The present invention relates to compositions and methods of treating a disease, such as diabetes, by implanting encapsulated biological material into a patient in need of treatment. This invention provides for the placement of biocompatible coating materials around biological materials using photopolymerization while maintaining the pre-encapsulation status of the biological materials. Several methods are presented to accomplish coating several different types of biological materials. The coatings can be placed directly onto the surface of the biological materials or onto the surface of other coating materials that hold the biological materials. The components of the polymerization reactions that produce the coatings can include natural and synthetic polymers, macromers, accelerants, cocatalysts, photoinitiators, and radiation. This invention also provides methods of utilizing these encapsulated biological materials to treat different human and animal diseases or disorders by implanting them into several areas in the body including the subcutaneous site. The coating materials can be manipulated to provide different degrees of biocompatibility, protein diffusivity characteristics, strength, and biodegradability to optimize the delivery of biological materials from the encapsulated implant to the host recipient while protecting the encapsulated biological materials from destruction by the host inflammatory and immune protective mechanisms without requiring long-term anti-inflammatory or anti-immune treatment of the host.

Description of the Invention

SUMMARY OF THE INVENTION

In one embodiment, the invention is directed to a composition for cellular therapy, which includes a plurality of encapsulating devices including a polyethylene glycol (PEG) coating, said PEG having a molecular weight between about 900 and about 3,000 Daltons; and a plurality of cells encapsulated in the encapsulating devices, wherein said composition has a cell density of at least about 100,000 cells/ml. In a preferred embodiment, the encapsulating devices are microcapsules. In a more preferred embodiment, the microcapsules are conformally coated cell aggregates. Preferably, the cell aggregates are pancreatic islets with a cell density which is at least about 6,000,000 cells/ml.

In a preferred embodiment, the cell is neurologic, cardiovascular, hepatic, endocrine, skin, hematopoietic, immune, neurosecretory, metabolic, systemic, or genetic. Preferably, the cell is autologous, allogeneic, xenogeneic or genetically-modified. In a most preferred embodiment, the cell is an insulin producing cell.

In another aspect, the invention is directed to a therapeutically effective composition which includes a plurality of encapsulating devices having an average diameter of less than 400 .mu.m, where the encapsulating devices include encapsulated cells in an encapsulation material, and the composition comprises at least about 500,000 cells/ml. In a more preferred embodiment, the average diameter of the encapsulating device is less than 300 micron. In yet a more preferred embodiment, the average diameter of the encapsulating device is less than 200 micron. In yet a more preferred embodiment, the average diameter of the encapsulating device is less than 100 micron. And in yet a more preferred embodiment, the average diameter of the encapsulating device is less than 50 micron.

In yet another embodiment, the invention is directed to a therapeutically effective composition including a plurality of encapsulating devices having an average diameter of less than 400 .mu.m, where the encapsulating devices include encapsulated cells in an encapsulation material, and the composition has a ratio of volume of encapsulating device to volume of cells of less than about 20:1. In a more preferred embodiment, the composition has a ratio of volume of encapsulating device to volume of cells of less than about 10:1. In a yet more preferred embodiment, the composition has a ratio of volume of encapsulating device to volume of cells of less than about 2:1.

In another embodiment, the invention is directed to using a therapeutic composition as described herein in a method which includes the step of implanting the composition into an implantation site in an animal in need of treatment for a disease or disorder.

In a preferred embodiment, the invention is directed to a method of using the therapeutic composition which includes encapsulating devices with a polyethylene glycol (PEG) coating having a molecular weight between 900 and 3,000 Daltons, where the composition has a cell density of at least about 100,000 cells/ml in a method which includes the step of implanting the composition into an implantation site in an animal in need of treatment for a disease or disorder. Preferably, the implanting is an injection.

In preferred embodiments, the disease or disorder is neurologic, cardiovascular, hepatic, endocrine, skin, hematopoietic, immune, neurosecretory, metabolic, systemic, or genetic. In a most preferred embodiment, the disease is an endocrine disease which is diabetes.

In a preferred embodiment, the animal is from an Order of Subclass Theria which is Artiodactyla, Carnivora, Cetacea, Perissodactyla, Primate, Proboscides, or Lagomorpha. Preferably, the animal is a Human.

In a preferred embodiment, the implantation site is subcutaneous, intramuscular, intraorgan, arterial/venous vascularity of an organ, cerebro-spinal fluid, or lymphatic fluid. More preferably, the implantation site is subcutaneous. In a most preferred embodiment, the method includes implanting encapsulated islets in a subcutaneous implantation site.

In a preferred embodiment, the method of implanting the composition into an implantation site in an animal in need of treatment for a disease or disorder also includes the step of administering an immunosuppressant or anti-inflammatory agent. Preferably, the immunosuppressant or anti-inflammatory agent is administered for less than 6 months. More preferably, the immunosuppressant or anti-inflammatory agent is administered for less than 1 month.

In another preferred embodiment, the invention is directed to using a therapeutic composition which includes a plurality of encapsulating devices having an average diameter of less than 400 .mu.m, where the encapsulating devices include encapsulated cells in an encapsulation material and the composition has at least about 500,000 cells/ml, in a method which includes the step of implanting the composition into an implantation site in an animal in need of treatment for a disease or disorder. Preferably, the implantation is an injection.

