EP2180890A1 - Methods of inhibiting or suppressing cellular proliferation - Google Patents
Methods of inhibiting or suppressing cellular proliferationInfo
- Publication number
- EP2180890A1 EP2180890A1 EP07837460A EP07837460A EP2180890A1 EP 2180890 A1 EP2180890 A1 EP 2180890A1 EP 07837460 A EP07837460 A EP 07837460A EP 07837460 A EP07837460 A EP 07837460A EP 2180890 A1 EP2180890 A1 EP 2180890A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- group
- formula
- independently represents
- cell
- polymer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/12—Ketones
- A61K31/121—Ketones acyclic
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/12—Ketones
- A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
- A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/74—Synthetic polymeric materials
- A61K31/765—Polymers containing oxygen
- A61K31/77—Polymers containing oxygen of oxiranes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- Antiproliferative agents are compounds that can inhibit or suppress cellular proliferation (i.e, growth and multiplication of the cells).
- the use of antiproliferative agents to treat or prevent a wide variety of proliferative (e.g., hyperproliferative) conditions and/or diseases has been disclosed. Examples of such conditions and/or diseases include, but are not limited to, hyperplasia of soft and hard tissues, malignant tumors such as cancer, skin keloids, fibrosis, and surgical adhesions.
- Antiproliferative agents can also be used to prevent cell proliferative processes resulting in in-stent restenosis (e.g., associated with coronary stents), pannus overgrowth on prosthetic heart valves (e.g., associated with sewing rings), urethral stenosis (e.g., associated with prostatic hyperplasia), and stenosis associated with surgical anastomosis.
- in-stent restenosis e.g., associated with coronary stents
- pannus overgrowth on prosthetic heart valves e.g., associated with sewing rings
- urethral stenosis e.g., associated with prostatic hyperplasia
- stenosis associated with surgical anastomosis e.g., associated with surgical anastomosis.
- Cytostatic agents are compounds that can inhibit or suppress cellular proliferation (i.e, growth and multiplication of the cells) without compromising the cell's viability and functionality. Few drugs have proven to have an effective cytostatic window with minimal cytotoxic potential.
- the method includes delivering into or proximate a cell at least one antiproliferative agent selected from the group consisting of: a compound of the formula (Formula I):
- each X independently represents NR 5 , CR 5 R 6 , SiR 5 R 6 , S, a sulfur-bonded group, a phosphorus-bonded
- each Y independently represents O, NR 5 ,
- each n is independently from 0 to 5; each R 1 independently represents an organic group; each R 2 , R 3 , R 4 , R 5 , and R 6 independently represents H or an organic group; and R 1 , R 2 , R 3 , R 4 , R 5 , and/or R 6 can optionally be joined to each other to form one or more rings.
- the method includes locating at least one polymer proximate a tissue; allowing the at least one polymer to hydrolyze to form at least one antiproliferative agent of Formula I, Formula II, and/or Formula III; and delivering the at least one antiproliferative agent into or proximate a cell.
- the method includes: providing a medical device including at least one biodegradable polymer; positioning the at least one biodegradable polymer proximate a tissue; allowing the at least one biodegradable polymer to biodegrade to form at least one antiproliferative agent of Formula I, Formula II, and/or Formula III; and delivering the at least one antiproliferative agent into or proximate a cell.
- the method including delivering into or proximate a cell at least one antiproliferative agent selected from the group consisting of: a ketal and/or a hemiketal of a compound of Formula I and/or Formula II.
- the method includes delivering into or proximate a cell at least one prodrug that can release an antiproliferative agent of Formula I, Formula II, and/or Formula III.
- Figure 1 is a graphical representation of the cytotoxicity of normal human dermal fibroblast (NHDF) cells exposed to various concentrations of 5,6-dihydroxy-hexan-2-one for 24 hours and 48 hours as described in Example 3.
- NHDF normal human dermal fibroblast
- Figures 2-6 are pictures that visually display the physical cellular response of normal human dermal fibroblast (NHDF) cells to various concentrations of 5,6-dihydroxy-hexan-2-one as described in Example 3.
- NHDF normal human dermal fibroblast
- Figures 7-16 are pictures that visually display the physical cellular response of normal human dermal fibroblast (NHDF) cells to various concentrations of 5,6-dihydroxy-hexan-2-one as described in Example 4.
- NHDF normal human dermal fibroblast
- Figure 17 is a graphical representation of normal human dermal fibroblast (NHDF) cell numbers measured after 48 hours exposure to different concentrations of 5,6-dihydroxy-hexan-2-one as described in Example 5.
- NHDF normal human dermal fibroblast
- Figure 18 is a graphical representation of coronary artery smooth muscle cell (CASMC) numbers measured after 48 hours exposure to different concentrations of 5,6-dihydroxy-hexan-2-one as described in Example 5.
- CASMC coronary artery smooth muscle cell
- Figure 19 is a graphical representation of viable coronary artery smooth muscle cell (CASMC) numbers measured after 48 hours exposure to different concentrations of 5,6-dihydroxy-hexan-2-one as described in Example 5.
- CASMC coronary artery smooth muscle cell
- Figures 20(a)-(l) are pictures that visually display the migration of coronary artery smooth muscle cells (CASMC) ( Figures 20(a)-(f)) and normal human dermal fibroblast (NHDF) cells ( Figures 20(g)-(l)) exposed to various concentrations of 5,6-dihydroxy-hexan-2-one after 48 hours exposure, then cultured an additional 72 hours after removal of the 5,6- dihydroxy-hexan-2-one as described in Example 5.
- CASMC coronary artery smooth muscle cells
- NHDF normal human dermal fibroblast
- Figure 21 is a graphical representation of human
- Glioblastoma/Astrocytoma U87 cell numbers measured after being exposed to 5,6-dihydroxy-hexan-2-one for 48 hours as described in Example 6.
- Time 0 refers to the replacement of the test agent with supplemented growth media.
- Figure 22 is a graphical representation of human umbilical vein endothelial cell (HUVEC) numbers measured after being exposed to 5,6- dihydroxy-hexan-2-one for 48 hours as described in Example 6. Time 0 refers to replacement of the test agent with supplemented growth media.
