FR2596762A1 - NUCLEIC ACID DERIVATIVES - Google Patents

Abstract

THE INVENTION CONCERNS A DERIVATIVE OF NUCLEIC ACIDS CHARACTERIZED IN THAT, IN A POLYMER OF NUCLEIC ACIDS, A PART OF THE PURINE OR PYRIMIDINE CYCLES WHICH ARE A CONSTITUENT ELEMENT THEREOF IS SUBSTITUTED BY OR ONE OR MORE SULFUR ATOMS WHERE THESE SULFUR ATOMS SUBSTITUTE FORM BRIDGES BY A DISULFIDE LINK, THEREFORE RESULTS BETTER CONFORMITY.

Description

l. The present invention relates to nucleic acid derivatives having

  useful physiological activity like

pharmaceutical products.

  The nucleic acid is composed of sugars such as ribose which are linked to a purine or pyrimidine ring and which are

  arranged in chains through phosphoric acid.

  Among nucleic acids, RNA (ribonucleotide polymer) is a compound of high molecular weight in

  the chain from which ribose is contained as sugar and the sugar moieties are combined by diester bonds of phosphoric acid.

  The double chain has a steric spiral structure consisting of a combination of the purine or pyrimidine ring part of the base (for example inosine, cytidine, uri15 dine, etc.) which constitutes the nucleic acid. Nucleic acids with double strands exhibit useful physiological activity and therefore numerous studies have been devoted to them. Among these are natural RNA (for example, RNA from a virus) and synthetic double-stranded RNA such as polyinosinic acid-polycytidylic acid derivatives 2.

  (hereinafter abbreviated as "poly I. poly C"), derivatives of polyadenylic-polyuridylic acid etc. have been studied by many researchers (e.g. Declark et al .: Texas Reports on Biology and Medicine, 41, 77, 1982).

  10 3. r 6 R ONH OCH HO-PI = O <Nx_> HO-P

0 R 25 HO-P = O0

OCH, o Ro

0O R

  / O Ribonucleic acid (R = OH, R '= H); DMsoxyribonucleic acid (R = H, R '= CH-) 35 l0

HO-P-OH.C

2 I OH (5'-cytidylic acid) HN O

HO-P-OH C

  2 0 OH OH OH (5 -inosinic acid) 4. Among these, many acid derivatives

  nucleic RNA were prepared by synthesis and their physiological activities were studied.

  Poly I. poly C is a substance with high activity; its usefulness has been evaluated and studies have been devoted to it. A compound in which only part of the poly I-poly C cytidine is transformed into uridine (i.e. a poorly matched RNA) has been studied due to

  its physiological activity similar to that of poly I-poly C 10 (see Japanese patent 50/082226).

  Studies on polycytidylic acid have also continued and it has been reported (British Patent Application No. 2,038,628) that when a mercapto (-SH) group is introduced (50% or more) in place of

  -NH2 group of the pyrimidine ring of polycytidylic acid, physiological activity increases.

  The conventional physiologically active substances listed above can be expected to exhibit

  useful effects although their toxicity, as observed with poly I.poly C, is undeniable (Declard et al.

  Infect. Immuni., 6, 344, 1972) and some improvements are necessary. We also wish

  increase their physiological activity.

  During the studies pursued for many years25 by the Applicant, the latter has found, although by pure coincidence, that RNA could form derivatives stabilized by certain covalent bonds. When we measured the physiological activity of these, we found that they had a much stronger activity than the phy30 siologically active substances with very low toxicity, and the

  present invention has been realized.

  The characteristic of the present invention is that, thanks to the combination by a bridge of covalent bonds or the substitution of part of the molecular structure of the nucleic acid by one or more SH groups, one obtains

  a nucleic acid in stable form.

  5. The nucleic acid derivatives of the present invention can be prepared by synthesis from a nucleic acid base into which an -SH group can be introduced. A particular example of the nucleic acid derivatives of the present invention is that having a double spiral structure complementary to a chalne compound (referred to as "poly C" in this specification) which is a polymer of the cytidylic acid and another chain compound (referred to as "poly I" herein) which is a polymer of inosinic acid. Although this compound has a structure similar to that of the poly I-poly C already indicated, the nucleic acid derivatives of the present invention still exhibit

  characteristics for the following reasons.

  The nucleic acid derivatives of the present invention form derivatives by partial substitution with a sulfur atom (SH group), by a transverse bond

  (S-S bond) or by the simultaneous presence of two substituents.

  The sulfur atoms present in the nucleic acid derivatives of the present invention can be introduced, before or after the preparation of the polymer, using an enzymatic reaction. The introduction is carried out by substituting the nucleic acid base with an -SH group, so that an -NH2 group of a pyrimidine ring of cytidylic acid is

  substituted by a group -SH. The substituent radical is called 4thiouridylic acid.

S HNt

$

0 oHi N

HO -; - OH C '

OH

OH OH

  5'-4-thiouridylic acid 6. The -SH group introduced is then oxidized by a suitable process as indicated below, so that

that an S-S bridge can be formed.

