activity assay method. The sulfurized poly C-poly I derivative used as sample I for the test is an SH-substituted derivative, while sample II is an S-S-substituted derivative. Both have an n-value of 13 while the residue numbers in the molecule are 50-2000.

Lymphocytes obtained from human peripheral blood (10 cells per ml) were treated with the sample (10 micrograms / ml) in a culture fluid (RPMI 1640; 35% FCS) for 2 hours. The reacted culture fluid was removed, and the cells were again transferred into a fresh culture fluid (RPMI 1640; 20% FCS), incubated for 20 hours, after which the supernatant was subjected to a conventional measurement method for the interferon antivirus activity 8700016 i * v 10 - Using sindbi's virus and FL cells (compare Rinsho Kensa, 2j3, 1726, 1984). The result is shown in the following table. The interferon titer was measured by a dilution concentration using a CPEI ^ q (Cytopathogenic Effect Inhibition 50) method. Conventional poly I-poly C was used as a control sample.

Concentrations (yg / ml) 10 100 200 500 10 Test Sample I 100> 6400> 6400> 6400

Test Sample II 100> 6400> 6400> 6400

Control sample 100 800 1600> 6400 (Interferon titer: units / ml)

It is clear that the nucleic acid derivatives of the present invention exhibit strong interferon inducer activity.

Of the sulfurized poly C-poly I nucleic acid derivatives of the present invention obtained in the example given later, one with an n value of 20 and a molecular size of 50-300 residue numbers and another with an n value of 20 and a molecular size of 200-2000 residue numbers taken as test samples and subjected to the same physiological activity tests, yielding similar results. Such results were also obtained - with the nucleic acid derivative (n-value of 20 and molecular size not less than 20,000 residue numbers) for molecular weight reduction.

(2) TNF (Tumor Necrosis Factor) -inducing ability

Rabbit germinate vesicle macrophages, 10 to 14 days previously treated with BCG, were adjusted to 2 x 10 per ml in an RPMI 1640 medium to which 10% FCS had been added, of which 1 ml was transferred to a plastic dish and grown in a carbon dioxide gas incubator (5% CC ^) in the presence or not of the sample at 37 ° C.

The cell culture supernatant liquid after 2 or 8 hours was subjected to the TNF activity test. The result is ®70 00 16 - 11 - listed below. The TNF activity values were determined. by measuring the cell hindering activity for LM cells after 72 hours using the dye pick-up method and the dilution at 50% cell hinder was taken as 5 titer. The fact that cellular interference was due to the TNF was confirmed by its activity neutralization, using monoclonal antibodies on rabbit TNF.

TNF activity Neutralization by (2 h) (8 h) anti-TNF antibody

Control - - - 10 LPS 32> 64. *

Test sample I 32> 64 +

Test sample II 32> 64 +

Control sample 32> 64 + LPS was used at a concentration of 1 µg / ml and the test and control samples both had concentrations of 10 µg / ml. The test and control samples used were the same as those in (1). It is clear that the TNF-inducing activity of the nucleic acid derivatives of the present invention is strong.

(3) The TNF-inducing activity in vivo

Meth A tumor (2 x 10 ^) was transplanted behind the abdominal skin of BALB / c mice (6 to 8 weeks old; male), with drugs administered on the second and twelfth days after transplantation, while blood was being taken 1 hour after the second administration and the TNF activity in the serum was measured. The necrosis phenomenon that occurred on the cancer-bearing part was then also measured. The results are shown below. The values in the table were used at the same titer as previously in (2).

8700011 ..... x.

'* «- 12 -

Treatment TNF activity Necrosis (first) (second) in serum BCG LPS 192 observed BCG Test sample I 48 observed 5 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 observed

Both the test and control samples were the same 10 as those in the previously described test (1). It is clear that the TNF-inducing activity of the nucleic acid derivatives of the present invention is strong in vivo.

(4) Inducing activity of Interleukin 1.

Normal human peripheral blood, to which heparin 15 was added, was separated from mononuclear particles using density centrifugation methods with Ficoll-Hypaque (Farmacia, Ficoll-paque, trademark), then the number was adjusted to 5 x 10 / ml in a 10% FCS added RPMI medium, transferred to a plastic dish, incubated for 1 hour at 37 ° C, and the cohesive cells used as an interleukin 1 producing source.

