JPH07501204A - Topical oligonucleotide therapy - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Topical oligonucleotide therapy Kourifu! scenery The present invention provides in vivo pre-selected focus sites useful for the treatment of diseases. s) relates to a method for reaching antisense oligonucleotides.

In recent years, oligonucleotides have been shown to clearly inhibit virus replication in tissue culture systems. For example, Zamecnik and 5tephenso have been confirmed to be able to n using Rous sarcoma virus (Rous 5 arcosa Virus), Confirming oligonucleotide-mediated inhibition of viral replication in tissue culture (P roc, Natl, ^cad, Sci, t+, sJ, 175,;280-28 4.1978). Zamecnic et al. 1 virus (currently old v-1) has been confirmed to be suppressed in tissue culture (Zame cnic et al, +Proc, Natl, Acad, Sci, U , S.A., 83; 4145-4146.1986). More oligonucleos Tide is specific by inhibiting translation of the protein encoded by the gene. It is also used to suppress the expression of non-viral genes.

Goodchild et al., using oligonucleotides in a cell-free system, It has been reported that Robulin synthesis can be inhibited (Arch, Bio chem, Biophys, 264i401-409.1988), anti Sense c-myb treatment inhibits human myeloid leukemia cell-like proliferation in vitro. (G, Anfossi, et al, Proc, Natl, Ac ad, Sci.

Ll, S, A,, 8 Shark; 3379.1989). The problem with this method is that Creotide is easily degraded or inactivated by intracellular nucleases. . To address this problem, some researchers have developed mutant oligonucleotides, i.e. A naturally occurring phosphodiester chain is replaced by another chain. A substance with a mutated intranucleotide linkage is used. For example, Ag rawa et al. used oligonucleotide phosphoramide and phosphorothioate. have been shown to enhance inhibition of IIIV-1 in tissue culture using Pro c, Natl, 8cad, Sci, U, S, A, 85; 7079-7 083°1988). 5arin et al. has been shown to enhance the suppression of HIV-1 (Proc, Natl.

Acad, Sci, U, S, 8, 85. ;744B-7451, 1988 ). Agrawal et al. in both early infection and chronically infected cell cultures using holotioate. has also been shown to inhibit HIV-1 replication (Proc, Natl.

Acad, Sci, U, S, A, 86; 7790-7794.1989 ). Using phosphorothioates of oligonucleotides, Leither et al. reported inhibition of influenza virus reproduction in tissue culture (Proc. Nat I.

Acad, Sci, U, S, 8, 87. ;3430-3434.1990).

Oligonucleotides with artificial linkages are resistant to degradation in vivo It has been confirmed that there is. For example, Shaw et al. Therefore, if the 3' end is protected, on the other hand, the unmutated oligonucleotide are more resistant to nucleases in vivo and are further capped. It has been shown that oligonucleotide phosphorothioates are not degraded in vivo. Reported (Nucleic Ac1ds Res, +1747-750. 1991).

Antisense oligonucleotides can selectively interfere with protein synthesis. While there are, and significant advances have been made to improve their intracellular stability, oligonucleotides In order for leotide to function in vivo, it must reach its intended intracellular site of action. The problem of what must be done has not been resolved. The intended therapeutic effect is systemic The oligonucleotide can be administered systemically. However, biological It is necessary or preferable to administer the oligonucleotide to a specific head within the In some cases, systematic administration is usually expected to fail.

If the target mRNA^ is present in normal cells as well as in the target tissue, and When antisense RNA that binds to induces an unnecessary physiological effect, This is especially true. In other words, locally significant Antisense oligonucleotides administered systemically in sufficient doses to have an effective effect Doses of cleotide may be toxic to patients.

How to limit the action of antisense oligonucleotides on target tissues Improving brings great benefits.

Such treatment guidelines include inducing restenosis that occurs following cardiovascular disorders. An example of this is inhibition of smooth muscle cell proliferation.

plays a major function in numerous pathological morphological changes, including arteriosclerosis and hypertension. The proliferation process of smooth muscle cells remains largely unknown. Proliferation of smooth muscle cells is Long-term disability for coronary and peripheral angioplasty, similar to coronary bypass grafting induce harm.

Vascular smooth muscle cells in adult animals are characterized by an abundance of contractile proteins. It has a distinct phenotype. This protein mainly acts on muscle actin and myosin. It is explained by S, M, Scl+wartz et al. (S, M, Schwartz, G.R., Ca5pbell.

J, H, Ca5pbell + Cr1c, Res, + 58 + 427 + 1 986). Furthermore, the endoplasmic reticulum is clearly missing. In vivo against injury Mature smooth moving cells when damaged or placed in in vitro cell culture (SMCs) undergo distinct phenotypic changes and lose their differentiation status. Thin The vesicles acquire large amounts of endoplasmic reticulum and begin to actively form extracellular matrix. . Furthermore, cells contain non-muscular actin and myosin, as well as R, J, and Dill. ey et al. (^theroscleosis, 63:99.1987) and P , Libby et al. (N. Engl., J. Med.

31 Needles 1493, 1987) began to express a number of new proteins, while Kuro-o et al. (J, Biol.

Chew, 264:1B272. 1989) Ya^J, CIow et al. As shown by Ce1l, Biol, IC.1939.1988), Expression of smooth muscle-specific contractile proteins such as smooth muscle myosin heavyweights and alpha-actin decreases.

The nuclear oncogene c-myb may play an important role in these changes. be. The oncogene is homogeneous with the transforming gene of avian myeloid saturation virus. moreover , despite considering that originally only blood stem cells are expressed, c-Bb was confirmed to exist in bird embryo fibroblasts as well as proliferating SMCs. (C, B, Thompson, et al, Nature+ 31 9:374.1986+ and C, F. Relly, et al., J. Biol, Chem, 264:69902.1989).

The human c-myb gene has been isolated, cloned, and sequenced ( Majelio, et at, Proc, Natl, Acad, S ci, IJSA+83:9636-9640.1986), c-myb Expression is growth dependent.

This gene is present only at low levels in stationary cells, but begins and rapid proliferation progresses, reaching a peak near stage 61, the final stage of the cell cycle. (C, F, Relly, et al, +J, Bio1.Chem, , 264:69902.1989), and furthermore, the expression of c-wyb is It has been shown that this is related to the differentiation state of . Myeloblast cells are induced to differentiate, As a result, the expression of c-myb is suppressed.

Heparin and proteoglycans closely related to heparin can be used both in vitro and in vitro. Blocks smooth muscle cell proliferation in vivo R, Guyton, et al. ciric, Res, + 46:625, 1980, and Tori, M.S.

Fr1tze, et al, J, Ce11. Biol,, 100:10 41.1985), this suppression occurs after the cell cycle! Occurs in the lllG1 stage and c-my b expression levels (but not c-fos or c-wyc levels). (C, R, Re1lLy+et al, +J, Bio1.Chem,, 26 Rainbow 6990.1989) and, in part, the expression of smooth muscle-specific contractile proteins. (M, to tlro-0+ et al-+J, Bio1.Che m,, 264:1989 and ^J, CIowes+ et al,, J , Ce1l.

Rial, 107:1939.1988). c-syb is resting smooth muscle Since it has been shown that it contains the initiation of cell proliferation, Parin may exert an antiproliferative effect through its action on c-myb. stomach Provides a method for delivering oligonucleotides to specific sites in vivo , thereby causing viral genes, oncogenes, diseases and other pathological conditions. It is an object of the present invention to provide local suppression of the expression of genes encoding proteins. It is.