  Derivatives having both SH and S-S groups in a molecule can be prepared either by partial oxidation of a compound in which an -SH group is

  introduced either by partial reduction of a compound into which an S-S group is introduced.

  The nucleic acid polymer indicated above (poly C) has a single strand. The single-stranded nucleic acid polymer after substitution of the SH or formation of the S-S bridge can be combined with a complementary single-stranded nucleic acid polymer by an appropriate method which will be indicated below to form multiple strands. Conversely, it is also possible that the stranded nucleic acid polymer

  single sulfur the first is associated with the complementary nucleic acid polymer to form multiple strands, after which the oxidation is carried out to give an S-S bridge.

  The nucleic acid derivatives thus prepared also appear among the nucleic acid derivatives having physiological activity which is the subject of the present invention. Thus, all the nucleic acid polymers belong to the present invention insofar as they contain nucleic acid bases into which -SH groups can be introduced. For example, the compounds obtained by disulfurization of nucleic acid polymers containing 4thiouridine, 2-thiouridine, 2-thioguanosine, 6mercaptopurine, 8-percaptopurine etc. come in

  in the field of the present invention.

   3 The nucleic acid derivatives of the present invention can be cut at their lateral phosphoric acid chain to give mass compounds

  lower molecular weight and these lower molecular weight compounds also belong to the present invention.

  Consequently, in this sense, there is a difference with the

poly I-poly C classic.

  7. Due to the characteristic of the nucleic acid derivatives of the present invention, namely substitution by SH, formation of transverse bond SS and formation of substances of low molecular weight by cleavage of the side chain phosphoric acid, it is possible for the first time to obtain the effect of the present invention, that (1) physiological activity can be increased

  and (2) security can be increased.

  The physiological activity of the nucleic acid derivatives of the present invention is very useful as pharmaceuticals. As will be described below, the nucleic acid derivatives of the present invention have a strong action as inducers of interferon. This action is only one of the various physiological actions of the nucleic acid derivatives of the present invention. With regard to the physiological actions of the nucleic acid derivatives of the present invention, several can be listed which will be indicated below. These include TNF production capacity, interleukin 1 production capacity, interleukin 2 production capacity, macrophage activation capacity, NK cell activation capacity, l action to inhibit the proliferation of tumor cells, the action to inhibit the proliferation of tumors in mice carrying cancers, the action of inhibiting the proliferation in shaved mice carrying cancers of tumor cells prevention of metastasis from tumor cells to the lungs, etc. The nucleic acid derivatives of the present invention are much safer than interferon inducers such as conventional poly I-poly C. Consequently, the compounds of the present invention can be used as agents

  antivirals and as anti-tumor agents.

  The expression "base pair" (abbreviated as "bp") frequently used to indicate the molecular size of a nucleic acid expresses the molecular size by the numbers of 8. bases in the nucleic acid (ie 10 bp denote a double strand polymer having ten bases) in each complementary strand. As reference is also made herein to polymers of nucleic acids other than double-stranded polymers, the term "number of radicals" will be used in place of bp. For example, "a number of radicals of 10" means a nucleic acid polymer

having 10 bases in a strand.

  The nucleic acid derivatives of the present invention contain substances having various species of molecular sizes and it is generally preferred that the size is not less than 50 radicals, for example from 10,000 radicals. The size can be even higher, for example 200,000 radicals will also be suitable and it is known that molecular size has little influence on the activity

  physiological of the present invention.

  To indicate the sulfur numbers in the nucleic acid derivatives of the present invention, the degree of sulfurization (n) is used herein. Cytidylic acid is transformed into 4thiouridylic acid by substitution of the group -NH2 in the pyrimidine ring by a group -SH and "n" indicates the numbers of cytidylic acid existing for a 4-thiouridylic acid. The nucleic acid derivatives of the present invention contain substances having many species of "n" and it is preferred that this n is not less than 6. When n is less than six, it has been confirmed that physiological activity decreases . Provided that n is equal to or greater than 6, there is no other limitation and n

  can reach 39, and it has little influence on the physiological activity of the present invention.

  When preparing nucleic acid derivatives

  of the present invention, various methods can be used.

  Poly C etc. which are the starting materials for the derivatives of the present invention are readily available. Poly C can easily be sulfurized, for example by reaction with a sulfurizing agent such as hydrogen sulfide. Under the effect of 9. this reaction, certain numbers of cytidylic acid in poly C can be transformed into 4thiouridylic acid. By varying reaction conditions such as reaction temperature and reaction time, poly Cs with the desired "n" values can be prepared. The sulfurized poly C can be combined with a poly I which is easily accessible by known methods. The sulfur poly poly I obtained can be introduced into the nucleic acid derivatives of the present invention by a disulfide formation reaction such as, for example, an oxidation

by iodine.

  The nucleic acid derivatives of the present invention can be obtained almost quantitatively

  by the above methods, and it is known that the overall yield is about 90%.

