WO1999029261A1 - Implantable drug delivery system - Google Patents

Abstract

This invention relates to an implantable drug delivery device and method of use thereof. More specifically, the invention relates to an implantable chamber for implantation into a patient for controlled delivery of a liposome- or ceramic-encapsulated therapeutic substance for enhancing the stability and lifetime of materials useful in medical therapy.

Description

IMPLANTABLE DRUG DELIVERY SYSTEM CROSS-REFERENCE

This application is a continuation-in-part of U.S. Patent Application Serial Number 08/463,368 filed June 5, 1995 which is a continuation-in-part of U.S. Patent Application Serial Number 08/272,189 filed July 8, 1994.

FIELD OF THE INVENTION

This invention relates to an implantable drug delivery device and method of use thereof. More specifically, the invention relates to an implantable chamber for implantation into a patient for controlled delivery of a liposome- or ceramic- encapsulated therapeutic substance and for enhancing the stability and lifetime of materials useful in medical therapy.

BACKGROUND OF THE INVENTION

"Slow-release" systems of drug delivery are useful for delivery of bioactive agents in a controlled manner using "encapsulate" systems. Such systems have the advantage of providing for controlled release of the bioactive agent over an extended period of time, and may allow for the administration of lower doses of bioactive agents, thus decreasing the toxic effects to the patient. Liposomes, microspheres and ceramic coatings represent three potential materials with which slow-release systems may be engineered.

Liposomes are well recognized as useful for encapsulation of drugs and other therapeutic agents and for carrying these agents to in vivo sites. For use in intravenous drug delivery, liposomes have the potential of providing a controlled "depot" release of a liposome-entrapped drag over an extended time period, and of reducing toxic side effects of the drug, by limiting the concentration of free drug in the bloodstream.

Liposome/drug compositions can also increase the convenience of therapy by allowing higher drug dosage and less frequent drug administration. For example, U.S. Patent No. 3,993,754 discloses an improved chemotherapy method in which an anti- tumor drug is encapsulated within liposomes and then injected. U.S. Patent No. 4,263,428 discloses an anti-tumor drug which may be more effectively delivered to selective cell sites in a mammalian organism by incorporating the drag within uniformly sized liposomes. Liposome delivery systems can have reduced toxicity, altered tissue distribution, increased drug effectiveness, improved therapeutic index and can be used to deliver a wide variety of drags. Liposome drug delivery systems are reviewed generally in Pomansky et al.

Although the encapsulation of therapeutic agents and biologically active materials in liposomes has significant potential for delivering such materials to targeted sites in the human body, major limitations have been encountered with the use of liposomes which preclude their broader use as general delivery systems. One limitation of intravenous liposome drag delivery which has been recognized for many years is the rapid uptake of blood-circulating liposomes by the mononuclear phagocytic system, also referred to as the reticuloendothelial system (RES). This system, which consists of the circulating macrophages and the fixed macrophages of the liver (Kupffer cells), spleen, lungs, and bone marrow, removes foreign particulate matter, including liposomes, from blood circulation with a half life on the order of minutes. Liposomes, one of the most extensively investigated particulate drag carriers, are removed from circulation primarily by Kupffer cells of the liver and to a lesser extent by other macrophage populations.

A variety of studies on factors which effect liposome uptake by the RES have been reported. Early experiments, using heterogeneous preparations of multilamellar liposomes containing phosphatidylcholine and cholesterol as their principal lipid constituents, demonstrated that these liposomes are rapidly removed from circulation by uptake into liver and spleen in a biphasic process with an initial rapid uptake followed by a slow phase of uptake. Half-life for removal of multilamellar liposomes from circulation was on the order of 5-15 minutes following intravenous injection. Negatively charged liposomes are removed more rapidly from circulation than neutral or postively charged liposomes. Small unilamellar liposomes are cleared with half- lives approximately three- to four-fold slower than MLV. Uptake of liposomes by liver and spleen occurs at similar rates in several species, including mouse, rat, monkey, and human.

One approach to increase liposome circulation time was to increase liposome stability in serum. This approach is based on studies which have shown that factors which decrease leakage of liposome contents in plasma also decrease the rate of uptake of liposomes by the RES. One factor contributing to this effect appears to be bilayer rigidity, which renders the liposomes more resistant to the destabilizing effects of serum components, in particular high density lipoproteins. Thus, inclusion of cholesterol in the liposomal bilayer can reduce the rate of uptake by the RES, and solid liposomes such as those composed of distearoylphosphatidylcholine (DSPC) or containing large amounts of sphingomyelin (SM) show decreased rate and extent of uptake into liver. However, this approach appears to have a limited potential for increasing liposome circulation times in the bloodstream.

Microspheres consisting of various materials have been utilized to encapsulate bioactive agents. Tabata, et al. (Phar. Res. 1989, 8:422-427), Oner, et al. (Phar. Res. 1993, 10:621-626) and Lou, et al. (Phar. Pharmacol. 1994, 47:97-102) have demonstrated the use of gelatin in preparing such micropheres. Microspheres have also been manufactured of poly(DL-lactic-co-glycolic acid) (PLGA) as demonstrated by Singh, et al. (Phar. Res. 1991, 8:958-961), Alonso, et al. (R. Pharm. Res. 1993, 10:945-953) and Chang, et al. (J. Pharm. Sci. 1996, 85(2):129-132). Ethylcellulose has been utilized, as shown by Watts, et al. (Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7:235-259) and Khawla, et al. (J. Pharm. Sci. 1996, 85(2):144-149). In addition, Aboofazeli, et al. (J. Pharm. Pharmacol. 1991, 43(supp.) 87), Shinoda, et al. (J. Phys. Chem. 1991, 989-998), and Ho, et al. (J. Pharm. Sci. 1996, 85(2):138-143) have synthesized polyglycerol fatty acid-based microemulsions.

Ceramics-based materials have also been utilized to carry bioactive agents. Radin, et al. (Biomaterials, 1997, 18(l l):777-782) demonstrated the use of calcium phosphate ceramic coatings as carriers of vancomycin. Rodriguez-Lorenzo, et al. (J. Biomed. Mater. Res. 1996, 30(4):515-522) prepared composite materials based on ceramic polymers for development of orthopedic surgery-related treatments. Steroids have been delivered using a ceramic device (Zafirau, et al. Biomed. Sci. Instram. 1996, 32:63-70). A hydroxyapatite ceramic matrix has also been shown to be useful for the continuous delivery of coumadin (Miteli and Bajpai, Biomed. Sci. Instram.,

1995, 31 :177-182). Hydroxyapatite and tricalcalcium phosphate ceramics have been utilized to deliver azidothymidine in a continuous system (Cannon and Bajpai,

Biomed. Sci. Instram. 1995, 31:159-164). Hydroxapatite ceramics have also been shown to be useful for the continuous delivery of heparin (Biomed. Sci. Instram.

1994, 30:169-174).

