Treatment method and product for uterine fibroids using purified collagenase

Abstract

To provide a method for treating a uterine fibroid.SOLUTION: A method comprises preparing an injectable or insertable formulation containing a uterine fibroid treatment agent in an amount effective to cause shrinkage of a uterine fibroid, and injecting or inserting the formulation into the uterine fibroid. The uterine fibroid treatment agent is collagenase.SELECTED DRAWING: Figure 17

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application is a continuation of U.S. Provisional Patent Application No. 61 / 790,07, filed March 15, 2013. This application claims priority from US Pat. No. 6,290,629, filed on Dec. 1, 2002, which is incorporated herein by reference. This shall be done.

[0002] [Field of the Invention] The present invention reduces and shrinks unwanted tissue, such as uterine fibroid tissue, and medical use for altering the viscoelastic properties of tissues, softening or removing such tissues; Therapeutic methods and products. [Background technology]

[0003] Uterine fibroid tumors (also called "uterine fibroids" or "leiomyomas") occur in 20% of women. It is a non-cancerous smooth muscle tumor of the uterine wall, occurring in 0-50% of cases and showing a surprisingly high cumulative incidence. Current research suggests that by the age of 50, 70-80% of women will develop uterine fibroids, commonly The incidence is higher in African-American women, who develop fibroids earlier than other racial groups. A significant number of people with uterine fibroids experience debilitating pelvic pain, severe and prolonged bleeding, and (which can lead to anemia and iron deficiency), bowel and bladder dysfunction, and Uterine fibroids can also cause lower back pain, frequent and urgent urination, and pain during intercourse. These can cause symptoms such as pain during intercourse (dyspareunia) and adverse effects on fertility. is associated with high morbidity rates of uterine bleeding and pain, with associated medical costs of approximately $100 million annually in the United States alone. The estimated cost is between $2.1 billion and $34.4 billion. Therefore, uterine fibroids are a reproductive risk factor. Postmenopausal women have a significant impact on the health and welfare of older women, as well as the economy. Fibroids generally shrink and very rarely cause any significant symptoms.

[0004] The etiology of this disease remains unknown, and therefore, there are no known ways to prevent uterine fibroids. There are several treatments available, but none can permanently remove fibroids. The only available treatment is a hysterectomy. The majority of hysterectomies performed each year in the United States are for uterine fibroids. Although hysterectomy obviously leads to the ultimate loss of fertility, Furthermore, this invasive surgical procedure is economically and socially The price to pay is high in both these and other respects. This invasive surgery involves long recovery times. The procedure can be lengthy, potentially resulting in serious postoperative complications, and can be physically uncomfortable. Therefore, this solution is far from ideal.

[0005] Other surgical procedures, such as myomectomy (surgical removal of fibroid tissue, preserving the remaining uterus) This method is commonly used, but the fibroids may be too large or too numerous to allow for sufficient normal In some cases, it may not be suitable if tissue cannot be preserved. In addition, approximately three-quarters of myomectomy surgeries are performed openly, with an abdominal incision. It is a surgical procedure, and so this procedure also carries the same risks of complications, discomfort, long recovery times, and and possible loss of fertility. Laparoscopic surgery to burn or freeze uterine fibroids Myolysis and cryomyolysis cause fibroids to shrink and die over time. However, multiple punctures of the fibroid are required to treat the entire tumor. This treatment requires a hole, and may cause postoperative adhesions. MRI-guided focused ultrasound is used to treat uterine fibroids. However, this procedure is very expensive and does not necessarily remove the fibroids permanently. During the embolization procedure, a catheter is inserted into the femoral artery and guided to the uterine fibroid artery. and injecting small particles into the fibroid arteries, cutting off the blood supply and killing the fibroid tissue. Although this procedure is less invasive than traditional surgery, postoperative pain is a frequent problem. In addition, this therapy, like hysterectomy, is the standard of care for women who do not wish to become pregnant in the future. Alternatively, MRgFUS can be performed using high-power ultrasound through the abdominal wall to It provides a non-invasive, fibroid-specific therapy that induces coagulation necrosis of fibroids. Real-time magnetic resonance imaging provides guidance and thermal monitoring. Surgical procedures to destroy uterine fibroids while undergoing surgery have major drawbacks, including the recurrence of fibroid tumors. Because of proliferation, it is often not completely successful.

[0006] Non-surgical drug-based medical treatments are available. Fibroids are caused by fibroid tumors themselves. It is often treated with medications that aim to treat the symptoms, not the body. In this case, doctors take a "wait and see" approach and wait until the condition affects the patient's ability to lead a normal life. Most fibroids do not cause symptoms unless they cause symptoms. However, even without a hysterectomy, fibroids, especially subserosal fibroids, can be difficult to treat. Fibroma may be associated with infertility.

[0007] Drugs that aim to shrink fibroid tumors or prevent them from growing in size Therapies have often been disappointing and have significant side effects. Drugs have been studied, including uterine Although sometimes effective in shrinking fibroids, many of these non-surgical treatments have systemic effects. These drugs are associated with side effects and are therefore not approved for clinical use. SPRMs are FDA-approved drugs that affect the endometrium. Only one drug, leuprolide acetate, is approved for shrinking uterine fibroids. This drug suppresses ovarian function (thus causing significant menopausal side effects). It is used as a short-term treatment to shrink fibroids before surgery. Other drug therapies, such as selective estrogen receptor modulators (SERMs), have been suggested, but clinical trials are limited. The results of the floor tests are disappointing.

[0008] Current treatment options for uterine fibroids are inadequate. is a sustainable alternative therapy for treating uterine fibroids that preserves the patient's uterus rather than undergoing laparotomy. There is a need, especially since treating uterine fibroids costs billions of dollars each year in healthcare costs. However, this condition remains a significant problem, and efforts to reduce or eliminate symptoms are underway. There is a need for treatments that provide fertility-preserving relief without the need for highly invasive procedures. exist. Summary of the Invention

[0009] The following brief summary is not intended to be exhaustive of all features and aspects of the present invention. , that the invention must include all features and aspects discussed in this summary It does not suggest that

[0010] Embodiments of the present invention provide a method for producing uterine fibroids that preserves the patient's uterus without the need for open surgery. The present invention provides advantageous formulations, compositions, and methods for treating tumors. Another potential advantage is that it exhibits improved retention of the agent in uterine fibroid tissue, thereby increasing delivery efficiency. Improved efficacy while simultaneously minimizing adverse effects such as non-specific damage and systemic effects. Injectable or insertable formulations are provided. These formulations, compositions, and methods The method comprises administering to the patient one or more uterine fibroid treatment agents, preferably at least purified collagenase. in an amount effective to shrink or eliminate fibroids exposed to the formulation, Implantable or insertable preparations are included.

[0011] The foregoing and other objects, features, and advantages of the present invention are apparent from and are not to be construed as limiting the present invention, as illustrated in the accompanying drawings, in which: The invention will become more apparent from the following more particular description of the preferred embodiments. Like reference features refer to the same parts throughout the different drawings. The drawings are to scale. No. 5,629,393, emphasis instead being placed upon illustrating the principles of the invention. .

[0012] The application file contains at least one drawing executed in color. Copies of any patent or patent application publication containing this application will be available upon request and payment of the necessary fee. It will be provided by the authorities upon payment. [Brief explanation of the drawings]

[0013] [Figure 1A.1B] 1A and 1B are electron micrographs (31,000x) showing collagen fibrils in a uterine fibroid (1A) and the corresponding myometrium (1B). [Figure 2]Photograph of a cube of uterine tissue. [Figure 3A.3B] Photographs of uterine tissue cubes during injection (FIG. 3A) and injected uterine tissue cubes during incubation (FIG. 3B). [Figure 4A.4B] Photographs of excised uterus showing uterine fibroids (FIG. 4A) and uterine fibroids during injection (FIG. 4B). [Figure 5] 1 is a pair of photographs showing vehicle (control) or collagenase injected fibroid tissue cubes after 48 hours of incubation. [Figure 6] Figure 6 is a series of four photomicrographs: Figures 6A and 6B show control tissue, and Figures 6C and 6D show collagenase-digested tissue. [Figure 7] 1 is a scanning electron micrograph of a BD 3D OPLA® scaffold. [Figure 8] FIG. 1 is a photomicrograph showing H&E staining of fibroid cells seeded on an OPLA scaffold and cultured for 9 days (Zeiss Axio Imager® wide-field fluorescence microscopy). [Figure 9] 9A and 9B are brightfield and fluorescent image overlays showing primary cultures of fibroid cells 8 days after static seeding (Zeiss Lumar® stereo images, stained with fluorescein-phalloidin for f-actin). [Figure 10] 10A and 10B are photomicrographs showing primary fibroid cells cultured on an OPLA scaffold and fixed in situ. Images 10A and 10B were acquired every 10 micrometers in the same field of view. [Figure 11] 1 is a bar graph showing the percentage of collagen types in fibroids. [Figure 12]Figure 12 shows SDS-PAGE analysis of collagen content in fibroid samples (Nydea Aviles and Sergey Leikin, NIH). Figure 12A shows total collagen under non-reducing conditions. Figure 12B shows total collagen under reducing conditions. Figure 12C shows a sample depleted of type V collagen by selective salting out. Figure 12D shows a sample enriched for type V collagen by selective salting out (Feng L, Leikin S et al., presented at the XXIInd Meeting of the Federation of European Connective Tissue Societies (FECTS), July 3-7, 2010). [Figure 13] This diagram illustrates how TGF-β may be involved in fibrosis. Fibrosis may be considered a form of altered tissue repair. Cellular injury in myometrial cells leads to activation of repair. In fibroids, wound healing stops in the proliferative phase and collagen accumulates. (Leppert et al. A new hypothesis about the origin of uterine fibroids based on gene expression profiling with microarrays. Am J Obstet Gyn 2006;95:415-20.) [Figure 14] Figure 14 shows structural changes associated with uterine fibroids. The change in cell shape from smooth muscle to fibroid cells, which appear to be myofibroblast-like cells, suggests that mechanotransduction plays a role in fibroid formation. Figure 14A shows phalloidin-stained fibroid cells at 40x magnification. Figure 14B shows phalloidin-stained sections from the corresponding myometrial specimen at 40x magnification. Figure 14C shows a fibroid specimen at 21,000x magnification, and Figure 14D shows the corresponding myometrial specimen at 21,000x magnification. Note the angular cell shape, decreased cytoplasm, and cleaved nuclei in fibroids (C) compared to myometrium (D). [Figure 15]This is a modified version of Figure 3 from Hinz and Gabbiani (Current Opinions in Biotechnology 2003, 14:38-46) showing how TGF-β is a key mediator of fibrosis. TGF-β stimulates the differentiation of fibroblasts into myofibroblasts, promotes collagen transfer, and stimulates extracellular matrix (ECM) accumulation. The diagram also illustrates the role of mechanotransduction and ECM in fibroid development. ECM accumulation exerts pressure on the cells, which leads to increased ECM production, which leads to mechanotransduction of increased collagen synthesis and deposition. Apoptosis of fibroid cells does not occur, and the cells produce more collagen. [Figure 16] This shows a series of representative gels of products from reverse transcription-PCR reactions of decorin, TGF-β1, TGF-β3, and interleukin-4. Pairs of leiomyoma (fibroid) and myometrial samples are as indicated. Leiomyoma-myometrial pairs were prepared as a 10-fold dilution of total RNA in each reaction. Negative controls were reactions containing primers but no RNA template. GAPDH was amplified as an internal control to assess amplification and similar amounts of RNA in the samples. Lane markers are indicated on the left edge of the gel. (Catherino et al. (2004) Genes, Chromosomes & Cancer 40:204-217.) [Figure 17] FIG. 1 shows the complex shear modulus (stiffness) of human fibroid tissue treated with purified clostridial collagenase. DETAILED DESCRIPTION OF THE INVENTION