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

  For example, when reacting 36 μl of uridine '-diphosphate (UDP) and 7 mM of 4-thiouridine-5'-diphosphate (4-SDH UDP) at 37 ° C. for 5 hours in 150 mM of Tris 25 buffer (pH 8.2) using 0.5 unit / ml of polynucleotide phosphorylase (PNPase, type 15, PL Biochemical), a heteropolymer containing 4-thiouridine is obtained for 13 uridine radicals with

  a yield of about 50%. When 2-thiouridine-5'diphosphate is used in place of 4-thiouridine-diphosphate, a poly U containing 2-thiouridine is obtained.

  As soon as a polymer of nucleic acids containing -SH groups has been prepared, the following derivatives can be obtained by the same operation as in the case of the sulfur-containing poly Ipoly C described in the examples. More specifically, both sulfur polyuridylic acid and equimolar polyadenylic acid are dissolved in a neutral aqueous solution 10.

  and they are subjected to annealing for the formation of a complex.

  Thus, after having dissolved in water 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 for the disulfurization of poly Ipoly C, that is to say oxidized with a 1N iodine solution to give a derivative

  poly A-poly U having a disulfide bridge. The yield after annealing is about 80%, and the overall yield is about 40%.

  The disulfide complex is carefully dialyzed

  against an aqueous solution and lyophilized to give a white and fibrous solid.

  The type of steric structure of acid derivatives

  nucleic acid of the present invention, determined by their characteristic S-S bond, is not always clear.

  The nucleic acid derivatives of the present invention give certain clear fusion curves as described below and, therefore, manifestly exhibit stable fundamental structures.

  Regarding the melting temperature at 50% (Tm value), it is 59.0 C in poly I-poly C in neutral medium in the presence of sodium ion O, 1M, while in the case 25 d 'a SH substituted nucleic acid derivative containing 4-thiouridine at a rate of n = 20 and a SS substituted nucleic acid derivative, the values are 59.5 C and 59.3 C, and accordingly , they are stabilized to the same degree or more. In the case of a nucleic acid derivative

  substituted by OH containing uridine at a rate of n = 20 (as above), the value is only 53.1 C.

  So the introduction of sulfur atoms is more

  effective in stabilizing the macromolecular structure of a nucleic acid than with other substituted derivatives such as those substituted by NH2 or by OH.

  The resistance to hydrolysis by 11. RNAase A (an enzyme which breaks down RNA) was then compared with respect to the time ratio until decomposition reached 50%. Thus, under certain conditions of enzymatic reaction, the time for poly I-poly C is 50 minutes 5 while those for 4-thiouridine (group SH) and its nucleic acid derivative (n = 20; containing SS) are 60 minutes; on the contrary, those for nucleic acid derivatives (n = 20)

  containing uridine (OH group) are only 20 minutes. It is therefore clear that the sulfur atom has a substituent ef10 even in biochemical stability.

  When the nucleic acid derivatives of the present invention are administered in the form of pharmaceutical products, they are administered to animals and to humans as they are.

  or in the form of a pharmaceutical composition containing for example 0.1 to 99.5% (preferably 0.5 to 90%) of ingredient in a non-toxic and inert pharmaceutically acceptable excipient.

  As excipients, one or more diluents, fillers and other auxiliaries to liquid, solid or semi-solid pharmaceutical preparations can be used. It is desirable that the pharmaceutical compositions be administered as a unit dose. The nucleic acid derivatives of the present invention can be administered orally, parenterally, locally or rectally. They are, of course, administered in forms suitable for each route of administration. For example, they are administered as oral administration, injection, inhalation, eye lotion, ointment, suppository, etc. We particularly prefer

  parenteral or local administration.

  It is desirable that the dose as a drug for malignant tumors is determined taking into account the patient's condition (e.g., age, body weight, etc.), the route of administration and the type and the degree of the disease. In general, 0.05 to 1000 mg is used at the

  As the effective amount of the nucleic acid of the present invention for adults, and preferably, 0.5 to 50 mg is commonly administered as an intravenous infusion.

  12. In some cases a lower dose is sufficient and in other cases higher doses are necessary. It is also possible to administer the drug one to several times a day or at intervals of one to several days. The dose as a remedy for benign tumors or for diseases caused by viruses is preferably determined taking into account the type and degree of the disease,

  the route of administration, the patient's condition, etc. and in general, the dose is usually 20 to 100% of that administered for the above malignant tumors.

  Physiological activity of the nucleic acid derivatives of the present invention:

  The physiological activity of the nucleic acid derivatives of the present invention is illustrated below.

  (1) Interferon inducer activity: With regard to the sulfur poly C-poly I derivative which is one of the nucleic acid derivatives of the present invention, its action as an interferon inducer has been determined by a method for determining antiviral activity. The sulfur poly C-poly I derivative used as sample I for the test is a derivative substituted by SH while sample II is a derivative substituted by S-S. Both have an n value of 13 and radical numbers from 50 to 2,000. Lymphocytes (106 cells per ml) obtained from human peripheral blood were treated for 2 hours with the sample (10 micrograms / ml ) in a culture liquid (RPMI 1640; 20% FCS). The reacted culture liquid is removed; the cells are again suspended in a fresh culture liquid (RPMI 1640;% of FCS), incubated for 20 hours and the supernatant liquid is subjected to an ordinary method of measuring the antiviral activity of the interferon using the Sindbis virus and the FL cell (see Rinsho Kensa, 28, 1726, 1984). The result is given in the following table. The titer of 13. interferon is measured by a dilution method using the CPEI50 method (Inhibition of the cytopathogenic effect). Sample I is an S-S substituted nucleic acid derivative containing 4-thiouridine at a rate of n = 20

  S while sample II is its derivative substituted by SH.

