JPH09176038A - Antiinflammatory antisense medicine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the above medicine comprising the composite product of a synthetic amino acid with a specific antisense oligonucleotide and useful for the therapy of disease states such as inflammatory diseases such as chronic rheumatoid arthritis, septicemia shock and AIDS, etc. SOLUTION: This antiinflammatory antisense medicine comprises the composite product of (A) a synthetic polyamino acid or its derivative with (B) an antisense.oligonucleotide complementary to a part or the whole base sequence of a mRNA coding a physiologically active substance relating to human inflammatory diseases (e.g., human chronic rheumatoid arthritis). The component A is preferably a nucleic acid-bound compound comprising the sequence of repeated lysine and serine residues or a nucleic acid derivative. The derivative of the component A is preferably the polyethylene glycol-blocked product of the synthetic polyamino acid.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an anti-inflammatory antisense drug. More specifically, the present invention relates to rheumatoid arthritis, peridontitis, nephritis, ulcerative colitis, arteriosclerosis, inflammatory diseases such as psoriasis, septic shock, pathological conditions such as Crohn's disease and AIDS, and refractory liver diseases. The present invention relates to an antisense drug that specifically blocks gene expression of physiologically active substances involved in pathological conditions in liver transplantation.

[0002]

2. Description of the Related Art Conventionally, an antisense method has been known as a method for controlling the functional expression of chromosomal DNA. This antisense method uses DNA or RNA (antisense strand) having a complementary base sequence in part or in whole with mRNA (sense strand) transcribed from chromosomal DNA encoding specific protein synthesis information. , A method of blocking protein synthesis information from mRNA by utilizing the fact that the sense strand and the antisense strand bind to each other by complementarity. In addition, mainly from the viewpoint of stability,
In the examples so far, antisense DNA is often used as a binding sequence to the sense strand.

Currently, in order to block the function of mRNA, an antisense strand complementary to the entire sequence is not required, and a partial sequence that controls protein expression from mRNA may be used as a targeting site. It is believed to be effective. That is, such a targeting site may be a splicing site, a capping site, or an AUG initiation codon.
The vicinity of the n) site is often selected, and a relatively high antisense effect is obtained especially for the AUG initiation codon site. Considering the three-dimensional structure of mRNA, it is generally considered that antisense DNA is likely to bind to a single-stranded region such as a loop structure or a bulge structure.

By the way, the history of medicines derived from natural products has undergone a drastic change with the progress of science, and one of the trends in drug discovery toward the new century is to certainly target genes. Is flowing out. According to the progress of the "Human Genome Project" aimed at elucidating the entire base sequence of human genes, it is said that a complete genome sequence including all human genes will be determined around 2010. This enormous amount of information has spurred analysis research on diseases thought to involve gene deletion or abnormal expression,
It is believed that it will redefine the concept of treatment.
Against the background of such research, gene therapy and antisense therapy have come into the spotlight as a new century therapy (Cook, ST, Ann. Rev. Pharm. 32, 329-37.
6, 1992; Tidd, DM, Anticancer Res. 10, 1169-118.
2, 1990).

Due to problems such as bioethics, gene therapy is limited to single gene diseases, cancer, AIDS and the like. On the other hand, antisense DNA can be captured as a compound similar to conventional synthetic medicines, and its effects have been reported not only in vitro but also in vivo, and the search for its possibility is new. Entering the research stage.

The possibility that antisense DNA as a drug was proposed was preceded by a report in 1978 that synthetic antisense DNA was added from the outside to suppress transformation of Rous sarcoma virus (Zamecnik PC a
nd Stephenson ML, Proc.Natl. Acad. Sci. USA. 7
5, 280-284, 1978). It was suspected that DNA, a polyanion, would be taken up into cells, but after that,
It was reported one after another that the antisense DNA blocked the function of the endogenous gene in the cultured cell system, and the expectation as a drug has been increased. However, phosphodiester type DNA (hereinafter abbreviated as D-oligo) existing in nature has a fatal defect as a drug that is decomposed in a short time by nuclease or the like. For this reason,
Chemical modification of DNA has been attempted in order to improve biological stability. Among them, phosphorothioate type nucleotides (hereinafter abbreviated as S-oligo) are stable and have high biological activity. It is this type that is currently in clinical trials.

The condition of antisense DNA as a drug is that it has a base sequence specificity for mRNA,
Stable transport into cells, access to mRNA targeting site, formation of stable duplex, stable presence in cells for a certain period of time, toxicity, no side effects, metabolization to some extent,
It is economical and so on. Above all, it is an essential condition that the protein is transported into the cell in a stable form. DNA
Alternatively, RNA is quantitatively decomposed by nuclease (hereinafter abbreviated as DNase and RNase, etc.) and the half-life in blood is extremely short, within 1 minute. For stabilization, particularly, S-oligo in which one of the oxygen atoms of the phosphate group is substituted with S- and methylphosphonate in which a CH ₃ group is substituted are mainly used. In addition, modification of the sugar moiety, modification of the 3'and 5'ends, and modification of the 2'position are also considered (Goodchild. J. Bioconjug Chem. 1, 165-187, 199).
0). Recently, Peptide Nucleic Acid (PNA) has been synthesized in which phosphate bond is changed to peptide bond.
It was reported that it had stronger binding to DNA and RNA than antisense DNA and was resistant to nucleases (Hanvey, L. et al., Science 258, 1481-148).
5 (1992)), there are no reports of biological activity. It is reported that those designed to form a base pair at the 3'end to form a loop structure, dumbbell-shaped ones, and ring-closed ones are also stable.

[0008] However, these modified products have many problems such as the cost involved in their synthesis and the extremely difficult purification thereof, and it is difficult to apply them to pharmaceuticals at present. Further, as described above, S-oligos have been extensively used in the studies so far, but it is also a big problem that this type of oligonucleotide has low binding specificity to the sense strand. In this sense, antisense D
When NA is applied to medicine, D-oligo is necessarily considered to be the most suitable one.

