WO1997044057A1 - Systeme et procede d'apport de cytokines au moyen de cellules de secretion de cytokines encapsulees - Google Patents

Systeme et procede d'apport de cytokines au moyen de cellules de secretion de cytokines encapsulees Download PDF

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Publication number
WO1997044057A1
WO1997044057A1 PCT/US1997/008475 US9708475W WO9744057A1 WO 1997044057 A1 WO1997044057 A1 WO 1997044057A1 US 9708475 W US9708475 W US 9708475W WO 9744057 A1 WO9744057 A1 WO 9744057A1
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patient
ifn
cells
encapsulated cells
biocompatible
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PCT/US1997/008475
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English (en)
Inventor
Joseph P. Hammang
E. Edward Baetge
Patrick Aebischer
Seth A. Rudnick
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Cytotherapeutics, Inc.
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Application filed by Cytotherapeutics, Inc. filed Critical Cytotherapeutics, Inc.
Priority to AU32074/97A priority Critical patent/AU708536B2/en
Priority to EP97927663A priority patent/EP0906116A1/fr
Priority to CA002252665A priority patent/CA2252665A1/fr
Publication of WO1997044057A1 publication Critical patent/WO1997044057A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0085—Brain, e.g. brain implants; Spinal cord

Definitions

  • This invention relates to controlled, sustained delivery of cytokines I directly to the CNS using encapsulated cytokine-secreting cells
  • Cytokines include interferons, such as IFN- ⁇ , IFN- ⁇ and IFN- ⁇ , which are useful in the treatment of a number of human viral infections and diseases, cancers and for affecting immunomodulation in the patient Other
  • cytokines contemplated here include lvmphokines, interleukins (e g IL-10). and molecules such as the TGF- ⁇ famiK of proteins
  • Cvtokmes have been studied to treat a number of diseases including viral infections (rabies) tumors (hairy cell leukemia), and nervous system disorders of viral or suspected viral etiology (herpes zoster infections, multiple sclerosis and I I subacute sclerosing panencephalitis (SSPE)
  • cytokines do not cross the blood brain barrier well, delivering drugs to their appropriate site of action without adverse side effects is a major challenge Svstemic delivery of cvtokmes for treatment has been problematic In addition to inflammatory responses resulting from administration of cytokines,
  • BetaseronTM an E coli-produced unglycosylated LFN- ⁇ from Berlex/Chiron was approved and launched in 1993 for treating relapsing or remitting multiple sclerosis ("MS”) IFN- ⁇ has demonstrated efficacy in delaying onset of episodes, decreasing the severity of episodes, and slowing growth of lesions.
  • Interferon beta is postulated to boost suppressor T-cell activity/number, thereby inhibiting a subset of myelin basic protein-specific T cells
  • Systemic subcutaneous delivery of BetaseronTM was accompanied by significant side effects like inflammation, pain at the injection site, injection-site necrosis, and flu-like symptoms
  • patient receiving systemic delivery may develop antibodies to LFN- ⁇
  • Such side effects can be quite severe since injections are given every other day.
  • side effects have included abnormal liver function and severe depression
  • the dosage of BetaseronTM administered is typically 8 x 10 6 IU or 250 ⁇ g every other day BetaseronTM is administered with carrier proteins such as 15mg of albumin or dextrose
  • MS is generally characterized by demyelination of CNS axons resulting in focal sclerotic lesions in the spinal cord, optic nerve and brain Symptoms of MS may include muscle weakness, loss of coordination, vertigo, numbness, tingling, speech disturbances, and varying degrees of visual impairment These initial symptoms often progress to acute muscle wasting, loss of vision, confinement to bed or wheelchair, aphasia, paralysis, and ultimately death. MS affects an estimated 250,000 people in the United States and is one of the most common causes of permanent neurological disability among young adults
  • a glycosylated recombinant human IFN- ⁇ , differing by one amino acid from Betaseron, is also expected to be available for treatment of patients that are suffering from chronic and progressive MS
  • the encapsulated cell approach described here is expected to mitigate the foregoing difficulties by local delivery of a much lower dose of a continuously-synthesized cytokine from a retrievable implant directly to the CNS SUMMARY OF THE INVENTION
  • This invention provides novel methods and devices for the delivery of cytokines directly to the central nervous system (“CNS").
  • this invention provides a novel approach employing polymer-encapsulated cells to release human cytokines directly to the CNS.
  • the cells may be xenogeneic or allogeneic.
  • Each device typically contains about 10 3 - 10 7 genetically-modified cells surrounded by a semipermeable membrane, and is implanted directly into the CNS, providing for slow continuous release of at least one cytokine.
