WO1997010840A1

WO1997010840A1 - Anti-inflammatory antisense drug

Info

Publication number: WO1997010840A1
Application number: PCT/JP1996/002682
Authority: WO
Inventors: Shuji Sato; Takeshi Goto; Akira Wada; Yousuke Suzuki; Shinichi Kawai; Yutaka Mizushima
Original assignee: Hisamitsu Pharmaceutical Co., Inc.; Ltt Institute, Co., Ltd.
Priority date: 1995-09-18
Filing date: 1996-09-18
Publication date: 1997-03-27
Also published as: AU7000496A; JPH09176038A

Abstract

An anti-inflammatory antisense drug which comprises a complex of a synthetic polyamino acid consisting of repeated sequences of lysine and serine residues or polyethylene glycol modifications thereof and an antisens oligonucleotide complementary to the partial or whole base sequence of an mRNA encoding physiologically active substances participating in human inflammatory diseases such as interleukin-1β, tumor necrotizing factor or α-cyclooxygenase. This drug enables efficacious treatment of inflammatory diseases such as rheumatoid arthritis, paradentitis, nephritis, ulcerous colitis, arterial sclerosis and psoriasis, septic shock, Crohn's disease, AIDS, intractable hepatic diseases, pathologies in association with liver transplant, etc.

Description

Description Anti-inflammatory antisense drug Technical field

The present invention relates to an anti-inflammatory antisense drug. More specifically, this invention relates to inflammatory diseases such as rheumatoid arthritis, periodontitis, nephritis, ulcerative colitis, arteriosclerosis, psoriasis, septic shock, Clone disease and AIDS The present invention relates to an antisense drug that specifically blocks gene expression of a physiologically active substance involved in a refractory liver disease or a pathological condition in liver transplantation. Background art

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

At present, to block the function of mRNA, it is not necessary to have an antisense strand complementary to the entire sequence, and a partial sequence that controls the expression of protein from mRNA is targeted at the targeting site. It is considered effective to do so. That is, as such a targeting site, (1) a splicing site, (2) a cabling site. (3) A vicinity of an AUG initiation codon (initiation codon) site is often selected, and in particular, an AUG initiation site. A relatively high antisense effect has been obtained for the codon region. The three-dimensional structure of the mRNA must also be considered, and it is generally thought that antisense DNA is likely to bind to a single-stranded region such as a loop structure or a bulge structure. Meanwhile, the history of drugs derived from natural products has undergone a dramatic change with the advancement of science, and one of the trends in drug discovery toward the new century is definitely targeting genes. It is flowing out to the one that did. According to the progress of the “Hugenome Project” aimed at elucidating the entire nucleotide sequence of the human gene, it is said that a complete genomic sequence including all human genes will be determined around 2010. ing. This vast amount of information is thought to spur analysis and analysis of diseases that may be related to gene deficiency or abnormal expression, and redefine the concept of treatment. Against the backdrop of this research momentum, gene therapy antisense therapy has been in the spotlight as a new century of therapy (Cook, ST, Ann. Rev. Pharm. 32, 329 — 376, 1992; Tidd, DM, Anticancer Res. 10, 1169-1182, 1990).

遗 Gene therapy is limited to mono-gene disease, cancer, AIDS, etc. due to problems such as biopsy. In contrast, antisense DNA can be captured as a compound similar to conventional synthetic drugs, and its effects in vivo as well as in vitro have been reported. Sex search is entering a new research phase.

The possibility of using antisense DNA as a drug was proposed in 1978, when a report was made in 1978 that synthetic antisense DNA was added from the outside to suppress the transformation of Rous sarcoma virus. Pioneer (Zamecnik PC and Stephenson ML, Proc.

Nat 1. Acad. Sci. USA. 75, 280-284, 1978). It was questioned that DNA, which is a polyanion, was taken up into cells.However, it was subsequently reported in cultured cell lines that antisense DNA blocked the function of endogenous genes, Expectations have increased. However, naturally occurring phosphodiester type DNA

(Hereinafter abbreviated as D-oligo) has a fatal drawback as a drug that is degraded in a short time by nucleases and the like. For this reason, attempts have been made to chemically modify DNA to increase its biological stability. Among them, phosphorothioate-type nucleotides (hereinafter abbreviated as S-oligo) are particularly stable. Has high biological activity. This type is currently in clinical trials. ₍ The condition of antisense DNA as a drug is that it has: 1) nucleotide sequence specificity for mRNA, and 2) it is stably transported into cells. And ③ mRNA target グ Being able to form stable duplexes, 存在 Being able to remain stable in cells for a certain period of time, ⑥ No toxicity and side effects, ⑦ Being metabolized to some extent, ⑧ Being economical, etc. Is raised. In particular, it must be transported into cells in a stable form. DNA or RNA is quantitatively degraded by nucleases (hereinafter abbreviated as DNase and RNase, respectively) and the like, and the blood half-life is extremely short, within 1 minute. For the purpose of stabilization, the mainstream is methylphosphine, in which one of the oxygen atoms of the phosphoric acid group is substituted with S—, which is substituted with an S—oligo CH ₃ group. In addition, modifications of the sugar moiety, modifications of the 3 'and 5' ends, and modifications of the 2 'position have been considered (Goodchild. J. Bioconjug Chem. 1, 165-187, 1990). Recently, Peptide Nucleic Acid, which has been changed from a phosphate bond to a peptide bond, is used.

It has been reported that (PNA) has been synthesized, has stronger binding to DNA and RNA than antisense DNA, and is more resistant to nucleases (Hanvey, L. et al., Science 258). , 1481-1485 (1992)), reports of biological activity do not yet exist. It has been reported that those designed to form a loop structure by forming a base pair at the 3 'end, and those with a dumbbell type or a ring-closed structure are also stable.