Furthermore, low membrane permeability of nucleic acids such as DNA has been regarded as a problem from the beginning, and in consideration of the intracellular stability problem of the above-mentioned phosphodiethyl-type oligonucleotide, the key for future antisense drugs. It is no exaggeration to say that is the method of intracellular delivery (delivery method). As can be seen from successful cases in the culture system, it is certain that oligonucleotides such as DNA will be taken up into the cells, mainly by endocytosis, and about 80
The membrane protein of kDa is considered as a putative receptor. Modified oligonucleotides are also mainly incorporated by endocytosis, but many details are unknown,
The amount transferred into cells is still small. Delivery methods have been devised to enhance low membrane permeability, but there are still points to be solved including problems such as toxicity.

On the other hand, for the treatment of inflammatory diseases, steroids and non-steroidal anti-inflammatory agents have been widely used in recent years. Steroids remarkably improve various symptoms in various inflammatory diseases, but their effects gradually decrease with administration, and risk of inducing coronary artery insufficiency, peptic ulcer, cataract, sepsis, and easy infection as side effects. There are problems such as In addition, non-steroidal anti-inflammatory drugs temporarily suppress inflammatory symptoms, but they do not fundamentally treat inflammatory diseases. Therefore, it is very effective,
At present, there is a demand for the development of a therapeutic agent for inflammatory diseases, which has a long-lasting therapeutic effect and is highly safe.

[0011] In particular, rheumatoid arthritis is a chronic inflammatory disease of unknown cause, in which the synovial membrane is the main site of lesion. The lesion site sometimes does not stop at the synovium of the joint, and the inflammation that initially occurs in the synovium eventually causes the destruction of cartilage and bone, eventually leading to the destruction of joint tissue throughout the body. Empirical factors strongly influence conventional treatments for rheumatoid arthritis, and nonsteroidal anti-inflammatory analgesics have been used as the first-line drugs.
However, recently, the role of non-steroidal anti-inflammatory analgesics in the treatment of rheumatoid arthritis is diminishing. Although it can be expected to have an analgesic effect by its administration, it is becoming common knowledge that non-steroidal anti-inflammatory analgesics have no anti-rheumatic effect, and moreover, non-steroids represented by gastrointestinal disorders, renal function decline, etc. This is because it has become clear that the side effects of anti-inflammatory analgesics cannot be ignored clinically.

Under these circumstances, early use of new antirheumatic drugs such as methotrexate, mizoribine, and FK506 is being carried out. The following mechanisms and the like are known for various inflammatory diseases such as rheumatoid arthritis. That is, inflammation is a biological reaction for maintaining homeostasis against invasion of a respiratory substance. Although various cells are recruited to the inflammatory response, cytokines are one of the mediators between these cells and play an important role in the inflammatory response. Various cytokines are produced at the site of inflammation, and regulate the inflammatory reaction in the inflammatory lesion as well as in the whole body while forming a cytokine cascade. Among these cytokines, Tumor Necrosis Factor (hereinafter abbreviated as TNF) is a cytokine produced by cells such as macrophages. Originally found as a substance that damages tumors, this TNF is now being widely understood as a cytokine involved in the body defense reaction through inflammation. T
The existence of the NF gene has been clarified in a wide range of mammals such as humans, pigs, cows, rabbits and mice, and their primary structures have been determined. According to this, homology of about 80% is conserved in the amino acid sequences among the animals, suggesting that TNF is an extremely important physiologically active substance for the living body. Human TNF precursor is 23
It has 3 amino acid residues and is 155 or 15 in the mature form.
It is composed of 7 amino acids. Although its molecular weight is 17 kDa, it forms a 45 kDa trimer in vivo. It is believed that sugar chains are present in mouse TNF,
It does not exist in humans, and sugar chains are not essential for its activity expression in mouse TNF.

TNF is expressed by inoculating BCG-sensitized animals with Lipopolysaccharide (hereinafter abbreviated as LPS). Experimentally, the TNF production preparation state of macrophages can be produced by various methods, and an appropriate stimulus (for example, bacterial cells or LPS) can be generated.
Cell components such as TN, which peak around 2 hours
It can lead to the production of F. In addition, TNF production can be reproduced in an in vitro experimental system using human macrophage cells (for example, U937 strain).

The cells on which TNF acts are diverse. For example, TNF produced from macrophages acts on neutrophils and vascular endothelial cells, resulting in the progression of inflammation from the beginning. Then, the inflammation is terminated and repaired by acting on fibroblasts and hepatocytes. The inflammatory reaction progresses while many cells and mediators interact with each other and usually disappears at a constant rate.However, if there is a substance that should be an antigen and the amount is a certain amount or more, the information is Is handed over to the immune system. TNF is thus also involved in the transition from a non-specific biological defense reaction to a specific defense reaction. Besides these, TNF is also known to act on, for example, osteoblasts, osteoclasts, adipocytes, epithelial cells, pituitary gland, and especially synovial cells.

Further, as the etiology of inflammatory diseases such as rheumatoid arthritis, various abnormalities of immune response system-inflammatory reaction are mentioned. For example, TNF-α which is one of TNF is mentioned.
Besides, it is considered that oncogenes such as c-fos, human interleukin-1β (hereinafter abbreviated as IL-1β), and cytokines such as IL-6 are involved. Furthermore, in recent years, prostaglandins (hereinafter, abbreviated as PG) are one of the mediators of inflammatory diseases.
And the role of the synthase is attracting attention. That is, 1
Since it was reported by Vane et al. In 1997 that the mechanism of action of non-steroidal anti-inflammatory analgesics such as aspirin lies in the inhibition of PG synthesis, research on non-steroidal anti-inflammatory analgesics has been carried out by PG.
It has been pursued in relation to synthetic inhibition. However, in recent years, there are two types of cyclooxygenase (hereinafter abbreviated as COX) that biosynthesize PG, and the COXs that have been studied so far are mainly physiologically existing COXs. It was found that COX-2, which is an enzyme called -1, is separately produced, and which is produced in free cells by various stimuli, cytokines, etc. and is greatly related to inflammation and tissue inhibition (Van
e, J., Nature, 367, 215-216, 1994: Xie, W., Robert
son, D., and Simmons, D., Drug Devel. Res., 25, 249
-265, 1992). The gene of this COX-2 is COX
Unlike -1, it is an enzyme newly produced by stimulation of IL-1. For example, according to the report of Lee et al., COX-1 in macrophages is invariable by LPS stimulation and is detected at a constant concentration regardless of stimulation or inflammation, but COX-2 is normal in its gene. Is hardly demonstrated, and it is increased by stimulation with LPS, and its expression is completely suppressed by dexamethasone. In recent years, human COX-2 cDNA has been cloned, whereby the regularity of COX-2 production in inflammatory cells is being elucidated.