Kosho Ω Kaname Leopard The present invention provides in vivo, locus, preferential targeting nucleic acids or antibodies. a nucleic acid sequence that is complementary to at least a portion of the antisense oligonucleotide. Using surgical or catheter administration methods of specific oligonucleotides It also includes administration directly to the target tissue. The oligonucleotide is Metsenger 11N^ (IR This is an antisense sequence specific for NA). antisense oligonucleotide hybridizes to the target TlRNA and thereby releases its encoded protein. It inhibits translation into white matter. In this way, the method of the present invention can be applied to selected genes. Therefore, expression of the encoded protein is suppressed. Furthermore, the dynamic Experiments have shown dramatic local therapeutic effects in vivo.

Preferably, the oligonucleotides of the invention are resistant to cellular nucleases and in vivo. resistant to degradation and/or expansion by other enzymes present. It is mutated into. This can be accomplished by methods known in the art. for example , one or more internal artificial chains, such as substituting sulfur for phosphate in the chain. Integrates intranucleotide linkages and/or has a capping structure by blocking the 3' end of the oligonucleotide, etc. can. Oligonucleotides of the invention preferably contain about 14 to 38 nucleotides. nucleotides in length, more preferably 15 to 30 nucleotides in length. .

Oligonucleotides to suppress expression of selected proteins in surrounding regions , administered locally. In a preferred embodiment, antisense oligonucleotides The electrode is placed in a biocompatible matrix or carrier and applied locally to the tissue surface. to be administered. The matrix or carrier is poly(propylene oxide). Hydrogel materials such as (ethylene oxide) gels can be used. For example, room temperature or is a liquid at temperatures below and becomes a gel at or above body temperature. It is a substance.

In this usage, oligonucleotides are mixed with a hydrogel material and the mixture is surgically Alternatively, administer to the required site via catheter. Oligonucleotides also gel can be supplied into solution by liquefying, that is, cooling. Ru. It is then retained at the site of administration as a gelled solid. Other carriers available For example, liposomes, microcapsules, red blood cells, and these It is possible to raise analogues of . Oligonucleotides can be administered locally by direct injection. It can be administered manually or using devices such as implanted stents or catheters. It can also be delivered directly to the affected area using an infusion pump. stomach.

The method of the present invention requires that in order to inhibit the expression of a gene encoding a protein, Useful as well as regulating non-encoding DNA that is normal sequence . Because antisense oligonucleotides reach specific, defined locations, Additionally, these oligonucleotides can be used in vivo even when systemic administration is not possible. It is possible to use For example, systemically administered oligonucleotides by endonucleases that disable them before they reach their target sites. It may become inactive. have harmful or toxic effects on the patient Large doses of oligonucleotides may be necessary for successful systemic therapy. It is considered to be Anti-antibody Using oligonucleotide therapy by delivering sense sequences, a large number of specific The methods of the present invention provide a means for treating certain diseases.

Brief description of the drawing Figures IA and B show antisense NMMHC (A) and antisense at various concentrations. Count the number of Sv-smooth muscle cells (SMC) treated with c-myb (B). This is a graph showing the results of counting.

Figures 2A and B show antisense IINMMHc (8) and c-myb (B) treatment. SV40TL-3MC cells, BC3H1 cells, horse aorta SMCs and Mouse aorta S? A graph showing the results of counting the number of cells for each IC. It is f.

Figure 3A shows S cells treated with antisense c1yb and NMMHC at different time intervals. It is a graph showing the effect on proliferation of V-3MC cells.

Figure 3B shows non-mutated antisense cells 16 and 40 hours after removing growth inhibition. SV-5MC treated with sc-myb (bright bars) and NMMHC (dark bars). This is a bar graph showing the effect of

Figure 4 shows sense c-*yb (S Myb), antisense c-wyb (^S M yb) and heparin-treated SV-5MC cells. c-&Iyb compared to untreated (control) and growth-suppressed (G^) cells. It is a bar graph showing the results of RNA dot plot.

Figure 5 shows oligonucleotides from Pluronic® 127 gel matrix. It is a graph showing the release rate of cleotide.

Figure 6 is a graph showing the release rate of oligonucleotides from the EVAc matrix. It's rough.

Figure 7 shows a delivery system for the administration of oligonucleotides to an injured artery. using Pluronic gel and ethylene vinyl acetate matrix (EVAc). We investigated the effects of antisense c-syb (AS Myb) on rat arteries. FIG. 3 is a bar graph showing the endo#/medium ratio as a measure of intimal proliferation.

Figure 8 compares a gel that does not contain Physalis, a gel that contains sense c-myb, and an untreated artery. To do this, antisense c-vyb was added to rat arteries using Pluronic gel. It is a bar graph of the effect when administered to.

Figure 9 shows the effect on intraarterial cell proliferation after balloon angioplasty treatment. , a mixture of antisense c-syb and human NMMHC (200 μM each) was added to the It is a bar graph showing the results of administration into the heron artery.

Mitsuho m sucking of genes encoding proteins using antisense oligonucleotides. A method for inhibiting expression is described. This method is used in vivo to It is based on local administration of oligonucleotides. oligonucleotide Preferably, the mixture with the graft &11 tissue or gel is administered directly to the target tissue. or by direct administration or injection. Furthermore, oligonucleotides cells are treated in vivo to be resistant to degradation and alteration by intracellular enzymes. It is managed.

Oligonucleotide′ Therapeutic approaches using antisense oligonucleotides disrupt gene function by interfering with translation of the encoded protein It is based on the principle that it is possible. This is the method transcribed from genes. complementary to at least a portion of Tsenger RN^ (sRNA); This can be achieved by supplying oligonucleotides of appropriate length. Ann The antisense strand hybridizes to mRNA and binds +nRN to destroy it. Target ^. Thereby, ribosemal translation and subsequent Protein synthesis is hindered.

The specificity of antisense oligonucleotides is determined by the specificity of the oligonucleotide on the target nucleic acid. Ha toson-Crick base pair form between a heterocyclic base and a complementary base For example, a nucleotide sequence with a length of 16 nucleotides is derived from , appears to occur randomly every approximately 41k or 4×109. Therefore, this It is predicted that there is only one such sequence, even in the human genome.

In contrast, a nucleotide sequence with 10 nucleotides is about 416 or appears approximately every 1×10h nucleotide. Such sequences There are thousands of times more than Nomu. Therefore, longer length oligonucleotides are shorter More specific than long oligonucleotides and requires no hybridization It is less likely to induce toxic complications. Therefore, the originality of the present invention Preferably, the gonucleotide has a length of 14 bases or more. About 14? Preferably, the oligonucleotide consists of about 38 bases, and more preferably about 15 to 38 bases. Particularly preferred are 30 base oligonucleotides.

Oligonucleotide sequences are selected based on sequence analysis of the gene to be inhibited. be done. Gene sequences can be obtained, for example, by isolation and sequencing of the gene, or if known. This can be determined from the literature, if any. The oligonucleotide sequence is sense j sequence, this sequence is complementary to the coding strand of the molecule. It has a sexual nature. The sequence of such oligonucleotides at least corresponds to the gene sequence. There is a substantial match for a portion of the column. and mRNA transcribed from the gene is complementary to

Viral and microbial genes essential for infection and replication, and related to disease processes. Genes encoding proteins related to diseases such as autoimmune disorders and cardiovascular diseases Regulatory sequences that control the expression of proteins involved in diseases and other disorders. To suppress the expression of which, oligonucleotide therapy can be used.

