GB2185026A - Nucleic acid derivatives having sulphur substitution - Google Patents

Abstract

The derivatives comprise nucleic acid polymers (e.g. single or double strand ribo or deoxyribo nucleotide polymers) in which some of the purine and/or pyrimidine rings are substituted with sulphur (e.g. by replacement of -NH2 groups by -SH groups), which sulphur substituents may be linked to form disulphide bonds. The sulphur substituted derivatives appear to more stable than non-substituted nucleic and polymers. The derivatives appear to act as immunostimulants and neoplastic proliferation inhibitors.

Description

SPECIFICATION Nucleic acid derivatives The present invention relates to nucleic acid derivatives having useful physiological activity as pharmaceuticals.

Basically, nucleic acids are composed of sugars, such as ribose, bonded'to purine or pyrimidine rings and arranged in chains via phosphoric acid (phosphate) linkages.

Among nucleic acids, RNA (ribonucleotide polymer) is a high molecular compound having a chain structure containing ribose as sugar, the sugar moieties being limited by divalent phosphate linkages.

Thus ribonucleic acids may be represented by the formula:

Ribonucleic acid (R=OH, R'=H); Deoxyribonucleic acid (R=H, R'=CH3) The double chain of a ribonucleotide is of spiral form due to combination between purine or pyrimidine ring moieties of the bases (e.g. inosine, cytidine, uridine, etc.) constituting the nucleic acid. Nucleic acids having double strands exhibit useful physiological activity and, accordingly, have been widely studied.

Natural nucleic acids (e.g. RNA derived from viruses and synthetic double strand RNA such as polyinosinic acid/polycytidylic acid derivatives (hereinafter simply referred to as "poly-l'/poly-C"), polyadenylic acid/polyuridylic acid derivatives, etc. have been widely studied (e.g. Declark, et. al: Texas Reports on Bilogy and Medicine, 41, 77, 1982).

5'-Cytidylic acid has the formula:

and 5'-inosinic acid has the formula:

As noted above, many RNA nucleic acid deriatives have been synthesized and their physiological activities studied. Poly-l/poly-C is a substance having high activity and its usefulness has been evaluated and studies have been conducted thereon. A compound in which only a few parts of the cytidine units in a poly-l/poly-C are changed to uridine units (i.e. mismatched RNA) has been studied because of its physiological activity similar to that of the poly-l/poly-C (cf.

Japanese Laid Open Specification No. 50/082226).

Studies on polycytidylic acid are continuing and there is a report (British Patent Application No.

2,038,628) stating that, when mercapto groups (-SH) are introduced (to an extent of 50% or more) in place of the -NH2 groups in pyrimidine ring of polycytidylic acid, the physiological activity increases.

The conventional physologically active substances as noted above can be expected to exhibit useful effects though their toxicity as evidenced by poly-l/poly-C cannot be denied (Declard et al: Infect. Immuni., 6, 344, 1972) and some improvidment is desired. It is also desired to increase their physiological activity.

It has now been found, in accordance with the present invention, that RNA can form stabilized derivatives of high conformation by means of certain covalent bonds. The physiological activity of these derivatives is greater than comparable with conventional physiologically active substances, with reduced toxicity.

The feature of the present invention is that, as a result of the combination by a bridge of a covalent bond or the substitution of a part of the molecular structure of nucleic acid with SH group(s), higher conformation of nucleic acid which can be present in stable form is obtained.

According to the invention there is provided a nucleic acid derivative comprising a nucleic acid polymer in which some of the purine and/or pyrimidine rings present as constituent units of the polymer are substituted with sulphur, which sulphur substituents may be linked to form disulphide bonds.

Nucleic acid derivatives of the present invention can be synthesized from nucleic acid bases to which an -SH group can be introduced. A specific example of nucleic acid derivative of the invention is that having a complementary double spiral structure of a chain compound (hereinafter simply refered to as "poly-C") which is cytidylic acid polymer and another chain compound (hereinafter simply referred to as "poly-l") which is an inosinic acid polymer. Though this compound has a structure similar to the known poly-l poly-C, the nucleic acid derivatives of the present invention exhibits other characteristics for the following reasons.