[0016]

Due to recent advances in molecular biology, immunology, etc., analysis of the etiology and pathophysiology of various inflammatory diseases including rheumatoid arthritis is rapidly progressing as described above. , Anti-cytokine therapy, anti-adhesion molecule therapy, monoclonal antibody therapy, oral peptide therapy, T cell vaccination, etc. based on these findings,
New therapeutics with more theoretical backing are being developed.

On the other hand, as a human disease targeted by the antisense method, a viral disease is mentioned at the top of the list because the relationship between its cause and the target gene is clear, and early antisense therapy , Cytomegalovirus, herpes virus, human immunodeficiency virus, papilloma virus, vesicular stomatitis virus (VS
V) is effective against infectious diseases. Also,
Antisense therapy targeting the translocated bcr-abl gene, which is the cause of chronic myelogenous leukemia, or ICAM-1, which is one of the adhesion molecules that influence inflammation or cancer metastasis, is also effective. .

However, in general, the maximum effect can be expected when there is a 1: 1 correspondence between the etiology and the gene, due to the characteristics of the antisense method, rather than the presence of a plurality of genes that cause the disease. Therefore, in order to apply the antisense method to the treatment of inflammatory diseases, it is necessary to grasp the disease state accurately and
Identification of the causative gene is essential. Furthermore, it is also necessary to specify the expression mechanism of the gene and select an appropriate site for blocking the expression.

The present invention has been made in view of the above circumstances, and is an inflammatory disease such as rheumatoid arthritis, periodontitis, nephritis, ulcerative colitis, arteriosclerosis, psoriasis, and septic shock. , A novel anti-inflammatory drug whose main component is antisense DNA that specifically blocks the expression of physiologically active substances involved in Crohn's disease, AIDS, and the like.

[0020]

As a first invention for solving the above problems, the present invention provides a synthetic polyamino acid or a derivative thereof and an mRNA encoding a physiologically active substance involved in human inflammatory disease. Provided is an anti-inflammatory antisense drug, which comprises a complex with an antisense oligonucleotide complementary to a part or the whole base sequence.

In the anti-inflammatory antisense drug of the first invention, the synthetic polyamino acid is a nucleic acid conjugate comprising a repeating sequence of a lysine residue and a serine residue, and its derivative is A preferred embodiment is a modified block of a synthetic polyamino acid polyethylene glycol (hereinafter abbreviated as PEG). The present invention also provides, as a second invention, a complex of a synthetic polyamino acid or a derivative thereof and an antisense oligonucleotide complementary to a partial or entire base sequence of mRNA encoding human IL-1β. An anti-inflammatory antisense drug characterized by: In this second invention, an antisense oligonucleotide complementary to a part or the whole base sequence of the above-mentioned mRNA encoding IL-1β is an oligo having the part or the whole base sequence of SEQ ID NO: 2 or 4. A preferred embodiment is a nucleotide.

As a third invention, a synthetic polyamino acid or a derivative thereof and an mRN encoding human TNF.
Antisense complementary to part or all of the base sequence of A
Provided is an anti-inflammatory antisense drug, which comprises a complex with an oligonucleotide. In this third invention, the human TNF is human TNF-α,
An antisense oligonucleotide complementary to a part or the whole base sequence of mRNA encoding this human TNF-α has a part or the whole base sequence of any one of SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10. The preferred embodiment is an oligonucleotide.

Furthermore, as a fourth invention, a complex of a synthetic polyamino acid or a derivative thereof and an antisense oligonucleotide complementary to a partial or entire base sequence of mRNA encoding a series of PGE ₂ synthases. An anti-inflammatory antisense drug comprising: In the fourth invention, the PGE ₂ synthase is COX-2, and m encoding the COX-2.
A preferred embodiment is that the antisense oligonucleotide complementary to a part or the whole base sequence of RNA is an oligonucleotide having a part or the whole base sequence of SEQ ID NO: 12.

A nucleic acid complex of a synthetic polyamino acid is composed of irregular or regular repeating water-soluble amino acid serine residue and cationic amino acid lysine residue. The constituent molar ratio of serine residue and lysine residue is about 1: 1,
Its molecular weight is about 3000-50000. Such a polyamino acid is, for example, poly-lysine: serine (hereinafter, PLS) which forms a complex with an oligonucleotide in a homogeneous system.
Abbreviated as: Patent WO95 / 09009). In addition, as the derivative of the synthetic polyamino acid, in particular, a modified PEG block of PLS (hereinafter,
(Abbreviated as PLSP) can be exemplified as a novel one, and this PLSP can be produced by, for example, the method of Example 1 below. PLS and PL
The structure of SP can be illustrated, for example, as the chemical formulas of FIGS.

Further, an antisense oligonucleotide complementary to an mRNA encoding a physiologically active substance involved in inflammatory diseases, or a modified oligonucleotide with a synthetic polyamino acid such as PLS or its derivative (eg, PLSP) is known. Method (Rajendra, BR et a
l., Human Genetics, 55, 3633, 1980, Lim, F. and Su
n, A., M., Science, 210, 908, 1980).

[0026]

Embodiments of the present invention will be described below in detail with reference to examples, but the present invention is not limited to the following examples. Reference Example <Synthesis and Purification of Oligonucleotides> The oligonucleotides of SEQ ID NOs: 1 to 15 in the sequence listing were
A synthesizer (Type 380B manufactured by Applied Biosystems)
Was synthesized using Phosphoramidite method using t-butyldimethylsilyl group as a protecting group for 2'-hydroxyl group (Nucl
eic Acid Res., vol.17, 7059-7071, 1989), and the oligonucleotides were purified by the literature (Nucleic Acid Res.
Res., Vol.19, 5125-5130, 1991).