  • the preferred implant sites are intraventricular or intrathecal. Other implant sites are also contemplated, provided that delivery is to the central nervous system (“CNS”), and not through a route of administration that leads to the above-mentioned side effects.
  • CNS central nervous system
  • a cytokine is delivered at a dosage sufficient to maintain a measurable concentration in the cerebrospinal fluid ("CSF") that provides a therapeutic effect.
  • CSF cerebrospinal fluid
  • the semipermeable membrane prevents immunologic rejection of the cells and interposes a physical barrier between the encapsulated cells and the patient. Moreover, the device may be retrieved from the patient.
  • Figure 1 is a plasmid map of vector pcDNA3-IFNb 1-123.
  • Figure 2 is a plasmid map of vector pcDNA3-IgSP-LFNb 1-124.
  • This invention contemplates delivery of proteins that, due to their cytokine-like properties, would be expected to produce the above-noted side effects when administered outside the CNS. Delivery of cytokines and like molecules directly to the CNS, may avoid their known and undesired side effects when administered outside the CNS, as well as allow continuous and more consistent dosing.
  • At least one cytokine is delivered intrathecally using encapsulated cells
  • Cells are genetically engineered to stably express and release recombinant human cytokines into the central nervous system ("CNS").
  • the living cells are encapsulated in one or more semipermeable polymer capsules and surgically inserted (under local anesthesia) in the spine, such that the cytokine is delivered into the CNS without having to cross the blood brain barrier
  • Drug can be delivered more consistently than with other dosing methods(e.g , tablets, injections), thus improving efficacy and diminishing side effects
  • cytokine generally means a soluble molecule that mediates interactions between cells and includes the family of molecules typically referred to as cytokines in the art Specifically, cytokines contemplated here include lymphokines, interferons (LFNs), colony stimulating factors (CSFs), interleukins (ILs) (including IL-10), CD antigens and tumour necrosis factors (TNFs) In particular, we prefer delivery of interferons IFN- ⁇ , IFN- ⁇ , IL-10, and the TGF- ⁇ family.
  • LFNs interferons
  • CSFs colony stimulating factors
  • ILs interleukins
  • TNFs tumour necrosis factors
  • Interferons are useful in the treatment of a number of human viruses, cancers and for immunomodulation.
  • the following diseases may be candidates for treatment with LFN- ⁇ or IFN- ⁇ :
  • the supply of IFN- ⁇ was not continuous.
  • the dosage range used was about 100-fold higher than that contemplated in the present invention « leading to potential undesired side effects
  • cytokine Any suitable cytokine may be delivered according to this invention
  • Primary cells that secrete such a cytokine may be used
  • cells may be genetically engineered to secrete such a cytokine, using techniques well known in the art
  • cytokines modified by attachment of one or more polyethylene glycol (PEG) or other repeating polymeric moieties
  • PEG polyethylene glycol
  • full length recombinant human LFN- ⁇ is delivered
  • IFN- ⁇ we prefer delivery of up to 1 4 ⁇ g/day /patient, more preferably between 0 005 - 1 4 ⁇ g/day, most preferably between 0 1-1 0 ⁇ g/day
  • IFN- ⁇ we prefer delivery of up to 0 02 ⁇ g/day/patient, more preferably between 0 0009 - 0 02 ⁇ g/day, most preferably between 0 001-0 01 ⁇ g/day
  • IL- 10 we prefer delivery of up to 21 3 ⁇ g/day/patient, preferably between 1 3-21 3 ⁇ g/day, most preferably between 5 0-10 0 ⁇ g/day
  • TGF- ⁇ we prefer delivery of up to 4.2 ⁇ g/day/patient, preferably between 0.042-4.2 ⁇ g/day, most preferably between 0 1-1.0 ⁇ g/day
  • CSF cerebrospinal fluid
  • IFN- ⁇ When delivered to a site that produces a measureable CSF concentration, we prefer delivery of IFN- ⁇ sufficient to maintain a measurable concentration of up to 10 ng/ml in the CSF, preferably between 0.62-10 0 ng/ml in the CSF When delivered to a site that produces a measureable CSF concentration, we prefer delivery of IFN- ⁇ sufficient to maintain a measurable concentration of up to 0.15 ng/ml in the CSF, preferably between 0 009-0 15 ng/ml in the CSF.
  • LL-10 When delivered to a site that produces a measureable CSF concentration, we prefer delivery of LL-10 sufficient to maintain a measurable concentration of up to 130 ng/ml in the CSF, preferably between 13-130 ng/ml in the CSF.
  • TGF- ⁇ When delivered to a site that produces a measureable CSF concentration, we prefer delivery of TGF- ⁇ sufficient to maintain a measurable concentration of up to 2.6 ng/ml in the CSF, preferably between 0 42-2 6 ng/rril in the CSF.
  • a lower dosage is particularly useful because cytokines are often involved in cell-mediated immune responses
  • cell-mediated immunopathology can occur.
  • a granuloma may cause a bulky space- occupying lesion, impairing the function of sensitive tissues such as brain, retina and nerve.