Animal models have also been utilized to demonstrate the usefulness of ceramics-based delivery systems. The sustained release of progesterone and estradiol has been shown in adult female rats using such devices (Benghuzzi, et al. 1993. Biomed Sci. Instram. 29:51-58). Anticancer drugs have been delivered to mice using porous calcium hydroxyapatite ceramic implanted into back muscle (Uchida, et al. 1992, 10(3):440-445). And, zinc calcium phosphate ceramics have been demonstrated to be useful for delivering insulin in vivo to rats (Arar and Bajpai, Biomed. Sci. Instram. 1992, 28:173-178). Despite limited success in increasing the half-life of bioactive agents in the bloodstream using encapsulates such as those described above, a method for increasing circulation time of drags encapsulated in liposomal, microsphere or cerarmics-based materials would enhance the useful application of such encapsulates for treatment of various disease states.

SUMMARY OF THE INVENTION

The present invention provides an implantable drag delivery system and use thereof. The chamber has walls of a semi-permeable material of sufficient porosity to permit release of liposome encapsulated therapeutic material in a controlled manner. The system comprises an implantable chamber and encapsulated bioactive agents, i.e., therapeutic material such as IL-2 and/or GM-CSF immunopotentiating molecules. The use ofthe implantable chamber to house encapsulated therapeutic agents prolongs circulation time ofthe bioactive agent in a controlled manner.

The present invention also provides a method for delivering therapeutic material to a mammal in need of such material by implanting one or more of implantable chambers which contain one or more therapeutic biological agents as an encapsulated suspension into a mammal. The implantable chamber containing the encapsulated bioactive agent increases the length of time during which the bioactive or therapeutic agent is present in the patient by preventing rapid dispersion from the administration site to other parts of the body and rapid uptake and elimination of the compound by phagocytic cells of the immune system and other clearance systems of the body.

Accordingly, one objective of the present invention is to provide a system for controlled drag delivery. The system comprises an implantable chamber which includes encapsulated bioactive agents such as therapeutic material, preferably an immunopotentiating molecule and even more preferably a cytokine such as IL-2 or

GM-CSF.

Another objective of the present invention is to provide a method for delivering a therapeutic material to a mammal in need of such material comprising implanting a chamber containing a encapsulated bioactive agent such as a therapeutic material.

These and other objects of the invention will become apparent in light of the detailed description below.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a diagram of the chamber used in a preferred embodiment of the invention.

Figure 2 is a time course of release of IL-2 from liposomes loaded into an implantable chamber. Figure 3 is a demonstrates the appearance of tumor in mice following treatment.

Figure 4 demonstrates the effect of devices containing IL-1RA on close vascular structure formation. DETAILED DESCRIPTION OF THE INVENTION

The references listed within this application are incorporated into this application in their entirety.

The present invention provides an implantable drug delivery system and method for delivering therapeutic material to a mammal using the same. The implantable drag delivery system includes an implantable chamber containing encapsulated therapeutic agents. Generally, the chamber prevents rapid dispersion of the therapeutic agent from the administration site and clearance by the body.

Chambers which could be useful in the present invention include without limitation: Agarose microcapsules (Iwata et al., J. Biomed. Mater. Res. 26, p. 967- 977 (1992); J. Bioact. and Comp. Polymers 3, p. 356-369 (1988), and Depuy et al., J. Biomed. Mater. Res. 22, p. 1061-1070 (1988)); Hollow fibers of XM50 (Winn et al., J. Biomed. Mater. Res. 23, p. 31-44 (1989) and Airman et al, Proc. of Third Meeting of ISAO, Supp. 5, p. 776-779 (1981); Diabetes, 35, p. 625-633 (1986)); Alginate- polylysine (Wong et al., Biomat., Art. Cells and Immob. Biotech. 19, p. 675-686 (1991)); Microcapsules of alginate-polylysine (O'Shea et al., Biochim. et Biophy. Acta 804, p.133-136 (1984), Sun et al, App. Biochem. and Biotech. 10, p. 87-99 (1984), Chicheportiche et al., Diabetologia 31, p. 54-57 (1988) and Goosen et al., U.S. Patent No. 4,689,293, August 25, 1987; U.S. Patent No. 4,487,758, December 11, 1984; U.S. Patent No. 4,806,355, Febraary 21, 1989, and U.S. Patent No. 4,673,566, June 16, 1987); Chitosan-alginate (McKnight et al, J. of Bioact. and Comp. Poly. 3, p. 334-355 (1988)); Polyacrylonitrile or other ultrafiltration membranes in a U-shaped device (Moussy et al., Artif Org. 13, p. 109-115 (1989), Lepeintre et al., Artif. Org. 14, p. 20-27 (1990), and Jaffrin and Reach, U.S. Patent No. 4,578,191, March 25, 1986); Hollow fibers of polyacrylonitrile (Aebischer et al, Biomat. 12, p. 50-56 (1991) and Lacy et al., Science 254, p. 1782-1784 (1991)); Track-etched polycarbonate membrane diffusion chambers (Gates and Lazarus, Lancet, Dec. 17, p. 1257-1259 (1977)); Polymerized 2-hydroxyethyl methacrylate pHEMA membrane devices (Ronel et al., J. Biomed., Mater., Res, 17, p. 855-864 (1983)); Microcapsules of polyacrylates (Douglas and Sefton, Biotech and Bioeng. 36, p. 653-664 (1990); Trans Am. Soc. Artif. Inter. Org. 35, p. 791-799 (1989)); Acrylic copolymer hollow fibers (Lanza et al., Proc. Natl. Acad. Sci. 88, p. 11100-11104 (1991)); Intravascular devices (Berguer, U.S. Patent No. 4,309,776, January 12, 1982) and Gaskill, U.S.

Patent No. 4,911,717, March 27,1990); Cationic-anionic crosslinked membranes, e.g. chitosan and polyaspartic or polyglutamic acid (Jarvis, U.S. Patent No. 4,803,168, Febraary 7, 1989); Surface-conforming bonding bridge layer of a multifunctional material and semipermeable polymer layer for cell encapsulation (Cochram, U.S.

Patent No. 4,696,286, September 29, 1987); Vascular perfusion devices (Chick et al,

U.S. Patent No. 5,002,661, March 26,1991); Barium-alginate cross-linked microcapsules (Zekorn et al, Acta. Diabetol. 29, p. 99-106 (1992)), other membrane devices (Ward et al, Fourth World Biomat. Con., Berlin, p. 152, April 24-28, 1992)) and other encapsulation devices (Aebischer, WO 94/07999; U.S. Patent No. 5,283,187; WO 93/00128; WO 93/00127; WO 93/00063; WO 92/19195; WO 91/10470; WO 91/10425; WO 90/15637; WO 90/02580). In the event that the particular chambers described above are not permeable enough to allow egress of a particular therapeutic material, one skilled in the art will understand that the permeability of such chambers may be altered without changing the basic design of such chambers.