SEQ ID NO: 1 is a known oligonucleotide consisting of 20 bases, which is a sense DNA chain corresponding to 20 bases including the initiation codon of IL-1β gene which is one of the physiologically active substances of rheumatoid arthritis. , SEQ ID NO: 2 is an antisense DNA complementary thereto
Is a chain. Further, SEQ ID NO: 3 is a sense DNA strand corresponding to 20 bases containing the untranslated region of the same IL-1β gene, and SEQ ID NO: 4 is an antisense D complementary thereto.
It is an NA chain (JP-A-6-41185).

Further, SEQ ID NO: 5 is a sense DNA strand corresponding to 20 bases including the initiation codon of TNF-α gene which is also one of the physiologically active substances of rheumatoid arthritis, and SEQ ID NO: 6 is Complementary antisense DNA strand, SEQ ID NO: 7 is a sense DNA strand corresponding to 20 bases containing the splicing site of the TNF-α gene (1624-1643 position of TNF-α gene sequence), and SEQ ID NO: 8 is Antisense DNA complementary to this
The strand, SEQ ID NO: 9 has 20 bases containing the splicing site of the TNF-α gene (2161-
2180th), and SEQ ID NO: 10 is an antisense DNA strand complementary thereto.

Further, SEQ ID NO: 11 is a sense DNA strand corresponding to a 20-base region containing the initiation codon of COX-2 involved in the immune mechanism of inflammation such as human rheumatoid arthritis, and SEQ ID NO: 12 is Is an antisense DNA strand complementary to. Furthermore, SEQ ID NO: 1
3 is a sense strand corresponding to 20 bases including the initiation codon of mouse IL-1β gene, and SEQ ID NO: 14 is an antisense DNA strand complementary thereto. Further, SEQ ID NO: 15 is an antisense strand corresponding to 20 bases including the initiation codon of mouse TNF-α gene. Example 1 <Synthesis and Purification of PEG-Modified PLS> 1.0 g of ε-carbobenzoxylidine-N-carboxylic acid anhydride (manufactured by Sigma) and 1.0 g of benzylserine-N-carboxylic acid anhydride (manufactured by Sigma) (Manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 30 ml of N, N-dimethylformamide (DMF: manufactured by Wako Pure Chemical Industries Ltd.), and 15 ml of chloroform (manufactured by Wako Pure Chemical Industries Ltd.) was added. One end methoxy One end amino group of polyethylene oxide (molecular weight 5000: manufactured by NOF CORPORATION) 4.0 g was dissolved in chloroform 15 ml, and the solution was mixed with ε-carbobenzoxylidine-N-carboxylic acid anhydride and benzylserine-N. -Added to the carboxylic anhydride solution. After 26 hours, the reaction mixture was added dropwise to 330 ml of diethyl ether, and the precipitated polymer was collected by filtration, washed with diethyl ether (manufactured by Wako Pure Chemical Industries, Ltd.), and then dried in vacuum to obtain a hydrobromic acetic acid solution (wa Deprotection was carried out with Kojun Pure Chemicals Co., Ltd. and poly-lysine: serine PEG block copolymer (PLS
P) was obtained. Example 2 <Preparation of antisense drug against IL-1β> Poly-
A copolymer of lysine: serine (PLS: manufactured by Sigma) or PLSP synthesized / purified in Example 1 and an antisense oligonucleotide (SEQ ID NO: 2) prepared in Reference Example
And either one of 4) and 4) was formed into an ionic complex by a known method.

That is, an antisense tube is placed in an ultrafiltration tube (manufactured by Japan Millipore Limited: ultrafree C3-GC UFC3 TGC 00 filter centrifuge tube) having a membrane fractionation capability so that the formed complex does not permeate. A mixed solution of the oligonucleotide and PLS or PLSP was added and centrifuged. The concentration of oligonucleotide is 1
It was added to be nM, 10 nM and 100 nM. In addition, the oligonucleotide of SEQ ID NO: 4 is 10 μm
M concentration was also prepared. Under the above conditions, the liberated oligonucleotide was filtered to the lower layer, and PLS and antisense-oligonucleotide complex (hereinafter abbreviated as antisense-PLS) and PLSP and antisense.
Oligonucleotide complex (hereinafter Antisense-PL
Abbreviated as SP).

The formation of turbidity or precipitate due to the formation of these complexes was examined spectroscopically by measuring the absorbance of the solution at 360 nm. During the experiment, the complex was mainly dissolved in a phosphate buffer (pH = 7.2) having an ionic strength of 0.02. As a result, the antisense oligonucleotide used this time and PLS or PLS
When forming a complex with P, no cloudiness was observed in the solution system.

The concentration of the oligonucleotide was determined from the absorbance at 260 nm. As a result, it was confirmed that the oligonucleotide in the formed complex had each of the above concentrations. The concentration of each charge neutralization in the formation of the ionic complex was calculated from the number of charges obtained from the molecular weight of PLS or PLSP and the oligonucleotide. In addition, antisense-PLS and antisense-PL
Capillary electrophoresis (multi-channel capillary electrophoresis device: CA)
PI-3000, MCPD-3600 SPECTRO MULTI CHANNEL DETECTOR
Equipment, manufactured by Otsuka Electronics Co., Ltd.). As a result, the peak of the complex was the same as the peak of uncharged phenylalanine. Example 3 <Effect of anti-inflammatory antisense drug on cultured cells>
The inhibitory effect on IL-1β production of the antisense-PLS and antisense-PLSP complexes prepared in Example 2 was examined in a cultured cell line.