[0014] Collagen is the main structural component of mammalian organisms and is the major component of the skin and other parts of the animal body. It accounts for the majority of the protein content. It is used in various skin conditions such as burns, surgery, infections, and accidents. Trauma results in the abnormal accumulation of fibrous tissue that is rich in collagen and exhibits increased proteoglycan content. In addition to replacing damaged or destroyed normal tissue, Excessive and disfiguring deposits of new tissue may form during the healing process. Diseases and conditions include excessive collagen deposition and abnormalities of collagen-rich fibrous tissue. Such diseases and conditions are collectively referred to herein as "collagen-mediated It is called a "sexual disease."

[0015] Uterine fibroids are characterized by excessive collagen deposition and abnormal accumulation of collagen-rich fibrous tissue. It is now known that fibroids are a collagen-mediated disease associated with There is considerable variation in growth rate and the expression of ECM proteins or Altered gene expression of genes involved in M ​​synthesis and secretion in fibroids compared with myometrium Microarray studies have shown that ECM (cell adhesion molecules) account for a large proportion of Dysregulation of the extracellular matrix (ECM) may be a contributing factor to this condition.

[0016] Transforming growth factors (TGF) play a role in fibroid development. In fibroids, the expression of other molecules is altered as well. In this case, dermatopontin expression was decreased, and fibronectin and glycosaminoglycan (G AG) is increased, and alpha 11 integrin, a collagen-binding integrin, is expressed. In addition, fibroids are resistant to apoptosis.

[0017] Recent studies have shown that the accumulation of extracellular matrix (ECM) and cell proliferation It has been shown that this leads to the formation of fibroids. With this in mind, please refer to Figure 1. The appearance and structure of collagen fibrils in uterine fibroids. The intercellular orientation is shorter and more randomly aligned compared to collagen fibrils in the myometrium. The fibrils were not aligned or parallel, but were adjacent. In the myometrium, the fibrils are well packed and oriented parallel to each other, which is This finding was characteristic of collagen-containing tissue. Cleaved nuclei) have been found in uterine fibroids. The appearance is that the nuclear membrane is bent and envaginated due to cell contraction caused by tension fibers. This indicates that the

[0018] Therefore, the present invention provides collagenase, an enzyme with the specific ability to digest collagen. The enzyme is used to treat uterine fibroids. The degradation of collagen leads to collagen dissolution. It not only induces mechanotransduction but also reduces cell contraction associated with mechanotransduction. This breaks the cycle of increased collagen secretion and uterine fibroid growth. uterine fibroids are rich in denatured collagen, consistent with fibrosis and stiffness. The rigid extracellular matrix (ECM) exerts forces on individual cells. Notransduction alters cell signaling and prevents apoptosis. Thus, collagen deposition continues (see Figure 15). Uterine fibroids grow at different rates. It has been suggested that mechanical transduction of tumors is responsible for the variability in growth rate. do.

[0019] Described herein is a purified collagenase to avoid systemic side effects and harm to other tissues. Local delivery of the composition reduces the symptoms of uterine fibroids and shrinks uterine fibroids. to reduce the stiffness and mechanical stress of uterine fibroid tissue and / or to increase the uterine fibroid Embodiments of the invention are described for performing treatments to remove fibroma. Generally, preferred Some methods involve administering the treatment using a syringe and needle under ultrasound or other visualization. Purified collagenase is directly injected into the uterine fibroid tissue. , preferably in a delivery vehicle such as a nanocarrier or other protective or sustained release carrier. .

[0020] The center of a fibroid is more fibrous and contains smaller capillary beds than the periphery, and Due to the dense vascular capsule surrounding fibroid tumors, systemic therapy is limited by therapeutic intra-tissue radiotherapy. Although it is highly likely that the drug will not be delivered to the fibroid center, there is a high possibility that systemic effects will occur. Therefore, medical therapy has not been successful in uterine fibroids. Local injection of therapeutic agents allows precise placement of drugs in tissues, increasing the potential for systemic effects. The width can be reduced.

[0021] Based on their location, uterine fibroids can be classified as subserosal, intramural, submucosal, pedunculated submucosal, or uterine fibroid. They are classified into several types, including fibroids in situ and fibroids of the broad ligament. Any and all of these uterine fibroids are contemplated for treatment using the present invention.

[0022] Myometrial hyperplasia is a condition that can mimic symptoms of uterine fibroids, and these tumors Myometrial hyperplasia may be a precursor to uterine cancer. Myometrial hyperplasia has irregular areas of hypercellularity and This structural change indicates an increased uterus / cell ratio, resulting in a swollen, hard, and enlarged uterus. The condition often leads to hysterectomy. Deeper MMH have lower cellularity. Therefore, this condition is also amenable to the methods and compositions of the present invention. Compositions can be used for treatment.

[0023] Local treatment of uterine fibroids with collagenase injections is available under ultrasound guidance during an outpatient or office visit. This method can be used to treat a wide range of diseases, from small to large. Treating medium-sized fibroids or asymptomatic fibroids that are not currently treated at all This allows clinicians to prevent potentially debilitating conditions and Preserving fertility and preventing large fibroids, which may require hysterectomy Therefore, the method of the present invention can eliminate all symptoms of uterine fibroid disease. It is contemplated that the compounds may be useful in the treatment of any stage or type of disease.

[0024] Collagenase for use in accordance with the present invention may be derived from mammalian (e.g., human, porcine), thyroid, or Shellfish (e.g., crab, shrimp), fungal, and bacterial (e.g., Clostridium (Cl ostridium, Streptomyces, Pseudomonas Pseudomonas, Vibrio, or Achromobacter - Contains iophagus (fermented by Achromobacter iophagus) Collagenase can be obtained from any convenient source, including collagenase isolated from natural sources. It may be a crude collagenase or may be genetically engineered / recombinant. A common source of is bacterial fermentation processes, particularly Clostridium histolyticum (C C. histolyticum is derived from the fermentation of Crude collagenase obtained from the plant can be purified by protein purification techniques, including chromatography. Purification can be performed using any of several techniques known in the art. Collagenase compositions useful for the present invention may utilize any commercially available or isolated collagenase activity. For example, purified or mixed proteins can be prepared. Collagenase was obtained from Biospecifics Technologies, Lynbrook. , may be provided by the State of New York.

[0025] The preferred collagenase for use in the present invention is derived from C. histolyticum, i.e., I Type II and Type III collagenases are produced by C. histolyticum. The practical advantage of this approach is that C. histolyticum can be grown in large quantities in simple liquid media. The key is to produce large amounts of proteolytic enzymes at all times and secrete them into the culture medium. The medium for C. histolyticum fermentation uses bovine products, which contain agents that cause morphologic encephalopathy (TSEs; e.g., proteins associated with bovine spongiform encephalopathy or "mad cow disease"); There is a risk of contamination with bovine products (e.g., cereals containing gluten). Therefore, it is advisable to avoid such bovine products. Preferred. Animal product-free systems are preferred. H4 strains of Clostridium histolyticum are Originally developed in 1956, it served as a source of cells for culture. This strain, and the ABC Clostridium histolyticum master Strains derived from the H4 strain designated as cell bank (deposited as ATCC 21000) was developed using animal products and is suitable for use in the present invention.

[0026] U.S. Patent No. 7,811,560 discloses a method for producing collagenase. The method described in this paper uses a soybean-derived fermentation medium to produce highly purified collagen. Enzymes I and II were produced separately. This patent also includes a porcine-derived product. A method for producing highly purified collagenase using culture media is disclosed. Any of these methods are suitable for use in the present invention. No. 0086971 includes plant-derived peptones, including soybean-derived peptones, or plant-derived peptones. Numerous fermentation recipes based on pork and fish gelatin are disclosed. The methods described are suitable for producing clostridial growth and collagenase activity. These methods are also suitable for and contemplated for use in the present invention. However, any method known in the art for producing collagenase enzymatic activity may be used. can be done.

[0027] In a preferred culture method, peptones are derived from soybeans, broad beans, peas, potatoes, and The peptones are derived from a plant source selected from the group consisting of pea, pea extract, pea syrup, pea extract ... Oxoid VG100 Plant Peptone No. 1 (VG100) derived from peas Oxoid VG200 Plant Peptone Phosphate Broth (VG200), animal-free Merck TSB CASO-Bouillion (TSB), Invitrog Soybean Peptone No. 110 Papain Digest (SP6), Fluka Broad Bean Peptone (BP), Organotechnie plant peptone E1 (E1P) from potato; BBL Phytone™ peptone, and BD Difco Phytone ( The compound may be selected from the group consisting of:

[0028] In a preferred embodiment of the present invention, a single type of peptone is present in the nutritional composition of the present invention. The peptones present are BP, E1P, soy peptone E110, VG100, and VG2 00, and the peptone concentration in the composition is about 5% weight / volume. In yet another highly preferred embodiment of the invention, the nutritional compositions of the present invention contain a single type of Peptone is present, and the peptone is BPL phytone peptone or Difco S The peptone concentration in the composition is about 10 ~13% w / v.