- HUVEC human umbilical vein endothelial cell
- Figures 23(a)-(l) are pictures that visually display the migration over time of human umbilical vein endothelial cells (HUVEC) after 48 hours exposure to various concentrations of 5,6-dihydroxy-hexan-2-one as described in Example 6.
- HBVEC human umbilical vein endothelial cells
- Figures 24(a)-(l) are pictures that visually display the migration over time of human Glioblastoma/Astrocytoma U87 cells after 48 hours exposure to various concentrations of 5,6-dihydroxy-hexan-2-one as described in Example 6.
- Figures 25(a) and (b) are graphical illustrations of cell counts and cell viability, respectively, for human coronary artery endothelial cells (HCAEC) exposed to 5,6-dihydroxy-6-phenyl-hexan-2-one for 24 hours and 48 hours, then allowed to grow for 96 hours after removal of 5,6- dihydroxy-6-phenyl-hexan-2-one as described in Example 7.
- HCAEC human coronary artery endothelial cells
- Figures 26(a) and (b) are graphical illustrations of cell counts and cell viability, respectively, for human coronary artery endothelial cells (HCAEC) exposed to 5,6-dihydroxy-heptan-2-one for 24 hours and 48 hours, then allowed to grow for 96 hours after removal of 5,6-dihydroxy- heptan-2-one as described in Example 7.
- HCAEC human coronary artery endothelial cells
- Figures 27(a) and (b) are graphical illustrations of cell counts and cell viability, respectively, for human coronary artery endothelial cells (HCAEC) exposed to 5,6-dihydroxy-5-methyl-hexan-2-one for 24 hours and 48 hours, then allowed to grow for 96 hours after removal of 5,6- dihydroxy ⁇ 5-methyl-hexan-2-one as described in Example 7.
- HCAEC human coronary artery endothelial cells
- Figures 28(a) and (b) are graphical illustrations of cell counts and cell viability, respectively, for human coronary artery endothelial cells (HCAEC) exposed to 2-(2',3'-dihydroxypropyl)-cyclohexanone for 24 hours and 48 hours, then allowed to grow for 96 hours after removal of 2- (2',3'-dihydroxypropyl)-cyclohexanone as described in Example 7.
- HCAEC human coronary artery endothelial cells
- Figures 29(a) and (b) are graphical illustrations of cell counts and cell viability, respectively, for human coronary artery endothelial cells (HCAEC) exposed to 2,3-dihydroxypropyl acetate for 24 hours and 48 hours, then allowed to grow for 96 hours after removal of 2,3- dihydroxypropyl acetate as described in Example 7.
- HCAEC human coronary artery endothelial cells
- Figures 30(a) and (b) are graphical illustrations of cell counts and cell viability, respectively, for human coronary artery endothelial cells (HCAEC) exposed to 5,6-dihydroxy-hexan-2-one for 24 hours and 48 hours, then allowed to grow for 96 hours after removal of 5,6-dihydroxy- hexan-2-one as described in Example 7.
- HCAEC human coronary artery endothelial cells
- the present disclosure provides methods of inhibiting or suppressing cellular proliferation.
- the methods include the delivery of at least one antiproliferative agent as described herein into or proximate a cell (e.g., into or near a tissue, region, or organ).
- the antiproliferative agent e.g., a cytostatic agent
- the inhibition or suppression of cellular proliferation upon delivery of the at least one antiproliferative agent into or proximate the cell is irreversible.
- the antiproliferative agent can inhibit or suppress cellular proliferation upon delivery into or proximate a wide variety of cell types.
- the antiproliferative agent can be a compound of the formula
- each n is independently from 0 to 5; each R 1 independently represents an organic group; each R 2 , R 3 , R 4 , R 5 , and R 6 independently represents H or an organic group; and R 1 , R 2 , R 3 , R 4 , R 5 , and/or R 6 can optionally be joined to each other to form one or more rings.
- the wavy bonds in the formulas herein are used to indicate unspecified stereochemistry. It should be noted that antiproliferative agents of Formula I, Formula II, and Formula III are intended to include any dimers and/or trimers of the indicated compounds that may exist either independently or in equilibrium with compounds of the indicated formulas.
- each X and Y independently represents
- each n is 1 ; each R 1 independently represents a C1-C10 organic group; each R 2 , R 3 , R 4 , R 5 , and R 6 independently represents H or a C1- C10 organic group; and R 1 and R 5 can optionally be joined to each other to form a ring.
- each X and Y independently represents
- each n is 1; each R 1 independently represents a phenyl group (and preferably a phenyl ring) or a C1-C4 aliphatic or alicyclic group (and preferably a C1-C4 aliphatic or alicyclic moiety); each R 2 , R 3 , R 4 , R 5 , and R 6 independently represents H, a phenyl group, or a C1-C4 aliphatic or alicyclic group (and preferably H, a phenyl group, or a C1-C4 aliphatic or alicyclic moiety); and R 1 and R 5 can optionally be joined to each other to form a five- or six-membered ring.
- the antiproliferative agent can be a ketal or hemiketal of a compound of Formula I or Formula II, and/or a hydrolysis product thereof.
- the ketone group in Formula I or Formula Il can react with one or more alcohols (including diols or polyols) to form a ketal or hemiketal.
- the diol group of Formula I can react with a ketone or aldehyde to form a cyclic ketal or acetal.
- a cyclic hemiketal can be formed by the cyclization of a compound of Formula I.
- a cyclic hemiketal formed by the cyclization of a compound of Formula I can be represented, for example, by the formulas wherein each X, Y, n, R 1 , R 2 , R 3 , and R 4 is defined as disclosed herein above for compounds of Formula I.
- the antiproliferative agent can be a prodrug that can release (e.g., upon metabolism and/or hydrolysis) a compound of Formula I, Formula II, and or Formula III.
- a ketal of a compound of Formula I (as described herein) can be hydrolyzed to release a compound of Formula I.
- organic group is used for the purpose of this invention to mean a hydrocarbon group that is classified as an aliphatic group, cyclic group, or combination of aliphatic and cyclic groups (e.g., alkaryl and aralkyl groups).
