Brachytherapy composition and related methods

Abstract

A brachytherapy composition fabricated from mammalian tissue is provided. Methods of processing a mammal's mammalian tissue to form a brachytherapy composition are provided. Regenerative methods are also provided.

Description

[0001] BRACHYTHERAPY COMPOSITION AND RELATED METHODS

[0002] BACKGROUND OF THE INVENTION

[0003] Human placental tissue has been utilized for various purposes over the past century for regenerative medicine purposes. There remains a need, however, for regenerative products and methods of producing novel human mammalian tissue products, as well as regenerative products derived from alternative sources.

[0004] SUMMARY OF THE INVENTION

[0005] The present invention is generally directed to a brachytherapy composition, and processes for producing the brachytherapy composition. The brachytherapy composition exhibits various regenerative medical properties. The brachytherapy composition may be placed in or around various tumors or cancerous tissue of a patient in need. The brachytherapy composition as provided herein is particularly advantageous in that the composition may be placed at or around a treatment area to aid in the healing cascade. The brachytherapy composition will stay at the implantation site and not easily migrate or move away from the tumor. The brachytherapy composition is also bioresorbable and, thus, may be resorbed by the body.

[0006] According to one aspect, a brachytherapy composition is provided. The brachytherapy composition includes at least one dehydrated mammalian tissue. According to one embodiment, the dehydrated mammalian tissue is molded into a substantially round or a substantially cylindrical shape. According to one embodiment, the brachytherapy composition further includes at least one bioresorbable bead. According to one embodiment, the bioresorbable bead includes a radiotherapy source. According to one embodiment, the radiotherapy source is a radionuclide selected from the group consisting of Cesium-131 , Cesium-137, Cobalt-60, lridium-192, lodine-125, Palladium-103, Ruthernium-106, and Radium- 226. According to one embodiment, the at least one dehydrated mammalian tissue includes dehydrated mammalian placental tissue comprising collagen I, collagen IV, elastin, laminin, fibronectin and hyaluronic acid. According to one embodiment, the mammalian tissue is porcine placental tissue.

[0007] According to another aspect, a method of treating a tumor is provided that includes the step of placing the brachytherapy composition in or next to a tumor or cancerous tissue. According to one embodiment, the tumor is located on or around the cervix, prostate, breast, skin, brain, eye, head, neck, mouth, respiratory tract, digestive tract, or urinary tract.

[0008] According to one aspect, a kit is provided. The kit includes a brachytherapy composition as provided herein and instructions for use.

[0009] According to one aspect, a method of preparing a brachytherapy composition is provided. The method includes the steps of: a) introducing from about 1 mL to about 40 mL of from about 1 M to about 4M sodium chloride solution per gram of mammalian tissue to the mammalian tissue; b) decanting the sodium chloride; c) rinsing the sodium chloride from the mammalian tissue with water; d) introducing from about 5mL to about 25 mL of a detergent solution per gram of mammalian tissue to the mammalian tissue; e) decanting the detergent solution; f) rinsing the detergent solution from the mammalian tissue with water; g) introducing from about 5 mL to about 15 mL of from about 0.1 M to about 1 .0M sodium hydroxide per gram of mammalian tissue to the mammalian tissue; h) rinsing the sodium hydroxide from the mammalian tissue with water; i) introducing from about 5 mL to about 50mL of buffer solution per gram of mammalian tissue to the mammalian tissue; j) decanting the buffer solution; k) measuring the pH of the mammalian tissue; l) repeating steps i), j) and k) until the pH of the mammalian tissue is between about 6.8 and about 7.2; m) rinsing the buffer solution from the mammalian tissue with water; n) milling the mammalian tissue into one or more fibers; o) forming the one or more fibers into at least one bead; and p) desiccating the at least one bead to form a brachytherapy composition.

[0010] According to one embodiment, the step of forming the one or more fibers into a bead includes forming the bead around a bioresorbable bead. According to one embodiment, the bioresorbable bead comprises at least one bioresorbable polymer. According to one embodiment, the method further includes the steps of: q) packaging the brachytherapy composition; and r) terminally sterilizing the packaged composition. According to one embodiment, the terminal sterilization is e-beam irradiation, gamma irradiation, peracetic acid treatment or vaporized peracetic acid (VPA) treatment. According to one embodiment, the detergent solution comprises at least one anionic detergent and at least one protease enzyme. According to one embodiment, the detergent solution comprises sodium linear alkylaryl sulfonate, phosphates, carbonates and at least one protease enzyme. According to one embodiment, the mammal is a pig. According to one embodiment, the pig is not genetically modified to halt or reduce expression of a functional alpha-1 ,3 galactosyltransferase gene. According to another aspect, a porcine brachytherapy composition is provided that is produced by any of the aforementioned methods.