  As a control sample, a conventional poly I-poly C was used.

  Concentrations (micrograms / ml) 100 o 200 5o00 Test sample I 100> 6400> 6400> 6400 Test sample II 100> 6400> 6400> 6400 Sample control 100 800 1600> 6400

  (Interferon titer: units / ml).

  It is found that the nucleic acid derivative of the present invention has a strong action as an inducer of interferon. Among the sulfur-containing poly C-poly I nucleic acid derivatives of the present invention obtained in the above example, that having 20 for the value of n and from 50 to 300 radicals for the molecular size, and another having 20 for the n value of 200 to 2000 radicals for molecular size were taken as test samples and subjected to the same physiological activity tests and gave the same results. The same result was also obtained with the

  derivative of nucleic acids (n value of 20 and molecular size not less than 20,000 radicals) before the reaction to reduce molecular dimensions.

  (2) Ability to induce TNF (necrosis factor

tumors).

  Macrophages of germinal vesicles from rabbits pre-treated with BCG 10 to 14 days previously were removed, adjusted to 2 × 10 per milliliter in RPMI 1640 medium supplemented with 10% FCS, one milliliter thereof was placed. on a plastic Petri dish and cultivated 14.

  in a carbon dioxide incubator (5% C02) in the presence or absence of the sample at 37 C.

  The cell culture supernatant after 2 or 8 hours was subjected to the TNF activity test. The results are given below. The TNF activity values were determined by measuring the cell inhibition activity for the LM cell after 72 hours using the dye binding method, and the 50% cell inhibition dilution. has been given as the title. The fact that this cell inhibition was due to TNF was confirmed, using a monoclonal antibody for the

  TNF rabbit, by its neutralization of activity.

  i TNF Neutralisa activity (2 h) i (8 h), tion by 1 anti-TNF antibody

Witness -

LPS 32> 64 I +

  Test sample I 32> 64 + Test sample II 32> 64 I + Control sample 32> 64 + The 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 in (1). It is noted that the TNF induction activity of the derivative

  of the nucleic acid of the present invention is high.

  (3) TNF-inducing activity in vivo Meth A tumors (2 x 105) were transplanted behind the skin of the abdomen of BALB / c mice (6 to 8 weeks old, males); the drugs were administered on the 2nd and 12th days after the transplant; blood was taken one hour after the second administration, and the activity of TNF in the serum was measured. The phenomenon of necrosis on the cancerous part which subsequently appeared was also observed. The results are given 15. below. The values in the table correspond to the same

title in (2) below.

  Treatments Necrosis activity (1st) (2nd) TNF in the (1st) (2nd) dried, BCG LPS 192 serum Observed BCG Test sample I 48 Observed BCG Test sample II 50 Observed BCG Control sample 24 Observed Sample Sample test I test I 12 Observed Sample Test sample II test II 24 Observed

15. ... _ __ _ _ _ __ _ _ _ __ _ _

  The test and control samples were the same as in (1) above. It is observed that the production capacity of TNF in vivo of the nucleic acid derivative of the present invention is high.

  (4) Interleukin 1 induction activity.

  Normal human peripheral blood supplemented with heparin was separated from mononuclear particles by a centrifugation method using a Ficoll-Hypaque apparatus (Farmacia, Ficoll-paque, registered trademark), the numbers were adjusted to 5 × 10 6 / ml in a RPMI medium supplemented with 10% FCS, placed in a plastic petri dish, incubated at 37 ° C. for one hour, and the

  cohesive cells were used as the source of interleukin 1 production.

  Drug was added to the 5 x 10 isolated particles / ml, incubated at 37 ° C for 24 hours, and the activity of interleukin 1 in the supernatant was measured using a fixation method. H-thymidine in PHA-stimulated mouse thymus cells using the thymus cell proliferation capacity as a target. The result is given in the following table. The control was physiological saline. The concentration of interleukin was 1.25 units / ml and that of test samples I and II and the control sample was 100 grams / ml. Samples I and II and the control sample used were the same as those used in (1) above. Samples used Amount incorporated (DPM (x104)) Control 3370 Test sample I 11384 Test sample II 11560 Control sample 8397 Interleukin 1 12517 It is found that the nucleic acid derivative of the present invention exhibits a strong activity of induction

of interleukin 1.

  (5) Interleukin 2 induction activity.

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

  At 5 x 10 6 lymph particles / ml, the drug was added, the mixture was incubated at 370C for 2448 hours, and the activity of interleukin 2 in the supernatant was determined in an incorporation method 25 of H-tymidine using CTLL-2 or NK-7 which is a proliferative cell strain dependent on interleukin 2 using the proliferation capacity as a target. The result is given in the following table. The control is a physiological saline solution. Interleukin 2 was at a concentration of 5 units / ml and the test samples I, II and the control sample were at the same concentration of 100 grams / ml. The test and control samples used

were the same as in (1).

