Microspheres for active embolization

专利摘要:
The present invention relates to an injectable composition comprising a biocompatible, swellable, substantially hydrophilic, non-toxic, substantially spherical polymeric material carrier capable of efficiently delivering bioactive therapeutic factor (s) for use in color conversion drug therapy. will be. The invention also relates to a method of color conversion gene therapy using such injectable compositions, in particular for the treatment of angiogenic and non-angiogenic dependent diseases.
公开号:KR20030011803A
申请号:KR1020027012622
申请日:2001-03-23
公开日:2003-02-11
发明作者:보겔진마리；보쉐티에기스토
申请人:바이오스피어 메디칼 인코포레이티드；
IPC主号:

专利说明:

MICROSPHERES FOR ACTIVE EMBOLIZATION}
[2] 1. Angiogenesis-dependent disease
[3] Angiogenesis-dependent diseases (ie, diseases requiring or causing vasculature growth) make up a significant portion of all diseases for medical treatment. Cancer, for example, is the second leading cause of death in the United States, accounting for more than one fifth of all deaths. Briefly, cancer is most typically characterized by uncontrolled division of cell populations that form one or more tumors. These tumors are also characterized by the internal growth of the vasculature provided by various blood circulation factors that allow for sustained tumor growth. Cancer is generally diagnosed more easily than in the past, but many forms are still incurable, even if detected early.
[4] Various cancer treatment methods are currently in use, including, for example, various surgical procedures. However, if only treated with surgery, many patients (especially some types of cancer, such as breast, brain, colon and liver cancer patients) will experience cancer recurrence. Thus, in addition to surgery, many cancers are also treated with combination therapies involving cytotoxic chemotherapeutic agents (eg vincristine, doxorubicin, taxel, vinblastine, cisplatin, methotrexate, 5-FU, etc.) and / or radiation therapy. do. However, one difficulty with this approach is that radiation therapy and chemotherapeutic agents are toxic to normal tissues and often cause life-threatening side effects. In addition, this approach often has a very high failure rate / release rate.
[5] In addition to surgical, chemo- and radiation therapies, other therapies have been attempted that use an individual's own immune system to remove cancer cells. For example, some have suggested using bacterial or viral components as adjuvant to stimulate the immune system to destroy tumor cells (see, generally, "Principles of Cancer Biotherapy," Oldham (ed.), Raven Press, New York). , 1987). Such agents are generally useful as adjuvants and non-specific stimulators in animal tumor models, but have not yet been proven to be generally effective in humans.
[6] One additional limitation of current methods is that local recurrence and local disease suppression remain a dangerous risk in the treatment of malignant diseases. In particular, a total of 630,000 patients (in the United States) each year have local disease (no evidence of distant metastatic spread at publication); This accounts for 64% of all patients diagnosed as malignant (not including melanoma skin cancer or in situ carcinoma). For the vast majority of these patients, surgical resection of the disease provides the greatest therapeutic opportunity and in fact 428,000 will be cured after the initial treatment. Unfortunately, 202,000 (or 32% of all patients with localized disease) will relapse after initial treatment. Among the relapsed patients, the number of relapses due to local recurrence of the disease amounts to 133,000 patients (or 21% of all patients with localized diseases) each year. The number of relapses due to long-distance spread of the disease is 68,000 per year (11% of all patients with local disease). Approximately an additional 102,000 patients will die each year as a direct consequence of not inhibiting the local growth of the disease. Examples of cancers that present problems faced by patients are breast cancer and liver cancer.
[7] Breast cancer
[8] This problem is particularly evident in breast cancer, a disease of approximately 186,000 women in the United States each year, with a 50-year mortality rate. Surgical resection of the disease through radical mastectomy, modified radical mastectomy or breast tumor resection is still a support for treating the disease. Unfortunately, 39% of patients treated with breast tumor resection alone will show recurrence of the disease, and surprisingly 25% of patients found to have histologically removed tumors at the marginal margin. As many as 90% of these local recurrences will occur within 2 cm of the preceding excision site.
[9] Liver cancer
[10] In 1999, more than 1.2 million people died of primary liver cancer, most of which occurred in Asia. Primary liver cancer refers to liver cancer formed in the liver rather than earlier cancer cells migrated from some other cancerous part of the body to the liver. Patients with some forms of hepatitis, including viral diseases that cause liver inflammation, are known to be at high risk for primary liver cancer. The incidence of primary liver cancer is expected to increase dramatically in the United States, with estimates that more than 4 million people are currently positive for hepatitis C.
[11] More than 70% of primary liver cancers are inoperable and undergo radiation or chemotherapy. The treatment options currently available are:
[12] Chemotherapy: Chemotherapy seeks to inhibit cancer by killing rapidly dividing cancer cells. However, many noncancerous cells in the body, such as bone marrow cells, also divide rapidly and are therefore very susceptible to unreasonable death by the chemotherapy. Thus, a dose sufficient to eradicate cancer often results in life-threatening side effects due to the destruction of noncancerous cells.
[13] Chemical embolization and various treatments under development: For example, subcutaneous ethanol infusion is a painful process that acts only on small tumors that are limited to less than 3-4 cm.
[14] Transplantation: Transplantation is also available therapy, which is expensive and limited by the availability of organs and still recurs tumors. There is also a risk of recurrence associated with invasive surgery.
[15] Moreover, chemotherapy may also impair the body's natural anti-tumor defenses.
[16] Uterine fibroids
[17] Suitable examples of noncancerous tumors are uterine fibroids, also known as leiomyomas. It is a noncancerous tumor consisting of several types of muscle fibers and fibrous connective tissue. The cause of uterine fibroids is unknown. Most patients with uterine fibroids are initially asymptomatic and remain untreated until the patient has experienced abnormal bleeding, frequent urination, pain, bloating and difficulty in pregnancy. Approximately 25 million women in the United States have uterine fibroids, of which about 5.5 million have enough symptoms to seek treatment each year. To date, women suffering from uterine fibroids have options that are nearly difficult to survive, such as hysterectomy, myoma ablation, and medical care and “waiting to see”. Currently available therapies for the treatment of uterine fibroids include significant shortcomings, such as temporary or permanent infertility in women of childbearing age, long recovery periods, adverse physiological effects that can lead to early menopause, medications, surgical procedures, and frequent And high costs, including long hospitalization costs, discomfort and side effects from invasive surgical procedures and hormone therapy, and / or risk of recurrence of fibroma.
[18] As a result, there is a significant need for uniformly effective treatment programs, especially for patients with breast cancer, liver cancer, pancreatic cancer and uterine fibroids.
[19] Manual embolization
[20] Despite limited success, one method that has been attempted to treat tumors is manual embolization. Briefly, blood vessels that nourish the tumor are deliberately blocked by injecting embolic material into the blood vessels. In this regard various substances, for example hair, cotton, steel spheres, plastic or glass beads, tantalum powder, silicone compounds, radioactive particles, sterile absorbent gelatin sponges, as well as autologous substances such as fat, blood clots and minced muscle fragments (Sterispon, Gelfoam), oxidized cellulose (Oxycel), steel coils, alcohols, lyophilized human transdermal membranes (Lyodura), microfibrous collagen (Avitene), collagen fibrils (Tachotop), polyvinyl alcohol sponges (PVA; Artificial materials such as Ivalon), barium-infused silicon spheres (Biss) and detachable balloons have been tried. Although the size of tumor metastasis can be temporarily reduced using such methods, the tumor typically responds to the growth of new blood vessels into the tumor.
[21] A problem associated with cancerous tumor formation is the development of cancerous blocking, which inhibits the flow of substances through body pathways such as bile ducts, organs, esophagus, vasculature and urethra. One design, a stent, has been developed to keep open the passage blocked by a tumor or other material. Typical examples of conventional stents include wall stents, striker stents, giant turco stents and palma stents. However, a major problem with stents is that they do not prevent the internal growth of tumors or inflammatory substances through their gaps. If such a substance reaches the interior of the stent and damages the stent's sight, the body passage through which the stent is inserted may be blocked. In addition, the presence of the stent in the body may cause reactive or inflammatory tissue (e.g. blood vessels, fibroblasts). , Leukocytes) may enter the stent turine, causing partial or complete closure of the stent.
[22] 2. Therapeutic or Active Embolization
[23] Administration of cytotoxic drugs into the vicinity of the tumor increases the ratio of tumor to normal tissue delivery. Such topical administration can be carried out by delivering the drug directly through the blood supply into the tumor or into the body cavity where a particular tumor is located. Topical drug perfusion has the advantage of increasing peak drug concentrations into target tissues, but exposure is limited to the first passage of blood through the organ being perfused. Portions of the drug that are not taken by the first pass circulate systemically and dissolve in normal tissues.
[24] Therapeutic vascular occlusion (embolization) is a technique used to treat some pathological conditions in the same place. This is generally done by the use of a catheter, which makes it possible to position the particulate occlusion agent (embolus) in the circulation under phase control. It also relates to vasculature of various processes, namely tumors, vascular malformations, hemorrhagic processes and the like. Significantly in the case of tumors, vascular occlusion can suppress pain, limit blood loss during surgical intervention following embolization, or even cause tumor necrosis and prevent surgery. In the case of vascular malformations, this can normalize blood flow to normal tissue, aid surgery, and limit the risk of bleeding. In the bleeding process, vascular occlusion reduces flow, facilitating the absence of arterial gap (s).
[25] Moreover, depending on the pathology being treated, embolization can be performed not only temporarily but also for permanent purposes.
[26] Different types of embolisms are known in the art. In particular, liquid preparations (acrylic glues, gels, viscous suspensions, etc.) or particulate preparations (various polymers, meninges, gelatin sponges, spheres, balloons, spirals, etc.) may be included. The main disadvantage of known liquid embolisms is their toxicity to tissues, which can cause necrosis and the risk of catheter sticking. Another limitation to liquid embolism is that it only works in a passive manner and cannot be used for drug delivery.
[27] The dual action of local distribution of the drug to the target site and minimizing loss from the site can be achieved by microspheres. When introduced through the local artery, such microspheres are trapped within the vasculature of the tissue, where they release their drug load. Such dual action is called active embolization. Microspheres can be of solid or porous composition and can be prepared to contain drug molecules dispersed in solution or in solid form (Zimmer and Kreuter, 1995). Microspheres have special use in the treatment of tumors located in organs, such as the liver, supplied by a single introducer arterial blood supply (Chen et al. 1994). This is of greatest value if the tumor of the target organ is the only site requiring treatment.
[28] While studies relating to the use of microspheres for therapy based on color phone drugs are known, relatively few studies have been performed on microspheres for use in gene therapy. For example, glass beads are commonly used to selectively extract DNA from heterogeneous mixtures through electrostatic forces. Hydroxyapatite has also been used for purification of nucleic acids (Kumazawa et al. 1992) and hydroxyapatite spheres have been used to slowly release doxorubicin by direct implantation into liver tumors via ultrasound induction, but other studies It has been found that such specific matrices may not actually be suitable for exposure to mammalian tissue (Dass et al., 1997a, 1997b). DNA was also maintained on PDB microspheres with diethylaminoethyl (DEAE) functionality on the surface (Katz et al., 1990; Maa et al., 1990).
[29] Thus, there is a proven need for further development of microspheres for gene delivery. A major advantage of interest is the ability to specifically target anticancer agents to the tumor vasculature by the bloodstream. Furthermore, blocking tumor blood supply, with simultaneous cessation of nutrient supply and waste removal, is presumably the most desirable result for the destruction of tumor cells.
[30] Summary of the Invention
[31] In one embodiment, the present invention provides compositions and methods for delivering drugs, vaccines, polynucleotides, and diagnostic or imaging agents to mammals using a wide variety of polymeric materials as carriers for such agents. In a preferred embodiment, the present invention provides microparticles, in particular microspheres, for the delivery of these agents. In a most preferred embodiment, the present invention provides microparticles for polynucleotide delivery using a transfection agent.
[32] More specifically, in a preferred embodiment, the polymeric material carrier for use in the present invention includes any particles capable of "carrying" or related to the biologically active therapeutic factors and transfection agents of the present invention. Preferred polymeric materials are based on substantially spherical, substantially hydrophilic, inert and ionic crosslinked polymers of sufficient size to perform embolization and release bioactive therapeutic factors. In a preferred embodiment, the bioactive therapeutic factor is physically bound to the transfection agent and the transfection agent is also linked to the microparticles.
[33] In one embodiment, the invention provides methods and devices for using such compositions for the treatment of a variety of other angiogenesis-dependent diseases, including bioactive therapeutic compositions as well as cancer and precancerous diseases. Within one aspect of the invention there is provided a composition comprising (a) a bioactive therapeutic factor, (b) a polymeric substance carrier, and (c) a transfection agent.
[34] Within another preferred aspect of the present invention there is provided a composition comprising (a) a polynucleotide encoding a bioactive therapeutic factor, (b) a polymeric material carrier, and (c) a transfection agent.
[35] Within another preferred aspect of the invention there is provided a composition comprising (a) a polynucleotide encoding a bioactive therapeutic factor, (b) a polymeric material carrier, and (c) a lipopolyamine transfectant.
[36] A wide variety of molecules as bioactive therapeutic factors, such as but not limited to angiogenesis inhibitors, antitumor agents, peptides and peptide analogs, antibodies or fragments thereof, vaccines, enzymes, nucleic acids, RNA and DNA of natural or synthetic origin For example recombinant RNA and DNA and antisense RNA and DNA; Hammerhead RNAs, ribozymes, antigenic nucleic acids, single and double stranded RNAs and DNAs and homologues thereof can be used within the scope of the present invention.
[37] Polymeric materials of the present invention include, but are not limited to, acrylic polymers, vinyl alcohol polymers, acrylate polymers, poly (lactic acid), polyacrylamides, poly (anhydrides), polysaccharides, silicones or mixtures thereof. More preferably, the polymeric material is an acrylic based substantially hydrophilic crosslinked copolymer such as polyacrylamide and derivatives thereof, polyacrylates and derivatives thereof, as well as polyallyl and polyvinyl compounds. These polymers are all crosslinked to be stable and non-resorbable.
[38] Within various embodiments of the present invention, a bioactive therapeutic factor is physically associated with a transfection agent and the bioactive therapeutic factor-transfectant complex is physically associated with a polymeric carrier. The bioactive therapeutic factor is preferably adsorbed by well known association forces for liquid adsorption chromatography, including but not limited to association forces such as ion exchange, hydrophobicity, molecular recognition or combinations thereof. In one preferred embodiment, the bioactive therapeutic factor is associated with the transfection agent to form a complex and then mixed or contacted with the polymeric carrier of the present invention.
[39] In one aspect of the invention, a given bioactive therapeutic factor is mixed with a transfection agent to form a complex in the liver, wherein the transfection agent imparts specific properties, eg increased hydrophobicity, to the complex.
[40] In another aspect of the invention, the polymeric material carrier is mixed with a sufficient amount of transfective bioactive therapeutic factor. The physical association between the bioactive therapeutic factor and the polymeric material carrier is a combination of ionic and hydrophobic associations that can be enhanced by the addition of salts such as, but not limited to, sodium chloride or contrast media such as barium or iodide containing salts. The result is.
[41] Within various embodiments of the present invention, the bioactive therapeutic factor-transfectant complex adsorbed on the surface of the embolic material is progressively desorbed once delivered to a suitable site, resulting in various mechanisms, for example, but not limited to spontaneous intracellular. Delivery to peripheral cells by migration, receptor-mediated endocytosis, endosomal degradation and membrane stabilization, or a combination thereof. Desorption of the bioactive therapeutic factor is induced by natural components of the biological liquid that weaken the adsorption strength between the factor and the embolic material until full desorption of the factor is achieved. Desorption of such bioactive therapeutic factors can also be controlled by the use of hydrolysable bonds in vivo, such as but not limited to ester or oxide bonds. In other various embodiments, desorption of a bioactive therapeutic factor can also be controlled by the use of a peptide associated with said factor, wherein the peptide bond can be cleaved by cellular proteolytic enzymes.
[42] Within another aspect of the present invention there is provided a method for color conversion of blood vessels, comprising administering a therapeutically effective amount of a bioactive therapeutic agent to the blood vessels of a patient in need thereof so as to effectively occlude the blood vessel. Within one embodiment, the bioactive therapeutic factors are delivered simultaneously or sequentially to blood vessels that nourish the tumor.
[43] Within another aspect of the invention, a therapeutically effective amount of a composition comprising a polynucleotide encoding a bioactive therapeutic factor is administered to a patient in need thereof for the treatment of an tumorigenic, angiogenic-dependent disease. A method of color conversion is provided wherein the polynucleotide is associated with a transfection agent and the polynucleotide-transfecting agent is further associated with a polymeric material carrier to effectively occlude the blood vessel.
[44] Within another more preferred aspect of the invention, the disease comprises administering to a patient in need thereof a therapeutically effective amount of a composition comprising a polynucleotide encoding a bioactive therapeutic factor to a patient in need of treatment of an tumorigenic, angiogenic-dependent disease. A method of color-changing blood vessels in a cell, wherein the polynucleotide is associated with a lipopolyamine transfectant and the associated polynucleotide-transfectant is further associated with substantially hydrophilic microspheres to effectively occlude the blood vessel. .
[45] Within another aspect of the invention, a therapeutically effective amount of a composition comprising a viral vector or viral type particle containing a polynucleotide encoding a bioactive therapeutic factor is administered to a patient in need of treatment of an oncogenic, angiogenic-dependent disease. Providing a method of color shifting blood vessels in the disease, wherein the viral vector or virus-like particle is associated with a transfection agent and the associated viral vector-transfection agent is substantially effective to effectively occlude the blood vessel. It is further associated with microspheres which are hydrophilic.
[46] Within another more preferred aspect of the invention, a therapeutically effective amount of a composition comprising a viral vector or viral type particle containing a polynucleotide encoding a bioactive therapeutic factor in a patient in need of treatment of tumorigenicity, angiogenesis-dependent disease Providing a method of color shifting blood vessels in the disease, wherein the viral vector or virus-type particle is associated with a lipopolyamine transfectant and the associated viral vector-transfection agent is Further associated with substantially hydrophilic microspheres to effectively occlude.
[47] Within another aspect of the invention, a therapeutically effective amount of a composition comprising a viral vector or viral type particle containing a polynucleotide encoding a bioactive therapeutic factor is administered to a patient in need of treatment of an oncogenic, angiogenic-dependent disease. Providing a method of color shifting blood vessels in the disease, wherein the viral vector or viral particles are associated with a transfection agent and the associated viral vector or viral particles are substantially effective to effectively occlude the blood vessels. It is further associated with microspheres which are hydrophilic.
[48] Within yet another aspect of the invention, the disease comprises administering to the patient in need of such treatment a non-tumorigenic, angiogenic-dependent disease a therapeutically effective amount of a drug with the polynucleotide encoding the bioactive therapeutic factor. A method of inhibiting angiogenesis in a patient of the subject, wherein the polynucleotide is associated with a transfection agent, and the associated polynucleotide-transfecting agent is further associated with a polymeric carrier so that new blood vessel formation is inhibited. . In another aspect of the invention, such a drug may be incorporated into the polymeric material carrier so as not to interfere with the delivery of the bioactive therapeutic factor administered conventionally.
[49] Within yet another aspect of the invention, a therapeutically effective amount of a drug in combination with a polynucleotide encoding a bioactive therapeutic factor associated with a transfection agent is administered to a patient in need thereof for the treatment of a non-tumorigenic, non-angiogenic-dependent disease. A method of treating a patient of said disease, comprising administering said drug, wherein said drug is administered concurrently to ameliorate symptoms of said non-tumorigenic, non-angiogenic-dependent disease. It is incorporated into the polymeric material carrier so as not to interfere with the delivery of a bioactive therapeutic factor associated with the material carrier.
