JP2002255856A - Disease treatment by drip infusion at specific site of cell or specific transformation of cell and kit for such treatment - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for giving an effective therapy in clinical symptoms, effective in treatments of recurrent thrombosis, other ischemic symptoms including unstable angina, myocardial infarction or chronic tissue ischemia, systemic and genetic diseases or cancer. SOLUTION: This kit is to treat a patient requiring a therapy, and is characterized by including a catheter means and a solution containing an enzyme and a low irritating detergent, and by that (i) the catheter means is constituted so as to be inserted into a blood vessel, and further equipped with a main catheter body including a balloon element capable of being inserted into the blood vessel and constituted so as to be able to expand the wall of the blood vessel at a suitable position in the blood vessel for maintaining the main catheter body, and a means for delivering the solution into the above blood vessel, and (ii) the above solution is a physiologically permissible solution.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the treatment of diseases by site-specific instillation or transformation of cells and a kit for the treatment.

[0002]

BACKGROUND OF THE INVENTION Effective treatment of many systemic and hereditary diseases is an important issue for modern medicine. I remedy
The ability to be delivered to a specific site in vivo may be advantageous, for example, in the treatment of local disease. In addition, being able to perfuse the therapeutic agent through the circulatory system would be effective, for example, in treating systemic diseases.

[0003] For example, it would be desirable to administer an anti-tumor agent or toxin in close proximity to a tumor in a stable manner. Similarly, it may be desirable to perfuse insulin, for example, in the blood of a patient suffering from diabetes. However, for many therapeutic agents, there is no satisfactory method for either site-specific or systemic administration.

[0004] For many diseases, it may also be desirable to cause defective endogenous gene expression, exogenous gene expression, or endogenous gene suppression, whether local or systemic. Again, the goal has not been achieved.

[0005] In particular, the etiology of atherosclerosis is threefold.
It is characterized by two basic biological processes. These include 1) proliferation of intimal smooth muscle cells with macrophage accumulation, 2) formation of a large amount of connective tissue matrix by the expanded smooth muscle cells, and 3) predominantly intracellular and peripheral connective tissue. The accumulation of lipids in the form of cholesterol esters and free cholesterol.

[0006] Endothelial cell injury is an early event, which is manifested by disruption of the endothelial permeability barrier, alteration of the non-thrombogenic properties of the endothelial surface, and promotion of the endothelial procoagulant properties. Monocytes migrate between endothelial cells, become active as capture cells, and differentiate into macrophages.

[0007] Macrophages are then converted to platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EFG) and transforming growth factor α (TGF).
Synthesis and secretion of growth factors, including -α). These growth factors are quite potent in stimulating the migration and proliferation of fibroblasts (or fibroblasts) and smooth muscle cells in atherosclerotic plaques. In addition, platelets can interact with damaged endothelial cells and activated macrophages, enhancing growth factor assimilation and thrombus formation.

[0008] Two major problems in the clinical management of cardiovascular disease include thrombosis in acute myocardial ischemia and restenosis following cardiovascular formation (PTCA). Both
It represents common cellular events including endothelial damage and the release of potent growth factors by activated macrophages and platelets. Cardiovascular formation causes destruction of atherosclerotic plaques and detachment of endothelium. This vascular wound promotes platelet aggregation and thrombus formation at the PTCA site. Furthermore,
Mitogen release from platelets and macrophages, smooth muscle cell proliferation and monocyte penetration leads to restenosis.

[0009] Empirical medical treatment with antiplatelet agents cannot prevent this problem, and one-third of patients with PTCA develop the above problem. The solution for restenosis is to prevent platelet aggregation, thrombus formation and smooth muscle cell proliferation.

[0010] Thrombus formation is also a significant cellular event in the transition from stable to unstable heart disease. Etiology may include acute endothelial cell damage and / or plaque destruction that promotes the detachment of adherent endothelial cells and also exposes underlying phage foam cells. This provides an opportunity for circulating platelets to adhere, aggregate, and form thrombi.

[0011] Tissue plasminogen activator (tP
Intravenous administration of a thrombolytic agent such as A) results in lysis of the thrombus in about 70% of patients who have experienced acute myocardial infarction. However, about 30% of patients failed reperfusion and about 25% of patients who resumed initial perfusion of infarct-related arteries.
% Have experienced a recurrence of the thrombus within 24 hours. Thus, effective treatments for thrombotic recurrence remain important challenges facing medical organizations today.

[0012]

As noted above, effective treatment for thrombotic recurrence is not the only significant treatment problem that exists today. Besides, unstable angina,
The challenge is to treat myocardial infarction or other ischemic conditions, including chronic tissue ischemia, or to treat systemic and inherited diseases or cancers. These are anticoagulants, vasodilators,
The treatment can be effected by administering to the patient an angiogenic agent, a growth factor inhibitor or a growth inhibitor. In all of these clinical conditions, the need for effective treatment is still felt strongly.

[0013]

DISCLOSURE OF THE INVENTION Accordingly, it is an object of the present invention to provide a novel method for site-specific administration of a therapeutic agent.

It is another object of the present invention to provide a method for perfusing a therapeutic agent in the bloodstream of a patient.

[0015] Another object of the present invention is to provide a method for inducing the expression of a foreign gene in a patient.

Another object of the present invention is to provide a method for inducing expression of a defective endogenous gene in a patient.

Another object of the present invention is to provide a method for suppressing the expression of an endogenous gene in a patient.

Another object of the present invention is to provide a method for site-specific replacement of damaged cells in a patient.

It is another object of the present invention to provide a method for treating a disease by administering a therapeutic agent at a specific site or perfusing the therapeutic agent into the bloodstream of a patient.

Another object of the present invention is to provide a method for treating a disease by inducing the expression of a foreign gene or the expression of a defective endogenous gene in a patient, or suppressing the expression of an endogenous gene.

Another object of the present invention is to provide a method for treating a disease by site-specific replacement of damaged cells in a patient.

Another object of the present invention is to provide a kit for instilling normal or transformed cells into a specific site of a patient.

Another object of the present invention is to provide a kit for site-specifically transforming cells in vivo.