For cells, human macrophage system U937
Cells (Dainippon Pharmaceutical Co., Ltd.) were used. Cell culture is 10%
FCS (Fetal Calf Serum: Sanko Junyaku Co., Ltd.) and 100
unit / ml penicillin (Life Technology)
Then, using RPMI medium (manufactured by Nikken Biomedical Research Institute) containing 100 ug / ml streptomycin (manufactured by Life Technology Co., Ltd.), it was carried out at 37 ° C. under 5% CO ₂ . The number of cultured U937 cells was determined by visually counting after staining with trypan blue.

At the time of measurement, 0.15 ml of medium containing U937 cells adjusted to 3 × 10 ⁵ cells / ml was added to a 96-well microplate. For these cells, 1 ng / ml of 12-o-tetradecanoyl-phorbol-
13-acetate (TPA: manufactured by Wako Pure Chemical Industries, Ltd.) and 1
After adding μg / ml LPS (manufactured by Sigma), antisense-PLS or antisense-PLSP prepared in Example 2 was added. As a control, sense D
NA chain (SEQ ID NOS: 1 and 3) and PLS or PLSP
Complex with (sense-PLS and sense-PL, respectively)
SP), antisense DNA strand alone and sense DNA
Chains alone were also added respectively.

After incubating for 24 hours, -70 ° C
Frozen well in, then repeat freezing three times,
250 μl of the supernatant was gently collected. These are equalizers (E
LISA) kit (IL-1β ELISA System: Amersham), and then data analysis was performed using a microplate reader (Bio-Rad). as a result,
The anti-inflammatory antisense drug of the present invention, especially IL-1β
As shown in FIG. 2, the complex of antisense DNA (SEQ ID NO: 2) with respect to the initiation codon of the gene and PLS or PLSP suppressed the production of IL-1β by 100% in the nanomolar (nM) order. That is, at an antisense DNA concentration of 100 nM, 100% IL-
1β production suppressed, 50% IL-1β at 10 nM concentration
Production was suppressed, and at 1 nM concentration, IL-1β production was suppressed by 20%.

On the other hand, antisense D for the untranslated region
In the case of NA (SEQ ID NO: 4), a carrier-specific effect was observed, and only the complex with PLSP was I on the nM order.
An L-1β production inhibitory effect was observed. That is, FIG.
As shown in, in the case of antisense-PLSP,
90% or more at an antisense DNA concentration of 10 μM, 10
At 0 nM, IL-1β production was suppressed by 80% or more, and 1
30% IL at 0 nM concentration and less than 20% IL at 1 nM concentration
-1β production was suppressed. On the other hand, in the case of antisense-PLS, at a concentration of 10 μM, a little less than 50%, 100%
At a concentration of nM or less, suppression of IL-1β production of about 10% was only observed.

It should be noted that, in the same low concentration range, the binding specificity was remarkably exhibited in the antisense strand and the sense strand. Comparative Example 1 By the same method as in Example 3, the IL-1β production inhibitory effect of phosphorothioate type antisense nucleotides (hereinafter abbreviated as S-antisense) was measured. The results are as shown in FIGS. 2 and 3. It is said that this S-antisense can obtain stable and high biological activity in the same evaluation system, but the S-antisense (SEQ ID NO: 2) against the initiation codon of the IL-1β gene is shown in FIG. As shown, about 20 at 10 μM concentration
% IL-1β production inhibitory effect. Further, in the case of S-antisense (SEQ ID NO: 4) for the untranslated region, as shown in FIG. 3, about 10% inhibition of IL-1β production was shown at a concentration of 10 μM. Comparative Example 2 In the same manner as in Example 3, IL of a complex of antisense DNA (SEQ ID NO: 2) and lipofectin (manufactured by Gibco) (hereinafter abbreviated as antisense-lipofectin).
The -1β production inhibitory effect was measured. The results are as shown in FIG. Although this lipofectin has been hitherto highly evaluated as a carrier for oligonucleotides, antisense-lipofectin has a considerably high antisense concentration (50 μM), but IL
The -1β production inhibitory effect was about 20%. Also neg
No difference was observed in the inhibitory effect even when compared with the complex of sense DNA (SEQ ID NO: 1) and lipofectin (hereinafter abbreviated as sense-lipofectin) used as an implicit control, and the difference in oligonucleotide binding specificity was observed. Was not seen. Example 4 <Preparation of antisense drug against TNF-α> TNF was prepared in the same manner as in Example 2 except that the antisense oligonucleotides of SEQ ID NOs: 6, 8 and 10 were used.
Antisense drug against -α (antisense-PLS
P and antisense-PLS).

For these antisense drugs, the turbidity and the formation of precipitates, the concentration of oligonucleotide, the charge thereof and the like were measured in the same manner as in Example 2. Example 5 <Effect of anti-inflammatory antisense drug on cultured cells>
The antisense-PLS and antisense-PLSP conjugates prepared in Example 4 were examined for their TNF-α production inhibitory effect using the same method as in Example 3.

However, as controls, a complex of sense DNA strand (SEQ ID NO: 5) and PLS or PLSP (sense-PLS and sense-PLSP, respectively), antisense DNA strand alone and sense DNA strand alone were used, respectively. I was there. Also, the effect measurement Elisa kit is TNF
-Α ELISA System (manufactured by Funakoshi) was used. As a result, as shown in FIG. 5, the complex of antisense DNA (SEQ ID NO: 6) with PLSP to the initiation codon of TNF-α gene produced TNF-α when the concentration of antisense DNA was 100 pM. Was suppressed by 80% and 60% was suppressed at a concentration of 10 pM, but at a concentration lower than that, suppression of TNF-α production was not observed.
It should also be noted that significant binding specificity was also observed on the antisense and sense strands within the same low concentration range. As a synthetic polyamino acid that forms a complex with an antisense oligonucleotide, PLSP showed a higher TNF-α production inhibitory effect than PLS.