- suitable organic groups for monomers and polymers of this invention are those that do not interfere with the ring opening polymerization reaction disclosed herein.
- aliphatic group means a saturated or unsaturated linear or branched hydrocarbon group. This term is used to encompass alkyl, alkenyl, and alkynyl groups, for example.
- alkyl group means a saturated linear or branched monovalent hydrocarbon group including, for example, methyl, ethyl, n-propyl, isopropyl, terf-butyl, amyl, heptyl, and the like.
- alkenyl group means an unsaturated, linear or branched monovalent hydrocarbon group with one or more olefinically unsaturated groups (i.e., carbon-carbon double bonds), such as a vinyl group.
- alkynyl group means an unsaturated, linear or branched monovalent hydrocarbon group with one or more carbon-carbon triple bonds.
- cyclic group means a closed ring hydrocarbon group that is classified as an alicyclic group, aromatic group, or heterocyclic group.
- alicyclic group means a cyclic hydrocarbon group having properties resembling those of aliphatic groups.
- aromatic group or “aryl group” means a mono- or polynuclear aromatic hydrocarbon group.
- heterocyclic group means a closed ring hydrocarbon in which one or more of the atoms in the ring is an element other than carbon (e.g., nitrogen, oxygen, sulfur, etc.).
- group and “moiety” are used to differentiate between chemical species that allow for substitution or that may be substituted and those that do not so allow for substitution or may not be so substituted.
- group when the term “group” is used to describe a chemical substituent, the described chemical material includes the unsubstituted group and that group with nonperoxidic O, N, S, Si, or F atoms, for example, in the chain as well as carbonyl groups or other conventional substituents.
- moiety is used to describe a chemical compound or substituent, only an unsubstituted chemical material is intended to be included.
- alkyl group is intended to include not only pure open chain saturated hydrocarbon alkyl substituents, such as methyl, ethyl, propyl, ferf-butyl, and the like, but also alkyl substituents bearing further substituents known in the art, such as hydroxy, alkoxy, alkylsulfonyl, halogen atoms, cyano, nitro, amino, carboxyl, etc.
- alkyl group includes ether groups, haloalkyls, nitroalkyls, carboxyalkyls, hydroxyalkyls, sulfoalkyls, etc.
- the phrase “alkyl moiety” is limited to the inclusion of only pure open chain saturated hydrocarbon alkyl substituents, such as methyl, ethyl, propyl, terf-butyl, and the like.
- the antiproliferative agent can be delivered into or proximate the cell by a wide variety of methods.
- the antiproliferative agent can be delivered topically, by inhalation, by contact with mucuous tissues, by injection, and combinations thereof.
- the antiproliferative agent can be delivered directly to the cell (e.g., as a suspension, dispersion, or emulsion) by injection via a needle or catheter.
- Other embodiments include delivery of the antiproliferative agent in semisolid and/or solid formulations designed to provide continuous and/or controlled release of the antiproliferative agent into the tissue-biomaterial interface or surrounding tissue.
- Other applications include the use of antiproliferative agents as components of cell, drug, and/or gene therapy formulations, as adjuvants for therapeutic potential. Further applications include, for example, limiting tumor growth.
- the antiproliferative agent can be delivered in suspension or solution into the extracellular space from which it can be diffused, distributed, contacted with, and/or internalized by cells and/or tissue.
- the antiproliferetive agent can be delivered as a component of a hydrogel that solidifies upon contact with living tissue for release to targeted cells and/or tissues.
- the antiproliferative agent can be delivered in a solid form (e.g., films, pellets, microspheres, and the like, that include, among other things, the antiproliferative agent), for release to targeted cells and/or tissues as biodegradation occurs.
- the antiproliferative agent can be delivered using a liposome (e.g., by diffusion from the liposome).
- the antiproliferative agent can be combined with at least one cell type, either to optimize the delivery of cells or to protect the implanted cells from surrounding physiological and/or pathological events such as inflammation and/or rejection, preferably increasing the therapeutic potential of cell-based therapies.
- the antiproliferative agent can be combined with at least one cell type, at least one therapeutic drug, and/or at least one molecular material designed to modify the expression of a certain gene affecting the etiology of a given therapy.
- At least one antiproliferative agent as described herein can be disposed in a polymer, and the polymer can be located proximate a cell and allowed to deliver the at least one antiproliferative agent into or proximate the cell.
- the term "disposed" is intended to be broadly interpreted as inclusive of dispersed, dissolved, suspended, or otherwise contained at least partially therein or thereon.
- the polymer can deliver the at least one antiproliferative agent by a variety of mechanisms including, for example, delivery of the at least one antiproliferative agent from pores in the polymer, diffusion of the at least one antiproliferative agent through the polymer, delivery of the at least one antiproliferative agent through degradation of the polymer, or combinations thereof.
- a medical device including at least one antiproliferative agent as described herein and the medical device can be located proximate a cell and allowed to deliver the at least one antiproliferative agent into or proximate the cell.
- the medical device including the at least one antiproliferative agent can include a polymer having the at least one antiproliferative agent disposed therein.
- polyurethanes e.g., polyether urethanes, polyester urethanes including polycaprolactone urethanes
- polyureas e.g., polyurethane-ureas
- polyesters e.g., polyethylene terephthalate
- poly(beta-aminoesters) polycarbonates
- poly(meth)acrylates polysulfones
- polyimides polyamides
- epoxies polyacetals
- polyketals polyorthoesters
- vinyl polymers polyanhydrides, polytriazoles, silicone rubber, natural rubber, rubber latex, synthetic rubbers, polyether-polyamide block copolymers, polyester-polyether copolymers, and combinations and/or copolymers thereof.
- Exemplary polyesters include, for example, linear aliphatic polyester homopolymers (e.g., polyglycolide, polylactide, polycaprolactone, and polyhydroxybutyrate) and copolymers (e.g., poly(glycolide-co-lactide), poly(glycolide-co-caprolactone), poly(glycolide-co- trimethylenecarbonate), poly(lactic acid-co-lysine), poly(lactide-co- urethane), poly(ester-co-amide)).