[0011] DETAILED DESCRIPTON OF THE INVENTION

[0012] The present disclosure will now be described more fully hereinafter with reference to exemplary embodiments thereof. These exemplary embodiments are described so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art. Indeed, the present disclosure may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements.

[0013] As used in the specification, and in the appended claims, the singular forms “a”, “an”, “the”, include plural referents unless the context clearly dictates otherwise. As used in the specification, and in the appended claims, the words "optional" or "optionally" mean that the subsequently described event or circumstance can or cannot occur.

[0014] As used herein, the term “brachytherapy” refers to a form of therapy where a composition as provided herein is placed inside, on or next to the area requiring treatment. An area requiring treatment may include a tumor or cancerous tissue.

[0015] As used herein, the terms “birth tissue” and “placental tissue” include, but are not limited to, elements of a mammalian placental organ such as, for example, mammalian placental membrane, mammalian amnion, mammalian chorion, mammalian intermediate layer, mammalian placental globe, mammalian umbilical cord, or a combination thereof.

[0016] As used herein, the term “placental membrane” refers to the full, intact placental membrane including the amnion and chorion layers that are obtained from a mammal such as, for example, a pig or human.

[0017] As used herein, the term “mammalian tissue” includes one or more of mammalian placental tissue (as defined herein), mammalian dermis, mammalian pericardium, mammalian small intestinal submucosa, mammalian tendon, mammalian ligament, mammalian abdominal viscus, mammalian abdominal diaphragm, mammalian bladder, mammalian artery, mammalian vein or any combination thereof.

[0018] As used herein, the terms “pig” and “porcine” may be used interchangeably.

[0019] As used to herein, the term “birth tissue composition” refers to a construct that is applied onto or around an injured area of a mammalian body.

[0020] As used herein, the term “wound” refers to an injured area of the body.

[0021] The brachytherapy composition as provided herein may aid in the healing cascade or healing process. The brachytherapy composition may be utilized to arrest tumor progression, reduce tumor size, or eliminate tumors. According to a particular embodiment, the brachytherapy composition is fully resorbed by the mammal's body during the healing process. The present disclosure further relates to methods for processing mammalian tissue to produce a brachytherapy composition. According to one embodiment, the brachytherapy composition includes dehydrated mammalian tissue that is processed as provided herein.

[0022] According to one embodiment, the brachytherapy composition may be used for a variety of regenerative medicine purposes. According to one embodiment, the regenerative medical use is for treatment for treatment of tumors. Such tumors may be located on or within various parts of a mammalian body including, but not limited to, the cervix, prostate, breast, skin, brain eye, head, neck, respiratory tract, digestive tract, urinary tract, or soft tissues. According to a particular embodiment, the tumor may be located in, for example, the lip, mouth, tongue, pharynx, trachea, bronchi, esophagus, gall bladder, bile duct, anus, rectum, bladder, urethra, penis, uterus, vagina, or vulva.

[0023] The brachytherapy composition may include one or more of mammalian tissue including mammalian placenta, mammalian dermis, mammalian pericardium, mammalian small intestinal submucosa, or any combination thereof. According to a particular embodiment, the brachytherapy composition may include any variety of mammalian placental tissue such as, for example, mammalian placental membrane, mammalian amnion, mammalian chorion, mammalian placental globe, mammalian umbilical cord or a combination thereof. According to one embodiment, the brachytherapy composition includes any variety of porcine birth tissue such as, for example, porcine placental membrane, porcine amnion, porcine chorion, porcine placental globe, porcine umbilical cord or a combination thereof.

[0024] A method of preparing a brachytherapy composition is provided. According to one embodiment, the method steps as provided herein may be applied to any mammalian tissue suitable for a brachytherapy composition. According to one embodiment, the method steps as provided herein may be applied to placental tissue. According to one embodiment, the method includes the step of collecting the mammalian tissue from a mammal. According to a particular embodiment, the method includes the step of collecting the mammalian tissue from a female mammal. According to one embodiment, the method includes the step of collecting the mammalian tissue from a pig. According to a particular embodiment, the method includes the step of collecting the mammalian tissue from a female pig. According to one embodiment, the pig is not genetically modified to halt or reduce expression of the functional alpha- 1 ,3 galactosyltransferase gene. According to a particular embodiment, when placental tissue is collected, the placental membrane may be collected with the umbilical cord attached. Potential mammalian tissue donors are screened and tested to exclude any donors that may present a health risk. According to a particular embodiment, when placental tissue processed, the placental tissue is recovered from a full-term delivery of one or more offspring such as an infant or piglet(s). According to one embodiment, the method further includes the step of placing the mammalian tissue in a transport container. According to one embodiment, the method further includes the step of placing the mammalian tissue in a transport container containing a transport solution.