However, these modified products have many problems such as the cost involved in their synthesis and the fact that their purification is extremely difficult, and their application to pharmaceuticals is difficult at present. is there. In addition, S-oligos have been used extensively in previous studies as described above, but this type of oligonucleotide is also likely to have low binding specificity to the sense strand. This is a problem. In this sense, D-oligo is necessarily considered to be the most suitable for applying antisense DNA to pharmaceuticals.

In addition, the low permeability of nucleic acids such as DNA has been regarded as a problem from the beginning, and considering the intracellular stability of the phosphogetyl-type oligonucleotide described above, future antisense It is no exaggeration to say that the key to pharmaceuticals lies in the intracellular transfer method (delivery method). As can be seen from successful examples in the culture system, it is certain that oligonucleotides such as DNA are taken up into cells, and are mainly taken up by endcytosis, and about 8 OkD. The membrane protein in a is considered as the estimated receptor. Modified oligonucleotides are also mainly obtained through end-site analysis. Although the details are unknown, there are still a lot of unknown details, and the transfer amount into cells is still small. A delivery method has been devised to enhance low membrane permeability, but there are still points to be resolved including problems such as toxicity.

On the other hand, in the treatment of inflammatory diseases, steroids and non-steroid anti-inflammatory drugs have been widely used in recent years. Steroids significantly improve the symptoms of various inflammatory diseases, but their effects gradually decrease with administration, and side effects include coronary artery insufficiency, peptic ulcer, cataract, sepsis, and susceptibility to infectious diseases. There is a problem that there is a risk of triggering. Non-steroid anti-inflammatory drugs also temporarily suppress inflammatory symptoms, but do not fundamentally cure inflammatory diseases. Therefore, at present, there is a demand for the development of a therapeutic agent for inflammatory diseases which has high efficacy, sustains its therapeutic effect and is highly safe.

In particular, rheumatoid arthritis is an unexplained chronic inflammatory disease with the synovium as the main lesion. The affected area sometimes does not stop at the joint synovium, and the inflammation that initially develops in the synovium causes the destruction of bones and bones, eventually leading to the destruction of the whole body. Empirical factors are strongly involved in the treatment of osteoporosis, and non-steroid anti-inflammatory analgesics have been used as first-line drugs. However, recently, although the role of non Suteroi de anti-inflammatory agent in the treatment of a chronic rheumatoid Umachi is analgesic effect by _c its administration in shrinking can be expected, antirheumatic effect on non-steroid anti-inflammatory drugs It has become common knowledge that there is no clinical benefit, and it has become clear that the side effects of non-steroid anti-inflammatory analgesics, such as gastrointestinal disorders and renal dysfunction, cannot be ignored clinically. It is.

Under these circumstances, early use of new antirheumatic drugs, such as methotrexate, mizoribine, FK506, and the like, is being carried out.

The following mechanisms are known for various inflammatory diseases such as rheumatoid arthritis. In other words, inflammation is a biological reaction to maintain homeostasis in response to inflammation. Various cells are recruited to the inflammatory response, and cytokines are one of the mediators between these cells and play an important role in the inflammatory response. Various cytokines are produced at sites of inflammation and regulate the inflammatory foci and systemic inflammatory response while forming rhino-i and force inskade. this Among these cytokines, Tumor Necrosis Factor (hereinafter abbreviated as TNF) is a cytokinin produced by cells such as macrophages. This TNF is now being understood as a site that is involved in biological defense reactions through inflammation, which was initially found as a substance that damages tumors. Genes of TNF have been clearly identified in a wide range of mammals, including humans, bushes, porcupines, magpies, and mice, and their primary structures have also been determined. According to this, the amino acid sequence between each animal has a homology of around 80% conserved, suggesting that TNF is an extremely important physiologically active substance in living organisms. The human TNF precursor has 233 amino acid residues, and is composed of 155 or 157 amino acids in the mature form. Although its molecular weight is 17 kDa, it forms a 45 kDa trimer in vivo. It is thought that sugar chains are present in mouse TNF <, which is not present in humans. In mouse TNF, sugar chains are not essential for the expression of their activities.

TNF is expressed by inoculating BCG-sensitized animals with lipopolysaccharide (hereinafter abbreviated as LPS). Experimentally, macrophages can be prepared for TNF production by various methods, and TNF peaks at around 2 hours by an appropriate triggering stimulus (eg, bacterial cells—cell components such as LPS). Can be produced. Also, human Bok macro Roff Aji system cells (e.g., U937 strain, etc.) can also reproduce the production of TNF in in vitro experimental systems using _c

TNF acts on a wide variety of cells. For example, TNF produced from macrophages acts on neutrophils and vascular endothelial cells, leading to the development of inflammation from the outset. Then, by acting on fibroblasts and hepatocytes, the inflammation ends and goes toward repair. The inflammatory response progresses in a way that many cells and mediators interact with each other, and usually disappears at a constant rate.However, if there is a substance to be an antigen and its amount is more than a certain level, the information Is then passed on to the immune system. TNF is thus also involved in the transition from a non-specific host defense response to a specific defense response. In addition, TNF is known to act on, for example, osteoblasts, osteoclasts, adipocytes, epithelial cells, pituitary gland, and especially synovial cells.