A preferred chamber for use in the present invention is a device comprising a chamber which includes a wall comprising (a) a first zone of a first porous material defining a chamber wherein the first porous material is permeable to the flow of nutrients from the host to the chamber and products from the chamber to the host and impermeable to the host immune cells; and (b) a second zone of a second porous material proximal to host tissue, the second porous material having a nominal pore size ranging from about 0.6 to about 20 μm and comprising frames of elongated strands that are less than 5 μm in all but the longest dimension wherein the frames define apertures which interconnect to form three dimensional cavities which permit substantially all inflammatory cells migrating into the cavities to maintain a rounded morphology and wherein the second zone promotes vascularization adjacent but not substantially into the second zone upon implantation into the host. Examples of such chambers are described, for instance, in U.S. Patent Nos. 5,453,278; 5,314,471; 5,593,440; 5,344,454; 5,653,756; 5,545,223; 5,569,462; 5,421,923; 5,549,675 and U.S. Patent application serial Nos. 08/210,068; 08/484,011; 08/485,632; 08/480,198; 08/481,886; 08/356,787; 08/461,471; 08/413,309; 08/594,120; 08/462,252;

08/462,249; 08,463,368; 08/320,199; 08/446,210; and 08/654,729, which are incorporated by reference in their entirety.

The porous wall comprises a material selected from the group consisting of polyethylene, polypropylene, polytetrafluoroethylene (PTFE), cellulose acetate, cellulose nitrate, polycarbonate, polyester, nylon, polysulfone, mixed esters of cellulose, polyvinylidene difluoride, silicone and polyacrylonitrile. The first and second zones of porous material may be made of the same or different material. In another embodiment of the invention, the first porous material is further impermeable to humoral immune factors.

Preferably, the material for the second zone that results in formation of close vascular stractures includes approximately 50% of the pores with average size of approximately 0.6 to about 20 μm. The structural elements which provide the three dimensional conformation may include fibers, strands, globules, cones or rods of amorphous or uniform geometry which are smooth or rough. These elements, referred to generally as "strands," have in general one dimension larger than the other two and the smaller dimensions do not exceed five microns.

In one arrangement, the material consists of strands that define "apertures" formed by a frame of the interconnected strands. The apertures have an average size of no more than about 20 μm in any but the longest dimensions. The apertures of the material form a framework of interconnected apertures, defining "cavities" that are no greater than an average of about 20 μm in any but the longest dimension. In this arrangement, the material for the second zone has at least some apertures having a sufficient size to allow at least some vascular stractures to be created within the cavities. At least some of these apertures, while allowing vascular structures to form within the cavities, prevent connective tissue from forming therein because of size restrictions.

A particularly preferred device (as shown in Figure 1) comprises two bilayer membranes (1) surrounded by a polyester mesh (2) sonically welded together, with a port (3) for access to the lumen (4). Each bilayer comprises a 5 μm PTFE membrane manufactured by Gore, Flagstaff, Arizona, Product No. L31324 and a 0.45 μm PTFE membrane manufactured by Millipore, Bedford, Massachusetts, Product No.

SF1R848E1. At one end there is a polyester (PE 90 ID 0.034" by OD 0.050") port to permit access to the interior of the device for loading cells. The device has an interior lumen having a volume generally ranging from 2 μl to about 100 μl, preferably 4.5 μl to 40 μl, and most preferably 40 μl. This device is described in copending application serial number 08/179,860 filed January 11, 1994 and copending application serial number 08/210,068 filed March 17, 1994. Previous studies have shown that this device has the advantage (though not required for all embodiments of the present invention) of being able to protect allograft tissue from immune rejection for extended periods (Carr-Brendel et al., J. Cellular Biochem. 18A, p. 223 (1994) and Johnson et al., Cell Transplantation 3, p. 220 (1994)).

Liposomes are unilamellar or multilamellar lipid vesicles which enclose a fluid space. The walls of the vesicles are formed by a bimolecular layer of one or more lipid components having polar heads and non-polar tails. In an aqueous (or polar) solution, the polar heads of one layer orient outwardly to extend into the surrounding medium, and the non-polar tail portions of the lipids associate with each other, thus providing a polar surface and a non-polar core in the wall of the vesicle. Unilamellar liposomes have one such bimolecular layer, whereas multilamellar liposomes generally have a plurality of substantially concentric bimolecular layers. Methods for preparing liposomes and for encapsulating therapeutic materials in the liposomes are known in the art (Liposome Technology, 1984; Bergers, 1993; Elorza, 1993; Allen, 1993). Liposomes can be utilized as microspheres composed of gelatin as described by Tabata, et al. (Phar. Res. 1989, 8:422-427), Oner, et al. (Phar. Res. 1993, 10:621-626), and Lou, et al. (L. Phar. Pharmacol. 1994, 47:97-102); poly(DL- lactic-co-glycolic acid) (PLGA) as described by Singh, et al. (Phar. Res. 1991, 8:958- 961), Alonso, et al. (R. Pharm. Res. 1993, 10:945-953), and Chang, et al. (J. Pharm. Sci. 1996 85(2): 129-132); or ethylcellulose as described by Watts, et al. (Critical Reviews in therapeutic drug carrier systems, 1990, 7:235-259) and Khawla, et al. (J. Pharm. Sci. 1996, 85(2): 144-149). Polyglycerol fatty acid ester based microemulsions may also be utilized as described by Aboofazeli, et al. (J. Pharm. Pharmacol. 1991, 43(supp.):87), Shinoda, et al. (J. Phys. Chem. 1991, 989-998), and Ho, et al. (J. Pharm. Sc. 1996, 85(2):138-143). Additionally, ceramic-based materials have been utilized to encapuslate bioactive agents. Calcium phosphate ceramic coatings (Radin, et al. Biomaterials,

1997, 18(l l):777-782), ceramic polymers (Rodriguez-Lorenzo, et al. J. Biomed.

Mater. Res. 1996, 30(4):515-522) and other ceramic devices have been demonstrated to be useful Zafirau, et al. Biomed. Sci. Instram. 1996, 32:63-70). In addition, hydroxyapatite-based ceramic materials have been shown to be useful by Miteli and

Bajpai (Biomed. Sci. Instram. 1995, 31:177-182), Cannon and Bajpai (Biomed. Sci

Instram. 1995, 31 :159-164) and Abrams (Biomed. Sci. Instram. 1994, 30:169-174).

Certain of these systems have also been demonstrated to be effective for encapsulating materials using animal models. Estrogen (Biomed Sci. Instram. 29:51-58), anticancer drags (Uchida, et al. 1992, 10(3):440-445) and insulin (Arar and Bajpai, Biomed. Sci. Instram. 1992, 28:173-178) have each been shown to be amenable to delivery when encapsulated in ceramics-based materials.

Any of the above-described types of materials (i.e., liposomes, microspheres or ceramic-based materials) may be utilized to encapsulate bioactive agents in practicing the present invention. It is to be understood by one skilled in the art that the present invention encompasses any of the wide variety of materials with which a bioactive agent or agents may be encapsulated in practicing the present invention.