  • the cell types that can be employed for encapsulated cell therapy within the scope of this invention include cells from allogeneic, autologous and xenogeneic sources.
  • One of the advantages of this encapsulated approach rests with the immunoisolatory properties of the membranes of this invention, and their ability to support cells that otherwise would not be appropriate for transplantation (i.e., non-human sources, immortalized and/or tumor cell lines).
  • a particular advantage to using xenogeneic over allogeneic cells is that in the unlikely event of membrane failure, the xenogeneic cells are more likely to be targeted for destruction by the immune system when compared to allogeneic cells. Furthermore, xenogeneic sources are easy to obtain and their use precludes the necessity for human tissue which is difficult to obtain and whose use is fraught with societal and ethical considerations. In addition, human tissue may contain adventitious agents that are more readily transmitted to the transplantation recipient. Finally, use of xenogeneic tissue and cell lines for transplantation in humans removes the risks associated with the handling and processing of human tissue.
  • the preferred cells chosen for the gene transfer technique are L6 or C2C12 mouse myoblast cells, baby hamster kidney (BHK) cells or rat insuloma cells (RIN) cells.
  • BHK or RIN cells are preferred, a wide variety of cells may be used. These include well known, publicly available immortalized cell lines as well as dividing primary cell cultures. Examples of suitable publicly available cell lines include, Chinese hamster ovary (CHO), mouse fibroblast (L-M), NLH Swiss mouse embryo (NLH/3T3), African green monkey cell lines (including COS-1, COS-7, BSC-1, BSC-40, BMT-10 and Vero), rat adrenal pheochromocytoma (PC 12 and PC12A), AT3, AtT-20, C6 glioma, astrocytes and other fibroblast cell lines.
  • CHO Chinese hamster ovary
  • L-M mouse fibroblast
  • NLH Swiss mouse embryo NLH/3T3
  • African green monkey cell lines including COS-1, COS-7, BSC-1, BSC-40, BMT-10 and Vero
  • PC 12 and PC12A rat adrenal pheochromocytoma
  • AT3, AtT-20 C6 glioma
  • Primary cells that may be used include adrenal chromaffm cells, neural progenitor cells and neural stem cells (Reynolds and Weiss, Science. 255, pp. 1707-1710 (1992); Richards et al., PNAS 89, pp. 8591-8595 (1992); Ray et al., PNAS 90, pp. 3602-3606 (1993)), primary fibroblasts, Schwann cells, ⁇ -TC cells, Hep-G2 cells, oligodendrocytes and their precursors, and the like.
  • a gene of interest i.e., encoding an IFN- ⁇ , IFN- ⁇ , IL-10, or TGF- ⁇
  • a suitable expression vector can be inserted into a suitable expression vector by using standard techniques. It will be appreciated that more than one gene may be inserted into a suitable expression vector.
  • the expression vector containing the gene of interest may then be used to transfect the cell line to be used in the methods of this invention
  • Standard transfection techniques such as calcium phosphate co-precipitation, DEAE-dextran transfection or electroporation may be utilized
  • Commercially available mammalian transfection kits may be purchased from e.g , Stratagene
  • Suitable promoters include, for example, the early and late promoters of SV40 or adenovirus and other known non-retroviral promoters capable of controlling gene expression
  • Useful expression vectors may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences, such as various known derivatives of SV40 and known bacterial plasmids, e g , pUC, pBlueScriptTM plasmids from E. coli including pBR322, pCRl, pMB9, pUC, pBlueScriptTM and their derivatives
  • Expression vectors containing the geneticin (G418) or hygromycin drug selection genes are also useful These vectors can employ a variety of different enhancer/promoter regions to drive the expression of both a biologic gene of interest (e g , IFN- ⁇ , IFN- ⁇ , LL-10 or TGF- ⁇ ) and/or a gene conferring resistance to selection with toxin such as G418 or hygromycin B
  • the promoter is selected from the following group.
  • hDBH human dopamine beta hydoxylase
  • hTH human tyrosine hydroxylase
  • hPNMT human phenylethanaolamine N-methyltransferase
  • mGFAP mouse glial fibrillary acidic protein
  • MBP myelin basic protein
  • rnNF-L mouse neurofilament-light subunit
  • hPo the human promoter for the gene encoding the major myelin glycoprotein in the peripheral nervous system
  • Increased expression can be achieved by increasing or amplifying the copy number of the transgene encoding the desired biologically active molecule(s), using amplification methods well known in the art
  • amplification methods include, e.g., DHFR amplification (see, e.g., Kaufman et al , United States patent 4,470,461) or glutamine synthetase ("GS”) amplification (see, e g., United States patent 5,122,464, and European published application EP 338,841)
  • the human IFN- ⁇ gene was expressed using the commercially available pcDNA-3 expression vector
  • the LFN- ⁇ expression vector was transfected into baby hamster kidney (BHK) cells using a standard calcium/phosphate transfection procedure and selected with increasing concentrations of methotrexate (1 to 200 ⁇ M) over 8 weeks to produce stable expressers Following this selection, the BHK engineered cells were maintained in vitro in 50 ⁇ M methotrexate