Another object of the present invention is to provide the following items 1 to 17. 1. A kit for treating a disease in a patient in need of treatment comprising a catheter means and a solution containing an enzyme or a mild detergent, wherein the catheter means is configured to be insertable into a blood vessel. A main catheter body including a balloon element configured to be insertable into the blood vessel and expandable in the wall of the blood vessel to hold the main catheter body in place within the blood vessel; Means for delivering a solution to the blood vessel provided, (ii) the solution is a physiologically acceptable solution. 2. The solution is dispase, trypsin, as the enzyme,
Item 2. The kit according to Item 1, comprising at least one enzyme selected from the group consisting of collagenase, papain, pepsin, chymotrypsin and lipase. 3. The solution is NP-40, Triton X100,
Item 2. The kit according to item 1, comprising at least one detergent selected from the group consisting of deoxycholate and SDS. 4. The main catheter body is configured to be inserted into a blood vessel and spaced apart from each other to form a chamber within the blood vessel and expand the wall of the blood vessel to hold the main catheter body in place. Item 2. A kit according to item 1, comprising means comprising two balloon elements arranged, said means for delivering a solution into said chamber being arranged between said balloon elements. 5. The kit of claim 1, wherein said means for delivering said solution into said blood vessel comprises a plurality of aperture means. 6. A kit for treating a disease in a patient in need of treatment, said kit comprising a catheter means and a physiologically acceptable solution, wherein (i) said catheter means is configured to be insertable into a blood vessel. Further comprising a main catheter body including a balloon element configured to be insertable into the blood vessel and capable of inflating the wall of the blood vessel to hold the main catheter body in place; and a main catheter body. Means for delivering a solution into the blood vessel, wherein (ii) the physiologically acceptable solution comprises heparin, poly-L-lysine, polybrene, dextran sulfate, a polycationic material and a divalent material. A kit comprising at least one substance selected from the group consisting of antibodies. 7. Item 7. The kit according to Item 6, wherein the physiologically acceptable solution further contains DNA. 8. Item 7. The kit according to Item 6, wherein the physiologically acceptable solution further contains a growth factor. 9. A method for treating a disease in a patient in need of treatment, comprising expressing an exogenous therapeutic protein in cells attached to a wall or organ or tissue of a blood vessel of the patient, the protein being used to reduce the disease. A method comprising treating. 10. Item 10. The method according to Item 9, wherein the disease is an ischemic disease, a vasomotor disease, diabetes, a malignant tumor, AIDS, or a genetic disease. 11. Item 9: The disease is a systemic disease.
The method described in. 12. The foreign therapeutic agent protein is TPA and a modified product thereof, urokinase, streptokinase, acidic fibroblast growth factor, basic fibroblast growth factor;
Tumor necrosis factor α, tumor necrosis factor β, transforming growth factor α, transforming growth factor β, atrial natriuretic factor, platelet-derived growth factor, endothelin, insulin, diphtheria toxin, pertussis toxin, cholera toxin, soluble CD4 10. The method according to item 9, which is one kind of protein selected from the group consisting of and a derivative thereof, and a growth hormone. 13. Item 10. The method according to Item 9, wherein the cells are selected from the group consisting of endothelial cells, vascular smooth muscle cells, fibroblasts, connective tissue cells, macrophages, monocytes and parenchymal cells. 14． A method for treating a disease, comprising instilling cells into a specific site. 15. Item 15. The method according to Item 14, wherein the cell is a transformed cell. 16. Item 14. The cell is a normal cell.
The method described in. 17． A method for treating a disease, comprising transforming cells in vivo in a site-specific manner.

The above and other objects of the present invention, which will become apparent through the following detailed description of the present invention, are as follows:
(I) site-specific instillation of normal (untransformed) or transformed cells into a patient, or (ii)
A method comprising either site-specifically transforming a patient's cells and (b) (i) site-specific instillation of normal or transformed cells or (ii) for site-specific transformation of cells. Have been found by the present inventors to be achieved by a kit comprising

[0027] Site-specific instillation of normal cells can be used to replace damaged cells, and instillation of transformed cells expresses defective or foreign genes or suppresses endogenous gene production. Can be used for Cells can be instilled into the patient's vascular wall, causing a constant perfusion of the therapeutic agent in the bloodstream.

[0027]

BEST MODE FOR CARRYING OUT THE INVENTION
In one embodiment, the invention is directed to infusing normal or transformed cells or transforming cells for diseases such as genetic diseases, systemic diseases, cardiovascular diseases, specific organ diseases or tumors. Used to treat.

Cells that can be instilled in the method of the present invention include endothelium, smooth muscle, fibroblasts, monocytes, macrophages and parenchymal cells. These cells have a therapeutic or diagnostic effect and are capable of producing proteins that are both naturally occurring and can arise from recombinant genetic material.

Reference is now made to the drawings. Throughout the drawings, the same reference numerals indicate the same or corresponding parts. In particular, FIG. 1 illustrates the practice of the present invention using a catheter having an embodiment as described in U.S. Pat. No. 4,636,195, which is hereby incorporated by reference. FIG. This catheter is
Normal or genetically modified cells can be applied to the vessel wall or used to introduce vectors for local transformation of the cells. In the figure, reference numeral 5 denotes a blood vessel wall. This figure shows the catheter body 4 held in place by the inflation of the inflatable balloon means 1 and 2.
Is shown. The portion of the catheter body 4 located between the balloon means 1 and 2 is provided with an infusion port means 3. The catheter may further be provided with a guide wire means 6. FIG. 2 shows the use of a similar catheter, except that the catheter shown in FIG. 1 has only a single inflatable balloon means 2 and a plurality of infusion port means 3. Different in that. The catheter can include up to twelve separate infusion port means 3, five of which are shown.

When delivered to an organ, the catheter
It can be introduced into the main artery that supplies tissue. After temporary occlusion of the arterial circulation, cells containing the recombinant gene or vector can be introduced through a central infusion port. In this way, cells or vector DNA can be delivered to large amounts of parenchymal tissue distributed through the capillary circulation. In addition, recombinant genes can also be introduced into the vasculature using double balloon catheter technology in the arterial circulation proximal to the target organ. In this way, the recombinant gene is
Related tissues can be secreted directly into the perfusing circulatory system or synthesized directly in organs.

In one embodiment, the therapeutic agent is secreted by vascular cells that supply specific organs affected by the disease. For example, ischemic cardiomyopathy can be treated by introducing vascular factors into the cardiovascular system.
The method can also be used for peripheral or cerebral vascular diseases where vascular-derived factors can improve circulation to the brain or other tissues. Diabetes mellitus can be treated by introducing glucose-responsive insulin-secreting cells into the portal circulation, where the liver usually shows higher insulin levels than other tissues.