The drug was added to 5 x 10 / ml individual particles, incubated for 24 hours at 37 ° C, after which the interleukin 1 activity in the supernatant liquid was determined by measuring the H-thymidine incorporation method in PHA-stimulated mouse thymus cells utilizing the proliferation capacity of the thymus cells as a target. The result is shown in the following table. The control sample was physiological saline. The concentration of interleukin was 1.25 units / ml and that of test samples I and II and of the control sample was 100 gamma / ml. The test samples I and II used and the control sample were the same as the samples used in (1) above.

8700010 - 13 - *. 4

Samples used Recording quantity (DPM (10})

Control 3370

Test Sample I 11384

Test Sample II 11560 5 Control Sample 8397

Interleukin ... 1 12517

Clearly, the nucleic acid derivatives of the present invention exhibit a strong interleukin 1 inducing activity.

«10 (5) Inducing activity of Interleukin 2.

Lymph particles obtained from spleen cells of BALB / c mice were used as a source of interleukin 2 production.

The drug 15 was added to 5 x 10 lymph particles / ml, the mixture was incubated for 24-48 hours at 37 ° C and the interleukin 2 activity in the supernatant fluid was determined by an H-thymidine incorporation method using CTLL-2 or NK-7, which are cell strains that proliferate in dependence on inter-leukin 2 using the proliferation potential as a target. The result is shown in the following table. The control sample is a physiological saline solution. The interleukin 2 had a concentration of 5 units / ml and both test samples I and II and the control sample 25 had the same concentration of 100 gamma / ml. The test and control samples used were the same as in (1).

Samples Amount absorbed (DPM (x10 ^))

Control 230

Test Sample I 3247 30 Test Sample II 3320

Control sample 653

Interleukin 2 3778

Clearly, the nucleic acid derivatives of the present invention exhibit strong interleukin 2-inducing 870 0 018-14 activity.

(β) Macrophage activation

Samples were administered intraperitoneally to BALB / c mice (7-10 weeks old; male), collecting cells separated from the peritoneum 3 to 5 days after administration, separating plastic cohesive cells (mainly macrophage) as effector cells and macrophage activation was examined by an H-thymidine isolation method using Meth-A tumor-10 cells as a target (E / T ratio was 15-20: 1). The result is shown in the following table. The control was 0.2 ml physiological saline. 50 µg / mouse was administered in each case from test sample I and II or control sample. The test and control samples used were the same as those 15 in (1).

Cytotoxicity% was calculated according to: (experimental value) - (background) .nf) (100% 3h-1 release) - (background) x

Samples% Cytotoxicity

Control 0.5 20 Test sample I 10.3

Test Sample II 10.8

Control sample 5.7

Clearly, the nucleic acid derivatives of the present invention show strong macrophage activation.

(7) NK Cell Activation

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

8700016 - 15 - * 51

Samples (pg / ml) Melt% (% Cr Isolation)

Control 30

Test Sample X (10) 47 (30) 55 5 (100) 57 (300) 50 (500) 30

Test Sample II (10) 51 (30) 59 10 (100) 63 (300) 60 (500) 45

Control sample (10) 52 (30) 60 15 (100) 65 (300) 62 (500) 52

It is understood that the nucleic acid derivatives of the present invention exhibit NK cell activation.

(8) Inhibitory effect on the proliferation of tumor cells. The inhibitory effect on the proliferation of cell line tumor cells was measured. Cells (3 x 10 µ / ml) were treated for 48 hours in a culture fluid containing 10% FCS along with samples (10 µg / ml and 100 µg / ml) and then incubated with tritium-labeled thymidine for 48 hours. The inhibition rate was given by an amount of thymidine incorporated in a control that had not been treated with the sample. The result is given below.

The test and control samples were the same as those in (1).

3700018 —--— -16-

Inhibition%

Cells Test Sample I Test Sample II Control Sample 100 10 100 10 100 10 µg / ml NAMALWA 65.6 55.7 61.2 46.4 56.9 37.4 RAJ1 68.3 64.6 67.8 61.5 74.7 69.4 5 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 clear that the nucleic acid derivatives of the present invention have an inhibitory effect on the proliferation of tumor cells.