Oligonucleotides useful in the present invention include multiple known techniques for nucleic acid synthesis. It can be synthesized by The oligonucleotides are preferably as detailed in the Examples. Automatic synthesizer model 8700 (Milligen-Biosea) explained in detail Automated synthesizers such as R.C.R.H., Burlington, MA) or controlled ABI model 38 using H-phosphorylation chemistry on porous glass (CPG) It can be successfully synthesized using an automatic synthesizer such as OB. oligonucleoside A detailed description of the H-phosphonate approach for thiophosphorylation synthesis can be found in Ag rawal and Tang (Tetrahedron L etters+31 Near 541-7544.1990). disclosed here The contents are also included in the present invention. Oligonucleoside methylphosphonate esters, phosphorodithionates, phosphoramidites, phosphate esters and The synthesis of bridged phosphorothioates is known from the prior art. Examples of the following documents do. Tetrahedron Letters+28:3539, 1987, N1elsen et al, Tetrahedron Letters 29:2911゜1988, Jagar et al., Biochemist ory 匡7237, 1988, Uzanskyet al, Tetrahe drone Letters, 2 losses 3401, 1987, Bannwarth+ He1v.

Chtm, Acta, July 1517, 1988, Crossstick an d Vyle, Tetrahedron Letters, 30:4693,19 89, ^grawa1. et al, , Proc, Natl, 8cad, Sc i.

USA, 8.1401-1405.1990゜Contents described in documents above Even if it is, it is integrated with the present invention. Synthesis or preparation methods other than the above methods is also available. Although ribonucleic acid (RNA) can also be synthesized and supplied, the preferred In examples, the oligonucleotide is deoxyribonucleic acid (DNA).

Oligonucleotides useful in the present invention preferably target intracellular nucleases. Designed to resist degradation due to In vivo degradation of oligonucleotides Produces oligonucleotide degradation products with reduced length. Such a partition is a non- It is preferable to bind by specific hybridization. and standard length It is desirable to have substantially reduced effectiveness against the other party. This way Uni, oligonucleotides are resistant to degradation in living organisms and can reach target cells. It is preferable to use otide. The oligonucleotides of the present invention are natural phosphors. replacing a fodiester linkage with one or more artificial intranucleotide linkages by replacing phosphoric acid with sulfur in the chain, for example. It can show even more resistance to decomposition. Examples of chains that can be used as, phosphorothioate, methylphosphonate, sulfone, sulfate, ketyl , phosphorodithioates, various phosphoramidates, phosphate esters, Includes bridged phosphorothioates and bridged phosphoramidates. Other Nucles Since interocdinal linkages are known in the prior art, such above illustrations are not limiting. This is not a practical example, but a demonstration example. As a known document, rcohen+Trendsi n.Biotechnology (1990). Hoss Oligonucleotides with one or more substitutions for the nucleotide chain of the hodiester synthesis of oligonucleotides with mixed intranucleotide linkages. are known from the prior art, including synthetic steps for making them.

Oligonucleotides can be "capped" or have 5' or 3' ends. Attaching similar groups to nucleotides makes them resistant to elongation by intracellular enzymes. can create resistance. Appl as a reagent for capping ied Biosystems+Tnc,, Foster C1ty, C^, Products sold under the product name 11w1no-Link 11 (Registered Trader 1) can be purchased on the market. The camping method is, for example, Show et al. , Nucleic Ac1ds Res, 1 flood 747-750.1991, Agrawal+et al,, Proc, Natl, Acad, Sci, U SA, Dandan (17) Near 595-7599.1991. Below The contents described in the above documents are also integrated with the present invention.

Oligonucleotide 1゛ According to the present invention, the in vitro By limiting the amount of antisense compound that acts on the target site in the body, The original binding characteristics of antisense oligonucleotides, which are characterized by the pairing of groups. The opposite sex is encouraged. (so that the oligonucleotide can be used to achieve the desired effect) It is administered locally. The concentration of oligonucleotide at the desired site is determined by significantly higher than if the nucleotide were administered systemically. and therapeutic effect can be achieved using significantly lower total doses. Locally high concentration of oligonucleotides promotes penetration into target cells and effective blocking of translation of target nucleic acid sequences.

The oligonucleotide is localized by a method suitable for local administration of the drug. can be reached. For example, oligonucleotide solutions can be injected directly into the topical area. It can also be delivered by intravenous drip using an infusion pump. Sara Oligonucleotides can also be combined with implantable devices. This outfit When placed at the desired site, oligonucleotides at surrounding sites are released. It's about to be served.

The most preferred administration of oligonucleotides is using hydrogel materials. . Hydrogels are non-inflammatory and biodegradable. Many of these materials are known, including those prepared from natural and synthetic polymers. preferable In embodiments, the method is a liquid at a temperature below body temperature; At temperatures around that, gelation occurs, forming semisolid hydrogels that retain their shape. It uses a hydrogel that

Preferred hydrogels include ethylene oxide-propylene oxide. Polymers with repeating units of

The properties of the polymer depend on the molecular weight of the polymer and the particles in the polymer. preferred hide Logel contains about 10% to about 80% ethylene oxide and about 20% to about 90% by weight Contains propylene oxide. Particularly preferred hydrogels have approximately 70 χ ports. Contains polypropylene oxide of 30x polyethylene oxide. Interest Hydrogels that can be used include, for example, RASF Corp., Parsippany It is available from +NJ under the trade name Pluronic.

In an embodiment of the invention, the hydrogel is cooled in a liquid state, and one gram of the hydrogel is cooled. Mix oligonucleotides to a concentration of 1 mg oligonucleotide per ram. . The resulting mixture can then be used, for example, by spraying, intraoperative application or atomization, or catheterization. It is administered to the surface of the treatment area using a tube or endoscopy. When the polymer warms up It solidifies into a gel, and the oligonucleotide is stored for a period of time determined by the composition of the gel. After this period, it diffuses from the gel into surrounding cells.

Oligonucleotides are available commercially and are available in libraries published in the scientific literature. Using implants, which are posomes, microcapsules and implantable devices It can also be administered by means. For example, polyacid anhydrides, polyorthoesters, Polylactic acid, polyglycolic acid, and their copolymers, collagen, protein polymers, Biodegradable materials such as rimers, ethylene vinyl JL/acetyl-(EVAc), Non-products such as polyvinyl acetate, ethylene vinyl alcohol, and their derivatives Biodegradable materials can be used for local delivery of oligonucleotides . The use of melting or solvent evaporation techniques on materials that polymerize or solidify, or materials and machinery. Oligonucleotides can be integrated into the material by chemical mixing etc. . In one embodiment, the oligonucleotide is a silica gel coated dextrin. Implantable devices such as runs, stems, catheters) are mixed into or coated with and administer.

As described in the Examples below, the dosage of oligonucleotide is Depends on the size of the tablet and the purpose for which it is administered. Generally, the dosage range is calculated based on the surface area of the tissue being treated. The effective amount of oligonucleotide is , somewhat dependent on the length and chemical composition of the oligonucleotide, but typically It ranges from 30-3000 μg per square centimeter of surface. rat model antisense in hydrogel to blood vessels injured by balloon angioplasty According to the calculations used to administer +syb, the dose per square centameter of tissue is The amount of oligonucleotide administered was approximately 320 μg, and at this dose c-+wyb Suppresses the expression of gene products. Oligonucleotides have both therapeutic and prophylactic purposes may be administered systemically to patients. For example, c-myb, NMMHC and and/or PCNACN antisense oligonucleotides may be used in angioplasty or may be administered to patients who are at risk for restenosis versus other methods of There is.

Oligonucleotides can be administered by any effective method, e.g., parenterally ( intravenously, intraperitoneally, intramuscularly) or orally, nasally, or Are there any other routes that are acceptable for otide administration and circulation into the patient's bloodstream? There is administration of these. Systemic administration of oligonucleotides is preferably in addition to local administration. However, it is beneficial even without topical administration. In general, human adults - It is effective for this purpose if administered within the range of 0.11-10.