In addition, the pathogenesis of inflammatory diseases such as rheumatoid arthritis includes various immune response systems-abnormalities of inflammatory reactions.For example, other than TNF-α, one of TNF, In addition, it is thought that oncogenes such as c-fos, cytokins such as human interleukin-1β (hereinafter abbreviated as IL-1β) and IL-16 are involved. . Furthermore, in recent years, the role of prostaglandin (hereinafter abbreviated as PG) and its synthase has been attracting attention as one of the mediators of inflammatory diseases. That is, since non-steroidal anti-inflammatory analgesics such as aspirin were reported by Vane et al. In 1971 to suppress PG synthesis, the non-steroid anti-inflammatory analgesic Drug research has been pursued in relation to PG synthesis inhibition. However, in recent years, there are two types of cycloxygenase (hereinafter, abbreviated as C0X), which is an enzyme that synthesizes PG, and C0X, which has been studied so far, is mainly used in physiology. Is an enzyme called C0X-1 that is already present in the body, and is produced by free cells and the like by various irritating and cytokins, and has a great effect on inflammation and tissue inhibition. The relevant COX-2 was found to exist (Vane, J., Nature, 367, 2 15-216, 1994: Xie. W., Robertson, D., and Simmons, D., Drug Devel. Res. ., 25, 249-265, 1992). This COX-2 gene is different from COX-1 in that it is an enzyme newly produced by stimulation of IL-11. For example, according to the report of Lee et al., C 0 X-1 in macrophages is invariable by LPS stimulation, and is detected at a constant concentration regardless of stimulation or inflammation. In the normal state, 2 has almost no evidence of its gene, and its expression is increased by LPS stimulation, and its expression is completely suppressed by dexamethasone. In recent years, the cDNA of human C0X-2 has been cloned, which is elucidating the regularity of C0X-2 development in inflammatory cells.

With the recent advances in molecular biology and immunology, the etiology and pathology of various inflammatory diseases, including rheumatoid arthritis, is rapidly progressing as described above, and based on these findings New therapies with more theoretical backing are being developed, such as anti-cytokine therapy, anti-adhesion molecule therapy, monoclonal antibody therapy, oral peptide therapy, and T-cell vaccine.

On the other hand, as for human diseases targeted by the antisense method, viral diseases are at the top of the list because the relationship between the cause and the target gene is clear, and early antisense therapy was used. Is a site Megalovirus, Hellswill , Human immunodeficiency virus, papi mouth virus, and vesicular stomatitis virus (VSV). Antisense therapies targeting the translocated bcr-abl gene, which causes chronic myeloid leukemia, or ICAM-1, one of the adhesion molecules that affect inflammation or cancer metastasis, are also available. Effective.

However, in general, the greatest effect can be expected when the etiology corresponds to the genes on a 1: 1 basis due to the characteristics of the antisense method, rather than multiple genes that cause the disease. Therefore, in order to apply the antisense method to the treatment of inflammatory diseases, it is essential to identify the causative gene as well as accurately understand the pathology. In addition, it is necessary to identify the expression mechanism of the gene and to select an appropriate site for shutting down the expression. Disclosure of the invention

The present invention has been made in view of the circumstances described above, and includes inflammatory diseases such as rheumatoid arthritis, periodontitis, nephritis, ulcerative colitis, arteriosclerosis, psoriasis, and septicemia. The purpose of the present invention is to provide a new anti-inflammatory drug containing antisense DNA as a main component that specifically blocks the expression of a physiologically active substance involved in shock, Crohn's disease, AIDS, and the like.

The present invention provides, as a first invention for solving the above-mentioned problems, a synthetic polyamino acid or a derivative thereof, and an mRNA encoding a physiologically active substance involved in a human inflammatory disease. The present invention provides an anti-inflammatory antisense drug comprising a complex with an antisense 'oligonucleotide complementary to a part or the entire nucleotide sequence of the anti-inflammatory antisense drug. 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 derivatives thereof. However, the preferred embodiment is a block modification of polyethylene glycol (hereinafter abbreviated as PEG) of synthetic polyamino acid.

Further, the present invention provides, as a second invention, a synthetic polyamino acid or a derivative thereof, which is complementary to a part or the whole nucleotide sequence of mRNA encoding human IL-1 / 3. An anti-inflammatory agent comprising a complex with a nucleic acid or an oligonucleotide. Provide antisense drugs. In the second invention, an antisense oligonucleotide complementary to a part or the entire nucleotide sequence of mRNA encoding IL-1 / 3 is represented by SEQ ID NO: 2. Alternatively, the preferred embodiment is an oligonucleotide having a part or the entire nucleotide sequence of 4.

Further, as a third invention, a synthetic polyamino acid or a derivative thereof and an antisense oligonucleotide complementary to a part or the entire nucleotide sequence of human TNF-encoding mRNA. An anti-inflammatory antisense drug comprising a complex with leotide is provided. In the third invention, the human TNF is human TNF-α, and a part or all of the mRNA encoding the human TNF-α is complementary to the entire nucleotide sequence. The antisense oligonucleotide is a polynucleotide having part or all of the nucleotide sequence of SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. This is the preferred mode.

Also of al, in the fourth invention, the synthetic poly A Mi Roh acid or a derivative thereof, a part of a series of PGE ₂ synthase co one sul mRNA A properly is complementary to the entire nucleotide sequence An anti-inflammatory antisense drug comprising a complex with an antisense oligonucleotide is provided. In the fourth invention, PGE ₂ synthase, a C OX 2, the C 0 X- 2 of code sul m RNA - complementary Anchisensu-cage to properly even some entire nucleotide sequence In a preferred embodiment, the gonionucleotide is an oligonucleotide having a part or the entire nucleotide sequence of SEQ ID NO: 12. BRIEF DESCRIPTION OF THE FIGURES

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

FIG. 2 is a graph showing the effect of suppressing the production of IL-1 / 3 by an anti-inflammatory antisense drug using the DNA chain of SEQ ID NO: 2 and a specific example.

FIG. 3 is a graph showing the IL-13 production inhibitory effect of an anti-inflammatory antisense drug using the DNA chain of SEQ ID NO: 4 and a comparative example.

Figure 4 shows anti-inflammatory antisense drugs using the DNA strand of SEQ ID NO: 2 and comparison It is a graph which shows the production inhibitory effect of IL-11 ^ by an example.

