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WO2002038130A1 - Chambre utilisee pour le criblage in vivo de composes modulant l'angiogenese et la croissance tumorale - Google Patents

Chambre utilisee pour le criblage in vivo de composes modulant l'angiogenese et la croissance tumorale Download PDF

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Publication number
WO2002038130A1
WO2002038130A1 PCT/US2001/047124 US0147124W WO0238130A1 WO 2002038130 A1 WO2002038130 A1 WO 2002038130A1 US 0147124 W US0147124 W US 0147124W WO 0238130 A1 WO0238130 A1 WO 0238130A1
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WIPO (PCT)
Prior art keywords
chamber
fibrin
matrix composition
tissue growth
porous surfaces
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PCT/US2001/047124
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English (en)
Inventor
Zishan Haroon
Charles S. Greenberg
Mark W. Dewhirst
Original Assignee
Duke University
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Publication date
Application filed by Duke University filed Critical Duke University
Priority to US10/416,603 priority Critical patent/US20040043462A1/en
Priority to AU2002227294A priority patent/AU2002227294A1/en
Publication of WO2002038130A1 publication Critical patent/WO2002038130A1/fr

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    • 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
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue

Definitions

  • the present invention pertains generally to methods and articles for in vivo screening of candidate compounds. More particularly, the present invention pertains to a chamber that can be used to study angiogenesis and/or tumor growth in vivo and to evaluate candidate compounds for an ability to modulate angiogenesis and/or tumor growth.
  • T-ZC - tumor cell-containing chambers ⁇ l - microliter(s)
  • VEGF vascular endothelial growth factor
  • angiogenesis plays a role in tissue growth, and in particularly wound healing as an example of tissue growth.
  • angiogenesis means the generation of new blood vessels into a tissue or organ.
  • angiogenesis is normally observed in wound healing, fetal and embryonal development and formation of the corpus luteum, endometrium and placenta. Uncontrolled angiogenesis is associated with tumor metastasis
  • Angiogenic stimulants induce the endothelial cells to migrate through the eroded basement membrane.
  • the migrating cells form a "sprout" off the parent blood vessel, where the endothelial cells undergo mitosis and proliferate.
  • the endothelial sprouts merge with each other to form capillary loops, creating the new blood vessel.
  • Persistent, unregulated angiogenesis occurs in a multiplicity of disease states, and abnormal growth by endothelial cells supports the pathological damage seen in these conditions.
  • the diverse pathological disease states in which unregulated angiogenesis is present have been grouped together as angiogenic-dependent or angiogenic-associated diseases. It is also recognized that angiogenesis plays a major role in the metastasis of a cancer. If this angiogenic activity could be repressed or eliminated, then the tumor, although present, would not grow. In the disease state, prevention of angiogenesis could avert the damage caused by the invasion of the new microvascular system. Therapies directed at control of the angiogenic processes could lead to the abrogation or mitigation of these diseases.
  • chamber 10 comprises a housing 12, a lower cover slip 14 and an upper cover slip 16.
  • a plurality of regularly spaced pores 18, numbering only ten or less, are formed in upper cover slip 16 using a 20-gauge needle. Pores 18 are about 0.8 mm in diameter. A fibrin gel is added to the internal void space 20 of the chamber 10. Thus, pores 18 are present on only one side, which severely limits applications for chamber 10. Moreover, while angiogenesis was studied by Dvorak et al. via implantation of chamber 10 in an animal subject, the focus of the assay methods were for use in demonstrating the contributions of fibrinogen and related proteins to angiogenesis. Therefore, chamber 10 of Dvorak et al.
  • chamber 10 of Dvorak et al. has never been used and indeed, cannot be reliably used to grow tumors or any other tissue because the presence of pores 18 on only one side creates long oxygen diffusion distances and limited access to blood vessels.
  • a chamber for in vivo delivery of an active agent comprises: (a) a housing having at least two porous surfaces, the at least two porous surfaces disposed on substantially opposite sides of the housing from each other; (b) an internal void space within the housing; and (c) a matrix composition comprising an active agent, the matrix composition disposed within the internal void space.
  • the matrix composition further comprises a tissue growth modulating agent.
  • the tissue growth modulating agent is fibrin
  • the matrix composition further comprises a stabilizing agent.
  • the matrix composition comprises an effective amount of a tissue growth modulating agent and a stabilizing agent in an amount sufficient to retard degradation of the tissue growth modulating agent.
  • the tissue growth modulating agent comprises fibrin.
  • the tissue growth modulating agent comprises fibrin and the stabilizing agent comprises phenylmethylsulfonylfluoride (PMSF), N-caproic acid, or combinations thereof.
  • PMSF phenylmethylsulfonylfluoride
  • a method of screening a candidate compound for tissue growth modulating activity is also disclosed.
  • the method comprises: (a) providing a chamber comprising: (i) a housing having at least two porous surfaces, the at least two porous surfaces disposed on substantially opposite sides of the housing from each other, (ii) an internal void space within the housing, and (iii) a matrix composition comprising a tissue growth modulating agent, the matrix composition disposed within the internal void space; (b) implanting the chamber into a test animal; (c) administering a candidate compound to the test animal; (d) extracting the chamber after a time suitable for measurement of tissue growth; and (e) evaluating tissue growth in the chamber to thereby determine the tissue growth modulating activity of the candidate compound.
  • the tissue growth modulating agent is fibrin
  • the matrix composition further comprises a stabilizing agent.
  • a method of generating tissue growth in a vertebrate animal comprises: (a) providing a chamber comprising: (i) a housing having at least two porous surfaces, the at least two porous surfaces disposed on substantially opposite sides of the housing from each other, (ii) an internal void space within the housing, and (iii) a matrix composition comprising an tissue growth modulating agent, the matrix composition disposed within the internal void space; (b) implanting the chamber in the vertebrate animal; and (c) generating tissue growth in the vertebrate animal through the implanting of the chamber.
  • Figure 1 is a top perspective view of a prior art chamber 10 as disclosed by Dvorak, H. F., et al., Laboratory Investigation 57(6): pp. 673- 686 (1987).
  • Figure 2 is a side elevation view of a chamber 110 of the present invention.
  • Figure 3 is a cross sectional view along port 120 of chamber 110 of the present invention.