[0029] A preferred method for isolating collagenase involves removing unwanted contaminating proteins such as clostripain. Clostripain, a cysteine ​​protease, is avoided. It is believed to be the main cause of enzyme degradation and instability during clostridial cultures. If such proteases are present in the crude collagenase mixture, Special care must be taken to neutralize ATPases, including leupeptin. and the use of protease inhibitors such as HCl and cooling solutions in specially designed cold rooms. All purification steps were carried out using PEG-4000 to reduce protease activity. Therefore, the preferred isolation method involves the use of the following two techniques to avoid clostripain: Use one of the following: Remove clostripain as early as possible in the purification process; reduces clostripain production during the fermentation stage.

[0030] A preferred collagenase composition is a collagenase composition for culturing C. histolytica in an animal-derived component-free culture medium. It is produced by fermenting Triticum and is substantially free of clostripain. "Substantially free" means that collagenase is not present in the total collagen. Less than 10 U / mg of genase, more preferably less than 5 U / mg, most preferably Contains about 1 U / mg or less of clostripain and / or has a higher clostripain activity than a reference standard Visible bands representing rostripain and / or degradative collagenases were detected by SDS-PAGE. It indicates that it does not appear on the gel.

[0031] A preferred method for purifying collagenase is to culture C. histolyticum as described herein. This medium comprises using a "low glucose" medium described in More preferably, less than about 1 g / L, and even more preferably, less than about 0.5 g / L glucose High salt concentrations in the growth medium can increase the production of glucose in the culture. This reduces the amount of clostripain present in C. histolyticum cultures. The preferred medium for cultivation is one with a total salt content of greater than about 5 g / L (or 0.5 w / v%), more preferably or greater than about 7.5 g / L (or 7.5%) total salts, more preferably greater than about 9 g / L (or 9%) or more. It is contemplated that any salt thereof may be used in the present invention. In a preferred embodiment, Chloride, phosphate, or sulfate salts can be used. In a more preferred embodiment , salts include sodium chloride, potassium chloride, monosodium phosphate, disodium phosphate, Sodium triphosphate, monopotassium phosphate, potassium diphosphate, tripotassium phosphate, potassium chloride The compound may be calcium, magnesium sulfate, or various combinations thereof. In an embodiment, the potassium diphosphate may be about 0.1 to 0.3%, and the potassium phosphate may be about 0.75% to 0.175%, and sodium phosphate may be about 0.2 to 0. 5%, and / or sodium chloride is about 0.15-0.35%. Preferably, the medium contains magnesium sulfate, riboflavin, niacin, Vitamins including calcium pantothenate, pimelic acid, pyridoxine, and thiamine Further includes:

[0032] In another preferred embodiment, the nutritional components are present in an amount of 0.5 to 5%, more preferably about 1 to 4% The yeast extract may be contained in an amount of about 1.5 to 2.5%, most preferably about 1.5 to 2.5%. Cole Parmer (Vernon Hills, IL) and Fisher Science Available from a variety of suppliers, including Entific (Pittsburgh, PA) is.

[0033] In a more preferred embodiment of the present invention, the pH of the medium is between pH 7 and pH 8. A pH of 0.2 to about pH 7.7 is even more preferred, and most preferably about pH 7.4.

[0034] Collagenases contemplated for use in the present invention include any collagenase that is active under the required conditions. However, preferred compositions are those that provide the desired or even maximum synergistic effect. A mass ratio of collagenase I and collagenase B modified or optimized to provide Preferably, collagenase I and collagenase II are produced in culture. Collagenase I and collagenase II were purified separately from the crude collagenase mixture. II are recombined in an optimized constant mass ratio. A preferred embodiment is about 0.5 0.6 to 1.3, more preferably 0.8 to 1.2, most preferably 0.6 to 1.5, more preferably 0.6 to 1.3, even more preferably 0.8 to 1.2, most preferably Preferably, it contains a 1:1 ratio by weight of collagenase I to collagenase II, but any combination is acceptable. A combination of or any single collagenase activity can be used.

[0035] A preferred method for producing collagenase contemplated for use in the present invention is from non-mammalian or The method comprises fermenting C. histolyticum in a non-animal medium, the culture supernatant being substantially The collagenase thus produced is isolated and purified. and, together with the substantially clostripain-free, optimized constant mass ratio The composition used in the present invention comprises a mixture of collagenase I and collagenase II of Crude collagenase obtained from the fermentation of C. histolyticum is a dye-ligand parent. affinity chromatography, heparin affinity chromatography, ammonium sulfate precipitation, hydroxyl Apatite chromatography, size exclusion chromatography, ion exchange chromatography Various methods known to those skilled in the art, including chromatography, and / or metal chelate chromatography In addition, collagenase purification methods are described in, for example, U.S. Pat. The one described in Patent No. 7,811,560 is known.

[0036] Both collagenase I and collagenase II are metalloproteases and They require tightly bound zinc and loosely bound calcium for their function. Collagenase has broad specificity for all types of collagen. Collagenase II hydrolyzes the triple helix region of collagen under physiological conditions. Each collagenase exhibits different specificity (e.g. , each with a different preferred cleavage target amino acid sequence), together form collagen Collagenase II exhibits synergistic activity against type II and type I collagenases. As reported in the literature, collagenase I reacts with all kinds of synthetic peptide substrates. It shows higher activity than

[0037] Preferred collagenases are collagenase ABC I and collagenase ABC II. It is composed of two microbial collagenases, called collagenase I and ABC. "Collagenase ABC I" and "Collagenase ABC I" mean the same thing and may be used interchangeably. Similarly, the terms "collagenase II," "ABC II," and "collagenase" can be used interchangeably. The terms "proteinase ABC II" and "proteinase ABC II" refer to the same enzyme and can be used interchangeably. The collagenases are secreted by bacterial cells. Preferably, these collagenases are chromatographically It was isolated and purified from the supernatant of Clostridium histolyticum culture by the Rafi method. Both collagenases are specialized proteases and have the same EC number (E.C3.4.2). However, collagenase or collagen derived from other sources may Combinations of enzymes are contemplated for use in the present invention. Collagenase ABC I is A single polypeptide consisting of approximately 1000 amino acids with a molecular weight of 115 kDa Collagenase ABC II has a molecular weight of 110 kDa. It has a single polypeptide chain consisting of approximately 1000 amino acids.

[0038] Collagenase acts by hydrolyzing the peptide bond between Gly-Pro-X where X is often proline or hydroxyproline. Collagenase I acts at the ends of the triple helical domain, whereas collagenase II cleaves internally Hydrolysis continues over time until all bonds are broken.

[0039] Preferably, the collagenase product is at least 95% pure collagenase, or More preferably, the collagenase product is substantially free of other contaminating proteases. 97% pure, and most preferably 98% or greater, as determined by one or more of the following: Pure. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAG) E); high-performance liquid chromatography (HPLC); reverse-phase HPLC; or enzyme assay The preferred collagenase product is essentially free of clostripain, and purification is preferably Preferably, it is carried out in the absence of leupeptin. The product has at least one specification selected from Table 1 below.

[0040] [Table 1]

[0041] The collagenase products described for use herein are useful for treating collagen, including uterine fibroids. Other corticosteroids that can be treated with the compositions of the present invention are useful for treating HIV-mediated diseases. Examples of antigen-mediated diseases include, but are not limited to, the following: Dupuyt's disease Itren's disease; Peyronie's disease; frozen shoulder (adhesive capsulitis), keloid; tennis elbow (lateral epicondyle) inflammation; scar tendon; glaucoma; herniated disc; adjunct to vitrectomy hypertrophic scars; pressure scars such as those resulting from inflammatory acne; postoperative adhesions; common acne acne; lipomas, and disfiguring conditions such as wrinkles, cellulite formation, and neoplastic fibrosis .

[0042] In addition to use in the treatment of certain collagen-mediated disorders, the compositions of the present invention may also be used to treat tissue It is also useful for dissociating proteins into individual cells and cell populations, making it suitable for a wide variety of diagnostic and diagnostic applications. These applications include the development of microvascular implants for seeding small diameter synthetic vascular grafts. Vascular endothelial cells, hepatocytes for gene therapy, drug toxicity screening and extracorporeal liver support devices cells for cartilage regeneration, and islets of Langerhans for the treatment of insulin-dependent diabetes mellitus. Enzyme therapy involves isolating multiple types of cells for a variety of uses, including the isolation of fragmented cells. Effective against extracellular matrix proteins and proteins that maintain cell-cell contact. In general, the compositions of the present invention are useful in any application where cell removal or extracellular matrix modification is desired. It is useful for various applications.

[0043] Collagenase compositions according to the present invention comprise a therapeutically effective amount of the described collagenase composition. or a pharmaceutical collagenase preparation in a therapeutically effective amount is provided to patients in need thereof. A "therapeutically effective amount" of a compound, composition, or formulation is intended for administration to any subject. Therapeutic efficacy is achieved in the treated population at a reasonable benefit / risk ratio applicable to any medical treatment. The therapeutic effect includes, but is not limited to, the following: Shrinkage or reduction in size of one or more uterine fibroids (including removal of fibroids) , liquefaction, partial liquefaction, or reduced rigidity (increased flexibility) of or around uterine fibroids or a reduction in pressure, a change in viscoelastic properties, or a reduction in symptoms such as pain and bleeding.

[0044] The therapeutic effect may be objective (i.e., measurable by some test or marker). ), or subjective (i.e., the subject gives an indication of or perceives an effect). The effective dose may be determined by the clinician or the patient. However, the compositions of the present invention may vary in their effectiveness and in their potential use in combination with other active agents. The total daily usage of the product will be determined by your physician within the scope of sound medical judgment. It will be understood that the particular therapeutically effective dose level for any particular patient will depend on the individual being treated. the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used the patient's age, weight, general health, and diet; the time of administration of the particular compound used; the route of administration and rate of excretion; duration of treatment; and the specific compound used in combination or with other compounds. It depends on various factors, including the drugs sometimes used; and similar factors well known in the medical field. It will be.

[0045] The term "patient" or "patient in need" refers to a patient with a uterus and uterine fibroids or symptoms thereof. Such a "patient" or "patient in need" includes any mammal having the disease. are used in livestock such as horses and pigs, companion animals such as dogs and cats, and mice, rats, and Preferred patients include humans or any mammal, including laboratory animals such as rabbits and rabbits. The subject is a human female of childbearing age.