2,596,762 17.

  Samples Amount incorporated (DPM (x103)) Control 230 Test sample I 3247 Test sample II 3320 Control sample 653 Interleukin 2 3778 It is found that the nucleic acid derivative of the present invention exhibits a strong induction activity 10

interleukin 2.

  (6) Activation of macrophages Samples were administered to Balb / c mice (7-10 weeks old, male) intraperitoneally; cells exuding from the peritoneum 3-5 days after administration have been collected; cells adhering to the plastic (mainly macrophages) were separated as effector cells and activation of macrophages was studied by a 3H-thymidine isolation method using Meth-A tumor cells as target (the ratio E / T being 15-20: 1). The results are given in the following table. The control consisted of 0.2 ml of physiological saline solution. 50 micrograms / mouse of test sample III or sample was administered

  witness, respectively. The test and control samples used were the same as in (1).

  The percentage of cytotoxicity was calculated by the formula: (experimental value) - (background) x 100 (100% 3H release) - (background) 18. Cytotoxicity samples% Control 0.5 Test sample I 10, 3 Test sample II 10.8 Control sample 5.7 It is found that the nucleic acid derivative of

  the present invention exhibits strong activation of macrophages.

  (7) Activation of NK cells Activation of NK cells in human peripheral blood was determined by measuring the inhibition activity using K562 as a target cell by a method of isolating 51Cr. The results are given below. The control used was physiological saline. The test and control samples were the same as in (1) above.

25 30 35

  Samples (micrograms / ml) Fusion% (% of 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 found that the nucleic acid derivative of

  the present invention presents an activation of an NK cell.

  (8) Activity inhibiting the proliferation of

tumor cells.

  The inhibitory action of the proliferation of tumor cell 19. cells was measured. Cells (3 x 104 / ml) were treated for 48 hours in a culture liquid containing 10% FCS at the same time as samples (10 micrograms / ml and 100 micrograms / ml), then incubated for 48 hours with tritium-labeled thymidine. The percentage inhibition was given by the amount of thymidine incorporated into a control which was not treated by the sample. Results are given

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

  Inhibition Cells% Sample Sample Control samples i test I test. II 1100 10 100 10 microram ms / ml iNAMALWA 65.6 55.7 61.2 46.4 56.9 37.4 RAJl 68.3 64.6 67.8 61.5 74.7 69.4

L929 36.9 35.7 36.8 34.7 32.7 20.5

RAMOS 40.8 42.2 45.5 40.5 40.1 52.4

  It is found that the nucleic acid derivative of the present invention has an inhibitory activity for

  proliferation of tumor cells.

  (9) Action inhibiting the proliferation of a

  tumor in mice carrying cancers.

  The inhibitory activity of tumor proliferation was measured by the following method in mice carrying Meth-A cancer which is the same type of transplanted cancer. The Meth-A cells (3 × 10 / 0.1 ml) were suspended in physiological saline solution, injected hypodermically into Balb / c mice (5 weeks old, male) and after 2 days, we started the administration

  of the drug three times a week for two weeks.

  On the second day after final administration, the cells

  tumors were extracted and their weight was determined.

  The results are given below. The test and control samples were the same as in (1) above.

  20. Samples Average Inhibited Number (micrograms / mouse) of standard deviation% ris Test sample I (10) 7 2.09 + 0.15 3

(30) 7 1.52 + 0.28 30

(100) 5 0.96 - 0.43 36

  Test sample II (10) 7 2.10 0.27

(30) 7 1.62 + 0.25

(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

15 20 25

  It is found that the derivative 'nucleic acids of

  the present invention inhibits the proliferation of tumor cells in cancer mice.

  (10) Proliferation inhibition action

  in shaved mice carrying cancer from human tumor cells.

  2.5 x 106 HeLa S strain cells from human cervical cancer are transplanted and

  2.5 x 106 cells of the Hep-2 strain originating from a cancer of the throat under the skin of the abdomen and, from the 7th to the 10th day after the transplantation, the living cohesion of the tumor is confirmed. Then, twice a week for 4 weeks, 100 Ig / mouse of the test sample (intravenously) and 25 mg / kg of 5-FU (intraperitoneal) are administered. The tumor is extracted four weeks after administration and weighed. The results are given in the following table. The test sample used is the same as in (1) above.

  21. 10 (HeLa S) Sample Weight (g) + standard deviation Inhibition rate (%) + Control 3.85 + 0.23 f + Test sample I 1.57 - 0, 19 59 Test sample II 1.55 + 0.20 60

-FU 2.06 - 0.35 47

  (Hep-2) Sample Weight (g) - standard deviation Inhibition rate (%) Control 0, 88+ 0.07 -Test sample I 0.38 - 0.07 57 Test sample II 0, 35 + 0.05 60

-FU 0.56 - 0.12 36

  It is found that the nucleic acid derivative of the present invention has a strong inhibiting action

of proliferation.

  (II) Inhibitory action against metastasis of

tumor cells in the lung.