Nucleic acid derivatives of the present invention form derivatives by partial substitution with sulphur atoms (SH groups) and/or by cross-linkage-(S-S bonds).

The sulphur atoms in the nucleic acid derivatives of the invention can be introduced, before or after preparation of the polymer, by utilizing an enzyme reaction. The introduction is conduced by substituting the nucleic acid base with -SH group, e.g. by replacing as -NH2 group in a pyrimidine ring of cytidyic acid by an -SH group. The resultant substituted residue is 4thiouridylic acid.

The introduced -SH may be further oxidized by a suitable method, as discussed below, to form S-S bonds.

Derivatives having both SH and S-S groups in a molecule can be prepared either by a partial oxidation of a compound containing -SH groups or by a partial reduction of a compound containing S-S groups.

The above nucleic acid polymer (poly-C) has a single strand. The single strand nucleic acid polymer, after introduction of the SH substitution or S-S bond briding can be associated with a complimentary single strand nucleic acid polymer, by a suitable method as discussed below, to form multiple strands. Conversely, a firstly-sulphurized single strand nucleic acid polymer may be associated with a complimentary nucleic acid polymer to form multiple strands followed by oxidation to give S-S bond bridges. The nucleic acid derivatives thus prepared are also included in the nucleic acid derivatives of the present invention. Thus, all nucleic acid polymers are included in the invention provided they contain nucleic acid bases to which -SH groups have been introduced.For example, the compounds obtained by disulphidation of nucleic acid polymers containing 4-thiouridine, 2-thiouridine, 2-thioguanosine, 6-mercaptopurine, 8-mercaptopurine, etc. are included in the invention.

The nucleic acid derivatives of the invention can be cleaved at their phosphoric acid side chains to give lower molecular weight compounds and such lower molecular compounds are within the invention. Accordingly, in this sense, there is a difference as compared with conventional poly-l/poly-C materials.

Due to a result of the characteristic feature of the nucleic acid derivatives of the present invention, i.e. substitution 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) physiological activity can be increased and (2) safety can be increased.

The physiological activity of the nucleic acid derivatives of the invention makes them useful as pharmaceuticals. As will be described later, the nucleic acid derivatives of the invention exhibit a strong action as interferon inducers. This action is only one of various physiologically activities of the nucleic acid derivatives of the invention. Other physiological activities of the nucleic acid derivatives of the invention include TNF-producing ability, interleukin 1 producing ability, interleukin 2 producing ability, macrophage activating ability, activating ability on NK cell, inhibiting action for proliferation of tumour cells, inhibition action for proliferation of tumours in cancerbearing mice, inhibition action for proliferation in nude mice bearing cancer of human tumour cells, and preventing action for matastasis of tumour cells in the lung.

The nucleic acid derivatives of the invention are generally safer as compared with interferon inducers such as conventional poly-l/poly-C. Accordingly, the compounds of the invention are useful as antiviral and antitumour agents.

The term "base pair" ("bp") frequently used to indicate the molecular size of nucleic acids serves to indicate the molecular size by the numbers of bases in the nucleic acid in each complimentary strand (i.e. 10 bp means a double strand polymer having ten bases). Since nucleic acid polymers other than double strand polymers are also referred to in the present specification, the term "residue numbers" in place of "bp" will be used. Thus, for example, "10 residue numbers" means a nucleic acid polymer having 10 bases in a strand.

Nucleic acid derivatives of the invention contain substants of various 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 larger, e.g. up to 200,000 residue numbers and it is known that the molecular size has little influence on physiological activity.