Many different compounds which are bioactive agents can be encapsulated in materials such as those described above in practicing the present invention. Such compounds include but are not limited to antibacterial compounds such as gentamycin, antiviral agents such as rifampacin, antifungal compounds such as amphoteracin B, anti-parasitic compounds such as antimony derivatives, tumoricidal compounds such as adriamycin, anti-metabolites, peptides, proteins such as albumin, toxins such as diptheriatoxin, enzymes such as catalase, polypeptides such as cyclosporin A, hormones such as estrogen, hormone antagonists, neurotransmitters such as acetylcholine, neurotransmitter antagonists, glycoproteins such as hyaluronic acid, lipoproteins such as alpha-lipoprotein, immunoglobulins such as IgG, immunomodulators such as interferon or interleuken, vasodilators, dyes such as Arsenazo III, radiolabels such ¹⁴C , radio-opaque compounds such as ⁹⁰Te, fluorescent compounds such as carboxy fluorescein, receptor binding molecules such as estrogen receptor protein, anti-inflammatories such as indomethacin, antiglaucoma agents such as pilocarpine, mydriatic compounds, local anesthetics such as lidocaine, narcotics such as codeine, vitamins such as alpha-tocopherol, nucleic acid bases (components) such as thymine, polynucleotides such as RNA polymers, psychoactive or anxiolytic agents such as diazepam, mono-, di- and polysaccharides, etc. A few of the many specific compounds that can be entrapped are pilocarpine, a polypeptide growth hormone such as human growth hormone, bovine growth hormone and porcine growth hormone, indomethacin, diazepam, alpha-tocopherol itself and tylosin.

Antifungal compounds include miconazole, terconazole, econazole, isoconazole, tioconazole, bifonazole, clotrimazole, ketoconazole, butaconazole, itraconazole, oxiconazole, fenticonazole, mystatin, naftifine, amphotericin B, zinoconazole and ciclopirox olamine, preferably miconazole or terconazole.

Antiasthmatics such as melairoterenol, aminophylline, theophylline, terbutaline, norepinephrine, ephedrine, isoproternol, adrenalin; Cardiac glycosides such as digitalis, digitoxin, lanatoside C, digoxin; Antihvpertensives such as apresoline, atenolol, captopril, reserpine; Antiparasitics such as praziquantel, metronidazole, pentamidine, ivermectin; Nucleic Acids and Analogs such as DNA, RNA, methylphosphonates and analogs, Antisense nucleic acids; Antibiotics such as penicillin, tetracycline, amikacin, erythromycin, cephalothin, imipenem, cefotaxime, carbenicillin, ceftazidime, kanamycin, tobramycin, ampicillin, gentamycin, cefoxitin, cefadroxil, cefazolin, other aminoglycosides, amoxicillin, moxalactam, piperacillin, vancomycin, ciprofloxacin, other quinolones; Vaccines such as other recombinant, killed and live vaccines and antigenic material for use as vaccines, antigenic material for the treatment of allergies, influenza, respiratory syncytial virus, HIV vaccine, Hemophilus influenza vaccines, Hepatitis A, B, C vaccines, mumps, rubella, measles, tetanus, malaria vaccines, herpes, cancer vaccines, Anti-leu-3a vaccine; Monoclonal Antibodies (human, mouse other species-derived and/or recombinant and/or fusions and/or fragments thereof) such as OKT3, OKT4, HA-IA, Anti-Carcino-Embryonic Antigen Antibodies, Anti-Ganglioside Antibodies: Anti GD2, Anti GM2, Anti GD3, Anti GM3, Urinary Tract- Associated Antigen-related antibodies, Anti-Il-2 Receptor, Chimeric Anti-Leu-2, Anti-IL-2 receptor, Anti-Leu-2, Chimeric Anti-Leu-3a, Chimeric L6, MAb-L6, Radiolabeled L6, Centorex, Centoxin, Panorex, Anti-LPS, Immunotoxin, Anti-tumor necrosis factor, Anti-pseudomonas, Anti-tumor necrosis factor, OncoRad 103, OncoScint CR1O3, OncoScint OV103, OncoScint PR356,

OncoTher 130, KS 1/4-DAVLB, ADCC agent, Murine monoclonal antibodies to human B-cell lymphomas (anti-idiotypes), Murine monoclonal antibody

(lMelpgl)(anti-idiotype) against murine monoclonal antibody to melanoma-associated antigen, Anti-B4-blocked ricin, Anti-My9-blocked ricin, ImunuRaid-CEA, MAb against colorectal, ovarian, and lung cancers, rhenium- 186 MAb, Orthoclone OKT®,

E5O, LYM-1, TNT, XomaZyme ®-791, XomaZyme ® CD5 Plus, XomaZyme ®-

CD7 Plus, Xoma ®-Mel; Proteins and Glycoproteins such as lymphokines, interleukins - 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, cytokines, GM-CSF, M-CSF, G- CSF, tumor necrosis factor, inhibin, tumor growth factor, Mullerian inhibitors substance, nerve growth factor, fibroblast growth factor, platelet derived growth factor, coagulation factors (e.g. VIII, IX, VII), insulin, tissue plasminogen activator, histocompatibility antigen, oncogene products, myelin basic protein, collagen, fibronectin, laminin, other proteins made by recombinant DNA technology, erythropoietin, IL-3/GM-CSF fusion proteins, Monoclonal antibodies, Polyclonal antibodies, antibody-toxin fusion proteins, antibody radionuclide conjugate, Interferons, Fragments and peptide analogs, and analogs of fragment of proteins, peptides and glycoproteins, Epidermal growth factor, CD4 receptor and other recombinant receptors, receptor agonists, other proteins isolated from nature, Antidiuretic hormone, oxytocin, adrenocorticotropin Hormone, calcitonin, follicle stimulating hormone, luteinizing hormone releasing hormone, luteinizing hormone, gonadotrophin, transforming growth factors, streptokinase, Human Growth Hormone, Somatotropins for other species, including, but not limited to: 1) porcine, 2) bovine, 3) chicken, 4) sheep, 5) fish, Growth Hormone releasing hormones for humans and various animal species, Glucagon, Desmopressin, Thyroid Releasing Hormone, Thyroid Hormone, Secretin, Magainins, Integrins, Adhesion Peptides, including, but not limited to, those having the Arginine-Glutamine-Aspartic Acid sequence, Super Oxide Dismutase, Defensins, T-Cell Receptors, Bradykinin antagnoists, Pentigetide, Peptide T, Antinflammins, Major Histocompatibility (MHC) complex components and peptides targeted to the MHC, Protease inhibitors, Lypressin, Buserelin, Leuprolide, Nafarelin, Deslorelin, Goserelin, Historelin, Triptorelin, LHRH antagonists, HOE-2013, Detirelix, Org-30850, ORF-21243, Angiotensin Converting Enzyme inhibitor Peptide, Renin inhibitory peptides, Ebiratide (HOE-427), DGAVP,

Opiate receptor agonists and antagonists, including, but not limited to: 1)

Enkephalins, 2) Endorphins, E-2078, DPDPE, Vasoactive intestinal peptide, Atrial

Natriuretic Peptide, Brain Natriuretic Peptide, Atrial Peptide clearance inhibitors, Hirudin, Oncogene Inhibitors, Other Colony Stimulating Factors; Neurotransmitters such as Dopamine, Epinephrine, Norepinephrine, acetylcholine, Gammaamino butyric acid; Others such as amino acids, vitamins, cell surface receptor blockers;