[0046] The pharmaceutical compositions of the present invention are preferably administered intravenously into the uterine fibroid tissue to be treated or intrauterine fibroid tissue. By direct injection, insertion, or implantation onto the tumor tissue, i.e., into the tissue to be treated. Other contemplated methods of administration include, but are not limited to: This includes: vaginal instillation or application to affected tissue, during surgery (laparoscopy or Instillation or application of liquid, fluid or other preparations into the affected tissue (e.g., hysteroscopy, etc.) Local administration to fibroid tissue by spraying or painting a gel formulation.

[0047] The formulations of the present invention can be delivered to uterine tissue by a variety of routes and in a variety of forms using a variety of devices. In some embodiments, the formulation is injected / inserted into a simple needle (e.g., 10 gauge or larger). needle below) and a sample pusher (e.g., a mandrel or modified obturator) For example, according to one embodiment, the formulation (e.g., Solid or semi-solid preparations in rod or other shapes, such as beads, suspensions, gels, or polymers ) is placed in a needle, syringe, or other chamber connected to a needle. Once the needle is positioned at the desired depth and location, use the pusher to push the sample through the needle and into the tissue. In some embodiments, the sample pusher is provided with a retaining clip or Alternatively, a hollow end is provided to preserve the sample until the time of delivery.

[0048] In yet another embodiment, the formulations according to the present invention can be administered by needles or other means, as known in the art. It is injected / inserted by jet injection without a physical delivery channel. Use a pressure system (e.g., a mechanical system or a gas such as helium, nitrogen, or carbon dioxide) The formulation should be produced at a rate fast enough to allow the formulation to penetrate the tissue to the desired depth. Jet injection devices can be used to deliver, for example, pre-filled or non-pre-filled drugs. With the drug cartridge being a cartridge, it may be disposable or reusable Examples of jet injectors include Bioject, a company based in New Jersey, USA. PowderJect, a registered trademark of PowderJect, a UK company In other embodiments, a section of the fibroid is excised and then a device (e.g., a biopsy device or tissue) that inserts a dosage form into the tissue shredding or laser irradiation) are used.

[0049] Formulations for delivering collagenase to patients are generally injectable or implantable products. agent, or any fluid, liquid, solid, semi-solid, gel, or other substance as described herein It is contemplated that the collagenase may include other compositions suitable for administering the collagenase to the tissue to be treated. The formulations of the present invention may be formulated by any method known in the pharmaceutical art. Therefore, any injection known in the art and compatible with collagenase activity can be used. Depot release of active collagenase agents can be used. or formulations that provide sustained release are contemplated. In particular, injectable depot compositions or sustained release compositions are contemplated. Although a release composition is preferred, any implantable formulation may be used. The composition creates or forms a depot effect in which the active agent resides in the tissue to which it is administered, The active agent is released over a period of time to provide continuous treatment of the tissue. Immediate release injectable formulations, which release activity immediately upon administration, are also contemplated for use in the present invention. These formulations are known in the art and one of ordinary skill in the art would be able to identify suitable formulations for use in the present invention. It can be configured as follows.

[0050] In some embodiments, the injectable or insertable formulations of the present invention are solid, semi-solid, or or highly viscous fluids, which improve dose retention in tissues and thereby improve the delivery of therapeutic agents. Improved delivery efficiency and / or minimized adverse effects such as unintended non-specific tissue damage "High viscosity" and other such terms are used herein to refer to a viscosity at, for example, 37°C. B with a CPE-40 conical spindle set at 0.5 rpm Any of several standard techniques, including the Brookfield kinematic viscometer HBDV-II+CP type Used to describe fluids with a viscosity greater than 1000 cps, measured by any "Low viscosity" fluids have viscosities below this value.

[0051] In some embodiments, the formulations according to the present invention are injected into a patient in a fluid state, whereby: into a form that is more easily retained, e.g., a solid form (the injected liquid is converted to a solid) that injected semi-solids are converted into solids, and liquids are converted into gels. (including the conversion of injected liquids into semi-solids, the conversion of injected semi-solids into , transformation into a semi-solid with increased yield stress and / or viscosity, and the liquid becoming a gel. or high viscosity fluids (including low viscosity fluids being converted into high viscosity fluids) , and the transformation of highly viscous fluids into more viscous fluids) in vivo To transform (or be transformed).

[0052] A preferred formulation for injection into uterine fibroids uses a carrier or nanocarrier. Suitable carriers include those that maintain active collagenase in the tissue and potentially inactivate collagenase. Protecting the active antibody from the action of certain tissues or tissue components and stabilizing the enzyme in the tissue for treatment solid or semi-solid pellets, beads, or gel-forming polymers for rapid release; and highly viscous liquids. Any suitable formulation capable of protecting and containing the active compound in place. Injectable dosage forms can be used. In mammals, C. histolyticum collagenase The enzyme is rapidly inhibited in the bloodstream by serum. Administration under conditions where collagenase can be inactivated or orally can result in collagenase being inactivated by digestive enzymes. This is problematic because it can decompose.

[0053] Nanocarriers are used to deliver and protect drug therapeutic agents (e.g., proteins) from degradation. In addition, nanocarrier formulations are used to prevent the drug from diffusing from the injected fibroids. and distribution away, prolonging release and delaying inactivation, thus Any such nanocarrier known in the art is preferred as it reduces the frequency of repeat injections. can be used in the present invention. Some of these nanocarriers are also called thermoresponsive delivery systems. can be.

[0054] Atrigel® is a biocompatible, water-miscible organic solvent (e.g., N-methyl water-insoluble biodegradable polymers (e.g., poly(2-pyrrolidone, NMP)) Contains poly(lactic-co-glycolic acid) (PLGA). At the time of use, collagenase is added to form a solution or suspension. Both the lactide-glycolide molar ratio (L:G ratio) influence drug delivery. In clinical studies using L:G ratios of 50–85:15 and polymer concentrations of 34–50% demonstrated that the depot formulation was maintained for more than three months.

[0055] ReGel® is a polymeric polymer consisting of PLGA-PEG-PLGA or PEG-PLGA-P EG repeats, PLGA and polyethylene glycol (PEG, 1000 Da or 1450 Da). eGel® is formulated as a 23% by weight solution of the copolymer in an aqueous medium. When the drug is added to the solution and the temperature is raised to 37°C, the entire system gels. The degradation of el® into end products of lactic acid, glycolic acid, and PEG is This occurs over a period of 1-6 weeks, depending on the molar composition of the hormone. Chemically distinct drugs, such as GLP-1, are incorporated into each It can be released from ReGel®.

[0056] LiquoGel™ provides mechanically independent drug delivery pathways: entrapment and covalent binding. This carrier can be used to deliver two or more drugs to the tumor site. LiquoGel™ is a thermogelling N-isopropyl acrylamide. Biodegradable macromers of poly(lactic acid) and 2-hydroxyethyl methacrylate; aqueous acrylic acid (to maintain solubility of degradation products); and LiquoGel (trademark) is a tetrameric copolymer of multifunctional hyperbranched polyglycerol. ) is formulated as a 16.9 wt% copolymer solution in an aqueous medium. It gels under physiological conditions and decomposes within 1 to 6 days to release the drug content.

[0057] Any of the above carriers can be used as nanocarriers in the present invention. Therefore, a preferred nanocarrier is hyperbranched polyglycerol (HPG), which has many desirable characteristics. HPG grows by incomplete formation of branching units in a convenient one-step reaction. The previous problem of high molecular weight polydispersity in their production has been overcome. The resulting polymers have numerous modifiable surface functional groups, as well as the ability to bind drugs. Other polymer approaches require fewer synthesis steps and These characteristics cannot be easily provided without significantly increasing the cost and consequently the cost. HPG polymers are based on glycerol and have the same structure as polyethylene glycol. It is biocompatible because of its similarity to the

[0058] Further components can be added to this polymer, thus modifying HPG polymers and HPG copolymers. It is contemplated that these additional moieties or monomers may be optionally added to the mer. can include, for example, cross-linked moieties, biodegradable moieties, and thermo-responsive moieties. The hydrogel responds to the temperature of the body and the required fluid volume is injected from a syringe maintained below body temperature. Upon warming, the mechanical properties increase, thereby constraining the material at the injection site. Poly(N-isopropylacrylamide) (poly- NIPAAm) is a thermoresponsive polymer with a lower critical solution temperature (LCST) of approximately 32°C. Therefore, the copolymer of HPG and NIPAAm is a polymer that can be used in the present invention. The nanocarriers are designed to be flexible and are preferred for this purpose. can be customized to capture small drug molecules, large proteins, or mixtures of components. The nanocarriers can be biodegraded and gel at body temperature, allowing for delayed release.

[0059] In a preferred embodiment of the present invention, the formulation exists as a liquid at temperatures below body temperature and The temperature at which the transition from liquid to gel occurs is called the LCST. In some cases, the LCST is not a specific temperature, but may be within a narrow temperature range. The material is referred to as an LCST material. Typical LCSTs in the practice of the present invention are, for example, 10 As a result, formulations injected below the LCST will remain in the body at temperatures above the LCST. The mixture is heated to a temperature above 100° C., thereby causing a liquid-to-gel transition.

[0060] Suitable LCST materials for use in the present invention include polyoxyethylene-polyoxypropylene. Two acceptable compounds are propylene (PEO-PPO) block copolymers. , pluronic acids F127 and F108, which have 12,600 and 13,000 kJ / kg, respectively. and a PEO-PPO block copolymer with a molecular weight of 14,600. Each of these is available from BASF (Mount Olive, NJ). Pluronic acid F108 at a concentration of 20-28% in phosphate buffered saline (PBS) is a preferred One useful preparation is 22.5% pluronic acid in PBS. A preparation of 22% pluronic acid F108 in PBS was incubated at 37°C for 1 hour. Pluronic acid F127 at a concentration of 20-35% in PBS is a suitable LC Another example of an ST material is a preparation of 20% pluronic acid F127 in PBS, which is heated at 37°C. The typical molecular weight is 5,000 to 25,000, and the LCST is For the two specific compounds mentioned, the values ​​are 12,600 and 14,600. Generally, other PEO-PPO block copolymers are biodegradable and decompose at body temperature. The present invention also provides for the use of substances that exist as gels and as liquids below body temperature. For more information on LCST materials, see U.S. Patent No. 6,565,530 (B2). Nos. 6,544,227(B2) and 6,544,227(B3).