  • Figure 4 is a cross sectional view along port 120 of chamber 110 of the present invention, wherein chamber 110 further comprises matrix composition 122.
  • Figure 5 is a top perspective view of chamber 110 of the present invention.
  • Figure 6 is a bottom perspective view of chamber 110 of the present invention.
  • Figure 7 is a schematic diagram depicting the role of fibrin in wound healing.
  • Figure 8 is a schematic diagram depicting the role of fibrin in wound healing and in tumor growth.
  • Figure 9 is a line graph depicting that there was no significant body weight lost with SUGEN 5416 treatment and chamber implantation in test animals (solid line) as compared to control animals (broken line).
  • Figure 10 is a bar graph depicting that SUGEN 5416 treatment causes significant tumor growth delay in test animals (shaded bar) as compared to control animals (open bar).
  • Figure 11 is a bar graph depicting that microvessel density did not change with SUGEN 5416 treatment in tumor cell containing chambers of the present invention isolated from test animals (shaded bar) as compared to control animals (open bar).
  • Figure 12 is a line graph depicting more residual D-dimer retention in tumor cell-containing chambers of the present invention isolated from test animals treated systemically with SUGEN 5416 (solid line) as compared to control animals (broken line).
  • Figure 14 is a bar graph depicting that SUGEN 5416 inhibited granulation tissue formation in fibrin-containing chambers of the present invention implanted in test animals (shaded bar) as compared to fibrin- containing chambers of the present invention implanted in control animals (open bar).
  • Figure 15 is a bar graph depicting that SUGEN 5416 inhibited neovascularization in fibrin-containing chambers of the present invention from test animals (shaded bar) as compared to fibrin-containing chambers of the present invention from control animals (open bar).
  • Figure 16 is a line graph depicting an increase in residual D-dimer retention in fibrin-containing chambers of the present invention implanted in animals treated with SUGEN 5416 (solid line) as compared to fibrin- containing chambers of the present invention implanted in control animals
  • Figures 17A and 17B are photographs showing gross examination of fibrin-containing chambers of the present invention as employed in Laboratory Examples 7-9, showing more influx of blood vessels in the controls (Figure 17A) than SU5416 treated chambers that appeared paler in color ( Figure 17B). Fibrin is inherently pale yellow in color and lack of blood vessels in SU5416 treated chambers results in the paler appearance of the chambers. Arrows indicate apparent blood vessel growth.
  • the granulation tissue in controls ( Figure 17C) is distinctly more than SU5416 treated chambers ( Figure 17D).
  • Figures 17G and 17H are photomicrographs showing that TG activity results in formation of isopeptide bonds that can be probed with a specific monoclonal antibody. Decreased isopeptide bond formation was found in extracellular matrix of SU5416 treated fibrin-containing chambers of the present invention as employed in Laboratory Examples 7-9 ( Figure 17H) in comparison to controls ( Figure 17G).
  • Figure 19 depicts a Western blot for TG. Control tissues show the full length TG at 80kd and multiple fragments that are typical in wound healing tissues for this enzyme. Tissues from SU5416 treated fibrin-containing chambers of the present invention as employed in Laboratory Examples 7-9 exhibit only one band for full length TG with no fragments suggestive of occupation of its nucleotide binding site.
  • Figures 20A and 20B are photographs showing gross examination of fibrin-containing chambers of the present invention as employed in Laboratory Example 10, showing more influx of blood vessels in the controls ( Figure 20A) than angiostatin treated chambers (1 ⁇ M angiostatin) that appeared paler in color ( Figure 20B). Fibrin is inherently pale yellow in color and lack of blood vessels in angiostatin treated chambers results in the paler appearance of the chambers.
  • Figures 20C and 20D are photomicrographs showing depth (represented by line with arrowheads at each end) of granulation tissue developed inside fibrin-containing chambers of the present invention as employed in Laboratory Example 10, which was used as a measure for the healing response.
  • the granulation tissue in controls ( Figure 20C) is distinctly more than angiostatin treated chambers (1 ⁇ M angiostatin) ( Figure 20D).
  • Figure 21 is a bar graph depicting that angiostatin inhibited granulation tissue formation in fibrin-containing chambers of the present invention implanted in test animals (shaded bar) as compared to fibrin- containing chambers of the present invention implanted in control animals (open bar).
  • Scale depth of granulation tissue (X10 microns).
  • Figures 22A and 22B are photomicrographs showing depth (represented by line with arrowheads at each end) of granulation tissue developed inside fibrin-containing chambers of the present invention as employed in Laboratory Example 11 , which was used as a measure for effect on tumor growth.
  • the granulation tissue in controls ( Figure 22A) is observably different than in SOD mimetic treated chambers ( Figure 22B).
  • Scale depth of granulation tissue (X10 microns); p ⁇ 0.01.
  • the present invention pertains to a chamber that can be used to deliver an active agent in vivo.
  • the chamber is employed in an in vivo screening assay for novel compounds that modulate tissue growth, such as but not limited to compounds that enhance or inhibit angiogenesis and/or compounds that inhibit the growth of neoplastic tissue.
  • a method of generating tissue growth in a vertebrate animal is thus also provided in accordance with the present invention.
  • the present invention also provides an in vivo screening assay method that can be used to identify compounds that enhance angiogenesis or to identify compounds that inhibit angiogenesis.
  • the present invention provides the first in vivo assay that can be used to evaluate angiogenesis enhancing compounds that has been provided in the art.
  • a chamber of the present invention preferably comprises a fibrin containing matrix composition in that fibrin is a preferred tissue growth modulating agent when the desired tissue growth is blood vessel growth.
  • the present invention provides an in vivo screening assay method for compounds that inhibit tumor growth or that inhibit the growth of new blood vessels to a tumor.
  • the present invention provides a screening assay method that can also be used to identify antiangiogenic agents and antitumor growth agents.
  • a candidate compound in each embodiment of the assay method of the present invention, can be administered to a test animal subject either systemically or locally by including the candidate compound within the matrix composition in a chamber of the present invention.
  • the chamber of the present invention provides for the use of small amounts of candidate compound, which can be very beneficial in the case of a rare, scarce and/or expensive candidate compound.
  • the ability to evaluate the activity of one or more candidate compounds in an in vivo setting can alleviate the currently observed bottleneck in the drug development process at the preclinical and clinical stages. Accordingly, the chamber and in vivo tissue growth assay method of the present invention solve a long felt and continuing need in the art.