Antiarrhythmics such as propanolol, atenolol, verapamil; Antianginas such as isosorbide dinitrate; Hormones such as thyroxine, corticosteroids, testosterone, estrogen, progesterone, mineralocorticoid; Antidiabetics such as Diabenese, insulin; Antineoplastics such as azathioprine, bleomycin, cyclophosphamide, vincristine, methotrexate, 6-TG, 6-MP, vinblastine, VP-16, VM-26, cisplatin, 5-FU, FUDR, fludarabine phosphate; Immunomodulators such as interferon, interleukin-2, gammaglobulin, monoclonal antibodies; Antifungals such as amphotericin B, myconazole, muramyl dipeptide, clotrimazole, ketoconozole, fluconazole, itraconazole; Tranquilizers such as chlorpromazine, benzodiazepine, butyrophenones, hydroxyzines, meprobamate, phenothiazines, thioxanthenes; Steroids such as preunisone, triamcinolone, hydrocortisone, dexamethasone, betamethasone, preunisolone; Antihistamines such as pyribenzamine, chlo heniramine, diphenhydramine; Sedatives and Analgesics such as morphine, dilaudid, codeine, codeine-like synthetics, demerol, oxymorphone, phenobarbital, barbiturates, fentanyl, ketorolac; Vasopressors such as dopamine, dextroamphetamine; Antivirals such as acyclovir and derivatives, Gancyclovir and phosphates, Winthrop-51711, rifavirlin, rimantadine/amantadine, azidothymidine and derivates, adenine arabinoside, amidine- type protease, inhibitors; RadionucUdes such as Technetium, Indium, Yttrium, Gallium; and Radiocontrasts such as Gadolinium chelates, Iohexol, Ethiodol, Iodexinol.

The amount of encapsulated bioactive agent administered will generally be dependent on the desired targeted blood concentration levels. The entrapment of two or more compounds simultaneously may be especially desirable where such compounds produce complementary or synergistic effects. The following examples illustrate model systems with which biologically active substances such as liposome-entrapped immunomodulatory molecules, e.g. cytokines, and unengineered tumor cells (live or irradiated) can be effectively delivered using an implantable chamber such as the TheraCyte® system, a cell transplant system (Baxter Healthcare Corp., Round Lake, IL). This system has several advantages over other immunotherapy approaches currently under clinical trials. First, there is a reduced risk of tumor formation in the host as the tumor cells introduced into the host for immunization are sequestered and cannot escape from the device.

Second, freely injected liposomes are frequently taken up by macrophages and the lymphatic system; use of the immunoisolation device may permit maintenance of cytokine secretion for extended periods of time by preventing this uptake. Finally, the cells and remaining therapeutic material can be removed quantitatively at the end of the therapy.

The following Examples are for illustrative purposes only and are not intended, nor should they be construed as limiting the invention in any manner. Those skilled in the art will appreciate that variations and modifications can be made without violating the spirit or scope ofthe invention.

Example I MATERIALS AND METHODS Proteins. Recombinant murine Interleukin-2 (rm IL-2) and recombinant murine granulocyte-macrophage colony stimulating factor (rm GM-CSF) were obtained from R & D Systems (Minneapolis, MN).

Lipids. L-α Phosphatydylcholine (PC, from egg yolk) solution in chloroform and L-α Phosphatydyl-DL-Glycerol (PG, from egg yolk) solution in chloroform:methanol (98:2) were obtained from Sigma Chemical Company (St.

Louis, MO).

Preparation of Liposomes. Liposomes were prepared by evaporating the organic solvents from the lipid mixture of L-α PC and L-α PG, followed by rehydration of lipids in an aqueous solution containing cytokines. Briefly, PC and PG solutions were mixed (9:1) in a round bottom flask. A thin film of dry lipids was formed, by rotary evaporator (Rotavap-R, Buchi), at 45°C under vacuum. The dry film of lipid was mechanically dispersed using sterile glass beads (5 mm, Kimble

Glass Inc., Germany) in a solution of cytokines (400 units IL-2/ml or 500 units GM-

CSF/ml) in hydration buffer (10 mM sodium acetate pH = 5.0, 100 mM NaCl and 270 mM glycerol). This lipid dispersion was achieved at 45°C by rotary evaporation (without vacuum). Finally, the liposome suspension was subjected to an ultrasonic shock for 1 min. at 45°C in the pulse mode using a Branson-2200 Ultrasonic Cleaner

(Branson Ultrasonic Corporation, CT). This treatment resulted in a liposomal suspension with a mean particle size of 10.26 ± 0.1 μ.

Preparation of Ceramic-Encapsulates. Ceramic-based encapsulates of bioactive agents for practicing the present invention may be prepared as described herein or as calcium phosphate ceramic coatings as described by Radin, et al. (Biomaterials, 1997, 18(11):777-782), as ceramic polymers as described by Rodriguez Lorenzo, et al. (J. Biomed. Mater. Res. 1996, 30(4):515-522), or as hydroxyapatite- based ceramic materials (Biomed. Sci. Instram, 1995, 31 :177-182; Biomed. Sci. Instram. 1995, 31 :159-164; Biomed. Sci. Instram. 1994, 30:169-174). Animal models may also be utilized as described herein or as described in Biomed Sci. Instram. 29:51-58; Uchida, et al. 1992, 10(3):440-445; and Arar and Bajpai, Biomed. Sci. Instram. 1992, 28:173-178. Other materials known by those skilled in the art may also be suitable for practicing the present invention. Cell Lines and Animals. Mouse colon carcinoma cell line (MCA-38) was provided by Dr. Augusto Ochoa (NCI, Frederick, MD). These cells were cultured in RPMI-1640/HEPES (Irvine Scientific, CA) media supplemented with 10% heat- inactivated fetal bovine serum (FBS, Harlan Bioscience Products, Indianapolis, IN), 1% L-Glutamate (stock 200 mM, Sigma Chemical Company, St. Louis, MO), 1% sodium pyravate (stock 100 mM, Sigma Chemical Company, St. Louis, MO), 0.1% b- mercaptoethanol (Sigma Chemical Company, St. Louis, MO), 1% Penicillin (10,000 U/ml)/Streptomycin (10 mg/ml) mixture (Sigma Chemical Company, St. Louis, MO) and 1% non-essential amino acid mixture (stock= 100X, Sigma Chemical Company, St. Louis, MO). These cells were maintained at 37°C in a humidified atmosphere with 5% CO₂. The IL-2 dependent murine cytotoxic T-cell line, CTLL-2, was obtained from

American Type Culture Collection (ATCC, Rockville, MD) and was cultured in RPMI-1640/HEPES (Irvine Scientific, CA) media supplemented with 10% heat- activated FBS (Harlan Bioscience Products, Indianapolis, IN), 1% L-Glutamate (stock=200 mM, Sigma Chemical Company, St. Louis, MO), 1% Sodium pyravate (stock=100 mM, Sigma Chemical Company, St. Louis, MO) and rm IL-2 (10 units/ml; R & D Systems, Minneapolis, MN). These cells were maintained at 37°C in a humidified atmosphere with 5% CO₂.