- Polymers used in the methods disclosed herein can be biostable or biodegradable.
- Polymers used in the methods disclosed herein having antiproliferative agents disposed therein can be prepared by a wide variety of methods known in the art.
- such compositions can be prepared by solution processing, milling, extruding, polymerizing components in the presence of an antiproliferative agent, and combinations thereof.
- At least one polymer can be located proximate a tissue and allowed to hydrolyze to form at least one antiproliferative agent, which can be delivered into or proximate a cell.
- the at least one polymer can be a polyester, a polyorthoester, a polyketal (e.g., as described in U.S. Application Serial No. 1 1/706,508, filed 15 February 2007), or a combination thereof that can form at least one antiproliferative agent upon hydrolysis (e.g., biodegradation).
- Polymers used in the methods disclosed herein can be used as, for example, tissue bulking agents, tissue replacement agents, tissue repair agents, surgical void fillers, agents used to prevent surgical adhesions, or combinations thereof.
- a "polyketal” refers to a homo- or co-polymer that includes two or more (i.e., a plurality) of ketal repeat units.
- a "ketal" repeat unit is a unit including a ketal-containing group that is repeated in the polymer at least once.
- a ketal group is a group that includes an -C-O-C(M)(N)-O-C- functionality with the proviso that neither M nor N is hydrogen (e.g., an acetal-containing group) or oxygen (e.g., an orthoester-containing group).
- M and N are attached to the carbon atom of the ketal group via a carbon-carbon bond.
- Exemplary polyketal polymers include two or more cyclic oxygen- containing repeat units selected from the group consisting of: a repeat unit of the formula (Formula IV):
- each X independently represents NR 5 , CR 5 R 6 , SiR 5 R 6 , S, a sulfur-bonded group, a phosphorus-bonded
- each Y independently represents O, NR 5 ,
- each n is independently from 0 to 5; each R 1 independently represents an organic group; each R 2 , R 3 , R 4 , R 5 , and R 6 independently represents H or an organic group; and R 1 , R 2 , R 3 , R 4 , R 5 , and/or R 6 can optionally be joined to each other to form one or more rings.
- each X and Y independently represents
- each n is 1 ; each R 1 independently represents a C1-C10 organic group; each R 2 , R 3 , R 4 , R 5 , and R 6 independently represents H or a C1- C10 organic group; and R 1 and R 5 can optionally be joined to each other to form a ring.
- each X and Y independently represents
- each n is 1 ; each R 1 independently represents a phenyl group (and preferably a phenyl ring) or a C1-C4 aliphatic or alicyclic group (and preferably a C1-C4 aliphatic or alicyclic moiety); each R 2 , R 3 , R 4 , R 5 , and R 6 independently represents H, a phenyl group, or a C1-C4 aliphatic or alicyclic group (and preferably H, a phenyl group, or a C1-C4 aliphatic or alicyclic moiety); and R 1 and R 5 can optionally be joined to each other to form a five- or six-membered ring.
- the polyketal polymer can further include repeat units selected from the group consisting of crosslinkable repeat units, crosslinked repeat units, repeat units having imagable groups, repeat units having latent reactive sites, and combinations thereof.
- the polyketal polymer can further include repeat units selected from the group consisting of alpha-hydroxy alkanoates, beta-hydroxy alkanoates, gamma-hydroxy alkanoates, delta-hydroxy alkanoates, epsilon-hydroxy alkanoates, glycols, carbonates, acetals, and combinations thereof.
- the polyketal polymers disclosed herein can include a single cyclic oxygen-containing repeat unit (i.e., a homopolymer), or two or more different repeat units (i.e., a copolymer).
- the two or more different repeat units can all be different cyclic oxygen-containing repeat units of Formula IV and/or Formula V, or alternatively, one or more cyclic oxygen-containing repeat units of Formula IV and/or Formula V in combination with one or more repeat units that are not of Formula IV or Formula V (e.g., lactide repeat units, glycolide repeat units, butyrolactone repeat units, valerolactone repeat units, caprolactone repeat units, cyclic carbonate repeat units such as trimethylene carbonate and 1 ,2-O- isopropylidene-[D]-xylofuranose-3,5-cyclic carbonate, cyclic ether repeat units such as ethylene oxide, cyclic acetals such as 1,3-dioxolane, and combinations
- the polyketal polymer can be a copolymer.
- the copolymer can be a random copolymer, an alternating copolymer, a block copolymer, a graft copolymer, or a combination thereof.
- the copolymer is a block copolymer
- at least one block of the block copolymer can be a polyketal block including the two or more repeat units selected from the group consisting of repeat units of Formula IV, repeat units of Formula V, and combinations thereof.
- At least one other block of the block copolymer includes repeat units such as alpha-hydroxy alkanoates, beta- hydroxy alkanoates, gamma-hydroxy alkanoates, delta-hydroxy alkanoates, epsilon-hydroxy alkanoates, carbonates, acetals, or combinations thereof.
- at least one other block of the block copolymer is a polyorthoester block.
- at least one other block of the block copolymer is a poly(alkyleneglycol) block including alkylene glycol repeat units.
- the presently disclosed polyketals include polymers that are not converted under physiological conditions to acidic products.
- the present invention provides polyketal polymers that can biodegrade at a sufficiently high rate to enable them to be considered for use in specific applications.
- biodegradable and “bioerodible” are used interchangeably and are intended to broadly encompass materials including, for example, those that tend to break down upon exposure to physiological environments.
- R 1 in each of the polyketal repeating units disclosed herein represents an organic group which is advantageous in providing polymers with useful biodegradability.
- polysaccharides are structures in which R 1 represents hydrogen. Although polysaccharides are useful biomaterials (e.g., useful in biomedical applications), they typically do not rapidly biodegrade in physiologic environments.
- the polyketal polymers disclosed herein are biodegradable.
- the average molecular weight (and preferably the weight average molecular weight) of the polymers disclosed herein is at least 1000 Daltons, and sometimes at least 2000 Daltons, 5,000 Daltons, or even 10,000 Daltons or more. Average molecular weights of the polymers disclosed herein can be as high as desired for specific applications.