[0025] According to one embodiment, the method further includes the step of rinsing the mammalian tissue with water. According to a particular embodiment, the water is sterile water. According to a particular embodiment, the water is type 1 water. According to one embodiment, the method further includes the step of removing a substantial portion of any residual moisture present on the mammalian tissue.

[0026] According to one embodiment, the method further includes the step of freezing the mammalian tissue including any umbilical cord that may have been collected. According to one embodiment, the mammalian tissue may be kept frozen until further processing is needed. According to one embodiment, the method further includes the step of removing the frozen, bagged mammalian tissue from the freezer and thawing in a refrigerator for about three (3) to five (5) days. According to one embodiment, the method further includes the step of thawing the mammalian tissue at ambient temperature.

[0027] According to one embodiment, the method includes rinsing the mammalian tissue with water. According to a particular embodiment, the water is sterile water. According to one embodiment, the water is type 1 water. According to one embodiment, the method includes draining the mammalian tissue. According to one embodiment, the method includes the step of opening any tube-shaped mammalian tissue, such as placental tissue, so the mammalian tissue will lie flat onto a cutting surface. According to a particular embodiment, when placental tissue is collected, the method includes separating the placental membrane from the umbilical cord. According to one embodiment, the method includes the step of dividing the mammalian tissue into pieces. According to one embodiment, a rotary cutter or other suitable cutter is used to cut the pieces.

[0028] According to one embodiment, the method optionally includes the step of removing Wharton’s jelly and excess fluids from any collected placental tissue to produce cleaned placental tissue. According to one embodiment, the method includes the step of weighing the cleaned mammalian tissue on a tared balance.

[0029] According to one embodiment, the method includes the step of treating the mammalian tissue with a bioburden reduction solution. According to a one embodiment, the bioburden reduction solution is sodium chloride. According to one embodiment, the method includes the step of adding from about 1 mL to about 40 mL of from about 1 M to about 4M sodium chloride solution per gram of mammalian tissue to the cleaned mammalian tissue. According to one embodiment, the method includes the step of adding from about 5 mL to about 25 mL of 3M sodium chloride solution per gram of mammalian tissue to the cleaned mammalian tissue. According to one embodiment, the method includes the step of adding about 20 mL of 3M sodium chloride solution per gram of mammalian tissue to the cleaned mammalian tissue.

[0030] According to one embodiment, the method includes the step of immersing the mammalian tissue in the sodium chloride solution from about thirty minutes to about two hours. According to a preferred embodiment, the method includes the step of immersing the mammalian tissue in the sodium chloride solution for about one hour. According to one embodiment, the method includes the step of shaking the mammalian tissue in the sodium chloride solution from about thirty minutes to about two hours at about 50 RPM to about 100 RPM. According to a preferred embodiment, the method includes the step of shaking the mammalian tissue in the sodium chloride solution for about one hour at about 50 RPM to about 100 RPM. According to one embodiment, the mammalian tissue is shaken on an orbital shaker table.

[0031] According to one embodiment, the method includes the step of decanting the sodium chloride. According to one embodiment, the method includes the step of rinsing the mammalian tissue with water. According to a particular embodiment, the water is sterile water. According to one embodiment, the water is type 1 water. According to one embodiment, the method includes the step of rinsing the placental membrane with from about 5 mL to about 25 mL of water per gram of mammalian tissue. According to a particular embodiment, the method includes the step of rinsing the mammalian tissue with about 20 mL of water per gram of mammalian tissue. According to one embodiment, the mammalian tissue is rinsed one time. According to one embodiment, the mammalian tissue is rinsed at least two times. According to one embodiment, the mammalian tissue is rinsed at least three times. According to one embodiment, the method includes the step of removing excess fluids from the mammalian tissue.

