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(12) United States Patent (10) Patent No.: US 6,495,161 B1 Soon-Shiong Et Al

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(12) United States Patent (10) Patent No.: US 6,495,161 B1 Soon-Shiong Et Al USOO64951.61B1 (12) United States Patent (10) Patent No.: US 6,495,161 B1 Soon-Shiong et al. (45) Date of Patent: Dec. 17, 2002 (54) CYTOPROTECTIVE BIOCOMPATIBLE 5,545,423 A * 8/1996 Soon-Shiong et al. ...... 424/484 CONTAINMENT SYSTEMS FOR 5,700,848 A 12/1997 Soon-Shiong et al. ......... 522/7 BIOLOGICALLY ACTIVE MATERIALS AND 5,759,578 A * 6/1998 Soon-Shiong et al. ...... 424/484 METHODS OF MAKING SAME 5,788.988 A * 8/1998 Soon-Shiong et al. - - - - - - 424/484 OTHER PUBLICATIONS (75) Inventors: Patrick Soon-Shiong, Malibu; Neil Desai, Los Angeles; Nilesh Ron, Culver Lim and Sun, “Microencapsulated Islets as Bioartificial City; Andrew Sojomihardjo S., West Endocrine Pancreas,” Science, 210:908-910 (1980). Covina; Roswitha Heintz, Los Angeles; * cited b Francesco Curcio, Westlake Village, all cited by examiner of CA (US) Primary Examiner Dameron L. Jones (74) Attorney, Agent, or Firm-Stephen E. Reiter; Foley & (73) Assignee: VivoRx, Inc., Santa Monica, CA (US) Lardner (*) Notice: Subject to any disclaimer, the term of this (57) ABSTRACT past l5SSh i. adjusted under 35 In accordance with the invention, there are provided a -- y yS. methods, capsules, and delivery Systems useful in preparing biological containment Systems with properties (e.g., (21) Appl. No.: 09/264,187 mechanical Strength, capsule permeability and porosity, (22) Filed: Mar. 9, 1999 desired controlled release rates of the biologic or compo nents Secreted by the biologic, and immunoreactivity) that (51) Int. CI.7 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A61 K 9/48 Ca be varied to adapt to a broader range of physiological (52) U.S. Cl. ........................................ 424/451; 424/450 conditions than known biological containment Systems. (58) Field of Search ................................. 424/450, 451, There are also provided methods of making capsules con 424/489, 455, 456, 459, 460, 463, 461, taining cell aggregates therein, as well as the capsules 462, 484 formed thereby, which are useful as a quantitatively plentiful and low cost alternative to usage of freshly harvested cell (56) References Cited aggregates (e.g., islets from pancreas), Since the latter are U.S. PATENT DOCUMENTS usually available only in limited numbers. 4,352,883 A * 10/1982 Lim ........................... 435/178 26 Claims, No Drawings US 6,495,161 B1 1 2 CYTOPROTECTIVE BOCOMPATIBLE employed, imperfections in the microcapsule membrane CONTAINMENT SYSTEMS FOR (allowing exposure of poly-L-lysine to the in vivo BIOLOGICALLY ACTIVE MATERIALS AND environment), failure of the microcapsule membrane to METHODS OF MAKING SAME completely cover the cells being encapsulated (thereby allowing exposure of the cells to the in Vivo environment), and the like. FIELD OF THE INVENTION Accordingly, there is a need in the art for new and better The present invention relates to new forms of biocom capsules for the encapsulation of biologically active mate patible containment Systems that envelop encapsulated or rials. In addition, there is a need for new methods of making free cells or other biologically active materials. In a particu capsules that encapsulate biologically active materials while lar aspect, the present invention relates to a System that permitting variation of certain properties (e.g., mechanical provides an immune barrier for the cells or other biologi Strength, capsule permeability and porosity, desired con cally active materials. In another aspect, the present inven trolled release rates of the biologic or components Secreted tion relates to a System that provides enhanced migration by the biologic, and immunoreactivity) across broad perfor and aggregation of the cells or other biologically active 15 mance ranges to address variable physiological conditions. materials within the containment System. In a further aspect, Further, there is a need for new methods of facilitating the present invention relates to a System that provides formation of and delivery Systems for cell aggregates. enhanced transfer of the Secretions of cells or other biologi cally active materials out of the containment System. BRIEF DESCRIPTION OF THE INVENTION In accordance with the present invention, capsules (e.g., BACKGROUND OF THE INVENTION microcapsules and macrocapsules) have been developed for Microencapsulation of cells (e.g., pancreatic islets) by an the encapsulation of biologically active materials therein. alginate-PLL-alginate membrane (i.e., an alginate-poly-L- Invention capsules comprise at least one biocompatible lysine-alginate membrane) is a potential method for preven gellable material, wherein at least the outer layer of the tion of