Jiou↓■Danyo The method of the invention can be applied to a variety of methods that involve or are based on the expression of proteins by genes. It can be used to treat diseases. This book is especially useful for vascular diseases and diseases, especially vascular restenosis. The method is useful. The following non-limiting example provides an antisense oligonucleotide that Vascular re-narrowing following vascular injury, such as induced by balloon revascularization procedures. An example of its use in the very clear inhibition or prevention of stenosis is shown. this is, using locally delivered antisense has been implicated in vascular restenosis. By inhibiting the expression of genes encoding identified proteins. This has been achieved. This gene is c-myb, non-muscle myosin heavy chain (N? IMHC) and growing cell nuclear antigen (PNAC). However, the present invention The method has many other uses.

Expression of specific genes in specific 1114M is determined by transcription of target genes - RNA inhibited by an oligonucleotide having a nucleotide sequence complementary to It will be done. Both c-myb and non-muscle myosin proteins are involved in smooth muscle cell growth. It is clear that it is clearly related to the antisense Inhibition of production of these proteins by oligonucleotides may be related to smooth muscle proliferation. angioplasty, restenosis, and arteriosclerosis, hypertension, and temporary methods for treating chronic disease processes such as hypertension and proliferative glomerular kidney disease. provide. Examples of other conditions that can be treated by the method of the invention include: The following primary diseases are listed. Acute respiratory distress syndrome, idiopathic pulmonary fibrosis, emphysema , - secondary pulmonary hypertension. These symptoms may occur, for example, when combined with an aerosol from an inhaler. can be treated with appropriate antisense that can be administered topically. Ru. These diseases affect the alveoli (air side), the underlying basement membrane and smooth muscle cells, and What is the pathological fact that is occurring between the epithelial cell surface (blood side) and the nearby epithelial cell surface? ! ! sloppy Induced by stacking. Alveolar macrophages, through T cell receptors, Collects white blood cells that recognize specific antigens, become activated, and stimulate fibroblasts. It has been speculated that a variety of substances, such as PDGF, can be produced. white blood The spheres gradually overwhelm the existing anti-proteases and become alveolar phenomocytes. Secretes proteases that cause damage. Fibroblasts induce fibrosis Secretes extracellular matrix. PI) selected growth factors such as GP; Subsequent blood flow that is secondary to causing damage to the alveolar membranes Decreased oxygen induces smooth muscle proliferation. This compresses the blood vessels of the microvascular system. , reduces blood flow in the lungs. This further reduces oxygen transport in the blood. Up The events that occur on the molecules outlined are the All tissue events that initiate blood coagulation. The presence of selectins and integrins, as well as the presence of progenitors, contribute to the endothelial Induces cell surface activation. These selectin and integrin surface receptors The target causes white blood cells to adhere to microvasculature endothelial cells, and then to these cells. Like other molecules that cause damage, proteases are released, causing fluid to enter the alveoli. Let it accumulate. The above events also trigger thrombus formation in the microvasculature due to blood vessel occlusion. becomes. The end result of these processes is that oxygen exchange is further disrupted.

Antisense oligonucleotides that reach the alveolar/microvascular region locally are targets can be interfered with using the pathological theory outlined above.

This is due to the cDNA sequence of all selected targets. It is. In this way, antisense molecules specific to mRNA transcribed from genes Ligonucleotides may inhibit alveolar macrophages by preventing the initiation of the above events. In addition to inhibiting phage T cell receptor production, it also exhibits the following effects. alveolar white Inhibits protein production by preventing activation of blood cells and prevents damage to alveolar membranes inhibition of elastase production, recruitment of leukocytes or consequent Inhibition of PDGF production by preventing fibrosis, c to suppress SMC proliferation -inhibits myb production and prevents adhesion of leukocytes to alveolar microvascular endothelial cells Inhibition of production of p-selectin, e-selectin or various integrins for , tissue factors for inhibiting microvascular thrombosis and inhibition of FAI-1 production. harm.

As an additional example, tissue factor (TF) requires activation of the coagulation system. to show that Targeting 5RNA or DNA of TF fragment at the site of thrombus formation Topical administration of antisense can prevent further aggregation. This treatment may be used as an adjunct to or as an alternative to systemic anticoagulant therapy, or can be employed after thrombolytic therapy, thereby avoiding systemic side effects be able to.

Plasminogen activator inhibitor (FAI-1) is a tissue plasminogen It is known to reduce the local concentration of protein activator (TPA). Hi The cDNA sequence of FAI-1 is known. -RNA or DNA of PAI-1 Local administration of antisense that targets . It is used to naturally dissolve blood clots without causing systemic side effects. This results in the production of a considerable amount of TP^.

The combination of antisense TF and antisense PA[-1 can induce local thrombolysis. To maximize the effectiveness of treatment of various diseases, including after therapy and after prophylactic angioplasty procedures. It may be effective for

Many other vascular diseases require identification of target DNA or mRNA sequences. Therefore, it is possible to treat in a manner similar to that described above. anti Sense therapy is useful in treating the following diseases: Myocardial disorders, peripheral muscle disorders , peripheral angiogenesis, thrombophlebitis, cerebrovascular disease (stroke, embolism), vasculitis (temporal artery inflammation), angina and Budd-Chiari syndrome.

The following examples are illustrative of the invention and do not limit the scope of the invention. It is not intended.

Implementation ± Hayashi Kangdan Soguta Nuki SV40LT-SMC (rat smooth muscle cells, C, Relly, Merck+ 5harp and Doh + me, West Po1nt, distribution from PA heat-inactivated fetal bovine serum (Gibco-BRL, Bethes Culture using Dulbecco's modified Eagle's medium (DMEM) containing 10x of da, MO). fed. BC3H1 mouse smooth muscle cells (ATCC CRL 1443. Ameri can Type Culture Co11ection.

Rockville, MD) heat-inactivated fetal bovine serum (FBS) Culture was performed using DMEM containing χ. Cell culture was performed at 37°C in a 5χ carbon dioxide atmosphere. I went with Iki.

Primary aortic smooth muscle cells (SMCs) were derived from Sprague-Dawley rats ( In vitro transplantation technique was performed from FVB mice (average weight 350 g) and FVB mice (average weight 50 g). isolated using (Ross, J., Ce1l Biol., 50:172-18 6.1971).

The cultures exhibited the characteristic morphological features of vascular SMCs (spindle-shaped and hill-shaped). pattern). Vascular SMCs recognize the presence of smooth muscle α-actin isoforms. Confirmed by Xan analysis.

Primary aortic SMCs were used for secondary passage.

Antisense and sense 18-mer thiophosphorylated oligonucleotides are AB I DNA Synthesized using a synthesizer. Oligonucleotides are deprotected on-device TE (10mM Tris+ pH7.5+1sM EDT^, pi+ 8.0) and quantified using an absorbance meter and gel electrophoresis. Next arrangement A column was adopted.

Antisense c1yb oligonucleotide Mongou No. 1”- GTGTCGGGGTCTCCGGGC antisense NMM) IC oligonucleotide Ochido Kai IILLI- CATGTCCTCII: ACCTTGGA antisense thrombomodulin ( TM) Oligonucleotide y Death Violet”- 2 compared to ACCCAGAAAGAAAATCCCAAG mouse c-myb Antisense human c-myb oligonucleotide sequence with a mismatch of JAJ L”- GTGCCGGGGTCTTCGGGC SEQ ID NO: 1 is a mouse c-myb nucleus. It is a complementary strand of octide 4-22 (Bender et al, Natl, 8c ad, Sci, USA Black 3204-3208.1986).

SEQ ID NO: 2 is the complementary strand of human NMM11C-^nucleotides 232-250. (Simons et al, Cir, Res, 6 losses 530-539. 1991), SEQ ID NO: 3 is the complementary strand of mouse TM 4-25 (Ditt man and Majerus.