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

The inhibition of tumor proliferation was measured by the following methods in Meth-A cancer-bearing mice, which is the transplanted, same type of cancer. Meth-A-

C

15 cells (3 x 10 / 0.1 ml) were suspended in physiological saline, hypodermally injected into BALB / c mice (5 weeks old; male) and after 2 days the drug was started three times a week for 2 weeks. On the second day after the last administration, the tumor cells were extracted and their weight measured. The result is shown below. The test and control samples were the same as those in (1) above.

Samples (µg / mouse) Number of mice Mean ± S.D. Inhibition%

Test Sample I (10) 7 2.09 ± 0.15 3 25 (30) 7 1.52 ± 0.28 30 (100) 5 0.96 ± 0.43 56

Test Sample II (10) 7 2.10 ± 0.27 (30) 7 1.62 ± 0.25 (100) 7 0.97 ± 0.30 30 Control Sample (10) 7 2.12 ± 0.27 2 ( 30) 8 1.38 ± 0.38 36 (100) 8 1.08+ 0.27 50 870 0 0 1 6 k. ..____ - 17 - '

Clearly, the nucleic acid derivatives of the present invention inhibit the proliferation of tumor cells in cancer-bearing mice.

(10) Proliferation inhibitory activity in nude mice bearing cancer from human tumor cells.

g 2.5 x 10 cells of the strain HeLa S ^, obtained from the human cervix, and the strain Hep-2, obtained from throat cancer cells, were transplanted under the skin of the abdomen and, on the seventh to tenth day after the transplantation, a live cohesion of the tumor was confirmed. Then, 100 µg / mouse test sample (intravenous) and 25 mg / kg 5-FU (intraperitoneal) were administered twice a week for four weeks. After the fourth week after administration, the tumors were extracted and the weight measured. The result is shown in the following table. The test sample used was the same as that in (1).

(HeLa S3)

Sample Weight (g) ± S.D. Inhibition rate

Control 3.85 + 0.23 20 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)

Sample Weight (g) ± S.D. Inhibition Rate 25 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 36

It is clear that the nucleic acid derivatives of the present invention exhibit a strong inhibitory action on proliferation.

% »8700016 - 18 - (11) Inhibitory activity against the metastasis of tumor cells to the lung.

In C57BL / 6 mice (male; 5 weeks old), 5 intravenous, 2 x 10 B16F10 cells, which are transplantable mela-5 noma of the same type, were transplanted and the number of nodules (number of colonies) spread to the lung in the second week after transplantation was counted. The sample was administered intravenously 24 hours before transplantation of the B16F10 melanoma. The number of cases was 9. The result is shown below. The test and control samples used were the same as those in (1).

Samples (yg / mouse) Number metastasized to lung. cells ± S.D.

Control 112 ± 17

Test Sample I (1) 33 ± 6 * 15 (10) 21 ± 10 * (100) 4 ± 2 *

Test Sample II (1) 21 + 6 * (10) 15 + 4 * (100) 4 ± 1 * 20 Control Sample (1) 36 ± 16 * (10) 3 + 0.7 * (100) 7 ± 3 *

It is clear that the nucleic acid derivatives of the present invention exhibit an inhibitory activity against the metastasis of tumor cells to the lung.

The safety of the nucleic acid derivatives of the present invention will be explained below. A sulfurized poly C-poly I derivative, which is one of the nucleic acid derivatives of the present invention, was taken and its cytotoxic activity on bone marrow main cells examined. The sulfurized poly C-poly I derivative used had an n value of 20 and a molecular size of 50-2000 residues.

The sample was injected intravenously into mice (8 weeks old; male; each group comprising 5 mice) 8700015 - ^ - - 19 - at a dose of 100 μg / mouse, and its bone marrow cells were collected after 24 hours. The cells were fixed and stained with Giemsa. The cells of the smeared sample were viewed under a microscope and the degree of reticulocyte occurrence was determined as a percentage. The known poly I-poly C was used as a control. A physiological saline solution was administered to the control. The result is given in the following table.

Erythrocyte cells (%) 10 Control 40

Test sample I 38

Test sample II 39

Control sample 11

It is clear that the nucleic acid derivatives of the present invention have a high safety.