[50] Within another preferred embodiment, the compositions of the present invention contain (a) a polynucleotide encoding a bioactive therapeutic factor, (b) a polymeric carrier, and (c) a lipopolyamine transfectant to inhibit the formation of new blood vessels. Together with lighter d-group transition metals (eg vanadium species, molybdenum species, tungsten species, titanium species, niobium species or tantalum species).
[51] In another preferred embodiment, the compositions of the present invention comprise (a) a polynucleotide encoding a bioactive therapeutic factor, (b) a cationic crosslinked microparticle, and (c) a lipopolyamine transfectant with a transfection promoter. Include.
[52] In another preferred embodiment, a method is provided for delivering a polynucleotide to a mammalian host comprising administering to the diseased mammal a substantially hydrophilic polymeric material associated with the polynucleotide and the transfection agent. An embodiment of this method is the administration of a substantially hydrophilic polymeric material associated with the polynucleotide and a transfection agent for gene therapy, wherein the polymeric material microspheres are of sufficient size for color conversion of blood vessels at the site of administration. The polymeric material is not sufficient to color the blood vessels at the site of administration but is of sufficient size to anchor to the tumor.
[53] Within another preferred embodiment, a mammalian host comprising administering to a mammal with an angiogenic dependent disease a substantially hydrophilic polymeric material associated with a bioactive therapeutic factor capable of expressing an angiogenesis inhibitor. An active color conversion method is provided wherein the bioactive therapeutic factor is associated with a transfection agent.
[54] Within another aspect of the invention, a polymeric material carrier organism associated with a transfection agent as described above to inhibit local recurrence of cancer at the site and formation of new blood vessels at the resected edge of the tumor following ablation. Provided is a method of treating a tumor resection site comprising administering an active therapeutic factor.
[55] In other aspects, the method comprises delivering a therapeutically effective amount of a composition comprising a drug together with a polynucleotide encoding a bioactive therapeutic factor to effectively occlude the blood vessel into a blood vessel of a non-tumorigenic, angiogenesis-dependent disease. It provides a method of color conversion of the blood vessels.
[56] Within another aspect of the invention, the bioactive therapeutic factors are encoded such that the vessels of tumorigenic, angiogenic-dependent disease are effectively occluded and the polynucleotides encoding the bioactive therapeutic factors are delivered into cells for expression. It provides a method for color conversion of the blood vessel comprising delivering a therapeutically effective amount of a composition comprising a drug together with a polynucleotide.
[57] Within another aspect of the invention there is provided a method for active color conversion of a mammalian host comprising administering to a diseased mammal a substantially hydrophilic polymeric material associated with a bioactive therapeutic factor. The active therapeutic factor is associated with a transfection agent.
[58] Within another aspect of the invention there is provided a microparticle suitable for active color conversion comprising a polymeric material capable of color conversion of blood vessels, wherein the polymeric material is bound to a transfection agent and the transfection agent is Bound to dielectric material.
[59] Within other aspects, for example, including but not limited to, administering a therapeutically effective amount of a bioactive therapeutic factor to the eye such that the formation of new blood vessels is inhibited in a patient in need of treatment of the angiogenic disease. Provided are methods for treating angiogenic diseases of organs.
[60] The present invention provides compositions and methods suitable for the treatment of cancer as well as other non-tumorigenic angiogenesis-dependent diseases, and also provide other related advantages. Such cancers include, but are not limited to, liver, ovary, kidney, pancreas, prostate, skin cancer, head and neck tumors, breast tumors and Kaposi's sarcoma, and a superficial form of bladder cancer. However, in addition to cancer, many other non-tumorigenic angiogenesis-dependent diseases characterized by abnormal growth of blood vessels can also be treated with the bioactive therapeutic factors or compositions of the present invention. Typical examples of such non-neoplastic angiogenesis-dependent diseases include, but are not limited to, hypertrophic scars and keloids, proliferative diabetic retinopathy, rheumatoid arthritis, arteriovenous malformations, atherosclerotic plaques, delayed wound healing, hemophilic joints, non-binding Fractures, Osier-Weber syndrome, psoriasis, purulent granulomas, scleroderma, trachoma, hypermenstrual and vascular adhesions.
[61] Within another embodiment of the invention, there is provided a composition comprising: (a) suspension of microspheres suitable for color conversion; And (b) a transfection agent suitable for delivering genetic material to the cell. Within another further embodiment of the invention, a transfection agent associated with a polynucleotide containing a polymeric material in one vial and encoding a bioactive therapeutic factor is contained in another vial and the contents of the two vials. It is mixed together to provide a kit for preparing a pharmaceutical composition. Within another further embodiment of the invention, the polymeric material is contained in one vial, the transfection agent is contained in another separate vial, and the polynucleotide encoding the bioactive therapeutic factor is another separate Provided are kits that are contained in vials and that mix the contents of all three vials together to produce a pharmaceutical composition. Within another further embodiment of the present invention, there is provided a composition comprising: (a) a suspension of microspheres suitable for color conversion; And (b) a kit in which the components of the transfection agent suitable for delivering the genetic material to the cell are in one vial.
[62] These and other aspects of the invention will be apparent from and elucidated with reference to the following detailed description and the accompanying drawings. In addition, various references are hereafter incorporated by reference in their entirety.
[1] The present invention provides compositions and methods for the treatment of diseases including cancer and various other angiogenesis-dependent diseases, and more particularly bioactive therapeutic factors, microspheres (coated or otherwise contained with such factors) as carriers. It relates to a composition comprising together with a method of using such a composition for active color phone therapy.
[63] Justice:
[64] As used herein, "substantially spherical" is generally defined as a volume that provides the lowest external surface area in a shape that is close to a perfect sphere. Specifically, "substantially spherical" in the present invention means that when looking at any cross section of the particle, the difference between the mean versus diameter and mean small diameter is less than 20%. The surface of the microspheres of the present invention appears smooth when magnified up to 1000 times. The microspheres of the present invention may include other materials disclosed and defined herein in addition to the particles.
[65] As used herein, "cell adhesion promoter" means any substance that promotes or enhances adhesion of cells to the surface of microspheres due to their presence in or associated with microspheres. Such materials are often proteins associated with the surface of microspheres in a covalently bonded or interpenetrated polymeric manner.
[66] As used herein, "therapeutic agent" refers to any substance that provides a therapeutic effect on the progression of angiogenesis-dependent disease or provides a biological or physiological response to the angiogenesis-dependent disease. Examples of therapeutic agents are anti-inflammatory agents that prevent or reduce the effects of inflammation associated with angiogenesis-dependent diseases.
[67] As used herein, a "chemical alteration" is characterized by chemical properties such that the microspheres have the ability to perform other actions once injected into the body, in addition to tumor color change, during the production of microspheres or by mixing or contacting with various agents or tissues. And characteristics are changed.
[68] As used herein, "stabilizer" or "stabilizer" refers to the stability of compositions, prodrugs, targeting ligands, and / or other bioactive therapeutic factors disclosed herein, such as mixtures, suspensions, emulsions, dispersions, vesicles, and the like. It refers to any substance that can improve. The definition of “stabilizer” includes several bioactive therapeutic factors and prodrugs of the present invention. In addition, the definition of "stabilizer" includes some of the transfection agents of the present invention. Improved stability involves, for example, maintaining a relatively balanced state, and can be exemplified by, for example, increased resistance of the composition to breakdown, degradation, disintegration, and the like. For example, unbound cation heads of transfection agents reduce transfection efficiency as seen in compounds such as C ₁₂ GluPhC _n N ⁺ and C ₁₄ GluPhC _n N ⁺ , while DOTB with the shortest spacing Compounds such as / DOSC produce the best surface charge density and the best transfection efficiency.
[69] In the preferred embodiments, including microspheres having bioactive therapeutic factors, prodrugs and / or other bioactive agents, the stabilizing compound releases some bioactive therapeutic factors, prodrugs and / or bioactive agents from the microspheres. May act to form or stabilize the microspheres in such a way as to minimize or substantially (completely) prevent the release until desired. The term "substantially" as used herein in the context of preventing the release of bioactive therapeutic agents, prodrugs and / or bioactive agents from microspheres is at least about 50%, preferably at least about 60% of the above contents. Above, more preferably at least about 70%, even more preferably at least about 80%, even more preferably at least about 90%, or remain associated on the surface of the microspheres until desired to be released or treated on the other hand. In the case of drug formulations it is meant to contain in the microspheres. In a particularly preferred embodiment, at least about 95% of the bioactive therapeutic factors, prodrugs and / or bioactive agents remain associated on the surface of the microspheres until release is desired or on the other hand in the case of a therapeutic drug formulation. It contains in microspheres. The bioactive therapeutic factors, prodrugs and / or bioactive agents may also remain completely contained in or contained within the microspheres (ie, about 100% of the surface of the microspheres) until they are desired to be released. Remaining associated with the phase or contained within the microspheres.
[70] Examples of typical stabilizing materials are lipids, proteins, polymers, carbohydrates and surfactants. The resulting mixtures, suspensions, emulsions, etc. may occupy walls (ie, films, membranes, etc.) in the periphery of the bioactive therapeutic factors or bioactive agents, or in some cases may be substantially free of walls or membranes. The stabilizing material may optionally form droplets. The stabilizing material may also comprise salts and / or sugars. In some embodiments, the stabilizing material may be crosslinked substantially (completely). The stabilizing material may have a neutral, positive or negative charge.
[71] As used herein, “crosslinked” and “crosslinked” generally refer to two or more stabilizing materials, such as lipids, proteins, polymers, carbohydrates, surfactant stabilizing materials, bioactive therapeutic factors and / or bioactive agents. Refers to binding by one or more crosslinks. The crosslinking may comprise one or more elements, groups or compounds, and generally couples atoms from the first stabilizing material molecule to atoms of the second stabilizing material molecule. The crosslinking may comprise covalent and / or non-covalent associations. Any of various elements, groups and / or compounds may form crosslinks in crosslinks and the stabilizing material may be crosslinked either naturally or through synthetic means. For example, crosslinking can occur naturally in materials formed from peptide chains that are bound by disulfide bonds of cystine residues, such as in keratin, insulin and other proteins. On the one hand, crosslinking combines a compound such as a stabilizing material with a chemical change that can act as a crosslinking agent that can cause a reaction, for example, by exposure to heat, high energy irradiation or the like by suitable chemical alteration. This can be done by. For example, crosslinking with sulfur forming disulfide bonds, crosslinking with organic peroxides, crosslinking of unsaturated materials by high energy irradiation, crosslinking with dimethylol carbamate, and the like. In some cases, the stabilizing compound, the bioactive therapeutic agent and / or the bioactive agent may be substantially crosslinked.
[72] The term "substantially" as used herein means that at least about 50% of the stabilizing compounds contain crosslinking crosslinks. If desired, at least about 60%, 70%, 80%, 90%, 95%, or even 100% of the stabilizing compounds contain such crosslinking crosslinks. On the other hand, the stabilizing material may not be crosslinked, ie at least about 50% of the stabilizing compound is free of crosslinking crosslinking, and in some cases about 60%, 70%, 80%, 90%, 95% of the stabilizing compound Above, or even 100% is free of crosslinking crosslinking.
[73] As used herein, “associated” refers to the binding of a bioactive therapeutic agent, transfection agent, and microsection of the invention. Such associations may or may not form complexes in the case of bioactive therapeutics and transfection agents. Such associations include, but are not limited to, covalent, non-covalent as well as ionic interactions, electrostatic interactions, van der Waals forces, hydrogen bonds, hydrophilic interactions, and hydrophobic interactions, each of which is further defined below. It includes meetings.
[74] As used herein, "covalent association" refers to intermolecular associations or bonds, including the sharing of electrons in a bond orbital of two atoms.
[75] As used herein, "non-covalent association" refers to intermolecular interactions between two or more separate molecules that do not include covalent bonds. Intermolecular interactions vary depending on various factors, for example the polarity of the molecules involved and the charge (positive or negative, if present) of the molecules involved. Non-covalent association is selected from ionic interactions, dipole-dipole interactions, van der Waals forces, and combinations thereof.
[76] As used herein, “ion interaction” or “electrostatic interaction” refers to intermolecular interactions between two or more molecules, each of which is either positively or negatively charged. Thus, for example, "ion interaction" or "electrostatic interaction" refers to the attraction between the first positive charge molecule and the second negative charge molecule. Examples of ionic or electrostatic interactions are the attraction between negatively charged stabilizing materials such as dielectric materials and positively charged lipids such as cationic lipids, for example lauryltrimethylammonium bromide.
[77] As used herein, "van der Waals forces" refers to the attraction between non-polar molecules due to quantum mechanics. Van der Waals forces are generally associated with momentary dipole moments induced by neighboring molecules and accompanied by changes in the electron distribution.
[78] As used herein, "hydrogen bond" refers to the attraction or crosslinking that can occur between a negatively charged atom, such as a hydrogen atom covalently bonded to oxygen, sulfur or nitrogen, and another negatively charged atom. The hydrogen bond may occur between the hydrogen atom of the first molecule and the negative electrode atom of the second molecule (molecular hydrogen bond). The hydrogen bonds can also occur between hydrogen atoms contained in single molecules and negative electrons (intramolecular hydrogen bonds).
[79] As used herein, “hydrophilic interaction” refers to a molecule or moiety that can substantially bind, absorb, and / or dissolve with water. This may result in swelling and / or reversible gel.
[80] As used herein, “hydrophobic interaction” refers to a molecule or moiety that is not substantially bound, absorbed and / or dissolved with water.
[81] For the sake of clarity, the description of the invention is divided into the following subsections, without limitation.
[82] The present invention provides a safe and effective method of color phone gene therapy, which is useful for the treatment of cancer and various other angiogenesis-dependent diseases. The method of color phone gene therapy as such is therefore advantageous to physicians and / or surgeons, such as but not limited to intermediate radiologists. The methods and compositions of the present invention can be used by a surgeon before, during or after surgery.
[83] In gene therapy, genes are simply delivered to the patient's cells as molecules of DNA or RNA. The genes form part of an expression making-cassette of a nucleic acid comprising the gene of interest and a promoter / enhancing factor that highly regulates the expression level of the gene in the target cell. DNA is transcribed by enzymes into RNA messenger molecules in cells, which form a template for translating the gene sequence into protein products. The protein then provides a function in the target cell or in surrounding tissue to correct cell detection or to kill cells such as tumor cells.
[84] Thus, genes and cell therapies consist of introducing genetic information into cells or diseased organs to ensure expression of the protein of interest or to correct deficiencies or abnormalities (mutations, abnormal expressions, etc.). Such genetic information can be introduced into cells extracted from an organ ex vivo, and then the modified cells can be reintroduced into the body, or directly into suitable tissues in vivo. Various techniques for the transfer of this genetic information, in particular complexes of DNA and DEAE-dextran (Pagano et al., J. Virol. 1 (1967) 891), complexes of DNA and nuclear proteins (Kaneda et al., Science 243 (1989) 375), complexes of DNA and lipids (Felgner et al., PNAS 84 (1987) 7413), complexes of DNA and polylysine, use of liposomes (Fraley et al., J. Biol. Chem. 255 ( Various transfection techniques are disclosed, including 1980) 10431).
[85] Embolization is a partial or complete occlusion of a bloody vessel. Therapeutic embolization is a procedure in which the blood flow is blocked to obstruct the artery or vein to correct dysfunction, such as arteriovenous malformation, or to stop the supply of essential elements to solid tumor / cancer growth. Most commonly therapeutic embolization is a "passive" action in the sense that the active molecules are not accompanied and / or delivered to where the embolic material is deposited.
[86] The present invention relates to "active" embolization that cooperates with a decrease in blood flow by localized delivery of transfected genetic material that acts on the reduction or elimination of cancer cells for the same purpose.
[87] Within one aspect of the invention there is provided a composition comprising (a) a bioactive therapeutic factor, (b) a microsphere carrier, and (c) a transfection agent. In a preferred embodiment, the present invention provides a composition for infusion comprising (a) a bioactive therapeutic factor, (b) a microsphere carrier, and (c) a transfection agent, for the treatment of cancer and various other angiogenesis-dependent diseases. Provided are methods of gene therapy for administration to a mammal.
[88] In another preferred embodiment, the invention provides a composition for infusion comprising (a) a bioactive therapeutic factor, (b) a microsphere carrier, and (c) a transfection agent for the treatment of cancer and various other angiogenesis-dependent diseases. A method of gene therapy is administered to a mammal in need thereof, wherein the composition for infusion is administered directly to a tumor or diseased tissue through a suitable graduated needle.
[89] In yet another preferred embodiment, the present invention provides a composition for infusion comprising (a) a bioactive therapeutic factor, (b) a microsphere carrier, and (c) a transfection agent for cancer and various other angiogenesis-dependent diseases. Provided is a method of gene therapy administered to a mammal in need thereof, wherein said infusion composition is delivered to a tumor or diseased tissue by infusion into a vasculature system.
[90] Within another aspect of the invention, a new blood vessel at the tumor resection site at the resection edge of the tumor following resection of an antitumor drug, such as but not limited to Taxol, doxorubicin, cisplatin, paclitaxel, Provided is a method of treating said site, comprising administering to inhibit the formation of and local recurrence of cancer.
[91] The polymeric carrier or preferably microspheres of the present invention may also be chemically modified to contain a therapeutic effect, an vasculizing effect, an angiogenic effect, visualization properties, or a combination thereof. In one embodiment, the chemical modification of the microspheres of the present invention is a particle made of crosslinked polymers such that the microspheres contain chemicals having various properties within their structure and have unique characteristics related to surface covalent bonds. It may be possible by the fact that it includes. Chemical alteration of the microspheres of the invention may also occur through interactions between the microspheres and neighboring cells and tissues after administration.
[92] The method of the present invention has the following advantages: (1) The injected material does not migrate easily within the tissue originally injected, so that the intended gene therapy is achieved without repeated administration or adverse effects on the patient; (2) the injected material is not readily cleaved, substituted or removed biochemically or through the immune or lymphatic system, thus the method lasts more effectively; (3) the materials are of sufficient size to be injected through a needle of 18 to 26 gauge or 30 gauge or less, thus the method is more accurate and effective and less invasive to the patient; (4) the injected particles are not flexible or brittle and are easily injected without breaking, thus providing easy and safe injection; (5) The injected particles are not irregularly shaped and do not clump together, thus providing easy and accurate injection. These benefits, either alone or together, improve treatment effectiveness and are safer, more convenient and comfortable for the patient.
[93] 1. Polymeric material
[94] 1.1 Microparticles
[95] For delivery of the polynucleotide or other genetic material of the present invention, any polymeric material capable of associating with the polynucleotide or other genetic material and transfection agent and targeting the site of action may be used. In a preferred embodiment, the material carries polynucleotides or other genetic material and should be color converted. Preferably, microparticles, most preferably microspheres, are used in the present invention.