[0032] According to one embodiment, the method includes the step of placing the mammalian tissue in from about 5 mL to about 25 mL of a detergent solution. According to a particular embodiment, the method includes the step of placing the mammalian tissue in about 20 mL of a detergent solution. According to a particular embodiment, the method includes the step of placing the mammalian tissue in about 20 mL of a detergent solution per gram of mammalian tissue. According to one embodiment, the detergent is present at a concentration of about 0.25% to about 3% w / v. According to one embodiment, the detergent is present at a concentration of about 1% w / v. According to a particular embodiment, the detergent solution includes at least one anionic detergent and at least one protease enzyme. According to one embodiment, the detergent solution includes sodium linear alkylaryl sulfonate, phosphates, carbonates and at least one protease enzyme. According to one embodiment, the detergent solution is commercially available under the trade name Tergazyme™. According to one embodiment, the detergent solution is a 1 % Tergazyme™ solution.

[0033] According to one embodiment, the method includes the step of immersing the mammalian tissue in the detergent solution for from about one hour to about three hours. According to a particular embodiment, the method includes the step of immersing the mammalian tissue in the detergent solution for about two hours. According to one embodiment, the method includes the step of shaking the mammalian tissue in the detergent solution for from about one hour to about three hours at about 50 RPM to about 100 RPM. According to one embodiment, the method includes the step of shaking the mammalian tissue in the detergent solution for about two hours at about 50 RPM to about 100 RPM. According to one embodiment, the mammalian tissue is shaken on an orbital shaker table.

[0034] According to one embodiment, the method includes the step of decanting the detergent solution. According to one embodiment, the method includes the step of rinsing the mammalian tissue with from about 5 mL to about 25 mL of water per gram of mammalian tissue. According to one embodiment, the method includes the step of rinsing the mammalian tissue with about 20 mL of water per gram of mammalian tissue. According to a particular embodiment, the water is sterile water. According to one embodiment, the water is type 1 water. According to one embodiment, the mammalian tissue is rinsed one time. According to one embodiment, the mammalian tissue is rinsed at least two times. According to one embodiment, the mammalian tissue is rinsed at least three times. According to one embodiment, the method includes the step of removing excess fluids from the mammalian tissue.

[0035] According to one embodiment, the method includes the step of treating the mammalian tissue with a viral inactivation solution. According to a one embodiment, the viral inactivation solution is sodium hydroxide. According to one embodiment, the method includes the step of adding or introducing from about 5mL to about 15 mL of about from about 0.1 M to about 1 .0M sodium hydroxide per gram of mammalian tissue. According to one embodiment, the method includes the step of adding or introducing from about 5mL to about 15 mL of 0.25M sodium hydroxide per gram of mammalian tissue. According to one embodiment, the method includes the step of adding or introducing about 10 mL of 0.25M sodium hydroxide per gram of mammalian tissue.

[0036] According to one embodiment, the method includes the step of immersing the mammalian tissue in the sodium hydroxide for about 15 minutes to about 45 minutes. According to one embodiment, the method includes the step of immersing the mammalian tissue in the sodium hydroxide for about 20 minutes. According to one embodiment, the method includes the step of shaking the mammalian tissue in the sodium hydroxide for about 15 minutes to about 45 minutes at about 50 RPM to about 100 RMP. According to one embodiment, the method includes the step of shaking the mammalian tissue in the sodium hydroxide for about 20 minutes at about 50 RPM to about 100 RPM. According to one embodiment, the mammalian tissue is shaken on an orbital shaker table. The sodium hydroxide may then be decanted. According to one embodiment, the steps of adding sodium hydroxide, shaking and decanting may be repeated as many times as necessary to inactivate any viruses present in the mammalian tissue to produce a mammalian tissue that is substantially void of viruses. According to one embodiment, the steps of adding sodium hydroxide, shaking and decanting may be repeated once. According to one embodiment, the steps of adding sodium hydroxide, shaking and decanting may be repeated twice.

[0037] According to one embodiment, the method includes the step of rinsing the mammalian tissue with water. According to a particular embodiment, the water is sterile water. According to one embodiment, the water is type 1 water. According to a particular embodiment, the step of rinsing the mammalian tissue with sterile water is carried out for up to about 10 minutes. According to one embodiment, the method includes the step of removing excess fluids from the mammalian tissue.

[0038] According to one embodiment, the method includes the step of adding or introducing from about 5 mL to about 50 mL of buffer solution per gram of mammalian tissue. According to a particular embodiment, the method includes the step of adding or introducing about 20 mL of buffer solution per gram of mammalian tissue. According to one embodiment, the method includes the step of immersing the mammalian tissue in the buffer solution. According to one embodiment, the method includes the step of shaking the mammalian tissue in the buffer solution for about 5 minutes to about 45 minutes at about 50 RPM to about 100 RPM.