Nucl, Ac1d Res, 17:802.1989). Sequence number 4 is It is a complementary strand of mouse c-myb, and has two base mismatches compared to mouse c-myb. are doing. The NMMHC sequence has the highest homology with known non-muscle myosin sequences. They were also selected from nearby areas. The sequence is human NMMHC-B (Simons et al., ditto), and one nucleotide differs from chicken NM phylum H. C-8 and NMMHC-B differ by two nucleotides. Corresponding sense array was used as a control.

Example 1: c-myb using antisense oligonucleotides in vitro and inhibition of NMMHC Proliferation courage Both cell lines, similar to early passage cells of primary aortic SMCs, have 6-well cells. IOXFBS-D in (Coastar, Cambridge, M^) MEM (Niha 202FBS-DMEM for BC3H1 cells) per 1 well Seeding was performed at a cell density of 25,000 cells. Phosphate the cells the next day After washing twice with buffered saline (PBS), the medium was diluted with 0,5χ FBS, which is a growth-inhibiting medium. - Replaced with DMEM medium. Cells were maintained for 96 hours in anti-proliferation medium. Next The medium was replaced with 10χ or 2oχ FBS-DMEH, and the synthetic c-1Ilyb , NMMHC antisense and sense oligonucleotides were added. The cells are Grown for 72 hours, trypsinized and counted in a Coulter counter.

Two cell lines and SMC cells were grown in 10χ and 202FBS-DMEM. and oligonucleotides were added. The number of cells is 5-8 as described above. Counted after days. Each experiment was performed in triplicate and at least two additional times. I did it repeatedly. The data are expressed as the average Hanato standard deviation value.

The results (see Figure 1) are as follows. c-myb (SEQ ID NO: 1) in vitro. The antisense oligonucleotide to NMMHC (SEQ ID NO: 2) is substantially significantly suppressed cell proliferation. On the other hand, sense oligonucleotides showed no effect and only The results were similar to those obtained using Udon Tris-EDTA M serum.

The oligonucleotides used were human/chicken IIMMHc or mouse cm It was derived from the nucleotide sequence of yb cDN^. , 18 bases When two bases of the c-n+yb antisense sequence of Column number 4), antisense oligonucleotides completely lose their antiproliferative effect. We were able to demonstrate the importance of the specificity of leotide. The results of this test are as follows: Ru.

77 Chisense c-myb 475,600 +25,000 cells, Mismatch Antisense c-myb 958,800±12.000 cells, sense c-my b 935+200±22.000 cells. In this way, mismatched antisense c -myb (SEQ ID NO: 4) cannot reliably inhibit the proliferation of SMC cells. Antise Sense and Sense Phosphorothiolate Thrombomodulin (TM) Oligonucle Reotide had no effect on SMC proliferation (antisense T?l 364, 580±19,000 cells, SenseTM 376.290±11, 000 cells ).

Acts directly on N M l’l II C compared to c-lock yb The inhibitory effect of antisense thiophosphorylated oligonucleotides is clearly concentration dependent. (antisense NMMHC; the inhibition rates of 2μ and 25μH were They are 32χ and 65χ. Antisense c-myb; 2μ and 25μ-, respectively The inhibition rates are 33χ and 50χ). of the relative proportions of these two messages. , a rough prediction shows that: c-dense yb mRNA is index Only extremely low concentrations are obtained in actively proliferating SMCs (0% of poly4+ RNA ．． 01%), but NMMHC mRNA is present at clearly high levels.

Observed concentration dependence of two antisense oligonucleotides involved in growth inhibition The gender was consistent with the relative abundance of the two mRNAs.

Antiproliferative effects of antisense and sense phosphorothiolate oligonucleotides used primary rat and mouse aortic SMCs as well as BC3111 cells. Evaluation was made using the line. The following data were obtained. In other words, the proliferation of the three types of cells is Although clearly suppressed by sulforothiolate antisense, sense NMMH C or c-Bb oligonucleotides (Fig. 2).

Antisense c-myb oligonucleotides were isolated from rat aortic SMCs and rat S Mouse aortic SMCs and mouse BC3 old cells compared to V40LT-5MCs showed a stronger antiproliferative effect (Fig. 2B). The difference in growth inhibition depends on the selected range. So, there is a mismatch of antisense nucleotides between the rat and mouse c-+wyb sequences. Most of this is due to the size of the chi.

antisense NMM) IC or c-myb phosphorothiolate oligonucleo Achieving maximum growth inhibition by contacting SV40LT-SMC with The minimum time required for this has been determined. In the studies presented above, cells were The growth-inhibiting effect of the cells was observed from the time of transfer from the growth-inhibiting medium to 10χ FBS-DME. Continuous contact with oligonucleotide for 72 hours until counting and measurement I let it happen. In the next experiment, SV40LT-SMCs were treated with antisense oligonucleotide for a certain period of time, then washed twice with PBS. Replaced with fresh 10x FBS-DMEH without oligonucleotide, 72 The growth inhibitory effect was evaluated by counting the number of cells after a period of time. Shown in Figure 3A The results show that to produce a significant anti-proliferative effect, a period of 1 hour or less; Add 25 μH of antisense NMMHC or c-myb oligonucleotide. There were 61 good things that happened. antisense NMM) IC oligonucleo The degree of growth inhibition after 2 hours of treatment with Tido was determined by continuous contact for 72 hours. The results were comparable (65x inhibition of cell growth).

Antisense c-myb oligonucleotide mixture achieved antiproliferative effect in 4 hours. Ta. This effect was observed after 72 hours of continuous contact (cell increase 50x inhibition of growth).

Antisense phosphorothiolate oligonucleotide growth inhibition is easily reversible We conducted an experiment to see if it was possible.

In the final step of this experiment, SV40LT-3M was released from growth inhibition and then 5μ-antisense or sense NMM) IC or c-myb oligonucleotide It was exposed to leotide for 4 hours. Cells were subsequently washed twice with PBS and Fresh 10χFBS-DME without oligonucleotides? Replace with l medium, Cell numbers were measured 3 and 5 days later. antisense NMMHC or c-a+ Proliferation of SV40LT-SMCs treated with yb was significantly affected by the corresponding sense oligonucleotide. When compared to Otide, after 3 days there was a clear increase in proliferation of approximately 65χ and 50χ, respectively. The data revealed that this indicates early inhibition. But antise 3 days of SV40LT-SMC treated with NMMHC or c-myb The doubling time between day 5 and day 5 is In this case, it was the same after 22 hours.

16 hours after release from growth inhibition, 25μ native antisense NMM)I Treatment of SV-40LT-SMC with c- or c-iyb oligonucleotides , no discernible antiproliferative effect was obtained at 72 hours.

On the other hand, if two oligonucleotides were brought into contact at the same level for 40 hours, In this case, an inhibitory effect similar to that observed using thiophosphorylated derivatives was obtained (Fig. 3B ).

Antiproliferation of antisense NMMHC and c-myb thiophosphorylated oligonucleotides The importance of growth inhibition on sexual vigor was evaluated. 10 μH antisense NMMHC or exposed to c-Bb oligonucleotide to increase cell numbers for 72 hours and 1 When counted at 2020 hours, 5V40LT-5I'IC grew exponentially. Ta. Antisense N? Treatment of Sl'IC with IMHC oligonucleotides No growth inhibitory effect was shown at either time point. However, antisene Contact with sc-myb oligonucleotide inhibited proliferation by 19% at 72 hours. The growth was suppressed by 40X at 12,020 hours.