  C57BL / 6 mice (males, 5 weeks old) 205 are transplanted intravenously 2 × 10 B16F10 cells

  which are a transplantable melanoma of the same type and we count the number of nodes that swarmed towards the lung (number of colonies) the second week after transplantation.

  The sample is administered intravenously 24 hours before transplantation of B16F10 melanoma. There were 9 cases. The results are given below. The test and control samples used are the same as in (1) above 22. Sample (micrograms / sou- Number of nodes that have risen) swarmed towards the lung + standard deviation Control 112 - 17 Test sample I (1) 33 + 61

(10) 21 10M

(100) 4 -2

  Test sample II (1) 21 + 6K (10) 15 - m

(100) 4 - 1

+ Control sample (1) 36 + 16K

) 3 0.7

(100) 7 - 3

  The nucleic acid derivative of the present invention is found to have an inhibitory action against metastasis from tumor cells to the lung.

  Safety of the Nucleic Acid Derivatives of the Present Invention The safety of the nucleic acid derivative of the present invention will be illustrated below. The poly C-poly I sulfur derivative, which is one of the nucleic acid derivatives of the present invention, is subjected to an examination of its cytotoxic effect for the main cells of the bone marrow. The sulfurized poly C-poly I derivative used has a value

  n of 20 and a molecular size of 150 to 2 OO0 radicals.

  The sample is injected intravenously into mice (8 weeks old; males, each group includes mice) at a dose of 100 micrograms / mouse and, after 24 hours, bone marrow cells from those are collected. -this. The cells are fixed and stained with Giemsa. 30 Smear cells are examined under a microscope and the

  degree of appearance of reticulocytes is counted as a percentage.

  A known poly I-poly C is used as a control. Physiological saline is administered to the witness. The results

  are given in the following table.

  23. Erythrocytes (%) Control 40 Test sample I 38 Test sample II 39 Control sample 11 It is found that the derivative of nucleic acids

  the present invention has high security.

  We will now study the pyrogenic effect of the nucleic acid derivatives of the present invention.

  Injection of poly I-poly C in vivo is known to be pyrogenic. The pyrogen test on rabbits has shown that poly I-poly C causes on average an increase in body temperature of 1.45 C. On the contrary, the nucleic acid derivatives of the present invention (both the SH substances than the SS substances in the above cytotoxicity experiment) show only an average increase in body temperature of only 0.25 C, and the result of the pyrogenic test cited is negative. In these experiments, three rabbits were used per group, and a solution of the sample (0.2 microgram / kg) in 10 ml of physiological saline was injected intravenously behind the ears of rabbits and the temperature body 4 hours after injection

has been measured.

  It is found that the nucleic acid derivatives of

  the present invention have very high security.

  The results of the acute toxicity test of the nucleic acid derivatives of the present invention are as follows. With respect 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 administered drug and the LD50 is not less than 394 mg / kg.

  The effect was appreciated by the fact that the mouse was dead or alive one week after the intravenous injection. The same assay using other mice (C57BL6 and Balb / c) 24. also gave the result that the nucleic acid derivative (the same as above) of the present invention exhibits lower toxicity than the poly I-poly C. It is found that the security of the nucleic acid derivatives of the present invention is very high. LD50 Sou- Route of administration- 5 Sou- Route of administration- Sample Sample Sample test che tration I trial II intravenous dY control> 394 mg / kg> 394 mg / kg 132 mg / kg

10 ....

  (Working example) The present invention will be further illustrated by means of examples of the process for the preparation of acid derivatives

  nucleic acids of the present invention, these examples not being limiting.

EXAMPLES

  (1) Synthesis of sulfur-containing polycytidylic acid Poly C (0.5 g) is dissolved in a mixed solvent of 4 ml of water and 2 ml of pyridine; the solution is placed in a 30 ml stainless steel reaction tube with 5 ml of liquid hydrogen sulfide and reacted at 37 ° C for 6 hours. The pressure in the sealed tube during

  the reaction is 20 to 22 kg / cm2.

  After the reaction, the reaction tube is cooled to 25 ° C or less to reduce the pressure sufficiently and the reaction tube is opened. An excess of unreacted hydrogen sulfide is removed; the sulfur poly C solution is passed through a 50 ml round bottom flask and the unreacted hydrogen sulfide is removed in vacuo. The solution obtained is dialyzed three times against Tris buffer (pH 7.5) containing

  liters of 50 mM sodium chloride, and the transparent liquid obtained is still lyophilized to give 0.47 g of white fibrous substance.

  The ultraviolet absorption spectra of the substance obtained were measured in a neutral aqueous solution, and the results were obtained in FIG. 1. It is known that the maximum absorption wavelength of the acid 25. cytidylic is 271 nm and the absorption wavelength of 4-thiouridylic acid in which an -NH2 group in position 4 of the pyrimidine ring of cytidylic acid is

  substituted by a group -SH is 330 nm.

  From the peak height ratio in Figure 1, it has been confirmed that there is a 4-thiouridylic acid

for 13 cytidylic acid.