[0061] Pharmaceutical formulations of the collagenase compounds of the present invention may be prepared in one or more pharmaceutically acceptable carriers. or a collagenase composition formulated with an excipient. The term "pharmaceutically acceptable carrier or excipient" refers to a non-toxic, inert, solid, semi-solid, or any type of filler, diluent, encapsulating material, vehicle, solvent, or formulation, whether liquid or solid It means an auxiliary agent and can be used in individual dosage forms or in bulk. Other dosage forms for producing pharmaceutical formulations are also contemplated for use in the present invention. Suitable collagenase formulations include, but are not limited to, the following: Multi-dose or single-dose lyophilized or other dry powders for reconstitution, ready for injection individual dose units (preferably also containing one or more preservatives), frozen units dosage form, or preparation in any manner known in the art. The formulations may also be provided in the form of a kit. The kit may include collagenase in solid form, a liquid or or solvents, as well as syringes and needles, especially dedicated syringes and needles for administering to uterine fibroids. The formulation may include any necessary equipment for administration, such as a syringe and / or needle. The product may be purified by any method known in the art, such as by filtration through a bacteria-retaining filter. They may be sterilized by any method or produced under aseptic conditions. Other methods include: This includes exposing the formulation or its components to heat, radiation, or ethylene oxide gas. can be.

[0062] Some examples of substances that can serve as pharmaceutically acceptable carriers are known in the art. Injectable vehicles such as those known in the art include, but are not limited to, the following: These include: sterile water, buffer solutions, saline solutions such as saline or Ringer's solution, fever Non-toxic substances such as water, ethyl alcohol, and non-toxic oils, or the like, may be used in the present invention. Any solvent compatible with injection or other modes of administration as described herein.

[0063] In addition, any solid excipient known in the art may be used, for example, as a vehicle or filler. Lactose, glucose, and sucrose may be used in pharmaceutical preparations with the present invention. sugars such as corn starch and potato starch; starches such as cellulose; Contains microcrystalline cellulose, sodium carboxymethylcellulose, ethylcellulose, and derivatives thereof, such as cellulose acetate; powdered tragacanth; malt; gelatin; gum; talc glycols such as propylene glycol; ethyl oleate and ethyl laurate esters; and agar, etc. can be used. Magnesium hydroxide and aluminum hydroxide Aluminum, citric acid, and acid or alkaline compounds such as phosphates or carbonates Buffers compatible with the active compound and method of use are contemplated for use, including lubricants, emulsifiers, wetting agents, suspending agents, binders, disintegrating agents, preservatives or antibacterial agents, antioxidants, Sustained-release excipients and non-toxic compatible excipients such as coating agents (e.g., sodium lauryl sulfate) sodium and magnesium stearate), as well as colorants, flavors, viscosity enhancers, and and bioadhesives, etc. may be used according to the judgment of a formulation specialist.

[0064] For example, one or more biodegradable binders are typically used in dosage forms having solid characteristics. When used, a wide range of biodegradable binder concentrations may be included in the formulations of the present invention. is based on, among other considerations, the desired physical characteristics of the resulting dosage form. and the characteristics of the selected uterine fibroid treatment agent (e.g., dilution, release delay, etc., That is, various amounts can be used based on desired / acceptable conditions. The concentration range of the combination is typically about 1 to 80% by weight of the biodegradable binder, more typically Approximately 5-50% by weight. "Biodegradable" materials dissolve when placed in tissue, such as uterine tissue. , degradation, resorption, and / or other degradative processes. When the formulation according to the invention contains Typically, at least a 10% weight loss occurs, more typically after 4 days in tissue. The preferred raw materials for use in connection with the present invention are Degradable binders include, but are not limited to, biodegradable organic compounds such as glycerin, and Biodegradable polymers or any known disintegrant compound known in the pharmaceutical arts are included. do.

[0065] When used, viscosity control agents typically impart a high degree of viscosity to the formulation, for example. and an amount effective to provide a formulation with a desired viscosity, for example, about 5,000 to 1,000 mg of PEG-1000. 200,000 cps, more typically about 10,000 to 100,000 cps, and even more Typically present in an amount effective to provide a viscosity of about 20,000 to 40,000 cps. By providing a formulation with a viscosity within these ranges, the formulation can be injected using conventional injection devices. (e.g., a syringe) can be used to inject into tissue, such as uterine tissue. The formulation has a high viscosity, which improves retention in the tissue at the injection site. The concentration of viscosity control agents may vary widely. Generally, the total concentration of viscosity control agents is about 1 In many embodiments, the viscosity control agent is of natural or synthetic origin. The polymer may be a polymer that is typically biodegradable. However, in some embodiments, the dimethylaminobenzoate is water soluble and / or hydrophilic. When organic solvents such as dimethyl sulfoxide (DMSO) are used as liquid components, viscosity control is required. The viscosity modifier may be relatively hydrophobic. Polymeric viscosity modifiers include homopolymers, copolymers, and the like. polymers, and polymer blends.

[0066] Examples of viscosity control agents for practicing the present invention include, but are not limited to: Included: cellulose polymers and copolymers, such as cellulose ethers, e.g. , methylcellulose (MC), hydroxyethylcellulose (HEC), hydroxypropyl Pillcellulose (HPC), Hydroxypropylmethylcellulose (HPMC), Methyl Hydroxyethyl cellulose (MHEC), methylhydroxypropyl cellulose (MH PC), carboxymethylcellulose (CMC) and its derivatives, including, for example, sodium salts various salts, hydroxyethyl carboxymethyl cellulose (HECMC) and its various salts, carboxymethyl hydroxyethyl cellulose (CMHEC) and its various salts; Other polysaccharides and polysaccharide derivatives, e.g., starch, hydroxyethoxybenzoates, HES, dextran, dextran derivatives, chitosan, and alginate acid and its various salts, carrageenan, xanthan gum, guar gum, gum arabic, Various gums, including karaya gum, guddi gum, konjac, and tragacanth gum glycosaminoglycans, and proteoglycans, such as hyaluronic acid and its salts; Heparin, heparin sulfate, dermatan sulfate, proteins such as gelatin, collagen , albumin, and fibrin, other polymers such as carboxyvinyl polymers, and their salts (e.g., carbomer), polyvinylpyrrolidone (PVP), polyacrylic Acrylic acid and its salts, polyacrylamide, polyacrylic acid / acrylamide copolymer , polyalkylene oxides, such as polyethylene oxide and polypropylene oxide; and poly(ethylene oxide-propylene oxide) (e.g., pluronic acid), poly Polyoxyethylene (polyethylene glycol), polyethyleneamine, and polypyridin Polyvinyl alcohol, polymethaphosphate (chloride salt), polyvinyl alcohol, Further salts and copolymers other than those mentioned, as well as blends of the foregoing (with the same monomers) (including mixtures of polymers containing the polymer but with different molecular weights), etc. Many of these are useful as binders.

[0067] In other embodiments of the invention, the formulation or carrier may be administered either prior to use or in vivo. Crosslinking is beneficial in that it acts, for example, to improve formulation retention (e.g., By providing a harder / viscous material and / or by adapting the polymer to specific environments This is advantageous when the formulation is crosslinked in vivo. The crosslinking agent is generally administered either before or after the formulation is injected or inserted according to the present invention. Depending on the nature of the formulation and crosslinker, the formulation may be, for example, a solid, semi-solid, or It may be converted into a body or into a highly viscous fluid.

[0068] Crosslinkers suitable for use in the present invention include any suitable crosslinker, including ionic and covalent crosslinkers. For example, in some embodiments, the formulations of the present invention may include any non-toxic crosslinking agent, e.g. For example, polymers ionically crosslinked with polyvalent metal ions. Suitable crosslinking ions include , calcium cation, magnesium cation, barium cation, strontium cation ions, boron cations, beryllium cations, aluminum cations, iron cations, copper cations cations selected from the group consisting of cations, cobalt cations, lead cations, and silver cations. Polyvalent anions include phosphate anion, citrate anion, borate anion, and anion, succinate anion, maleate anion, adipate anion, and oxalate More broadly, bridging anions are generally polybasic organic acids or Ionic crosslinking is achieved by, for example, dissolving an ionically crosslinkable polymer in a solvent. by contacting the cations with an aqueous solution containing ions that are dissolved in water, as known in the art. It can be implemented in.

[0069] In some embodiments, for example, a multifunctional crosslinker that is reactive with functional groups in the polymer structure. It includes polymers that can be covalently crosslinked using a crosslinking agent. any compound having at least two functional groups that react with functional groups in the polymer; The various polymers described herein may be crosslinked, both covalently and ionically. It may be done.

[0070] Suitable polymers for ionic and / or covalent crosslinking include, for example, but are not limited to, the following: Select from the list: Polyacrylate; Poly(acrylic acid); Poly(methacrylic acid) ;Polyacrylamide;Poly(N-alkylacrylamide);Polyalkylene oxide Poly(ethylene oxide); Poly(propylene oxide); Poly(vinyl alcohol) ;Poly(vinyl aromatic);Poly(vinylpyrrolidone);Poly(ethyleneimine);Poly( Ethyleneamine; Polyacrylonitrile; Poly(vinyl sulfonic acid); Polyamide; Poly L-lysine; Hydrophilic polyurethane; Maleic anhydride polymer; Protein; Collagen Gen; Cellulose polymer; Methylcellulose; Carboxymethylcellulose; Dextrose Orchid;Carboxymethyl dextran;Modified dextran;Alginate;Alginic acid; Pectinic acid; Hyaluronic acid; Chitin; Pullulan; Gelatin; Gellan; Xanthan; carboxymethyl starch; hydroxyethyl starch; chondroitin sulfate; guar; de Starch; and salts, copolymers, mixtures, and derivatives thereof.

[0071] In one preferred embodiment, the collagenase is in a solution of lactose, sucrose, or any It is formulated as a lyophilized injectable composition formulated with a suitable sugar. The lagenase composition is a frozen formulation of sucrose, Tris at a pH level of about 8.0. It is a dry injectable composition. Most preferably, 1.0 mg of the drug substance of the present invention is about 8. 60 mM sucrose, 10 mM Tris at a pH of 0.0 (e.g., in a formulation buffer of approximately 20. It is formulated in 5 mg / mL sucrose and 1.21 mg / mL Tris).