Preferably, the disease or disorder is neurologic, cardiovascular, hepatic, endocrine, skin, hematopoietic, immune, neurosecretory, metabolic, systemic, or genetic. In a most preferred embodiment, the disease is diabetes.

Preferably, the animal is from an Order of Subclass Theria which is Artiodactyla, Carnivora, Cetacea, Perissodactyla, Primate, Proboscides, or Lagomorpha. More preferably, the animal is a Human.

Preferably, the implantation site is subcutaneous, intramuscular, intraorgan, arterial/venous vascularity of an organ, cerebro-spinal fluid, or lymphatic fluid. More preferably, the implantation site is subcutaneous. In a most preferred embodiment, the method includes implanting encapsulated islets in a subcutaneous implantation site.

In a preferred embodiment, the method of implanting the composition into an implantation site in an animal in need of treatment for a disease or disorder also includes the step of administering an immunosuppressant or anti-inflammatory agent. Preferably, the immunosuppressant or anti-inflammatory agent is administered for less than 6 months. More preferably, the immunosuppressant or anti-inflammatory agent is administered for less than 1 month.

In another embodiment the invention is directed to a method of encapsulating a biological material which includes the steps of: adding a solution which includes a first buffer to the biological material; centrifuging the biological material to form a pelleted biological material; removing supernatant; adding a solution which includes a photoinitiator dye conjugated to a cell adsorbing material to the pelleted biological material; resuspending and incubating the pelleted biological material with the solution including the photoinitiator dye conjugated to the cell adsorbing material for an effective amount of time; centrifugating mixture; removing the solution including the photoinitiator dye conjugated to the cell adsorbing material; resuspending the pelleted biological material with a second solution including a second buffer; centrifugating and removing the second buffer; resuspending and mixing the biological material with a photoactive polymer solution; and irradiating the resuspended biological material with a photoactive polymer solution with an energy source to form an encapsulated biological material. Preferably, the encapsulated biological material is a PEG conformal coated islet allograft.

Preferably, the cell adsorbing material is a polycationic polymer. In a preferred embodiment, the polycationic polymer is a PAMAM Dendrimer. In an alternate preferred embodiment, the polycationic polymer is poly(ethyleneimine).

Preferably, the biological material is an organ, tissue or cell. More preferably, the tissue is a cluster of insulin producing cells. More preferably, the cell is an insulin producing cell.

In a preferred embodiment, the first and second buffer is 1 to 200 mM. More preferably, the first and second buffer is 10 to 50 mM. More preferably, the first and second buffer is 20 mM.

In a preferred embodiment, the photoinitiator is carboxyeosin, ethyl eosin, eosin Y, fluorescein, 2,2-dimethoxy, 2-phenylacetophenone, 2-methoxy, 2-phenylacetophenono, camphorquinone, rose bengal, methylene blue, erythrosin, phloxine, thoionine, riboflavin or methylene green. More preferably, the photoinitiator is carboxyeosin.

In a preferred embodiment, the photoactive polymer solution includes a polymerizable high density ethylenically unsaturated PEG and a sulfonated comonomer. In a more preferred embodiment, the polymerizable high density ethylenically unsaturated PEG is a high density acrylated PEG. Preferably, the polymerizable high density acrylated PEG has a molecular weight of 1.1 kD.

In a preferred embodiment, the sulfonated comonomer is 2-acrylamido-2-methyl-1-propanesulfonic acid, vinylsulfonic acid, 4-styrenesulfonic acid, 3-sulfopropyl acrylate, 3-sulfopropyl methacrylate, or n-vinyl maleimide sulfonate. In a more preferred embodiment, the sulfonated comonomer is 2-acrylamido-2-methyl-1-propanesulfonic acid.

In a preferred embodiment, the photoactive polymer solution also includes a cocatalyst which is triethanolamine, triethylamine, ethanolamine, N-methyl diethanolamine, N,N-dimethyl benzylamine, dibenzyl amino, N-benzyl ethanolamine, N-isopropyl benzylamine, tetramethyl ethylenediamine, potassium persulfate, tetramethyl ethylenediamine, lysine, omithine, histidine or arginine. More preferably, the cocatalyst is triethanolamine.

In a preferred embodiment, the photoactive polymer solution also includes an accelerator which is N-vinyl pyrrolidinone, 2-vinyl pyridine, 1-vinyl imidazole, 9-vinyl carbazone, 9-vinyl carbozol, acrylic acid, n-vinylcarpolactam, 2-allyl-2-methyl-1,3-cyclopentane dione, or 2-hydroxyethyl acrylate. More preferably, the accelerator is N-vinyl pyrrolidinone.

In a preferred embodiment, the photoactive polymer solution also includes a viscosity enhancer which is selected from the group including natural and synthetic polymers. In a more preferred embodiment, the viscosity enhancer is 3.5 kD PEG-triol or 4 kD PEG-diol.

In a preferred embodiment, the photoactive polymer solution also includes a density adjusting agent. More preferably, the density adjusting agent is Nycodenz or Ficoll.

In a preferred embodiment, the photoactive polymer solution also includes a "Good" buffer. In a more preferred embodiment, the "Good" buffer is HEPES or MOPS. In a most preferred embodiment, the "Good" buffer is MOPS.

In a preferred embodiment, the energy source is an Argon laser.

In a preferred embodiment, the biological material for the encapsulation method is neurologic, cardiovascular, hepatic, endocrine, skin, hematopoietic, immune, neurosecretory, metabolic, systemic, or genetic.