[96] A wide variety of polymeric carriers, such as but not limited to poly (ethylene-vinyl acetate) (40% crosslinked), poly (D, L-lactic acid) oligomers and polymers, poly (L-lactic acid) oligomers and polymers , Poly (glycolic acid), copolymers of lactic acid and glycolic acid, poly (caprolactone), poly (valerolactone), poly (anhydrides), poly (caprolactone) or poly (lactic acid) and polyethylene glycol, polysaccharides , Copolymers with silicones and mixtures thereof can be used in the present invention.
[97] 1.2 Microspheres
[98] For the drug delivery aspect of the present invention, the preferred polymeric material is microspheres. In drug delivery embodiments, the use of transfection agents is optional.
[99] Preferably the microbeads or microspheres (collectively referred to herein as microspheres) for use in the present invention are based on biocompatible, hydrophilic, substantially spherical non-toxic polymers. The microspheres are injectable through a needle of 18 gauge or less and cannot be removed by the immune or lymphatic system. The polymer may preferably be coated with reagents that promote cell adhesion. Living cells can also be attached to microspheres to form layered cells, where they can be combined with surrounding tissues to enhance the long term stability of the beads.
[100] The microspheres of the present invention comprise an elastomer selected from the group consisting of elastomers, preferably acrylic polymers, vinyl alcohol polymers, acrylate polymers, polysaccharides, silicones and mixtures thereof. More preferably, the hydrophilic copolymers that can be used for such applications are acrylic copolymers such as polyacrylamide and derivatives thereof, polyacrylates and derivatives thereof, as well as polyallyl and polyvinyl compounds. In another embodiment, the microspheres of the present invention may also be based on the combination of a cationic hydrophobic polymer with a polyvinyl acetate. These polymers are all crosslinked so as to be stable and not reabsorbable, and may contain other chemicals in their structure that exhibit certain properties such as chemotactic effects, promotion of cell adhesion to cells or tissues.
[101] Microspheres intended to be implanted at various locations in the body, preferably via infusion, in accordance with the present invention comprise non-resorbable hydrophilic polymers containing materials suitable for cell adhesion, and are localized by radiation prior to or during intervention. It may further contain radiopaque molecules or other labeling reagents in order to facilitate.
[102] Microspheres for use in the present invention are nontoxic to tissues and cells, biocompatible, and adhere to a variety of cells and tissues by cell growth promoted by the microspheres at the site of implantation. In addition, the microspheres are non-resorbable and non-biodegradable and therefore stable, durable and will retain their general shape and position once implanted at the desired site. In alternative embodiments, the microspheres of the present invention are resorbable and therefore biodegradable. Such resorbable and biodegradable microspheres are based on, for example and without limitation, polysaccharides and polysaccharide derivatives.
[103] In general, the microspheres for use in the present invention may have any shape, with substantially spherical microspheres being preferred. Microspheres for use in the present invention may have a diameter in the range of about 10 μm to about 2000 μm. Preferably, the microspheres for use in the present invention to which cells adhere to the surface will have a diameter in the range of 40 μm to 1000 μm.
[104] The elastic microspheres of the present invention may be injected through a needle of preferably 18 gauge or less and removed through macrophages or other elements of the immune or lymphatic system. Preferred mean diameters of the microspheres in this case are from about 40 μm to about 400 μm, more preferably from about 50 to about 200 μm. In the most preferred embodiment, the average diameter of the injectable microspheres is in the range of about 70 to about 120 μm.
[105] In another aspect of the invention, the subset of microspheres for use in the present invention is based on non-toxic, biocompatible, swellable, hydrophilic, substantially spherical particles comprising various polymers. The swellable microspheres are crosslinked polymers that are highly absorbent and therefore can swell upon contact with an aqueous medium under certain conditions. As will be understood by those skilled in the art, the degree of swelling of the crosslinked polymer varies with the nature and degree of crosslinking of the polymeric material. The properties of the solvent in which the microspheres are suspended or in contact with the microspheres, such as salt and ion concentrations and pH levels, also affect the degree of swelling.
[106] By carefully adjusting the size and degree of swelling of some crosslinked and swellable polymers, the microspheres can be used to achieve color conversion and gene transfer. According to the invention, the polymeric material with high water absorption is first selected. The swelling properties of these polymers can be further manipulated by controlling the degree of crosslinking, which can be done chemically or via irradiation, as known to those skilled in the art.
[107] More importantly, the swelling of the microspheres comprising the polymer can be further controlled by adjusting the solvent in which the microspheres are suspended. This is done in two steps disclosed herein. First, prior to injection, the size of the microspheres is carefully adjusted using a suitable solvent, salt concentration and pH level depending on the specific microspheres used. The microspheres prior to injection may have their original size or may swell to a certain degree due to contact with the solvent. By adjusting the pre-infusion swelling, microspheres can be easily injected through a needle of 30 gauge or less. Secondly, the microspheres can be swelled to a predetermined size after the injection and upon contact with the tissue at the injection site, or the size can be maintained prior to its injection, either of which is fixed to the injection site The desired color conversion gene therapy effect is achieved. The degree of swelling before injection and thus the swelling after injection is determined by the nature and location of the particular microspheres used and the skin defect to be treated.
[108] Microspheres for use in the present invention have a diameter in the range of about 10 to about 400 μm before swelling. Preferably, prior to swelling, the diameter of the microspheres is about 10 to about 200 μm, most preferably about 10 to about 120 μm. After injection and swelling, the microspheres have an average diameter of at least about 40 μm, preferably at least about 50 μm, and more preferably at least about 70 μm. The microspheres of the present invention can swell up to about 15 times their original size. The fully swollen size of the microspheres after injection is controlled by the various means discussed above, so that the microspheres are fixed at the injection site without causing any potential damage to the tissue. Moreover, the fully swollen size of the microspheres after injection is predetermined based on factors such as the physiological conditions of the injection site, the original microsphere size, the solvent used and the swelling before injection of the microspheres. Thus, specific infusion schemes can be envisioned according to the specific embolization gene therapy needs of the case. The size and nature of these microspheres allow microspheres to be easily injected through needles of 30 gauge or less, but are large enough to be anchored to the injection site and will not be digested or eliminated by macrophages or other elements of the immune system. To provide.
[109] Possible variations of the microspheres of the invention include replacing the microspheres with any biocompatible, non-toxic, non-resorbable polymeric particles, membranes, fibers or other solid substrates treated with reagents that promote cell adhesion. The present invention also includes the use of embolic materials which may be liquid gelable solutions disclosed in US Pat. No. 5,925,683, which is incorporated herein by reference. The present invention also includes linear soluble polymers that crosslink in situ after injection to form a solid cell adhesion promoting filler. Also contemplated herein are the preparation and / or injection of empty microspheres (microballoons) made previously or produced in place using a suitable catheter.
[110] The microspheres or other solid substrates for use in the present invention are flexible, so that they can easily pass into the injection device and small catheter without being permanently altered, but also myotrophic stresses occurring during and after the implantation process. Resistant to. It is also thermally stable, allowing for easy, convenient sterilization and freezing storage.
[111] Microspheres or other solid substrates for use in the present invention are also stable in suspension, which allows formulation and storage of the microspheres or other solid substrates in suspension and infusion with different liquids. More specifically, the hydrophilic nature of the microspheres allows the microspheres to adhere to suspensions, in particular sterile and pyrogenic (pyrogenic sources), while avoiding the formation of aggregates or adhesion to the walls of containers and implanting devices such as catheters, syringes, needles and the like. Non-containing) can be added in the form of infusion.
[112] In one embodiment, preferred microspheres of the invention are hydrophilic and cationic. The microspheres preferably comprise neutral hydrophilic monomers, bifunctional monomers, one or more monomers having a cationic charge, and optionally functionalized monomers which make it possible to detect the microspheres. The microspheres may also include one or more cell adhesion promoters and labeling reagents. The copolymer preferably copolymerizes about 25 to about 98 weight percent neutral hydrophilic acrylic monomer, about 2 to about 50 weight percent bifunctional monomer, and one or more monomers having about 0 to about 50 weight percent cationic charge It is a hydrophilic acrylic copolymer comprising in the form. By way of example, the copolymers disclosed in French Patent No. 2,378,808, incorporated herein by reference, can be used according to the invention to prepare basic microsphere copolymers. As the hydrophilic acrylic monomer, acrylamide and its derivatives, methylacrylamide and its derivatives or hydroxymethyl methacrylate can be used. Examples of bifunctional monomers include, but are not limited to, N, N'-methylene-bis-acrylamide, N ', N'-diallyltarthiamide or glyoxal-bis-acrylamide. In addition, monomers having cationic charges include, but are not limited to, those having tertiary or quaternary amine functionalities, preferably diethylaminoethyl acrylamide, methacrylamidopropyl trimethylammonium or acrylamidoethyl triethylammonium. . In a particularly preferred embodiment, copolymers comprising from about 25 to about 98 weight percent methacrylamide, from about 2 to about 50 weight percent N, N-methylene-bis-acrylamide are used.
[113] In another related aspect of the present invention, the hydrophobic or ionic properties of the embolic material can be altered as deemed necessary, for example, by introducing non-limiting hydrocarbon chains and / or hydrophilic ionizable chemical groups. Such alteration of the embolic material increases the adsorption intensity between the bioactive therapeutic factor and the transfection agent, which is sufficient to control the time period for delivery of the bioactive therapeutic factor. Such adjustment of the adsorption intensity between the bioactive therapeutic factor and the transfection agent can be used to control the absolute amount of bioactive therapeutic factor associated with the embolic material.
[114] In one particularly advantageous embodiment of the invention, it is also possible to increase the stability of the microspheres by reticulating the coalescing agent. By way of example, in the case of gelatin, a network forming agent can be selected from bifunctional chemical agents (e.g., glutaraldehyde, formaldehyde, glyoxal, etc.) reacting on gelatin amines.
[115] Another embodiment of the invention is to have microspheres visible under the light source and within the body. For example, it is also possible to label the microspheres after synthesis. This can be done, for example, by implanting fluorescent labeling derivatives (eg fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (RITC), etc.). The functionalized monomer is generally obtained by chemically coupling the monomer with a marker (which may be a chemical dye such as Cibacron Blue or Procion Red HE-3B, which enables direct visualization of microspheres). (Boschetti, J. Biochem-Biophys. Meth., 19: 21-36 (1989)). Examples of functionalized monomers that can be used for this type of label include N-acryloyl hexamethylene civacron blue or N-acryloyl hexamethylene procion red HE-3B; Magnetic resonance imaging agents (erbium, gadolinium or magnetite); Contrast agents, for example barium or iodine salts (eg acrylamino-e-propion-amido) -3-triiodo-2,4,6-benzoic acid, see Boschetti et al., Bull. Soc. Chim., No. 4 France, (1986). In the case of barium or magnetite salts, this can be introduced directly into the initial monomer solution in powdered form.
[116] Various types of cell adhesion promoters well known in the art can be used in the present invention. In particular, cell adhesion promoters can be selected from collagen, gelatin, glucoseaminoglycans, fibronectin, lectins, polycations (eg, polylysine, chitosan, etc.), and any other natural and synthetic biological cell adhesion agents.
[117] Preferably, the cell adhesion promoter is present in an amount of about 0.1 to 1 g per ml of microspheres precipitated in microspheres or other solid substrates.
[118] Microspheres are prepared by suspension polymerization, drop-by-drop polymerization, or any other method known to those skilled in the art. The manner of preparation of the selected microspheres will usually vary depending on the properties desired for the resulting microspheres, for example microsphere diameter and chemical composition. The microspheres of the present invention can be prepared by standard polymerization methods disclosed in the art (e.g., E. Boschetti, Microspheres for Biochromatography and Biomedical Applications.Part I, Preparation of Microbeads, which is incorporated by reference herein). In: Microspheres, Microencapsulation and Liposomes, John Wiley & Sons, Arshady R., Ed., Vol. 2, p. 171-189 (1999). Microspheres are prepared starting from an aqueous solution of monomers containing coalescing agents such as collagen (gelatin is denatured collagen). The solution is then mixed with a suitable non-aqueous solvent to prepare a suspension of droplets, which is then turned into a solid gel by the polymerization of monomers using a suitable catalyst. The microspheres are then collected by filtration or centrifugation and washed.
[119] Cell adhesion promoters or labeling reagents are introduced onto the microspheres by well-known chemical coupling procedures (called "ligand immobilization") for affinity chromatography. Another method of introduction is to diffuse in the gel network constituting the microspheres and then trap the diffused molecules in place by precipitation or chemical crosslinking.
[120] The microspheres of the invention are also described in the art, for example in French Patent No. 2,378,808, US Patent No. 5,648,100; 5,635,215; And 5,648,100, each of which is incorporated herein by reference. Generally, solution polymerization of the monomers is carried out at temperatures in the range of about 0 to about 100 ° C. and about 40 to about 60 ° C. in the presence of a polymerization initiator.
[121] The polymerization initiator is advantageously chosen among redox systems. In particular, alkali metal persulfates and N, N, N ', N'-tetramethylethylenediamine or dimethylaminopropionitrile, organic peroxides such as benzoyl peroxide or even 2,2'-azo-bis-isobuty Combinations with ronitrile can be used. The amount of initiator used is selected by one skilled in the art with respect to the amount of monomers sought and the rate of polymerization. The polymerization can be carried out in lumps or as emulsions.
[122] In the case of mass polymerization, aqueous solutions containing different dissolved components and initiators are polymerized in a homogeneous medium. This makes it possible to obtain an aqueous gel mass and then separate it into microspheres, for example by passing it through a screen of screens.
[123] Emulsification or suspension polymerization is the preferred production method since it is possible to directly obtain microspheres of a desired size. This can be done as follows: An aqueous solution containing different dissolved constituents (eg different monomers, cell adhesion) is mixed with a liquid organic phase which is not miscible with water by stirring and optionally in the presence of an emulsifier. The stirring speed is adjusted to obtain an aqueous phase emulsion in an organic phase which forms drops of the desired diameter. The polymerization is then initiated by the addition of an initiator. This may involve exothermic reactions and then the temperature of the reaction medium may be measured.
[124] As the organic phase, vegetable or mineral oils, some petroleum distillates, chlorinated hydrocarbons or mixtures of these different solutions can be used. Furthermore, if the polymerization initiator comprises a plurality of components (redox system), one of them can be added to the aqueous phase before emulsification.
[125] The microspheres obtained as above can then be recovered by cooling, decantation and filtration. These are then separated by size category and washed to remove residuals of any secondary products.
[126] The polymerization step may be followed by the reticulation step of the cell adhesion agent and the labeled reagent step if the putative microspheres are presumably distinguishable by transplantation after synthesis.
[127] 2. Bioactive Therapeutic Factors
[128] Bioactive therapeutic factors of the present invention include one or more of the following substances: antitumor agents, angiogenesis inhibitors, hormones and steroids, vitamins, peptides and peptide analogs, antibodies or fragments thereof, cell division inhibitory factors, vaccines, enzymes, Allergen inhibitors, circulatory drugs, anti-tuberculosis agents, antiviral agents, angina inhibitors, antibacterial agents, and antifungal agents, anti-inflammatory agents, antiprobiotics, rheumatoid inhibitors, anesthetics, cardiovascular glycosides, sedatives, local anesthetics, antihistamines, radiosensitizing agents, General anesthetics, or combinations thereof.
[129] 3. Bioactive Therapeutic Factors for Use in Gene Therapy
[130] For polynucleotide-based embolization gene therapy, the polynucleotide may encode any bioactive therapeutic factor of the invention, wherein the polynucleotide is associated with a transfection agent of the invention. Genetic materials encoding bioactive therapeutic factors of the present invention include, for example and without limitation, nucleic acids, polynucleotides, RNAs and DNAs of natural or synthetic origin, such as recombinant RNAs and DNAs and antisense RNAs and DNAs; Hammerhead RNAs, ribozymes, antigenic nucleic acids, single and double stranded RNAs and DNAs and their homologues such as phosphorothioate and phosphorodithioate oligodeoxynucleotides. Types of genetic material that can be used include, for example, genes delivered on expression vectors such as plasmids, phagemids, cosmids, yeast artificial chromosomes (YACs), and viruses carrying genetic material as well as defective or "helper" viruses. There are mold particles. Such polynucleotides may also be used in conjunction with other elements such as, but not limited to, tissue specificity enhancers, nuclear localization signals, and the like.
[131] In addition, the genetic material may be combined with a protein or other polymer, for example. For example, in one embodiment, the present invention also provides the use of targeting polyclonal and monoclonal antibodies with polymeric material carriers, bioactive therapeutic factors and transfection agents. Some examples of genetic therapeutics that can be applied using the microspheres of the invention include DNA encoding at least a portion of the HLA gene, DNA encoding at least a portion of dystrophin, at least a portion of a cystic fibrosis transmembrane regulator (CFTR) There is an antisense oligonucleotide capable of binding to DNA encoding the DNA, DNA encoding at least a portion of IL-2, DNA encoding at least a portion of the TNF, DNA encoding at least a portion of Ras. DNA encoding several proteins can be used for the treatment of many different types of diseases. For example, tumor necrosis factor and / or interleukin-2 can be provided for the treatment of advanced cancer; HDL receptors can be provided for the treatment of liver disease; Thymidine kinase can be provided for the treatment of ovarian cancer, brain tumors or HIV infection; HLA-B7 can be provided for the treatment of malignant melanoma; Interleukin-2 may be provided for the treatment of neuroblastoma, malignant melanoma or kidney cancer; Interleukin-4 may be provided for the treatment of cancer; HIV envelope can be provided for the treatment of HIV infection; Antisense ras / p53 may be provided for the treatment of lung cancer; Adenosine deaminase can be provided for the treatment of ADA defects; Factor VIII may be provided for the treatment of hemophilia B. See, eg, Science 258, 744-746.
[132] 4. Bioactive Therapeutic Factors for Use in Drug Therapy
[133] For drug-based embolization aspects of the invention, the bioactive therapeutic factor comprises one or more of the following agents associated with the microspheres of the invention.
[134] Anti-tumor agents are for example, but not limited to, platinum compounds (eg, spiroplatin, cisplatin and carboplatin), adriamycin, mitomycin c, ansamitocin, bleomycin, bleomycin sulfate, cytosine arabinoside, arabin Nosyl adenine, mercaptopolylysine, vincristine, busulfan, chlorambucil, melparan (e.g. PAM, L-PAM or phenylalanine mustard), mercaptopurine, mitotan, procarbazine hydrochloride, dactinomycin ( Actinomycin D), daunorubicin hydrochloride, doxorubicin hydrochloride, taxol, mitomycin, plicamycin (mithramycin), aminoglutetidemide, estramestine phosphate sodium, flutamide, leuprolide acetate, methi Estrol acetate, tamoxifen citrate, testosterone, trirostane, amsacrine (m-AMSA), asparaginase (L-asparaginase) , Erwina asparaginase, etoposide (VP-16), interferon α-2a, interferon α-2b, teniposide (VM-26), vinblastine sulfate (VLB), vincristine sulfate, methotrexate and carbox Contains gellesin.
[135] Blood products include, for example and without limitation, erythropoietin, non-oral iron, hemin and hematoporphyrin and derivatives thereof.
[136] Biological response modifiers include, for example and without limitation, muramyldipeptides, muramyltripeptides, lymphokines (e.g. bacterial endotoxins such as lipopolysaccharides, macrophage activating factors), bacterial subunits (e.g. Mycobacteria, corynebacteria), synthetic dipeptides, N-acetyl-muramil-L-alanyl-D-isoglutamine and prostaglandins.
[137] Antifungal agents include, for example and without limitation, ketoconazole, nystatin, griseofulvin, flucitocin (5-fc), myconazole, amphotericin B, lysine and beta-lactam antibiotics such as sulfazincin.