The GM-CSF dependent murine cell line, C2GM, was provided by Dr. T. Michael Dexter (Christie CRC Research Center, Manchester, England). Cells were grown in Fischers medium (Sigma Chemical Company, St. Louis, MO) containing 20% (vol/vol) horse serum (Sigma Chemical Company, St. Louis, MO) and rm GM- CSF/ml (50 units/ml; R & D Systems, Minneapolis, MN). These cells were maintained at 37°C in a humidified atmosphere with 5% CO₂. Female C57/BL6 mice were obtained from Harlan Sprague Dawley

(Indianapolis, IN) and were housed, treated with anesthetics and analgesics (on as needed basis) and euthanized based on standard procedures.

Cytokine Bio-Assays. The activity of rm IL-2 and its efficiency of incorporation into liposomes was measured in a cell proliferation assay using the IL-2 dependent murine cytotoxic T-cell line, CTLL-2. Liposomes containing IL-2 were disrupted in RPMI-1640 media containing 25 mM SDS using an ultrasonic bath (Branson-2200) for 15 minutes at 45°C. In the first stage of the assay, CTLL-2 cells were harvested by centrifugation at 1500 RPM/5 min./4°C and washed three times with basal RPMI-1640 media. These cells were resuspended in RPMI-1640 media at a cell density of 10,000 cells/50 μl and incubated at 37°C in humidified atmosphere with 5% CO₂, for 1.5 hours to deplete any remaining (intracellular) IL-2. These cells (50 μl/well) were then incubated (37°C/humidity/5% CO₂,) in a 96 well tissue culture plate with 100 μl of RPMI-1640 media containing different concentrations of rm IL-2 (0.0-5.0 units/ml) as standard or with various dilutions of the disrupted liposomes. After 12-16 hours, 25μl of RPMI-1640 media containing 0.5 μCi of ³H_rthymidine (Amersham, Arlington Heights, IL) was added to each well for 6 hr, after which the cells were harvested using the PHD cell harvester (Cambridge Technology Inc.,

Watertown, MA).

A similar cell-proliferation assay was employed for determining the biological activity of rm GM-CSF, using a GM-CSF dependent murine cell line, C2GM. Liposomes containing rm GM-CSF were disrapted (as described above) and incubated

(37°C/humidity/5% CO₂) with GM-CSF depleted C2GM cells (50,000) cells/well) in

Fischers media supplemented with 20% Horse serum. After 48 hr, 25 ml of C2GM media containing 0.5 mCi of ³H-thymidine was added to each well and the cells were incubated (37°C/humidity/5% CO₂) for another 3 hr. Finally the cells were harvested using the PHD cell harvester.

Quantitation of ³H-thymidine incorporation was obtained using a Pharmacia Wallac-1410 liquid scintillation counter (WALLAC, Inc., Gaithersburg, MD). These counts were directly proportional to the levels of IL-2 or GM-CSF present. The data were fit to a four parameter curve using DeltaSOFT 11 (BioMetallics, Inc., Princeton, NJ) from which the activities and encapsulation efficiencies were determined.

Cytokine ELISA. For determining the concentration of rm IL-2 and rrnGM- CSF commercially available ELISA kits (Endogen, Cambridge, MA) were used. Assays were performed as per the instruction provided by the supplier. Data were analyzed using the DeltaSOFT II program (BioMetallics, Inc., Princeton, NJ). In vitro Time Course of Release of Cytolάnes from Liposome loaded into the

Devices: 20 μl of liposome encapsulated IL-2 were incubated in 1.5 ml of saline (Baxter Healthcare Corp., IL) at 37°C in a six well tissue culture plate. Samples were withdrawn at different time periods and stored at -70°C. After withdrawing each sample the devices were washed in saline solution and transferred to a new well containing 1.5 ml of saline. After all samples were collected, the concentration of cytokines released was determined using commercially available ELISA as described above.

Tumor Initiation. For in situ pre-existing tumor experiments, tumors were initiated in C57/BL6 mice as follows: Exponentially growing MCA-38 cells were harvested by brief trypsinization, washed twice with Hanks balanced salt solution (HBSS, Sigma Chemical Company, St. Louis, MO), and resuspended in sterile saline solution at a cell density of 1000 cells/50μl. Mice were injected intramuscularly with

50μl of this cell suspension in the right flank. Three days post tumor initiation, devices were implanted as indicated in the results section.

For resection experiments, MCA-38 cells were resuspended at a cell density of 10⁶ cells/50 μl of saline. Animals were injected in the dorsal subcutaneous space

(near the neck) with 50 μl of this cell suspension. Animals were checked regularly for the presence of palpable tumor. Once palpable tumor was present (usually 10-14 days post injection) an incision was made adjacent to the primary tumor and the tumor was removed by blunt dissection. The wound site was sealed with wound clips and swabbed with providone-iodine solution (Baxter Healthcare Corp., IL). The devices were implanted into experimental arnmals as indicated in individual experiments.

Devices. Ported 4.5 μl trilayer, sonically welded devices (PDO4.5C) were obtained from Baxter Gene Therapy Unit (Round Lake, IL). Devices were sterilized in 70% ethanol followed by serial soaking, three times with 20 minute incubation each, in sterile saline to remove remaining ethanol. Sterilized devices were stored in sterile saline solution and were implanted within 48h. 20 μl trilayer sonically welded devices (PD20F) were also obtained from Baxter Gene Therapy Unit (Round Lake, IL) and sterilized as described above.

Cell Preparation for Device Loading. MCA-38 cells were trysinized from the tissue culture flasks and resuspended in fresh tissue culture media (4°C). A 10 μl aliquot was removed to calculate cell density and the remaining cell suspension was irradiated at 3500 rads using a ⁶⁰Co source. The irradiated cells were resuspended in fresh growth media at a cell density of 10⁶ cells/5 μl and 2xl0⁶ cells/5 μl, and kept on ice until loading. Devices were loaded under sterile conditions. Briefly, devices were massaged using a cotton gauze to remove saline and any air bubbles trapped inside the device. For loading 4.5 μl devices containing cells or liposomal preparations, 1 ul of tissue culture media was taken up in a 10 μl Hamilton syringe followed by 5 μl of desired cell suspension or 2.5 μl of liposomal suspension. Devices were squeezed from the lateral edges using a forceps to open the lumen. The needle of the Hamilton syringe was inserted via the port into the lumen to about 2/3 of the length of the device and the contents were released as the needle was withdrawn. A mixture of MCA-38 cells and liposomal suspension was prepared in a sterile Eppendorf tube and was used to load devices that received both cells and liposomes. Devices were sealed by injecting silicon adhesive (Silastic, Dow Coming, MI) into the port using a syringe and a 23 gauge blunt needle. The port of each sealed device was immersed in 70% ethanol followed by three washes in sterile saline. The sealed devices were stored in growth media at 37°C in humidified atmosphere with 5% CO₂ until implanted.