  • a chamber of the present invention comprises a fibrin containing matrix composition, and the matrix composition also comprises a cell or cells.
  • active agent refers to compounds, molecules, or other substances that modulate, mediate, impart or otherwise affect responses or signals in a biological system in vitro or in vivo.
  • a representative active agent comprises a tissue growth modulating agent.
  • a cell can also comprise an active agent in that a cell is capable of sending and receiving chemotactic, chemokinetic and other biological signals and responses.
  • tissue growth modulating agent is meant to refer to an active agent that acts to stimulate or inhibit the growth of cells or tissues in culture or in vivo. Such an agent can thus also be referred to as a "tissue growth stimulating agent” or as a “tissue growth inhibiting agent”.
  • a preferred "tissue growth modulating agent” is an active agent that modulates (i.e. stimulates or inhibits) angiogenesis.
  • an agent is also referred to herein as an “angiogenesis modulating agent", or depending on the activity of the agent, as an “angiogenesis stimulating agent” or as an “angiogenesis inhibiting agent”.
  • cytokine refers to any secreted polypeptide that affects the functions of cells and is a molecule that modulates interactions between cells in the immune, inflammatory or hematopoietic response.
  • a cytokine includes, but is not limited to, monokines and lymphokines, regardless of which cells produce them.
  • a monokine is generally referred to as being produced and secreted by a mononuclear cell, such as a macrophage and/or monocyte.
  • Lymphokines are generally referred to as being produced by lymphocyte cells.
  • cytokines include, but are not limited to, Interleukin-1 (IL-1), lnterleukin-6 (IL- 6), Tumor Necrosis Factor-alpha (TNF- ⁇ ) and Tumor Necrosis Factor beta (TNF- ⁇ ).
  • chemokine refers to any secreted polypeptide that affects the functions of cells and is a molecule which modulates interactions between cells in the immune, inflammatory or hematopoietic response, similar to the term “cytokine” above.
  • a chemokine is primarily secreted through cell transmembranes and causes chemotaxis and activation of specific white blood cells and leukocytes, neutrophils, monocytes, macrophages, T-cells, B-cells, endothelial cells and smooth muscle cells.
  • chemokines include, but are not limited to, interleukin-8 (IL-8), interleukin-12 (IL-12), neutrophil attractant/activation protein-2 (NAP-2), growth regulated chemokine (GRO) ⁇ , ⁇ and y, interferon- gamma induced protein 10 kD (IP-10), macrophage inflammatory protein (MIP) -1a and -1b, and monocyte chemoattracctant protein (MCP) 1 , 2 and 3.
  • IL-8 interleukin-8
  • IL-12 interleukin-12
  • NAP-2 neutrophil attractant/activation protein-2
  • GRO growth regulated chemokine
  • IP-10 interferon- gamma induced protein 10 kD
  • MIP macrophage inflammatory protein
  • MCP monocyte chemoattracctant protein
  • neoplasm is meant to refer to an abnormal mass of tissue or cells. The growth of these tissues or cells exceeds and is uncoordinated with that of the normal tissues or cells and persists in the same excessive manner after cessation of the stimuli that evoked the change.
  • neoplastic tissues or cells show a lack of structural organization and coordination relative to normal tissues or cells that usually result in a mass of tissues or cells that can be either benign or malignant.
  • Representative neoplasms thus include all forms of cancer, benign intracranial neoplasms, and aberrant blood vessels such as arteriovenous malformations (AVM), angiomas, macular degeneration, and other such vascular anomalies.
  • AVM arteriovenous malformations
  • angiomas macular degeneration
  • macular degeneration macular degeneration
  • neoplasm includes any neoplasm, including particularly all forms of cancer. This includes, but is not limited to, melanoma, adenocarcinoma, malignant glioma, prostatic carcinoma, kidney carcinoma, bladder carcinoma, pancreatic carcinoma, thyroid carcinoma, lung carcinoma, colon carcinoma, rectal carcinoma, brain carcinoma, liver carcinoma, breast carcinoma, ovary carcinoma, and the like. This also includes, but is not limited to, solid tumors, solid tumor metastases, angiofibromas, retrolental fibroplasia, hemangiomas, Karposi's sarcoma and the like cancers which require neovascularization to support tumor growth.
  • treating a neoplasm includes, but is not limited to, halting the growth of the neoplasm, killing the neoplasm, reducing the size of the neoplasm, or obliterating a neoplasm comprising a vascular anomaly.
  • Halting the growth of the neoplasm refers to halting any increase in the size of the neoplasm or the neoplastic cells, or halting the division of the neoplasm or the neoplastic cells.
  • Reducing the size of the neoplasm relates to reducing the size of the neoplasm or the neoplastic cells.
  • compositions, carriers, diluents and reagents are used interchangeably and represent that the materials are capable of administration to or upon a vertebrate animal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.
  • candidate compound or “candidate substrate” is meant to refer to any compound wherein the characterization of the compound's ability to modulate tissue growth, and preferably to modulate angiogenesis, is desirable.
  • candidate compounds or substrates include xenobiotics such as drugs and other therapeutic agents, carcinogens and environmental pollutants, as well as endobiotics such as steroids, fatty acids and prostaglandins.
  • endothelium means a thin layer of flat epithelial cells that lines serous cavities, lymph vessels, and blood vessels.
  • target cell and “target tissue” refer to a cell or to a tissue for which it is desired to produce a chemotactic, chemokinetic and other biological signal or response.
  • a “target tissue” can be a neoplastic tissue in which it is desired to retard or inhibit angiogenesis.
  • a “target tissue” can comprise a tissue in which stimulation of tissue growth is desired, such as an injured or diseased tissue.
  • a “target cell” is thus preferably a cell within such tissues.
  • chamber 110 comprises a housing 112. Chamber 110 further comprises at least two porous surfaces
  • housing 112 can thus serve as a support that is disposed between porous surfaces 114 and 116.
  • housing 112 comprises a ring.
  • housing or ring 112 contacts porous surfaces 114 and 116 along a periphery of porous surfaces 114 and 116.
  • Ring 112 can have an inside diameter ranging from about 5 to about 15 millimeters, and corresponding outside diameters ranging from about 6 to about 20 millimeters.