[138] Hormones and steroids include, but are not limited to, growth hormones, melanocyte stimulating hormones, corticotrophic hormones, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, cortisone, cortisone acetate, hydrocortisone, hydrocortisone acetate, hydrocortisone cypionate, Hydrocortisone sodium phosphate, hydrocortisone sodium succinate, prednisone, prednisolone, prednisolone acetate, prednisolone sodium phosphate, prednisolone tebutate, prednisolone pivalate, triamcinolone, triamcinolone acetonide, triamcinolone hexaacetonyl hexaacetonitrile Methylprodinisolone Acetate, Methylprodinisolone Sodium Succinate, Plow Solid, Beclomethasone Di Ropionate, Betamethasone Sodium Phosphate, Betamethasone, Betamethasone Disodium Phosphate, Betamethasone Sodium Phosphate, Betamethasone Acetate, Betamethasone Disodium Phosphate, Chloroprednisone Acetate, Corticosterone, Desoxycorticosterone Pivalate, Desoxycorticosterone Pivalate, Des Oxymethasone, estradiol, fludrocortisone, fludrocortisone acetate, dichlorisone acetate, fluorohydrocortisone, fluoromessolone, fluprednisolone, paramethasone, paramethasone acetate, androsterone, fluoxymesterone, aldosterone, Metandrostenolone, Methylandrostendiol, Methyl Testosterone, Norethaneandrolone, Testosterone, Testosterone Enanthate, Testosterone Propionate, Equilenin, Equilin, Estradiol Ben Eate, estradiol dipropionate, estriol, estrone, estrone benzoate, acetoxypropenolone, anazestone acetate, chlormadinone acetate, fluororosetone acetate, hydroxymethylprogesterone, hydroxymethylprogesterone acetate , Hydroxyprogesterone, hydroxyprogesterone acetate, hydroxyprogesterone caproate, melengestrol acetate, normesterone, pregnenolone, progesterone, ethynyl estradiol, mestranol, dimethosterone, etisterone, ethino Diol diacetate, noethyne drone, noethyne drone acetate, noethysterone, fluorinolone acetonide, fluandrenonelone, flunisolidide, hydrocortisone sodium succinate, methylprodinisolone sodium succinate, prednisolone force Yttrium sodium, triamcinolone acetonide, hydroxydione sodium spironolactone, oxandrolone, oxymetholone, prometholone, testosterone cypionate, testosterone phenylacetate, estradiol cypionate, and noethinodrel do.
[139] Vitamins include, but are not limited to, cyanocobalamine neinoic acid, retinoids and derivatives thereof such as retinol palmitate, alpha-tocopherol, naphthoquinone, cholecalciferol, folic acid and tetrahydrofolate.
[140] Peptides and peptide homologs include, but are not limited to, manganese superoxide dismutase, tissue plasminogen activator (t-PA), glutathione, insulin, dopamine, RGD, AGE, RGE, KGD, KGE or KQAGDV (GPIIBIIIa receptors). Peptide ligands, opiate peptides, enkephalins, endorphins and their analogs, human chorionic gonadotropin (HCG), corticopropine releasing factor (CRF), cholecystokinin and its analogs, bradykinin And homologues and promoters and inhibitors thereof, elastin, vasopressin, pepsin, glucagon, substance P, integrin, captopril, enalapril, ricinopril and other ACE inhibitors, corticotrophin (ACTH), oxytocin, calcitonin, IgG or Fragments thereof, IgA or fragments thereof, IgM or fragments thereof, ligands for effector cell protease receptors (all subtypes), thrombin, strepto Nase, urokinase, t-PA and all active fragments or homologues, protein kinase C and binding ligands thereof, interferon (alpha-, beta-, gamma-interferon), colony stimulating factor (CSF), granulocyte colony stimulating factor (GCSF), Granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF), nerve growth factor (NGF), platelet derived growth factor, lymphotoxin, epidermal growth factor, fibroblast growth factor, vascular endothelial growth factor , Erythropoietin, transforming growth factor, oncostatin M, interleukin (1,2,3,4,5,6,7,8,9,10,11 and 12), metalloprotein kinase ligand, cola Geneases and agonists and antagonists.
[141] Antibodies used in the present invention include, for example, non-limiting substantially purified antibodies or fragments thereof, such as non-human antibodies or fragments thereof. In various embodiments, the substantially purified antibodies or fragments thereof of the invention can be human, non-human, chimeric and / or humanized antibodies. Such non-human antibodies may be goat, mouse, sheep, horse, chicken, rabbit or rat antibodies. On the other hand, non-human antibodies of the invention may be chimeric and / or humanized antibodies. Fully human antibodies are particularly preferred for the treatment of human patients. Chimeric antibodies are molecules in which different parts are derived from different animal species, such as a murine mAb and a variable region derived from a human immunoglobulin constant region (Cabilly et al., Incorporated herein by reference). Patent 4,816,567; and US Pat. No. 4,816,397 to Boss et al. In addition, non-human antibodies contemplated within the scope of the present invention may be polyclonal antibodies or monoclonal antibodies. Any of the antibodies of the invention can be bound to a therapeutic moiety or detectable substance. Non-limiting examples of detectable materials that can bind to the antibodies of the invention are enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials and radioactive materials.
[142] The present invention provides the use of targeting polyclonal and monoclonal antibodies in combination with polymeric material carriers, bioactive therapeutics and transfection agents. Such antibodies are useful in the methods of the present invention and allow targeted delivery of bioactive therapeutic factors and polymeric materials bound to transfection agents to the site of action. As used herein, the term "monoclonal antibody" or "polyclonal antibody composition" refers to a population of antibody molecules containing only one species of antigen binding site capable of immunoreacting with a particular epitope. Polyclonal antibodies can be prepared by immunizing a suitable subject as an immunogen with a polypeptide of the invention as described above. Preferred polyclonal antibody compositions are, for example, those selected from among antibodies against a polypeptide or polypeptides of a receptor on a cancer cell surface of a tumor treated by the composition of the invention.
[143] Cell division inhibitory factors include, but are not limited to, esturamustine and its phosphorylated derivatives, esturamustine-phosphate, doxorubicin, amphetyl, combretastatin A4 and colchicine.
[144] Vaccines include, but are not limited to, pneumococcal vaccine, polio vaccine, anthrax vaccine, tuberculosis (BCG) vaccine, hepatitis A vaccine, cholera vaccine, A, C, Y meningococcal vaccine, W135 vaccine, plague vaccine, rabies ( Human diploid) vaccine, yellow fever vaccine, Japanese encephalitis vaccine, typhoid (phenol and heat-lethal) vaccine, hepatitis B vaccine, diphtheria vaccine, tetanus vaccine, pertussis vaccine, H. influenza b vaccine, polio vaccine, measles vaccine, pandemic Mumps vaccine, rubella vaccine, chickenpox vaccine, Streptococcus pneumoniae Ty (live mutant bacterium) vaccine, Vi (Vi capsular polysaccharide) vaccine, DT (denatured toxin) vaccine, Td (denatured toxin) vaccine, aP ( Inactive bacterial antigen / axula (DtaP) vaccine, Hib (bacterial polysaccharide-protein conjugate) vaccine, hepatitis B virus (inactive serum-derived viral antigen / recombinant antigen) vaccine, influenza vaccine, rotavirus vaccine, respiration Syndrome Virus (RSV) Vaccine, Human Astrovirus Vaccine, Rotavirus Vaccine, Hepatitis A and B Human Influenza Virus Vaccines, Hepatitis A Virus Vaccine, Live Attenuated Parainfluenza Virus Type 3 Vaccine, Enterovirus Vaccine, Retrovirus Vaccines and picornavirus vaccines.
[145] Diseases that can be treated with the compositions and methods of the present invention include, but are not limited to, for example, liver, kidney, acute lymphoblastic leukemia, acute myeloid leukemia, Ewing's sarcoma, gestational stromal carcinoma, Hodgkin's disease, non-Hodgkin Lymphoma, Burkitt's lymphoma Diffuse large cell lymphoma, follicular mixed lymphoma, lymphoblastic lymphoma, rhabdomyosarcoma, testicular carcinoma, Wilm's tumor, anal carcinoma, bladder carcinoma breast carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia , Head and neck carcinoma, lung (small cell) carcinoma, multiple myeloma, non-Hodgkin's lymphoma, cystic lymphoma, ovarian carcinoma, brain tumor (astrocytoma), cervical carcinoma, colorectal carcinoma, hepatocellular carcinoma, Kaposi's sarcoma, lung (non-small cell) ) Carcinoma, melanoma, pancreatic carcinoma, prostate carcinoma, soft tissue carcinoma, breast carcinoma, colorectal carcinoma (stage III), osteogenic sarcoma, ovarian carcinoma (stage III), testicular carcinoma or these There are combined.
[146] Enzymes include, for example and without limitation, alkaline phosphatase, cyclooxygenase type I and antagonists and agonists.
[147] Allergen inhibitors include, for example and without limitation, amelexanox.
[148] Coagulation inhibitors include, for example and without limitation, phenprocumon and heparin.
[149] Cyclic drugs include, for example and without limitation, propranolol.
[150] Antituberculous agents include, for example and without limitation, para-aminosalicylic acid, isoniazid, capreomycin sulfate cycloserine, ethambutol hydrochloride ethionamide, pyrazineamide, rifampin, and streptomycin sulfate.
[151] Antiviral agents include, for example and without limitation, acyclovir, amantadine azitimidine (AZT or zidovudine), ribavirin and vidarabine monohydrate (adenine arabinoside, ara-A).
[152] Angina therapeutics include, for example and without limitation, diltiazem, nifedipine, verapamil, erythritol tetranitrate, isosorbide dinitrate, nitroglycerin (glyceryl trinitrate) and pentaerythritol tetranitrate do.
[153] Antibiotics include, for example, dapsone, chloramphenicol, neomycin, cefaclo, cephadroxyl, ceparexin, cepradine erthromycin, clindamycin, lincomycin, amoxicillin, ampicillin, bacampicillin, carbenicillin, dicloxacillin Lean, cyclocycline, picloxacillin, hetacillin, methicillin, naphcillin, oxacillin, penicillin G, penicillin V, ticarcillin, rifampin and tetracycline.
[154] Anti-inflammatory and analgesic agents are for example diflunisal, ibuprofen, indomethacin, meclofenamate, mefenamic acid, naproxen, oxyfenbutazone, phenylbutazone, pyrocampam, sulfindac, tolmethine, aspirin and salicylic Includes the rate.
[155] Probiotic agents include, for example and without limitation, chloroquine, metronidazole, hydroxychloroquine, quinine and meglumine antimonate.
[156] Rheumatoid inhibitors include, for example and without limitation, renisilamine.
[157] Anesthetics include, for example and without limitation, analgesics and opioids, such as codeine, heroin, methadone, morphine and opium.
[158] Cardiac glycoside agents include, for example and without limitation, desranoside, digitoxin, digoxin, digitarine and digitaris.
[159] Neuromuscular blockades include, but are not limited to, ataccurium mesylate, galamine triethiodide, hexafluorenium bromide, metocurin iodide, pancuronium bromide, succinylcholine chloride (succinatemedium chloride) Tubulocurin chloride and becuronium bromide.
[160] Sedatives (hypnotics) include, for example and without limitation, amobarbital, amobarbital sodium, aprobarbital, butabarbital sodium, chloral hydrate, ethclobinol, ethinamate, flulazepam hydrochloride, glutetimide , Metotrimethrazine hydrochloride, metyprilon, midazolam hydrochloride paraaldehyde, pentobarbital, pentobarbital sodium, phenobarbital sodium, secobarbital sodium, talbutal, temazepam and triazolam.
[161] Local anesthetics include, for example and without limitation, bupivacaine hydrochloride, chloroprocaine hydrochloride, ethidocaine hydrochloride, lidocaine hydrochloride, mepivacaine hydrochloride, procaine hydrochloride and tetracaine hydrochloride.
[162] General anesthetics include, for example and without limitation, dropperidol, etomidate, fentanyl citrate with dropperidol, ketamine hydrochloride, methohexyl sodium and thiopental sodium.
[163] Radioactive particles or radioactive ions include, for example and without limitation, strontium, rhenium, yttrium, technetium and cobalt.
[164] Preferably, the bioactive therapeutic factor is an antitumor agent, hormone, steroid, antifungal agent, peptide or peptide analog. More preferably, the bioactive therapeutic factor is dexamethasone, amphotericin B, adriamycin, mitomycin c, taxol, doxorubicin or tissue plasminogen activator (t-PA). The bioactive therapeutic factors used in the present invention are preferably very active at low concentrations. The targeting aspect of the present invention keeps the effective concentration at the site of treatment undiluted in the body, thereby lowering the dose applied to the treatment. The amount of bioactive therapeutic factor of the invention administered to a patient varies, for example, depending on the particular bioactive therapeutic factor used, the method in which the bioactive therapeutic factor is administered, and the age, sex, weight and physical conditions of the patient. In general, treatment begins with a small dose, which can then be increased in small increments until the desired effect is achieved under certain circumstances. In addition, those skilled in the art will appreciate reference materials such as Physician's Desk Reference, Medical Economics Company, Montvale N.J. 07645-1742 can be used to determine suitable bioactive therapeutic factors in an appropriate amount, and thus the corresponding prodrugs of the invention that can be administered to a patient in the case of therapeutic drugs and gene therapy. In accordance with the present invention, the prodrug is delivered to the patient (eg a local site of the patient), for example, to treat a patient's symptoms (ie, disease state, disease, disorder, etc.). The prodrugs can be used as such or incorporated into other embodiments such as emulsions.
[165] 5. Transfection Agents for Polynucleotide Delivery
[166] Nucleic acids, polynucleotides, RNA and DNA of natural or synthetic origin, such as recombinant RNA and DNA and antisense RNA and DNA; Genetics comprising hammerhead RNA, ribozymes, antigenic nucleic acids, single and double stranded RNA and DNA and their analogs, alone or in combination with other elements such as but not limited to tissue specificity enhancers, and nuclear localization signals The material can be introduced into eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” refer to various techniques known in the art for introducing foreign nucleic acids into host cells, including but not limited to calcium phosphate or calcium chloride coprecipitation, DEAE- Dextran is used to refer to mediated transfection, lipofection, or electroporation. Suitable methods for transformation or transfection of host cells are disclosed in Sambrook, et al., And other experimental manuals above.
[167] For stable transfection of mammalian cells, it is known that only small cell fractions can incorporate foreign DNA into its genome, depending on the expression vector and transfection technique used. To identify and select these integrations, genes encoding selectable markers (eg for antibiotic resistance) are generally introduced into the host cell along with the gene of interest. Preferred selectable markers are those that confer resistance to drugs such as G418, hygromycin and methotrexate. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (eg, cells with selectable marker genes will survive while other cells will die).
[168] In order to obtain efficient in vivo delivery of the bioactive therapeutic compositions of the invention, various transfection agents are used. Typical examples of suitable transfection agents for use in the methods of the invention include, but are not limited to, calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, quaternary ammonium amphoteric DOTMA ((dioleoyloxypropyl) Trimethylammonium bromide, marketed by GIBCO-BRL as lipofectin (Felgner et al., (1987) Proc. Natl. Acad. Sci. USA 84, 7413-7414; Malone et al., (1989) Proc. Natl Acad.Sci. USA 86 6077-6081); Lipophilic glutamate diesters with trimethylammonium head pendant (Ito et al. (1990) Biochem. Biophys. Acta 1023, 124-132); Metabolic parent lipids, such as cationic lipids dioctadecylamido glycylspermine (DOGS, Transfectam, Promega) and dipalmitoylphosphatidyl ethanol amylspermine (DPPES) (JP Behr (1986) Tetrahedron Lett. 27 5861-5864, JP Behr et al. (1989) Proc. Natl. Acad. Sci. USA 86, 6982-6986); Metabolic quaternary ammonium salts (DOTB, N- (1- [2,3-dioleoyloxy] propyl) -N, N, N-trimethylammonium methylsulfate (DOTAP) (Boehringer Mannheim), polyethyleneimine (PEI), Dioleoyl esters, ChoTB, ChoSC, DOSC) (Venentis et al. (1990) Biochim. Inter. 22, 235-241); 3-beta [N- (N ', N'-dimethylaminoethane) -carbamoyl] cholesterol (DC-Chol), dioleoylphosphatidylethanolamine (DOPE) / 3beta [N- (N', N'-dimethyl) Aminoethane) -carbamoyl] cholesterol DC-Chol (1: 1 mixture) (Gao et al., (1991) Biochim, Biophys. Acta 1065, 8-14), spermine, spermidine, lipopolyamine (Behr et al., Bioconjugate Chem, 1994, 5: 382-389), lipophilic polylysine (LPLL) (Zhou et al., (1991) Biochim. Biophys. Acta 939, 8-18), [[(1,1 , 3,3-tetramethylbutyl) cresoxyl] ethoxy] ethyl] dimethylbenzylammonium hydroxide (DEBDA hydroxide) (containing excess phosphatidylcholine / cholesterol) (Ballas et al., (1988) Biochim. Biophys Acta 939, 8-18), cetyltrimethylammonium bromide (CTAB) / DOPE mixture (Pinnaduwage et al., (1989) Biochim. Biophys. Acta 985, 33-37), DOPE, CTAB, mixed with phosphatidylethanolamine Of glutamic acid with DEBDA, didodecylammonium bromide (DDAB) and stearylamine Lipophilic diesters (TMAG) (Rose et al., (1991) Biotechnique 10, 520-525), DDAB / DOPE (TransfectACE, GIBCO BRL) and oligogalactose containing lipids (Remy et al., Will be issued) There is this.
[169] Also included in the present invention is the use of various transfection enhancers to increase the efficiency of delivery of bioactive therapeutic factors into cells. Suitable transfection enhancers include, but are not limited to, for example, DEAE-dextran, polybrene, lysosomal-destructive peptides (Ohmori NI et al., Biochem Biophys Res Commun 1997 Jun 27; 235 (3): 726-9), Chondroitin-based proteoglycans, sulfated proteoglycans, polyethyleneimine, polylysine (Pollard H et al. J Biol Chem, 1998 273 (13): 7507-11), integrin-binding peptide CYGGRGDTP, linear dextran non-saccharides Glycerol, a cholesteryl group bound to the 3'-terminal nucleoside linkage of an oligonucleotide (Letsinger, RL 1989 Proc Natl Acad Sci USA 86: (17): 6553-6), lysophosphatid, lysophosphatidylcholine Lysophosphatidylethanolamine and 1-oleoyl lysophosphatidylcholine.
[170] Examples of suitable suitable transfection agents include, but are not limited to, US Pat. No. 5,171,678 to Behr et al., Dated Dec. 15, 1992, US Pat. No. 5,476,962 to Dec. 19, 1995, And Lipopolyamine disclosed in US Pat. No. 5,616,745 to April 1, 1997, to Bear et al., Which are incorporated herein by reference.
[171] Particularly preferred transfection agents of the present invention are U.S. Patent Nos. 5,171,678 to Behr et al., Dec. 15, 1992; U.S. Patent Nos. 5,476,962, Dec. 19, 1995, and 1997. Lipopolyamines of Formula (I) as disclosed in US Pat. No. 5,616,745 to Bear et al.
[172] Lipopolyamines of formula (I) are particularly useful as vectors for transfection of eukaryotic cells. Lipopolyamines of formula (I) have the property of forming unstable single lamellar nanoparticles in ion media when dispersed in water and strongly associated with plasmid or oligonucleotide DNA via their cationic moiety to fill them and cover them with a lipid layer. By using excess cationic charge relative to nucleic acid, the lipid / DNA complex can be adsorbed onto the cell membrane, thereby promoting the uptake of DNA by the cell.
[173] Such lipopolyamines of formula (I) may further transfect weak cells (eg, but not limited to pituitary mesenchymal or anterior lobe cells, chromophoreic cells, peripheral or central nerves).
[174] 6. Recombinant Expression Vector
[175] Another aspect of the invention relates to the transfer of vectors with or without color telephones. Preferably the expression vector contains a nucleic acid encoding a bioactive therapeutic factor or polypeptide (or a portion thereof) of the invention. As used herein, the term "vector" refers to a nucleic acid molecule capable of delivering another nucleic acid to which it is bound. One type of vector is a "plasmid," which refers to a circular double stranded DNA ring capable of connecting additional DNA segments. Another type of vector is a viral vector, where additional DNA segments can be linked into the viral genome. Specific examples of viral vectors include, but are not limited to, adenovirus and retroviral vectors for genetic methods using the microspheres and transfection agents of the present invention. Also contemplated herein is the use of viral type particles containing bioactive therapeutic factors, wherein the viral type particles are physically bound to the transfection agent, which is also bound to microspheres. Also contemplated herein is the use of viral type particles containing bioactive therapeutic factors, wherein the viral type particles are physically bound to the transfection agent, which is also bound to microspheres. Such viral type particles can be designed using polyethyleneimine (PEI) bound to the integrin-binding peptide CYGGRGDTP through the formation of disulfide bridges. Such PEI / RGD-containing peptides / complexes share adenovirus constituent properties such as size and centrally protected cores as well as initial properties such as cell invasion mediated by integrin and acid-triggered endosomal escape. .
[176] Some vectors can autonomously replicate in host cells into which they are introduced (eg bacterial vectors and episomal mammalian vectors with replication of bacterial origin). Other vectors (eg, non-episomal mammalian vectors) integrate into the genome of the host cell upon introduction into the host cell and thereby replicate with the host genome. Moreover, some vectors, expression vectors, can control the expression of genes to which they are functionally bound. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors). However, the present invention seeks to include such other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which function equally.
[177] The recombinant expression vector of the present invention comprises the nucleic acid of the present invention in a form suitable for expression of the nucleic acid in a host cell. This means that the recombinant expression vector comprises one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed, selected based on the host cell used for expression. Within a recombinant expression vector, "functionally linked" means that the nucleotide sequence of interest (eg in an in vitro transcription / translation system or in a host cell into which the vector is introduced) is controlled in a manner that allows for expression of the nucleotide sequence. Bound to the sequence (s). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (eg polyadenylation signals). Such regulatory sequences are disclosed, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those that control the constitutive expression of nucleotide sequences in many types of host cells and those that manipulate nucleotide sequences (eg, tissue-specific regulatory sequences) only in a few host cells. Those skilled in the art will recognize that the design of the expression vector may vary depending on factors such as the choice of host cell to be transformed, the level of expression of the protein of interest, and the like. Expression vectors of the invention can be introduced into a host cell to produce a protein or peptide encoded by a nucleic acid, such as a fusion protein or peptide, as disclosed herein.
[178] The recombinant expression vectors of the invention are designed to express the polypeptide of the invention in prokaryotic cells (e.g. E. coli) or eukaryotic cells (e.g. insect cells (baculovirus expression vector), yeast cells, or mammalian cells). can do. Suitable host cells are further discussed in the Goidel literature above. On the one hand, recombinant expression vectors can be transcribed and translated in vitro, for example, using T7 promoter regulatory sequences and T7 polymerase.
[179] In another embodiment, nucleic acids of the invention are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors are pCDM8 (Seed (1987) Nature 329: 840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6: 187-195). When used in mammalian cells, the regulatory function of the expression vector is often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and simian virus 40. See Sambrook et al., Chapters 16 and 17, above for other expression systems suitable for both prokaryotic and eukaryotic cells.