  • encapsulation of cell lines engineered in this fashion allows for the selection of cells over several months for stable expression of the DHFR and the IFN- ⁇ gene before transplantation
  • the selected cells can then be analyzed for IFN- ⁇ transgene stability and gene expression by Southern and Northern blot analysis, respectively
  • other suitable molecules may also be delivered to the CNS.
  • Such molecules include growth or trophic factors, other cytokines, lymphokines, hormones, and neurotransmitters.
  • Co-delivery can be accomplished in a number of ways.
  • Cells may be doubly transfected to secrete both molecules of interest.
  • cells may be transfected with a single construct containing both genes.
  • Also contemplated is encapsulation of two separately transfected cells or cell lines, each secreting one of the desired molecules. Separate cell lines can be encapsulated in separate devices and both devices can be implanted in the patient. The cells or cell lines may be the same or different.
  • This invention also contemplates use of a "suicide" gene in the transformed cells.
  • the cell carries the Herpes simplex thymidine kinase gene (HSV-tk) as a safety measure, permitting the encapsulated cells to be killed in vivo by treatment with ganciclovir.
  • HSV-tk Herpes simplex thymidine kinase gene
  • a "suicide" gene is known in the art. See., e.g., Anderson, published PCT application WO 93/10218; Hamre, published PCT application WO 93/02556.
  • the recipient's own immune system provides a first level of protection from adverse reactions to the implanted encapsulated cells if the cells are xenogeneic.
  • the polymer capsule is preferably immunoisolatory (see infra).
  • the presence of the TK gene (or other suicide gene) in the expression construct adds an additional level of safety to the recipient of the implanted cells.
  • the HSV-tk gene is inserted into the cytokine expression construct.
  • This suicide gene allows elimination of the transfected cells upon ganciclovir administration.
  • a typical dosage of ganciclovir required to "kill" the cells is approximately 5 mg/kg.
  • the transduced cells are surrounded with a microporous or permselective membrane which permits the diffusion of small molecules such as nutrients and trophic factors into and out of the polymer capsule.
  • the capsule membrane also permits the molecule of interest to easily diffuse from the capsule into the surrounding host tissue or cerebrospinal fluid.
  • the implant may be retrieved if necessary or desired. Such retrievability may be essential in many clinical situations.
  • Numerous encapsulation devices are known, having various outer surface morphologies. Capsules have been categorized as Type 1 (Tl), Type 2 (T2), Type 1/2 (Tl/2) or Type 4 (T4) depending on their outer surface morphology.
  • a biocompatible capsule means that the capsule, upon implantation in a host mammal, does not elicit a detrimental host response sufficient to result in the rejection of the capsule or to render it inoperable, for example through degradation.
  • an immunoisolatory capsule means that the capsule upon implantation into a mammalian host minimizes the deleterious effects of the host's immune system on the cells within its core.
  • the jacket of the capsule should provide a physical barrier sufficient to prevent detrimental immunological contact between the isolated cells and the host's immune system.
  • the thickness of this physical barrier can vary, but it will always be sufficiently thick to prevent direct contact between the cells and/or substances on either side of the barrier.
  • the thickness of this region generally ranges between 5 and 200 microns; thicknesses of 10 to 100 microns are preferred, and thickness of 20 to 75 microns are particularly preferred.
  • MWCO molecular weight cutoff
  • capsules are suitable for delivery of molecules according to this invention.
  • the capsule of this invention will be similar to those described in U.S. patent nos. 5,158,881, 5,284,761, 5,389,533, 5,283,187, 4,976,859 and 4,968,733, incorporated herein by reference.
  • Useful biocompatible polymer capsules comprise (a) a core which contains a cell or cells, either suspended in a liquid medium or immobilized within an immobilizing biocompatible matrix, preferably comprising a hydrogel or extracellular matrix components, and (b) a jacket comprising a membrane which does not contain isolated cells, and which is biocompatible.
  • the jacket is immunoisolatory and is sufficient to protect the cells in the core from detrimental immunological attack.
  • hydrogel refers to a three dimensional network of cross-linked hydrophilic polymers.
  • the network is in the form of a gel, substantially composed of water, preferably but not limited to gels being greater than 90% water. Compositions which form hydrogels fall into three classes. The first class carries a net negative charge (e.g., alginate).