  • ring 112 can have a width ranging from about 1 to about 5 mm.
  • ring 112 comprises an inert, non-immunogenic, non-pyrogenic material suitable for implantation into an animal subject.
  • ring 112 can comprise a metal (e.g., gold), a plastic material, a fiberglass material, a resinous material such as that sold under the registered trademark PLEXIGLAS® by Rohm & Haas of Philadelphia, Pennsylvania, or other suitable material.
  • surfaces 114 and 116 are preferably substantially permeable for the entire area of surfaces 114 and 116, i.e., an area extending substantially to the perimeter of each surface.
  • surfaces 114 and 116 can comprise a mesh.
  • the mesh can comprise any inert, non-immunogenic, non-pyrogenic material suitable for implantation into an animal subject, such as nylon, cotton, polyester (e.g., that sold under the registered trademark DACRON® by E.I. du Pont de Nemours and Company of Wilmington, Delaware), or other natural or other synthetic fiber.
  • Preferred meshes comprise a pore size ranging from about 150 to about 200 micrometers. The use of a mesh facilitates removal of tissue from chamber 110, as discussed in the Laboratory Examples.
  • chamber 110 further comprises an internal void space
  • void space 118 defined by ring 112 and porous surfaces 114 and 116.
  • the depth of internal void space 118 ranges from about 1 to about 3 millimeters (which corresponds to the height of ring 112).
  • the depth of internal void space 118 is about 2 mm.
  • Internal void space 118 is accessible via a port 120 that proceeds from the exterior of chamber 110 to internal void space 118 of chamber 110.
  • the diameter of port 120 preferably ranges from about 1 millimeter to about 3 millimeters.
  • a matrix composition 122 is loaded into chamber 110 via port 120.
  • Matrix composition 122 comprises an active agent as defined herein.
  • port 120 can be sealed closed, such as via an adhesive or heat, after loading matrix composition 122 into chamber 110.
  • matrix composition 122 can be loaded into chamber 110 prior to attaching one of the porous surfaces 114 or 116 and port 120 can be omitted from chamber 110.
  • the chamber 110 is prepared and loaded with matrix composition 122, it can be treated in accordance with any standard sterilization technique prior to use in an animal as described below.
  • chamber 110 comprises a matrix composition 122 that can optionally be loaded into chamber 110 via port 120.
  • Matrix composition 122 comprises an active agent as defined herein. As disclosed in the Laboratory Examples, it is preferred that the matrix composition comprises a gel.
  • matrix composition 122 can comprise any suitable substrate or scaffolding, whether natural, synthetic or combination thereof, as would be apparent to one of ordinary skill in the art after review of the disclosure of the present invention.
  • suitable substrates including but not limited to collagen, laminin, agar, agarose, and the basement membrane derived biological cell culture substrate sold under the registered trademark MATRIGEL® by Collaborative Biomedical Products, Inc. of Bedford, Massachusetts comprise suitable substrate or scaffolding material.
  • Synthetic matrix materials, substrate materials or scaffolding materials which are typically made from a variety of materials such as polymers, are also within the scope of the present invention.
  • Matrix composition 122 can comprise any suitable growth media, buffer solutions, biological reagents or gelling reagents.
  • a representative growth media is Dulbecco's Modified Eagle's Medium (DMEM), as disclosed in the Laboratory Examples.
  • DMEM Dulbecco's Modified Eagle's Medium
  • Thrombin is a representative biological reagent, as it is used to drive the formation of fibrin from fibrinogen.
  • component materials within a particular matrix composition 122 can be varied through the inclusion of different agents and combinations thereof as necessary to assess a particularly response or to accomplish a particular result.
  • Matrix composition 122 can further comprise a stabilizing agent so that the tissue growth modulating agent (e.g., fibrin) will not be degraded in an in vivo setting.
  • protease inhibiting agents can comprise the stabilizing agent and representative protease inhibiting agents include phenylmethylsulfonylfluoride (PMSF), N-caproic acid, aprotinin (a non-specific inhibitor) and plasminogen and activators 1 and 2 (specific protease inhibitors).
  • PMSF phenylmethylsulfonylfluoride
  • N-caproic acid N-caproic acid
  • aprotinin a non-specific inhibitor
  • plasminogen and activators 1 and 2 specific protease inhibitors
  • the concentration of stabilizing agents is adjusted so that a tissue growth modulating agent, such as fibrin, remains stable in the matrix composition but the stability of the tissue growth modulating agent, such as fibrin, is not enhanced so as to prevent tissue growth processes from proceeding.
  • the concentration of the stabilizing agent for fibrin maintains the stability of fibrin while allowing remodeling of the fibrin to promote tissue growth in a chamber 110 of the present invention.
  • PMSF and N-caproic acid comprise preferred stabilizing agents for a fibrin containing matrix composition 122 of the present invention.
  • the PMSF is present in a molar concentration ranging from 0.5 to 50 ⁇ M PMSF, with 5 ⁇ M PMSF comprising a preferred molar concentration; and N-caproic acid is present in a concentration ranging from about 0.1 to about 10 mM, with a concentration of about 1 mM comprising a preferred concentration.
  • the tissue growth modulating agent is present in an effective amount.
  • an "effective" amount refers to one that modulates (i.e. promotes or inhibits) tissue growth in a given setting, and preferably in an in vivo setting.
  • an effective amount of fibrin preferably ranges from about 0.5 to about 10 mg/ml, more preferably from about 1 to about 7.5 mg/mL, and even more preferably from about 2.5 to about 5 mg/ml.
  • matrix composition 122 comprises a tissue growth modulating agent as an active agent.
  • a preferred tissue growth modulating agent is an active agent that modulates (i.e. stimulates or inhibits) angiogenesis.
  • Such an agent is also referred to herein as an "angiogenesis modulating agent", or depending on the activity of the agent, as an “angiogenesis stimulating agent” or as an “angiogenesis inhibiting agent”.
  • Representative tissue growth modulating agents include but are not limited to fibrin, fibrinogen, transforming growth factor (TGF), vascular endothelial growth factor (VEGF), chemokines, cytokines and/or any other growth factor as would be apparent to one of ordinary skill in the art after reviewing the disclosure of the present invention presented herein.
  • a tissue growth modulating agent in matrix composition 122 comprises fibrin.