In addition to providing localized concentrations of the therapeutic agent, the method of the present invention allows the delivery of high concentrations of viral and other vectors to a particular circulatory system, thereby reducing the amount of recombinant gene. It can also be used to deliver to tissues. This method can also be used to treat organ-specific protein deficiencies. For example, in the liver, α-antitrypsin inhibitor deficiency or hypercholesterolemia can be treated by introducing an α-antitrypsin or LDL receptor gene. Further, the method can be used to treat malignant diseases. Secretion of certain recombinant toxin genes into the circulation of inoperable tumors has a therapeutic effect.

Examples of this include auditory tumors and certain hemangiomas that cannot be resected otherwise.

[0034] In a clinical setting, the therapeutically modified gene is introduced into cells that supply the circulatory system of the organ concerned. While the arterial and capillary circulations are preferred places for the introduction of such cells, the venous system is also suitable.

In application to the treatment of local vascular injury,
The present invention provides for expression of a protein that ameliorates said condition in situ. In one embodiment, the vascular cells are used as a carrier to carry a therapeutic agent, as they are found at such vascular sites.

That is, in one embodiment, the present invention provides a method for treating a therapeutic agent (ie, protein, growth factor) to a local area of vascular injury.
Altering the genes of endothelium and other vascular cells to transfer, ie, body wall cell gene therapy. For successful use of gene transfer in cells, four conditions must be met. That is, first, the gene to be transplanted into the cell must be identified and isolated. Second, the gene to be expressed must be cloned and genetically engineered. Third, the gene must be introduced into the cell in an expressed or functional form. Fourth, the genetically modified cells must be placed in the vascular area where they are needed.

According to the present invention, the modified cells or suitable vectors can be introduced percutaneously or intravenously, by surgery, and adhere to a portion of the patient's vascular wall. Alternatively, some of the cells present on the patient's vascular wall are transformed with the desired genetic material or by directly applying the vector. In some cases, to replace lost or damaged cells on the vascular surface,
Vascular cells that have not been genetically modified can also be introduced by the above method.

In accordance with the present invention, any blood vessel can be treated, ie, arteries, veins, and capillaries. These blood vessels can be in or near any organ of the human or mammalian body.

(Introduction of Normal Cells or Genetically Modified Cells into Blood Vessels) The above-mentioned deafness of the present invention can be explained as follows. I. Establishment of Endothelial Cells or Other Vascular Cells in Tissue Culture First, a cell line is established and stored in liquid nitrogen.
Prior to cryopreservation, aliquots were taken for infection or transfection with a vector, virus or other material containing the desired genetic material.

Endothelial cells or other vascular cells are described in W.
Ford et al., In Vitro, 17, 40 (198
Using the technique already described in 1) above,
Can be induced by enzymatic action. Cut out blood vessels
And flip it over a stainless steel bar to remove 0.1%
Psin, Ca with 0.125% EDTA⁺⁺-And Ma
⁺⁺-Hanks Balanced Salt Solution (BSS), pH 8, free
Incubated at 37 ° C for 10 minutes. Cells (0.
4-1.5 × 10⁶) Was collected by centrifugation and 10
% Fetal bovine serum, endothelial cell growth cofactor (ECGS, C
olaborative Research, Wal
tam, MA) (25 μg / ml), heparin (15
U / ml) and gentamicin (50 μg / ml).
Medium 199 (GIBCO). In advance
75cm coated with ratine (2mg / ml in distilled water)
^TwoCells were added to the tissue culture flasks from. Cells spread all over
Cells were fed into the above medium every 2 days until the end.

After culturing for 2 weeks, ECGS and heparin can be omitted from the medium when porcine endothelium is cultured. If vascular smooth muscle cells or fibroblasts are desired, heparin and ECGS can be completely excluded from the culture process. Aliquots of cells were stored in liquid nitrogen by resuspending up to about 10 ⁶ cells in 0.5 ml of fetal calf serum on ice. 1
Add an equal volume of ice-cold fetal calf serum containing 0% DMSO,
Cells were harvested using a pre-cooled screw-top cone (Cor).
ning) and transferred to a freezing tube. Prior to prolonged storage in liquid nitrogen, the cells were transferred to a -70 C freezer for 3 hours.

The cells were then infected with a vector containing the desired genetic material.

(II. Introduction of Cells Expressing Normal or Foreign Protein into the Vascular System) Introduction of Cells Expressing Related Proteins by Catheterization Patients were prepared surgically or percutaneously for catheterization strictly adhered to sterile techniques. After appropriate anesthesia, a cutting procedure is performed on the target vessel or a needle is inserted into the target vessel. A catheter (USCI, Billeric) as described in U.S. Pat. No. 4,636,195, which is hereby incorporated by reference, pierces a blood vessel (5).
a, available from MA) is advanced into the vessel (5) by means of a guide wire (6) under fluoroscopic guidance, if necessary (FIG. 1). The catheter means (4) is designed to introduce infected endothelial cells into independent areas of the artery. The catheter has a proximal balloon means (2) and a distal balloon means (1) (e.g., each balloon means can be about 3mm long and about 4mm wide) with a fixed length between the balloons Catheter means.
A length of catheter means between the balloons has port means connected to the infusion port means (3). As the proximal and distal balloons are inflated, a central space is created in the vessel and infected cells can be instilled through the port. The area of the blood vessel is identified by anatomical landmarks, and the proximal balloon means (2) may be dispase, trypsin, by mechanical trauma (eg, by forced passage of a partially inflated balloon catheter within the blood vessel). Swelled to denature the endothelium by mechanical wounding in combination with small amounts of proteolytic enzymes, such as collagenase, papain, pepsin, chymotrypsin or cathepsin, or by incubation with the proteolytic enzymes alone. In addition to proteolytic enzymes, lipases can also be used. The area of the blood vessel is furthermore NP-40, Trit
It can also be exfoliated by treating with a mild detergent such as on X100, deoxycholate or SDS.

Substantially complete loss of endothelium for cell transfer and about 20 to 9 from the vessel wall for direct infection.
Exfoliation conditions are adjusted so that 0%, preferably 50-75% of cells are lost. In some cases, cell ablation may not be necessary. The catheter is then advanced so that the infusion port means (3) is placed in the ablated area of the endothelium. The infected, transfected or normal cells are then instilled into the independent part of the artery for 30 minutes. Where blood vessels have spread to organs that can tolerate some ischemia, such as skeletal muscle, distal perfusion is not a major problem, but if necessary use a catheter that can be perfused by an external shunt or distally Perfusion can be restored. After instillation of infected endothelial cells, the balloon catheter is removed and the perforated site of the artery and the site of local skin incision are repaired. If distal perfusion is required, another catheter designed to allow distal perfusion can be used.