RNA separation Cho*zynski and 5ach i method (J, Ce11. Physio Proliferation induction with 101101FBS-D Total cell 12N8 was measured from SV40LT-SMCs cultured for 24 hours. Ta. RNA was quantified using a spectrophotometer. Then dot plot the total 10 μg. plotted on nitrocellulose using the device. The plot is 10χ dext Randomly primed c-IIIyb,N in lan sulfate and 40χ formamide MMHC, large T antigen, GAP[lH and 7M probe at 42°C. Hybridization was allowed for 16 hours. Northern plot and RNA dot plot is 50°C and 0.5XSSC for c-a+yb and 55°C and 0.5XSSC for NM-C. and Q, 5XSSC, 55°C and 0.2XSSC for large T antigen, GAO 50°C and 0.2XSSC for Ptl, 50°C and 0.2 for TM The final wash performed in X5SC was washed in SSC. Northern plot is autoradio I did an ophthalmology. RNA plot was performed using Vestascove 603 analyzer. (Bestagen Jaltham, MA) was used to calculate the large T antigen count. The normalized c-myb or GAPDH counts were used for quantification. Total The numbers are expressed as total counts for each dot. All experiments were repeated twice. Next place Results were obtained for cells treated with Sense clyb oligonucleotide (25 μM), antisense c-myb oligonucleotide (25 μM), heparin ( 100 μg/ml), cells were cultured until they reached confluence and left undisturbed for 2 days. did.

Cells contacted with antisense c-Ilyb oligonucleotides were exposed to radiation. IIN eight-dot plot hybridization using sex-labeled c-myb probe There was a clear decrease in the amount of c-Bb mes- sage as measured by Ta. The results are shown in Figure 4.

Figure 4 shows the number of dots in each, corresponding to the amount of IINA in each sample. Ta. This sample is: sense c-+wyb (S-Myb) and Synchronized growth SV-5M treated with antisense c-n+yb (A-Myb) C, heparin-inhibited (heparin), growth-arrested (GA) cells.

Cell proliferation is dependent on heparin, which is present in an amount comparable to that of the c-IIlyb message. antisense c-myb suppresses the effect to the same extent as using antisense c-myb. in the same way , antisense NMMIIC oligonucleotides were detected in Northern Broth. causes a significant attenuation of the NMMIIC message. antisense c-Bb The amount of c-myb protein in cells treated with Analysis showed that this protein was found in cells treated with antisense NMMHC-8. It was a non-muscle myosin protein.

C-ll1b and J myosin are very stiff.

Yariba next day SV40LT-SMC was incubated at room temperature for 15 minutes using 2χ formaldehyde IPBS. Immobilize, infiltrate with 2χ Toridoa X-100/PBS, and 1χBSA/PBS Wash 3 times and dilute 1:250 or 1:1000 with 1χBSA/PBS. The cells were exposed to anti-chocolate or anti-NMMHC antiserum for 2-4 hours. anti-yb antiserum Cambridv, eRe5search Laboratories (Wi 1+*tngton, DE). Mouse c-myb amino acid residue 33 Synthetic peptide corresponding to 2-345 old 5-Thr-Cys-5er-Tyr-P Using ro-Gly-Trp-old 5-5er-Dinghr-5er-11e-Vat It was obtained by immunizing rabbits.

Anti-nonmuscle myosin antiserum was obtained from R5Aderstein and JS Sellers ( LMC.

Kindly provided by N11 (Bethesda, MD). This antiserum is purified human It was prepared by immunizing rabbits with platelet myosin. This antiserum was Monospecificity was confirmed by tanblond analysis. The cells are Washed three times with 1xBS/PBS to remove excess primary antibody. then 1 Second antibody (Organon Teknik) diluted 100 times with χBSA/PBS Rhodamine-conjugated goat obtained from A.A., Durham, NC. anti-rabbit IgG and FTTC-conjugated sheep anti-rabbit rgG). Then, incubation was performed for 2 hours. After washing 3 times with 1χBSA/PBS The samples were tested on a Nikon Optiphoto fluorescence micrograph.

NMM) Ic and c-myb induced by antisense oligonucleotides mRNA1l! ! A decrease in degree was shown as a decrease in the level of specific proteins. child To confirm the effect of SV40LT-5MC cells at low density (10,000/ cm2), 2-well glass slide (Nunc, Inc, +Naperv ille, IL). Grow in 0.5χ FBS-DflEM for 96 hours. 25μ antisense or sense NMMIIC or c-ta 10χFBS-DIIIEH supplemented with yb thiophosphorylated oligonucleotide. Moved. After 24 hours (c-myb) or after 72 hours (NMMMC), SMC using specific antiserum against Nl'lMIIG or c-myb. , observation by indirect immunofluorescence microscopy was performed. antisense oligonucleotide In most treated cells, compared to sense oligonucleotide treatment, N? I It was clearly established that the concentration of MIIC or c-myb protein was dramatically reduced. I could recognize it. However, the special cells in each panel have a substantial It was clear that he had the quantity. This observation suggests that this subpopulation may be affected by growth inhibition. escape before the maturation of the ration and subsequently occur at the level of target mRNA5. The increase is probably secondary. - the secondary antibody is not present or When there is a large amount of purified antigen with minimal background signal, unprocessed The specificity of the immunofluorescence method is demonstrated by carrying out the above method on a physical SMC.

Example 2: Release of oligonucleotides from polymerized matrices PlurOniC゛Lumatoto Cus 4 (Dt　’　j　’9.9　k, 9C-Steel yb and NMMHC antisense oligonucleotides (Materials and Methods) (ethylene oxide-propylene oxide) polymer containing prepared to test the oligonucleotide release rate from the matrix. Ta. The test sample was sterilized PIur in a scintillation vial. onic127 powder (BASF Corp, Parsippany, N, J, ) and 3.25 ml of purified water. Thaw and cool on ice with shaking. So I went.

For these solutions, oligonucleotide (5,041a+1500 μm) 500 μm of a sterile aqueous solution containing was added. The final gel is a polymer 25χ (W/W ) and l+sg/g of oligonucleotide.

The release rate of the gel containing oligonucleotides was determined over time by replacing the gel with PBS. Determined by measuring the absorbance (OD260) of each 4 as shown in Figure 5. Results from two test gels showed that the oligonucleotide was released from the gel within 1 hour. It showed that.

EVAC matrix oligonucleotide 1 ethylene vinyl acetate The release of oligonucleotides from EVAc is shown below.

The matrix was constructed using the method disclosed by Murray et al. (InVi tro, 19, 743-748.1983).

Ethylene vinyl acetate (EVAc) copolymer (ELAVAX 40P, Du Pont Chemicals Jilmington, DE) has a liquid capacity of lO It was dissolved in dichloromethane to give χ. Bovine serum albumin and oligonucleotides Reotide was dissolved together in deionized water in a ratio of 1000-2000:1. , frozen in liquid nitrogen. It was then freeze-dried to form a dry powder. powder In order to obtain a shape in which particles with a diameter of 400 microns or less are uniformly distributed, Shattered. A known amount of powder was placed in a 221 glass scintillation vial. (W/14) Combined with 4-4-1O of EVAc copolymer solution. The vial is Stir for 10 seconds to obtain a homogeneous suspension of drug particles in the polymer solution. mixed. This suspension was transferred to a glass mold and placed on a board of dry ice. Precooled. After freezing the mixture, let it stand for 10 minutes, remove the mold, and -20" It was left standing on a wire mesh in the freezer of C for 2 days. This slab contains residual dichloromethane. To remove the tan, store at 23°C2 for an additional 2 days in a vacuum of 600 mTorr. Allowed to dry for a while. After completely drying, use 113 corkpolar, 5++w Xo, A circular slab of 8+ms was cut out.

The results shown in Figure 6 indicate that 34χ of the oligonucleotide was released within 48 hours. It shows Goshichi.

Example 3: Oligonucleotides for c-myl) and NMMHC inhibition in rats In vivo administration of otide ■、Animal model Using Balloon Stripping of Rat Carotid Arteries as an In Vivo Thrombus Formation Model there was. Rats were anesthetized with Nembutal (50 mg/kg). Left carotid artery incision A 2P Fogarty catheter was introduced into the carotid artery through arteriotomy. Ta. Insert the catheter up to the aortic arch, inflate the balloon, and cut the catheter into the artery. It was pulled back to the incision site. This was repeated twice. Next, pull the balloon and move Renbe. It was left inside the vein to stop the bleeding and the wound was closed.