  • fibrin is prepared in matrix composition 122 using reaction of thrombin and fibrinogen in a suitable medium, as disclosed in the Laboratory Examples.
  • fibrin plays a wide- ranging role in wound healing and in tumor biology. First of all, it impedes blood loss. Additionally, fibrin induces platelets to release growth factors and trigger repair process. Fibrin also serves as a provisional matrix for cell migration and attracts other wound healing cells. Fibrin is also readily remodeled and promotes tissue retraction. In tumor biology, tumor cells induce fibrin formation by tissue factor pathway. Tissue factor is also induced by VEGF, while fibrin induces VEGF. VEGF alters vascular permeability and enhances fibrin formation.
  • the platelet and coagulation system upon the occurrence of a wound and tissue injury, the platelet and coagulation system is activated. This induces increased levels and activity of VEGF and TGF ⁇ , which then results in increased cellular hyperpermeability and fibrin formation from fibrinogen and thrombin. This leads to an influx of inflammatory and endothelial cells at the site of injury, followed by angiogenesis and fibrinolysis. Inflammation responses and cellular cell proliferation are also observed.
  • a cell can comprise an active agent in that a cell is capable of sending and receiving chemotactic, chemokinetic and other biological signals and responses.
  • Cells can be either naturally occurring cells or transfected cells produced in accordance art recognized techniques. Indeed, the preparation of recombinant vectors is well known to those of skill in the art and described in many references, such as, for example, Sambrook et al. (1992), Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), incorporated herein in its entirety.
  • the cell comprises a cell from a target tissue, such as a cell from a neoplasm or from a tissue in which the stimulation of growth is desired.
  • a target tissue such as a cell from a neoplasm or from a tissue in which the stimulation of growth is desired.
  • the matrix composition can further comprise a suitable pharmaceutically acceptable carrier.
  • Suitable pharmaceutical compositions in accordance with the invention will generally comprise an effective amount of the desired active agent admixed with an acceptable pharmaceutical diluent or excipient, such as a sterile aqueous solution, to give an appropriate final concentration with respect to the active agent.
  • an acceptable pharmaceutical diluent or excipient such as a sterile aqueous solution
  • Such formulations will typically include buffers such as phosphate buffered saline (PBS), or additional additives such as pharmaceutical excipients, stabilizing agents such as bovine serum albumin (BSA), or salts such as sodium chloride.
  • PBS phosphate buffered saline
  • stabilizing agents such as bovine serum albumin (BSA)
  • salts such as sodium chloride.
  • compositions are further rendered pharmaceutically acceptable by insuring their sterility, non-immunogenicity and non-pyrogenicity.
  • Such techniques are generally well known in the art as exemplified by Remington's Pharmaceutical Sciences, 16th Ed. Mack Publishing Company (1980), incorporated herein by reference. It should be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by United States Food and Drug Administration (FDA) Office of Biological Standards.
  • FDA United States Food and Drug Administration
  • the method comprises: providing a chamber of the present invention; implanting the chamber into a test animal; administering a candidate compound to the test animal; extracting the chamber after a time suitable for measurement of tissue growth; and evaluating tissue growth in the chamber to thereby determine the tissue growth modulating activity of the candidate compound.
  • the tissue growth modulating activity comprises angiogenesis modulating activity or wound healing modulation activity.
  • the tissue growth modulating agent is fibrin, fibrinogen, transforming growth factor, or combinations thereof.
  • the matrix composition can further comprise a cell.
  • the cell can comprise a cell from a target tissue, such as a neoplasm.
  • the test animal can further comprise a neoplasm, and in this case, the chamber can be implanted in the neoplasm.
  • the candidate compound can be systemically administered to the animal subject, or alternatively, the candidate compound can be administered in the chamber as a component of the matrix composition.
  • the candidate compound can also be administered to the test animal by collecting serum from a human subject at a time after the human subject received a candidate tissue growth modulating compound, adding the serum to the chamber, and implanting the chamber in the animal subject.
  • the method can further comprise implanting two or more chambers into the test animal.
  • the method can further comprise implanting two or more test chambers into the test animal, wherein a different candidate compound is inserted in each chamber.
  • the chamber can be incubated in the test animal for any length of time, so long as the time is sufficient to provide for cell growth/wound healing processes to proceed.
  • the chamber is incubated in the test animal for about 5 to about 15 days, more preferably about 10 to about 12 days.
  • tissue is readily harvested from the chamber by cutting out the mesh surfaces of the chamber. Tissue invades and pervades the chamber due to the porous surfaces and thus, in situ conditions are closely approximated for histology and tumor biology analysis.
  • the evaluation of tissue growth can be accomplished by any suitable or desired technique, including but not limited to: histology, immunohistochemistry, confocal imaging, magnetic resonance imaging, assessment of tumor growth, assessment of vascular density, immunoblotting, assessment of cell migration rate, assessment of cell death, assessment of hypoxia, assessment of vascular permeability, and combinations thereof.
  • a candidate substance identified according to the screening assay described herein has an ability to modulate tissue growth, and preferably has an ability to modulate angiogenesis.
  • Such a candidate compound can have utility in the treatment of disorders and conditions associated with the biological activity abnormal tissue growth, including neoplastic growth.
  • Candidate compounds can be hydrophobic, polycyclic, or both, molecules, and are typically about 500-1 ,000 daltons in molecular weight.
  • D. Method of Generating Tissue Growth are provided.
  • a method of delivering an active agent to a vertebrate animal is provided. The method comprises providing a chamber as disclosed herein; and delivering the active agent to the vertebrate animal by implanting the chamber in the vertebrate animal.
  • a method of generating tissue growth in a vertebrate animal comprises: providing a chamber of the present invention; implanting the chamber in the vertebrate animal; and generating tissue growth in the vertebrate animal through the implanting of the chamber.
  • the tissue growth that is generated comprises angiogenesis.
  • the active agent comprises a cell, a tissue growth modulating agent, or combinations thereof.
  • the cell can comprise a cell from a target tissue.
  • the tissue growth modulating agent can optionally comprises fibrin, fibrinogen, transforming growth factor, a chemokine, a cytokine or combinations thereof.
  • a preferred subject is a vertebrate animal subject.
  • a preferred vertebrate animal is warm-blooded vertebrate animal, and a preferred warmblooded vertebrate animal is a mammal.
  • a preferred mammal is a human.