B. In vivo direct introduction of recombinant genes into cells on the vessel wall or into cells perfused by a specific circulatory system: infection or transfection of cells on the vessel wall and organs. use. Instead of using infected cells, a high titer of the desired viral vector for transduction of genetic material (10 ⁵ -10 ⁶ particles / ml) or the DNA complexed with the delivery vector is transferred using double balloon catheterization. Instillation directly into the vessel wall. This vector is instilled in a medium containing serum and polybrene (10 μg / ml) to increase the efficiency of the infection. In the cavity formed by the catheter for an appropriate time (0.
After incubation (2 to 2 hours or more), the medium was drained and washed briefly with phosphate buffered solution to restore arterial circulation. A similar procedure is used for post-operative recovery.

The vascular surface can be treated by mechanical ablation alone or in combination with small amounts of proteolytic enzymes (eg dispase, trypsin, collagenase or cathepsin) or by incubation with these proteolytic enzymes alone. .
The denudation conditions are adjusted so that cells are appropriately lost from the vessel wall.

The viral vector or the DNA-vector complex is composed of polybrene (10 μg / ml), polybrene (10 μg / ml), which is used to enhance the efficiency of infection by enhancing the attachment of the virus particles to the original serum and related target cells. -L
An attachment molecule such as lysine, dextran sulfate or any biologically suitable polycationic substance, or
Use of the purified virus or complex in Dulbecco's modified Eagle's medium containing the envelope glycoprotein of the virus or vector and a hybrid antibody to the relevant target cells in the blood vessel wall or in tissue extending through the blood vessel And instill it. Hybrid antibodies to the viral or vector envelope glycoprotein and related target cells can be produced in one of two ways.
Antibodies to different epitopes can be chemically cross-linked (G. Jung, CJ Honsik, R. et al.
A. Reisfeld and H.C. J. Muller-Eb
erhard, Proc. Natl. Acad. Sc
i. USA, 83, 4479 (1986); D. S
taerz, O .; Kanagawa and M.K. J. Bev
an, Nature, 314, 628 (1985); Perez, R .; W. Hoffman, J .;
A. Titus and D.M. M. Segal, J.M. Exp.
Med. 163, 166 (1986)), or hybrid hybridomas can be used to bind biologically (UD Staerz and MJ, Bev.
an, Proc. Natl. Acad. Sci. US
A, 83, 1453 (1986); Mils
tein and A.I. C. Cuello, Nature, 3
05, 537 (1983)). After 0.2-2 hours or more of incubation in the central space of the catheter, the medium is drained, lightly washed with phosphate buffered saline, and circulation is restored.

Different catheter configurations can be used (see FIG. 2) and different infusion protocols can be used. This second method involves the use of a single balloon means (2) having a plurality of port means (3). Multiple ports (3) allow retroviruses to be delivered under high pressure into the partially denuded arterial segment. The vascular surface is treated as described above and the defective vector is introduced using similar attachment molecules. In this case, the use of a high pressure delivery system optimizes the interaction of the vector with cells in adjacent vascular tissue.

The present invention further provides for the use of growth factors delivered locally or systemically by catheter to increase the efficiency of the infection. In addition to retroviral vectors, herpes virus, adenovirus or other viral vectors are suitable vectors for the method of the present invention.

It is also possible to transform cells in an organ or tissue. Direct transformation of organ or tissue cells can be achieved by one of two methods. In the first method, high pressure transfection is used. High pressure can cause the vector to move through the vessel wall and into the surrounding tissue. In the second method, the infection is caused directly into the surrounding tissue by injection into the capillary bed, possibly following a leak-causing injury.

[0051] The time required for instillation of the vector or cell will depend on the particular embodiment of the method of the invention used. That is, for instilling the vector in cells or blood vessels, 0.01 to 12 hours is appropriate, preferably 0.1 to 6 hours, and most preferably 0.2 to 2 hours. Alternatively, for high pressure infusion of vectors or cells, shorter times may be preferred.

(Acquisition of cells used in the present invention) The term "genetic material" is generally used to describe a DNA encoding a protein.
Point to A. The term further includes RNA when used in RNA viruses or other RNA-based vectors.

Transformation is the process by which a cell is introduced by a foreign gene directly by infection, transfection or other means of uptake.

The term “vector” is well understood and the term “cloning vehicle” is often used.
Is synonymous with The vector may be a non-chromosomal duplex DN containing an intact replicon such that the vector replicates when transferred into a unicellular organism by, for example, a transformation process.
A. Viral vectors can include retroviruses, adenoviruses, herpes viruses, papovaviruses or other natural virus variants. Vector also means a preparation of DNA that contains a chemical or a substance that can be taken up by cells.

In another embodiment, the present invention provides for inhibiting the expression of a gene. Four methods can be used to achieve this goal. These are antisense agents (antisense agents).
t), ie, synthetic oligonucleotides that are complementary to mRNA (Maher III, LJ and Dolnic).
k, B. J. Arch, Biochem. Biophy
s. , 253, 214-220 (1987) and Zam.
ecnik, P .; C. Et al., Proc, Natl. Aca
d. Sci. , 83, 4143-4146 (198).
6)) or the reverse complementary sequence of the gene (reverse
plasmid (Iz)
ant, J. et al. H. And Weintraub, H .; , Sc
issue, 229, 345-352 (1985); C
, 36, 1077-1015 (1984)). Furthermore, RNA having a catalytic function called ribozyme can specifically cleave RNA sequences (Uhlenbeck, OC,
Nature, 328, 596-600 (1987),
Haseloff, J .; And Gerlach, W .;
L. , Nature, 334, 585-591 (198
8)). A third method involves "intracellular immunity" in which analogs of intracellular proteins can specifically interfere with the function of the intracellular proteins (Friedman, AD, Trie).
Zenberg, S.M. J. And McKnight, S .;
L. , Nature, 335, 452-454 (198).
8)). This will be described later in detail.

The first method can be used to specifically eliminate transcripts in cells. Transcript loss can be confirmed by S1 nuclease analysis, and expression of the binding protein as determined using functional analysis. Single-stranded oligonucleotide analogs can be used to interfere with the processing or translation of transcription factor mRNA. Briefly, synthetic oligonucleotides or thiol-derived analogs (20-50 nucleotides) can be made that are complementary to the coding strand of the target gene. These antisense agents are mRNA
Can be prepared for various regions. They are complementary to the 5 'untranslated region of the gene, the translation start site followed by 20-50 base pairs, the central coding region or the 3' untranslated region. The antisense agent can be incubated with the transfected cells prior to activation. The efficacy of antisense antagonists against various portions of the messenger RNA can be compared to determine whether the specific region is more effective at preventing the expression of such genes.