The cell number and the volume of liposomal preparations loaded into 20 μl device was increased to 1 x 10⁷ cells and 5 or 10 μl of liposomal suspension. The 20 μl devices were loaded by a non-contact method using the bag system. The bag system is composed of a device enclosed in a polyethylene envelope such that the port of the device extends out of the envelope. For loading the devices, the 0.04 inch I.D. end of a tapered silicon collar (Baxter Gene Therapy Unit, Round Lake, IL) was connected to the device port. A 25 μl Hamilton syringe (with blunt end) containing cell suspension or the liposomal preparation was pushed into the 0.024 inch I.D. side of the silicon collar until the Hamilton needle reached the lumen of the device (just pass the duckbill of the device). The contents were released into the lumen of the device. The Hamilton syringe was then pulled out ofthe lumen but not entirely out of the silicon collar. Devices were sealed by injecting silicon adhesive (Silastic, Dow Corning, MI) into the port using a syringe and a sharp needle. The device port was then cut to separate the bag from the syringe, and the bagged device was stored at 37°C in humidified atmosphere with 5% CO₂ until implanted. At the time of implant the bag was swabbed with 70% ethanol, and was then cut open to remove the device. Device Implantation. Each animal received two devices at ventral subcutaneous sites. Animals receiving implants were anesthetized in accordance with standard procedures, by intraperitoneal injection of 0.1-0.2 ml of a mixture of Xylazine (Burns Veterinary Supply, Inc., Rockville Center, NY) and Ketamine (Fort Dodge Laboratories, Inc., IA) in sterile saline (0.75 ml Xylazine + 1.0 ml Ketaset +2.25 ml saline). The abdominal area was swabbed with providone-iodine solution (Baxter Healthcare Corporation, IL). Using sterile surgical instruments, a ventral midline incision was made through the dermal layer and pockets were made on either side of the incision using blunt dissection. One device was placed into each pocket between the skin and muscle layers with port facing towards the tail. The incision was closed with the wound clips and swabbed with providone-iodine solution.

Immediately following the surgery, animals were kept on a heated (37°C) pad for 30 minutes to promote faster recovery.

Example 2 Sterility of Liposomes and Encapsulation of Cytokine Proteins into Liposomes.

PC and PG stocks used for the preparation of liposomes were in organic solvents and the liposome preparation was carried out under sterile conditions. No bacterial or fungal growth was seen in liposomes plated on LB/agar plates.

The biological activity of the rm IL-2 entrapped in liposomes and its efficiency of encapsulation into liposomes was determined using an IL-2 dependent murine cell line (CTLL-2) mediated bio-assay. A similar bio-assay was performed using murine GM-CSF dependent cell line (C2GM) to estimate the biological activity of liposome GM-CSF. To determine the encapsulation efficiency of rm GM- CSF into liposomes, a commercially available ELISA was employed. Both rm IL-2 and rm GM-CSF molecules maintained their biological activity after encapsulation into liposomes. The biological activity of these molecules was comparable to that of stock solutions of these cytokines that were used for the entrapment into lipid vesicles. Using a PC:PG ratio of 9:1, and lipid:aqueous ratio of 50 mg lipid/ml aqueous solution containing 100 mg cytokine/ml, we obtained high efficiency of entrapment (90% ± 5%) for rm IL-2. A similar high efficiency of entrapment into liposomes was obtained for GM-CSF using PC:PG ratio of 9:1 and lipid.aqueous ratio of 20 mg lipid/ml aqueous solution containing 50 mg cytokine/ml. Based upon this level of incorporation 1 ml of the liposome-IL-2 suspension was equivalent to 400 units of IL-2 and 1 ml of the liposome GM-CSF suspension was equivalent to 500 units of GM-CSF.

Example 3 In vitro Time Course of Release of IL-2 from Liposomes. The in vitro time course of release of IL-2 from the liposomal preparation in an implantable chamber was measured. The data illustrated in Figure 2 indicates an initial burst in the release of IL-2 following injection after which 10-12 units of IL-

2/ml/20h were released for up to 5-6 days.

Example 4 Effect of Implantation of 4.5 μl Devices Containing Liposome Encapsulated IL-2 or GM-CSF on in situ Pre-existing Tumors.

The effect of liposome encapsulated cytokines on in situ pre-existing tumors was tested. Three days after initiation of tumor, animals were implanted with two 4.5 μl devices containing both irradiated MCA-38 cells and liposomes containing either IL-2 or GM-CSF in various combinations as outlined in Table 1. With liposomes containing IL-2, optimal results (80% tumor free survival for >60 days) were obtained by implanting tumor cells (2 x 10⁶ cells) and liposomal preparations in separate devices. However, when using liposomes containing GM-CSF, the most effective treatment for delaying and preventing the tumor growth at the challenge site was to combine tumor cells and the liposomal preparations within the same implantable chamber (80% tumor free survival for >60 days). Treatment involving administration of tumor cells and GM-CSF in separate implantable chamber was also found to be effective (60% tumor free survival for > 60 days). In all experiments, all of the control animals (that did not receive any implant) developed tumors within 14 days. These results suggested that an anti-tumor response can be generated using a completely closed system when both cells and liposome encapsulated cytokines are used.

Example 5

Effect of Implantation of 4.5 μl Devices Containing Liposome Encapsulated

IL-2 or GM-CSF after Resection of Existing Tumors. The effect of soluble cytokine (GM-CSF) in preventing tumor reformation after surgical resection was initially tested. After resection of primary tumor, C57/B6 mice were implanted with two 4.5 μl devices, each containing 1 x 10⁶ irradiated MCA-38 cells and given an injection of 1000 units of rmGM-CSF (in sterile saline) at the time of implant. The cytokine was also injected at the implant site weekly for three weeks. These animals developed tumors at the same rate as control animals that did not receive implants. Liposome-encapsulated cytokines were then tested. In these experiments, tumors were resected and the animals were implanted as outlined in Table 2. In the group that received irradiated cells and slow release preparations of IL-2 (mixed together), 60% ofthe animals were tumor free for >60 days while 80% ofthe animals that received irradiated cells and slow release GM-CSF preparations (mixed together) remained tumor free for >60 days. Control animals that did not receive any treatment

(no implant) after surgical resection of their tumor mass all developed tumors by day

14.

Treatment of animals with tumor cells in a first implantable device and GM- CSF in a second implantable device following tumor resection resulted in 40%> tumor-free animals for > 60 days. This indicates that treatment using separate implantable chambers containing tumor cells and an immunopotentiating molecule, in this case GM-CSF, is an effective treatment following tumor resection. In contrast, none of the resected animals survived when IL-2 was used in place of GM- CSF. Without being bound by any theory of operation for this invention, it is believed that no local effect is required when GM-CSF is used. In the case of IL-2, however, positive results are obtained when both the tumor cells and IL-2 are physically together at the same time. The reasons are unknown.