- the average molecular weight (and preferably the weight average molecular weight) of the polymers disclosed herein is at most 10,000,000 Daltons, and sometimes at most 5,000,000 Daltons, 2,000,000 Daltons, or even 1 ,000,000 Daltons.
- the polydispersity index (PDI) of the polymers disclosed herein is at most 3, and sometimes at most 2.5, and other times at most 2.0.
- polymers used in the methods disclosed herein can be blended with another polymer (e.g., the same or different) to provide the desired physical and/or chemical properties.
- polymers used in the methods disclosed herein e.g., polyketal polymers
- another polymer e.g., the same or different
- two polyketal polymers having different molecular weights can be blended to optimize the release rate of a biologically active agent.
- two polyketal polymers having different repeat units can be blended to provide desired physical and/or chemical properties.
- a polyketal polymer can be blended with another polymer that is not a polyketal polymer to provide desired physical and/or chemical properties.
- Polymers used in the methods disclosed herein can be used in various combinations for various applications. They can be used for replacements for nucleus pulposis in intervertebral disc repair procedures. They can be used as tissue adhesives or sealants. They can be used as surgical void fillers, for example, in reconstructive or cosmetic surgery (e.g., for filling a void after tumor removal). They can be used to repair aneurysms, hemorrhagic stroke or other conditions precipitated by failure of a blood vessel. They can be used to prevent surgical adhesions. They can be used to limit tumor growth.
- Polymers used in the methods disclosed herein can be used in injectable compositions.
- injectable compositions could be used, for example, as void fillers (e.g., in cosmetic or reconstructive surgery, such as serving as a replacement for the nucleus pulposis) or as an injectable drug delivery matrix.
- one or more polymers can be combined with a solvent such as N-methyl-2-pyrrolidone or dimethylsulfoxide (DMSO), which are fairly biocompatible solvents. The solvent can diffuse away after injection and the polymer can remain in place.
- a solvent such as N-methyl-2-pyrrolidone or dimethylsulfoxide (DMSO), which are fairly biocompatible solvents. The solvent can diffuse away after injection and the polymer can remain in place.
- DMSO dimethylsulfoxide
- injectable materials can be applied to a desired site (e.g., a surgical site) using a syringe, catheter, applicator, or by hand.
- injectable compositions could include crosslinkers (such as diacrylates), plasticizers (such as triethyl citrate), lipids (soybean oil), poly( ethylene glycol) (including those with the ends blocked with methyls or similar groups), silicone oil, partially or fully fluorinated hydrocarbons, N-methyl-2-pyrrolidone, or mixtures thereof.
- crosslinkers such as diacrylates
- plasticizers such as triethyl citrate
- lipids such as poly( ethylene glycol) (including those with the ends blocked with methyls or similar groups)
- silicone oil such as silicone oil, partially or fully fluorinated hydrocarbons, N-methyl-2-pyrrolidone, or mixtures thereof.
- Polymers used in the methods disclosed herein can be used in combination with a variety of particulate materials.
- they can be used with moisture curing ceramic materials (e.g., tricalcium phosphate) for vertebroplasty cements, bone void filling (due to disease such as cancer or due to fracture).
- They can be used in combination with inorganic materials such as hydroxyapatite to form pastes for use in bone healing, sealing, filling, repair, and replacement.
- They can be used as or in combination with polymer microspheres that can be reservoirs for a biologically active agent such as a protein, DNA plasmid, RNA plasmid, antisense agent, etc.
- polymers used in the methods disclosed herein can be used in combination with other materials to form a composite (e.g., a polymer having an additive therein).
- composites can include a wide variety of additives, and particularly particulate additives, such as, for example, fillers (e.g., including particulate, fiber, and/or platelet material), other polymers (e.g., polymer particulate materials such as polytetrafluoroethylene can result in higher modulus composites), imaging particulate materials (e.g., barium sulfate for visualizing material placement using, for example, fluoroscopy), biologically derived materials (e.g., bone particles, cartilage, demineralized bone matrix, platelet gel, and combinations thereof), and combinations thereof.
- Additives can be dissolved, suspended, and/or dispersed within the composite. For particulate additives, the additive is typically dispersed within the composite.
- Polymers used in the methods disclosed herein can be combined with fibers, woven or nonwoven fabric for reconstructive surgery, such as the in situ formation of a bone plate or a bone prosthesis.
- one or more polymers used in the methods disclosed herein can be shaped to form a medical device, preferably a biodegradable medical device.
- the one or more polymers can be shaped by methods known in the art including compression molding, injection molding, casting, extruding, milling, blow molding, or combinations thereof.
- a "medical device” includes devices that have surfaces that contact tissue, bone, blood, or other bodily fluids in the course of their operation, which fluids are subsequently used in patients. This can include, for example, extracorporeal devices for use in surgery such as blood oxygenators, blood pumps, blood sensors, tubing used to carry blood, and the like which contact blood which is then returned to the patient.
- medical devices can include biodegradable nasal and sinus stents.
- medical devices can include chronically removable pacemaker leads.
- a medical device can also be fabricated by polymerizing components in a suitable mold.
- Polymers used in the methods disclosed herein can also be coated onto a substrate if desired.
- a coating mixture of the polymer can be prepared using solvents such as toluene, chloroform, tetrahydrofuran, perfluorinated solvents, and combinations thereof.
- Preferred solvents include those that can be rendered moisture-free and/or those that have no active hydrogens.
- the coating mixture can be applied to an appropriate substrate such as uncoated or polymer coated medical wires, catheters, stents, prostheses, penile inserts, and the like, by conventional coating application methods. Such methods include, but are not limited to, dipping, spraying, wiping, painting, solvent swelling, and the like.
- the solvent is preferably allowed to evaporate from the coated substrate.
- the materials of a suitable substrate include, but are not limited to, polymers, metal, glass, ceramics, composites, and multilayer laminates of these materials.
- the coating may be applied to metal substrates such as the stainless steel used for guide wires, stents, catheters and other devices.