On the other hand, antisense DNA (SEQ ID NO: 8, 1) for the splicing site of TNF-α gene
The inhibitory effect on TNF-α production by the complex of 0) and PLSP was almost the same as that of the complex of antisense DNA (SEQ ID NO: 6) with respect to the initiation codon and PLSP, as shown in FIG. The antisense effect on this splicing site indicates that the oligonucleotide was efficiently transferred into the nucleus of the cell. Comparative Example 3 In the same manner as in Example 5, T of a complex of antisense DNA (SEQ ID NO: 6) and lipofectin (manufactured by Gibco) was used.
The NF-α production inhibitory effect was measured. The results are as shown in FIG. 7, and although the antisense-lipofectin had a considerably high antisense concentration (50 μM), the TNF-α production inhibitory effect was about 20%.
In addition, no difference was observed in the binding specificity of the oligonucleotide. Furthermore, although not shown in FIG.
S-antisense (SEQ ID NO: 6) by the same method as
The effect of suppressing TNF-α production was also measured. As a result, this S-antisense had a TNF-α production inhibitory effect of about 20% even at a concentration of 10 μM. Example 6 <Preparation of antisense drug against COX-2> An antisense-PLSP against COX-2 was prepared in the same manner as in Example 2 except that the antisense oligonucleotide of SEQ ID NO: 11 was used.

About this antisense drug, Example 2
In the same manner as above, turbidity and precipitate formation, oligonucleotide concentration, its charge, etc. were measured. Example 7 <Effect of anti-inflammatory antisense drug on cultured cells>
Antisense prepared in Example 6-PLX COX-
2 The production inhibitory effect was examined in a human bone cell culture system. As human bone cells, human synovial cells prepared from a tissue excised from a knee joint of a rheumatoid patient were used, and the culture was performed under the same conditions as the U937 cells of Example 3, and the number of cells was counted.

For measurement, 2.8 × 10 ⁴ pieces / ml
96 ml of 0.3 ml medium containing human synovial cells prepared in
Add to the well microplate (Falcon) and add 2
The cells were cultured for 4 hours. Then, gently remove the supernatant,
The adhered cells were washed with 0 μl of RPMI medium and subjected to the following tests. The antisense DNA strand alone and 0.15 ml of the antisense-PLSP solution prepared in Example 6 were added to each well, and 30 minutes later, 1 ng / ml of IL was added.
-1β (Genezyme, Lot. B41399) was added as needed. Antisense DNA strand and antisense-
Several kinds of PLSP were prepared from 1 nM to 10 μM (final concentration). After incubation for 2 hours, 3 of the supernatant
After collecting 00 μl and diluting if necessary, PGE ₂ Elisa Kit (Japan Perceptive Limited, No. 8-
6801).

As a control, S-antisense and gene transfer of Comparative Example 1 (manufactured by Wako Pure Chemical Industries, Ltd .: Co
de 074-03621) and an antisense DNA strand complex (hereinafter abbreviated as antisense-gene transfer)
Was similarly measured. Gene Transfer
It is a liposome-based carrier and has hitherto been highly evaluated as an oligonucleotide carrier.

The results are shown in FIG. 8. S-antisense did not suppress PGE ₂ production at all. Similarly, in the case of antisense-gene transfer,
No inhibition of PGE ₂ production was observed. On the other hand, antisense-PLSP, which is the anti-inflammatory antisense drug of the present invention, effectively suppressed the production of PGE _{2 in} the μM order. That is, 92% of P (when IL-1β was added)
GE ₂ production was suppressed.

It should be noted that the binding specificity between the antisense strand and the sense strand in inhibiting PGE ₂ production was remarkably exhibited within the concentration range tested. Example 8 <Uptake of anti-inflammatory antisense drug into cultured cells>
Antisense-PLSP prepared in the same manner as in Example 6
The uptake efficiency of each of these into the cultured cells was tested.

First, the antisense DNA strand was labeled by the following method. DNA (1.83 OD ₂₆₀ unit, 5p
mol) 27.3 μl, autoclaved distilled water 1
5.7 μl, 10 × Kinase buffer (250 mM Tris-HCl buffer: pH 7.6), 100 mM DTT, T4
Polynuclease solution (100 units / μl), γ-
After mixing 1 μl of ³² P-ATP, the mixture was reacted at 37 ° C. for 1 hour. For labeling with ³² P, a T4 polynucleotide kinase kit (Takara Shuzo Code No. 2030) was used.

Human macrophage U937 cells as in Example 2 and human synovial cells as in Example 7 were used as the cultured cells. Under the same conditions as in Example 7, 1 × 10 ⁶
Cells / ml of U937 cells were cultured in a glass tube (Falcon 2058, 6 ml-12 × 75). In the case of human synovial cells, 50 μl of antisense-PLSP solution adjusted to the desired concentration was added and cultured, and U93
In case of 7 cells, 1 ng / ml of 12-o-tetradecan
After adding oyl-phorbol-13-acetate (TPA: manufactured by Wako Pure Chemical Industries, Ltd.), antisense-PLSP (final concentration: 1 ng / ml to 10 μg / ml) was added and the culture was continued. After culturing for a predetermined time, put the culture tube in 25
After centrifugation at 1500 rpm for 10 minutes at 1500 ° C. to precipitate the cells, the supernatant was gently removed. Then, 3 ml of the same PBS solution as used in Example 7 was added to the cells attached to the bottom of the tube, and the cells were washed twice,
The cells were lysed by adding 500 μl of 1% SDS (sodium dodecyl sulfate). Finally, 10 ml of hyperflora solution was added to all of the dissolved solutions, and measurement was performed with a liquid scintillator.

As a control, as in Example 7, S-
Antisense and antisense-gene transfer were similarly measured. The results are shown in FIGS. 9 and 10.
In the case of S-antisense and antisense-gene transfer, no antisense DNA was incorporated into U937 cells (Fig. 9) or human synovial cells (Fig. 10). It was

On the other hand, the antisense-PLSP of the present invention
Was confirmed to be taken up into cells over time. That is, in the culture for up to 3 hours, 100 times or more DNA was incorporated into the cells as compared with S-antisense, and 50 times or more as compared with antisense-gene transfer. Example 9 <Lethal suppressive effect of anti-inflammatory antisense drug on endotoxin-induced shock model mouse> SEQ ID NO: 1
Antisense-PLSP against mouse IL-1β and mouse TNF-α were prepared in the same manner as in Example 2, except that oligonucleotides 3, 14, and 15 were used.
Antisense-PLSP was prepared and its lethal suppressive effect on endotoxin-induced shock was examined using model mice.