The pyrogenic activity of the nucleic acid derivatives of the present invention will be explained below.

It is known that injecting the poly I-poly C in vivo is pyrogenic. A pyrogen test in rabbits 20 has shown that the poly I-poly C yields an average increase in body temperature of 1.45 ° C. On the other hand, the nucleic acid derivatives of the present invention (both the SH and also the SS-substituted derivatives in the above cytotoxic experiment) gave only a body temperature increase of 0.25 ° C on average and the result of the pyrogen test was negative. In those experiments, 3 rabbits per group were used and a solution of the sample (0.2 µg / kg) in 10 ml physiological saline was injected intravenously under the rabbits ear and the body temperature was measured 4 hours after the injection .

Clearly, the nucleic acid derivatives of the present invention are very safe.

The result of the acute toxicity test of the nucleic acid derivatives of the present invention is given below. With respect to normal ddY mice, the dose of the nucleic acid derivatives of the present invention is limited by the upper limit of the solubility of the drug 8700016 ** - 20 - and the LD 2 value was not less than 394 mg / kg. The effect was assessed by whether the mouse was dead or alive at 1 week after the intravenous injection. The same assay, performed on other murine 5 strains (C57BL6 and BALB / c) also resulted in the nucleic acid derivatives (the same as above) of the present invention having less toxicity than poly I-poly C.

It is clear that the safety of the nucleic acid derivatives of the present invention is very high.

- LU50: -

Strain Route of administration Test sample I Test sample II Control sample ddY intravenous> 394 mg / kg> 394 mg / kg 132 mg / kg

The present invention will be further elucidated hereinbelow using examples which relate to the method of preparation of the nucleic acid derivatives of the present invention and it will be understood that the present invention is not limited to these examples.

EXAMPLE I

Synthesis of sulfurized polycytidylic acid.

Poly C (0.5 g) was dissolved in a mixed solvent of 4 ml of water and 2 ml of pyridine, after which the solvent was transferred into a 30 ml stainless steel reaction tube together with 5 ml of liquid hydrogen sulphide and allowed to react for 6 hours. at 37 ° C. The pressure in the sealed tube during the reaction was 20-22 kg / cm2.

After the reaction, the reaction tube was cooled to 0 ° C or lower to sufficiently reduce the pressure and the reaction tube was opened. An excess of the unreacted hydrogen sulfide was removed, the sulfurized poly C solution was transferred to a 50 ml round bottom flask and the unreacted hydrogen sulfide was removed in vacuo. The resulting solution was dialyzed three times against a Tris buffer (pH 7.5) containing 10 l of 50 mM sodium chloride, and the resulting transparent liquid was further lyophilized to yield 0.47 g of white fibrous substance.

8700018 - 21 -

The ultraviolet absorption spectrum of the obtained substance was measured in a neutral aqueous solution and the result thereof is shown in Fig. 1. It is known that the maximum absorption wavelength of cytidylic acid is 271 nm and that the absorption wavelength of 4-thiouridylic acid, in which the The N 2 group at the 4 position of the pyrimidine ring of cytidyl acid has been replaced by the. SH group is 330 nm.

From the ratio of the peak heights in Figure 1, it was determined that one 4-thiouridylic acid is present relative to 13 cytidyl acid units.

Similarly, while the reaction temperatures and reaction times in the stainless steel tube were varied, sulfurized polycytidyl acids were obtained with the degree of sulfurization shown in the following table. The degree of sulfurization is represented here by "n", and this n value means the number of cytidyl acid units per 4-thiouridyl acid unit.

Response temperature Response time n 45 ° C 12 h 1 20 45 6 6 + 1 37 6 13 ± 2 37 4 26 ± 2 37 2.5 39 ± 2

EXAMPLE II

Preparation of Double Strand Nucleic Acid Derivatives

The sulfurized poly C and poly I obtained in Example I were dissolved in equimolar amounts in a Tris buffer containing 50 mM sodium chloride, to a concentration of 10-20 mg / ml and using a water bath the temperature was gradually increased from room temperature up to 68 ° G. The mixture was held at 68 ° C for about 10 min and then left to stand at room temperature and then stored at 4 ° C.