- Organic substrates that may be coated with the polymers used in the methods disclosed herein include, but are not limited to, polyether- polyamide block copolymers, polyethylene terephthalate, polyetherurethane, polyesterurethane, other polyurethanes, silicone, natural rubber, rubber latex, synthetic rubbers, polyester-polyether copolymers, polycarbonates, and other organic materials.
- Additives that can be combined with a polymer used in the methods disclosed herein to form a composition include, but are not limited to, wetting agents for improving wettability to hydrophobic surfaces, viscosity and flow control agents to adjust the viscosity and thixotropy of the mixture to a desired level, antioxidants to improve oxidative stability of the coatings, dyes or pigments to impart color or radiopacity, and air release agents or defoamers, cure catalysts, cure accelerants, plasticizers, solvents, stabilizers (cure inhibitors, pot-life extenders), and adhesion promoters.
- wetting agents for improving wettability to hydrophobic surfaces include, but are not limited to, wetting agents for improving wettability to hydrophobic surfaces, viscosity and flow control agents to adjust the viscosity and thixotropy of the mixture to a desired level, antioxidants to improve oxidative stability of the coatings, dyes or pigments to impart color or radiopacity, and air release agents or defo
- the polymers used in the methods disclosed herein can include one or more biologically active agents different than the one or more antiproliferative agents described herein.
- a biologically active agent is intended to be broadly interpreted as any agent capable of eliciting a response in a biological system such as, for example, living cell(s), tissue(s), organ(s), and being(s).
- Biologically active agents can include natural and/or synthetic agents.
- a biologically active agent is intended to be inclusive of any substance intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease or in the enhancement of desirable physical or mental development and conditions in a subject.
- subject as used herein is taken to include, but is not limited to, humans, sheep, horses, cattle, pigs, dogs, cats, rats, mice, birds, reptiles, fish, insects, arachnids, protists (e.g., protozoa), and prokaryotic bacteria.
- the subject is a human or other mammal.
- a preferred class of biologically active agents includes drugs.
- drug means any therapeutic agent.
- Suitable drugs include inorganic and organic drugs, without limitation, and include drugs that act on the peripheral nerves, adrenergic receptors, cholinergic receptors, nervous system, skeletal muscles, cardiovascular system, smooth muscles, blood circulatory system, synaptic sites, neuro-effector junctional sites, endocrine system, hormone systems, immunological system, reproductive system, skeletal system, autocoid systems, alimentary and excretory systems (including urological systems), histamine systems, and the like.
- Such conditions, as well as others, can be advantageously treated using compositions as disclosed herein.
- Suitable drugs include, for example, polypeptides (which is used herein to encompass a polymer of L- or D- amino acids of any length including peptides, oligopeptides, proteins, enzymes, hormones, etc.), polynucleotides (which is used herein to encompass a polymer of nucleic acids of any length including oligonucleotides, single- and double- stranded DNA, single- and double-stranded RNA, DNA/RNA chimeras, etc.), saccharides (e.g., mono-, di-, poly-saccharides, and mucopolysaccharides), vitamins, viral agents, and other living material, radionuclides, and the like.
- polypeptides which is used herein to encompass a polymer of L- or D- amino acids of any length including peptides, oligopeptides, proteins, enzymes, hormones, etc.
- polynucleotides which is used herein to encompass a polymer of nu
- antithrombogenic and anticoagulant agents such as heparin, Coumadin, protamine, and hirudin
- antimicrobial agents such as antibiotics
- antineoplastic agents and antiproliferative agents such as etoposide, podophylotoxin
- antiplatelet agents including aspirin and dipyridamole
- antimitotics (cytotoxic agents) and antimetabolites such as methotrexate, colchicine, azathioprine, vincristine, vinblastine, fluorouracfl, adriamycin, and mutamycinnucleic acids
- antidiabetic such as rosiglitazone maleate
- anti-inflammatory agents such as heparin, Coumadin, protamine, and hirudin
- antimicrobial agents such as antibiotics
- antineoplastic agents and antiproliferative agents such as etoposide, podophylotoxin
- antiplatelet agents including aspirin and dipyridamole
- Anti-inflammatory agents for use in the present invention include glucocorticoids, their salts, and derivatives thereof, such as Cortisol, cortisone, fludrocortisone, Prednisone, Prednisolone, 6a- methylprednisolone, triamcinolone, betamethasone, dexamethasone, beclomethasone, aclomethasone, amcinonide, clebethasol and clocortolone.
- glucocorticoids such as Cortisol, cortisone, fludrocortisone, Prednisone, Prednisolone, 6a- methylprednisolone, triamcinolone, betamethasone, dexamethasone, beclomethasone, aclomethasone, amcinonide, clebethasol and clocortolone.
- Preferred classes of drugs include, for example, Plasmid DNA, genes, antisense oligonucleotides and other antisense agents, peptides, proteins, protein analogs, siRNA, shRNA, miRNA, ribozymes, DNAzymes and other DNA based agents, viral and non-viral vectors, liposomes, cells, stem cells, antineoplastic agents, antiproliferative agents, antithrombogenic agents, anticoagulant agents, antiplatelet agents, antibiotics, anti-inflammatory agents, antimitotic agents, immunosuppressants, growth factors, cytokines, hormones, and combinations thereof.