  In the same way, the reaction temperatures and the reaction time of the stainless steel tube were varied and polycytidylic acids were obtained having the degrees of sulfurization indicated in the following table. The degree of sulfurization is indicated here by "n", this "n" denoting

  the numbers of cytidylic acid for a 4-thiouridylic acid.

  Reaction temperature Reaction time n C 12 hours 1

6 6 1

37 6 13 + 2

37 4 26 2

37 2.5 39 - 2

* (2) Preparation of double stranded nucleic acid derivatives: Poly sulfur and poly C sulfur obtained in (1) above are dissolved in the same molar quantities, in a Tris buffer containing 50 mM of chloride sodium to obtain a concentration of 10 to 20 mg / ml, and in a water bath the temperature is gradually raised from room temperature to 68 C. The mixture is kept at 68 C for approximately 10 minutes; let it sit until it 30

  return to room temperature and store at 4 C.

  The solution obtained is lyophilized, which gives 1.2 g of nucleic acid derivative having SH groups under

the shape of a white solid.

  Then add thereto about 10 times its volume (in molar ratio) of a 1N iodine solution (mixture

  a third of a mole of iodine and 2/3 of a mole of sodium iodide).

  26. The mixture is vigorously stirred to homogenize it and it is left to stand at 0 ° C. for 1 hour. The reaction solution is carefully dialyzed against water until

  let the yellow color of the iodine disappear.

  The solution thus obtained is lyophilized, which gives 0.98 g of a white solid. The fact that the solid obtained contains an S-S bond is confirmed in the following manner. 0.1 g of this solid is dissolved in 10 ml of Tris buffer (pH 7.5) containing 0.03 M sodium sulfite, stored at room temperature for 0 to 7.5 hours and the spectra are examined each time. UV absorption. The results are given in FIG. 2. It is known that the S-S bonds are generally reversible and that the S-S bond cuts by reduction to give an S-H group. Over time, the shoulder at 310 nm which is an absorption wavelength of the SS bond decreases while an absorption at 330 nm, due to the substance -SH (4-thiouridine) increases and, at the end 7.5 hours, the degree of absorption at 330 nm becomes identical to that of the sulfur-containing poly C 20 before oxidation. It is therefore clear that the S-S link

  was quantitatively reduced in group -SH.

  (3) Crosslinked structure and biological activity Among the nucleic acid derivatives into which the sulfur atoms are introduced, the methods of synthesis and the physiological activities of test sample I (of which all the SH groups are of the type reduced) and test sample III (of which all the SS groups are of the oxidized type) were described as above. The crosslinking substances of the nucleic acid derivatives described herein are the compounds in which part of the sulfur atoms introduced into the same molecule exhibit a disulfide bond between the molecules or in the molecules. For example, crosslinking derivatives having 80% of SH groups and 20% of S-S groups in the same molecule can form a multistrand crosslinking structure in 20% of the whole. In other words, the poly C having 27. 1000 bp for example, has a cross-linking structure in ten parts. Any nucleic acid derivative having various numbers

  crosslinking can easily be prepared by partial oxidation of the substance SH under moderate conditions or by partial reduction of the substance S-S under moderate conditions.

  The synthesis conditions are the same as those described

in the examples.

  The ratio of SH group numbers to SS group numbers, i.e. the degree of crosslinking, can be calculated as a ratio of the values obtained by dividing the ultraviolet absorbance at 310 nm from the SS group and 330 nm from the SH group, respectively, by the molecular absorption coefficients. Another solution is as follows: the nucleic acid derivatives are RNA-ase (for example, ribonuclease P1), the decomposed product is subjected to high performance liquid chromatography (HPLC) of the reverse phase system, and the 4-thiouridine (or 4-thiouridylic acid) and a substance

  bis formed by a disulfide bond thereof are separated and their amounts are determined.

  With regard to physiological activities, it has been found that nucleic acid derivatives having any degree of crosslinking from 1 (substance totally in SS form; for example, test sample II) to 0 (substance totally in SH form; for example, test sample I)

  have the same activity and the same safety as the test samples I and II mentioned above.

  This fact suggests that even if these nucleic acid derivatives may be partially oxidizable or reducible, they may exhibit the same stable physiological activity

than the initial derivatives.

  (4) Transformation into derivatives of low molecular mass The nucleic acid derivative containing SH (1 g) obtained in (2) above is dissolved in 120 ml of water and 30 ml of chloride solution are added. 5M sodium and 150 ml of 28. formaldehyde. The mixture is stirred vigorously to obtain a uniform solution. The solution is heated at 80 ° C. for 8 hours; carefully dialyzed against water, and

  it is lyophilized, which gives 0.95 g of white solid.

  When the nucleic acid derivative containing SS bonds obtained above is subjected to a transformation into a low molecular weight derivative as above,

gets the same result.

  This derivative is subjected to a high performance liquid chromatography 10 by gel filtration and the diagram obtained is given in FIG. 3. The conditions are as follows: TSK G4000SW gel (0.65 x 60 cm), eluate: Tris HC1 buffer 50 mM

  (pH 7.5) containing 0.5M sodium chloride.