200 μl of an antisense-PLSP solution prepared to a desired concentration with physiological saline was used to prepare BALB / c male mice aged 8 to 10 weeks (Japan SLC: about 20 g).
To the tail vein. Immediately after that, endotoxin (LPS, Lot N prepared at physiological saline equivalent to 20 mg / kg)
o. 68692 WE coli055: B5. Difco) Solution 2
00 μl was intraperitoneally administered to mice, and the number of surviving solids was counted over time.

The results are as shown in FIGS. First, as shown in FIG. 11, when antisense-PLSP against IL-1β was used, the survival rate of mice increased depending on the concentration of the administered antisense drug. In particular, 100 mg / kg concentration of antisense-P
When LSP was administered, no deaths were observed for 40 hours.

Further, as shown in FIG. 12, 10 mg /
The effect of the case where the antisense-PLSP at a concentration of kg was administered at once and the case where the antisense-PLSP at a concentration of 5 mg / kg was administered twice (total 10 mg / kg) were compared. 12 sense drugs
It was found that administration in two divided doses at a time interval was more effective than single administration.

FIG. 13 shows the difference in effect between the case where antisense-PLSP was administered into the tail vein and the case where it was administered intraperitoneally. Although antisense-PLSP did not show a lethal suppressive effect when administered intraperitoneally, it showed an excellent lethal suppressive effect when administered intravenously into the tail vein. Therefore, the antisense drug of the present invention has different effects depending on the administration route. Was found to occur.

Further, as shown in FIG. 14, IL-1
When the effects of the antisense drug against β and the antisense drug against TNF-α were compared, 30 mg / kg
When administered at a concentration, the antisense drug against TNF-α had a higher lethal suppression effect, and the mouse survival rate after 40 hours was 60%. From the above test results using model mice, the antisense drug of the present invention is
It was confirmed that the ivo model experimental system also showed excellent drug efficacy.

[0055]

INDUSTRIAL APPLICABILITY As described above in detail, according to the present invention, an anti-inflammatory antisense drug which binds to sense RNA in a primary structure-specific manner and exerts a sufficient physiologically active substance inhibitory effect in a very low concentration range. Will be provided. By this, inflammatory diseases such as rheumatoid arthritis, periodontitis, nephritis, ulcerative colitis, arteriosclerosis, psoriasis, diseases such as septic shock, Crohn's disease, AIDS, or intractable liver disease or liver transplant Effective treatment for pathological conditions becomes possible.

[0056]

[Sequence list]

SEQ ID NO: 1 Sequence length: 20 Sequence type: Nucleic acid Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Sequence including the initiation codon of human IL-1β gene GCAGCCATGG CAGAAGTACC 20 SEQ ID NO: 2 Sequence length: 20 Sequence type: Nucleic acid Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Sequence of antisense strand of SEQ ID NO: 1 Sequence GGTACTTCTG CCATGGCTGC 20 SEQ ID NO: 3 Sequence length: 20 Sequence type: Nucleic acid Sequence type: Other Nucleic acid Synthetic DNA Sequence characteristics Sequence containing untranslated region of human IL-1β gene AGAGAGCTGT ACCCAGAGAG 20 SEQ ID NO: 4 Sequence length: 20 Sequence type: Nucleic acid Sequence type: Other nucleic acid Synthetic DNA Sequence characteristic SEQ ID NO: Antisense strand sequence 3 CTCTCTGGGT ACAGCTCTCT 20 SEQ ID NO: 5 Sequence length: 20 Sequence type: Nucleic acid Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics human TNF -A sequence including the initiation codon of the α gene CCCTGGAAAG GACACCATGA 20 SEQ ID NO: 6 Sequence length: 20 Sequence type: Nucleic acid Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics SEQ ID NO: 5 antisense strand sequence TCATGGTGTC CTTTCCAGGG 20 SEQ ID NO: 7 Sequence length: 20 Sequence type: Nucleic acid Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Splicing site of human TNF-α gene (1624-
164th position) CCAGGCAGTC AGTAAGTGTC 20 SEQ ID NO: 8 Sequence length: 20 Sequence type: Nucleic acid Sequence type: Other nucleic acid Synthetic DNA Sequence features SEQ ID NO: 7 antisense strand Sequence GACACTTACT GACTGCCTGG 20 SEQ ID NO: 9 Sequence length: 20 Sequence type: Nucleic acid Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Splicing site (2161− of human TNF-α gene
2180th) sequence CTCCCTCCAG CAAACCCTCA 20 SEQ ID NO: 10 Sequence length: 20 Sequence type: Nucleic acid Sequence type: Other nucleic acid Synthetic DNA Sequence features SEQ ID NO: 9 antisense strand Sequence TGAGGGTTTG CTGGAGGGAG 20 SEQ ID NO: 11 Sequence length: 20 Sequence type: Nucleic acid Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Sequence including initiation codon of human COX-2 gene TGCCCGCCGC TGCGATGCTC 20 SEQ ID NO: 12 Sequence length: 20 Sequence Type: Nucleic acid Type of sequence: Other nucleic acid Synthetic DNA Sequence characteristics Antisense strand of SEQ ID NO: 11 Sequence GAGCATCGCA GCGGCGGGCA 20 SEQ ID NO: 13 Sequence length: 20 Sequence type: Nucleic acid Type of sequence: Other nucleic acid Synthetic DNA Sequence features Sequence including the initiation codon of mouse IL-1β gene GCAGCTATGG CAACTGTTCC 20 SEQ ID NO: 14 Sequence length: 20 Sequence : Nucleic acid Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Sequence number 13 antisense strand Sequence GGAACAGTTG CCATAGCTGC 20 SEQ ID NO: 15 Sequence length: 20 Sequence type: Nucleic acid Sequence type: Other nucleic acid Synthetic DNA sequence Features of 20-base antisense strand sequence including the initiation codon of mouse TNF-α gene ATCATGCTTT CTGTGCTCAT 20

[Brief description of the drawings]

FIG. 1 is a chemical formula (a) of PLS and a chemical formula (b) of PLSP that can be used in the present invention.