[0072] Preferred collagenase compositions for use in the present invention have at least 1000 SRC units. at least about 700 SRC units / mg, more preferably at least about 1500 SRC units / mg A mixture of collagenase I and collagenase II with a specific activity of 0.000 SRC units / mg One SRC unit is equivalent to 1000 kJ / min of rat tail collagen at 25°C and pH 7.4. It will be solubilized in a ninhydrin reactant equivalent to 1 nmole of leucine. Collagenase is similarly described in ABC units. This potency assay for collagenase is performed at pH 7. Digestion of native collagen (derived from bovine tendon) at 2°C and 37°C for 20-24 hours The number of cleaved peptide bonds was determined by reaction with ninhydrin. Subtract the amino groups released by the ATP digestion control. One net ABC unit of collagen is 1 It solubilizes ninhydrin reactants equivalent to 1.09 nmoles of leucine per minute. One SRC credit is equivalent to approximately 6.3 ABC credits or 18.5 GPA credits. In one embodiment, the injectable collagenase contains approximately 2800 SRC units per milligram. It will contain the rank.

[0073] Doses contemplated for administration by direct injection into uterine fibroid tissue are determined based on the tissue being treated. Doses will vary depending on the size of the tissue and the judgment of the treating physician. Typically, 1 cm of tissue to be treated 3 Approximately 0.06 mg collagenase to approximately 1 mg collagen per genase, or 1 cm of tissue to be treated 3 Approximately 0.1 mg collagenase per 100ml to approximately 0.8 mg mg collagenase, or 1 cm of tissue to be treated 3 Approximately 0.2 mg of collagenase per Approximately 0.6 mg of collagenase.

[0074] Also contemplated are formulations containing additional active agents or drugs. Optional additional agents that may be included in the formulation for administration or separate administration include, for example, uterine Current or future treatments for shrinking, treating, or eliminating fibroids or their symptoms Any pharmaceutical agent known in the art to aid in the implementation of the method, for example: and, in the same or separate administrations, one or more fibroid therapeutic agents, such as those May be co-administered with: aromatase inhibitors (e.g., letrozole, anastrozole) , and exemestane, a progesterone receptor agonist and Desirable hormones (e.g., progesterone, progestin, mifepristone, levonorgestrel levonoergestrel, norgestrel, asoprisnil, ulipristal, and acetic acid Ulipristal acetate, telepristone, selective estrogen receptor modulator Agents (SERMs) (e.g., benzopyrans, benzothiophenes, chromans, indoles, Naphthalene, triphenylethylene compounds, arzoxifene, EM-652, CP3 36,156, raloxifene, 4-hydroxytamoxifen, and tamoxifen ), gonadotropin-releasing hormone analogues (GnRHa) (e.g., 6-position is replaced with a D-amino acid) and / or the carboxyl-terminal Gly10-amide has been changed to an ethylamide Substituted GnRH agonist peptides or analogs, such as triptorelin, or GnRH antagonists such as cetrorelix, ganirelix, degarelix, and ozarelix antagonists), growth factor modifiers (e.g., TGFb neutralizing antibodies), leuprolide acetate, Nonsteroidal anti-inflammatory drugs, mTOR pathway inhibitors, WNT signaling pathway inhibitors, vitamins Vitamin D, vitamin D metabolites, vitamin D modulators, and / or additional anti-fibrotic compounds (e.g., pirfenidone and halofuginone).

[0075] Chemical ablative agents may also be included in the formulations of the present invention. In effective amounts, the compound causes tissue necrosis or shrinkage upon exposure. The irritant is added at a concentration appropriate for the condition, but which avoids inactivating the collagenase, according to the present technique. The amount to be used can be easily determined by one skilled in the art. Typical concentrations are The concentration range is about 1 to 95% by weight of the ablative agent, more typically about 5 to 80% by weight. Suitable ablative agents for use in the present invention include, but are not limited to: Osmotic stressors (e.g., sodium chloride or potassium chloride) salts of ethanol, etc.), organic compounds (e.g., ethanol), basic agents (e.g., sodium hydroxide, and potassium hydroxide), acidic agents (e.g., acetic acid and formic acid), enzymes (e.g., hyaluronidase, pronase, and papain), free radical generators (e.g., peroxides hydrogen peroxide and potassium peroxide), oxidizing agents (e.g., sodium hypochlorite, hydrogen peroxide , and potassium peroxide), tissue fixatives (e.g., formaldehyde, acetaldehyde , or glutaraldehyde), and / or blood coagulants (e.g., gengpin These agents may be used in combination with other collagenases of the same formula as long as they do not adversely affect the enzymatic activity of collagenase. The agent may be mixed with collagenase or may be administered separately at the same or different time points. It may also be administered to

[0076] The method according to the present invention can be used with any known treatment to control the symptoms caused by fibroids. For example, NSAIDs or other pain relievers may be used to reduce menstrual pain. Oral contraceptives can be prescribed to reduce uterine bleeding, and iron supplements can be given. If the condition of the uterus does not lead to the expulsion of the device, A bonorgestrel intrauterine device can be used to reduce bleeding and other symptoms. do.

[0077] The ability to non-invasively image the area in which the formulations of the present invention are being introduced and have been introduced. This is a useful diagnostic tool for practicing the present invention. In addition to the therapeutic agent and any of the various optional ingredients discussed above, the uterine fibroids of the present invention may be used. The formulation optionally guides the clinician in administering the collagenase compound to the fibroid or tissue being treated. one or more images to assist in the administration of an object and to determine that the administration has been correctly placed Non-invasive imaging techniques include magnetic resonance imaging (MRI), ultrasound, and the like. Suitable for use in imaging, fluoroscopy, and nuclear medicine. Any imaging agent known in the art may be used as part of the compositions and formulations of the present invention. It is possible.

[0078] Any real-time imaging technique can be used to guide injection or insertion in the present invention. For example, X-ray based fluoroscopy allows real-time patient monitoring of intra-patient motion. For visualization under fluoroscopy, the preparation typically contains In various embodiments of the present invention, the fibrous structure is provided with a higher X-ray absorption than the surrounding tissue. This is achieved through the use of contrast agents. Examples of contrast agents used in conjunction with fluoroscopy include: Contains metals, metal salts and oxides (especially bismuth salts and oxides), and iodine compounds. More specific examples of such contrast agents include tungsten, platinum, tantalum, iridium, zinc, gold, or other high density metals, barium sulfate, bismuth subcarbonate, bismuth trioxide, Bismuth oxychloride, metrizamide, iopamidol, sodium iothalamate, iodine These include iodomide sodium, and meglumine.

[0079] Ultrasound imaging and magnetic resonance imaging provide two-dimensional or three-dimensional images of body parts. Ultrasound and MRI can be used to observe the patient and physician without exposing them to harmful radiation. This is particularly advantageous as it provides detailed images of the area under examination. It is a useful diagnostic aid and can be used to more precisely control the amount and location of the formulations of the present invention. It can be used.

[0080] Suitable ultrasound imaging contrast agents for use in connection with the present invention include those having a maximum dimension (e.g., spherical) If shaped particles are used, the diameter is about 0.01 to 50 microns, more typically about It contains solid particles ranging from 0.5 to 20 microns. Both inorganic and organic particles Examples include calcium carbonate, hydroxyapatite, silica, These include poly(lactic acid) and poly(glycolic acid) microparticles / microspheres. As is known in the imaging arts, microbubbles are used as ultrasound imaging contrast agents. Ultrasound imaging contrast agents for use in connection with the present invention are preferably biocompatible. When solid particles are used, they are biocompatible and stable in the formulation. The concentration is typically about 0.01 to 10% by weight of the formulation, more typically about 0.05% by weight. % to 2% by weight.

[0081] For contrast-enhanced MRI, suitable contrast agents have a large magnetic moment and relatively short electron relaxation times. Based on these criteria, Gd(III), Mn(II), and Fe(II Gadolinium(III) is the most commonly used contrast agent among these three. It has the largest magnetic moment and is therefore widely used for MRI imaging. Gd-DTPA (a paramagnetic species with a ligand diethylenetriaminepentaacetic acid) Also suitable are chelates of paramagnetic ions such as gadolinium ions (which are often referred to as gadolinium ions). For more information, see, for example, "Implantable or insertable m edical devices visible under magnetic re U.S. Patent Application No. 2003-01008 entitled "Sonance Imaging" It can be found in issue 30.

[0082] The collagenase formulations described herein are preferably delivered through a hollow needle, such as a hollow needle or cannula. The delivery channel is used to inject into one or more individual uterine fibroid tumors. For example, administration may be by needle with conventional or specially designed syringes, cannulae, catheters, etc. It can be performed using manual, mechanical, hydraulic, pneumatic or other pressure Use of a force application source (e.g., a conventional syringe plunger, a pump, an aerosol, etc.) Alternatively, the formulation can be administered during laparoscopic surgery. and during hysteroscopic procedures, for example, can be administered intraoperatively via a trocar.

[0083] Injection routes include, for example, the intraperitoneal route, the transcervical route, and the transvaginal route. If the injection volume is of a different nature, it may depend on, for example, the size of the fibroid, as well as the type and type of therapeutic agent. The amount varies depending on the dosage and concentration, and typically ranges from 1.0 to 10.0 ml per injection. Similarly, formulations with solid properties (e.g., pellets or powders) may be used. If used, the amount of formulation injected / inserted will depend on, for example, the size of the fibroid, the treatment used, The amount of collagenase composition to be added will also depend on the type and concentration of the agent. Alternatively, multiple doses can be administered at a single injection site. Multiple injection / insertion sites can be established within a single fibroid, and the number of injections varies depending on the size of the fibroid. Depending on the size and shape, and the type and / or concentration of the therapeutic agent used, Multiple fibroids or a single fibroid can be treated.

[0084] In various embodiments, the injection / insertion device is guided to the fibroid site under image guidance. Image guidance includes, for example, direct visual guidance (e.g., laparoscopic guidance in transperitoneal procedures, transvaginal guidance, etc.). hysteroscopic guidance in surgical procedures) and indirect visual guidance (e.g., ultrasound guidance, fluoroscopic guidance, and / or or MRI guided).