  • the second class carries a net positive charge (e.g., collagen and laminin).
  • a net positive charge e.g., collagen and laminin.
  • extracellular matrix components include MatrigelTM and VitrogenTM. Fibroblasts generally survive well in a positively charged matrix and are thus suitably enclosed in extracellular-matrix type hydrogels.
  • the third class is net neutral in charge (e.g., highly crosslinked polyethylene oxide, or polyvinylalcohol). Any suitable matrix or spacer may be employed within the core, including precipitated chitosan, synthetic polymers and polymer blends, microcarriers and the like, depending upon the growth characteristics of the cells to be encapsulated.
  • the core may also be formed from cells encapsulated in microspheres.
  • the capsule may have an internal scaffold.
  • the scaffold may prevent cells from aggregating and improve cellular distribution within the device. See PCT publication WO96/02646.
  • polymers and polymer blends can be used to manufacture the capsule jacket, including polyacrylates (including acrylic copolymers), polyvinylidenes, polyvinyl chloride copolymers, polyurethanes, polystyrenes, polyamides, cellulose acetates, cellulose nitrates, polysulfones (including polyethersulfones), polyphosphazenes, polyacrylonitriles, poly(acrylonitrile/covinyI chloride), as well as derivatives, copolymers and mixtures thereof.
  • polyacrylates including acrylic copolymers
  • polyvinylidenes including acrylic copolymers
  • polyvinyl chloride copolymers polyurethanes
  • polystyrenes polyamides
  • cellulose acetates cellulose nitrates
  • polysulfones including polyethersulfones
  • polyphosphazenes polyacrylonitriles
  • poly(acrylonitrile/covinyI chloride) as
  • the capsule can be any configuration appropriate for maintaining biological activity and providing access for delivery of the product or function, including for example, cylindrical, rectangular, disk-shaped, patch-shaped, ovoid, stellate, or spherical. Moreover, the capsule can be coiled or wrapped into a mesh ⁇ like or nested structure. If the capsule is to be retrieved after it is implanted, configurations which tend to lead to migration of the capsules from the site of implantation, such as spherical capsules, are not preferred. Certain shapes, such as rectangles, patches, disks, cylinders, and flat sheets offer greater structural integrity and are preferable where retrieval is desired.
  • the implantable capsule is of a sufficient size and durability for complete retrieval after implantation.
  • Such macrocapsules have a core of a preferable minimum volume of about 1 to lO ⁇ l and depending upon use are easily fabricated to have a volume in excess of 10O ⁇ l.
  • the fiber will have an inside diameter of less than 1500 microns, preferably less than 300-600 microns.
  • the hydraulic permeability will be in the range of
  • glucose mass transfer coefficient of the capsule defined, measured and calculated as described by Dionne et al., ASAIO Abstracts, p. 99 (1993), and
  • any suitable method of sealing the capsules may be used, including the employment of polymer adhesives and/or crimping, knotting and heat sealing. These sealing techniques are known in the art.
  • any suitable "dry” sealing method can also be used. In such methods, a substantially non-porous fitting is provided through which the cell-containing solution is introduced. Subsequent to filling, the capsule is sealed. Such a method is described in copending United States application Serial No. 08/082,407, herein incorporated by reference.
  • the device has a tether that aids in retrieval.
  • tethers are known. See, e.g., WO 92/19195.
  • the encapsulation procedure is as follows:
  • the hollow fibers are fabricated from polyether sulfone (PES) with an outside diameter of 720 ⁇ m and a wall thickness of 100 ⁇ m (AKZO-Nobel)
  • Fiber material is first cut into 5 cm long segments and the distal extremity of each segment were sealed with a photopolymerized acrylic glue (LCM- 25, ICI). Following sterilization with ethylene oxide and outgassing, the fiber segments are loaded with a suspension of about 2 x 10 5 transfected cells/ ⁇ l in a collagen solution (Zyderm® soluble bovine collagen) via a Hamilton syringe and a 25 gauge needle through an attached injection port. The proximal end of the capsule is sealed with the same acrylic glue. A silicone tether (Specialty Silicone Fabrication, Taunton, USA) (LD: 690 ⁇ m; OD: 1.25 ⁇ m) is placed over the proximal end of the fiber allowing easy manipulation and retrieval of the device.
  • a silicone tether (Specialty Silicone Fabrication, Taunton, USA) (LD: 690 ⁇ m; OD: 1.25 ⁇ m) is placed over the proximal end of the fiber allowing easy manipulation and retrieval of the device
  • the methods and devices of this invention are intended for use in a mammalian host, recipient, patient, subject or individual, preferably a primate, most preferably a human.
  • Implantation of the BAO is performed under sterile conditions. Generally, the BAO is implanted at a site in the host which will allow appropriate delivery of the secreted product or function to the host and of nutrients to the encapsulated cells or tissue, and will also allow access to the BAO for retrieval and/or replacement.