  • the term "patient” is includes both human and animal patients, and veterinary therapeutic uses are provided in accordance with the present invention.
  • Warm-blooded vertebrate animals comprise preferred subjects for treatment in accordance with the methods of the present invention. Therefore, the invention concerns mammals and birds. Contemplated is the treatment of mammals such as humans, as well as those mammals of importance due to being endangered (such as Siberian tigers), of economical importance (animals raised on farms for consumption by humans) and/or social importance (animals kept as pets or in zoos) to humans, for instance, carnivores other than humans (such as cats and dogs), swine (pigs, hogs, and wild boars), ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), and horses.
  • mammals such as humans, as well as those mammals of importance due to being endangered (such as Siberian tigers), of economical importance (animals raised on farms for consumption by humans) and/or social importance (animals kept as pets or in zoos) to humans, for instance, carnivores other than
  • domesticated fowl i.e., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economical importance to humans.
  • livestock including, but not limited to, domesticated swine (pigs and hogs), ruminants, horses, poultry, and the like.
  • a method of generating new tissue growth in a vertebrate animal subject is provided.
  • Cells from a tissue or organ can be incorporated into a chamber of the present invention along with a matrix composition comprising, for example, fibrin.
  • Representative tissues include pancreas, liver, or other suitable tissues that will be apparent to one of ordinary skill in the art after review of the disclosure of the present invention herein.
  • the chamber loaded with a matrix composition, the matrix composition comprising a cell and fibrin can be implanted in any suitable location (e.g., subcutaneously or within a target tissue) within a vertebrate animal.
  • the matrix composition facilitates the recruitment of cells to the chamber to generate tissue growth within the chamber.
  • the cells that are included in the chamber are cells from the vertebrate animal subject so as to minimize problematic immunological responses to the chamber. Stated differently, the vertebrate animal subject recognizes the cells within the chamber as "self” as opposed to “non-self, and thus, problematic immune responses are avoided.
  • the internal void space of the chamber also facilitates new tissue growth by providing an enclosed space and scaffolding upon which tissue growth can occur.
  • the matrix composition comprises fibrin.
  • a matrix composition comprising fibrin can also be used in the treatment of cardiovascular disease.
  • a chamber loaded with matrix composition comprising fibrin can be implanted at a site of cardiovascular disease. New blood vessel growth is generated at the site of implantation of the chamber to thereby treat the cardiovascular disease at the site of implanting of the chamber.
  • Customized PLEXIGLAS® rings are prepared.
  • the rings have internal diameters of 10 and 5 mm and external diameters of 14 and 8 mm for rats and mice, respectively.
  • a 1 mm port is formed on a lateral surface of the ring and the port is used to load the ring with matrix composition.
  • MFTM cement available commercially, such as from MF Composites, Inc., of Mississauga, Ontario, Canada
  • the ring is then pressed against the nylon mesh (available commercially, such as from Millipore Corporation of Bedford, Massachusetts) and allowed to dry for about 4 hours.
  • the chamber is then cut from the nylon mesh around the periphery of the ring to produce a PLEXIGLAS® ring with one side covered with nylon mesh. The procedure is repeated for the other side.
  • the nylon mesh is inspected for proper bond with the rings.
  • the chambers are then sent for gas sterilization, or other suitable sterilization.
  • Phenylmethylsulfonylfluoride (PMSF) (A8456, Sigma Chemical Company of St. Louis, Missouri) N-Caproic acid (A2504, Sigma Chemical Company of St. Louis,
  • DMEM Dulbecco's Modified Eagle's Medium
  • the concentration of fibrinogen is measured by spectrophotometer. 50 ul of test and control solutions are diluted in 5 ml of de-ionized water. The diluted test solution is measured at 325 nanometers (nm) and 280 nm with control solution as a blank. The reading at 280 nm is subtracted from that at 325 nm. The result is multiplied by 50.5 and 2, and then divided by 1.62 for final fibrinogen concentration. The fibrinogen is diluted to about 5 mg/ml for experiments.
  • the sterilized chambers are placed on sterilized 20 millimeter diameter caps with the port of the chambers facing to the right side.
  • the caps provide a flat surface upon which the chambers are placed during loading to minimize leakage.
  • a 1 ml tuberculin syringe with 20-gauge needle is used to fill the chambers.
  • the chamber is held from the sides and pressed against the cap.
  • the fibrinogen solution is added from the right side through the port.
  • the solution is followed with 2-8 ⁇ l of thrombin from a pipette.
  • the chamber, or fibrin Z-chamber (F-ZC) is allowed to stand for about 15 minutes so the fibrinogen can gel into fibrin.
  • the chambers are implanted soon after preparation.
  • a tumor cell chamber, or tumor Z-chamber (T-ZC) is prepared by adding tumor cells to the fibrinogen solution before adding the solution to the chamber.
  • the tumor cells are embedded in fibrin gel.
  • Implantation is accomplished by making two 2 cm long incisions on the backs of the animals, followed by making blunt dissections on both sides to establish a pocket in the subcutaneous space. The chambers are inserted into these pockets. Clips are used to close the incision in rats and sutures are used for mice. A topical antibiotic, such as that sold under the registered trademark NEOSPORIN ® by Glaxo-Wellcome, Inc., is applied over the incision.
  • the sample can be put into a paraffin cassette and fixed in 10% neutral buffered formalin (NBF) for 24-36 hours.
  • NBF neutral buffered formalin
  • a drop of OCT can be added on top and then the sample can be partially fixed in liquid nitrogen. Then the sample is cut in half, embedded face down in OCT in a cryomold, and frozen with liquid nitrogen.
  • the nylon mesh is pulled apart and the tissue is scraped from the mesh and saved in a propylene tube after freezing it in liquid nitrogen.
  • SUGEN 5416 is described by Mendel et al., (2000) Anti-Cancer Drug Design 15:29- 41 , and its chemical name is 3-(2,4 ⁇ Dimethylpyrrol-5-yl)methylene-2- indolinone. SUGEN 5416 was administered at a concentration of 20 mg/kg.
  • SUGEN 5416 treatment retarded tumor growth so that only a 40% increase in tumor volume was observed in test animals as compared to about a 90% increase in tumor volume in control animals.
  • MMD microvessel density
  • SUGEN 5416 treatment produced an observed MVD per high power (200X) focal (HPF) of about 13, while in the control animals, MVD per HPF of about 15 was observed.