[0039] According to one embodiment, the method includes the step of shaking the mammalian tissue in the buffer solution for about 20 minutes at about 50 RPM to about 100 RPM. According to one embodiment, the mammalian tissue is shaken on an orbital shaker table. The buffer solution may then be decanted.

[0040] According to a one embodiment, the buffer solution is phosphate-buffered saline. According to one embodiment, the method includes the step of measuring the pH of the mammalian tissue after buffer solution treatment. According to one embodiment, the steps of adding buffer solution, shaking and decanting may be repeated until the pH of the mammalian tissue is between about 6.8 and about 7.2.

[0041] According to one embodiment, the method includes the step of rinsing the mammalian tissue with water. According to a particular embodiment, the water is sterile water. According to one embodiment, the water is type 1 water. According to one embodiment, the mammalian tissue is rinsed one time. According to one embodiment, the rinsing step is carried out multiple times. According to a one embodiment, the rinsing step is carried out at least twice. According to a one embodiment, the rinsing step is carried out at least three times. According to one embodiment, the method further includes the step of removing a substantial portion of any residual moisture present in the mammalian tissue.

[0042] According to one embodiment, the method optionally includes the step of dehydrating the mammalian tissue. According to one embodiment, the mammalian tissue may be dehydrated by any method known in the art, including, but not limited to, chemical dehydration (e.g., organic solvents), lyophilization, desiccation, oven dehydration and air drying. According to a preferred embodiment, the method includes the step of adding or introducing an alcohol to the mammalian tissue to cover the entire surface of the mammalian tissue (i.e., submerge the mammalian tissue). According to one embodiment, the method includes the step of adding or introducing from about 1 mL to about 10 mL of alcohol per gram of mammalian tissue. According to one embodiment, the method includes the step of adding or introducing about 5 mL of alcohol per gram of mammalian tissue. According to one embodiment, the mammalian tissue is fully submerged in the alcohol for from about one hour to about 24 hours. According to one embodiment, the mammalian tissue is not agitated while in contact with the alcohol. The alcohol may be any alcohol which is safe and appropriate for contact with mammalian tissue. According to a particular embodiment, the alcohol is ethanol. According to another embodiment, the ethanol is from about 90%-100% ethanol. According to a particular embodiment, the ethanol is 200 proof (i.e., absolute ethanol). According to one embodiment, the method includes the step of decanting or draining the alcohol from the mammalian tissue.

[0043] According to one embodiment, the optional dehydration method may be carried out by spreading the mammalian tissue onto a drying table. According to one embodiment, the mammalian tissue may be blotted with a micro fiber wipe or similar. The mammalian tissue may be spread in a manner so as to fully dehydrate the mammalian tissue while ensuring no wrinkles or bubbles are present.

[0044] According to one embodiment, the optional dehydration method may be carried out by lyophilizing the mammalian tissue. According to a particular embodiment, the method includes the steps of placing the mammalian tissue on a lyophilization tray and spreading the mammalian tissue out evenly. The tray containing the mammalian tissue may then be subject to a lyophilization drying cycle to produce a dehydrated mammalian tissue. The dehydrated mammalian tissue may be stored until further processing is required.

[0045] According to one embodiment, the method includes the step of cutting the mammalian tissue into a plurality of strips. According to one embodiment, the strips of mammalian tissue may be milled into one or more fibers. The fibers may be produced using milling / grinding instruments known in the art, including, but not limited to, oscillating mills, cryogenic mills, dispersers, and homogenizers. According to one embodiment, the milling step is carried out by running the mammalian tissue through an analytical mill at least once. According to one embodiment, the milling step is carried out by running the mammalian tissue through an analytical mill at least twice. According to one embodiment, the analytical mill is equipped with a screen to ensure output is of the desired fiber size. According to one embodiment, the milled mammalian tissue may be passed through a series of sieves to ensure desired fiber size. According to another embodiment, mammalian tissue may be reduced to fibers through hydrolyzation or solubilization followed by lyophilization and milling. According to one embodiment, multiple or serial milling steps / methods and multiple or serial size screening steps / methods may be utilized to render mammalian tissue to fibers within the following categories: (i) nano fibrils: 300 nanometers to 1000 nanometers; (ii) micro fibrils: 1 micrometer to 1000 micrometers; and (iii) macro fibrils: 1 millimeter to 10 millimeters.