FIG. 5 is a graph showing the effect of suppressing the 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 the production of TNF-α by an anti-inflammatory antisense drug using the DΝ chain of SEQ ID NO: 8 or 10, and a comparative example.

FIG. 7 is a graph showing the effect of suppressing the production of TNF-α by an anti-inflammatory antisense drug using the DΝ 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 DΝ chain of SEQ ID NO: 11 or 12 and a control.

FIG. 9 is a graph showing the uptake amount of an anti-inflammatory antisense drug into U933 cells using the DNA chain of SEQ ID NO: 11 or 12.

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

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

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

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

FIG. 14 is a graph showing the difference in the effect of the anti-inflammatory antisense drug using the DNA strands of SEQ ID NO: 2 and SEQ ID NO: 6 on the endotoxin-induced shock model against lethality. . BEST MODE FOR CARRYING OUT THE INVENTION

Nucleic acid composites of synthetic polyamino acids are composed of irregular or regular repetitions of water-soluble amino acid serine residues and cationic amino acid lysine residues. Serine The molar ratio of the residue to the lysine residue is about 1: 1 and its molecular weight is about 3000-50,000. Such a polyamino acid is, for example, a polylysine: serine (hereinafter, abbreviated as PLS) which forms a complex in a homogeneous system with an oligonucleotide. Patent WO95 / No. 0909) can be used. In addition, as a derivative of the synthetic polyamino acid, in particular, the above-mentioned modified PLS PEG block (hereinafter abbreviated as PLSP) can be exemplified as a novel one. Can be created, for example, by the method of Example 1 below. The structures of PLS and PLSP can be exemplified as, for example, the chemical formulas in FIGS. 1 (a) and 1 (b). In addition, antisense 'oligonucleotides, which are complementary to mRNA encoding bioactive substances involved in inflammatory diseases, or synthetic polyamino acids such as PLS or the like. Oligonucleotides modified with derivatives (eg, FLSP) can be prepared by known methods (Rajendra, BR et al., Human Genetics. 55, 3633, 1980, Lira, F. and Sun, A., M. , Science, 210, 908, 1980). Example

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples. The oligonucleotides used in the following Examples and Comparative Examples were synthesized and produced as follows. Synthesis and Purification of Oligonucleotides>

Oligonucleotides of SEQ ID NOS: 1 to 15 in the sequence listing were synthesized using a DNA synthesizer (Applied Biosystems, Inc. type 380B). Oligonucleotide was synthesized based on the phosphoramidite method (Nucleic Acid Res., Vol. 17, 7059-7071, 1989) using a t-butylmethylsilyl group as the protecting group for the 2'-hydroxyl group. Purification of the compound was performed according to the method described in the literature (Nucleic Acid Res., Vol. 19, 5125-5130, 1991).

SEQ ID NO: 1 is a known 20-nucleotide oligonucleotide, including the initiation codon of IL-11 / 9 gene, one of the physiologically active substances of rheumatoid arthritis. It is a sense DNA strand corresponding to 0 bases, and SEQ ID NO: 2 is complementary to this. Typical antisense DNA strand. In addition, SEQ ID NO: 3 is a sense DNA strand corresponding to 20 bases including the untranslated region of the same IL-11 gene, and SEQ ID NO: 4 is an antisense DNA strand complementary thereto (particularly, Kaihei 6 — 4 1 1 8 5). SEQ ID NO: 5 is a sense D-chain corresponding to 20 bases including the initiation codon of the TNF-gene, which is also one of the physiologically active substances of rheumatoid arthritis, and SEQ ID NO: 6 is The complementary antisense DNA sequence, SEQ ID NO: 7, has a nucleotide sequence corresponding to 20 nucleotides (the TNF-α gene sequence at positions 1624 to 1643) containing the TNF-α gene splicing site. SEQ ID NO: 8 is a complementary antisense DNA strand, and SEQ ID NO: 9 is 20 bases including the TNF-α gene splicing site (TNF-α gene sequence 2161- SEQ ID NO: 10 is the complementary antisense DNA strand.

Furthermore, 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. SEQ ID NO: 12 is the complementary antisense DNA strand ₍ also SEQ ID NO: 13 contains the initiation codon of the mouse〗 L-11β gene. SEQ ID NO: 14 is the sense strand corresponding to 0 bases, and SEQ ID NO: 14 is the complementary antisense DΝC strand, and SEQ ID NO: 15 is the mouse TNF-α 遗 gene initiator. Example 1 An antisense strand corresponding to 20 bases including a don.

Synthesis and purification of PEG-modified PLS>

ε One-potency lipobenzoxy lysine-1.0 g of carboxylic anhydride (manufactured by Sigma) and 1.0 g of benzylserine-N-carboxylic anhydride (manufactured by Sigma) in N, N-dimethyl The solution was dissolved in 30 ml of formamide (DMF: manufactured by Wako Pure Chemical Industries, Ltd.), and 15 ml of Kokuguchi Holme (manufactured by Wako Pure Chemical Industries, Ltd.) was added. One-terminal methoxy One-terminal amino group polyethylene oxide (molecular weight: 5000, manufactured by NOF Corporation) 4.0 g was dissolved in 15 ml of chloroform, and the solution was dissolved in ε-carbobenzoxylidine. N-one N—Carbo Acid anhydride and benzyl serine-N-potassium sulfonic acid were added to the solution. After 26 hours, the reaction mixture was dropped into 330 ml of getyl ether, and the precipitated polymer was collected by filtration, washed with getyl ether (manufactured by Wako Pure Chemical Industries, Ltd.), and then vacuumed. Then, deprotection was performed using a hydrogen bromide acetic acid solution (manufactured by Wako Pure Chemical Industries, Ltd.) to obtain a PEG block copolymer (PLSP) of poly-lysine: serine. Example 2

Preparation of antisense drugs for IL-1 / 3>

Poly-lysine: a copolymer of serine (PLS: Sigma) or PLSP synthesized in Example 1 and purified Z and either antisense 'oligonucleotide (SEQ ID NOS: 2 and 4) Were formed by a known method.