With regard to the amount of sulphur in the nucleic acid derivatives of the invention, the degree of sulphurization (n) is used in this specification. Cytidylic acid is converted to 4thiouridylic acid by replacing the -NH2 group in the pyrimidine ring with an -SH group and the number "n" indicates the number of cytidylic acid units per one 4-thiouridylic acid. The nucleic acid derivatives of the invention have varying degrees of sulphurization but it is preferred that n is not less than 6. If n is less than 6, it has been found that the physiological activity decreases.

If n is 6 or more, there will be no further limitation and n on physiological activity.

In manufacturing the nucleic acid derivatives of the invention, various methods can be used.

The poly-C and the like which are starting materials for the derivatives of the invention can be readily available. Poly-C may be easily sulphurized by, for example, reaction with a sulphurizing agent such as hydrogen sulphide. As a result of said reaction, some of the cytidylic acid units in the poly-C are changed to 4-thiouridylic acid units. By varying reaction conditions such as temperature and time, poly-C derivatives with desired "n" values can be prepared.

The sulphurized poly-C can be associated with poly-l, which is readily available by known methods. The sulphurized poly-C/poly-l obtained as such can be introduced to nucleic acid derivatives of the invention by a disulphide production reaction such as, for example, oxidation with iodine reagent.

The nucleic acid derivatives of the present invention can be obtained almost quantitatively by the above methods and the overall yield may be around 90%.

In the synthesis of other nucleic acids, such as poly-A/poly-U, having disulphide bridges, the polymer is first prepared by an enzyme reaction using a nucleic acid base containing sulphur atoms therein. Conventional methods for the manufacture of heteropolymers can be applied.

By way of typical example, when 36mM of uridine-5'-diphosphate (UDP) and and 7 mM of 4thiouridine-5'-diphosphate (4-SDH UDP) are reacted at 37"C for 5 hours in 150 mM of Tris buffer (pH 8.2) using 0.5 unit/ml of polynucleotide phosphorylase (PNPase, type 15, PL Biochemical), there is obtained a heteropolymer, containing one 4-thiouridine residue for 13 uridine residues, in around 50% yield. When 2-thiorudine-5'-diphosphate is used in place of 4-thiouridine-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 preparation as for the sulphurized poly-l/poly-C described in the Examples below. More specifically, both sulphurized polyuridylic acid and equimolar polyadenylic acid are dissolved in neutral aqueous solution and subjected to an annealing for a complex formation.

Then, after water-dissolving in an incubator, the temperature is gradually raised to 85"C, then heated for 10 minutes, and allowed to stand at room temperature.

The complex thus obtained is oxidized under the same conditions as in the disulphidation of poly-l/poly-C, i.e. oxidation with 1N iodine solution, to give a poly-A/poly-U derivative having a disulphide bridge. The yield after the annealing is about 80% and the overail yield is about 40%.

The disulphide complex is subsequently well dialyzed to an aqueous solution and lyophilized to give white and fibrous solid.

It is not always clear that the nucleic acid derivatives of the present invention is in what type steric structure by their characteristic S-S bond.

The nucleic acid derivatives of the invention give certain clear melting curves as discussed later and, accordingly, it is apparent that they exhibit stable fundamental structures.

With reference to the 50% melting temperature (Tm value), it is 59.0 C in the poly-C/poly-C under a neutral condition in the presence of 0. 1 M sodium ion while, in the cases of SHsubstituted nucleic acid derivative containing 4-thiouridine at the rate of n=20 and S-S substituted nucleic acid derivative thereof, the values are 59.5"C and 59.3"C and, accordingly, they are similarly or more stabilised. In the case of OH-substituted nucleic acid derivative containing uridine at the rate of n=20 the same as above, the value is as low as 53.1"C.

Thus the introduction of sulphur atoms are more effective 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 decomoses RNA) was compared in terms of the time ratio until the decomposition arrives at 50%. Thus, under certain enzymatic reaction conditions, poly l-poly C was 50 minutes while those of 4-thiouridine (SH group) and its nucleic acid derivative (n=20; 0 containing S-S) are 60 minutes and, on the contrary, those of nucleic acid derivatives (n=20) containing uridine (OH group) were as short as 20 minutes. It is therefore apparent that sulphur atoms exhibits substituting effect even in biochemical stability.