[0046] According to one embodiment, the resulting milled mammalian tissue may be placed in a bead press at least once to form a brachytherapy composition. According to one embodiment, the brachytherapy composition as provided herein may be mixed with hyaluronic acid, glycerol, collagen, or a combination thereof. The brachytherapy composition may then be mechanically pressed into small beads with a custom machined press mold. The beads may be of various sizes and shapes depending on where within the body the composition is needed. The bead may be substantially round or substantially cylindrical according to a particular embodiment. According to one embodiment, the resulting bead is desiccated for about 24 hours to about 72 hours at about 0.1 % to about 10% relative humidity. According to a particular embodiment, the resulting bead is desiccated for about 48 hours at about 1% relative humidity.

[0047] According to one embodiment, the step of forming the brachytherapy composition into a bead includes forming the bead around a bioresorbable bead with a custom machined press mold. According to one embodiment, the bioresorbable bead includes at least one bioresorbable polymer. The bioresorbable bead may include one or more compounds that break down or may be absorbed or otherwise disposed of by the human body, including, bioresorbable beads composed of polymers and monomers, e.g., ethane, paraffin, polyethylene, glycogen, propylene, vinyl chloride, tetrafluoroethylene, and monosaccharides. According to a particular embodiment, the bioresorbable bead includes one or more of chitosan and cellulose compounds that eventually break down into molecules that the human body can process. According to one embodiment, the bioresorbable bead includes a radiotherapy source. According to one embodiment, the radiotherapy source is a radionuclide such as, for example, Cesium-131 , Cesium-137, Cobalt-60, lridium-192, lodine-125, Palladium-103, Ruthernium-106, and Radium-226.

[0048] According to one embodiment, the at least one bioresorbable bead as provided herein is a microbead. According to one embodiment, the at least one bioresorbable bead is a nanobead. According to one embodiment, the at least one bioresorbable bead is from about 0.01 micrometer to about 500 micrometers in diameter. According to one embodiment, the at least one bioresorbable bead is from about 0.1 micrometer to about 250 micrometers in diameter. According to one embodiment, the at least one bead is a bioresorbable poly(2- hydroxyethyl methacrylate) nanobead. According to one embodiment, the at least one bead is a recombinant bioresorbable microbead, e.g., a microbead composed of protein disulfideisomerase (P4HB), poly-L -lactic acid (PLLA), or poly(lactic -co- -glycolic acid) (PLGA).

[0049] According to one embodiment, the brachytherapy composition as provided herein may be placed in a proper package. A suitable package includes a vial or a pouch. According to one embodiment, the chosen package may then be placed into and sealed within an outer package.

[0050] According to one embodiment, the method includes the step of terminally sterilizing the packaged brachytherapy composition. According to one embodiment, the method of terminal sterilization may be e-beam irradiation, gamma irradiation, peracetic acid treatment, vaporized peracetic acid (VPA) treatment, any combination thereof, or any other terminal sterilization method known in the art.

[0051] According to one embodiment, any dehydrated porcine placental membranes utilized in the brachytherapy birth tissue composition as provided herein include one or more of collagen I, collagen IV, elastin, laminin, fibronectin and hyaluronic acid. According to one embodiment, each of the one or more of collagen I, collagen IV, elastin, laminin, fibronectin and hyaluronic acid is present in the dehydrated porcine placental membrane in an amount that is different from a porcine placental membrane that is not processed according to one or more of the processing steps provided herein. According to one embodiment, collagen I is present in an amount of from about 0.001% w / w to about 99.9% w / w based on the total weight of the placental membrane. According to one embodiment, collagen IV is present in an amount of from about 0.001 % w / w to about 99.9% w / w based on the total weight of the placental membrane. According to one embodiment, elastin is present in an amount of from about 0.001% w / w to about 99.9% w / w based on the total weight of the placental membrane. According to one embodiment, laminin is present in an amount of from about 0.001% w / w to about 99.9% w / w based on the total weight of the placental membrane. According to one embodiment, fibronectin is present in an amount of from about 0.001% w / w to about 99.9% w / w based on the total weight of the placental membrane. According to one embodiment, hyaluronic acid is present in an amount of from about 0.001% w / w to about 99.9% w / w based on the total weight of the placental membrane.