[180] In another embodiment, the recombinant mammalian expression vector can preferentially express nucleic acid in a particular cell type (eg, express the nucleic acid using tissue-specific regulatory elements). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include albumin promoters (liver-specific; Pinkert et al. (1987) Genes Dev. 1: 268-277), lymph-specific promoters (Calame and Eaton (1988) Adv). Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Bltimore (1989) EMBO J. 8: 729-733) and immunoglobulins (Banerji et al. (1983) Cell 33: 729-740; Queen and Baltimore (1983) Cell 33: 741-748), neuron-specific promoters (e.g. neurofilament promoters; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas -Specific promoters (Edlund et al. (1985) Science 230: 912-916), prostate-specific promoters and / or enhancers (US Pat. Nos. 5,830,686 and 5,871,726, incorporated herein by reference) and mammary gland specific Promoters (eg, whey promoter; US Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally regulated promoters, such as the rat Hawks promoter (Kessel and Gruss (1990) Science 249: 374-379) and the α-fetal protein promoter (Campes and Tilghman (1989) Genes Dev. 3: 537-546) Is considered.
[181] The invention also provides a recombinant expression vector comprising a DNA molecule of the invention cloned into an expression vector in an antisense orientation. That is, the DNA molecule is operatively bound to a regulatory sequence in a manner that permits the expression of an RNA molecule that is antisense to the mRNA encoding a given polypeptide (by transcription of the DNA promoter).
[182] Regulatory sequences can be selected that are operatively bound to the cloned nucleic acid in an antisense orientation that controls the continuous expression of antisense RNA molecules in various cell types, for example, constitutive, tissue specific or cell type specific of antisense RNA. Viral promoters and / or enhancers that control expression, or regulatory sequences can be selected. The antisense expression vector may be in the form of a recombinant plasmid, phagemid or directed virus in which the antisense nucleic acid is produced under the control of a highly efficient regulatory region whose activity can be determined by the cell type into which the vector is introduced. A discussion of the regulation of gene expression using antisense genes is described in Weintraub et al. Reviews-Trends in Genetics, Vol. 1 (1) 1986.
[183] In one aspect of the invention, various cancers can be treated by feeding tissue specific enhancers and / or promoters to toxin genes on DNA templates to focus on expression of the cancer cell genes. For example, there are non-limiting diphtheria toxin genes in toxin genes. Intracellular expression of diphtheria toxin is known to kill cells. The use of some promoters may be tissue-specific, such as the use of pancreas-specific promoters for pancreatic cancer. Thus, the functional diphtheria toxin gene delivered to pancreatic cells can theoretically eradicate the entire pancreas. This strategy can be used for the treatment of pancreatic cancer. Tissue-specific enhancers cause diphtheria toxin expression only in pancreatic cells. Under the control of a tissue specific enhancer, the DNA / lipopolyamine / microsphere complex containing the diphtheria toxin gene will be introduced directly into the cannulated artery that enters the pancreas. The infusion will be performed according to some dosing schedule for as long as necessary to eradicate pancreatic tissue. In addition to diphtheria toxin, other lethal genes, such as lysine or cobra toxin factor or genes for enterotoxin, can be used with similar effects.
[184] Another specific example is the use of prostate specific antigen promoters / enhancers to direct the bioactive therapeutic factors of the present invention to the prostate of patients in need of treatment for pancreatic cancer. Specialized cancers can also be treated, for example, by delivery of genes that inhibit the cancerous properties of some cancers, such as the p53 gene, the retinoblastoma gene (and others of the gene family).
[185] 7. Adenovirus-Mediated Gene Delivery
[186] For the purpose of gene therapy, adenoviruses with deletions have been proposed as suitable vehicles. Adenoviruses are enveloped DNA viruses. Gene-transfer vectors derived from adenoviruses (so-called adenovirus vectors) have a number of features that make them particularly useful for gene delivery of such purposes. For example, the biology of adenoviruses is, in particular, independent of serious human pathologies in which the adenoviruses are highly efficient at introducing their DNA into host cells and infect a wide variety of cells. Can be produced, have a broad host range, can be produced relatively easily in large quantities, and can become replication defects, particularly by deletion of early-region 1 (E1) of the viral genome.
[187] The adenovirus genome is a linear double stranded DNA molecule of approximately 36,000 base pairs, with a 55-kDa terminal protein covalently attached to the 5 'end of each DNA strand. The Ad DNA contains about 100 base pairs of identical inverted terminal repeats (ITRs) with the correct length depending on the antigenic type. The origin of replication of the virus is precisely located inside the ITR at the genome end. DNA synthesis occurs in two stages. First, replication proceeds by strand substitution, resulting in a parentally substituted strand with the double daughter molecule. The substituted strands are single stranded and can form so-called "knob" intermediates, which allow for replication initiation and generation of double daughter molecules. Alternatively, replication can proceed simultaneously at both ends of the genome, eliminating the need for formation of the knob structure.
[188] During a productive infection cycle, the viral gene is expressed in two stages, ie the initial stage is the period until viral DNA replication, and the later stage coincides with the onset of viral DNA replication. During this initial stage only the initial gene product, encoded by the El, E2, E3 and E4 regions, is expressed, which expression performs a number of functions to prepare cells for the synthesis of viral structural proteins (Berk, 1986). . During later stages, later viral gene products are expressed in addition to the initial gene products, and host cell DNA and protein synthesis are stopped. As a result, the cells contribute to the production of viral DNA and viral structural proteins (Tooze, 1981).
[189] There are various adenovirus antigen types that differ somewhat in structure and properties. Among these antigenic types, the use of type 2 or 5 human adenoviruses (Ad2 or Ad5) or adenoviruses of animal origin (see WO 94/26914, which is incorporated herein by reference), is within the constitution of the present invention. desirable. Among adenoviruses of animal origin that can be used within the constitution of the present invention are dogs, cows, mice (e.g. Mav1, Beard et al., Virology 75 (1990) 81), sheep, pigs, birds or monkeys (e.g. Adenoviruses of SAV) origin are contemplated. Preferably the adenovirus of animal origin is a canine adenovirus, more preferably a CAV-2 adenovirus (for example strain Manhattan or A26 / 61 (ATCC VR-800)). Preferably, adenoviruses of human or dog or mixed origin can be used in the methods of the invention.
[190] Defective recombinant adenoviruses for use in the methods of the present invention can be prepared by any technique known to those skilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham, EMBO). J. 3 (1984) 2917). In particular, they can be produced by homologous recombination between adenoviruses and plasmids, in particular cassettes, containing the gene of interest. The homologous recombination occurs after cotransfection of the adenovirus and the plasmid into a suitable cell line. The cell line used preferably contains a sequence capable of (i) transforming by said elements, and (ii) complementing said defective adenovirus genome portion in an integrated form, preferably to avoid the risk of recombination. Should be. Examples of cell lines include human fetal kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59), in particular incorporating the left portion (12%) of the Ad5 adenovirus genome into the genome. Strategies for producing vectors derived from adenoviruses are also disclosed in WO94 / 26914 and FR 2,707,664, which are incorporated herein by reference.
[191] 8. Retroviral Particle-Mediated Gene Delivery
[192] Retroviral vectors are mammalian gene delivery vehicles that utilize features of the retroviral replication cycle such as stable co-linear integration of information delivered by the virus in the target cell chromosome and high infection efficiency. Retroviral vectors are becoming important tools for basic biotechnology and gene therapy research.
[193] Most of the retroviral vectors currently in use are derived from murine leukemia virus (MLV). MLVs are particularly suitable as vectors because of their well-reported transcription patterns and relatively simple unit gene structure in various cell types.
[194] Retroviral structure: Retroviruses belong to the enveloped virus. The bilipid envelope is derived from the host cell membrane and modified by the insertion of viral surface proteins (SU) and transmembrane proteins (TM). Matrix protein (MA) is located just below the outer membrane surrounding the inner core. The core consists of capsid protein (CA). Within the capsid are two copies of the retroviral genome that are attached to each other at the 5 'end via hydrogen bonding. The viral core particles also contain viral enzymes, ie reverse transcriptase (RT), protease (PR), and integrase enzyme (IN), and nucleocapsid protein (NC) bound to the viral genome. In addition to these proteins encoded by the virus, the virion also contains a number of tRNA molecules derived from a host cell tRNA population.
[195] Rat leukemia virus (MLV) is a simple retrovirus. Retroviruses have a characteristic genomic map, two long terminal repeats (LTRs) flanking the three structural genes gag, pol and env. The LTR has three regions, a U3 region containing an enhancer and a promoter element recognized by a cell transcriptor, an R region that plays an important role during reverse transcription and moreover also contains a polyadenylation signal, and a reverse transcription and retroviral genome. It can be subdivided into U5 regions containing sequences important for the packaging of. The LTR also contains important cis elements, inverted repeats, during the integration process.
[196] The integrated provirus produces two mRNA transcripts, full length mRNA encoding gag- and gag-pol polyproteins, and subsequent mRNA encoding envelope glycoproteins. The full length mRNA also acts as genomic RNA and contains three important cis elements in the 5 'untranslated sequence in addition to the components of the previously disclosed LTR residues. Located below the U5 region, the primer binding site (PBS) consists of 18 nucleotides complementary to the 3'-end of the primer tRNA molecule. Also, a pavement signal PSI is located in the 5 'untranslated region between the PBS and the beginning of the gag open reading frame. The 5 'untranslated region contains a dimeric binding domain that contributes to the dimerization of two viral genomes of virions. Directly above the U3 region is another important cisular region, the polypurine region (PP), which consists of the extension of the A and G residues. The element acts as a site to initiate plus-strand DNA synthesis during reverse transcription.
[197] Retroviral Life History: Two different mechanisms have been proposed to explain the invasion of viral particles into the host cytoplasm. Most retroviruses, including MLVs, are thought to enter the host cell through receptor-mediated endocytosis, the process by which the entire enveloped viral particle is absorbed into the endosome. Receptor molecules for homologous murine leukemia virus were cloned and identified as cationic amino acid carriers.
[198] After viral core particles enter the cytoplasm of the host cell, all enzymatic functions leading to the integrated double stranded DNA provirus are synthesized in the preceding host cell and managed by viral proteins involved in the virion. The fate of the viral protein after core particle invasion is not clear, but the reverse transcriptase, coenzyme, and capsid protein are retained with the RNA genome forming the nuclear protein complex where reverse transcription occurs. Recently, matrix proteins have also been found associated with these nucleoprotein complexes.
[199] Following reverse transcription, the nucleoprotein complex migrates to the host cell nucleus. The mechanism contributing to the nuclear localization is not clear, but evidence from Raus sarcoma virus (RSV) suggests that the IN protein is important because the RSV IN protein is localized in the nucleus when produced in mammalian cells. Invasion of the nucleoprotein complex into the nucleus requires indirect nuclear fission since the nucleoprotein complex is probably unable to produce a nuclear envelope. Once in the nucleus, integration by the IN protein is mediated. The IN protein recognizes the reverse of the conserved reverse at the end of the LTR and removes two bases from the 3 'hydroxyl end of both strands. The IN protein also promotes cleavage of the host DNA and mediates the binding between the proviral DNA and the host DNA. For the specificity of integration, no matching host DNA target sequence was found, but a tendency to integrate nearby DNase I-sensitive sites was reported.
[200] For simple retroviruses (including MLV), transcription and translation are performed with host cell groups. Complex viruses (including HIV and HTLV) encode cross-active proteins involved in transcriptional regulation. The assembly of MLV particles appears in the host membrane and the process is consistent with the germination process. In mammalian type B and C viruses (MMTV and HTLV, respectively), viral core particles are assembled in the host cell cytoplasm. Capsidation of viral genomic RNA is mediated through the binding of cis-acting capsidation signals and NC residues of the Gag- or Gag-Pol precursor protein.
[201] After germination, Gag- and Gag-Pol polyproteins are cleaved by viral protease (PR). Mutations in viral proteins alter the virion form as a whole. In addition to the proteolytic cleavage of viral polyproteins following germination of viral particles, genomic RNA also undergoes a mutation process to produce a dense dimeric genome.
[202] Retroviral Vectors: Retroviruses from which retroviral plasmid vectors can be derived include, but are not limited to, Molecular Rat Leukemia Virus, Spleen Necrosis Virus, Retroviruses such as Raus Sarcoma Virus, Harvey Sarcoma Virus, Avian Leukemia Virus, Long Sleeve Monkey leukemia virus, human immunodeficiency virus, adenovirus, myeloproliferative sarcoma virus and breast tumor virus. In one embodiment, the retroviral plasmid vector is derived from Moroni murine leukemia virus.
[203] The vector comprises one or more promoters. Suitable promoters that can be used include, but are not limited to retrovirus LTRs; SV40 promoter; And human cytomegalovirus (CMV) promoters (Miller et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or any other promoters such as cell promoters such as eukaryotic cell promoters, Non-limiting examples are histones, pol III, and β-actin promoters.Other viral promoters that may be used include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of suitable promoters will be apparent to those skilled in the art from the teachings contained herein.
[204] Nucleic acid sequences encoding bioactive therapeutic factors of interest are placed under the control of a suitable promoter. Suitable promoters that can be used include, but are not limited to, adenovirus promoters such as the adenovirus late promoter; Or heterologous promoters, such as cell expansion virus (CMV) promoters; Respiratory syncytial virus (RSV) promoter; Inducible promoters such as the MMT promoter, metallothionein promoter; Thermal promoters; Albumin promoter; ApoAI promoter; Human globin promoter; Viral thymidine kinase promoters such as the herpes single thymidine kinase promoter; Retrovirus LTRs (including the modified retrovirus LTRs described above); β-actin promoter; And human growth hormone promoters. The promoter may also be a natural promoter that controls genes encoding bioactive therapeutic factors of interest.
[205] The retroviral plasmid vector is used to transduce packaging cell lines to form producer cell lines. Examples of packaged cells that can be transfected include, but are not limited to, PE501, PA317.psi.-2, .psi.-AM, PA12, T19-14X, VT-19-17-H2, .psi.CRE, .psi. CRIP, GP + E-86, GP + envAm12, and DNA cell lines (Miller, Human Gene Therapy, Vol. 1, pg. 5-14 (1990), incorporated herein by reference). The vector can transduce the packaging cells by any means known in the art. Such means include, but are not limited to electroporation, the use of liposomes, and CaPO ₄ precipitation.
[206] The producer cell line produces infectious retroviral vector particles comprising polynucleotide sequence (s) encoding a bioactive therapeutic factor of interest. Such retroviral vector particles can then be used to transduce eukaryotic cells in vitro or in vivo. The transduced eukaryotic cells will express the polynucleotide sequence (s) encoding the bioactive therapeutic factor of interest. Eukaryotic cells that can be transduced include, but are not limited to, fetal stem cells, fetal carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.
[207] In one embodiment, retroviral plasmid vectors comprising polynucleotide sequence (s) encoding a bioactive therapeutic factor of interest can be coupled to lipids and then administered to a host as described above. In one preferred embodiment, the retroviral plasmid vector can be associated with a lipopolyamine transfectant of the invention to form a complex, which is then mixed with the microspheres of the invention and then administered to a patient in need of color conversion gene therapy. .
[208] 9. Antisense Gene Therapy
[209] The invention also includes delivering antisense nucleic acid molecules with or without embolization. An antisense nucleic acid molecule is a molecule that is complementary to a sense nucleic acid encoding a polypeptide of the invention, eg, complementary to the coding strand of a double stranded cDNA molecule or complementary to an mRNA sequence. Thus, antisense nucleic acids can hydrogen bond to sense nucleic acids. The antisense nucleic acid may be complementary to the entire coding strand or just a portion thereof, for example all or part of a protein coding region (or open reading frame). Antisense nucleic acid molecules can be antisense to all or part of the non-coding region of the coding strand of the nucleotide sequence encoding a polypeptide of the invention. The non-coding region ("5 'and 3' untranslated regions") are 5 'and 3' sequences flanking the coding region and not translated into amino acids.
[210] Antisense oligonucleotides can be about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length, for example. Antisense nucleic acids of the invention can be constructed using chemical synthesis and enzymatic linkage reactions using procedures known in the art.
[211] For example, antisense nucleic acids (eg, antisense oligonucleotides) can be prepared using natural or various modified nucleotides designed to increase the biological stability of the molecule or to increase the physical stability of the overlap formed between the antisense and sense nucleic acids. It can be synthesized chemically. For example, phosphorothioate derivatives and acridine substituted nucleotides can be used.
[212] Examples of modified nucleotides that can be used to prepare antisense nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (Carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylquaocin, inosine, N6-isopentenyladenin , 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methyl Aminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylquaosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyl Adenine, uracil-5-oxyacetic acid (v), waibutoxosocin, pseudouracil, quaosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thioura , 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino-3-N- 2-carboxypropyl) uracil, (acp3) w and 2,6-diaminopurine.
[213] On the other hand, the antisense nucleic acid can be biologically prepared using an expression vector in which the nucleic acid is subcloned in an antisense orientation (ie, RNA transcribed from the inserted nucleic acid is a target nucleic acid of interest as further disclosed in the subsections below). Antisense orientation).
[214] The expression of the antisense nucleic acid molecules of the invention is typically hybridized with or binds to genomic DNA and / or cellular mRNA encoding a given polypeptide of the invention to inhibit expression, for example by inhibiting transcription and / or translation. Prepared in situ or administered to the patient whenever possible. The hybridization can be carried out through specific interactions in the large grooves of the double helix with an antisense nucleic acid molecule that forms stable duplexes by conventional nucleotide complementarity or binds DNA duplexes, for example. . An example of a route of administration of an antisense nucleic acid molecule of the invention is the direct injection of an antisense nucleic acid molecule / lipopolyamine transfectant complex at a specific tissue site.
[215] On the one hand, antisense nucleic acid molecules can be modified for delivery to a given target cell and then administered systemically using the microspheres and transfection agents of the invention. For example, for systemic administration, the antisense molecule specifically binds to an antigen or receptor expressed on a given cell surface, for example by binding the antisense nucleic acid molecule to an antibody or peptide that binds to a cell surface receptor or antigen. Can be modified. Such peptides or antibodies can enhance the enhanced delivery obtained by the microspheres and transfection agents of the invention. The antisense nucleic acid molecule can also be delivered to cells using the vectors described above. In order to obtain sufficient intracellular concentration of the antisense molecule, a vector construct in which the antisense nucleic acid molecule is under the control of a strong pol II or pol III promoter is preferred. The antisense nucleic acid molecules of the invention may be α-anomeric nucleic acid molecules.
[216] α-anomeric nucleic acid molecules form specific double-stranded hybrids with complementary RNAs, wherein the strands are parallel to each other in opposition to conventional β-units (Gaultier et al. (1987) Nucleic Acids Res. 15 (6625-6641). The antisense nucleic acid molecule can also be a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15: 6131-6148) or a chimeric RNA-DNA homolog (Inoue et al. (1987) FEBS Lett. 215 : 327-330).
[217] The present invention further includes ribozymes. Ribozymes are catalytic RNA molecules with ribonuclease activity capable of cleaving single stranded nucleic acids with complementarity regions, for example mRNA. Thus, ribozymes (eg, hammerhead ribozymes (Haselhoff and Gerlach (1988) Nature 334: 585-591)) can be used to catalytically cleave mRNA transcripts to prevent translation of the protein encoded by the mRNA. It can be suppressed. A ribozyme having specificity for a nucleic acid molecule encoding a polypeptide of the invention can be envisioned based on the nucleotide sequence of the target gene of interest. For example, derivatives of tetrahimena L-19 IVS RNA complementary to the nucleotide sequence from which the nucleotide sequence of the active site is cleaved can be constructed (Cech et al. US Pat. No. 4,987,071; and Cech et al. US Pat. 5,116,742). On the other hand, mRNA encoding the bioactive therapeutic factors of the invention can be used to select catalytic RNA with specific ribonuclease activity from a pool of RNA molecules. See, eg, Bartel and Szostak (1993) Science 261: 1411-1418.
[218] The invention also includes nucleic acid molecules that form triple helix structures. For example, the expression of a bioactive therapeutic factor of the invention complements the regulatory region of a gene encoding a polypeptide (eg, promoter and / or enhancer) to form a triple helix structure that prevents transcription of the gene in the target cell. Can be inhibited by targeting a specific nucleotide sequence. In general, Helene (1991) Anticancer Drug Des. 6 (6): 569-84; Helene (1992) Ann. N.Y. Acad. Sci. 660: 27-36; And Maher (1992) Bioassays 14 (12): 807-15.