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

As to carriers, one or more liquid, solid or semisolid diluent, filler and other auxiliary agents for pharmaceutical preparations may be used. It is desired that the pharmaceutical compositions are administered in unit dosage form. Nucleic acid derivatives of the present invention may be given via mouth, tissue, local place, rectum. They are of course given by forms suitable for each administration route. For example, they are given as oral administration form, injection, inhalation, eye lotion, ointment, suppository, etc. It is especially preferred to give into tissue or locally.

It is desired that the dose as remedies for malignant tumor is regulated by considering the status of the patient (e.g. age, body weight, etc.) administration route, and type and degree of the disease. Generaly, 0.05 to 1000 mg at a time is used as an effective amount of the nucleic acid of the present invention to adults and, preferably, 0.5 to 50 mg of intravenous infusion is common. In some cases, less dose is sufficient and, in some other cases, more doses may be necessary. It is also possible that the administration is applied for one to several times a day or with an intermission of one to several days.

Dose as remedy for being tumor or for diseases caused by virus is preferably regulated by considering 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 fiftieth of the dose for the above malignant tumor.

Physiological Activity of Nucleic Acid Derivatives of the Present Invention: Physiological activity of the nucleic acid derivatives of the present invention is illustrated as hereunder.

(1) Interferon-inducing activity: With reference to sulphurized poly C-poly I derivative which is one of the nucleic acid derivatives of the present invention, its action as interferon-inducer was determined by an antiviral activity assay method. The sulphurized poly C-poly I derivative used as Sample I for the test is an SH substitute while Sample II is an S-S substitute. Both are with n value of 13 and with 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), incubated 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 CPElso (Cytopathogenic Effect Inhibition 50) method.

Sample I is an S-S substituted nucleic acid derivative containing 4-thiouridine at the rate of n=20 while Sample II is its SH-substitute. Conventional poly 1-Poly C was used as a control sample. inhibition 50) method. Conventional poly 1-poly C was used as a control sample.

Concentrations (micrograms/ml) 10 100 200 500 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 apparent that the nucleic acid derivative of the present invention exhibit strong action as an interferon inducer.

Among the sulphurized poly C-poly I derivative of nucleic acid derivatives of the present 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 2000 residue were taken as test samples and subjected to the same physiological activity tests to give similar result. The similar result was also obtained when the nucleic acid derivative (n-value of 20 and molecular size of not less than 20,000 residue numbers) before the lowmolecularization.

(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 2x 106 per milliliter in a 10% FCS-added RPMI 1640 medium, one milliliter of it was placed on a plastic dish, and cultured in a carbon dioxide gas incubator (5% CO2) in the presence or absence of the sample at 370C.

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 LM 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 neutralization.

TNF 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 + LPS was used at the concentration of 1 microgram/ml and the test and control samples were at the same concentrations 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 activity in vivo.

Meth A tumor (2 x 105) was transplanted behind the abdomen skin of BALB/c micr (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 was the same titer as used in the above (2).

Treatments TNF Activity (1st) (2nd) in Serum Necrosis BCG LPS 192 observed BCG Test Sample 1 48 observed BCG Test Sample il 50 observed BCG Control Sample 24 observed Test Sample I, Test Sample I 12 observed Test Sample II, Test Sample II 24 observed Both test and control samples were the same as those in the above (1). It is apparent that the TNF producing ability in vivo of the nucleic acid derivative of the present invention is strong.

(4) Inducing Activity of Interleukin 1.

Normal human heparin-added peripheral blood was separated from mononuclear particles by specific gravity centrifugation method using Ficoll-Hypaque (Farmacia, Ficoll-paque, trademark), the numbers were adjusted to 5 x 106/mI in a 10% FCS-added RPMí medium, placed in a plastic dish, incubated at 37 C for one hour, and the cohesive cells were used as an interleukin 1producing source.