[0052] A method of treating a tumor is also provided. According to one embodiment, the method includes the step of providing a brachytherapy composition as provided herein. According to one embodiment, the method includes the step of placing the brachytherapy composition in or around a tumor. According to one embodiment, the brachytherapy composition is introduced directly in contact with at least one tumor or introduced in close proximity or around at least one tumor. According to one embodiment, the tumor is benign, malignant, or premalignant. The brachytherapy composition may be guided to a particular location with the assistance of ultrasound, fluoroscopy, x-ray, MRI, or other imaging technique. According to one embodiment, the brachytherapy composition as provided herein may be delivered to a tumor site via one or more afterloading techniques.

[0053] A kit is also provided. The kit includes a brachytherapy composition as provided herein. The kit may also include instructions for use.

[0054] Although specific embodiments of the present invention are herein illustrated and described in detail, the invention is not limited thereto. The above detailed descriptions are provided as exemplary of the present invention and should not be construed as constituting any limitation of the invention. Modifications will be obvious to those skilled in the art, and all modifications that do not depart from the spirit of the invention are intended to be included with the scope of the appended claims.

[0055] EXAMPLE

[0056] Porcine placental membrane was processed according to the methods as provided herein. Particularly, porcine placental membrane was treated with a bioburden solution, treated with a detergent solution, treated with a viral inactivation solution and dehydrated according the methods as provided herein. The resulting dehydrated porcine placental membrane was analyzed to assess the presence of the following extracellular matrix components: collagen I, collagen IV, elastin, laminin, fibronectin, and hyaluronic acid (HA). An immunostaining procedure was carried out utilizing a primary antibody to detect the specific protein in the sample and then a secondary antibody was labeled with a fluorophore to detect any bound primary antibody. Utilizing this methodology enabled improved detection of the primary antibodies since two or more secondary antibodies can detect a single primary antibody and thereby increase the fluorescent signal for imaging.

[0057] The following primary antibodies were used:

[0058] • Rabbit Anti-Collagen I antibody (Cat #ab34710 Abeam, Cambridge, MA, USA)

[0059] • Rabbit Anti-Collagen IV antibody (Cat #ab6586 Abeam, Cambridge, MA, USA)

[0060] • Rabbit Anti-Elastin antibody (Cat #ab21610 Abeam, Cambridge, MA, USA)

[0061] • Rabbit Anti-Laminin antibody (Cat #ab11575 Abeam, Cambridge, MA, USA)

[0062] • Rabbit Anti-Fibronectin antibody (Cat #ab2413 Abeam, Cambridge, MA, USA)

[0063] • Mouse Anti-Hyaluronic Acid antibody (Cat#CAU29210 Biomatik, Wilimgton, DE, USA)

[0064] The following secondary antibodies were used:

[0065] • Anti-Rabbit 488 secondary antibody (provided by the Integrated Microscopy Center, The University of Memphis, Memphis, TN, USA). • Anti-Mouse 594 secondary antibody (provided by the Integrated Microscopy Center, The University of Memphis, Memphis, TN, USA).

[0066] One centimeter diameter samples were cut at random from ten sterile porcine placental membrane samples (approximately 2.5 cm diameter by <0.5mm thick). Triplicate samples were evaluated for each matrix component. Samples were attached to cover slips (approximately 2 cm diameter) using phosphate buffered saline (PBS). The samples were allowed to dry at ambient conditions overnight. Samples were then were soaked in 1% NP-40 (a detergent used to increase permeability of biological specimen for staining procedures) for 5 minutes and then rinsed three times with approximately 1 ml of PBS. The primary antibodies for collagen I, collagen IV, elastin, laminin, fibronectin, and hyaluronic acid (HA) were diluted 1 :20 in PBS. The samples were covered with 50 pl of the primary antibody dilutions and incubated overnight at 4°C. The next day, the samples were rinsed three times with approximately 1 ml PBS to remove unbound primary antibodies. The secondary antibodies were diluted 1 :50 in PBS and the samples were covered with 50 pl of the secondary antibodies. The anti-rabbit 488 secondary antibody was used for the collagen I, collagen IV, elastin, fibronectin, and laminin samples. The anti-mouse 594 secondary antibody was used for the hyaluronic acid (HA) sample. After 1 hour of incubation at 4°C, the samples were again rinsed three times with 1 ml PBS to remove unbound secondary antibodies. The coverslips were mounted to slides (7.5 cm long X 2.5 cm wide) using Slowfade Diamond Antifade Mountant (Fisher Scientific). Slides were examined using a confocal laser scanning microscope (Ti-E A1 rSi System, Nikon Instruments, Inc. Melville, NY, USA) at 20x magnification. Secondary only controls (sections stained only with the secondary antibodies) were used to adjust brightness and intensity to account for any background fluorescence due to non-specific antibody absorption on to samples. Brightness and intensity settings were kept uniform so that images from all groups could be compared. Samples were imaged using optical sectioning. For the optical sectioning, between 35 - 55 slices or planes of focus were collected starting at the surface and moving into the sample at approximately 0.51 pm intervals. The collected images were stacked to create a composite image. There were n=3 images collected for each extracellular matrix component evaluated.