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

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Then about ten times the volume (with a molar ratio) of 1N iodine solution (a mixture of 1/3 mol iodine and 2/3 sodium iodide) was added. The mixture was mixed well to make it uniform and left at 0 ° C for 1 hour. The reaction solution was dialyzed well against water until the yellow color of iodine disappeared.

The solution thus obtained was lyophilized to yield 0.98 g of white solid.

The fact that the resulting solid contained an S-S bond was confirmed by the following. 0.1 g of this solid was dissolved in 10 ml Tris buffer (pH 7.5) containing 0.03M sodium thiosulfate, stored at room temperature for 0-7.5 hours, and the ultraviolet absorption spectrum was mixed with intervals included. The result is shown in Figure 2. It is known that the S-S bond is usually reversible and the S-S bond splits by reduction to yield an SH group. As time passed, a shoulder peak at 310 nm, which is the absorption wave length of the. SS binding decreases as an absorbance peak at 330 nm from the SH group (4-thiouridine) increased and after 7.5 hours the absorbance at 330 nm had become that of the sulfurized poly C prior to oxidation. It is therefore clear that the S-S bond was quantitatively reduced to the SH group.

EXAMPLE III

Cross-linking structure and biological activity.

Of the nucleic acid derivatives into which sulfur atoms have been introduced, the synthesis methods and physiological activities of the test samples I (where all SH groups are of the reduced type) and the test sample II (where all SS groups are of the oxidized type) were described. The cross-linking substances of the nucleic acid derivatives described herein are the compounds in which a portion of the sulfur atoms introduced into the same molecule forms a disulfide bond between or within the molecules.

For example, the cross-linked derivatives with 80% SH groups and 20% S-S groups in the same molecule can form a many-strand cross-linking structure in 20% of the total. In other words, for example, in the case of 1000 bp poly C, it has a cross-linking structure at 10 parts. A nucleic acid derivative with various cross-linking numbers can easily be prepared by partial oxidation of the SH substance under mild conditions or by partial reduction of the S-S substance under mild conditions.

The synthesis conditions are the same as described in the examples.

The ratio of the number of SH groups and the number of SS groups, ie the degree of cross-linking, can be calculated as the ratio of the values obtained by dividing the ultraviolet absorbances at 310 nm of the SS, respectively. group and at 330 nm of the SH group 15 by the molecular absorption coefficients.

An alternative method is as follows. The nucleic acid derivative is decomposed by an RNA ase (for example, ribonuclease P1), after which the degraded product is subjected to a high performance liquid chromatography (HPLC) of the reverse phase system, and 4-thiouridine (or 4-thiouridylic acid) and one bis-substance formed by a disulfide bond is isolated and their amounts measured.

With regard to the physiological activities, it has been found that nucleic acid derivatives with a degree of cross-linking from 1 (SS groups only; e.g. test sample II) to 0 (SH groups only; e.g. test sample I) have the same activity and safety as those of the aforementioned test samples I and II.

This fact suggests that even if those nucleic acid derivatives are partially oxidizable or reducible, they exhibit the same stable physiological activity as the original derivatives.

EXAMPLE IV

Decrease in molecular weight.

The SH-containing nucleic acid derivative (1 g) obtained in Example II was dissolved in 120 ml of water and 30 ml of SM sodium chloride solution and 150 ml of formatm was added thereto. The mixture was stirred vigorously to obtain a uniform solution 8700016 - 24. The reaction solution was heated at 80 ° C for 8 hours, dialyzed well with water and freeze dried to yield 0.95 g of white solid.

When the S-S bond containing nucleic acid derivative obtained in Example II is subjected to a molecular weight reduction as above, the same result is obtained.

The resulting solid was subjected to HPLC 10 by gel filtration and the resulting pattern is shown in Figure 3. Using TSK gel G4000SW (0.65 x 60 cm) and 50 mM Tris-HCl buffer (pH 7.5), containing 0.5M sodium chloride, as eluents.

Each arrow in the figure shows the elution position of each standard size marker (the unit of this size marker was bp) and this figure 3 shows that there is a maximum distribution near 500 residue numbers and that it has a molecular size that varies between 150 to 1000 residue numbers. The result was in good agreement with the value obtained from an electrophoresis using polyacrylamide gel or agarose gel. As already mentioned, the molecular size in this description is indicated by the term "residue number" and, in Fig. 3, the "residue number" is in accordance with "bp" as a unit.