Drug was added to the 5 x 105/ml of single particle, incubated at 37"C for 24 hours, and the interleukin 1 activity in the supernatant liquid was determined by measuring a 3H-thymidine intake method into a PHA-stimulated mice thymus cells using a proliferation ability of the thymus cells as a target. The result is given in the following 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 ml. The test Samples I and II and control Sample used were the same as those used in the above (1).

Samples Used In-Take Amount (DPM (x 104)) Control 3370 Test Sample 1 11384 Test Sample II 11560 Control Sample 8397 Interleukin 1 12517 It is apparent that the nucleic acid derivative of the present invention exhibits strong interleukin 1 inducing activity.

(5) Inducing activity of Interleukin 2.

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

To 5 x 1 06/ml of lymph particles was added the drug, the mixture was incubated at 37"C for 24-48 hours, and the interleukin 2 activity in the supernatant liquid was determined by a 3Htymidine incorporation method using CTLL-2 or NK-7 which is cell strain proliferating depending upon interleukin 2 using the proliferation ability as a target. The result is given in the following table. The control is physiological saline solution. The interleukin 2 was in a concentration of 5 units/ml and both test 1,11 and control samples were in the same concentration of 100 gammas/ml. The test and control samples used were the same as those in (1).

Samples Incorporated Amount (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 derivative of the present invention exhibits strong interleukin 2 inducing activity.

(6) Macrophage Activation.

Samples were administered to Balb/c mice (7-10 weeks age; male) intraperitoneally, cells exuded from the peritoneum at 3 to 5 days after administration were collected, plastic-cohesive cells mainly macrophage) were separated as effector cells, and the macrophage activation was investigated by a 3H-thymidine isolation method using Meth-A tumor cell as a target (the E/T ratio being 15-20:1). The result is given in the following table. The control was 0.2 ml of physiological saline solution. Each 50 micrograms/mouse of test sample Ill or control sample was administered. The test and control samples used were the same as those in (1).

% Cytotoxicity was calculated by: (experimental value) - (background) x 100 (100% 3H release) - (background) Samples % Cytotoxicity Control 0.5 Test Sample 1 10.3 Test Sample II 10.8 Control Sample 5.7 It is apparent that the nucleic acid derivative of the present invention exhibits 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 5'Cr. The result is given below.

The control used was physiological saline solution. The test and control samples were the same as those in the above (1).

Samples (micrograms/ml) Melting % (%5'Cr Isolation) Control 30 Test Samples I (10) 47 (30) 55 (100) 57 (300) 50 (500) 30 Test Samples II (10) 51 (30) 59 (100) 63 (300) 60 (500) 45 Control Samples (10) 52 (30) 60 (100) 65 (300) 62 (500) 52 It is apparent that the nucleic acid derivative of the present invention exhibits NK cell activation.

(8) Inhibitory Action for Proliferation of Tumor Cells.

Inhibitory action for proliferation of cell line tumor cells was measured. Cells (3x 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 incubated for 48 hours with tritium-labelled thymidine. The inhibiton % was given by an incorporated amount of thymidine to a control which was not treated with the sample. The result is given below. The test and control samples were the same as those in the above (1).

Inhibition % Test Sample I Test Sample II Control Samples Cells 100 10 100 10 100 10 micrograms/ml NAMALWA 65.6 55.7 61.2 46.4 56.9 37.4 RAJI 68.3 64.6 67.8 61.5 74.7 69.4 L5929 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 derivative of the present invention exhibits 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 measured 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) and, after 2 days, started in administration of the drug three times a week for two weeks. On the second day after final administration, tumor cells were extracted and the weight was measured. The result is given below. The test and control samples were the same as those in the above (1).