  • Chambers containing a matrix composition comprising fibrin were used to assess wound healing in response to systemic treatment with
  • SUGEN 5416 at a concentration of 20 mg/kg. As shown in Figure 13, no significant body loss in test animals as compared to control (i.e., untreated) animals was observed with the SUGEN 5416 treatment and chamber implantation. Indeed, the mean relative weight for the test animals closely followed the mean relative weight of the control animals over the 10-12 day test period. SUGEN 5416 inhibited neovascularization in the fibrin-containing chambers. This observation was made upon inspection of the gross appearance of the chambers from treated animals as compared to those from control animals, and upon inspection of hematoxylin and eosin (H & E) top sections and cross sections.
  • H & E hematoxylin and eosin
  • This 1 :1 DMSO and CREMOPHOR ® vehicle was used as the control solution.
  • Daily injections were started 2 days before initial surgery for implantation of fibrin chambers of the present invention. Rats with fibrin chambers tolerated surgery and daily injections very well and both treated and control animals maintained their pre-treatment weights for the duration of the study.
  • Fibrin Z-Chambers F-ZC were employed in a fibrin gel based in vivo assay.
  • fibrinogen and thrombin are added to a dual porous chamber through a port and chambers are then implanted in the subcutaneous tissue of the rats and harvested at day 12 post-implantation to assess the wound healing response generated due to presence of fibrin.
  • These chambers are constructed from customized PLEXIGLAS ® rings with internal diameter of 10 mm and have an access port drilled on the side. The two open surfaces are covered by nylon mesh (pore size 180 microns, # NY8H04700, Millipore, Massachusetts) glued to the rings.
  • Fibrinogen (# 341578, CalBiochem, La Jolla, California) was prepared in DMEM (# 11995-065, Gibco BRL, Rockville, Maryland) at a concentration of 4 mg/ml and was converted to fibrin by addition of thrombin (#605160, CalBiochem, La Jolla, California) inside the chambers.
  • Fischer 344 rats were anesthetized, hair removed using the clippers and the surface was surgically prepared. Two small midline skin incisions were made in the dorsal region about 4 cm apart. Fascia was blunt dissected and small pockets were created on both sides along the midline incision. Thus, four F-ZCs were implanted per animal.
  • F-ZCs 5 animals were implanted for each group (treatment or control).
  • the F-ZCs were harvested on day 12-post surgery.
  • the tissues were cut out from the chamber and were either preserved in 10% formalin for paraffin embedding (2 chambers) and snap frozen in liquid nitrogen for western blots, ELISA and D-Dimer measurements (2 chambers).
  • the maximum depth of granulation tissue inside the F-ZC was measured from the H&E tissue sections to assess the degree of wound healing response. Two independent pathologists in a blinded fashion did all measurements.
  • Immunohistochemistry was carried out on paraffin embedded tissues for primary antibody against tissue transglutaminase (TG100, 1 :10, endothelial cell marker (Haroon, Z. A., et al., Faseb J (1999) 13(13):1787-95), non-reactive to Factor Xllla) (# MS-279, Neomarkers, Inc. of Union City, California) or Isopeptide (#814 MAM, 1 :75) CovalAB (Oullins, France) using procedures described previously. See Haroon, Z. A., et al., Faseb J (1999) 13(13):1787-95.
  • tissue transglutaminase TG100, 1 :10
  • endothelial cell marker (Haroon, Z. A., et al., Faseb J (1999) 13(13):1787-95)
  • non-reactive to Factor Xllla) # MS-279, Neomarkers, Inc
  • H&E Hematoxylin & Eosin
  • MT Masson's trichrome
  • TGF 61 ELISA Active TGF ⁇ 1 ELISA was carried out in triplicate using DUOSET ® kit (DY240, Genzyme Corporation, Cambridge, Massachusetts) as described previously (Danielpour, D., J Immunol Methods (1993) 158(1 ): 17-25) with tissue lysates from chamber contents generated as detailed above. Latent form of TGF ⁇ 1 was converted to immunoreactive form to ascertain total TGF ⁇ 1 content. The data is shown as pg per mg of protein in the tissues. D-Dimer Measurements. The MINIQUANTTM D-Dimer (Cat # 1447,
  • Biopool AB of Umea, Sweden was used to measure residual D-Dimer in the chamber content lysates generated as detailed above. The results are displayed in ng/ml. The measurements were carried out in duplicate. Each measurement is from pooled tissue for the treatment and control groups.
  • Microtiter Plate TG Assay was determined by quantitating the incorporation of 5-biotin (amido) pentylamine into N, N ' -dimethyl casein coated microtiter plate as described previously. See Slaughter T. F., Anal Biochem (1992) 205(1): 166-71.
  • the MVD in the granulation tissue of SU5416 treated F-ZC dropped significantly by 10% from an average value of 30 microvessels per unit area of control tissues (Table 1 ). This observation is consistent with other reports of minor reduction in MVD with marked anti-tumor activity of anti-angiogenic compounds. Lund, E. L., Bastholm, L., and Kristjansen P. E., Clin Cancer Res (2000) 6(3):971-8.
  • MT was utilized to assess collagen deposition and in semi-quantitative measurements observed significant decrease (>70%) with controls ( Figures 17E and 17F, Table 1 ).
  • Collagen production is primarily mediated by the pro-fibrogenic cytokine TGF ⁇ 1.
  • VEGF is known to induce TGF ⁇ 1 (Saadeh, P. B., et al., Am J Physioi (1999) 277(4 Pt 1 ):C628-37) and it was expected SU5416 mediated inhibition of VEGF signal transduction would reduce TGF ⁇ 1 production.
  • the levels of total and active TGF ⁇ 1 in the F-ZC tissue were measured by ELISA and it was found that active TGF ⁇ 1 was reduced 90% even though the total TGF ⁇ 1 was increased in treated F-ZC (Table 1 ).
  • TGF ⁇ 1 is activated from its latent to active form predominantly by a surface complex of uPAR, plasminogen, mannose-6-phosphate receptor and TG.
  • Kojima S., Nara, K., and Rifkin, D. B., J Cell Biol (1993) 121 (2):439-48. It was investigated which part of this pathway was being effected by SU5416. It was hypothesized that since plasmin production is up-regulated by VEGF (Baker, E. A., Bergin, F. G., and Leaper, D.