- BMP bone morphogenetic proteins
- rhBMP-2 recombinant human bone morphogenetic protein
- Suitable drugs can have a variety of uses including, but are not limited to, anticonvulsants, analgesics, antiparkinsons, antiinflammatories (e.g., ibuprofen, fenbufen, cortisone, and the like), calcium antagonists, anesthetics (e.g., benoxinate, benzocaine, procaine, and the like), antibiotics (e.g., ciprofloxacin, norfloxacin, clofoctol, and the like), antimalarials, antiparasitics, antihypertensives, antihistamines, antipyretics, alpha-ad renergic agonists, alpha-blockers, biocides, bactericides, bronchial dilators, beta-adrenergic blocking drugs, contraceptives, cardiovascular drugs, calcium channel inhibitors, depressants, diagnostics, diuretics, electrolytes, enzymes, hypnotics, hormones, hypo, hypo
- Certain preferred embodiments include a drug selected from the group consisting of podophyllotoxin, mycophenolic acid, teniposide, etoposide, trans-retinoic acids, 9-cis retinoic acid, 13-cis retinoic acid, rapamycin, a rapalog (e.g., Everolimus, ABT-578), camptothecin, irinotecan, topotecan, tacromilus, mithramycin, mitobronitol, thiotepa, treosulfan, estramusting, chlormethine, carmustine, lomustine, busultan, mephalan, chlorambucil, ifosfamide, cyclophosphamide, doxorubicin, epirubicin, aclarubicin, daunorubicin, mitosanthrone, bleomycin, cepecitabine, cytarabine, fludarabine, cla
- Certain preferred embodiments include a drug selected from the group consisting of salicylic acid, fenbufen, cortisone, ibuprofen, diflunisal, sulindac, difluprednate, prednisone, medrysone, acematacin, indomethacin, meloxicam, camptothecin, benoxinate, benzocaine, procaine, ciprofloxacin, norfloxacin, clofoctol, dexamethasone, fluocinolone, ketorolac, pentoxifylline, rapamycin, ABT-578, gabapentin, baclofen, sulfasalazine, bupivacaine, sulindac, clonidine, etanercept, pegsunercept, and combinations thereof.
- a drug selected from the group consisting of salicylic acid, fenbufen, cortisone, ibuprofen
- compositions including a biologically active agent and a polymer used in the methods disclosed herein can be prepared by suitable methods known in the art.
- such compositions can be prepared by solution processing, milling, extruding, polymerizing components in the presence of a biologically active agent, and combinations thereof.
- compositions including polymers used in the methods disclosed herein can further include additional components.
- additional components include fillers, dyes, pigments, inhibitors, accelerators, viscosity modifiers, wetting agents, buffering agents, stabilizers, biologically active agents, polymeric materials, excipients, and combinations thereof.
- Medical devices that include one or more polymers used in the methods disclosed herein and a biologically active agent can have a wide variety of uses.
- the biologically active agent is preferably disposed in the one or more polymers.
- such devices can be used to deliver a biologically active agent to a tissue by positioning at least a portion of the device including the one or more polymers proximate the tissue and allowing the one or more polymers to biodegrade and deliver the biologically active agent disposed therein.
- such devices can be used to control the release rate of a biologically active agent from a medical device by disposing the biologically active agent in at least one of the one or more polymers.
- the effects of the antiproliferative agents disclosed herein can be evaluated in vitro, for example, by using cultures or co-cultures of primary or commercially available cell lines.
- endothelial cells can be isolated from aortic or coronary arteries
- stem cells can be isolated from bone marrow, and myoblasts from skeletal muscle.
- adult stem cells, embryonic stem cells, somatic cells, cancer cell lines, or cells from any proliferative biopsy and/or tissue can be utilized to evaluate the effectiveness of the antiproliferative agents disclosed herein, and to direct the application of the methods disclosed herein.
- An antiproliferative agent with potential for biomedical applications can be evaluated by its effects in cell culture in vitro.
- a test agent e.g., by adding into culture medium
- the antiproliferative effect of the test agent can be confirmed by a reduced number of cells following a given time of exposure, in comparison to controls that do not have the test agent added into their media.
- the test agent e.g., a cytostatic agent
- the test agent does not evidence significant signs of cytotoxicity under conditions in which the test agent shows antiproliferative effects.
- the test agent is withdrawn from the cell culture wells, the cells preferably again exhibit normal morphology. Optimal concentration ranges can be estimated based on the above-described conditions.
- Evaluation of the effect in vivo can be done at several concentrations, the first of which is targeted at fine-tuning optimal concentrations and confirming cytostatic properties. Histological studies following acute and chronic exposure of the test agent is typically carried out. Certain antiproliferative agents disclosed herein have been shown to have acceptable cell compatibility in vitro, and at concentrations of from 1 to 10 mg/ml have evidenced antiproliferative effect on endothelial cell lines (e.g., human umbilical vein endothelial cells; HUVEC) and Glioblastoma/Astrocytoma cell lines (e.g., human Glioblastoma/Astrocytoma cell line U87). Interestingly at these concentrations the cell morphology appeared uncompromised, thus suggesting a cytostoatic effect.
- endothelial cell lines e.g., human umbilical vein endothelial cells; HUVEC
- Glioblastoma/Astrocytoma cell lines e.
- kits for evaluating cell growth or proliferation, cell metabolism, leakage of enzymes, and cytotoxicity can be used to evaluate the effect of test agents (e.g., cytostatic and/or cytotoxic responses).
- Methods as disclosed herein can be used with a wide variety of cell lines including, but not limited to, fibroblast cells, smooth muscle cells, tumor cells, endothelial cells, and the like, and combinations thereof.
- Preferred cell lines include, but are not limited to, normal human dermal fibroblast (NHDF) cells, coronary artery smooth muscle cells (CASMC), .
- NHDF normal human dermal fibroblast
- CASMC coronary artery smooth muscle cells
- human Glioblastoma/Astrocytoma U87 cells human umbilical vein endothelial cell (HUVEC), human coronary artery endothelial cells (HCAEC), and combinations thereof.
- Methods as disclosed herein can be used to treat or prevent a wide variety of proliferative (e.g. hyperproliferative) conditions and/or diseases.
- proliferative e.g. hyperproliferative
- diseases include, but are not limited to, hyperplasia of soft and hard tissues, malignant tumors such as cancer, skin keloids, fibrosis, and surgical adhesions.
- Methods as disclosed herein can also be used, for example, to prevent cell proliferative processes resulting in in-stent restenosis (e.g., associated with coronary stents), pannus overgrowth on prosthetic heart valves (e.g., associated with sewing rings), urethral stenosis (e.g., associated with prostatic hyperplasia), and stenosis associated with surgical anastomosis.
- Methods as disclosed herein can also be used to limit tumor growth.
- Methods as disclosed herein can also be used to prevent the occlusion of catheters, such as, for example, cerebrospinal fluid (CSF) shunts.
- CSF cerebrospinal fluid
- Methods as disclosed herein can be used, for example, to inhibit any area of the body as desired to retard excessive cell proliferation, including, for example, treatment of male sterility, inhibition of moles, prevention of excessive hair growth, and the like.