Numbers Samples of (micrograms/mouse) Mice Mean+S.E. Inhibition% Test Sample 1 (10) 7 2.09+0.15 3 (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 1.6210.25 (100) 7 0.97+0.30 Control Sample (10) 7 2.12+0.27 2 (30) 8 1.38+0.38 36 (100) 8 1.08+0.27 50 It is apparent that the nucleic acid derivative of the present invention inhibits the proliferation of tumor cells in cancer-bearing mice.

(10) Proliferation inhibiting action in nude mice bearing cancer from human tumor cells.

Each 2.5x 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 ,ug/mouce of test sample (intravenously) and 25 mg/kg of 5-FU (intraperitoneally) were administered twice a week for four weeks. Tumor was extrated on the fourth week after the administration and the weight was measured. The result is given in the following table. The test sample used was the same as that in the above (1).

(La S3) Sample Weight (g)+S.E. Inhibition Rate (%) Control 3.8510.23 - Test Sample 1 1.5710.19 59 Test Sample II 1.55+0.20 60 5-FU 2.0610.35 47 (Hep-2) Sample Weight (g)+S.E. Inhibition Rate (%) Control 0.8810.07 - Test Sample I 0.38t0.07 57 Test Sample II 0.3510.05 60 5-FU 0.5610.12 30 It is apparent that the nucleic acid derivative of the present invention exhibits strong inhibitory action for proliferation.

(11) Inhibitory Action against Metastasis of Tumor Cells to Lung.

To C57BL/6 Mice (male; 5 weeks age) was transplanted, to intravenous vein, 2x 105 B16F10 cells which are transplantable melanoma of the same type and the numbers of nodes moved to lung (numbers of colonies) on the second week after the transplantation were counted. The sample was administered intravenously at 24 hours prior to the transplantation of B16F10 melanoma. Numbers of the cases were 9. The result is given below. The test and control samples used were the same as those in the above (1).

Sample (micrograms/mouse) Numbers of Nodes moved to Lung sisS.E.

Control 112+17 Test Sample I (1) 33 to 6" (10) 21110* (100) 4+2* Test Sample II (1) 21+6" (10) 15+4* (100) 411* Control Sample (1) 361 16* (10) 3+0.7* (100) 7~3* It is apparent that the nucleic acid derivative of the present invention exhibits inhibitory action against metastasis of tumor cells to lung.

Safety of the Nucleic Acid Derivatives of the Present Invention: Safety of the nucelic acid derivatives of the present invention will be illustrated as hereunder.

Sulphurized poly C-poly I derivative which one of the nucleic acid derivatives of the present invention was taken and its cytotoxic effect to bone marrow main cells was examined. The sulphurized poly C-poly I derivative used was with n-value of 20 and molecular size of 150-2000 residues.

The sample was injected intravenously to mice (8 weeks age; male; each group comprises 5 mice) at the dose of 100 micrograms/mouse and, after 24 hours, bone marrow cells of them were collected. The cells were fixed and dyed with Giemsa. Cells of the smeared sample were observed with a microscope and the degree of appearance of reticulocytes was counted by %.

Known poly 1-poly C was used as a control. Physiological saline solution was administered to the control. The result is given in the following table: Erythrocyte Cells (%) Control 40 Test Sample 1 38 Test Sample II 39 Control Sample 11 It is apparent that the nucleic acid derivative of the present invention exhibits high safety.

Then pyrogenic effect of the nucleic acid derivatives of the present invention will be explained.

Injection of the poly 1-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 1-poly C gives 1.45"C of body temperature rise in average. On the contrary, the nucleic acid derivatives of the present invention (both SH and S-S substances in the abobe cytotoxic experiment) shows only 0.25"C rise in body temperature in average and the result of the pyrogenic test was negative. In those experiments, three rabbits 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.

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

The result of acute toxicity test of the nucleic acid derivatives of the present invention is given as follows. 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 effect was judged by the fact 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 nucleic acid derivative (the same as above) of the present invention show weaker toxicity than poly 1-poly C.