In a preferred embodiment, the biological material is from an animal of Subclass Theria of Class Mammalia. In a more preferred embodiment, the animal is from an Order of Subclass Theria which is Artiodactyla, Carnivora, Cetacea, Perissodactyla, Primate, Proboscides, or Lagomorpha. In a most preferred embodiment, the animal is a Human.

In another embodiment, the invention is directed to a composition for encapsulating biological material which includes a polymerizable high density ethylenically unsaturated PEG having a molecular weight between 900 and 3,000 Daltons, and a sulfonated comonomer. In a preferred embodiment, the polymerizable high density ethylenically unsaturated PEG is a polymerizable high density acrylated PEG. In a more preferred embodiment, the polymerizable high density acrylated PEG has a molecular weight of 1.1 kD.

In a preferred embodiment, the sulfonated comonomer is 2-acrylamido-2-methyl-1-propanesulfonic acid, vinylsulfonic acid, 4-styrenesulfonic acid, 3-sulfopropyl acrylate, 3-sulfopropyl methacrylate, or n-vinyl maleimide sulfonate. In a most preferred embodiment, the sulfonated comonomer is 2-acrylamido-2-methyl-1-propanesulfonic acid.

In a preferred embodiment, the composition for encapsulating biological material further includes a cocatalyst which is triethanolamine, triethylamine, ethanolamine, N-methyl diethanolamine, N,N-dimethyl benzylamine, dibenzyl amino, N-benzyl ethanolamine, N-isopropyl benzylamine, tetramethyl ethylenediamine, potassium persulfate, tetramethyl ethylenediamine, lysine, omithine, histidine or arginine. In a more preferred embodiment, the cocatalyst is triethanolamine.

In a preferred embodiment, the composition for encapsulating biological material further includes an accelerator which is N-vinyl pyrrolidinone, 2-vinyl pyridine, 1-vinyl imidazole, 9-vinyl carbazone, 9-vinyl carbozol, acrylic acid, n-vinylcarpolactam, 2-allyl-2-methyl-1,3-cyclopentane dione, or 2-hydroxyethyl acrylate. In a more preferred embodiment, the accelerator is N-vinyl pyrrolidinone.

In a preferred embodiment, the composition for encapsulating biological material is biocompatible with a score of at least about a "2". More preferably, the composition is biocompatible with a score of at least about a "2" in a mammal, even more preferably in a sub-human primate and most preferably in a human.

In a preferred embodiment, the composition for encapsulating biological material has the quality of permselectivity. More preferably, the permselectivity can be engineered by manipulating the composition.

In a preferred embodiment, the composition for encapsulating biological material has an allowance of cell functionality with a score of at least about a "2". In a more preferred embodiment, the allowance of cell functionality with a score of at least about a "2" is in a mammal, even more preferably in a sub-human primate and most preferably in a human.

In a preferred embodiment, the composition for encapsulating biological material further is biodegradable. More preferably, the composition is biodegradable in a mammal. Even more preferably, the composition is biodegradable in a sub-human primate. In a most preferred embodiment, the composition is biodegradable in a human.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

One preferred embodiment of the invention is related to compositions and methods of treating one or more diseases or disorders, such as neurologic (e.g., Parkinson's disease, Alzheimer's disease, Huntington's disease, Multiple Sclerosis, blindness, peripheral nerve injury, spinal cord injury, pain and addiction), cardiovascular (e.g., coronary artery, angiogenesis grafts, valves and small vessels), hepatic (e.g., acute liver failure, chronic liver failure, and genetic diseases effecting the liver), endocrine (e.g., diabetes, obesity, stress and adrenal, parathyroid, testicular and ovarian diseases), skin (e.g., chronic ulcers and diseases of the dermal and hair stem cells), hematopoietic (e.g., Factor VIII and erythropoietin), or immune (e.g., immune intolerance or auto-immune disease), in a subject in need of treatment comprising: providing cells or tissue, such as pancreatic islets, hepatic tissue, endocrine tissues, skin cells, hematopoietic cells, bone marrow stem cells, renal tissues, muscle cells, neural cells, stem cells, embryonic stem cells, or organ specific progenitor cells, or genetically engineered cells to produce specific factors, or cells or tissue derived from such; enclosing said cells or tissue within at least one encapsulating material, such as a hydrogel, made of physically or chemically crosslinkable polymers, including polysaccharides such as alginate, agarose, chitosan, poly(amino acids), hyaluronic acid, chondroitin sulfate, dextran, dextran sulfate, heparin, heparin sulfate, heparan sulfate, gellan gum, xanthan gum, guar gum, water soluble cellulose derivatives, carrageenan, or proteins, such as gelatin, collagen, albumin, or water soluble synthetic polymers with ethylenically unsaturated groups or their derivatives, such as poly(methyl methacrylate) (PMMA), or poly(2-hydroxyethyl methacrylate) (PHEMA), poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(thyloxazoline) (PEOX); or a combination of the above, such as alginate mixed with PEG, or more hydrophobic or water insoluble polymers, such as poly(glycolic acid) (PGA), poly(lactic acid) (PLA), or their copolymers (PLA-GA), or polytetrafluoroethylene (PTFE) and administering a therapeutically effective amount of said encapsulated cells or tissue to the subject in need of treatment via subcutaneous injection or implant, or directly into organs via either direct injection into the substance of the organ or injection through the vascular system of those organs.