In addition, RNA can function autocatalytically to trigger autolysis or to specifically cleave complementary RNA sequences (Uhlenbeck,
O. C. , Nature, 328, 596-600 (1
987), Haseloff, J. et al. And Gerlac
h, W.S. L. , Nature, 334, 585-591.
(1988), and Hutchins, C .; J. Et al.
Nucleic Acids Res. , 14,362
7-3640 (1986)). Successful RNA cleavage requires that the blanking region contain RNA
A hammerhead structure in which the sequence is preserved can be mentioned. The region adjacent to this catalytic domain is made complementary to a specific RNA, and thus ribozymes target specific cellular mRNAs. To inhibit the production of a particular target gene, the mRNA encoding this gene can be specifically cleaved using ribozymes. Briefly, any GUG sequence in an RNA transcript can serve as a target for cleavage by a ribozyme. These are GUs of RNA transcripts that can be used for DNA sequence analysis and specific cleavage.
Identified by G site scan. Sites in the 5 'untranslated region, the coding region and the 3' untranslated region can be targeted to determine if any one region is more effective in cleaving this transcript. Synthetic oligonucleotides encoding the 20 base pair complementary sequence upstream of the GUG site, the hammerhead structure, and the -20 base pair complementary sequence downstream of the site can be inserted into the relevant site of the cDNA. In this way, ribozymes can target the same cellular compartment as the endogenous message. Ribozymes can also be created that are inserted downstream of specific enhancers that provide high levels of expression in certain cells. Such a plasmid is a neomycin resistant plasmid (pSVZ-
Neo) or other selectable markers can be used to introduce into relevant target cells using electroporation and co-transfection methods. Expression of such transcripts can be confirmed by Northern blot and S1 nuclease analysis. If confirmed, mRNA expression was
Assessed by nuclease protection, it can be determined whether expression of such transcripts has reduced the steady state levels of the target mRNA and the genes it regulates. In addition, the amount of protein can be investigated.

The gene can also be inhibited by preparing a mutant transcript lacking the domains necessary for activation. Briefly, after identifying the domain, a mutant that cannot stimulate function is synthesized. This truncated gene product can be inserted downstream of the SV-40 enhancer in a plasmid containing the neomycin resistance gene (Mulligan, R. and Berg,
P. , Science, 209, 1422-1427.
(1980) (in separate transcription units)). This plasmid can be introduced into cells using G418 and selected. The presence of a mutant of this gene is
Nuclease analysis and immunoprecipitation (immunoprec)
It can be confirmed by an IPT.
The function of endogenous proteins in such cells can be evaluated in two ways. First, the expression of normal genes can be investigated. Second, the known function of such proteins can be evaluated. If the intercellular interference form of this mutation is toxic to the host, it can be introduced on an inducible control element such as a metallothionein promoter. After isolating a stable cell line, the cells can be incubated with Zn or Cd to express this gene. The effect on the host cell can then be evaluated.

Another approach to inactivating a particular gene is to overexpress a recombinant protein that antagonizes the expression or function of another activity. For example, if it is desired to reduce TPA expression (eg, in clinical manifestations of sporadic thrombolysis), one may overexpress the plasminogen activator inhibitor.

With recent advances in biochemistry and molecular biology, for example, retroviruses and plasmids
"Recombinant" vectors carrying NA or DNA have been constructed. For example, a recombinant vector is a heterologous R
It may include NA or DNA, ie, RNA or DNA encoding a polypeptide not normally produced by an organism susceptible to transformation by a recombinant vector. Recombinant RNA
And the production of DNA vectors is well understood,
There is no need to explain in detail. However, this process is briefly described for reference.

For example, a linear RNA having an end capable of cutting a retrovirus or plasmid vector and ligating it
Or obtain DNA. The ends are ligated to foreign RNA or DNA having complementary, similar ligation ends to obtain an intact replicon and a biologically functional recombinant RNA or DNA molecule having the desired phenotypic properties.

A variety of techniques are available for RNA or DNA recombination that facilitate the joining of adjacent ends of separate RNA or DNA fragments.

The foreign or donor RNA used in the present invention
Alternatively, the DNA is obtained from a suitable cell. Vectors are constructed using known techniques for obtaining transformed cells capable of in vivo expression of a therapeutic protein. Transformed cells are obtained by contacting the target cells with an RNA or DNA-containing preparation capable of transferring and incorporating RNA or DNA into the target cells. Such preparations include, for example, retroviruses, plasmids, ribosome preparations, or complexes of the plasmid with a polycationic agent (eg, poly-L-lysine, DEAC-dextran and a targeting ligand).

The present invention therefore provides for the genetic modification of cells as a method for delivering therapeutic or diagnostic agents to localized areas of a blood vessel for local or systemic purposes. The range of recombinant proteins that can be expressed in these cells varies. For example, vectors expressing proteins such as tPA, angiogenic or revascularizing growth factors used in the treatment of thrombosis and restenosis, and vasoactive factors to alleviate vasoconstriction or vasospasm. The gene transfer used can be mentioned. This technique can be further extended to gene therapy for local or systemic genetic or acquired diseases. The present invention can also be used to introduce normal cells to specific cell loss sites, for example, to replace damaged endothelium during angioplasty or catheterization procedures.

For example, when treating ischemic disease (thrombosis), cells are transformed using genetic material encoding tPA or its modification, urokinase or streptokinase. Ischemic organs (eg heart, kidney, intestine,
For the treatment of liver and other disorders, transforming growth factor α (TGF
Collateral remodeling substances such as -α), transforming growth factor β (TGF-β), angiogenic factors, tumor necrosis factor α, tumor necrosis factor β, acidic fibroblast growth factor or basic fibroblast growth factor Can be used. In the treatment of vasomotor disorders, genetic material encoding vasodilators or vasoconstrictors may be used. These materials are atrial natriuretic factors,
Contains platelet-derived growth factor or endothelin. In the treatment of diabetes, genetic material encoding insulin can be used.

The present invention can further be used for treating malignant tumors by placing transformed cells in the vicinity of the malignant tumor. In this application, genetic material encoding diphtheria toxin, pertussis toxin or cholera toxin may be used.

When using the present invention to treat AIDS,
Genetic material encoding soluble CD4 or a derivative thereof can be used. In the treatment of genetic disorders such as, for example, growth hormone deficiency, genetic material encoding a required substance (eg, human growth hormone) is used. All of these genetic materials are readily available to those skilled in the art.