[0085] As a specific example, visual guidance of the injection / insertion device may be achieved by using a scope positioned in the abdomen. The procedure is performed laparoscopically using a tube (eg, by insertion through a trocar). In this manner, a device (e.g., a delivery needle or cannula) is inserted percutaneously into the abdomen to It can be guided to the uterine fibroid under laparoscopic vision. Once it reaches the fibroid, it is preferable to is performed using fluoroscopy, MRI, or ultrasound (e.g., transvaginal ultrasound, transperitoneal ultrasound, abdominal ultrasound). Using intrathecal ultrasound or other methods, the tip of the delivery needle is guided to the desired location within the fibroid, and the needle is then inserted into the desired location. The preparation is injected or inserted into the fibroid at the point where it is needed. As long as there is a last, the location of the preparation within the fibroid will also be visualized.

[0086] The compositions and processes of the present invention will be better understood with reference to the following examples. However, they are intended as examples only and are not intended to limit the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Including, but not limited to, relating to the processes, formulations, and / or methods of the present invention. Such changes and modifications are beyond the spirit and scope of the present invention and the appended claims. It can be done without breaking down. [Example]

[0087] Basal collagenase production To prepare an animal-material-free clostridial cell bank, M. histolyticum cells were suspended in a medium containing vegetable peptone and optionally yeast extract. For example, one basic way to achieve this is as follows:

[0088] [Table 2]

[0089] Once an animal-material-free cell bank has been established, it is possible to culture the cells in any convenient medium known in the art, preferably a medium containing The cells can be grown or fermented in a medium of animal origin or a non-animal origin. Illustrative, non-limiting examples of such media are listed in Table 3 below. In addition, the fermentation process See Table 4 for illustrative, non-limiting general examples of steps.

[0090] [Table 3]

[0091] [Table 4]

[0092] After preparing the "second culture," collagenase I and collagenase II were incubated separately. and may be isolated and purified using any method capable of producing the desired product in a purity of at least 95%. This method can be used to purify the hydroxyapatite ( HA) one or more of the steps of chromatography, gel filtration, and ion exchange The gel filtration is preferably G75 gel filtration. The ion exchange is preferably anion exchange: Q-Sepharose chromatography. In addition, it requires less glucose and more It is preferable to culture Clostridium in a medium containing the salts of Protease inhibitors such as putin are not required. [Example]

[0093] Preparation of an animal-material-free clostridial cell bank The starting cell culture was Clostridium histolyticum ATCC 21000, strain 0 04. This was originally made from bovine-derived materials. First, Cells were grown in medium containing no ATP (M#1, Table 3). Briefly, the recipe is as follows: Contains 5g of phytone, 8.5g of yeast extract, and 1000mL of water. Adjust the pH with NaO. The temperature was adjusted to 7.30°C with H2O and the medium was sterilized at 121°C for 20 minutes. The starting material was inoculated into 300 mL of M#1 and incubated at 37°C for 24 hours (first Three milliliters of the first culture was transferred to 1000 mL of M#1 and incubated for 16 hours. The second culture was then aseptically centrifuged to pellet the cells. The cells were resuspended in 5 mL of M#1 with 5 mL of 20% glycerol. The suspension was slowly frozen and stored at -80°C. [Example]

[0094] Fermentation Process Clostridium histolyticum ATCC 21000, strain 004, was used as M#1. or M#2 and incubated at 37°C for 16 hours. Mix 10 milliliters of starter culture (M#1 or M#2) and 10 mL of Mg / vitamin solution. Solution (8g MgSO4, 1.2g ferrous sulfate, 0.05g riboflavin, 0.1g niacin Syn, 0.1g calcium pantothenate, 0.1g pimelic acid, 0.1g pyridoxine, and 0.1 g thiamine dissolved in 1100 mL water, then sterilized by 0.22 μm filtration. (prepared separately by adding 1 liter each of M#3 or M#4 (or their equivalents) The cells were transferred to a modified plate and incubated for 22 hours. Clostridium histolyticum was well tolerated. The cells grew rapidly and the OD600 reached >2.5. [Example]

[0095] General procedure for the isolation and purification of collagenase I and collagenase II.

[0096] [Table 5] [Example]

[0097] Ex vivo treatment of uterine fibroid tissue Fibroid tissue and myometrial samples were obtained from consenting women after hysterectomy and surgical procedures. The tissue samples were transported to the laboratory and separated into 1 cm 3Cut into cubes See Figure 2. These cubes were filled with purified collagenase dissolved in culture medium or serum. (0.06 or 0.2 mg in 100 μL) was injected, and then the , 72, or 96 hours. See Figure 3. The fibroid tissue was Due to constant variability, each treatment consisted of tissue from three different patients, with two treatments per treatment. Control fibroids and myometrial cubes were injected with vehicle or At the end of the incubation period, tissue samples were photographed and the macroscopic appearance was evaluated. The degree of liquefaction and softening was observed and recorded using a four-point subjective scale. Ta.

[0098] Samples were frozen for biomechanical evaluation (compression analysis). Histological examination and Masson's trichrome staining For color and picrosirius red staining, samples were fixed in formalin. The presence or absence of vasopressin was analyzed by light microscopy and assessed using computerized morphometry. The degree of resolution was determined. In the case of picrosirius red staining, polarized light microscopy was performed. The orientation of collagen fibers was determined. The samples were fixed with glutaraldehyde and then analyzed for electron microscopy. Postfixation with osmium tetroxide revealed collagen fibril orientation and evidence of fibril degradation. Further injections at a dose of 0.58 mg / injection (250 ul of 2.3 mg / ml) were administered. was carried out.

[0099] These ex vivo studies demonstrated softening and partial liquefaction of fibroid specimens after hysterectomy, and that purified collagenase is effective in reducing collagen content. The treated fibroid specimens were generally softer with a partially liquefied center. Masson's trichrome staining and picrosirius red staining of these tissues revealed that the vehicle-injected Compared with intact fibroid tissue, the tumors showed a dramatic subjective reduction in collagen content. [Example]

[0100] Ex vivo treatment of whole uterine fibroids The donor tissue must be able to provide legally valid consent and must be able to provide final cure for fibroids by hysterectomy. Hysterectomy specimens were obtained from four female adult patients aged 18 years or older who were planning to undergo treatment. After removal, the uterus was examined macroscopically by a surgical pathologist using standard procedures. Complete fibroids (submucosal ( fibroids (in contact with the endometrium), intramural (in the myometrium) fibroids, and subserosal (in contact with the uterine serosa) fibroids fibroids, or pedunculated fibroids (attached to the uterus by a stalk), if present, and available Where possible, a 0.5 cm section of endometrium was dissected from the specimen and placed in saline.

[0101] The tissue was immediately transported to the laboratory, washed, and purified Clostridium histolyticum collagenase PCHC (0.1 mg / 100 μl / cm 3 ) was injected. Optionally, higher concentrations Purified collagenase was used to reduce the injection volume. Dilute with 0.3 mg / mL calcium chloride dihydrate in sodium chloride and optionally add a marker Mixed with 1% methylene blue as a marker for the distribution of injected material in the fibroids and uterus. For fibroids, PCHC or vehicle was injected into the center of the specimen. See Figures 4A and 4B. The amount of collagenase injected was 100 mg / kg / day for fibroids (1-3%). The size of the tube depended on the size of the tube (approximately 2.5 cm diameter). Generally, approximately 818 μL of material was placed in a tube with a diameter of approximately 2.5 cm. The injection was performed on fibroids with a central focus on the entire treatment volume to minimize tissue resistance to injection. If this was not feasible due to resistance or other factors, multiple locations within the fibroid were injected. The fibroid tissue was then incubated in DMEM / F12 medium at 37°C for 24 hours. At least one fibroid with attached myometrium served as a control. This specimen received 100 mg of collagenase-free medium as a non-randomized placebo injection. % methylene blue was injected into the center of the fibroid.

[0102] Color photographs of the uterine, fibroid, and myometrial slices were taken before and after injection. Fibroid diameter was measured with a metric ruler.

[0103] At the end of the incubation period, the samples were visually reassessed for size, consistency, and hardness. Color photographs were taken and all measurements were taken during manual fibroid swelling and sectioning. The degree of liquefaction and softening was observed and recorded at four points. Subjective scales were used to record.

[0104] It was determined whether collagenase could penetrate the capsule and affect the adjacent myometrium. The fibroids and adjacent myometrial tissue, as well as the fibroids and tissue still attached Sections containing adjacent myometrium and / or endometrium, as well as sections containing only myometrium Histological examination, Masson's trichrome staining, and picrosirius red staining were performed. For staining and hematoxylin-eosin staining, the samples were fixed in formalin. The tissue was analyzed by light microscopy for the presence or absence of collagen and analyzed using computerized morphometry. The degree of resolution was assessed using polarized light microscopy with picrosirius red staining. The orientation of the collagen fibers was determined.

[0105] Exemplary Treatment Plans for Each Patient

[0106] Inject fibroid 1:8 18 µL of 1 mg / mL collagenase.

[0107] Fibroid 2: Inject 818 µL of 1 mg / mL collagenase.

[0108] Fibroid 3: Inject 818 µL of control vehicle.

[0109] From the fibroid capsule to the fibroid core, from the myometrium to the fibroid core, or from the endometrium The injection was made from the center of the fibroid to mimic the in vivo injection route. The fibroma was liquefied in the same manner as shown in Figure 5 (see below). [Example]

[0110] Biomechanical evaluation of human uterine fibroids after injection of purified clostridial collagenase. Two collagenases (ABC I and ABC II) isolated from Clostridium histolyticum Collagenase II and ABC II were mixed in a 1:1 mass ratio. It is a lyase with a wide range of hydrolytic activity and degrades type I and III collagen. The biomechanical properties of uterine fibroid tissue from control and collagenase-treated specimens were evaluated using Rheometr Lee analyzed.

[0111] Uterine fibroids produce approximately 70% of type I collagen compared with approximately 80% in the myometrium. Approximately 28% of type III collagen compared with approximately 20% in the myometrium, and It has been shown to contain approximately 5% type V collagen, compared with approximately 2% type I / III. , and are less common in the center and edges of fibroids compared with the myometrium (Feng et al. al,

[0112] Fibroid tissue was obtained from four different patients after surgery (hysterectomy or myomectomy). , cut into cubes (1cm 3 (n = 43). The tissue cubes were filled with 100 μL of purified Lagenase (0, 0.25, 0.5, 1.0, 2.0 mg / mL; n = 4 per dose) 14) was injected centrally and incubated at 37°C for 24, 48, or 96 hours. At the end of the incubation period, the cubes were cut in half and flash frozen in liquid nitrogen. The degree of softening and liquefaction of the samples was found to be different. The sample stiffness was measured dynamically (complex shear modulus (Pa) at 10 rad / s) and the viscosity of the material was measured. Both mechanical and elastic behavior were taken into account. At least two specimens from each tissue cube were (5 mm diameter punched out) were measured. Data were analyzed by two-way ANOVA and Dunnett's multiple Analysis was carried out using a comparative test.