Organs maybe selected from, but not limited to, liver, spleen, kidney, lung, heart, brain, spinal cord, muscle, and bone marrow. The subject in need of treatment may be selected from, but not limited to, mammals, such as humans, sub-human primates, cows, sheep, horses, swine, dogs, cats, and rabbits as well as other animals such as chickens, turkeys, or fish.

In a further embodiment of the invention, the encapsulated cell or tissue may be administered to a subject in need of treatment in combination with an immunosuppressant and/or an anti-inflammatory agent. The immunosuppressant may be selected from, but not limited to cyclosporine, sirolimus, rapamycin, or tacrolimus. The anti-inflammatory agent may be selected from, but not limited to, aspirin, ibuprofen, steroids, and non-steroidal anti-inflammatory agents. Preferably, the immunosuppressant and/or an anti-inflammatory agent is administered for six months following implantation or injection of the encapsulated cells or tissue. More preferably the immunosuppressant and/or an anti-inflammatory agent is administered for one month following implantation or injection of the encapsulated cells or tissue

In a preferred embodiment, encapsulated islets are implanted or injected subcutaneously or into liver or spleen. In one aspect of the invention, conformally coated islets are administered subcutaneously.

Preferably, the encapsulated material comprises acrylated PEG and at least one comonomer, such as N-vinyl pyrrolidinone, 2-vinyl pyridine, 1-vinyl imidazole, 9-vinyl carbazone, acrylic acid, and 2-allyl-2-methyl-1,3-cyclopentane dione, 2-acrylamido-2-methyl-1-propanesulfonic acid, vinylsulfonic acid, 3-sulfopropyl acrylate, 3-sulfopropyl methacrylate, and 2-acrylamido-2-methyl-1-propanesulfonic acid, plus N-vinyl pyrrolidinone. Most preferably the encapsulating material comprises acrylated PEG with 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) and/or N-vinyl pyrrolidinone (NVP) as a comonomer, to form encapsulated cells or tissue that are conformally coated by a process such as an interfacial photopolymerization process.

In some embodiments of the invention, the concentration of ingredients and composition of encapsulating solution may vary. Preferred concentration ranges are as follows.

For Buffer solution a preferred concentration is 1 to 200 mM, yet more preferred is 5 to 100 mM, and yet more preferred is 10 to 50 mM.

For CaCl.sub.2 a preferred concentration is 0.1 to 40 mM, yet more preferred is 0.5 to 20 mM, and yet more preferred is 1 to 5 mM.

For Manitol a preferred concentration is 10 mM to 6M, yet more preferred is 50 mM to 3M, yet more preferred is 100 mM to 1M, and yet more preferred is 200 to 300 mM.

For pH of CaCl.sub.2/Manitol solution a preferred value is 6 to 8, yet more preferred is 6.4 to 7.6, and yet more preferred is 6.6 to 7.4.

For DEN-EY a preferred concentration is 0.005 to 8 mg/ml, yet more preferred is 0.01 to 4 mg/ml, and yet more preferred is 0.05 to 2 mg/ml.

For DEN-EY conjunction level a preferred level is 0.15 to 68, yet more preferred is 1 to 34, and yet more preferred is 1.5 to 15.

For pH of macromer solution a preferred value is 6.5 to 9.5, yet more preferred is 7 to 9, and yet more preferred is 7.5 to 8.5.

For PEG TA a preferred concentration is 0.1 to 100%, yet more preferred is 0.2 to 50%, and yet more preferred is 1 to 25%.

For PEG TA a preferred density is 0.05 to 20 K, yet more preferred is 0.1 to 10 K, yet more preferred is 0.5 to 5 K, and yet more preferred is 0.8 to 2.5 K.

For PEG-triol a preferred concentration is 0.1 to 100%, yet more preferred is 1 to 75%, and yet more preferred is 2 to 50%.

For PEG-triol a preferred density is 0.15 to 70 K, yet more preferred is 0.3 to 35 K, yet more preferred is 1.5 to 15 K, and yet more preferred is 2.3 to 7.5 K.

For PEG-diol a preferred concentration is 0.1 to 100% yet more preferred is 1 to 75%, and yet more preferred is 2 to 50%.

For PEG-diol a preferred density is 0.2 to 80 K, yet more preferred is 0.5 to 40 K, yet more preferred is 1 to 20 K, and yet more preferred is 2 to 10 K.

For TEoA a preferred concentration is 5 mM to 2 M, yet more preferred is 10 mM to 1M, yet more preferred is 50 to 500 mM, and yet more preferred is 75 to 125 mM.

For AMPS a preferred concentration is 2 to 640 mg/ml, yet more preferred is 5 to 300 mg/ml, and yet more preferred is 10 to 150 mg/ml.

For NVP a preferred concentration is 0.01 to 40 .mu.l/ml, yet more preferred is 0.1 to 20 .mu.l/ml, and yet more preferred is 0.5 to 10 .mu.l/ml.

For Nycodenz a preferred concentration is 0.1 to 100%, yet more preferred is 1 to 50%, and yet more preferred is 5 to 25%.

For the Laser a preferred strength is 10 mW/cm.sup.2 to 4 W/cm.sup.2, yet more preferred is 25 mW/cm.sup.2 to 2 W/cm.sup.2, and yet more preferred is 75 mW/cm.sup.2 to 1 W/cm.sup.2.