According to another embodiment, the present invention provides a kit for treating a disease in a patient, the kit comprising a catheter and a solution containing an enzyme or a mild detergent, wherein the catheter comprises A main catheter body configured for insertion into a blood vessel, the catheter further comprising a balloon element inserted into the blood vessel and configured to expand the vessel wall to retain the main catheter body within the blood vessel. And, provided on the main catheter body, means for delivering the solution to the blood vessel,
Solutions containing enzymes or mild detergents are physiologically acceptable solutions. The solution may contain a proteolytic enzyme such as dispase, trypsin, collagenase, papain, pepsin or chymotrypsin. Lipases other than proteolytic enzymes may be used. As a mild detergent, the solution may contain NP-40, Triton X100, deoxycholate, SDS, and the like.

Alternatively, the kit comprises heparin, poly-L-
Physiologically acceptable solutions containing substances such as lysine, polybrene, dextran sulfate, polycationic materials or bivalent antibodies may be included. The solution may further comprise vectors or (normal or transformed) cells. According to yet another embodiment, the kit comprises a catheter, a solution containing an enzyme or a mild detergent, and a substance such as heparin, poly-L-lysine, polybrene, dextran sulfate, a polycationic material or a bivalent antibody. And optionally a solution that may contain the vector or cells.

The kit comprises a catheter having a single balloon and a central distal perfusion port, and introducing cells into a particular organ, or introducing a vector into a capillary bed, or perfused by the capillary bed. And an acceptable solution capable of introducing cells into a particular organ or tissue.

Alternatively, the kit may include a main catheter body having two spaced apart balloon elements configured to be inserted into a blood vessel,
The two balloon elements define a chamber within the vessel and allow the vessel wall to be inflated to hold the main catheter body. In this case, the means for delivering the solution into the chamber is located between the balloon elements. The kit may include a catheter having a plurality of port means for delivering a solution into a blood vessel.

Accordingly, the present invention provides a method for treating a patient's disease by adhering any cell to the wall of a patient's blood vessel or cells of an organ perfused by the blood vessel and expressing the foreign therapeutic protein in the cell. Depending on the method, the protein may be useful for treating a disease or for diagnostic purposes. The method of the invention can be used to treat diseases such as ischemic disease, vasomotor disease, diabetes, malignancy, AIDS or genetic diseases.

The present invention provides a method for treating diseases, which comprises treating TPA and its modifications, urokinase, streptokinase, acidic fibroblast growth factor, basic fibroblast growth factor, tumor necrosis factor α, tumor necrosis factor β, and transformed growth. Factor α, transforming growth factor β, atrial natriuresis factor, platelet-derived growth factor, endothelian, insulin, diphtheria toxin, pertussis toxin, cholera toxin, soluble CD4 and its derivatives, and growth hormone for treating diseases Outpatient therapeutic proteins such as can be used.

The present invention can also use foreign proteins of diagnostic value. For example, a marker protein such as β-galactosidase can be used to monitor cell migration.

It is preferable that cells expressing the foreign therapeutic agent protein be endothelial cells.

Other features of the present invention will become apparent in the description of the following examples which illustrate, but do not limit, the present invention.

The data reported below show that endothelial cell transfer and gene transplantation are possible, endothelial cells can be stably transplanted in situ into the arterial wall by catheterization, and β-galactosidase, a recombinant marker protein, can be used. It demonstrates that it can be expressed in vivo.

Because the atherogenesis of pigs is similar to humans, the Yucatan minipig (Wilmington,
Charles River Labor, MA
atories) was selected as an animal model (1). Primary endothelial cell lines were established from the internal jugular vein of an 8-month-old female minipig. The endothelial cell identity of this system is
This was confirmed by the fact that the cells exhibited growth characteristics and morphology typical of porcine endothelium in tissue culture. Endothelial cells also express the receptor for the acetylated form of low density lipoprotein (AcLDL), in contrast to fibroblasts and other mesenchymal cells (2). Analysis of AcLDL receptor expression by fluorescent AcLDL incorporation showed that over 99% of the cultured cells contained this receptor.

Two independent β-galactosidase expressing endothelial systems were isolated after infection with a mouse β-galactosidase transducing retroviral vector (BAG) that was replication defective and contains both the β-galactosidase gene and the neomycin resistance gene (BAG). 3). Cells having the ability to grow in the presence of G-418 were selected from cells containing this vector. According to histochemical staining, more than 90% of the selected cells synthesized β-galactosidase. The endothelial properties of these genetically modified cells were similarly determined by fluorescent Ac
Confirmed by analysis of LDL incorporation. Furthermore, Southern blot analysis confirmed infection with the BAG retrovirus, which revealed the presence of intact proviral DNA at a rate of about 1 copy / genome.

Since the endothelial cells obtained from this inbred line are syngeneic, they are applicable for investigations in two or more minipigs and were tested in nine different experimental animals. Under general anesthesia, the femoral artery and the iliac artery were exposed, and a catheter was introduced into the blood vessel (FIG. 1). The intimal tissue of the arterial wall was mechanically abraded by inserting a partially inflated balloon catheter firmly inside the vessel. The arteries are rinsed with heparinized saline and neutral protease, dispase (50 U / m
l) to remove residual luminal endothelial cells. After the catheter balloon was evacuated and blood flowed through the vascular segment, the residual enzyme was rapidly inactivated by α2 globulin in the plasma. Cultured endothelial cells expressing β-galactosidase were introduced using a specially designed arterial catheter (USCI, Billerica, Mass.) With two balloons and a central infusion port (FIG. 1).

When these balloons were inflated, a protective space was formed inside the artery, and cells were instilled into this space through the central port 3 (FIG. 1). These endothelial cells expressing β-galactosidase were incubated for 30 minutes to facilitate attachment to the dissected vessels. The catheter was then removed, the arterial branches connected, and the incision closed.

A segment of the artery inoculated with endothelium expressing β-galactosidase was removed 2-4 weeks later.
Arterial samples were examined visually after staining with X-gal dye, and multiple blue colored areas representing β-galactosidase activity were observed compared to arteries inoculated with uninfected endothelium. As a result of the light microscopic examination, β-galactosidase staining was mainly confirmed in endothelial cells in the intima of the blood vessel experimentally inoculated.