FIG. 2 is a graph showing the inhibitory effect on IL-1β production by an anti-inflammatory antisense drug using the DNA chain of SEQ ID NO: 2 and a comparative example.

FIG. 3 is a graph showing the inhibitory effect on IL-1β production by an anti-inflammatory antisense drug using the DNA chain of SEQ ID NO: 4 and a comparative example.

FIG. 4 is a graph showing the IL-1β production inhibitory effect of an anti-inflammatory antisense drug using the DNA chain of SEQ ID NO: 2 and a comparative example.

FIG. 5 is a graph showing the effect of suppressing production of TNF-α by an anti-inflammatory antisense drug using the DNA chain of SEQ ID NO: 6 and a comparative example.

FIG. 6 is a graph showing the effect of suppressing production of TNF-α by an anti-inflammatory antisense drug using a DNA chain of SEQ ID NO: 8 or 10 and a comparative example.

FIG. 7 is a graph showing the effect of suppressing production of TNF-α by an anti-inflammatory antisense drug using the DNA chain of SEQ ID NO: 6 and a comparative example.

FIG. 8 is a graph showing the amount of PGE ₂ produced by an anti-inflammatory antisense drug using the DNA strand of SEQ ID NO: 11 or 12 and a control.

FIG. 9 is a graph showing the uptake amount of anti-inflammatory antisense drug into U937 cells using the DNA strand of SEQ ID NO: 11 or 12.

FIG. 10 is a graph showing the amount of anti-inflammatory antisense drug incorporated into synovial cells using the DNA strand of SEQ ID NO: 11 or 12.

FIG. 11 is a graph showing the relationship between the lethality suppressive effect and the dose on the anti-inflammatory antisense drug using the DNA chain of SEQ ID NO: 2 and the endotoxin-induced shock model according to the comparative example.

FIG. 12 is a graph showing the relationship between the lethality suppressive effect and the usage in an endotoxin-induced shock model by an anti-inflammatory antisense drug using the DNA chain of SEQ ID NO: 2.

FIG. 13 is a graph showing the relationship between the lethal suppressive effect and the administration route on the endotoxin-induced shock model by the anti-inflammatory antisense drug using the DNA chain of SEQ ID NO: 2.

FIG. 14: DNA of each of SEQ ID NO: 2 and SEQ ID NO: 6
It is a graph which showed the difference of the lethal suppression effect with respect to the endotoxin induction shock model by the anti-inflammatory antisense drug which used the chain.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI Technical display location A61K 48/00 ABX A61K 48/00 ABX ACJ ACJ ACK ACK ACV ACV ADA ADA ADY ADY ADZ ADZ C07H 21 / 04 C07H 21/04 B C12N 15/09 9282-4B C12N 15/00 A (72) Inventor Akira Wada 1-25-11 Kannondai, Tsukuba City, Ibaraki Prefecture Hisamitsu Pharmaceutical Co., Ltd. Tsukuba Research Institute (72) Inventor Kayosuke Suzuki 1-25-11 Kannondai, Tsukuba-shi, Ibaraki Hisamitsu Pharmaceutical Co., Ltd. Tsukuba Research Laboratory (72) Inventor Shinichi Kawai 3-3-3 Denenchofu, Ota-ku, Tokyo (72) Inventor Yu Mizushima Tokyo 1-11-1 Umegaoka, Setagaya-ku, Tokyo

Claims (12)

[Claims] 1. A synthetic polyamino acid or a derivative thereof,
M encoding a physiologically active substance involved in human inflammatory disease
An anti-inflammatory antisense drug characterized by comprising a complex with an antisense oligonucleotide complementary to a part or the whole base sequence of RNA. 2. The anti-inflammatory antisense drug according to claim 1, wherein the synthetic polyamino acid is a nucleic acid conjugate or a nucleic acid derivative having a repeating sequence of a lysine residue and a serine residue. 3. The anti-inflammatory antisense drug according to claim 2, wherein the synthetic polyamino acid derivative is a polyethylene glycol block modified product of the synthetic polyamino acid. 4. The anti-inflammatory antisense drug according to claim 1, wherein the physiologically active substance involved in human inflammatory disease is a physiologically active substance involved in human rheumatoid arthritis. 5. A synthetic polyamino acid or a derivative thereof,
The anti-inflammatory antisense drug according to claim 1, which comprises a complex with an antisense oligonucleotide complementary to a part or the whole base sequence of mRNA encoding human interleukin-1β. 6. An antisense oligonucleotide complementary to a part or the whole base sequence of mRNA encoding human interleukin-1β is an oligonucleotide having a part or the whole base sequence of SEQ ID NO: 2 or 4. The anti-inflammatory antisense drug according to claim 5. 7. A synthetic polyamino acid or a derivative thereof,
The anti-inflammatory antisense drug according to claim 1, which comprises a complex with an antisense oligonucleotide complementary to a part or the whole base sequence of mRNA encoding human tumor necrosis factor. 8. The anti-inflammatory antisense drug according to claim 7, wherein the human tumor necrosis factor is human tumor necrosis factor-α. 9. An mR encoding human tumor necrosis factor-α
The antisense oligonucleotide complementary to a part or the whole base sequence of NA is an oligonucleotide having a part or the whole base sequence of any of SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10. Anti-inflammatory antisense drug. 10. A complex comprising a synthetic polyamino acid or a derivative thereof and an antisense oligonucleotide complementary to a partial or entire base sequence of mRNA encoding a series of prostaglandin E ₂ synthases. The anti-inflammatory antisense drug according to claim 1. 11. A prostaglandin E ₂ synthase,
The anti-inflammatory antisense drug according to claim 10, which is cyclooxygenase-2. 12. The antisense oligonucleotide complementary to a part or the whole base sequence of mRNA encoding cyclooxygenase-2 is an oligonucleotide having a part or the whole base sequence of SEQ ID NO: 12. Anti-inflammatory antisense drug.