rejection of foreign cells by the host's immune 25 capsule is covalently crosslinked and optionally polyioni System. By this technique, researchers are able to encapsu cally crosslinked (or, in the case of macrocapsules compris late living islets in a protective membrane that allows insulin ing microcapsules therein, either polyionically crosslinked, to be Secreted, yet prevents antibodies from reaching the covalently crosslinked, or both polyionically croSSlinked islets, causing rejection of the cells. This membrane (or and covalently crosslinked), but not ionically crosslinked. microcapsule) protects the islet from rejection and allows Surprisingly, invention capsules permit enhanced migration insulin to be Secreted through its "pores' to maintain the and aggregation of the biologically active material within diabetic in normal glucose control. the capsule and enhanced control over the release rates of the Successful transplants of microencapsulated cells have biologically active material or components Secreted by the not been clinically feasible to date due to fundamental 35 biologically active material, while decreasing the risk of problems of transplant rejection and/or fibrotic reaction to biomineralization due to ions required for ionic crosslinking the microcapsule. In the treatment of diabetes, Lim and Sun, and enabling the biologically active material contained 1980; Science 210:908, reported the first successful implan within the capsule to retain a significant proportion of the tation of microencapsulated islets and described normaliza functionality of the unencapsulated biologically active mate tion of blood Sugar in diabetic rats. 40 rial. However, for microencapsulated cells to be clinically In a further aspect of the present invention, there also have useful and applicable in humans, it is important that the been developed methods of making invention capsules. One capsule be biocompatible, allow adequate diffusion for the of the invention methods comprises Subjecting a capsule encapsulated cells to respond appropriately to a Stimulatory whose outer layer is ionically crosslinked and covalently Signal and to provide the encapsulated cells with necessary 45 crosslinked, and optionally polyionically crosslinked (or, in nutrients, and optionally be retrievable. Retrievability is the case of macrocapsules comprising microcapsules desirable for a variety of reasons, e.g., So that accumulation therein, ionically crosslinked and either polyionically of the implanted materials can be avoided, So that encapsu crosslinked, covalently crosslinked, or both polyionically lated cells can be removed from the recipient when no longer crosslinked and covalently crosslinked), to conditions Suf needed or desired (e.g., when the product(s) of the encap 50 ficient to disruptionic crosslinking in at least the outer layer Sulated cells are no longer needed, if the encapsulated cells thereof. Surprisingly, invention methods facilitate the rela fail to perform as desired, etc.), So that encapsulated cells tively rapid formation of invention capsules under condi can be removed if/when they become non-viable, and the tions which are not cytotoxic, while decreasing the risk of like. biomineralization caused by the presence of ions required Biocompatibility of encapsulated islets remains a funda 55 for ionic crosslinking and enabling the biologically active mental problem. The term “biocompatible” is used herein in material contained within the capsule to retain a significant its broad Sense, and relates to the ability of the material to proportion of the functionality of the unencapsulated bio result in long-term in Vivo function of transplanted biologi logically active material. cal material, as well as its ability to avoid a foreign body, Additional methods of making invention capsules com fibrotic response. A major problem with microencapsulation 60 prise Simultaneously Subjecting a droplet comprising a Sus technology has been the occurrence of fibrous overgrowth of pension of biologically active materials in a covalently the epicapsular Surface, resulting in cell death and early graft crosslinkable carrier to conditions Sufficient to prevent Sub failure. Despite extensive Studies, the pathological basis of Stantial dissociation thereof and Subjecting the droplet to this phenomenon in alginate based capsules remains poorly conditions Sufficient to induce Substantial covalent understood. However, several factors have recently been 65 crosslinking thereof. Surprisingly, these invention methods identified as being involved in graft failure, e.g., the gulu facilitate the relatively rapid formation of invention capsules ronic acid/mannuronic acid content of the alginate under conditions which are not cytotoxic, while reducing to US 6,495,161 B1 3 4 Substantially Zero the risk of biomineralization caused by
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