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

LD50 Strain Route of Administration Test Sample I Test Sample II Control Sample 25ddy intravenously > 394mg/kg 132 mg/kg 132mg/kg (Working Example) The present invention will be further illustrated by way of examples concerning the manufacturing method of nucleic acid derivatives of the present invention and it will be understood that the present invention is not limited thereto.

Examples.

(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 5 ml of liquid hydrogen sulphide, and made to react at 37"C 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 0 C 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 10 liters of 50mM sodium chloride and the resulting transparent liquid was further lyophilyzed to give 0.47 g of white fibrous substance.

The ultraviolet absorption spectra of the resulting substance were measured in a neutral aqueous solution to give the result of 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 -NH2 group at 4-position of pyrimidine ring of cytidylic acid was substituted with -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 4thiouridylic acid to 13 cytidylic acid.

By the same manner, the reaction temperatures and reaction 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 numbers of cytidylic acid to one 4-thiouridyic acid.

Reaction Temp. Reaction Time n 45"C 12 hours 1 - 45 6 6+1 37 6 13+2 37 4 26 + 2 37 2.5 39 + 2 (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 containing 50mM of sodium chloride to make the concentration of 10-20 mg/ml and, in the water bath, the temperature was gradually raised from the room temperature to 68"C. The mixture was maintained at 68"C for about 10 minutes, allowed to stand until it became to room temperature, and stored at 4"C.

The resulting solution is lyophilized to give 1.29 of a nucleic acid derivative having SH groups in white solid.

Then, to this was added about 10 times volume (by molar ratio) of 1N 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 0 C for 1 hour. The reaction solution was well dialyzed against water until the yellow colour of iodine vanished.

The solution thus obtained was lyophilyzed to give 0.98 g of white solid.

To fact that the resulting solid contains a S-S linkage was confirmed as follows. Thus, 0.1 g of this solid was dissolved in 10 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 absorptive 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 sholder peak of 310 nm which is an absorption wavelength due to the S-S bond decreased while an absorption at 330 nm which is due to -SH substance (4-thiouridine) incresed and, after 7.5 hours, the degree of absorption at 330 nm became to be identical with that of the sulphurized poly C before the oxidation. It is thereforeapparent that the S-S bond was quantitatively reduced to -SH group.

(3) Cross-Linking Structure and Biological Activity.

Among the nucleic acid derivatives into which sulphur atoms are introduced, synthetic methods and physiological activities of the test sample I (having all-reduced type SH groups) and the test sample Ill (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 1000 b.p. for example, it has a cross-linking structure at ten parts. Any nucleic acid derivatie having various cross-linking numbers may be easily prepared by a partial oxidation of the SH substance under a mild condition or by a partial reduction of the S-S substance under a mild condition. The synthetic condition 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 crosslinking, 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 a RNA-ase (e.g., ribonuclease P,), 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 sample II) to O (totally SH substance; e.g. test sample I) exhibits the similar activity and safety as those of the aforementioned 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.

(4) Low-Molecularization.

containing nucleic acid derivative (1 g) obtained in the above (2) was dissolved in 120 ml of water and 30 ml of 5M sodium chloride solution and 150 ml of formaldehyde were added thereto. The mixture was vigorously stirred to give uniform solution. The reaction solution was heated at 80"C for 8 hours, well dialyzed against. water, and lyophilyzed to give 0.95 g of white solid.

When the SS bond-containing nucleic acid derivative obtained in the above(z) is subjected to low molecularization the same as abode, the same result is obtained.

This was subjected to a high performance liquid chromatography by a gel filtration and the resulting pattern is given in Fig. 3. The condition applied was that TSK-gel G4000SW (0.65 x60 cm) was used and, as an eluate, 50mM Tris HCI 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 maximum distribution at around 500 residue numbers and that it has a molecular size which distributes 1 50 to 1000 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 following molecular sizes were obtained by the same manner.

Max.