That is, an ultrafiltration tube (Nippon Millipore Limited): Ultrafiltration C3-GC UFC3 TGC 00 filter with membrane fractionation ability through which the formed complex does not pass A mixture of antisense oligonucleotide and PLS or PLSP was placed in a centrifuge tube) and centrifuged. The oligonucleotides were added so that the concentrations were 1 nM, 100 nM, and 100 nM. In addition, the oligonucleotide of SEQ ID NO: 4 was also prepared at a concentration of 10 M. Under the above conditions, the released oligonucleotides are filtered to the lower layer, and the PLS and antisense ′ (oligonucleotide complex (hereinafter abbreviated as “antisense-PLS”) and PLSP and antisense are used. 'An oligonucleotide complex (hereinafter abbreviated as antisense-PLSP) was obtained.

In addition, turbidity or formation of a precipitate due to the formation of these complexes was examined spectrophotometrically by measuring the absorbance of the solution at 360 nm. In the experiment, the complex was mainly dissolved in a phosphate buffer (pH = 7.2) having an ion intensity of 0.02. As a result, no white turbidity was observed in the solution system when forming the complex of the antisense 'oligonucleotide used here and PLS or PLSP.

The concentration of the oligonucleotide was determined from the absorbance at 260 nm. As a result, it was confirmed that the oligonucleotides in the formed complex had the above concentrations. Also, the concentration of each charge neutralization in the formation of the ionic complex depends on PLS or PLS. Was calculated from the number of charges obtained from the molecular weights of PLSP and oligonucleotides. In addition, it was confirmed that each complex of antisense-PLS and antisense-PLSP is uncharged by charge-coupled electrophoresis (multi-channel electrophoretic device: CAPI-3000, MCPD-3600 SPECT). O 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

The inhibitory effect of the antisense-PLS and antisense-PLSP complex prepared in Example 2 on IL-1 / 3 production was examined in a cultured cell line.

Human macrophage U933 cells (Dainippon Pharmaceutical Co., Ltd.) were used as cells. Cell culture was performed using 10% FCS (Fetal Calf Serum: Sanko Junyaku), 100 unit Zml of penicillin (Life's Technology), and 1 OOug Zml. be sampled replica Bok My Thin using the RPMI medium (Nikken made by Institute for biomedical Research), including the (line-off ♦ made Techno Russia di one company), under the conditions of 3 7 ° C, 5% C 0 2 went. The number of cultured U933 cells was visually counted after staining with trypable.

In the measurement, medium was added 0. 1 5 m 1 comprising U 9 3 7 cells prepared in 3 X 1 0 ⁵ or Zm 1 to microphone Ropure bets 9 6 holes. To these cells, 1 ng / m 1 Cl 2 -o-tetradecanoy 1-phorbol — 13 — acetate (TPA: manufactured by Wako Pure Chemical Industries) and 1 gZm 1 of LPS (manufactured by Sigma) were added. Subsequently, the antisense-PLS or antisense-PLSP prepared in Example 2 was added. As controls, complexes of sense DNA strands (SEQ ID NOS: 1 and 3) with PLS or PLSP (sense-PLS and sense-PLSP, respectively), antisense DNA strand alone and sense DNA strand alone were also used. Each was added.

After incubation for 24 hours, the mixture was sufficiently frozen at 170 ° C., and then thaw-thawed three times, and 2501 of the supernatant was gently collected. These were prepared using an ELISA kit (1L-1 / S ELISA System: manufactured by Amersham), and then subjected to data analysis using a micro-mouth plate reader (manufactured by Bio-Rad). As a result, the complex of the anti-inflammatory antisense drug of the present invention, in particular, the antisense DNA (SEQ ID NO: 2) against the initiation codon of the IL-1 ^ gene and PLS or PLSP was obtained as shown in FIG. In the nanomolar (nM) order, the production of IL-1 / 3 was suppressed by 100%. That is, at an antisense DNA concentration of 100 nM, 100% of IL-1 production was inhibited, at a concentration of 100 nM, 50% of IL-1β production was inhibited, and at a concentration of 1ηΜ, 2%. 0% IL-11 production was suppressed.

On the other hand, in the case of antisense D Ν 非 to the untranslated region (SEQ ID NO: 4), a carrier-specific effect is observed, and only the complex with PLSP has an nM order of IL-11β The production inhibitory effect was observed. In other words, as shown in Fig. 3, in the case of antisense-PLSP, the production of IL-1 is suppressed by more than 90% at an antisense DNA concentration of 1 and at least 80% at 100 ηΜ, At 0 η Μ concentration, 30%. At 1 nM concentration, less than 20% IL-11 production was suppressed. In contrast, in the case of Antisense-PLS, suppression of IL-11 / 3 production was observed at less than 50% at a concentration of 10 M and at a concentration of less than Ι Ο ηηΜ by about 10%. I just stopped.