  • VEGF RTKs inhibition would lead to reduced plasmin levels although there are leads that suggest VEGF induction of plasmin production is independent of RTKs. See Kroon, M. E., et al., Thromb Haemost (2001 ) 85(2):296-302.
  • the fibrin inside the chambers is removed by plasmin producing D-Dimer (a fibrin degradation product), thus D-Dimer levels in the chambers would reflect the degree of plasmin activity.
  • the D-Dimer values in SU5416 treated tissues remained elevated (Table 1). The relatively high levels of D-Dimer in the treated group suggested that plasmin production was not the target of inhibition by SU5416.
  • TG is a multi-functional wound healing enzyme with GTP and ATP binding sites. Greenberg, C. S., Birckbichler, P. J., and Rice, R. H., Faseb J (1991) 5(15):3071-7.
  • TG cross-linking function (vital for TGF ⁇ 1 activation) is calcium dependent and is inhibited by ATP/GTP binding. Lai, T. S., et al., J Biol Chem (1998) 273(3): 1776-81 ; Lai, T. S., et al., J Biol Chem (1996) 271(49): 31191-5. It was postulated that SU5416 was reacting with ATP binding site, thereby inhibiting its cross-linking function. The interaction of SU5416 with TG was directly investigated. It was observed that SU5416 inhibited TG activity by more than 60% starting at 20 ⁇ M and reaching peak inhibition of 80% at 40 ⁇ M ( Figure 18).
  • the tissues were also probed with a monoclonal antibody directed towards the isopeptide bonds generated by TG to ascertain TG activity in the granulation tissue.
  • Factor Xllla can also generate isopeptide bonds in the granulation tissue (Gibran, N. S., Heimbach, D. M., and Holbrook, K. A., J Surg Res (1995) 59(3):378-86), but this would be limited to blood vessels and macrophages (sites of both Factor XIII and TG activity).
  • Decreased TG activity was expected to lead to low levels of isopeptide bonds in the ECM. Very low levels of isopeptide bonds were detected in the extracellular matrix of SU5416 treated tissues in comparison to controls, confirming this hypotheses ( Figures 17G and 17H).
  • TG antigen in the chamber tissues was next investigated. It was observed that TG production was decreased by more than 70% and the TG antigen was not proteolyzed in SU5416 treated tissues in western blot results ( Figure 19, Table 1). It has been reported earlier that TG is degraded in healing tissues (Haroon, Z. A., et al., Fasejb J (1999) 13(13): 1787-95) and TG degradation by trypsin is inhibited when the ATP/GTP binding sites are occupied (Lai, T. S., et al., J Biol Chem (1998) 273(3):1776-81 ). The relative lack of TG degradation suggests that SU5416 could have interacted with the ATP/GTP binding site of TG, shutting down the cross-linking function.
  • Table 1 presents a summary of various measurements carried out on paraffin embedded F-ZC and tissue lysates obtained from F-ZC. Data is presented with + standard error of mean values. Please refer to Examples 7-9 for experimental details and data generation.
  • Figures 20A and 20B are photographs showing gross examination of fibrin-containing chambers of the present invention in which angiostatin was administered in laboratory animals in accordance with techniques described herein above for Laboratory Examples 1-9.
  • Figures 20A and 20B show more influx of blood vessels in the controls ( Figure 20A) than angiostatin treated chambers (1 ⁇ M angiostatin), which appeared paler in color ( Figure 20B).
  • Fibrin is inherently pale yellow in color and lack of blood vessels in angiostatin treated chambers results in the paler appearance of the chambers.
  • Figures 20C and 20D are photomicrographs showing depth (represented by line with arrowheads at each end) of granulation tissue developed inside fibrin-containing chambers of the present invention. Depth granulation was used as a measure for the healing response. The granulation tissue in controls ( Figure 20C) is distinctly more than angiostatin treated chambers (1 ⁇ M angiostatin) ( Figure 20D). A reduction in healing response in the presence of angiostatin was thus observed.
  • Figure 21 is a bar graph depicting that angiostatin inhibited granulation tissue formation in fibrin-containing chambers of the present invention implanted in test animals (shaded bar) as compared to fibrin- containing chambers of the present invention implanted in control animals (open bar).
  • Scale depth of granulation tissue (X10 microns).
  • FIGS 22A and 22B are photomicrographs showing depth (represented by line with arrowheads at each end) of granulation tissue developed inside fibrin-containing chambers of the present invention in which superoxide dismutase (SOD) mimetics were administered to tumors in laboratory animals in accordance with techniques described herein above for Laboratory Examples 1-9.
  • the chambers of the present invention are referred to as tumor Z-Chambers, or T-ZC.
  • This Example was performed to assess the predictive value of fibrin-containing T-ZCs of the present invention where tumor reduction was expected to be minor (e.g. about a 15% reduction).
  • Figure 23 is a bar graph depicting the detection of a minor reduction in tumor growth after SOD mimetic administration. This reduction was detected T-ZCs of the present invention implanted in test animals

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Abstract

La présente invention concerne une chambre qui permet d'apporter in vivo un agent actif, cette chambre comprenant au moins deux surfaces poreuses qui sont situées à distance l'une de l'autre sensiblement sur les côtés opposés de l'enceinte, un espace vide interne présent dans l'enceinte et une composition de matrice renfermant un agent actif qui est placée dans l'espace vide interne. La présente invention concerne également des procédés thérapeutiques et de criblage dans lesquels on utilise la chambre, y compris des procédés de criblage in vivo d'agents modulant l'angiogenèse et/ou la croissance tumorale.
PCT/US2001/047124 2000-11-13 2001-11-13 Chambre utilisee pour le criblage in vivo de composes modulant l'angiogenese et la croissance tumorale WO2002038130A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5201728A (en) * 1991-05-03 1993-04-13 Giampapa Vincent C Subcutaneous implantable multiple-agent delivery system
US5964745A (en) * 1993-07-02 1999-10-12 Med Usa Implantable system for cell growth control

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5201728A (en) * 1991-05-03 1993-04-13 Giampapa Vincent C Subcutaneous implantable multiple-agent delivery system
US5964745A (en) * 1993-07-02 1999-10-12 Med Usa Implantable system for cell growth control

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