[219] In various embodiments, nucleic acid molecules of the invention can be modified, for example, at base residues, sugar residues or phosphate backbones to improve the stability, hybridization or solubility of the molecules. For example, the deoxyribose phosphate backbone of nucleic acids can be modified to produce peptide nucleic acids (Hyrup et al. (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used herein, the term “peptide nucleic acid” or “PNA” refers to a nucleic acid mimetic, eg, a DNA mimetic, in which the deoxyribose phosphate backbone is replaced with a pseudopeptide backbone and only four natural nucleobases are maintained. Refer. The neutral backbone of PNA has been shown to allow specific hybridization of DNA and RNA under low ionic strength conditions. Synthesis of PNA oligomers is described above in Hyrup et al. (1996) and Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93: 14670-675, which can be performed using standard solid phase peptide synthesis protocols.
[220] PNA can be used for the therapeutic and diagnostic purposes of the present invention. For example, PNA can be used as antisense or antigen for sequence-specific regulation of gene expression by inducing transcription or translation arrest or inhibiting replication. PNA may also be used, for example, in the analysis of single base pair mutations of a gene by PNA directed PCR restriction; As an artificial restriction enzyme when used in combination with other enzymes, eg S1 nuclease (see Hyrup (1996), supra); Or as a probe or primer for DNA sequence and hybridization (Hyrup et al. (1996) supra and Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93: 14670-). 675).
[221] In another embodiment, complexing with the PNA is accomplished by, for example, binding a lipophilic or other helper group to the PNA, or by forming a PNA-DNA chimera, or most preferably prior to association with the microspheres of the present invention. The use of lipopolyamines as transfection agents can be modified to enhance their stability or cellular uptake. For example, PNA-DNA chimeras can be prepared that can combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes such as RNAse H and DNA polymerase to interact with the DNA portion while the PNA portion provides high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of suitable lengths selected for base stacking, number of bonds between nucleobases and orientation (see Hyrup (1996), supra). Synthesis of PNA-DNA chimeras is described in Hyrup (1996) and Finn et al. (1996) Nucleic Acids Res. 24 (17): 3357-63. For example, DNA chains can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside homologs. 5 '-(4-methoxytrityl) amino-5'-deoxy-thymidine phosphoramidite can be used as a bond between PNA and the 5' end of DNA (Mag et al. (1989) Nucleic Acids Res. 17: 5973-88). The PNA monomers are then coupled in a stepwise manner to produce chimeric molecules with 5'PNA segments and 3'DNA segments (Finn et al. (1996) Nucleic Acids Res. 24 (17): 3357-63). On the one hand, chimeric molecules with 5'DNA segments and 3'PNA segments can be synthesized (Peterser et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119-11124).
[222] In other embodiments, the oligonucleotides may be added to other added groups such as peptides (eg, for targeting of host cell receptors in vivo) or reagents that facilitate delivery through cell membranes (eg Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86: 6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84: 648-652; PCT publication WO88 / 09810) or blood- Brain barrier layers (see, eg, PCT publication WO89 / 10134). Oligonucleotides can also be modified with hybridization-triggered cleavage reagents (Krol et al. (1988) Bio / Techniques 6: 958-976) or insertion reagents (Zon (1988) Pharm. Res. 5: 539-549). Can be. To this end, the oligonucleotides may be prepared before or after the association with the polyamine transfection agent and microspheres of the invention, followed by another molecule, such as a peptide, a hybridization triggered crosslinker, a carrier reagent, a hybridization-initiated Binding reagents and the like.
[223] 10. Active Embolization for Drug Delivery and Gene Therapy
[224] One aspect of the invention is the administration of drugs, vaccines and diagnostic or imaging agents to mammals using microspheres. Of course, this embodiment does not necessarily require the use of transfection agents. However, such reagents are selective due to their ability to associate with microspheres and bioactive agents, ie their ability to improve endocytosis or their ability to act as a linker.
[225] A method of color conversion of a blood vessel is provided, comprising delivering a therapeutically effective amount of a bioactive therapeutic factor transfectant microsphere composition to the blood vessel so that the blood vessel is effectively occluded within one aspect of the invention. A therapeutically effective amount suitable for vascular occlusion can be readily determined by the information provided above. Within a particularly preferred embodiment, the bioactive therapeutic factor transfectant microsphere composition is delivered to blood vessels that nourish the tumor.
[226] Briefly, there are a number of clinical situations (eg bleeding, tumor development) in which it is desirable to reduce or eliminate the blood supply to an organ or area. As disclosed in more detail below, this can be accomplished by injecting the bioactive therapeutic factor transfectant microsphere composition of the invention into a desired blood vessel through a selectively placed needle or catheter. The composition travels through the bloodstream until it is forced into the vasculature, whereby the blood vessels are physically (or chemically) occluded. Reduction or elimination of blood flow to certain areas results in infarction (cell death due to inadequate supply of oxygen and nutrients) or reduced blood loss from damaged blood vessels.
[227] For use in embolization, the bioactive therapeutic factor transfection microspheres of the present invention are preferably nontoxic, embolic, easy to inject vascular catheter, radiopaque, fast and permanent, sterile and effective It is readily available in different shapes or sizes at the time of the process. In addition, the composition preferably releases the bioactive therapeutic factors slowly (ideally over a period of weeks to months). Particularly preferred bioactive therapeutic factor compositions should have a predictable size of 15 to 200 μm after infusion into the pulse system. Preferably, it should not aggregate into larger particles in solution or once injected. In addition, preferred compositions should not change physical properties during storage prior to use.
[228] The embolization of the present invention aids in the treatment of neoplasms, namely (1) limited treatment of tumors (usually benign); (2) for color telephony before surgery; And (3) can be used in three or more main ways for a mitigating color phone. Briefly, benign tumors can sometimes be successfully treated by embolization alone. Examples of such tumors are tumors of vasculature origin (eg hemangiomas), endocrine tumors such as parathyroid adenoma and benign bone tumors.
[229] For other tumors (e.g., renal adenocarcinoma), surgery may be performed prior to surgical resection to reduce blood loss during surgery, shorten the duration of surgery, and reduce the risk of visible malignant cell dispersion by surgical manipulation of the tumor. Preoperative color phone calls can be used for hours or days. Many tumors, such as nasopharyngeal tumors, crystalline hematoma tumors, meningiomas, chemodectomas and vagus nerve neuromas, can be successfully colored before surgery.
[230] Saxon can also be used as a primary way of treating inoperable malignant diseases in order to prolong the survival time of patients with advanced disease. Color turnover can significantly improve the quality of life of patients with malignant tumors by alleviating unpleasant symptoms such as bleeding, vein occlusion and bronchial compression. However, the greatest benefit from palliative tumor color shift can be found in patients suffering from the humoral effects of malignant endocrine tumors, where metastasis from oncological tumors and other endocrine neoplasias, such as insulinoma and glyconoma, Although it may progress slowly, endocrine symptoms arising from the tumors will still cause great pain.
[231] In general, embolization using the bioactive therapeutic factor transfection microsphere compositions of the invention is typically performed in a similar manner regardless of site. Briefly, angiography of the area to be color shifted is performed by first injecting a radiopaque contrast agent through a catheter inserted into an artery or vein. The catheter can be inserted subcutaneously or by surgery. The blood vessels are then color shifted by refluxing the bioactive therapeutic factor transfection microsphere composition of the invention through the catheter until the flow stops. Occlusion can be confirmed by repeating the angiography.
[232] Embolization generally dispenses compositions containing bioactive therapeutic factors throughout the gap of the tumor or vascular mass to be treated. The physical volume of the embolic particles that block the arterial lumen obstructs the blood supply. The presence of bioactive therapeutic factor (s), in addition to the above effects, enhances the loss of vitality that blocks the blood supply by preventing the formation of new blood vessels that supply the tumor or vasculature.
[233] Thus, it is apparent that a wide variety of tumors can be color converted using the bioactive therapeutic factor transfection microsphere compositions of the present invention. Briefly, tumors are divided into two groups: benign and malignant. In benign tumors, cells retain their differentiated characteristics and do not divide in a completely uncontrolled manner. In malignant tumors, cells do not differentiate, do not respond to body growth and hormonal signals, and multiply in an uncontrolled manner; The tumor is invasive and can spread to distant sites (metastasis).
[234] Within one aspect of the invention, metastasis of the liver (secondary tumor) can be treated by embolization. Briefly, the catheter is advanced into the hepatic artery by inserting it through the femoral or bronchial artery and manipulating it through the arterial system under fluorescence induction. The catheter proceeds to the liver arterial tree as far as necessary to allow complete blockade of the blood vessels supplied to the tumor (s) while saving the supply of as many arterial branches as possible to normal tissue. Ideally this would be a partial hepatic artery branch, but it may be necessary to block the entire hepatic artery, or even multiple discrete arteries far from the gastrointestinal artery origin, depending on the extent of the tumor and its individual blood supply. Once the desired catheter position is reached, the artery is color-changed by injecting an angiogenesis inhibiting therapeutic composition through the arterial catheter until it stops flowing, preferably even 5 minutes after observation. Arterial occlusion can be confirmed by injecting a radiopaque contrast agent through the catheter and demonstrating by fluoroscopy or X-ray film that the vessel previously filled with the contrast agent is no longer filled with the contrast agent.
[235] Within another aspect of the invention, active therapeutic embolization can be used during surgery to remove tumors or lumps of blood vessels or cancerous organs. Still another aspect of the present invention relates to the use of active therapeutic embolization to prevent or ameliorate metastasis.
[236] As indicated above, both benign and malignant tumors can be color converted using the angiogenesis inhibiting therapeutic compositions of the invention. Typical examples of benign liver tumors are hepatocellular adenoma, cavernous angioma, and focal nodular hyperproliferation. Other benign tumors, which are rarer and often do not show clinical signs, can also be treated. This includes bile duct adenoma, bile duct cystic adenoma, fibroma, lymphoma, leiomyoma, mesothelioma, teratoma, myxoma and nodular regeneration hyperplasia.
[237] Malignant liver tumors are generally further classified into two categories, primary and secondary. Primary tumors arise directly from the tumors in which they are found. Thus, primary liver tumors originate from the cells that make up liver tissue (eg, hepatocytes and bile cells). Typical examples of primary liver malignancies that can be treated by arterial color conversion include hepatocellular carcinoma, cholangiocarcinoma, angiosarcoma, cell adenocarcinoma, squamous cell carcinoma and hepatocellular carcinoma.
[238] Secondary tumors or metastases are tumors that originate elsewhere in the body but subsequently spread to distant organs. Conventional pathways of metastasis follow direct growth to adjacent tissues, diffusion through pulsed or lymphatic systems, and along tissue surfaces or body space (peritoneal fluid, cerebrospinal fluid, etc.). Secondary liver tumors are one of the most common causes of death in cancer patients and the most common form of liver tumors. Virtually any malignant tumor can metastasize to the liver, but tumors most likely to spread to the liver include gastric, colon and pancreatic cancers; Melanoma; Lung, oropharyngeal and bladder tumors; Hodgkin's and non-hodgkin's lymphomas; Breast, ovarian and prostate tumors. The primary tumors mentioned above each have a number of different tumor types that can be treated by arterial color conversion (eg, 32 non-limiting reports of ovarian cancer have been reported).
[239] As indicated above, embolization using the bioactive therapeutic factor transfection microsphere compositions of the invention may also be applied to a variety of other clinical situations aimed at vascular occlusion. Within one aspect of the invention, arteriovenous malformations can be treated by administration of one of the bioactive therapeutic factor transfection microsphere compositions described above. Briefly, arteriovenous malformations (vascular malformations) refer to a group of diseases in which one or more (most typically many) abnormal communication between arteries and veins occurs such that the local tumor-like mass predominates the blood vessels. These diseases can be congenital or acquired.
[240] Within one embodiment of the present invention, arteriovenous malformations can be treated by inserting a catheter through the femoral or bronchial artery and progressing it into the supply artery under fluoroscopic guidance. The catheter is advanced as far as necessary to allow complete blockage of blood vessels supplied to the vasculature, preferably saving as much of the arterial branch as possible to normal tissue. Often multiple separate arteries may need to be blocked depending on the extent of the tumor and its individual blood supply). Once the desired catheter position is reached, each artery is color converted using the bioactive therapeutic factor transfectant microsphere composition of the present invention.
[241] Within another aspect of the present invention, color change may be performed to treat excessive bleeding conditions. For example, hypermenorrhea (excessive bleeding during menstruation) can be easily treated by color change of the uterine artery. Briefly, the uterine artery is the branch of the internal iliac artery on both the left and right sides. Within one embodiment of the present invention, a catheter can be advanced into each uterine artery by inserting it through the femoral or bronchial artery and manipulating it through the arterial system under fluoroscopic guidance. The catheter preferably proceeds as far as necessary to allow complete blockage of the blood vessels supplied to the uterus, while saving from supplying as many arterial branches as possible from the uterine arteries and supplying normal tissue. Ideally, both single uterine arteries may be color shifted, but often it may be necessary to block multiple separate arteries with separate blood supply. Once the desired catheter position is reached, each artery can be color shifted by administering the bioactive therapeutic factor transfectant microsphere composition described above.
[242] In a similar manner, arterial color change can be performed for a variety of other symptoms, such as but not limited to acute bleeding, vasculature, central nervous system disease, and hyperfunction.
[243] 10.1. Active color phone call in combination with gene therapy
[244] As indicated above, embolization using the bioactive therapeutic factor transfectant microsphere compositions of the present invention may also be applied to a variety of other clinical situations aimed at concurrent vascular occlusion and for performing gene therapy in patients.
[245] Within one preferred aspect of the invention there is provided a composition comprising (a) a bioactive therapeutic factor, (b) a polymeric carrier and (c) a transfection agent.
[246] Within another preferred aspect of the present invention there is provided a composition comprising (a) a polynucleotide encoding a bioactive therapeutic factor, (b) a polymeric material carrier, and (c) a transfection agent.
[247] Within a most preferred aspect of the invention there is provided a composition comprising (a) a polynucleotide encoding a bioactive therapeutic factor, (b) cationic crosslinked microspheres, and (c) a lipopolyamine transfectant.
[248] In a most preferred aspect of the invention, there is provided a composition comprising (a) a polynucleotide encoding a bioactive therapeutic factor, (b) cationic crosslinked microspheres, and (c) a lipopolyamine transfectant, wherein Polynucleotides include RNA and DNA of natural or synthetic origin, such as recombinant RNA and DNA and antisense RNA and DNA; Hammerhead RNA, ribozymes, antigenic nucleic acids, single and double stranded RNA and DNA and their analogs; Bioactive therapeutic factors include antitumor agents, angiogenesis inhibitors, hormones and steroids, vitamins, peptides and peptide analogs, antibodies or fragments thereof, vaccines, enzymes, allergen inhibitors, circulatory drugs, antituberculosis agents, antiviral agents, angina inhibitors, antigenic agents Animals, rheumatoid inhibitors, anesthetics, cardiovascular glycosides, sedatives, local anesthetics, and general anesthetics; Cationic crosslinked microspheres are based on ionic crosslinked polymers that release nontoxic, biocompatible, swellable or non-swellable, substantially spherical, hydrophilic, inert, bioactive therapeutic factors and have a size sufficient for color conversion, The polymer is preferably a sodium acrylate polymer, acrylamide and acrylamide derivative polymers, sodium acrylate and vinyl alcohol copolymers, saponified products of copolymers of vinyl acetate and acrylic esters, vinyl acetate and acrylic ester copolymers, vinyl acetate Consisting of methyl maleate copolymer, isobutylene-maleic anhydride crosslinked copolymer, starch-acrylonitrile graft copolymer and saponified product thereof, crosslinked sodium polyacrylate polymer and crosslinked polyethylene oxide It is selected from; Preferred examples of such suitable transfection agents include, but are not limited to, lipopolyamines and polyethyleneimines (PEIs). Particularly preferred transfection agents of the present invention are U.S. Patent Nos. 5,171,678 to Behr et al., Dec. 15, 1992; U.S. Patent Nos. 5,476,962, Dec. 19, 1995, and 1997. Lipopolyamines of Formula (I) as disclosed in US Pat. No. 5,616,745, issued April 1, 2010 to Bear et al., Each of which is incorporated herein by reference.
[249] Within the preferred and most preferred embodiments of the present invention, the bioactive therapeutic agent is physically associated with a transfection agent to form a complex and the bioactive therapeutic factor-transfectant is then physically associated with a polymeric carrier. do. The bioactive therapeutic factor is preferably adsorbed by associative force, which is well known in liquid adsorption chromatography, for example, but not limited to ion exchange, hydrophobicity, molecular recognition or combinations thereof. Certain bioactive therapeutic factors are mixed with transfection agents, which impart specific properties to the bioactive therapeutic factor-transfectants, eg increased hydrophobicity. Microspheres containing a color conversion material (e.g., Embosphere®) are mixed with a sufficient amount of a transfective bioactive therapeutic factor, wherein the physical association between the transfective bioactive therapeutic factor and the embolic material is an ionic and hydrophobic association. This can be further enhanced by the addition of salts such as, for example, but not limited to sodium chloride.
[250] Within the preferred and most preferred embodiments of the present invention, the bioactive therapeutic factor-transfectant complex adsorbed on or associated with the surface of the embolic material is progressively desorbed to various mechanisms, such as non- Limited spontaneous endocytosis, receptor-mediated endocytosis, endosomal degradation and cell membrane destabilization or combinations thereof are delivered to surrounding cells. Desorption of the bioactive therapeutic factor is induced by natural components of the biological liquid that weaken the adsorption strength between the embolic material and the bioactive therapeutic factor until full desorption of the factor is achieved.
[251] Within another aspect of the invention, a polynucleotide encoding a bioactive factor associated with a transfection agent in a blood vessel of a patient in need of treatment of a tumorigenic, angiogenic-dependent disease, wherein The polynucleotide encoding the associated bioactive factor is further associated with microspheres) delivering a therapeutically effective amount of the composition such that the vessel is effectively occluded and the tumorigenic, angiogenic-dependent disease is improved. Provided are methods for colorizing the blood vessels in the disease.
[252] 11. Diagnostic image
[253] As discussed above, the bioactive therapeutic factor transfectant microsphere compositions of the present invention may be used for diagnostic imaging, therapeutic imaging and therapeutic drug delivery, such as ultrasound (US) magnetic resonance imaging (I), nuclear magnetic resonance (NMR). It can be used in connection with drug delivery by computerized tomography (CT), electron spin resonance (ESR), nuclear medical imaging, optical imaging, elastomeric, ultrasound, radio frequency and microwave laser. The delivery vehicles and stabilizing agents of the present invention can be used in combination with conventional contrast agents, which serve to increase the effectiveness as various contrast agents, for example contrast agents for diagnostic and therapeutic imaging.
[254] Examples of suitable contrast agents for use with the stabilizing materials of the present invention include, but are not limited to, stable free radicals, such as stable nitroxides, as well as transition, lanthanide and actinide based elements, which are optionally in the form of salts or complexing agents, eg For example, it may be covalently or non-covalently bound to lipophilic derivatives or proteinaceous macromolecules thereof). Examples of preferred transition, lanthanide and actinide elements include Gd (III), Mn (II), Cu (II), Cr (III), Fe (II), Fe (III), Co (II), Er (II) ), Ni (II), Eu (III) and Dy (III). More preferably, the elements are Gd (III), Mn (II), Cu (II), Fe (II), Fe (III), Eu (III) and Dy (III), most preferably Mn ( II) and Gd (III). The elements may be in the form of salts, for example inorganic salts such as manganese salts such as manganese chloride, manganese carbonate, manganese acetate, and organic salts such as manganese gluconate and manganese hydroxyapatite. Other exemplary salts include salts of iron such as iron sulfite, and ferric salts such as ferric chloride.