In contrast, no similar evidence of staining was observed in control segments inoculated with endothelial cells without β-galactosidase. β-galactosidase staining may be evident in deeper intimal tissue, suggesting that the inoculated endothelium was trapped or migrated inside the previously damaged vessel wall. Local thrombosis was observed in the first two experimental animals. This complication was minimized in subsequent studies by administering acetylsalicylic acid prior to the endothelial cell transfer procedure and using anticoagulant heparin at the time of inoculation. In the case of thrombus formation, β-galactosidase staining was observed in endothelial cells extending from the vessel wall to the surface of the thrombus.

The first problem of in vivo gene transfer is to produce a replication-competent retrovirus from genetically engineered cells. In these tests, this potential problem was minimized by using a replication defective retrovirus. 2 in vitro
After 0 passages, no helper virus could be detected from these lines. Although the defective virus was used because of its high infection rate and stable uptake into the host cell genome (4), this gene transfer method can be adapted to other viral vectors.

The second problem is that the recombinant gene in vivo
o Lifetime of onset. As a result of this study, endothelial cell expression of β-galactosidase was found to be constant in the tested blood vessels up to 6 weeks after transduction.

As a result of these tests, it was demonstrated that genetically modified endothelial cells could be introduced into the vessel wall of the Yucatan minipig by arterial catheterization. in this way,
The present invention can be used for local biochemical treatment of vascular diseases by using a genetically modified endothelium as a vector.

A major complication of current methods of treating vascular disease, such as balloon angioplasty or graft insertion into diseased vessels, is the disintegration of atherosclerotic plaques and thrombus formation at local tissue wound sites (5). ). In part, this is mediated by endothelial cell damage (6). The data show that genetically modified endothelial cells can be introduced during treatment to minimize local thrombosis.

The method can also be used for other ischemic conditions including unstable angina or myocardial infarction. For example, an antithrombotic effect can be obtained by introducing cells expressing a gene encoding tissue plasminogen activator or urokinase. This method is also useful for treating chronic tissue ischemia. For example, angiogenesis or the assimilation of growth factors (7) stimulates the formation of collateral vessels in severely ischemic tissues such as the myocardium. Finally, the use of this improved method of endothelial cell gene transfer enables somatic gene replacement of systemic hereditary diseases.

(Experimental part :) Analysis of AcLDL receptor expression in normal and β-galactosidase transduced porcine endothelial cells.

Endothelial cell cultures derived from Yucatan minipigs were divided into two sublines and infected with BAG retrovirus or 3T3 fibroblast control and fluorescently labeled Ac
AcLDL receptor expression was analyzed using LDL.

Endothelial cells were removed from the external jugular vein using the neutral protease dispase (8). The venous segment is excised and dispase (5 in Hanks balanced salt solution).
0U / ml) and incubated at 30 ° C for 20 minutes. The endothelium obtained by this means was transformed into a medium 199 (GIBCO, Grand Island) supplemented with fetal calf serum (10%), 50 μg / ml endothelial cell growth co-factor (ECGS) and heparin (100 μg / ml).
d, N.C. Y. A) maintained during. These cells were infected with the BAG retrovirus and G-418 resistant cells were selected. The cell culture was (1,1′-dioctadecyl-3,
3,3 ′, 3′-Tetramethylindocarbocyanine perchlorate) (Dil) AcLDL (Biomedi
cal Technologies, Stoutto
n, MA) (10 μg / ml) for 4-6 hours at 37 ° C., followed by three rinses with phosphate buffered saline containing 0.5% glutaraldehyde. Cells were visualized by phase contrast and fluorescence microscopy.

B. Method for introducing endothelial cells by catheterization.

Endothelial cells were instilled using a double balloon catheter. Each catheter is 6mm long
And 5 mm wide proximal and distal balloons, the distance between the balloons is 20 mm. The central portion of the catheter has a 2 mm hole connected to the infusion port.
Inflating the proximal and distal balloons creates a central space where the infected cells can be instilled through the ports into individual segments of the vessel. Schematic diagrams of cell introduction by a catheter are shown in FIGS.

The breeding of the animals is described in "Principles
f Laboratory Animal Care "
And "Guide for the Care and
Use of Laboratory Animal
s "(NIH No. 80-23, revised in 1978)
Was carried out according to Female Yucatan Mini Pig (80 ~
100 kg) with pentobarbital (20 mg / kg)
Anesthetized, intubated and mechanically ventilated. The iliac and femoral arteries of these animals were surgically exposed under sterile conditions. The distal femoral artery was perforated and a double balloon catheter was advanced through the guidewire into the iliac artery. The external iliac artery was identified and the proximal balloon was partially inflated and moved distally and proximally to mechanically ablate the endothelium. The catheter was then positioned so that the central space was located in the area of the ablated endothelium and the two balloons were inflated. The exfoliated segments were irrigated with heparinized saline and the remaining adherent cells were removed by instillation of dispase (20 U / ml) for 10 minutes. The dissected vessels were further irrigated with heparin solution and BAG infected endothelial cells were instilled for 30 minutes. The balloon catheter was then removed and pre-operative blood flow was restored. Vascular segments were excised after 2-4 weeks. A portion of the artery was placed in 0.5% glutaraldehyde for 5 minutes, stored in phosphate buffered saline, and another portion was fixed in paraffin blocks for sectioning. The presence of a retrovirus expressing β-galactosidase was determined by standard histochemistry (19).

C. Endothelial cell in vitro and in
Vivo analysis. (A) Primary endothelial cells from Yucatan Mini Big, (B)
Subsystems induced by BAG retroviral vector infection, (C) segment of normal control artery, (D) BAG
Arterial segment infused with endothelium infected with retroviral vector, (E) microscopic cross section of normal control artery,
And (F) The β-galactosidase activity was examined by histochemical staining of the cross-section of the artery into which the endothelium infected with the BAG retrovirus vector was instilled with drip.

[0096] Prior to histochemical staining, endothelial cells in tissue culture were fixed in 0.5% glutaraldehyde. Infecting endothelial cells in vitro and in vivo using the enzymatic activity of E. coli β-galactosidase protein
o. β-galactosidase transduced Mo-M
uLV vectors (2) and (BAG)
Donated by Dr. Cepko. This vector is β-
Wild-type M as a galactosidase gene promoter
An o-MuLV LTR was used. The SV40 early promoter linked to the Tn5 neomycin resistance gene is G
-418 Provides a marker that confers drug resistance, is inserted downstream of the β-galactosidase gene, and selects cells containing a β-galactosidase expressing retrovirus. The defective retrovirus was prepared from fibroblast φam cells (3, 10) and maintained in Dulbecco's modified Eagle's medium (DMEM) and 10% bovine serum. Cells were passaged twice a week after trypsinization. 1
0 ⁴ was added to endothelial cells The supernatant having to 10 ⁵ / ml of G-418 resistant colonies titer 2/3 confluence, 8 [mu] g /
DM at 37 ° C. in 5% CO ₂ in the presence of
Incubated for 12 hours in EM and 10% bovine serum. The supernatant containing the virus was removed and the cells were further plated in medium 199 containing 10% fetal calf serum, ECGS (50 μg / ml) and endothelial cell conditioned medium (20%).
Maintained for 8 hours, then selected in G-418 (0.7 μg / ml in 50% racemic mixture). G-418 resistant cells were isolated and tested for β using standard histochemical staining (9).
-Galactosidase expression was analyzed. Cells that stably express the β-galactosidase enzyme were maintained in continuous culture so that they could be used as needed. Frozen aliquots were stored in liquid nitrogen.