For the light source a preferred time is 3 seconds to 20 minutes, yet more preferred is 6 seconds to 10 minutes, and yet more preferred is 12 seconds to 3 minutes.

In an embodiment, the encapsulating material comprises a hydrogel that forms a sphere around at least one cell or tissue. In a further embodiment, the encapsulating material is an alginate microcapsule, which is conformally coated with another encapsulating material comprising acrylated PEG. In one embodiment, a cell or tissue may be encapsulated in a biocompatible alginate microcapsule, wherein the alginate is made biocompatible by coating the alginate in a biocompatible material, such as PEG or hyaluronic acid, purifying the alginate and/or removing the poly-lysine and replacing it with PEG.

Most preferably the disease to be treated is diabetes, the cells or tissue comprise insulin producing cells or tissue, or cells or tissue derived from pancreatic cells or tissue, or cells derived from progenitor or stem cells that are converted into insulin producing cells, and the encapsulated cells or tissue are administered to the subject in need of treatment via subcutaneous or liver injection or implant.

According to an embodiment of the invention the microcapsules of encapsulated insulin-producing cells or tissue may have an average diameter of 10 .mu.m to 1000 .mu.m, preferably 100 .mu.m to 600 .mu.m, more preferably 150 .mu.m to 500 .mu.m, and most preferably 200 .mu.m to 300 .mu.m. In another embodiment, the invention relates to an insulin-producing cell or tissue encapsulated in microcapsules having a concentration of at least 2,000 IEQ (islet equivalents)/ml, preferably at least 9,000 IEQ/ml, and more preferably at least 200,000 IEQ/ml. In another embodiment of the invention, the volume of insulin-producing cells or tissue encapsulated in microcapsules administered per kilogram body mass of a subject may be 0.001 ml to 10 ml, preferably 0.01 ml to 7 ml, more preferably 0.05 ml to 2 ml. In a further embodiment of the invention, the ratio of microcapsule volume to insulin producing cell or tissue volume is less than 300 to 1, preferably less than 100 to 1, more preferably less than 50 to 1, and most preferably less than 20 to 1.

According to an embodiment of the invention, conformally coated insulin-producing cells or tissue may have an average membrane thickness of 1 to 400 .mu.m, preferably 10 to 200 .mu.m, and more preferably 10 to 100 .mu.m. In a further embodiment the invention relates to a conformally coated insulin-producing cell or tissue having a concentration of at least 10,000 IEQ/ml, preferably at least 70,000 IEQ/ml, more preferably at least 125,000 IEQ/ml, and most preferably at least 200,000 IEQ/ml. According to an embodiment of the invention the volume of the conformally coated insulin producing cell or tissue administered per kilogram body mass of a subject may be 0.01 to 7 ml, preferably 0.01 to 2 ml, and more preferably 0.04 to 0.5 ml. In another embodiment of the invention the ratio of conformal coating volume to insulin-producing cell or tissue volume is less than 13 to 1, preferably less than 8 to 1, more preferably less than 5 to 1, and most preferably less than 2.5 to 1.

According to an embodiment of the invention the microcapsules of encapsulated cells or tissue may have an average diameter of 10 .mu.m to 1000 .mu.m, preferably 100 .mu.m to 600 .mu.m, more preferably 150 .mu.m to 500 .mu.m, and most preferably 200 .mu.m to 300 .mu.m. In a further embodiment of the invention, the ratio of microcapsule volume to insulin producing cell or tissue volume is less than 300 to 1, preferably less than 100 to 1, more preferably less than 50 to 1, and most preferably less than 20 to 1.

According to an embodiment of the invention, conformally coated cells or tissue may have an average membrane thickness of 1 to 400 .mu.m, preferably 10 to 200 .mu.m, and more preferably 10 to 100 .mu.m. In another embodiment of the invention the ratio of conformal coating volume to cell or tissue volume is less than 13 to 1, preferably less than 8 to 1, more preferably less than 5 to 1, and most preferably less than 2.5 to 1.

An embodiment of the invention relates encapsulated cells or tissue where the cell density is at least about 100,000 cells/ml. Preferably, the encapsulated cell is conformally coated. More preferably, the cell is conformally coated with an encapsulating material comprising acrylated PEG. In a further embodiment, the invention is related to a method of treating diabetes in a subject comprising administering encapsulated islets where the cell density is at least about 6,000,000 cells/ml, preferably where the curative dose is less than about 2 ml per kilogram body mass of the subject.

Another embodiment of the invention is related to agricultural animals or pets, such as cows, sheep, horses, swine, chickens, turkeys, rabbits, fish, or dogs and cats; to change the growth rate, or alter the condition of the animal (e.g., increase meat or dairy production), or protect them from or treat them for different diseases. According to this embodiment, a method of providing cells or tissue to an agriculturally relevant animal comprises: a) providing a cell or tissue; b) enclosing said cell or tissue within at least one encapsulating material, such as a hydrogel, made of physically or chemically crosslinkable polymers, including polysaccharides such as alginate, agarose, chitosan, poly(amino acids), hyaluronic acid, chondroitin sulfate, dextran, dextran sulfate, heparin, heparin sulfate, heparan sulfate, gellan gum, xanthan gum, guar gum, water soluble cellulose derivatives, carrageenan, or proteins, such as gelatin, collagen, albumin, or water soluble synthetic polymers or their derivatives, such as methyl methacrylate (MMA), or 2-hydroxyethyl methacrylate (HEMA), polyethylene glycol (PEG), poly(ethylene oxide) (PEO), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(thyloxazoline) (PEOX); or a combination of the above, such as alginate mixed with PEG, or more hydrophobic or water insoluble polymers, such as poly(glycolic acid) (PGA), poly(lactic acid) (PLA), or their copolymers (PLA-GA), or polytetrafluoroethylene (PTFE); and c) administering said encapsulated cell or tissue to the subject in need of treatment via subcutaneous injection or implant, or directly into organs via either direct injection into the substance of the organ or injection through the vascular system of those organs. Definitions:

DETAILED DESCRIPTION

The present invention relates to methods of treating a disease or disorder by implanting encapsulated biological material into patients in need of treatment. Diabetes is of particular interest because a method is needed to prevent complications related to the lack of good glycemic control in insulin-requiring diabetics. Specifically, PEG conformally coated islet allografts in diabetic primates are shown herein to be successfully implanted in the subcutaneous site by injection and achieve relatively normal blood glucose values out to 220 days post-implant. The current complications of clinical islet transplantation and the significant risks and discomfort of continuous immunosuppression may be eliminated by applying the methods described herein to patients with insulin-requiring diabetes. In addition, encapsulated islet implants are expected to protect these insulin-requiring diabetic patients and prevent them from developing the complications from diabetes related to inadequate glycemic control in spite of exogenous insulin therapy.

Methods according to the present invention may provide therapeutic effects for a variety of diseases and disorders, in addition to diabetes, in which critical cell-based products lost by disease or disorder may be replaced through implantation of cells or tissue into the body. A preferred embodiment of the invention is the use of human insulin-producing cells from the pancreas, or cells derived from human insulin-producing cells from the pancreas, that are encapsulated as cell clusters for implantation into the subcutaneous site of insulin-requiring patients. Treatment of disease via encapsulated biological materials requires that the encapsulated material be coated with a biocompatible coating, such that the immune system of the patient being treated does not destroy the material before a therapeutic effect can be realized.

Permselectivity of the coating is a factor in the effectiveness of such treatments, because this regulates the availability of nutrients to the cells or tissue, and plays a role in preventing rejection of the biological materials. Permselectivity of the coating affects the nutrition available to the encapsulated cell or tissue, as well as the function of the cell or tissue. Permselectivity can be controlled by varying the components of the biocompatible coating or by varying how the components are used to make the cell coating. Treatment via injection of encapsulated biological materials according to the present invention provides a stable and safe method of treatment. Size of the implant and the site of implantation, as well as replenishment and/or replacement of the encapsulated materials is also a consideration of the methods described herein. These methods provide a treatment that has a wide range of applications in the treatment of disease at various sites of implantation, while avoiding complications associated with other treatment methods.

The conformal coatings described herein can be produced with different pore sizes that can be produced to limit access to the cells by proteins of widely varying molecular weights, including the exclusion of antibodies. This control allows for survival and maintained function of the encapsulated materials, while excluding components of the host immune system. The appropriate pore size of the conformal coating may be determined by routine experimentation for each cell or tissue type and the disease or disorder to be treated. The conformal coatings described herein provide a small encapsulated cell product with a minimal volume of the coating material, thus allowing the coated materials to be implanted into various sites of the body, including direct injection into the liver, spleen, muscle, or other organs, injection via vascular access to any organ, injection into the abdominal cavity, and implantation into a subcutaneous site.

An important factor for successful encapsulated cell therapy is that the permselective coating used to encapsulate the cells be inert in terms of causing inflammatory reactions in the host. Most previous encapsulating materials were not completely biocompatible. With some devices, not making a large scar is sufficient. However, when using the coating for permselective protection between the encapsulated cells and the host immune system, there cannot be any non-specific inflammatory reaction to the host's complement system or to macrophages. If this occurs, then the inflammatory and/or immune reaction is sufficient to release cytokines that readily cross the membrane and can cause the loss of the encapsulated cells. Most encapsulation technologies for islets, which have had difficulties in working appropriately, had non-specific inflammatory reactions due to biocompatibility reactions to the coating materials.

Problems such as chronic inflammation are significantly reduced due to the lack of host reaction to the biocompatible conformal coatings used to encapsulate cells and tissues used in the methods described herein. The components used to produce the conformal coating described herein have been shown to be completely biocompatible when injected into animals, such as, rodents, dogs, pigs, and primates.

We discovered that biocompatibility of hydrogels synthesized from highly acrylated PEG was exceptionally good, and much better than that shown with moderately acrylated PEG hydrogels. The highly acrylated PEGs were either obtained commercially, or home-made by acrylating corresponding PEGs. Hydrogels with highly acrylated PEGs were conformally coated on the surface of alginate microbeads using an interfacial photopolymerization technology. This discovery also can be extended to other biomedical, biotechnological and pharmaceutical areas where biocompatibility of the devices or formulations is of concern.

Some PEG conformal coatings described herein are biodegradable over time, thus allowing the body to safely break down the materials over the course of time and avoiding the need to retrieve the encapsulated materials, which is required by other treatments. Replacement of cells can be done whenever the previous dose of encapsulated materials has begun to lose function. Encapsulated islets may be expected to last two to five years or longer. In the case of subcutaneous injections, replacement of the encapsulated materials may simply be done via another percutaneous injection of new materials into the patient at a different site prior to loss of the previous dose. In the case of encapsulated islets, this replacement can be done prior to loss of function in the first dose of islets, without fear of low glucose values, because the encapsulated islets autoregulate themselves to prevent hypoglycemia. Different implant timing may have to be determined for treating diseases and disorders using cells or tissues that do not autoregulate the release of their product.