It should be noted that within the same low concentration range, the binding specificity was remarkably exhibited in the antisense chain and the sense chain. Comparative Example 1

In the same manner as in Example 3, the inhibitory effect of phosphorothioate-type antisense nucleotide (hereinafter, abbreviated as S-antisense) on IL-1 / 3 production was measured. The results are as shown in FIGS. The S-antisense is said to be capable of obtaining a stable and high biological activity in the same evaluation system. <The S-antisense to the initiation codon of the IL-11 / S gene (SEQ ID NO: 2) ) Showed only about a 20% IL-11 production inhibitory effect at 10 M concentration, as shown in FIG. In the case of S-antisense (SEQ ID NO: 4) for the untranslated region, as shown in FIG. 3, at a concentration of 10 M, IL- IL production was inhibited by about 40%. Was. Example 2 In the same manner as in Example 3, IL of the complex of antisense DNA (SEQ ID NO: 2) and ribofectin (manufactured by Gibco) (hereinafter, abbreviated as antisense-livofectin) was used. The effect of inhibiting 13 production was measured. The results are as shown in FIG. This lipofectin has been highly evaluated as a carrier for oligonucleotides, and antisense lipofectin has a considerably higher antisense concentration (50%). M), the effect of inhibiting 〖L-1 / 3 production was about 20%. In addition, there is a difference in the inhibitory effect when compared with the complex of sense DNA (SEQ ID NO: 1) and ribofectin (hereinafter abbreviated as sense-libofectin) used as a negative control. No difference was found in the binding specificity of oligonucleotides. Example 4

Antisense drugs against TNF-α (antisense-PLSP and antisense) were prepared in the same manner as in Example 2 except that the antisense 'oligonucleotides of SEQ ID NOs: 6, 8 and 10 were used. PLS).

For these antisense drugs, turbidity and precipitate formation, the concentration of the oligonucleotide, the charge thereof, and the like were measured in the same manner as in Example 2. Example 5

The TNF-α production inhibitory effect of the antisense-PLS and antisense-PLS S complex prepared in Example 4 was examined using the same method as in Example 3.

However, as a control, a complex of sense D Ν (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. The ELISA kit for effect measurement was a TNF-a ELISA System (Funakoshi).

As a result, as shown in FIG. 5, the complex of antisense DNA (SEQ ID NO: 6) to the initiation codon of the TNF-α gene and PLSP When the concentration of DNA was 100 OpM, the production of TNF-α was suppressed by 80%, and at a concentration of 10 ρΜ, it was suppressed by 60%, but at a lower concentration, TNF-α was suppressed. No production suppression was observed. Remarkably, within the same low concentration range, remarkable angiogenesis specificity was also observed in the antisense strand and the sense strand. As a synthetic polyamino acid forming a complex with an antisense 'oligonucleotide, PLSP showed a higher TNF-α production inhibitory effect than pLS.

On the other hand, antisense DNA against the TNF-α gene splicing site

As shown in FIG. 6, the effect of the complex of (SEQ ID NOS: 8, 10) and PLSΡ on the inhibition of TNF-α production was shown by the antisense DNA against the initiation codon (SEQ ID NO: 6) and PLSΡ. Was almost the same as the composite. The antisense effect on the 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, the effect of the complex of antisense DNA (SEQ ID NO: 6) and lipofectin (manufactured by Gibco) on the inhibition of TNF-α production was measured. The results are as shown in FIG. 7. Antisense-lipofectin had an inhibitory effect on TNF-α production of about 20% even though the antisense concentration was very high (50%). In addition, there was no difference in the binding specificity of the oligonucleotide _c . Further, although not shown in FIG. 7, the S-antisense (SEQ ID NO: 6) was obtained in the same manner as in Example 5. ) Was also measured for its inhibitory effect on TNF-α production. As a result, this S-antisense had an inhibitory effect on TNF-α production of about 20% even at a concentration of 10%. Example 6

An antisense to C0X-2-PLSP was prepared in the same manner as in Example 2 except that the antisense 'oligonucleotide of SEQ ID NO: 11 was used.

For this antisense drug, turbidity and precipitate formation were observed in the same manner as in Example 2. The concentration of the oligonucleotide, its charge, etc. were measured.

Example 7

Of anti-inflammatory antisense drugs on cultured cells

The inhibitory effect of antisense-PLSP prepared in Example 6 on COX-2 production was examined in a human bone cell culture system.

As human bone cells, human synovial cells prepared from tissue resected from a knee joint of a rheumatoid patient were used, and the culture was performed under the same conditions as the U933 cells in Example 3. The number of cells was counted.

In the measurement, 2. Add the 8 X 1 0 of ⁴ / m 9 6 holes of medium 0. 3 ml containing human synovial cells prepared in 1 My Kuropure bets (manufactured Fuaruko down Corp.), 2 Culture was performed for 4 hours. Thereafter, the supernatant was gently removed, and the adhered cells were washed with 300 ^ 1 RPMI medium, and subjected to the following tests.

For each cell, the antisense DNA strand alone and the antisense DNA prepared in Example 6? Then, 0.15 ml of the solution of 8? Was added, and 30 minutes later, 1 ng / ml of IL-1 / 3 (Genezam, Lot. B41399) was added as needed. Antisense DNA strands and antisense—PLSPs are available in several concentrations from 1 nM to 10 M (final concentration). After 2 hours of incubation, collect the supernatants 301 and dilute if necessary, then use the PGE ₂ ELISA kit (Nippon Perceptive Limited, No. 8-6801) Was measured.

As a control, a complex (hereinafter referred to as A) of the S-antisense and gene transfer primers of Comparative Example 1 (Wako Pure Chemical Industries, Ltd .: Code 074-03621) and an antisense DNA chain was used. (Abbreviated as gene transfer). Genetransfer is a ribosome-based carrier, and has been highly evaluated as an oligonucleotide carrier.

Results are as shown in FIG. 8, S- A Nchise Nsu did not completely inhibit the production of PGE _2. Similarly, in the case of antisense-gene transfer, no suppression of PGE production was observed.

On the other hand, antisense-PLSP, the anti-inflammatory antisense drug of the present invention, effectively suppressed the production of PGE on the order of M. That is, 9 2% (IL— 1 PGE 2 production at the time of addition) was suppressed.

It should be noted that within the concentration range tested, the binding specificity of the antisense strand and the sense strand in inhibiting PGE 2 production was remarkable. Example 8

The efficiency of incorporation of the antisense-P LSP prepared in the same manner as in Example 6 into cultured cells was tested.