Example 6 Effect of Implantation of 20 μl Devices Containing Liposome-Encapsulated

Cytokines on Close Vascular Structure (CVS)

IL-1 is a cytokine primarily produced by macrophages and has broad biological activity including regulation of local and systemic inflammation. IL-1 receptor antagonist (IL-IRA) is a naturally-occurring, specific inhibitor of IL-1 activity. It is a soluble form of the IL-1 receptor and functions by blocking binding of IL-1 to its cell surface receptor (Dinarello, CA. 1996. J. Amer. Soc. Hematol. 67:2095-2147).

The effect of liposome encapsulated IL-IRA on the formation of close vascular stractures (CVS) around the Theracyte® device was tested. Animals (n=6) were implanted ventral SQ with one 20 μl device containing liposome-encapsulated IL-IRA and one empty device. The devices were explanted after three weeks and examined microscopically for the development of CVS. A positive CVS was scored if a blood vessel was observed within one cell width of the explanted device membrane. Figure 4 illustrates the results. There was a significant difference between the number of CVS observed surrounding empty devices and those containing IL-IRA (p=0.0002). Empty devices had a mean of 35.2 ± 9 CVS while those containing IL-IRA had 9.7 + 7. These data demonstrate that the materials released from the device of the present invention may affect the local environment surrounding the implant site. In particular, the data suggest that IL-1 may play a role in the formation of CVS induced by membrane-driven vascularization.

Example 7 Utilization of Ceramic-Encapsulated Bioactive Agents

The in vitro time course of release of a bioactive agent prepared as a ceramic encapsulate in an implantable chamber is measured. The data thus generated relates the time course of release of the bioactive agent from the chamber when prepared as a ceramic encapsulate. The effect of a ceramic-encapsulated bioactive agent on in situ pre-existing tumors is tested. Animals are implanted after tumor growth with at least one device containing both irradiated tumor cells and a ceramic encapsulated bioactive agent in various combinations as outlined for IL-2 and GM-CSF in Table 1.

The effect of the ceramic-encapsulated bioactive agent to prevent tumor reformation after resection is tested by implantion of a device containing irradiated tumor cells and an injection of bioactive agent at the time of implant. The bioactive agent is also injected at the implant site weekly for several weeks. These animals are then observed for development of tumors and compared to the growth of tumors in control animals that do not receive implants. The effect of a ceramic-encapsulated bioactive agent on the formation of close vascular structures (CVS) around the Theracyte® device is also tested. Animals are implanted ventral SQ with a device containing a ceramic-encapsulated bioactive agent and one empty device. The devices are explanted after several weeks and examined microscopically for the development of CVS. A positive CVS is scored if a blood vessel is observed within one cell width ofthe explanted membrane. The difference between the number of CVS observed surrounding empty devices containing the ceramic-encapsulated bioactive agent is determined. The data demonstrates whether the materials released from the device of the present invention may affect the local environment surrounding the implant site. In particular, the data may suggest that a particular bioactive agent is involved in the formation of CVS.

TABLE 1

Effect of 4.5 μl devices containing irradiated cells and slow release preparations of either IL-2 or GM-CSF on pre-existing tumors

^a One animal died tumor free within a couple of days of surgery. ^bData animals received half the number of cells (1 x 10⁶ total) ^cTwo animals were missing from the cage on day 15 (post implant).

TABLE 2

Effect of 4.5 μl devices containing irradiated cells and slow release preparations of either IL-2 or GM-CSF following tumor resection

Two animals died immediately after surgery due to anesthetic shock.

While a preferred form of the invention has been shown in the drawings and described, variations in the preferred form will be apparent to those skilled in the art. Hence, the invention should not be construed as limited to the specific form shown and described, but instead is as set forth in the claims.

Claims

WHAT WE CLAIM:

1. An implantable system for delivery of a therapeutic material to a mammal in need of such material comprising:

(a) an implantable semi-permeable chamber having a wall which is permeable to the flow of nutrients from the host to the chamber and products from the chamber to the host and impermeable to the host immune cells; and

(b) a therapeutically effective amount of a bioactive agent encapsulated in a material allowing for increased availability in the mammal.

2. The system of claim I wherein said therapeutic material is an immunopotentiating molecule.

3. The system of claim 1 wherein said therapeutic material is a cytokine.

4. The system of claim I wherein said therapeutic material is IL-2.

5. The system of claim I wherein said therapeutic material is GM-CSF.

6. The system of claim I wherein the wall comprises:

(a) a first zone of a first porous material defining a chamber wherein the first porous material is permeable to the flow of nutrients from the host to the chamber and products from the chamber to the host and impermeable to the host immune cells;

(b) a second zone of a second porous material proximal to host tissue, the second porous material having a nominal pore size ranging from about 0.6 to about 20 ╬╝m and comprising frames of elongated strands that are less than 5 ╬╝m in all but the longest dimension wherein said frames define apertures which interconnect to form three dimensional cavities which permit substantially all inflammatory cells migrating into the cavities to maintain a rounded morphology and wherein the second zone promotes vascularization adjacent to but not substantially into the second zone upon implantation into the host.

7. The system of claim I wherein the porous wall comprises a material that is selected from the group consisting of polyethylene, polypropylene, polytetrafluoroethylene (PTFE), cellulose acetate, cellulose nitrate, polycarbonate, polyester, nylon, polysulfone, mixed esters of cellulose, polyvinylidene difluoride, silicone and polyacrylonitrile.

8. The system of claim I wherein the first zone of porous material and second zone of porous material are made ofthe same material.

9. The system of claim I wherein the first zone of porous material and second zone of porous material are made of different material.

10. The system of claim I wherein the first porous material is further impermeable to humoral immune factors.

11. A method of controlled delivery of a therapeutic material to a mammal in need of such material comprising providing an implantable semi- permeable chamber and liposome encapsulated therapeutic material contained therein, the chamber having a wall of sufficient porosity to permit delivery of the therapeutic material in a controlled manner and implanting said chamber in said mammal.

12. The method of claim 11 wherein said therapeutic material is an immunopotentiating molecule.

13. The method of claim 12 wherein said therapeutic material is a cytokine.

14. The method of claim 12 wherein said therapeutic material is IL-2.

15. The method of claim 12 wherein said therapeutic material is GM- CSF.

16. An implantable system for delivery of a therapeutic material to a mammal in need of such material comprising: (a) at least two implantable semipermeable chambers each having a wall which is permeable to the flow of nutrients from the host to the chamber and products from the chamber to the host and impermeable to the host immune cells;

(b) wherein at least one of said chambers comprises a therapeutically effective amount of a bioactive agent encapsulated in a material allowing for increased availability in the mammal; and

(c) wherein at least another of said chambers comprises cells.

17. An implantable system for delivery of a therapeutic material to a mammal in need of such material comprising at least one implantable semi- permeable chamber having a wall which is permeable to the flow of nutrients from the host to the chamber and products from the chamber to the host and impermeable to the host immune cells wherein at least one of said chambers comprises a therapeutically effective amount of a bioactive agent encapsulated in a material allowing for increased availability in the mammal and cells.