(References) 1. J. S; Reitman, R .; W. Mahley,
D. L. Fly, Atherosclerosis 4
3, 119 (1982). 2. R. E. FIG. Pitos, T .; L. Innerarit
y, J. et al. N. Weinstein, R.A. W. Mahle
y, Arteriosclerosis 1,177
(1981); J. C. Van Berkel,
J. P. Kruijt FEBS Lett. 132,
61 (1981); C. Voyta, P .; A. Ne
land, D.C. P. Via, E .; P. Zetter,
J. Cell. Biol. , 99, 81A (abst
r. (1984); M. Wilson, D .;
E. FIG. Johnston, D .; M. Jefferson,
R. C. Mulligan, Proc. Natl. Ac
ad. Sci. U. S. A. , 84, 4421 (198
8). 3. J. Price, D. Turner, C.I. Cepk
o, Proc. Natl. Acad. Sci. U. S.
A. , 84, 156 (1987). 4. R. Mann, R.A. C. Mulligan, D.M. B
artimore, Cell 33, 153 (198
3); L. Cepko, B .; E. FIG. Roberts,
R. C. Mulligan, Cell 37, 1053
(1984); A. Eglitis, W.C. F. An
derson, Biotechniques 6,608
(1988). 5. S. G. FIG. Ellis, G .; S. Roubin. S.
B. King, J .; S. Douglas, W.C. S. We
intrau et al. , Circulatio
n 77, 372 (1988); Schwart
z, M .; G. FIG. Bourassa, J .; Lesperan
ce, H .; E. FIG. Aldridge, F .; Kazim, et
al. , N .; Engl. J. Med. 318,171
4 (1988). 6. P. C. Block, R.A. K. Myler, S .; S
tertzer, J.M. T. Fallon, N .; Eng
l. J. Med. 305, 382 (1981);
M. Steele, J.M. H. Chesebro, A .;
W. Stanson, Circ. Res. 57,105
(1985); R. Wilentz, T .; A. Sa
nborn, C.I. C. Handenschild, C.I.
R. Valeri, T .; J. Ryan, D .; P. Fax
on, Circulation 75, 636 (198
7); McBride, R .; A. Lange, L .;
D. Hillis, N.M. Engl. J. Med. 31
8, 1734 (1988). 7. J. Folkman, M .; Klagsbrunn, S
science 235, 442 (1987); J.
Leibovich, P .; J. Polverini,
H. Michael Shepard, D.M. M. Wi
seman, V .; Shivery, N.M. Nuseir,
Nature 329, 630 (1987); Fo
1kman, M .; Klagsbrunn, Nature
329, 671 (1987). 8. T. Matsumura, T .; Yamaka,
S. Hashizume, Y .; Irie, K .; Nitt
a, Japan. J. Exp. Med. 45,377
(1975); G. FIG. S. Thilo, S .; Mull
er-Kusel, D.A. Heinrich, I .; Kau
ffer, E. et al. Weiss, Artery, 8, 25a
(1980). 9. A. M. Dannenberg,
M. Suga, in Methods for Stu
dying Monoclear Pharmacy
tes, D.S. O. Adams, P .; J. Edelso
n, H .; S. Koren, Eds. (Academic
Press, New York 1981), pp3
75-395. 10. R. D. Cone, R .; C. Mulligan,
Proc. Natl. Acad. Sci. US ・ S ・
A. , 81, 6349 (1984). Of course, many modifications of the invention are possible in light of the above teachings. Therefore, it will be understood that the invention may be practiced otherwise than as specifically described in the present specification, within the scope of the appended claims.

The present invention will become more fully understood and the many advantages associated with the present invention will become more readily apparent if the present invention is better understood from the following detailed description, taken in conjunction with the accompanying drawings. Would.

[Brief description of the drawings]

1 and 2 are in accordance with the present invention for surgically or percutaneously implanting cells into blood vessels or for transforming cells present on the wall of a patient's blood vessel in vivo. FIG. 3 illustrates the use of a catheter.

FIGS. 1 and 2 are in accordance with the present invention for surgically or percutaneously implanting cells into blood vessels or for transforming cells present on the wall of a patient's blood vessel in vivo. FIG. 3 illustrates the use of a catheter.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 35/00 A61K 9/08 // A61K 9/08 47/20 38/43 47/28 38/46 47 / 34 38/48 A61M 25/00 410Z 47/20 A61K 37/48 47/28 37/547 47/34 37/54 (71) Applicant 597015922 Wolverine Tower, Room 2071, 3003 State Street, Ann Arbor, MI 48109-1280, U.S. Pat. S. A. (72) Inventor Elizabeth G. Nebel United States, Michigan 48105, Ann Arbor, Andover Road 3390 (72) Inventor Gary J. Neighbor United States, Michigan 48105, Ann Arbor, Andover・ Load ・ 3390 F term (reference) 4C076 AA11 BB17 CC11 CC13 CC14 CC27 CC29 DD04 DD70 EE23 FF68 4C084 AA27 MA66 NA10 ZA36 ZA39 ZA40 ZA54 ZB26 4C167 AA02 AA09 BB02 BB08 BB26 BB27 BB31 CC08 CC09 CC29 DD

Claims (1)

[Claims] Claims: 1. A kit for treating a disease in a patient in need of treatment, the kit comprising a catheter means and a solution containing an enzyme or a mild detergent, wherein the catheter means is inserted into a blood vessel. A main catheter body, the main catheter body including a balloon element configured to be capable of being inserted into the blood vessel and configured to expand the wall of the blood vessel to hold the main catheter body in place within the blood vessel. Means for delivering a solution to the blood vessel, provided on the main catheter body, and (ii) the solution is a physiologically acceptable solution.