  • implantation sites include the central nervous system, including the brain, and spinal cord.
  • Preferred sites in the brain include the striatum, the cerebral cortex, subthalamic nuclei and nucleus Basalis of Maynert.
  • Other preferred sites are the cerebrospinal fluid, most preferably the subarachnoid (intrathecal) space and the lateral ventricles.
  • capsular delivery of LFN- ⁇ , synthesized in vivo, to the brain ventricles, brain parenchyma, the intrathecal space or other suitable CNS location, in a dosage described supra, is desirable.
  • IFN- ⁇ has potential applications based on its anti-viral, anti- cancer, anti-tumor and immunomodulatory activities.
  • Such therapies potentially include malignant diseases such as osteosarcoma, glioma, and Hodgkin's disease, tumor therapy, viral infections, and demyelinating diseases, such as multiple sclerosis.
  • the actual dosage of IFN- ⁇ can be varied by implanting a fewer or greater number of capsules. We prefer implanting between 1 and 10 capsules, most preferably between 1 and 5 capsules.
  • the dose can be varied by increasing/decreasing the number of cells per capsule, as well as the output per cell (including choosing cells with a different output of IFN- ⁇ ), or any other suitable method.
  • capsular delivery of LFN- ⁇ synthesized in vivo, to the brain ventricles, brain parenchyma, the intrathecal space or other suitable CNS location, in a dosage described supra is desirable.
  • the actual dosage of LFN- ⁇ can be varied by implanting a fewer or greater number of capsules or other suitable method.
  • this embodiment is used in the treatment or prophylaxis of acute leukaemia, Hodgkin's disease, hepatitis, herpes keratitis, adenovirus conjunctivitis, ocular infections, infections caused by Epstein-Barr virus, cytomeglo virus, varicella zoster, herpes simplex virus- 1, herpes labialis, chronic hepatitis B infections, warts, condyloma acuminatum, juvenile laryngeal papillomatosis, hairy cell leukemia, coryza, subacute sclerosing panencephalitis (SSPE), amyotrophic lateral sclerosis, and malignant gliomas.
  • acute leukaemia Hodgkin's disease, hepatitis, herpes keratitis, adenovirus conjunctivitis, ocular infections, infections caused by Epstein-Barr virus, cytomeglo virus, varicella zoster
  • capsular delivery of IL-10 synthesized in vivo to the brain ventricles, parenchyma, the intrathecal space or other suitable CNS location in a dosage described supra is desirable.
  • the actual dosage of IL-10 can be varied by implanting a fewer or greater number of capsules.
  • this embodiment is used in the treatment of inflammatory disease.
  • capsular delivery of TGF- ⁇ synthesized in vivo to the brain ventricles, parenchyma, the intrathecal space or other suitable CNS location in a dosage described supra is desirable.
  • the actual dosage of TGF- ⁇ can be varied by implanting a fewer or greater number of capsules.
  • this embodiment is used in the treatment of multiple sclerosis, or inflammatory diseases.
  • Plasmid pLG104R containing the pre-interferon ⁇ l cDNA was obtained from American Type Culture Collection (ATCC, Rockville, Maryland). Due to the lack of convenient restriction sites in pLG104R, the preLFN ⁇ l coding region was generated by polymerase chain reaction (PCR) using oligonucleotides ohIFNBl-219 (SEQ. LD NO: 1 :
  • PCR reaction mixture containing 10 mM Tris.HCl (pH 8 3), 50 mM KC1, 800 of each nM dNTP, 2 mM MgCl 2 , 400 nM of primers ohIFN ⁇ l-219 and ohIFN ⁇ 1-220, and 2.5 units of Thermus aquatics (Taq) DNA polymerase (Boehringer Mannheim, Germany)
  • the PCR reaction mixtures were subjected to 30 amplification cycles consisting of.
  • the 589 bp pre-IFNBl PCR fragment was digested with restriction endonucleases BamHI and Hindlll and resolved on an 1% Trivie agarose gel (TrivieGen)
  • the 589-bp Hindlll/BamHI DNA fragment was excised and purified using the FMC SpinBind DNA purification kit (FMC BioProducts, Rockland, ME)
  • pcDNA3 expression vector was also digested with BamHI and Hindlll and purified from 1% SeaPlaque agarose (FMC BioProducts, Rockland, ME) using the FMC SpinBind DNA purification kit (FMC BioProducts, Rockland, ME)
  • the ligation mixture was transformed into E Coli DH5 ⁇ (Gibco BRL,
  • Oligonucleotides ohLFNB 1-221 (SEQ LD NO. 3 5'- CCC AAGCTTGCGTC ACCCCT AGAGTCGAGCTGT-3 ') and ohIFN ⁇ 1 -220 (SEQ LD NO: 2: 5'-CCCGGATCCTCAGTTTCGGAGGTAACCTGT-3') are specific for the IgG signal peptide sequence (IgSP) and the mature IFN ⁇ 1 sequence, respectively, and contain synthetic Hindlll and BamHI restriction sites at the 5' end, respectively Oligonucleotides ohLFN ⁇ 1-222 (SEQ ID NO 4 5'- GTTGTAGCTC ATCCTCTTGAACTCCAGGGG-3') and ohIFN ⁇ 1 -223 (SEQ ID NO.
  • oligonucleotide ohlFNB 1-222 has its 5 1 16 nucleotides identical to the IgSP sequence and its 3' 18 nucleotides identical to the mature IFNBl, and vice versa for ohLFNB 1-223
  • the first two PCR reactions were carried out using oligonucleotide pairs ohLFNBl -221/ohIFNB 1-222 and ohLFNB 1-223/ohTFNB 1-220 on templates pBS-IgSP-hPOMC ⁇ ACTH-029 and pLG104R plasmids, respectively