First, the antisense DNA strand was labeled by the following method. DNA (1.830 D26; unit, 5 pmol) 27.3〃1, autoclaved distilled water 15.71, 10X kinase buffer ( ₂₅ OmM tris) HCl buffer: p H 7. 6), l OO mM DTT, T 4 polynucleotidyl click Rare Ichize solution (1 0 0 unit / / 1), ₇ one ³² P - after mixing the ATP 1 〃 1, The reaction was performed at 37 ° C for 1 hour. In addition, T 4 polynucleotide (Code No. 2030, manufactured by Takara Shuzo) was used for labeling with ³² P.

Human macrophage U933 cells similar to those in Example 2 and human synovial cells similar to those in Example 7 were used as cultured cells. Under the same conditions as in Example II, 1 × 10 ^κ 7 ml of U933 cells were cultured in a glass tube (Falcon's 25058, 6 ml—12X75). For human synovial cells, add 50 1 of antisense-PLSP solution adjusted to the desired concentration, and incubate. For U933 cells, 1 ng / l of 12—o -After adding tetradecanoy 13- phorbol — 13-acetate (TPA: manufactured by Wako Pure Chemical Industries, Ltd.), add Dentisense PLSP (final concentration: 1 ngZm1 force, 10 g / m1). The culture was continued. After culturing for a predetermined time, the culture tube was centrifuged at 25 ° C. for 10 minutes at 150 rpm to precipitate cells, and then the supernatant was gently removed. Next, 3 ml of the same PBS solution as used in Example 7 was added to the cells adhered to the bottom of the tube, and the cells were washed twice, followed by 1% SDS (sodium dodecyl sulfate). The cells were lysed by adding 500 51. Finally, a 10 ml hyperflora solution was added to all of the lysates, and measurements were taken overnight at the liquid scintillator. Also, as a control, S-antisense and antisense-transfer were measured in the same manner as in Example 7.

The results are shown in FIG. 9 and FIG. 10. In the case of S-antisense and antisense-gene transfer, U9337 cells (FIG. 9) and human synovial cells (FIG. 9) were used. Even in FIG. 10), no antisense DNA was taken up into the cells at all, whereas it was confirmed that the antisense-PLSP of the present invention was taken up into the cells over time. That is, up to 3 hours of culture, up to 100 times more DNA than S-antisense, and more than 50 times more DNA than that of antisense gene transfection. Was. Example 9

Antisense-PLSP against mouse IL-1 / 3 and mouse TNF-α in the same manner as in Example 2 except that the oligonucleotides of SEQ ID NOS: 13, 14 and 15 were used. Anti-sense-PLSP was prepared, and its lethal effect on endotoxin-induced shock was examined using model mice.

Antisense-PLS II solution 2001, prepared to the desired concentration with physiological saline, was injected into the tail vein of male BALBZc mice (Japan SLC: approx. 20 g) at the age of 8 to 10 weeks. did. Immediately afterwards, endotoxin (LPS, Lot No. 68692 W. E. col i055: B5. Difco manufactured by physiological saline solution corresponding to 20 mg / kg) Solution 2

0 1 was administered intraperitoneally to mice, and the number of surviving individuals was counted over time.

The results are as shown in FIGS. First, as shown in Figure 11,

When antisense-PLSP to 1L-13 was used, the survival rate of the mouse increased depending on the concentration of the antisense drug administered. In particular, when antisense-P L SP was administered at a concentration of 10 O mg / kg, no deaths were observed for 40 hours.

In addition, as shown in Figure 12, antisense-PLSP at a concentration of 1 O mgZ kg was administered once and antisense-PLSP at a concentration of 5 mgkg was administered twice. When the effects of separate administration (total of 10 mg / kg) were narrowed down, the ability to administer the antisense drug of the present invention in two divided doses at 12-hour intervals was less than that of single administration. It turned out to be effective.

FIG. 13 shows the difference between the effects of administration of antisense-PLSP in the tail vein and intraperitoneal administration. Antisense-PLSP did not show a lethal inhibitory effect when administered intraperitoneally, but showed an excellent lethal inhibitory effect when administered in the tail vein. It was recognized that it caused a difference in the effect.

Furthermore, as shown in Figure 14, when comparing the effects of the antisense drug against IL-13 and the antisense drug against TNF-a, when administered at a concentration of 30 mg Z kg, TNF — The antisense drug against α was more effective in suppressing lethality, and the mouse survival rate after 40 hours was 60%.

From the test results using the above model mice, it was confirmed that the antisense drug of the present invention exhibited excellent drug efficacy even in an in vivo model experimental system. Industrial applicability

According to the present invention, there is provided an anti-inflammatory antisense drug which binds to sense RNA in a primary structure-specific manner and exerts a sufficient effect of suppressing a physiologically active substance in a very low concentration region. As a result, inflammatory diseases such as rheumatoid arthritis, periodontitis, nephritis, ulcerative colitis, arteriosclerosis, psoriasis, diseases such as septic shock, Crohn's disease and AIDS, or intractable diseases Effective treatment for hepatic disease and pathological conditions due to liver transplantation is possible.