[0113] Overall, the stiffness of the control fibroid cubes (6585 ± 707 Pa; n = 13) was significantly higher than that of the control fibroid cubes (6585 ± 707 Pa; n = 13) after treatment. This was greater than that of the quadrate (2003±275 Pa; n=30; p<0.0001). Furthermore, the stiffness of fibroid tissue was reduced in a time- and dose-dependent manner. , while treatment with 0.25 mg / mL did not reduce stiffness (5032 ± 1796 Pa). , treatment with 0.5 mg / mL reduced stiffness (2014 ± 1331 Pa; p ≤ 0. At 96 hours, both the 0.25 and 0.5 doses were effective (1720± 377 and 1072 ± 160 Pa; p ≤ 0.01). The placement reduced stiffness at 24 hours, but not significantly (2177 ± 37 and 2480 ± 10). ±984 Pa; n=4). However, the 1.0 and 2.0 mg / mL doses were significantly lower than the 48 time (3588 ± 637; p ≤ 0.05 and 1254 ± 445 Pa; p ≤ 0.01; n = 6), and 96 hours (921 ± 305 and 1350 ± 571 Pa; p ≤ 0.000 1; n = 10) was effective.

[0114] A torsional rheometer was used to measure a wide range of Our data demonstrate that a defined dose of purified Clostridium perfringens (C. perfringens) significantly increased tissue stiffness. Treatment of fibroid tissue with um collagenase significantly reduced the stiffness (modulus) of the tissue. See Figure 5, which shows the results after 48 hours of incubation. Collagen degradation in fibroid tissue is shown. The left photo shows a vehicle (control) injected mouse. The photograph on the right shows the tissue injected with collagenase. (Figures 6A and 6B) and collagenase-treated tissue (Figures 6C and 6D). Masson staining in panels A and C (left) shows that collagen is reduced. Picrosirus red staining clearly visualized under polarized light. (FIG. 6D) clearly shows that collagen fibers are degraded on the bottom right side. [Example]

[0115] Treatment of human uterine fibroids in a nude mouse model Three-dimensional organotypic cultures of human uterine fibroid cells are implanted subcutaneously into female nude mice. Using a xenograft mouse model, we have identified fibrous epithelial tumors with biological characteristics similar to fibroid tumors. Keloids, a skin disorder, have been successfully studied using this model. This demonstrates the in vivo effect of PCHC injection of the agent on fibroid tissue.

[0116] Polylactic acid sponge, other synthetic polylactic acid scaffolds, or any suitable commercially available scaffold The scaffolds were seeded with human uterine fibroid cells to form uterine fibroids that could be implanted into nude mice. Generate organotypic three-dimensional cultures of fibroid cells. These three-dimensional organotypic cultures (3D fibroids) , which represents a human fibroid, produces and contains extracellular matrix.

[0117] OPLA sponge (open-cell polylactic acid, BD Biosciences; Figure 7) This is a synthetic polymer scaffold synthesized from DL,L polylactic acid. The faceted structure is effective for culturing high-density cell suspensions. Cells are cultured in 3D under dynamic conditions. When seeded onto a sponge-like scaffold, a uniform cell population was observed throughout the sponge. This will result in a higher number of cells per sponge than static seeding. The molecular weight of PLA is 100-135 kD. They are 5 mm x 3 mm (0.04 cm 3 ) and the average pore size is 100 to 200 μm.

[0118] The cells and scaffold are subjected to shear forces and to the cells and / or scaffold. A fluid (culture medium) that allows for consistent cell suspension while minimizing gravity-induced settling of the fluid. The cell culture chamber of the bioreactor consists of a rotating chamber filled with (Synthecon Inc.) inside the rotating bioreactor chamber. Some cells are suspended in a virtually weightless state.

[0119] Primary human fibroid cells derived from hysterectomy specimens were statically cultured on OPLA sponges. or dynamically seeded and allowed to grow for 30 days to allow extracellular matrix production and assembly. Cells were grown throughout the scaffold, fixed in formalin, and then paraffinized. They can be embedded, thin-sectioned for observation, and optionally stained for multiple markers. See Figure 8. In the figure, a cell lattice is formed along the contour of the sponge-like scaffold. It has been shown that there are

[0120] Figure 9 shows a primary culture of fibroid cells after static seeding. The cells are fixed to the scaffold and observed in situ. The cells were then destained (Figure 9A) or stained for f-actin with fluorescent phalloidin (Figure 9B). The cells were uniformly distributed throughout the scaffold. The field is >1 mm thick, so not all cells are in focus. This indicates that cells are growing not only on the surface but also deep inside the scaffold. In Figure 10, the cell population is seen throughout the sponge-like scaffold. 10A and 10B) using a confocal microscope.

[0121] High-quality RNA was extracted from 3D cultures of fibroid cells in OPLA sponges and The expression of two genes of interest is verified using the fibroid-like protein versican and TGFβ3. It is known that fibroid tumors are highly expressed in tumor tissues and cells. Both the cell lines and primary cultures of fibroid cells in this 3D culture system express these two genes. It shows that it is expressed in large amounts.

[0122] [Table 6]

[0123] The patent and scientific literature cited herein establishes knowledge that is available to those skilled in the art. do.

[0124] While the present invention has been particularly shown and described with reference to preferred embodiments thereof, Those skilled in the art will appreciate that the present invention may be modified in various ways without departing from the scope of the present invention as encompassed by the appended claims. It will be understood that various changes can be made therein in form and detail.

Claims

1. A method for treating uterine fibroids, comprising administering to said patient a therapeutically effective amount of a compound effective in causing shrinkage of uterine fibroids. preparing an injectable or insertable formulation containing an effective amount of a uterine fibroid therapeutic agent; and a step of injecting or inserting the preparation into the uterine fibroids, is collagenase.

2. The collagenase is from a mammalian source, a crustacean source, a fungal source, or a bacterial source. The method of claim 1, wherein the compound is obtained from

3. 3. The method of claim 2, wherein the collagenase is obtained from a bacterial source.

4. 4. The method of claim 3, wherein the collagenase is obtained from a Clostridium.

5. 5. The method of claim 4, wherein the Clostridium is Clostridium histolyticum. How to do it.

6. 2. The method of claim 1, wherein the collagenase is a mixture of collagenase I and collagenase II.

5. The method according to claim 5.

7. The collagenase I and collagenase II are present in a mass ratio of about 0.5 to about 1.

5. The method of claim 6 .

8. 1 cm of tissue to be treated ３ About 0.06 mg to about 1 mg of collagenase is administered per The method of claim 1 .

9. 1 cm of tissue to be treated ３ About 0.1 mg to about 0.8 mg of collagenase is administered per The method of claim 8 .

10. 1 cm of tissue to be treated ３ About 0.2 mg to about 0.6 mg of collagenase is administered per The method of claim 8 .

11. 10. The method of claim 1, wherein the formulation is injected or inserted intraperitoneally into the fibroid. 。

12. 10. The method of claim 1, wherein the formulation is injected or inserted transvaginally into the fibroid.

13. 10. The method of claim 1, wherein the formulation is injected or inserted into the fibroid under image guidance. Law.

14. 14. The method of claim 13, wherein the image is at least one of a direct vision image and an indirect vision image. The method described.

15. The method of claim 14 , wherein the direct image is a scope image.

16. The method of claim 14, wherein the indirect vision image is an MRI image.

17. 17. The method of claim 16, wherein the formulation comprises an MRI contrast agent.

18. The method of claim 16 , wherein the indirect vision image is an ultrasound image.

19. 20. The method of claim 18, wherein the formulation comprises an ultrasound contrast agent.

20. The method of claim 14 , wherein the indirect vision image is a fluoroscopic image.

21. 21. The method of claim 20, wherein the formulation comprises an x-ray contrast agent.

22. The agent may be a chemical ablative, a nonsteroidal anti-inflammatory drug, an oral contraceptive, a GnR and further comprising an H agonist, antiprogestogen, or selective progesterone receptor modulator. The method of claim 1 .

23. 23. The method of claim 22, wherein the formulation comprises a chemical ablative agent.

24. 24. The method of claim 23, wherein the chemical ablative agent is a salt.

25. The chemical ablative agent is selected from an enzyme, an acid, a base, and an oxidizing agent. Item 24. The method according to item 23.

26. 10. The method of claim 1, wherein the formulation comprises multiple different uterine fibroid therapeutic agents.

27. 10. The method of claim 1, wherein the formulation is in a dosage form having a maximum dimension of between 1 mm and 20 mm. 。

28. 10. The method of claim 1, wherein the formulation is delivered to the fibroid through a hollow channel.

29. The method of claim 1 , wherein the formulation is encapsulated.

30. 10. The method of claim 1, wherein the formulation is a powder.

31. 31. The method of claim 30, wherein the powder is introduced into the fibroid by jet injection. Law.

32. The viscosity control agent provides a viscosity in the range of 10,000 cps to 50,000 cps. The method of claim 1 , wherein the compound is present in an effective amount.

33. The method of claim 1 , wherein the formulation is ionically crosslinked in vivo.

34. The method of claim 1 , wherein the formulation comprises an alginate polymer.

35. the formulation has a lower critical solution temperature (LCST) that is below the patient's body temperature, 10. The method of claim 1, wherein the agent is injected at a temperature below the lower critical solution temperature (LCST). Law.

36. The method of claim 1 , wherein the formulation comprises gelatin.

37. 1. A system for treating uterine fibroids, comprising: (a) causing shrinkage of uterine fibroids; an injectable or insertable formulation comprising an effective amount of a uterine fibroid therapeutic agent; and b) a device for injecting or inserting the formulation into the fibroids, A system in which the therapeutic agent is collagenase.

38. Collagenase, as well as nonsteroidal anti-inflammatory drugs, oral contraceptives, GnRH agonists, and one or more of an antiprogestogen and a selective progesterone receptor modulator. a fibroid injectable or insertable composition comprising an amount effective to cause shrinkage of fibroids; formulation.

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