When the reaction time and temperature of this decrease in molecular weight were varied, compounds of the following molecular sizes were obtained in the same manner.

Response- Response- Max. distribution Temperature distribution duration (bp) (bp) 30 0 hours - 2000 80 ° C 24 30 20 - 55 80 ° C 16 200 50 - 300 80 ° C 8 500 150 - 1000 60 ° C 8 1000 200 - 5000 35 60 ° C 12 600 50 - 2000

From the nucleic acid derivatives obtained in this example, those with 50-2000 residue numbers, based on the 8700016-25 * * molecular size distribution, were taken and their melting curves recorded.

The sample I or IX (0.7 ÖD / ml) in 10 mM Tris buffer (pH 7.5), containing 0.1M sodium chloride, was heated at a heating rate of 2 ° C / 4 min, of 20 ° C to 90 ° C, while the ultraviolet absorption at each temperature was measured at the wavelength of 260 nm, and an increase in ultraviolet absorption by hyperchromicity was represented by a relative ratio taking the state at 20 ° C as the basis. A Beckmann-DU-8B spectrophotometer was used for this measurement. The result is shown in Figure 4. The melting of the nucleic acid derivatives was observed gradually at 35-55 ° C and, at about 59 ° G, a sudden melting curve was obtained. At values above 70 ° C, the curve practically approached a plateau and this means the disappearance of a high dimensional structure and spiral structure formation by hydrogen bonding of the nucleic acid derivatives.

The 50% melting temperatures for samples I and II were 59.5 and 59.3 ° C, respectively. From the calculation of the increase / decrease ratio in ultraviolet absorption at 260 nm between 90 ° C and 25 ° C, it was determined that the hyperchromicity (the color darkening effect) for samples I and II was 43.5% and 42.4%, respectively. This indicates that the nucleic acid derivatives have very stable structures under physiological conditions.

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Claims (11)

Nucleic acid derivative, characterized in that in a nucleic acid polymer, a portion of the purine or pyrimidine rings, which are a component thereof, is substituted with one or more sulfur atom (s) or this or these sulfur atom (s) m (s) (a) bridge (s) form (s) through a disulfide bond, giving a higher conformation. Nucleic acid derivative according to claim 1, characterized in that the nucleic acid polymer is a single stranded ribonucleotide polymer. Nucleic acid derivative according to claim 2, characterized in that the single-stranded ribonucleotide polymer is a polycytidylic acid, which contains a certain ratio of sulfur atom-containing 4-thiouridylic acid due to the substitution of the N 11 group in the pyrimidine ring of one or more 15 more cytidyl sulfur (s) in the polycytidylic acid by an SH group. Nucleic acid derivative according to claim; 3, characterized in that the size of the polymer is 50-5000, calculated as the number of bases. Nucleic acid derivative according to claim 3 or 4, characterized in that in the polycytidyl acid there is one sulfur atom relative to 6-39 cytidyl acids. Nucleic acid derivative according to claim 1, characterized in that the nucleic acid polymer is a double stranded ribonucleotide polymer. Nucleic acid derivative according to claim 6, characterized in that the double-stranded ribonucleotide polymer is composed of polyinosinyl acid and polycytidyl acid, which contains a certain ratio of sulfur atom-containing 4-thiouridylic acid as a result of the substitution of the N1-group in the pyrimidine ring of one or more cytidyl sulfur (s) in the polycytidyl acid by an SH group. Nucleic acid derivative according to claim: 7, characterized in that the size of the polymer is 50-10,000, calculated as the number of base pairs. Nucleic acid derivative according to claim 7 or 8, characterized in that in the polycytidyl acid there is one sulfur atom relative to 6-39 cytidyl acids. 8700Ö1S - 27 - * Method for the preparation of a medicament, characterized in that a nucleic acid derivative according to any one of claims 1-9 is brought in a dosage form suitable for medical purposes. Shaped medicament, characterized in that it contains a nucleic acid derivative according to any one of claims 1-9 as active ingredient, as well as optionally pharmaceutically acceptable carriers. 87 0 0 0 16