Sequence listing

SEQ ID NO: 1

Array length: 20

Sequence type: nucleic acid

Sequence type: Other nucleic acids Synthetic DNA

Array features

Sequence containing the initiation codon of the human IL-1 / 3 gene

GCAGCCATGG CAGAAGTACC 20 SEQ ID NO: 2

Array length: 20

Sequence type: nucleic acid

Sequence type: Other nucleic acids Synthetic DNA

Array features

Antisense strand of SEQ ID NO: 1

Array

GGTACTTCTG CCATGGCTGC 20 SEQ ID NO: 3

Array length: 20

Sequence type: nucleic acid

Sequence type: Other nucleic acids Synthetic DNA

Array features

Includes untranslated region of human IL-1S gene

Array

AGAGAGCTGT ACCCAGAGAG 20 SEQ ID NO: 4 Array length: 20

Sequence type: nucleic acid

Sequence type: Other nucleic acid Synthetic DNA

Array features

Antisense strand of SEQ ID NO: 3

Array

CTCTCTGGGT ACAGCTCTCT 20 SEQ ID NO: 5

Array length: 20

Sequence type: nucleic acid

Sequence type: Other nucleic acid Synthetic DNA

Array features

Sequence containing initiation codon of human TNF-α gene

CCCTGGAAAG GACACCATGA 20 SEQ ID NO: 6

Array length: 20

Sequence type: nucleic acid

Sequence type: Other nucleic acid Synthetic DNA

Array features

SEQ ID NO: 5 antisense peptide

Array

TCATGGTGTC CTTTCCAGGG 20 SEQ ID NO: 7

Array length: 20

Sequence type: nucleic acid Sequence type: Other nucleic acid Synthetic DNA

Array features

Human TNF — Sequence containing the splicing site (positions 1624 to 1643) of the α gene

CCAGGCAGTC AGTAAGTGTC 20

SEQ ID NO: 8

Array length: 20

Sequence type: nucleic acid

Sequence type: Other nucleic acid Synthetic DNA

Array features

Antisense strand of SEQ ID NO: 7

Array

GACACTTACT GACTGCCTGG 20

SEQ ID NO: 9

Array length: 20

Sequence type: nucleic acid

Sequence type: Other nucleic acids Synthetic DNA

Array features

Human TNF — sequence containing the α gene splicing site (positions 2161–2180)

CTCCCTCCAG CAAACCCTCA 20

SEQ ID NO: 10

Array length: 20

Sequence type: nucleic acid

Sequence type: Other nucleic acids Synthetic DNA

Array features Antisense strand of SEQ ID NO: 9

Array

TGAGGGTTTG CTGGAGGGAG 20

SEQ ID NO: 1 1

Array length: 20

Sequence type: nucleic acid

Sequence type: Other nucleic acids Synthetic DNA

Array features

Sequence containing the initiation codon of human C 0 X-2 gene

TGCCCGCCGC TGCGATGGCTC 20 SEQ ID NO: 1 2

Array length: 20

Sequence type: nucleic acid

Sequence type: Other nucleic acids Synthetic DNA

Array features

Antisense strand of SEQ ID NO: 11

Array

GAGCATCGCA GCGGCGGGCA 20 SEQ ID NO: 1 3

Array length: 20

Sequence type: nucleic acid

Sequence type: Other nucleic acids Synthetic DNA

Array features

Sequence containing the initiation codon of mouse IL113 gene GCAGCTATGG CAACTGTTCC 20 SEQ ID NO: 14

Array length: 20

Sequence type: nucleic acid

Sequence type: Other nucleic acid Synthetic DNA

Array features

Antisense strand of SEQ ID NO: 13

Array

GGAACAGTTG CCATAGCTGC 20 SEQ ID NO: 15

Array length: 20

Sequence type: nucleic acid

Sequence type: Other nucleic acid Synthetic DNA

Array features

Mouse TNF — 20-base antisense chain containing the initiation codon for the α 遗 gene

Array

ATCATGCTTT CTGTGCTCAT 20

Claims

The scope of the claims

1 Antisense complementary to a part or all of the base sequence of mRNA encoding a synthetic polyamino acid or a derivative thereof and a bioactive substance involved in human inflammatory disease ♦ Oligo An anti-inflammatory antisense drug comprising a complex with a nucleotide.

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 comprising a repeating sequence of a lysine residue and a serine residue.

3.An anti-inflammatory antisense drug _{c according to} claim 2, wherein the synthetic polyamino acid derivative is a modified polyethylene glycol block of the synthetic polyamino acid.

4. The anti-inflammatory antisense drug according to claim 1, wherein the bioactive substance involved in human inflammatory disease is a bioactive substance relating to human rheumatoid arthritis.

5 Synthetic polyamino acid or a derivative thereof and antisense oligonucleotide complementary to part or all of the mRNA encoding human interleukin-1 / 3 2. The anti-inflammatory antisense drug according to claim 1, which is composed of a complex with a nucleotide.

Antisense oligonucleotide complementary to a part or the entire base sequence of mRNA coding for 1/3 of leucine-1 / 3 is identified as SEQ ID NO: 2 or 4. 6. The anti-inflammatory antisense drug according to claim 5, which is an oligonucleotide having a partial or full nucleotide sequence.

7 Complex of synthetic polyamino acid or its derivative with antisense complementary to part or all of the nucleotide sequence of mRNA encoding human tumor necrosis factor. Oligonucleotide complex 2. The anti-inflammatory antisense drug of claim 1, wherein the drug comprises:

8. The anti-inflammatory antisense drug according to claim 7, wherein the human tumor necrosis factor is human tumor necrosis factor-α.

9 Antisense 'oligonucleotides complementary to part or all of the mRNA encoding human tumor necrosis factor-1 alpha are SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 9. The anti-inflammatory antisense drug according to claim 8, which is an oligonucleotide having any one or all of the nucleotide sequences of 10. 1 0 synthetic poly § Mi Roh acid or a derivative thereof and a series of prostaglandin E ₂ synthase co one sul mR part of NA also rather are complementary Anchisen scan-O Li Gore j click the entire nucleotide sequence 2. The anti-inflammatory antisense drug according to claim 1, comprising a complex with leotide.

1 1 prostaglandin E ₂ synthase claim 1 0 of the anti-inflammatory antisense drug is consequent Rookishigena one Zee 2.

1 2 Chromoxygenase-2 An antisense that is complementary to a part or all of the mRNA that encodes a part of the mRNA, or an oligonucleotide, is a part of SEQ ID NO: 12 or The anti-inflammatory antisense drug according to claim 11, which is an oligonucleotide having an entire nucleotide sequence.