[0067] The sterilized porcine placental membranes showed positive staining for each of collagen I, collagen IV, elastin, laminin, fibronectin, and hyaluronic acid (HA). Collagen I and collagen IV showed the highest intensity of staining, although all antibodies did show positive staining. Based on the immunostaining, the porcine placental membranes processed according to the methods as provided herein contained collagen I, collagen IV, elastin, laminin, fibronectin, and hyaluronic acid (HA).

Claims

CLAIMSWe claim:1 . A brachytherapy composition comprising: dehydrated porcine placental tissue fibers molded into a substantially round or a substantially cylindrical shape; and at least one bioresorbable bead comprising a radiotherapy source, wherein the brachytherapy composition is adapted to be placed in or around a tumor or cancerous tissue of a patient in need of treatment.

2. The brachytherapy composition of claim 1 , wherein the radiotherapy source is a radionuclide selected from the group consisting of Cesium-131 , Cesium-137, Cobalt-60, Iridium- 192, lodine-125, Palladium-103, Ruthernium-106, and Radium-226.

3. The brachytherapy composition of claim 1 , wherein the dehydrated porcine placental tissue fibers comprise collagen I, collagen IV, elastin, laminin, fibronectin, and hyaluronic acid.

4. The brachytherapy composition of claim 1 , wherein the bioresorbable bead further comprises at least one bioresorbable polymer.

5. A method of preparing a brachytherapy composition, comprising the steps of: a) introducing from about 1 mL to about 40 mL of from about 1 M to about 4M sodium chloride solution per gram of mammalian tissue to the mammalian tissue; b) decanting the sodium chloride; c) rinsing the sodium chloride from the mammalian tissue with water; d) introducing from about 5mL to about 25 mL of a detergent solution per gram of mammalian tissue to the mammalian tissue; e) decanting the detergent solution; f) rinsing the detergent solution from the mammalian tissue with water; g) introducing from about 5 mL to about 15 mL of from about 0.1 M to about 1 .0M sodium hydroxide per gram of mammalian tissue to the mammalian tissue; h) rinsing the sodium hydroxide from the mammalian tissue with water;i) introducing from about 5 mL to about 50mL of buffer solution per gram of mammalian tissue to the mammalian tissue; j) decanting the buffer solution; k) measuring the pH of the mammalian tissue; l) repeating steps i), j) and k) until the pH of the mammalian tissue is between about 6.8 and about 7.2; m) rinsing the buffer solution from the mammalian tissue with water; n) milling the mammalian tissue into one or more fibers; o) forming the one or more fibers into at least one bead; and p) desiccating the at least one bead to form a brachytherapy composition.

6. The method of claim 5, wherein the step of forming the one or more fibers into a bead includes forming the bead around a bioresorbable bead.

7. The method of claim 6, wherein the bioresorbable bead comprises at least one bioresorbable polymer.

8. The method of claim 8, further comprising the steps of: q) packaging the brachytherapy composition; and r) terminally sterilizing the packaged composition.

9. The method of claim 8, wherein the terminal sterilization is e-beam irradiation, gamma irradiation, peracetic acid treatment or vaporized peracetic acid (VPA) treatment.

10. The method of claim 5, wherein the detergent solution comprises at least one anionic detergent and at least one protease enzyme.11 . The method of claim 5, wherein the detergent solution comprises sodium linear alkylaryl sulfonate, phosphates, carbonates and at least one protease enzyme.

12. The method of claim 5, wherein the mammal is a pig.

13. The method of claim 12, wherein the pig is not genetically modified to halt or reduce expression of a functional alpha- 1 ,3 galactosyltransferase gene.

14. A porcine brachytherapy composition produced by the method of claim 5.

15. A method of treating a tumor comprising the step of: placing the brachytherapy composition of claim 1 in or next to a tumor or cancerous tissue.

16. The method of claim 15, wherein the tumor is located on or around the cervix, prostate, breast, skin, brain, eye, head, neck, mouth, respiratory tract, digestive tract, or urinary tract.

17. A kit comprising a brachytherapy composition as provided in claim 1 ; and instructions for use.