  Each of the arrows in the figure shows the elution position of each standard size marker (the unit of this size marker is bp), and it appears from Figure 3 that it has a maximum distribution of about 500 radicals and that it has a molecular size that distributes 150 to 1000 radicals. The result agrees well with the value obtained from an electrophoresis using a polyacrylamide gel or an agarose gel. As has already been said, the molecular size is indicated in this specification by the expression "number of radicals" and in FIG. 3 the "number of radicals" is in agreement

with the "pb" as units.

  When varying the reaction time and the

  reaction temperature of this reaction for transformation into compounds of molecular weight, the substances having the following molecular dimensions are obtained in the same way.

  29. Distribution Time Temperature Distribution (bp) reaction max reaction (bp) - O h - 2,000

 C 24 30 10 - 55

 C 16 200 50 - 300

 C 8 500 150 -1000

 C 8 1000 200 -5000

  , ..... DTD: Among the nucleic acid derivatives obtained in accordance with the above example, those with

  numbers of radicals from 200 to 5000 and their melting curves have been determined.

  Sample I or II was heated (0.70D / ml) in 10 mM Tris buffer (pH 7.5) containing 0.1 M sodium chloride, raising the temperature by 2 C / 4 minutes by 20 C at 90 C. The ultraviolet absorbance was measured at each temperature at the wavelength of 260 nm, and an increase in ultraviolet absorbance by hyperchromicity was represented by relative ratio giving the state at 20 C as a base. . A Beckman DU-8B spectrophotometer was used for the measurement. The results are given in FIG. 4. The fusion of the nucleic acid derivatives was progressively observed from 35 to 55 ° C., and around 59 ° C., an abrupt fusion curve was obtained. At more than 70 ° C., the curve almost passes through a plateau and this means the disappearance of large structures and the formation of spiral structures 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 calculations based on the rate of increase / decrease of the ultraviolet absorbance at 260 nm between 90 C and 25 C, it was confirmed that hyperchromicity (color darkening effect) for samples I and He was, respectively,

  43.5% and 42.4%. This indicates that the nucleic acid derivatives have very stable structures under physiological conditions.

30.

  FIG. 1 represents ultraviolet absorption spectra of a sulfur polycytidylic acid obtained in (1) of the example. The ordinates and the abscissas are respectively the absorption and the wavelength (nm).

  FIG. 2 represents the ultraviolet absorption spectrum of the nucleic acid derivative obtained in (2) of the example. It is a UV spectrum of reduction by sodium thiosulfate. The ordinates and the abscissas represent the absorbance and the wavelength (nm) respectively. FIG. 3 represents the elution diagrams obtained by high performance liquid chromatography of a nucleic acid derivative of the present invention obtained in (3) of the example. The abscissa and the ordinate represent the elution time (in minutes) and the eluted quantity respectively. 15 Each of the arrows indicates the elution position of the marker

of dimensions.

  FIG. 4 represents the fusion curve of the nucleic acid derivative of the present invention in which the distribution of molecular sizes is from 50 to 2000 radicals. 20 The abscissa and the ordinate represent respectively the

  temperature (0C) and relative absorption.

  The present invention is not limited to the exemplary embodiments which have just been described, it is on the contrary subject to modifications and variants which will appear to those skilled in the art.

31.

Claims (5)

CLAIMS i - Derivative of nucleic acids, characterized in that, in a polymer of nucleic acids, part of the purine or pyrimidine rings which are a constituent element thereof is substituted by one or more sulfur atoms or in that this or these substituted sulfur atoms form bridges by a disulfide bond, from which a better conformation results.   2 - A nucleic acid derivative according to claim 1, in which this nucleic acid polymer is a   single strand ribonucleotide polymer.   3 - A nucleic acid derivative according to claim 2, in which this single-stranded ribonucleotide polymer is a poly C containing certain proportions of 4-thiouri15 dylic acid containing sulfur atoms resulting from the substitution of an -NH2 group of the pyrimidine ring of cytidylic acid in poly C by a group -SH.   4 - A nucleic acid derivative according to claim 3, wherein the length of the polymer is from 50 to 5000 calculated in base numbers.   - Nucleic acid derivative according to claim 3, in which the number of sulfur atoms is one for 6 to 39 cytidylic acids present in poly C. 6 - Nucleic acid derivative according to claim 4, in which the number of sulfur atoms is one for 6 to 39 cytidylic acids present in poly C.   7 - A nucleic acid derivative according to claim 1, wherein the nucleic acid polymer is a double stranded ribonucleotide polymer.   8 - A nucleic acid derivative according to claim 7, wherein the double stranded ribonucleotide polymer is composed of poly I and poly C containing certain proportions of 4-thiouridylic acid containing sulfur atoms   by substituting an -NH2 group in the pyrimidine ring of the cytidyl ac35 in poly C with an NH group.   32. 9 - A nucleic acid derivative according to claim 8, in which the length of the polymer is from 50 to 10,000,   calculated in number of base pairs.   - A nucleic acid derivative according to claim 8, in which the number of sulfur atoms is one for 6 to 39 cytidylic acids present in poly C. 11 - A nucleic acid derivative according to claim 9, in which the number of sulfur atoms is one for 6 to 39 cytidylic acids present in poly C.