++, oligonucleotide arrival Oligonucleotides can be used in hydrogel and implantable ethylene vinyl acetate ( EVAc) matrix was administered. Polyethylene oxide as hydrogel Side polyethylene oxide polymer (Pluronic 127+ BA SF, Parsipany+NJ) was used.

Pluronic gel matrix was prepared according to the method of Example 2. UV sterilized Weigh 1.25 g of PIuronic powder into a scintillation vial; 3. A sterile solution of Pluronic 127 was prepared by adding 25 grams of sterile water.

Cool the solution on ice while shaking to obtain a solution containing 27.7x of the polymer by weight. I made it. These solutions contain antisense c-Bb (see Example 1) oligo Add 500 μl of a sterile aqueous solution of nucleotide (5,041 mg 1500 μm). Ta. The final gel contained Pluronic polymer at 25χ (W/W) and 1 mg/g. Consists of oligonucleotides.

A 25χ (W/W) gel without drug was prepared as a control.

The EVAc matrix was prepared as disclosed in Example 2. Another 20g of oligonucleotides.

Immediately after balloon injury, 200 μl of Pluronic/oligonucleotide The solution (containing 200 μg of oligonucleotide) was administered to the adventitial surface of the artery. , a gel was formed. Antisense/EVAc matrix (oligonuclease) (containing 40 μg of otide) and drug-free gel were administered in the same manner.

II+, Measurement of effectiveness After 14 days, the animals were sacrificed and the carotid artery was injected with lactate Ringer's solution at a pressure of 120 mmHg. Dispersed by force. Both carotid arteries were removed and fixed in 3χ formalin. one Sections for light microscopy were prepared using standard methods. Visualize the slides and digitalize them digitalized using a digital computer system and a small plenimeter to create simulated blood. The extent of intimal proliferation was calculated (square + 11 RI).

The results are shown in FIG. untreated control animals, drug-free gel treatment; In physical animals, the restenosis is enlarged and a symmetrical pseudointima is formed along the intact segment of the injured artery. formed, the lumen is narrowed by 60χ, and the intima/inner canal ratio is 1.4. It was characteristic that there was

Antisense c-myh oligonucleotide-treated animals showed increased restenosis and borderline. Growth is minimal (less than 10% of the lumen) and this is due to direct contact with the oligonucleotide. The intima/inner canal ratio was 0.09. figure As shown in Figure 7, this result was particularly significant for antisense/Pluronic-treated animals. It was very clear. Intima/inner tube obtained using EVAc/antisense The ratio was 0.45. However, EVA containing 40//g oligonucleotide c matrix was 200 μg of oligonucleotide administered in Pluronic gel. Compared to leotide, the difference was clear.

Figure 8 shows the results when this experiment was expanded and 28 rats were treated in the same manner as above. It shows. Seven rats in each treatment group underwent balloon angioplasty. Then the movement The following treatments were performed on the vein wall.

Drug-free hydrogel (Pluronic 127 shown above), Hydrogel containing antisense c-myb, hydrogel containing antisense c-myb, Untreated. As shown in Figure 8, similarly high levels of pseudointimal proliferation were observed in antisense It was other than c-Bb treatment. Antisense C-+1lyb treatment reduces the level of proliferation. was dramatically lower.

Example 4: Inhibition of PCNA using antisense oligonucleotides Example 1 and Using the same method, the PCN^ antisense with the following sequence was isolated from 5V-3lI in culture. was administered to C cells.

force distribution m GAT CAG GCG TGCCTC ^^^ Set as negative control ance PCNA was used. NMMHC was used as a positive (inhibition) control. there was.

Negative controls did not suppress smooth muscle cell proliferation.

Antisense Nl'1 MHC-8 inhibited 52χ, antisense PCN^ inhibited 58 showed suppression of χ.

Example 5: Antisense oligonucleotides for inhibition of rabbit smooth muscle cell proliferation In vivo administration of New Zealand White Rabint (1-1, 5kg) with Ketamine and Zyra Anesthetized with a mixture of gin and the carotid artery was dissected as disclosed in Example 3.

A　5F　Insert a Swan-Ganz catheter and place it in the descending artery with a fluorescent guide. did. S@an-Ganz catheter is a vascular type with a 3.0+ue balloon. The normal iliac aorta was replaced with a wire for the catheter. Angioplasty was performed three times at 100 PSI per second. -o1insky catheter A total volume of 5 cc of oligonucleotide solution in physiological saline was added. outside bottom Physiological saline was injected as a control in the aorta. oligonucleotide is Antisense mouse c-Bb and human NMMI (C mixture (200 μM each) were added as above. It was used like this. The mixture was injected at a pressure of 5 atmospheres over 60 seconds. two cormorants The heron was treated with antisense oligonucleotide. Animals were sacrificed after 4 weeks; It was treated in the same manner as the rat artery described in Example 3.

The results are shown in Figure 9, and when compared with saline alone, antisense-treated rabbits It was confirmed that the pseudointima of the artery was reduced by 50x.

Example 6: Inhibition of proliferation of baboon smooth muscle cells using antisense oligonucleotides Using a method similar to that shown in Example 1, primary baboon smooth muscle cells (Ko, Hawk er, Emory (distributed from tlniversity) to antisense h)wy b (SEQ ID NO: 4) and H) were treated with NMMHC (SEQ ID NO: 2).

Cells were grown for 72 hours after oligonucleotide treatment and then as described in Example 1. Counted by method. NMMHC showed 65% growth inhibition against baboon cells. , c-syb showed 57.9% growth inhibition.

Deaf Those skilled in the art will appreciate that there are many equivalents, modifications, variations and modifications to the method of the present invention from the foregoing detailed description. and be understood. All such equivalents, modifications and variations are encompassed by the claims below. It is intended that

Sequence list (1) General information (ii) Name of the invention Bilocal oligonucleotide therapy (iii) Number of sequences: 5 (iv) Contact information: (C) Reference/Document number: Old T-5583CP2 (2) Between sequence number 1 Information to do (ti) Molecular species: I) NA (synthesis) (iii) Hybothetical: None (xi) Sequence: Sequence number I GTG TCG GGG TCT CCG GGC (2) Information regarding SEQ ID NO: 2 Information (ii) Molecular species: DNA (synthesis) (iii) Hybothetical: None (iv) Antisense: Yes (ix) Features: (xi) Sequence: Sequence number 2 Information regarding CAT GTCCTCCCACCT GGA (2) Sequence number 3 (ii) Molecular species = DNA (synthesis) (iit) Hybothetical: None (iv) Antisense: Yes (xi) Sequence: Sequence number 3 ACCCAG AAA GAA AAT CCCAAG (2) Regarding sequence number 4 information (it) Molecular species: DN^ (synthesis) (ii) Hybothetical: None (iv) Antisense: Yes (xi) Sequence: Sequence number 4 GTG CCG GGG TCT TCG GGC (2) Information regarding SEQ ID NO. 5 Information (it) Molecular species: DNA (synthesis) (iii) Hybothetical: None (iv) Antisense: Yes Standard name Tomi antisense PCNA (xi) Sequence: Sequence number 5 GAT CAG GCG TGCCTCAAFIG, IA 01igo concentration (uM) (As NMMHC) FIG,lB %CEuC0LJNT Special Table Sen7-501204 (13) FIG, 3A TIME ToWASHOUT (hr) FIG, 3B CONTROL 16 hrs 40 hrs TIME To WASHOtJ T FI G, 4 FIG.5 TIME (min) FIG.6 0 24 48 72% +20 144 TIME (hours) FIG.7 CONTROL Pluronic/ASMyb EVAC/ASMybFIG , 8 0CONTRCL TREA Engineering MENT GROLIPS FIG.9 Special Table Sen7-501204 (15) ■-1-11-PCT/υS 92105305 Continuation of front page (51) Int, C1,' Identification symbol Internal office reference number C12Q 1/68 9453-4B (81) Designated countries EP (AT, BE, CH, DE.