  • One hundred ng of template DNA was added to a 50 ⁇ l PCR reaction mixture containing 10 mM Tris HC1 (pH 8 3), 50 mM KC1, 800 of each nM dNTP, 2 mM MgC12, 400 nM of primers #1 and #2, and
  • the second PCR reaction was subjected to 30 amplification cycles consisting of: denaturation, 94 °C for 30 seconds (first cycle 2 minutes), annealing, 60°C 30 seconds (second to fourth cycles 37 °C 2 minutes), and extension, 72 C C 30 seconds (last cycle 2 minutes)
  • the 725 bp IgSP-mature IFN ⁇ 1 fusion PCR product and the cloning vectors pcDNA3 were digested with BamHI and Hindlll restriction enzymes and subsequently purified from 1% SeaPlaque agarose gel using the FMC SpinBind DNA purification kit (FMC BioProducts, Rockland, ME)
  • the ligation mixture was transformed into E. coli DH5 ⁇ (Gibco BRL, Gaithersburg, MD).
  • a cracking gel procedure Promega Protocols and Applications
  • the final plasmid constructions discussed above were all amplified in a standard E.coli strain (HB 1O1) and purified by the Qiagen-Plasmid Kit (Kontron)
  • the final plasmid is transfected into a line of baby hamster kidney cells (BHK) using standard calcium phosphate methodology
  • Gene amplification is performed in increasing concentrations of methotrexate (1-200 ⁇ M) over 8 weeks to produce stable amplified cell lines
  • the engineered BHK cells are maintained in vitro in 50-200 ⁇ M MTX IFN expression is observed in the absence of drug selection over three months inclusive, when assayed by Northern Blot analysis, bioassay, or ELISA
  • the encapsulation of cell lines engineered in this fashion allows for the selection of cells over several months for stable expression of the DHFR gene and gene of interest before transplantation
  • the selected cells can then be analyzed for transgene stability and gene expression by Southern and Northern blot analysis, respectively
  • the level of human IFN expression from each capsule is assayed (using ELISA or bioassay) before implantation
  • the protein is biologically active as determined by a bioassay Encapsulation
  • the encapsulation procedure is as follows:
  • the hollow fibers are fabricated from polyether sulfone (PES) with an outside diameter of 720 ⁇ m and a wall thickness of a 100 ⁇ m (AKZO-Nobel Wuppertal, Germany). These fibers are described in United States patents 4,976,859 and 4,968,733, herein incorporated by reference. In some studies, we prefer a PES#5 membrane which has a MWCO of about 280 kd. In other studies, we contemplate using a PES#8 membrane which has a MWCO of about 90 kd.
  • the devices we contemplate for these studies comprise: 1) a semipermeable poly (ether sulfone) hollow fiber membrane fabricated by AKZO Nobel Faser AG;
  • the preferred semipermeable PES#5 membrane has the following characteristics:
  • the components of the device are commercially available.
  • the LCM glue is available from Ablestik Laboratories (Newark, DE); Luxtrak Adhesives LCM23 and LCM24).
  • the tether material is available from Specialty Silicone Fabricators
  • the tether dimensions are 0.79 mm OD x 0.43 mm ID x length
  • Fiber material is first cut into 5 cm long segments and the distal extremity of each segment are sealed with a photopolymerized acrylic glue (LCM- 25, ICI).
  • a photopolymerized acrylic glue (LCM- 25, ICI).
  • the fiber segments are loaded with approximately 1 x 10 6 transfected cells in a collagen solution (Zyderm® soluble bovine collagen) via a Hamilton syringe and a 25 gauge needle through an attached injection port
  • the proximal end of the capsule is sealed with the same acrylic glue
  • the collagen matrix may also be formed from ZyplastTM or any other suitable matrix forming material
  • a silicone tether (Specialty Silicone Fabrication, Taunton, MA) (LD
  • the subarachnoid space is punctured with a 25G Tuohy needle and 12 ml of CSF is withdrawn A guide wire is introduced through the needle, and the needle is then retrieved A dilator is introduced over the guide wire to widen the ligamentum flavum The dilator is retrieved, and a cannula (4F) is introduced over the guide wire The guide wire is then retrieved The capsule is then pushed through the cannula and positioned in the subarachnoid space Finally, the cannula is retrieved See, e g , WO 94/15663 and U S patent 5,487,739, incorporated herein by reference If more than one capsule is implanted, the same procedure is repeated
  • the silicone tether is fixed at the lumbar fascia with 4-0 polypropylene (Prolene®)
  • the skin is closed with interrupted 4-0 nylon suture (Dermalon®)
  • the skin is re-opened at the same location and the 4-0 polypropylene is sectioned.
  • the capsule or capsules are retrieved by gently pulling on the silicone tether.
  • the biocompatibility, the histology and the cytokine release of the explanted capsule or capsules is examined before the re-implantation of one or more new devices in the patient, following the same protocols.
  • the patients are evaluated for side effects such as cough, weight loss, stomatitis, asthenia, and fever.