Reaction Reaction Distribution Distribution Temp. Time (bp) (bp) O hr - 2,000 80"C 24 30 10-55 80"C 16 200 50-300 80"C 8 500 150-1000 60"C 8 1000 200-5000 Among the nucleic acid derivative obtained by the above example, those with 200 to 5,000 residue numbers of molecular size distribution were taken and their fusing curves were measured.

The sample I or II (0.70D/ml) in 1OmM Tris buffer (pH 7.5) containing 0.1M sodium chloride was warmed, at the rising rate of 2"C/4 minutes, from 20"C to 90"C., the ultraviolet absorbancy at each temperature was measured at the wavelength of 260 nm, and an increase of ultraviolet absorbancy by a hyperchromicity was represented by a relative ratio giving the state at 20"C as a base. A Beckmann-DU-3B spectrophotometer was used for the measurement. The result is given in Fig. 4. Fusion of the nucleic acid derivatives was gradually observed during 35 to 55"C and, at around 59"C, a sudden fusion curve was obtained.At higher than 70"C, the curve nearly arrived at a pleateau and this means the disappearance of high dimension structure and spiral structure formation by hydrogen bond of the nucleic acid derivatives.

The 50% fusing temperatures for the samples I and II were 59.5 and 59.3"C, respectively. Out of the calculation from the increasing/decreasing rate in ultraviolet absorbancy at 260 nm between 90"C and 25"C, it has been confirmed that the hyperchromicity (colour darkening effect) for the samples I and II were 43.5% and 42.4% respectively. This indicates that the nucleic acid derivatives are with very stable structures under physiological conditions.

(4) Brief Explanation of the Attached Drawings: Figure 1 shows ultraviolet absorption spectra of sulphurized polycytidylic acid obtained in (1) of Example. The ordinate and abscissa are absorption and wavelength (nm), respectively.

Figure 2 shows ultraviolet absorption spectra of nucleic acid derivative obtained (2) of the Example. This is a UV patterns of reduction of sodium thiosulphate. The ordinate and abscissa are absorbancy and wavelength (nm), respectively.

Figure 3 shows elution patterns by high performance liquid chromatography of nucleic acid derivative of the present invention obtained in (3) of the Example. The abscissa and oridinate are elution time (minute) eluted amount, respectively. Each arrow indicates the eluting position of size marker.

Figure 4 shows fusion curve of the nucleic acid derivative 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.

Claims (10)

1. A nucleic acid derivative comprising a nucleic acid polymer in which some of the purine and/or pyridine rings present as constituent units of the polymer are substituted with sulphur, which sulphur substituents may be linked to form disulphide bonds.

2. A nucleic acid derivative as claimed in claim 1 in which the nucleic acid polymer is a single strand ribonucleotide polymer.

3. A nucleic acid derivative as claimed in claim 2 in which the single strand ribonucleotide polymer is a poly-C containing 2-thiouridylic units obtained by replacing -NH2 groups in the pyrimidine rings of cytidylic acid units in the poly-C, with -SH groups.

4. A nucleic acid derivative as claimed in claim 3 in which the length of the polymer is from 50 to 8000 calculated as base number.

5. A nucleic acid derivative as claimed in claim 3 or claim 4 containing one sulphur atom substituent per 6 to 39 cytidylic acid units present in the poly-C.

6. A nucleic acid derivative as claimed in claim 1 in which the nucleic acid polymer is a double strand ribonucleotide polymer.

7. A nucleic acid derivative as claimed in claim 6 in which the double strand ribonucleotide polymer is one compound of poly-l and poly-C and containing 4-thiouridylic acid units obtaining by replacing -NH2 groups in the pryimidine rings of cytidylic acid units in the poly-C, by -SH groups.

8. A nucleic acid derivative as claimed in claim 7 in which the length of the polymer is from 50 to 10,000 calculated as base pair numbers.

9. A nucleic acid derivative as claimed in claim 7 or claim 8 containing one sulphur atom substituent per 6 to 39 cytidylic acid units present in the poly-C.

10. A nucleic acid derivative as claimed in claim 1 substantially as hereinbefore described with reference to the examples.