[255] The elements may also be bound to complexing agents, for example lipophilic derivatives thereof, or proteinaceous macromolecules, for example via covalent or non-covalent association. Preferred complexing agents are, for example, diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclododecane-N, N ', N', N "' Tetraacetic acid (DOTA), 1,4,7,10-tetraazacyclododecane-N, N ', N "-triacetic acid (DOTA), 3,6,9-triaza-12-oxa-3, 6,9-tricarboxymethylene-10-carboxy-13-phenyltridecanoic acid (B-19036), hydroxybenzylethylenediamine diacetic acid (HBED), N, N'-bis (pyridoxyl-5-phosphate) ethylene Diamine, N, N'-diacetate (DPDP), 1,4,7-triazacyclononane-N, N ', N "-triacetic acid (NOTA), 1,4,8,11-tetraazacyclotetra Decane-N, N ', N ", N"'-tetraacetic acid (TETA), kryptand (macrocyclic complex) and desperioxamine. More preferably, the complexing agent is EDTA, DTPA, DOTA, DO3A and cryptstands, most preferably DTPA. Preferred lipophilic complexes include complexing agents EDTA, DOT Alkylated derivatives of A, such as N, N'-bis- (carboxydecylamidomethyl-N-2,3-dihydroxypropyl) ethylenediamine-N, N'-diacetate (EDTA-DDP); N, N'-bis- (carboxyoctadecylamidomethyl-N-2,3-dihydroxypropyl) ethylenediamine-N, N'-diacetate (EDTA-ODP); and N, N'-bis ( Carboxy-laurylamidomethyl-N-2,3-dihydroxypropyl) ethylenediamine-N, N'-diacetate (EDTA-LDP); and U.S. Patent No. 5,312,617, incorporated herein by reference. Examples of preferred proteinaceous macromolecules include albumin, collagen, polyarginine, polylysine, polyhistidine, γ-globulin and β-globulin, with albumin, polyarginine, polylysine and polyhistidine more preferred. Thus, suitable complexes include Mn (II) -DTPA, Mn (II) -EDTA, Mn (II) -DOTA, Mn (II) -DO3A, Mn (II) -cryptand, Gd (III) -DTPA, Gd (III ) -DOTA, Gd (III) -DO3A, Gd (III) -cryptand , Cr (III) -EDTA, Cu (II) -EDTA, or iron-desperioxamine, more preferably Mn (II) -DTPA or Gd (III) -DTPA.
[256] Nitroxide is a paramagnetic contrast agent that increases the relaxation rate of both T1 and T2 on MRI due to the presence of unpaired electrons in the nitroxide molecule. As is known to those skilled in the art, the paramagnetic efficacy of a compound provided as an MRI contrast agent is at least partially equal to the square of the number of unpaired electrons in the paramagnetic nucleus or molecule, and specifically the number of unpaired electrons. May be related. For example, gadolinium has seven unpaired electrons, while nitroxide molecules have one unpaired electron. Thus, gadolinium is generally a much stronger MRI contrast agent than nitroxide. However, another important variable for evaluating the effectiveness of the contrast agent, effective correlation time, imparts potential increased relaxation to the nitroxide. If the tumbling rate is slowed down by, for example, binding of the paramagnetic contrast to large molecules, it will tumbling slower and thereby energy will be more effectively delivered to promote relaxation of several quantums. However, in gadolinium, electron spin relaxation time is fast and will limit the degree to which slow rotation correlation time can increase relaxation. However, for nitroxide the electron spin correlation time is more advantageous and a significant increase in relaxation can be obtained by slowing the rotational phase correlation time of these molecules. While not wishing to be bound by any particular theory, nitroxide can be envisioned to optimize the correlation time produced by, for example, preparing all alkyl derivatives thereof and coating the periphery of the microspheres. Furthermore, the resulting contrast medium of the present invention may be considered as a magnetic sphere that is a geometric shape that maximizes relaxation.
[257] Exemplary superparamagnetic contrast agents suitable for use in the compositions of the present invention include metal oxides and sulfides, which have experienced magnetic regions, ferrous or ferrous magnetic compounds such as pure iron, magnetic iron oxide And, for example, magnetite, γ-Fe ₂ O ₃ , O ₃ , Fe ₃ O ₄ , manganese ferrite, cobalt ferrite and nickel ferrite. MR whole-body images can then be used to quickly screen the body, for example for thrombosis, and optionally to apply ultrasound to help thrombosis.
[258] Contrast agents such as the paramagnetic and superparamagnetic contrast agents described above can be used as components in the microspheres and / or stabilizing materials. In relation to vesicles, the contrast agent can be entrapped in its internal voids, administered as a solution with microspheres, combined with any additional stabilizing material, or coated on the surface or membrane of the vesicles. Mixtures of any one or more paramagnetic reagents and / or superparamagnetic reagents may be used in the compositions of the present invention. The paramagnetic and superparamagnetic reagents may also be administered separately if desired.
[259] If desired, the paramagnetic or superparamagnetic reagents can be delivered as alkylated or other derivatives incorporated into the lipid wall of the composition, in particular microspheres. In particular, the nitroxide 2,2,5,5-tetramethyl-1-pyrrolidinyloxy free radicals and the 2,2,6,6-tetramethyl-1-piperidinyloxy free radicals have various bonds, for example For example, acetyloxy bonds can form adducts with long-chain fatty acids at positions in the ring that are not occupied by methyl groups. Such adducts are very likely to be incorporated into the bioactive therapeutic factor transfectant microsphere compositions of the present invention.
[260] The stabilizing material and / or microspheres of the present invention, and in particular microspheres, can serve as an effective carrier of the above-mentioned supermagnetic reagents, as well as improve the magnetizing effect of the contrast agent. Superparamagnetic contrasting agents include metal oxides, especially iron oxides (except manganese oxides), and iron oxides (which have undergone magnetism) containing varying amounts of manganese, cobalt and nickel. These reagents are nano or microspheres and have very high bulk susceptibility and transverse relaxation rates. Larger particles, for example particles having a diameter of about 100 nm, have a much higher R ₂ relaxation compared to R ₁ relaxation. Smaller particles, for example particles having a diameter of about 10 to about 15 nm, have somewhat lower R ₂ relaxation, but have much more balanced R ₁ and R ₂ values. Much smaller particles, for example particles having a diameter of about 3 to about 5 ㎚ is gajina lower flaccid R _2, and has a phase estimation most balanced R ₁ and R ₂ relaxation rates. Ferritin can also be formulated to encapsulate a very high relaxation rate supermagnetic iron core.
[261] Iron oxide may simply be incorporated into the stabilizing material and / or microspheres. In the case of microspheres, preferably formulated from lipids, iron oxides may be incorporated into the walls of the microspheres, for example by adsorbing on the surface of the microspheres or by trapping them inside the microspheres.
[262] 12. Pharmaceutical Formulations
[263] Therapeutic Formulations: Polynucleotide Salts: Administration of a pharmaceutically acceptable salt of a polynucleotide encoding a bioactive therapeutic factor disclosed herein is within the scope of the present invention. Such salts may be prepared from pharmaceutically acceptable non-toxic bases such as organic bases and inorganic bases. Salts derived from inorganic bases include sodium, potassium, lithium, ammonium, calcium, magnesium and the like. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts such as primary, secondary and tertiary amines, basic amino acids and the like. For helpful discussion of pharmaceutical salts, see S.M. Berge et al., Journal of Pharmaceutical Sciences 66: 1-19 (1977), which is incorporated herein by reference.
[264] Polynucleotides encoding bioactive therapeutic factors, combined or associated with suitable transfection agents of the invention bound to microspheres suitable for infusion, may be prepared in unit dosage form in ampoules or in multiple dose containers. The polynucleotide may be present in such form as a suspension, solution or emulsion in an oily or preferably aqueous vehicle. Alternatively, the polynucleotide salt may be in lyophilized form, which is reconstituted with a suitable vehicle, eg, sterile pyrogen-free water, upon delivery. Both the lyophilized form and the liquid to be reconstituted comprise a medicament, preferably a buffer, in an amount necessary to appropriately adjust the pH of the solution being injected. For any non-oral use, especially when the formulation is to be administered intravenously, the total concentration of the solute should be adjusted such that the formulation is isotonic, hypotonic or weak hypertonic. Non-ionic substances, for example sugars, are preferred for controlling tonicity, with sucrose being particularly preferred. Any of these forms may further comprise a suitable formulation agent, for example starch or sugar, glycerol or saline. The composition per unit dose may contain between 0.1 and 99% polynucleotide material, whether liquid or solid.
[265] A unit dose ampoule or multiple dose container in which a polynucleotide encoding a bioactive therapeutic factor, mixed or associated with a suitable transfection agent of the invention bound to microspheres, is packaged prior to use is a pharmaceutically effective dose or several effective doses. Mixed with or associated with a suitable transfection agent of the present invention bound to microspheres, or mixed with a suitable transfection agent of the present invention bound to microspheres or solutions containing a polynucleotide encoding a bioactive therapeutic factor It may include a hermetically sealed container containing an amount of polynucleotides that associate with the bioactive therapeutic factors associated. A polynucleotide encoding a bioactive therapeutic factor, mixed or associated with a suitable transfection agent of the present invention bound to microspheres, is packaged as a sterile formulation, and the hermetically sealed container is used until the sterility of the formulation is used. Design to preserve
[266] Labels a container containing a polynucleotide encoding a bioactive therapeutic factor, mixed or associated with a suitable transfection agent of the present invention bound to microspheres, the label being prescribed by a government agency, such as the Food and Drug Administration. A notification, wherein the notification is made of a polynucleotide encoding a bioactive therapeutic factor, mixed or associated with a suitable transfection agent of the invention bound to microspheres, contained in the container for human administration, It reflects the approval of government agencies under federal law for use or sale. Federal law requires that federal agencies approve the use of drugs in the treatment of the human body. The responsibility for the enforcement rests with the Food and Drug Administration, which is the 21 U.S.C. 301-392, detailed in the appropriate rules for assuring such approval. Regulations for biological substances, including products made from animal tissues, are given in 42 U.S.C. It is presented under § 262. Similar approvals are required by most foreign countries. Regulations vary from country to country, but individual procedures are well known in the art.
[267] Dosage and Route of Administration: The dose administered varies depending on the number of treatments and the route of administration as well as the degree of symptoms and size of the patient to be treated. The regimen for continued therapy, including dose and withdrawal, may depend on initial response and clinical judgment. Although a non-oral route of infusion into the interstitial space of the tissue is preferred, other non-oral routes, such as inhalation of the aerosol formulation, may be required for specific administration, eg, to the mucous membranes of the nose, throat, bronchial tissues or lungs. . In a preferred protocol, a formulation comprising a polynucleotide encoding a bioactive therapeutic factor, mixed or associated with a suitable transfection agent of the invention bound to microspheres in an aqueous carrier, contains from 10 μl to about 1 ml per site to the tissue. Inject in quantities. The concentration of polynucleotide encoding a bioactive therapeutic factor in the formulation is from about 0.1 μg / ml to about 20 mg / ml.
[268] Preferred formulations for transfection of polynucleotides and peptides into cells include the cationic lipopolyamine transfection agents of the invention with or without an effective transfection promoting amount of lysophosphatid. The lisophosphatides may have neutral or negative head groups. Lysophosphatidylcholine and lysophosphatidylethanolamine are preferred, with 1-oleoyl lysophosphatidylcholine being particularly preferred. The lysophosphatid ligand is advantageously present in the formulation in a molar ratio of 0.5 lysolipids to cationic lipids. Novel cationic lipids of the invention Lyso forms of cationic lipids selected from DOTMA and DOTAP can also be used to increase the effectiveness of transfection. These lysoforms are advantageously present in the microsphere formulations of the present invention in an effective amount of about one third or less of the total cationic lipid.
[269] According to another aspect of the invention, there is provided a microsphere composition comprising a cationic lipid of the invention, wherein the cationic lipid is incorporated as a transfection agent associated with a bioactive therapeutic factor composition. The lipid of the microsphere composition may further comprise a neutral lipid species selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin and cholesterol. The preferred molar ratio of cationic to neutral lipid species in these microsphere formulations is from about 9/1 to 1/9; Particular preference is given to a molar ratio of about 5/5. The microsphere formulation may further comprise a lyso lipid selected from the group consisting of lysophosphatidylcholine, lysophosphatidylethanolamine and lyso form of cationic lipid species.
[270] According to yet another aspect of the invention, microspheres bound to bioactive therapeutic factors by any of the cationic and amphoteric lipids disclosed herein in combination with a pharmacologically effective amount of additional therapeutic agent, for example, a therapeutic drug It provides a pharmaceutical product comprising. Cationic or amphoteric lipids present in these compositions facilitate intracellular delivery of both the above bioactive therapeutic factors and / or additional active therapeutic agents. Products for topical, intestinal and non-oral use are provided. In one pharmaceutical product, the additional therapeutic agent is for example, without limitation, a steroid; Another therapeutic agent is, for example and without limitation, non-steroidal anti-inflammatory agent.
[271] In other pharmaceutical products of the invention, the additional therapeutic agent is an antiviral nucleoside analog or preferably a lipid derivative of an antiviral nucleoside analog, ie, a phosphatidyl derivative or a diphosphate diglyceride derivative. The antiviral nucleoside may be dideoxynucleoside, didehydronucleoside, halogenated derivative or azido-derivative of nucleoside, or acyclic nucleoside. In a preferred embodiment, the lipid derivative of the antiviral nucleoside is (3'-azido-3'-deoxy) thymidine-5'-diphospho-3- diacylglycerol (AZT diphosphate diglycerides). ) And dideoxythymidine diphosphate diglycerides. In a particularly preferred embodiment, the lipid derivative of the antiviral nucleoside is 1- (2-deoxy-2'-fluoro-1-beta-D-arabinofuranosyl) -5-iodoshitocin (FIAC Or acyclovir or strong cyclovir diphosphate diglyceride of 1 (2'-deoxy-2'-fluoro-1-beta-D-arabinofuranosyl) -5-iodouracil (FIAU) or Diphosphate diglyceride derivatives.
[272] 13. Kit
[273] The present invention relates to pharmaceutical packs and kits comprising one or more containers filled with one or more components of the compositions of the invention mentioned above. A form prescribed by a government agency coordinating the manufacture, use, or sale of the medicament or biological product, which reflects the approval of the government agency in the manufacture, use, or sale of products for human administration in connection with such container (s). There can be notice. Reagents for any of the assays or methods disclosed herein can also be included as elements of a kit.
[274] In one kit format, commercially available beads or microspheres of the invention are present in a physiologically suitable solution having a polynucleotide encoding the transfection agent and bioactive therapeutic factor of the invention, wherein Components are all in one vial. In another kit format, commercially available microspheres of the invention can be obtained in one vial. The polynucleotides encoding the bioactive therapeutic factors associated with the transfectants of the invention may be present in another vial, and then the microspheres are mixed with the polynucleotide transfection agent complex. Finally, in another kit format, commercially available beads or microspheres of the present invention are present in a physiologically suitable solution in one vial. The commercially available transfection agent may be present in another separate vial. Polynucleotides encoding the bioactive therapeutic factors of the invention may also be present in another vial. The three components from the separate vials can then be combined to form polynucleotide microspheres encoding the bioactive therapeutic factors associated with the transfectants of the invention.
[275] In yet another kit format, the beads or microspheres of the invention are present in a physiologically suitable solution having a polynucleotide encoding a bioactive therapeutic factor associated with a transfection agent in combination with a transfection enhancer. In yet another kit format, the beads or microspheres of the present invention are present in a physiologically suitable solution having a polynucleotide encoding a bioactive therapeutic factor associated with a transfection agent in combination with an absorption promoter of the bioactive therapeutic agent.
[276] The following examples are provided by way of example and not by way of limitation.
[277] Example 1
[278] In a beaker containing 100 ml of demineralized water, 58 g of sodium chloride and 27 g of sodium acetate are dissolved. 400 ml of glycerol are added followed by adjusting the pH to 5.9 to 6.1. Then 90 g of N-tris-hydroxy-methyl methylacrylamide, 35 mg of diethylaminoethylacryl-amide and 10 g of N, N-methylene-bis-acrylamide are added. Heat to 60-70 ° C. and add 100 moles of hot 100 mg 300 mg / gelatin solution. The total volume of the mixture is adjusted to 980 ml by addition of hot water followed by the addition of 20 ml of 70 mg / ammonium persulfate solution and 4 ml of N, N, N ', N'-tetramethylethylenediamine.
[279] The solution is poured into paraffin oil with stirring at 50-70 ° C. After a few minutes, the polymerization of acrylic monomers is manifested by an increase in temperature. The microspheres are then recovered by decantation, carefully washed, screened and sterilized in an autoclave in buffer medium.
[280] The microspheres have desirable properties for color conversion, including significant cationic charge and effective adhesive (gelatin or denatured collagen) after selective calibration.
[281] Example 2
[282] The procedure of Example 1 is carried out using triethylaminoethyl acrylamide instead of diethylaminoethyl acrylamide. After recovering the microspheres, gelatin is reticulated with 25% glutaraldehyde solution (100 mL for all microspheres). The treatment is carried out overnight at 4 ° C. Then it is washed with demineralized water.
[283] Examples 3 and 4
[284] The procedure of Examples 1 and 2 is carried out using 10 g of acrylic acid instead of 10 g of N-tris-hydroxymethyl methylacrylamide. The microspheres obtained have a high swellability which is adjustable by salt and ion concentrations and pH levels. The microspheres can advantageously be used while the user is looking directly at the time of handling.
[285] Examples 5 and 6
[286] The procedure of Examples 1 and 2 is carried out using 10 g of N-acryloyl hexamethylene procion red HE-3B instead of N-tris-hydroxymethyl methylacrylamide. The microspheres obtained are dark red due to the incorporation of acrylic dye into the polymer lattice. The microspheres can advantageously be used while the user directly looks at the handling.
[287] Examples 7 and 8
[288] 100 ml of the microspheres obtained according to Examples 1-4 are washed with 0.1 M borate buffer at pH 8 and then suspended in 50 ml of 5 mg / rhodamine isothiocyanate solution. The suspension is then stirred for at least 15 hours and then washed with neutral buffer to give a colorless supernatant.
[289] Examples 9 and 10
[290] Instead of 10 g of N-tris-hydroxymethyl methylacrylamide, 10 g of an X-ray impermeable monomer (acrylamido-3-propionamido) -3-triiodo-2,4,6-benzoic acid is used. By performing the process of Examples 1 to 4.
[291] The microspheres obtained have the property of absorbing X-rays and are therefore particularly important for the above trace in vivo after use in color conversion gene therapy.
[292] Examples 11-14
[293] Radiopaque soluble linear polymer, acrylamino-3-triiodo-2,4,6-benzopolyacid (Examples 11 and 12) or (acrylamino-3-propionamido) -3-in initial monomer solution 5 g of triiodo-2,4,6-benzopolyacid (Examples 13 and 14) were added to carry out the procedure of Examples 1-2.
[294] The polymers having molecular weights in excess of 100,000 Daltons are confined within the polymer lattice and can obtain radiopacity which can be used for in vivo tracking of color phone gene therapy without interfering with the general properties of the microspheres for the intended use. have.
[295] Examples 15 and 16
[296] Examples 1 and 2 were carried out by adding 200 g of barium sulfate powder to the initial monomer solution. The microspheres obtained are impermeable to both visible and X-rays.
[297] Examples 17 and 18
[298] Examples 1 and 2 were carried out by adding 50 mg of magnetite (Fe 3 O 4) to the initial monomer solution.
[299] The microspheres obtained have the property of being detected on magnetic resonance imaging (MRI).
[300] Example 21
[301] Preparation of injectable suspensions for use in color phone gene therapy
[302] A further embodiment of the present invention utilizes any of the microspheres of Examples 1-20 described above and encodes the microspheres with a polynucleotide encoding a p53 gene and a suitable promoter, wherein the polynucleotide is a transfection agent, for example Further mixing under the control of Transfectam® (associated with Biosphere Medical). Such microspheres / p53 gene / Tansfectam® compositions are useful for arterial color conversion and amelioration and subsequent removal of various cancers, such as liver, kidney and pancreatic cancers.
[303] Example 22
[304] Preparation of injectable suspensions for use in combination therapy of color phone gene therapy and angiogenesis inhibition
[305] A further embodiment of the invention uses an angiogenesis inhibitor using any microspheres of Examples 1-20 described above, wherein said microspheres are encoded by a polynucleotide, wherein the polynucleotide is a transfection agent, for example Transfectam And additional mixing with (registered trademark) (Biosphere Medical). Such microspheres / angiogenesis inhibitors / Tansfectam® compositions provide for the prevention of subsequent angiogenesis for arterial colorectalization and amelioration of complex cancers, as well as the subsequent development of various cancers such as liver, kidney and pancreatic cancer. It is useful for removal.
[306] The above-described embodiments of the present invention are merely exemplary and those skilled in the art will recognize or assure that many conventional experiments may be used that are equivalent to the specific procedures disclosed herein. All such equivalents are considered to be within the scope of this invention and covered by the following claims.
[307] The contents of all references disclosed herein are incorporated herein by reference. Other embodiments are within the scope of the following claims.