A factor in producing encapsulated cell products is the cell source. Cells may be primary cells, expanded cells, differentiated cells, cell lines, or genetically engineered cells. In the case of human islets, primary islets may be isolated from cadaver-donated pancreases; however, the number of human pancreata available for isolating islets is very limited. Alternative cell sources may be used to provide cells for encapsulation and injection.

One alternative source of cells, particularly insulin-producing cells, is embryonic stem cells. Human embryonic stem cells come from the very early fetus. They are only available when grown from frozen, fertilized human eggs collected from couples that have successfully undergone in vitro fertilization and no longer want to keep these fertilized eggs for future children. Embryonic stem cells have the ability to grow indefinitely, potentially avoiding the need for the mass of tissues required for transplantation. There are a series of steps required to differentiate these embryonic stem cells into insulin producing cells with clinical relevancy. A few studies have shown both mouse and human embryonic stem cells can produce insulin when treated under tissue culture with a variety of factors. Insulin-producing cells developed from embryonic stem cells may be an acceptable cell source for transplantation, and encapsulated cell or tissue implantation.

Cell Sourcing

Additional cell sources, organ specific progenitor cells from the brain, liver, and the intestine, have been shown to produce insulin. In order to produce insulin, each of these organ specific progenitor cells have undergone tissue culture treatments with a variety of growth and differentiation factors. Additional organ specific progenitor cells from many other organs such as bone marrow, kidney, spleen, muscle, bone, cartilage, blood vessels, and other endocrine organs may also be useful in providing insulin producing cells.

Pancreatic progenitor cells may be used according to the methods of the invention. The pancreas seems to have organ specific stem cells that can produce the three pancreatic cell types in the body under normal and repair conditions. It is believed the islet cells bud off from the duct cells to form the discrete islets. The insulin producing beta cells, as well as the other hormone producing cells, may form directly from differentiating duct cells or may form from pancreatic progenitor cells located amongst the duct cells. These pancreatic progenitor cells may be used to provide insulin-producing cells for encapsulation and implantation according to the methods described herein.

There has been a great deal of research on genetically inserting genes into non-insulin producing cells to make them produce insulin. Genetically engineered cells capable of insulin production may also be used for encapsulation and implantation according to the methods described herein.

The use of pig cells has commonly been considered as a source of islet cells for implantation in patients with diabetes. Over 90 million pigs are raised per year for meat production in the USA alone. Therefore, the number of islets to treat the millions of patients with insulin-requiring diabetes are readily available through large scale processing of adult pig pancreata into purified pig islets for encapsulation. One consideration limiting this choice is the recognition that pigs harbor an endogenous retrovirus (PoERV). There have been efforts to eliminate PoERV from strains of pigs. Virus-free pig xenograft islets may be readily encapsulated and available as a preferred cell source for the treatment of human diabetes.

Alternative xenograft sources for human implantation may be obtained from primary cells of species other than pigs. These other species could be agriculturally relevant animals such as beef, sheep, and even fish. With the ability to expand and differentiate insulin producing cells from pancreatic sources or other stem or progenitor cells, one can envision using insulin-producing cells from many other xenogeneic sources such as primates, rodents, rabbits, fish, marsupials, ungulates and others.

Disease Treatment

Diabetes and other diseases in which a local or circulating factor is deficient or absent can be treated according to the methods described herein. Encapsulated cell therapy may be applied in the treatment of neurologic, cardiovascular, hepatic, endocrine, skin, hematopoietic, and immune disorders and diseases. Neurologic diseases and injuries, such as Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple sclerosis, blindness, spinal cord injury, peripheral nerve injury, pain and addiction may be treated by encapsulating cells that are capable of releasing local and/or circulating factors needed to treat these problems. Cardiovascular tissue, such as the coronary artery, as well as angiogenic growth factor releasing cells used for restoring vascular supply to ischemic cardiac muscle, valves and small vessels may be treated. Acute liver failure, chronic live failure, and genetic diseases affecting the liver may be treated. Endocrine disorders and diseases, such as diabetes, obesity, stress and adrenal, parathyroid, testicular and ovarian diseases may be treated. Skin problems, such as chronic ulcers, and diseases of the dermal and hair stem cells can be treated. Hematopoietic factors such as Factor VIII and erythropoietin may be regulated or controlled by administering cells capable of stimulating a hematopoietic response in a patient. Encapsulated biological materials may also be useful in the production of bone marrow stem cells. Encapsulated materials, such as, antigens from primary cells or genetically engineered cells, may be useful in producing immune tolerance or preventing autoimmune disease. In addition, these materials may be used in vaccines.

Conformal Coating Components

Components of the coatings may be altered depending on the specific cell type and permselectivity desired. Various polymerizable monomers or macromers, photoinitiating dyes, cocatalysts, and accelerants may be used to produce conformally coated cells and tissues.