Abstract

Procédé et système d'administration continue de cytokines à un patient au moyen d'une capsule biocompatible contenant des cellules de sécrétion de cytokines implantée directement dans le système nerveux central.
PCT/US1997/008475 1996-05-21 1997-05-21 Systeme et procede d'apport de cytokines au moyen de cellules de secretion de cytokines encapsulees WO1997044057A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU32074/97A AU708536B2 (en) 1996-05-21 1997-05-21 System and method for delivery of cytokines using encapsulated cytokine-secreting cells
EP97927663A EP0906116A1 (fr) 1996-05-21 1997-05-21 Systeme et procede d'apport de cytokines au moyen de cellules de secretion de cytokines encapsulees
CA002252665A CA2252665A1 (fr) 1996-05-21 1997-05-21 Systeme et procede d'apport de cytokines au moyen de cellules de secretion de cytokines encapsulees

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US65190096A 1996-05-21 1996-05-21
US08/651,900 1996-05-21

Publications (1)

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AU (1) AU708536B2 (fr)
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Cited By (3)

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Publication number Priority date Publication date Assignee Title
WO2005039622A2 (fr) * 2003-10-21 2005-05-06 Medtronic Minimed, Inc. Modulation de l'expression de socs dans des schemas posologiques therapeutiques
US10653619B2 (en) 2009-03-23 2020-05-19 Medtronic, Inc. Drug depots for treatment of pain and inflammation
USRE48948E1 (en) 2008-04-18 2022-03-01 Warsaw Orthopedic, Inc. Clonidine compounds in a biodegradable polymer

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Publication number Priority date Publication date Assignee Title
JPS6110514A (ja) * 1984-06-26 1986-01-18 Toyo Tire & Rubber Co Ltd 生体内に生理活性物質を徐放する医薬
EP0290891A1 (fr) * 1987-04-29 1988-11-17 Massachusetts Institute Of Technology Système à libération retardée d'un médicament pour le traitement des troubles nerveux
EP0412554A2 (fr) * 1989-08-10 1991-02-13 Sumitomo Pharmaceuticals Company, Limited Préparation à libération retardée À  administrer dans le cerveau
WO1991010425A1 (fr) * 1990-01-08 1991-07-25 Brown University Research Foundation Systemes d'extrusion de capsules de cellules
WO1995028480A1 (fr) * 1994-04-15 1995-10-26 Biohybrid Technologies, Inc. Procedes d'utilisation de particules de gel non enrobees
WO1996040871A1 (fr) * 1995-06-07 1996-12-19 Cytotherapeutics, Inc. Organes bioartificiels comprenant des membranes a pores ouverts, semblables a de la mousse

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Publication number Priority date Publication date Assignee Title
JPS6110514A (ja) * 1984-06-26 1986-01-18 Toyo Tire & Rubber Co Ltd 生体内に生理活性物質を徐放する医薬
EP0290891A1 (fr) * 1987-04-29 1988-11-17 Massachusetts Institute Of Technology Système à libération retardée d'un médicament pour le traitement des troubles nerveux
EP0412554A2 (fr) * 1989-08-10 1991-02-13 Sumitomo Pharmaceuticals Company, Limited Préparation à libération retardée À  administrer dans le cerveau
WO1991010425A1 (fr) * 1990-01-08 1991-07-25 Brown University Research Foundation Systemes d'extrusion de capsules de cellules
WO1995028480A1 (fr) * 1994-04-15 1995-10-26 Biohybrid Technologies, Inc. Procedes d'utilisation de particules de gel non enrobees
WO1996040871A1 (fr) * 1995-06-07 1996-12-19 Cytotherapeutics, Inc. Organes bioartificiels comprenant des membranes a pores ouverts, semblables a de la mousse

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005039622A2 (fr) * 2003-10-21 2005-05-06 Medtronic Minimed, Inc. Modulation de l'expression de socs dans des schemas posologiques therapeutiques
WO2005039622A3 (fr) * 2003-10-21 2005-06-16 Medtronic Minimed Inc Modulation de l'expression de socs dans des schemas posologiques therapeutiques
US8034764B2 (en) 2003-10-21 2011-10-11 Medtronic Minimed, Inc. Modulation of SOCS expression in therapeutic regimens
USRE48948E1 (en) 2008-04-18 2022-03-01 Warsaw Orthopedic, Inc. Clonidine compounds in a biodegradable polymer
US10653619B2 (en) 2009-03-23 2020-05-19 Medtronic, Inc. Drug depots for treatment of pain and inflammation

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AU708536B2 (en) 1999-08-05
AU3207497A (en) 1997-12-09
CA2252665A1 (fr) 1997-11-27

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