DK, ES, FR, GB, GR, IT, LU, MC, NL, SE), 0A (BF , BJ, CF, CG, CI, CM, GA, GN, ML, MR, SN, TD, TG. ), AT, AU, BB, BG, BR, CA, CH, C3, DE.

DK, ES, FI, GB, HU, JP, KP, KR, LK, LU, MG, MN, MW, NL, No, PL, RO, RU, SD, SE, US (72) Inventor: Simmons, Mitchell USA 02167 Masachu -Setts, Chestnut Hill, 115 Grove Street FI (72) Inventor Edelman, Eraser USA 02146 Masachi 91 Baxter Road, Plutz Klein, (72) Inventor Langer, Robert Niss.

United States 02159 Massachusetts, Newton, Lombard Treat (72) Inventor: DeKyser, Gene Lac Brussels, Belgium Le 1080. A.B. Des Groyers Nationals.

Claims (40)

[Claims] 1. Preferentially inhibits translation of target nucleic acid sequences locally in vivo a tissue cell, comprising about 14-38 nucleobases, complementary to a target sequence; directly into local tissues within the mammalian body in an amount sufficient to penetrate the oligonucleotide. administered to hybridize with the target nucleic acid and inhibit intracellular translation of the target sequence. How to do it. 2. The oligonucleotide is in a physiologically acceptable solution and can be administered by injection. The method of claim 1, wherein the method of claim 1 is administered. 3. The solution is administered using an infusion pump, stent or catheter. Method of frame 1. 4. The method of claim 1, wherein the tissue comprises smooth muscle tissue. 5. The oligonucleotide is a group consisting of c-myb, NMMHC and PCNA. The method of claim 1 comprising an antisense sequence complementary to a gene sequence selected from . 6. oligonucleotides to resist degradation or extension by intracellular enzymes. How claim 1 is handled. 7. The treatment removes at least one backbone phosphodiester linkage of the oligonucleotide. The chain is phosphorothioate, methylphosphonate, sulfone, sulfate, ketyl, Phosphorodithioates, various phosphoramidates, phosphate esters, cross-linked a chain selected from the group consisting of phosphorothioate and bridged phosphoramidate chains; The method of claim 6 comprising replacing with strands. 8. The treatment produces 3' nucleotides with a structure that is resistant to the addition of nucleotides. The method of claim 6 comprising capping. 9. Approximately 30-3000μ of oligonucleotide per square centimeter of tissue surface area The method of claim 1, wherein the tissue is reached at a concentration between g. 10. The method of claim 1, wherein the target nucleic acid sequence comprises m-RNA. 11. m-RNA is derived from c-myb protein, non-muscle myosin heavy chain and PCNA. The method of claim 10 for encoding a protein selected from 12. Inhibits translation of target nucleic acid sequences preferentially and locally in vivo. A method in which an oligonucleotide consisting of about 14-38 nucleobases and complementary to a target sequence is used. The gonucleotide is deposited directly onto the surface of tissues at the site within the mammalian body, and permeate into tissue cells in a sufficient amount to bind to the target nucleic acid and inhibit its intracellular translation. Method. 13. Oligonucleotides are administered locally by injection, catheter, stent, or infusion. The method of Claim 12 to reach the location. 14. 13. The method of claim 12, wherein the oligonucleotide is incorporated into a carrier. 15. 15. The method of claim 14, wherein the carrier comprises an implantable matrix. 16. 15. The method of claim 14, wherein the carrier comprises a hydrogel. 17. Claim 1: The hydrogel is made of a material that is liquid at a temperature lower than 37°C. Method 6. 18. The hydrogel material is polyoxyethylene oxide-polypropylene oxide. The method of claim 17 comprising a side copolymer. 19. The polymer is about 10 to about 80% by weight polyethylene oxide and about 20% by weight polyethylene oxide. The method of claim 18 comprising about 90% by weight polyethylene oxide. 20. The polymer contains about 70% by weight polyethylene oxide and about 30% by weight polyethylene oxide. The method of claim 19, comprising lipropylene oxide. 21. The target nucleic acid is selected from the group consisting of c-myb, NMMHC and PCNA. 13. The method of claim 12, comprising an mRNA sequence transcribed from a gene. 22. 13. The method of claim 12, wherein the oligonucleotide is deposited extravascularly. 23. Oligonucleotides are deposited on or below the surface of the adventitia of the vasculature The method of claim 12. 24. In vivo, it inhibits smooth muscle cell (SMC) proliferation in targeted areas of the vasculature. an antisense oligonucleotide complementary to a nucleic acid required for and expressed in SMCs, oligonucleotides that initiate or support their proliferation and are selected within the SMC. Selective antisense - a drug with sufficient dosage to induce nucleic acid binding and inhibit proliferation for a sufficient period of time. A method comprising administering to the targeted area. 25. Is the nucleic acid a group consisting of c-myb protein, non-muscle myosin and PCNA? The method of claim 24, wherein the protein is encoded by an mRNA selected from the following. 26. Antisense oligo consisting of a carrier containing an antisense oligonucleotide Implants for sustained local delivery of nucleotides 27. 27. The implant of claim 26, wherein the carrier is a polymer. 28. The polymer is a hydrocarbon that is a liquid at temperatures below 37°C and a gel at body temperature. The implant according to claim 27, which is made of a gel material. 29. The hydrogel is a polyethylene oxide-polypropylene oxide copolymer. The implant of claim 28 which is a fusion. 30. About 30 to about 3000 oligonucleotides per square centimeter of tissue area Cresponses present in the polymer in sufficient concentration to be released to the surface to deposit μg device in system 26. 31. Encodes an mRNA selected from the group consisting of non-muscle myosin and PCNA oligonucleotides that are cell-permeable and resistant to intracellular degradation. Otide. 32. Proteins required to initiate or support smooth muscle cell proliferation Consisting of nucleotide bases that are complementary to intracellular nucleic acids expressing quality, penetrating cells Oligonucleotides that are durable and resistant to intracellular degradation. 33. The protein is composed of c-myb protein, non-muscle myosin or PCNA. Reme 32 oligonucleotide. 34. nucleotide sequences that are complementary to the physiologically acceptable carrier and tissue nucleotide sequences. Long-lasting antisense oligonucleotides in combination with oligonucleotides Implantation device to reach. 35. The carrier is polyacid anhydride, polyorthoester, polylactic acid, polyglycol Acid, poly(lactic acid-glycolic acid) copolymer, collagen, dextran, protein Polymer, ethylene vinyl acetic acid, polyvinyl acetate, polyvinyl alcohol , propylene glycol and ethylene oxide polymers and their derivatives. 35. The device of claim 34, comprising a polymer selected from the group consisting of: 36. Claim 34 wherein the target nucleotide sequence encodes a molecule that induces restenosis device. 37. Claim 34 wherein the target nucleotide sequence encodes a molecule that induces thrombosis. device. 38. The target nucleotide sequence encodes a molecule that causes lung disease. 34 devices. 39. The target nucleotide sequence encodes a molecule that causes cardiovascular disease. 34 devices. 40. For the manufacture of medicaments that inhibit translation of target nucleotide sequences in smooth muscle tissue , about 14 to about 38 nuclei that are complementary to the target nucleotide sequence of smooth muscle tissue. Oligonus with leotide base