权利要求:
Claims (44)
[1" claim-type="Currently amended] Biocompatible crosslinked and substantially hydrophilic polymers, and
One or more active ingredients, including drugs, vaccines, or any combination thereof
Microspheres suitable for active embolization, including.
[2" claim-type="Currently amended] The microspheres of claim 1 comprising at least one elastomer.
[3" claim-type="Currently amended] 3. The microspheres of claim 2 wherein the elastomer is selected from the group consisting of acrylic polymers, acrylamide polymers, vinyl alcohol polymers, acrylate polymers, polysaccharides, silicones and mixtures thereof.
[4" claim-type="Currently amended] The microspheres of claim 2 wherein the diameter ranges from about 10 μm to about 2000 μm.
[5" claim-type="Currently amended] The microspheres of claim 4 wherein the diameter ranges from about 50 μm to about 300 μm.
[6" claim-type="Currently amended] The microspheres of claim 1 which are swellable.
[7" claim-type="Currently amended] 7. Sodium acrylate polymer, acrylamide polymer, acrylamide derivative polymer or copolymer, sodium acrylate and vinyl alcohol copolymer, vinyl acetate and acrylic ester copolymer, vinyl acetate and methyl maleate copolymer, iso Microspheres comprising a polymer selected from the group consisting of butylene-maleic anhydride crosslinked copolymers, starch-acrylonitrile graft copolymers, crosslinked sodium polyacrylate polymers and crosslinked polyethylene oxides.
[8" claim-type="Currently amended] The microspheres of claim 6, wherein the diameter ranges from about 10 μm to about 400 μm before swelling.
[9" claim-type="Currently amended] The microspheres of claim 8 wherein the diameter ranges from about 10 μm to about 200 μm before swelling.
[10" claim-type="Currently amended] The microspheres of claim 6 wherein the diameter ranges from about 10 μm to about 2000 μm after swelling.
[11" claim-type="Currently amended] The microspheres of claim 1 wherein the drug is selected from the group consisting of antitumor, angiogenesis inhibitor, antifungal, antiviral, anti-inflammatory drug, antibacterial drug and antihistamine drug.
[12" claim-type="Currently amended] The vaccine of claim 1 wherein the vaccine is pneumococcal vaccine, polio vaccine, anthrax vaccine, tuberculosis (BCG) vaccine, hepatitis A vaccine, cholera vaccine, A, C, Y meningococcal vaccine, W135 vaccine, plague vaccine, rabies ( Human diploid) vaccine, yellow fever vaccine, Japanese encephalitis vaccine, typhoid (phenol and heat-lethal) vaccine, hepatitis B vaccine, diphtheria vaccine, tetanus vaccine, pertussis vaccine, H. influenza b vaccine, polio vaccine, measles vaccine, pandemic Mumps vaccine, rubella vaccine, chickenpox vaccine, Streptococcus pneumoniae Ty (live mutant bacterium) vaccine, Vi (Vi capsular polysaccharide) vaccine, DT (denatured toxin) vaccine, Td (denatured toxin) vaccine, aP ( Inactive Bacterial Antigen / Axula (DtaP) Vaccine, Hib (Bacterial Polysaccharide-Protein Combination) Vaccine, Hepatitis B Virus (Inactive Serum-Derived Virus Antigen / Recombinant Antigen) Vaccine, Influenza Vaccine, Rotavirus Vaccine, Respiratory Tract Happo Virus (RSV) vaccines, human astrovirus vaccines, rotavirus vaccines, human influenza A and B vaccines, hepatitis A virus vaccines, live attenuated parainfluenza virus type 3 vaccines, enterovirus vaccines, retrovirus vaccines, and Microspheres selected from the group consisting of picornavirus vaccines.
[13" claim-type="Currently amended] The microspheres of claim 1 further comprising an imaging agent or contrast medium selected from the group consisting of fluorescent marker derivatives, chemical dyes and magnetic resonance imaging agents.
[14" claim-type="Currently amended] The microspheres of claim 1 wherein the polymer comprises about 0.5 to about 20 molecular weight% crosslinker.
[15" claim-type="Currently amended] An injectable composition comprising the microspheres of claim 1 and a biocompatible carrier.
[16" claim-type="Currently amended] The composition of claim 15 comprising about 10 to about 90 weight percent microspheres and about 10 to about 90 weight percent biocompatible carrier.
[17" claim-type="Currently amended] The composition of claim 16 comprising about 10 to about 50 weight percent microspheres and about 50 to about 90 weight percent biocompatible carrier.
[18" claim-type="Currently amended] The composition of claim 15 which is a suspension of microspheres in a biocompatible carrier.
[19" claim-type="Currently amended] The composition of claim 18 wherein the biocompatible polymer is in an emulsion.
[20" claim-type="Currently amended] The composition of claim 18 wherein the biocompatible polymer is in an organic or non-aqueous solution.
[21" claim-type="Currently amended] 19. The composition of claim 18, wherein the biocompatible polymer is in an aqueous solution, an water-organic solution or a mixture thereof.
[22" claim-type="Currently amended] 19. The composition of claim 18, wherein the biocompatible carrier comprises a salt consisting of cations selected from the group consisting of sodium, potassium, calcium, magnesium, iron, zinc and ammonium in an amount of about 0.01 M to about 5 M.
[23" claim-type="Currently amended] The composition of claim 22 wherein the salt is supplied in the form of a contrast agent.
[24" claim-type="Currently amended] 19. The composition of claim 18, wherein the contrast agent is monomeric (acrylamido-3-propionamido) -3-triiodo-2,4,6-benzoic acid.
[25" claim-type="Currently amended] The composition of claim 15 injectable through a needle of up to about 18 gauge.
[26" claim-type="Currently amended] A method for active color conversion of mammals comprising administering to a mammal in need of color conversion treatment a microsphere comprising a biocompatible crosslinked and substantially hydrophilic polymer and one or more drugs, vaccines or combinations thereof.
[27" claim-type="Currently amended] 27. The method of claim 26, wherein the microspheres comprise one or more elastomers.
[28" claim-type="Currently amended] 28. The method of claim 27, wherein the elastomer is selected from the group consisting of acrylic polymers, acrylamide polymers, vinyl alcohol polymers, acrylate polymers, polysaccharides, silicones and mixtures thereof.
[29" claim-type="Currently amended] The method of claim 27, wherein the microspheres have a diameter in the range of about 10 μm to about 2000 μm.
[30" claim-type="Currently amended] The method of claim 29, wherein the microspheres have a diameter in the range of about 50 μm to about 300 μm.
[31" claim-type="Currently amended] 27. The method of claim 26, wherein the microspheres are swellable.
[32" claim-type="Currently amended] 32. The method of claim 31, wherein the microspheres comprise ionic polysaccharides and ionic synthetic polymers.
[33" claim-type="Currently amended] 33. The method of claim 32, wherein the ionic polysaccharide is selected from the group consisting of carboxymethyldextran, dextran sulfate, and alzenic acid.
[34" claim-type="Currently amended] 33. The method of claim 32, wherein the ionic synthetic polymer is a sodium acrylate polymer, acrylamide polymer, acrylamide derivative polymer or copolymer, sodium acrylate and vinyl alcohol copolymer, vinyl acetate and acrylic ester copolymer, vinyl acetate and methyl maleic An ester copolymer, isobutylene-maleic anhydride crosslinked copolymer, starch-acrylonitrile graft copolymer, crosslinked sodium polyacrylate polymer and crosslinked polyethylene oxide.
[35" claim-type="Currently amended] The method of claim 31, wherein the diameter of the microspheres ranges from about 10 μm to about 400 μm before swelling.
[36" claim-type="Currently amended] The method of claim 35, wherein the diameter of the microspheres ranges from about 10 μm to about 200 μm before swelling.
[37" claim-type="Currently amended] The method of claim 31, wherein the diameter of the microspheres ranges from about 10 μm to about 2000 μm after swelling.
[38" claim-type="Currently amended] 27. The method of claim 26, wherein the therapeutically active drug is an antitumor, angiogenesis inhibitor, antifungal, antiviral, anti-inflammatory drug, antibacterial drug and antihistamine drug, angiogenesis inhibitor, antitumor agent, hormone and steroid, vitamin, peptide and peptide analog. , Enzymes, allergen inhibitors, circulatory drugs, anti-tuberculosis agents, antiviral agents, angina inhibitors, probiotic, rheumatoid inhibitors, anesthetics, cardiovascular glycosides, sedatives, local anesthetics, general anesthetics.
[39" claim-type="Currently amended] The vaccine of claim 26 wherein the vaccine is pneumococcal vaccine, polio vaccine, anthrax vaccine, tuberculosis (BCG) vaccine, hepatitis A vaccine, cholera vaccine, A, C, Y meningococcal vaccine, W135 vaccine, plague vaccine, rabies ( Human diploid) vaccine, yellow fever vaccine, Japanese encephalitis vaccine, typhoid (phenol and heat-lethal) vaccine, hepatitis B vaccine, diphtheria vaccine, tetanus vaccine, pertussis vaccine, H. influenza b vaccine, polio vaccine, measles vaccine, pandemic Mumps vaccine, rubella vaccine, chickenpox vaccine, Streptococcus pneumoniae Ty (live mutant bacterial) vaccine, Vi (Vi encapsulated polysaccharide) vaccine, DT (denatured toxin) vaccine, Td (denatured toxin) vaccine, aP ( Inactive Bacterial Antigen / Axula (DtaP) Vaccine, Hib (Bacterial Polysaccharide-Protein Combination) Vaccine, Hepatitis B Virus (Inactive Serum-Derived Virus Antigen / Recombinant Antigen) Vaccine, Influenza Vaccine, Rotavirus Vaccine, Respiratory Tract Happo Virus (RSV) vaccines, human astrovirus vaccines, rotavirus vaccines, human influenza A and B vaccines, hepatitis A virus vaccines, live attenuated parainfluenza virus type 3 vaccines, enterovirus vaccines, retrovirus vaccines, and Method selected from the group consisting of picornavirus vaccines.
[40" claim-type="Currently amended] 27. The method of claim 26, wherein the microspheres also comprise a diagnostic or contrast medium selected from the group consisting of fluorescent marker derivatives, chemical dyes, and magnetic resonance imaging agents.
[41" claim-type="Currently amended] 27. The method of claim 26, wherein the polymer comprises about 0.5 to about 20 molecular weight% crosslinker.
[42" claim-type="Currently amended] 27. The method of claim 26, wherein administering comprises injecting into the area of the mammal in need of color conversion.
[43" claim-type="Currently amended] The microspheres of claim 1 wherein the antitumor drug is Taxol, doxorubicin, tamoxifen, or a combination thereof.
[44" claim-type="Currently amended] A method of active color conversion in a mammal comprising administering to the mammal in need of color conversion therapy a microsphere comprising a biocompatible crosslinked and substantially hydrophilic polymer and at least one drug, vaccine or combination thereof. Delivering said microspheres to the site of action using a target antibody.

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引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2000-03-24|Priority to US19189900P

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2000-03-24|Priority to US60/191,899

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2001-03-23|Application filed by 바이오스피어 메디칼 인코포레이티드

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2001-03-23|Priority to PCT/US2001/009619

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2003-02-11|Publication of KR20030011803A

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2008-07-01|First worldwide family litigation filed

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2008-12-10|Publication of KR100872884B1

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2008-12-10|Application granted

优先权:

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申请号 | 申请日 | 专利标题

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US19189900P| true| 2000-03-24|2000-03-24|

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US60/191,899|2000-03-24|

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PCT/US2001/009619|WO2001072281A2|2000-03-24|2001-03-23|Microspheres for active embolization|