Monomers or Macromers

Monomers or macromers are used as the building blocks to polymerize biocompatible coatings for use in methods disclosed herein. The monomers are small polymers, which are susceptible to polymerization into the larger polymer membranes of this invention. Polymerization is enabled because the macromers contain carbon-carbon double bond moieties, such as, acrylate, methacrylate, ethacrylate, 2-phenyl acrylate, 2-chloro acrylate, 2-bromo acrylate, itaconate, acrylamide, methacrylamide, and styrene groups. The monomers or macromers are non-toxic to biological material before and after polymerization.

Examples of monomers are methyl methacrylate (MMA) and 2-hydroxyethyl methacrylate (HEMA). Examples of macromers are ethylenically unsaturated derivatives of poly(ethylene oxide) (PEO), poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(thyloxazoline) (PEOX), poly(amino acids), polysaccharides such as alginate, hyaluronic acid, chondroitin sulfate, dextran, dextran sulfate, heparin, heparin sulfate, heparan sulfate, chitosan, gellan gum, xanthan gum, guar gum, water soluble cellulose derivatives and carrageenan, and proteins such as gelatin, collagen and albumin. These macromers can vary in molecular weight and number of branches, depending on the use. For purposes of encapsulating cells and tissue in a manner that has minimum tissue response, the preferred starting macromer is PEG--triacrylate with MW 1.1K. The molecular weight designation is an average molecular weight of the mixed length polymer.

Photoinitiating Dyes

The photoinitiating dyes capture light energy and initiate polymerization of the macromers and monomers. Any dye can be used which absorbs light having frequency between 320 nm and 900 nm, can form free radicals, is at least partially water soluble, and is non-toxic to the biological material at the concentration used for polymerization. Examples of suitable dyes are ethyl eosin, eosin Y, fluorescein, 2,2-dimethoxy, 2-phenylacetophenone, 2-methoxy, 2-phenylacetophenono, camphorquinone, rose bengal, methylene blue, erythrosin, phloxine, thionine, riboflavin and methylene green. To enhance the dye-cell surface binding, the dyes used here are conjugated to polymers that have strong interactions with cell surfaces, such as polycationic polymers, polymers with multiple phenylboronic acid groups attached. Examples of polycationic polymers include PAMAM dendrimer, linear, branched or dendritic poly (ethyleneimine) (PEI), polyvinylamine, polyallylamine, polylysine, chitosan, and polyhistidine. The preferred initiator dye is the carboxyeosin conjugated to PAMAM Dendrimer Generation 4.

Cocatalyst or Radical Generator

The cocatalyst is a nitrogen-based compound capable of stimulating the free radical reaction. Primary, secondary, tertiary or quaternary amines are suitable cocatalysts, as are any nitrogen atom containing electron-rich molecules. Cocatalysts include, but are not limited to, triethanolamine, triethylamine, ethanolamine, N-methyl diethanolamine, N,N-dimethyl benzylamine, dibenzyl amino, N-benzyl ethanolamine, N-isopropyl benzylamine, tetramethyl ethylenediamine, potassium persulfate, tetramethyl ethylenediamine, lysine, omithine, histidine and arginine. A preferred cocatalyst is triethanolamine.

Accelerator or Co-Monomer

The accelerator, which is optionally included in the polymerization mixture, is a small molecule containing an allyl, vinyl, or acrylate group, and is capable of speeding up the free radical reaction. Incorporating a sulfonic acid group to the accelerant also can improve the biocompatibility of the final product. Accelerators include, but are not limited to, N-vinyl pyrrolidinone, 2-vinyl pyridine, 1-vinyl imidazole, 9-vinyl carbazone, 9-vinyl carbozol, acrylic acid, 2-allyl-2-methyl-1,3-cyclopentane dione, 2-hydroxyethyl acrylate, 2-acrylamido-2-methyl-1-propanesulfonic acid, vinylsulfonic acid, 4-styrenesulfonic acid, 3-sulfopropyl acrylate, 3-sulfopropyl methacrylate, n-vinylcarpolactam, and n-vinyl maleimide sulfonate (from SurModics), with 2-acrylamido-2-methyl-1-propanesulfonic acid plus N-vinyl pyrrolidinone being the preferred combination of accelerators.

Viscosity Enhancer

To generate conformal coating without long tails on cell aggregates, the viscosity of the macromer solution may be optimized. This may be accomplished by viscosity enhancers which are added into the macromer solution. Preferred viscosity enhancers are PEG--triol with MW 3.5 kD and 4 kD PEG-diol.

Density Adjusting Agent

To generate conformal coating without long tails on cell aggregates, the density of the macromer solution may be optimized. This may be accomplished by adding density adjusting agents into the macromer solution. Preferred density adjusting agents are Nycodenz.TM. and Ficoll.TM..

Radiation Wavelength

The radiation used to initiate the polymerization is either longwave UV or visible light, with a wavelength in the range of 320-900 nm. Preferably, light in the range of 350-700 nm, and even more preferred in the range of 365-550 nm, is used. This light can be provided by any appropriate source able to generate the desired radiation, such as a mercury lamp, longwave UV lamp, He-Ne laser, or an argon ion laser or an appropriately filtered xenon light source.

 

Claim 1 of 36 Claims

1. A composition comprising: encapsulating devices comprising a conformal coating, and cell aggregates, wherein said composition has a cell density of at least about 100,000 cells/ml, wherein the conformal coating for the encapsulating devices comprises a polymerizable high density ethylenically unsaturated PEG having a molecular weight between 900 and 3,000 Daltons, and a sulfonated comonomer, and wherein the coating conforms to the size and shape of the cell aggregates.

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