- EXAMPLE 1 Incorporating 5,6-dihydroxyhexan-2-one into a polymer chain.
- EXAMPLE 2 Derivatizing 5,6 ⁇ dihydroxyhexan-2-one.
- the assay used was a non-radioactive cytotoxicity assay available under the trade designation CytoTox 96 from Promega Corporation (Madison, Wl), which was designed to determine the cytotoxicity of a substance by quantitatively measuring the presence of lactate dehydrogenase (LDH) in cell culture supernatant.
- the enzymatic assay utilizes the LDH released in the supernatant in the conversion of a tetrazolium salt substrate into a red formazan product. This resulting product is measured using a standard 96 well plate reader where the absorbance recorded at 490 nanometers is directly proportional to the number of lysed cells.
- the Normal Human Dermal Fibroblasts were available from Clonetics Corporation (San Diego, CA).
- the 5,6-dihydroxy-hexan-2-one (1.2 grams) was dissolved in 12 ml of phosphate buffered saline and was filter sterilized through a 0.22 micrometer syringe filter prior to use. The 5,6-dihydroxy-hexan-2-one was further diluted to 1 ⁇ ', 10 , 10 , 10 , 10°, and10° using serial dilutions for the evaluation.
- NHDF were seeded at 45,000 cells/well in 24 well plate(s). Cells were grown for 72 hours, 48 hours of which was done in the presence of the test chemicals. 24 hours after seeding, 5,6-dihydroxy-hexan-2-one was added to the cell culture plates (2 ml/well). Assay plates were pulled for analysis at 24 and 48 hours after addition of the chemical compound. Samples were analyzed on the above-described assay as provided in the instructions for use of the assay.
- Figure 1 is a graphical representation of the cytotoxicity of fibroblast cells exposed to various concentrations of 5,6-dihydroxy-hexan-2-one for 24 hours and 48 hours. Negligible levels (from 0 to 2.5% on a scale of 0 to 100%) were observed for controls and test groups. The results illustrated in Figure 1 indicate no increase in cytotoxicity as a result of exposing the cells to the various concentrations of 5,6-dihydroxy-hexan- 2-one for 48 hours.
- the cytotoxicity assay revealed no significant cytotoxicity in response to the presence of 5,6-dihydroxy-hexan-2-one, at 10 "1 concentration, indicating minimal release of LDH attributable to cell death. However the ability for cells to proliferate in the presence of 5,6-dihydroxy-hexan-2- one was significantly affected. Furthermore, the culture of previously exposed cells in a media without 5,6-dihydroxy-hexan-2-one indicated a persistent negative effect on proliferation. This may suggest accumulative anti-proliferative effect or the possibility of agent remaining in the in vitro system.
- EXAMPLE 4 Examination of the cell growth effects upon 24 hour and 48 hour exposure of Normal Human Dermal Fibroblasts (NHDF) to 5,6-dihydroxy-hexan-2-one. After the 24 hour or 48 hour exposures, the 5,6-dihydroxy-hexan-2-one was removed and the cells were allowed to grow for an additional 96 hours, then evaluated for growth.
- NHDF Normal Human Dermal Fibroblasts
- the assay used was a luminescent cell viability assay available under the trade designation CellTiter-Glo from Promega Corporation (Madison, Wl). This assay determines the number of viable cells present based on the amount of adenosine triphosphate (ATP) present from the cell cytoplasm. The concentration of ATP present is proportional to the measured amount of a luminescent signal. The amount of ATP shows the existence of metabolically active cells, and hence, the number of viable cells.
- the Normal Human Dermal Fibroblasts were available from Clonetics Corporation (San Diego, CA).
- the 5,6-dihydroxy-hexan-2-one (1.2 grams) was dissolved in 12 ml of phosphate buffered saline and was filter sterilized through a 0.22 micrometer syringe filter prior to use. The 5,6-dihydroxy-hexan-2-one was further diluted to 10 , 10 , and 10 using serial dilutions for the evaluation.
- NHDF were seeded at 45,000 cells/well in four 24 well plates (two testing cell growth and two to re-seed after 24 and 48 hours without 5,6- dihydroxy-hexan-2-one). 24 hours after seeding, 5,6-dihydroxy-hexan-2- one was added to the cell culture plates (2 ml/well) at a concentration of
- Figure 7 (10 i-1 , 24 hours), Figure 8 (10 -2 , 24 hours), Figure 9 (10 v3 , 24 hours), Figure 10 (control, 24 hours), Figure 11 (10 ⁇ ⁇ 48 hours), Figure 12 (10 "2 , 48 hours), Figure 13 (10 "3 , 48 hours), and Figure 14 (control, 48 hours) visually display the physical cellular response of NHDF to various concentrations of 5,6-dihydroxy-hexan-2-one.
- Table 2 represents a cell count taken of the different concentration groups after 96 hours of growing without the presence of 5,6-dihydroxy- hexan-2-one. Although, a slight reduction of cell growth was observed in the 10 "2 group, the cell count matched what was visually seen, as did the 10 ⁇ 1 and 10 "3 concentrations. The 10 "1 group showed significant inhibition of cell growth and had roughly the same amount of cells that were previously seeded. The cells in the 10 "1 group have proven to have an impaired ability for cell proliferation or growth.
- EXAMPLE 5 Evaluation of the effects of 5,6-dihydroxy-h ⁇ xan-2-one on cell proliferation, viability, and migration of normal human dermal fibroblast (NHDF) cells and coronary artery smooth muscle cells (CASMC) performed in vitro.
- NHDF human dermal fibroblast
- CASMC coronary artery smooth muscle cells
- a luminescent cell viability assay available under the trade designation CellTiter-GIo from Promega Corporation (Madison, Wl) uses the amount of ATP present in the cell cytoplasm as an indicator for metabolic activity.
- the amount of ATP present is proportional to the measured amount of a luminescent signal and therefore the amount of viable cells.
- NHDF human dermal fibroblast
- CASMC coronary artery smooth muscle cells
- Figure 17 is a graphical representation of fibroblast cell numbers measured after 48 hours exposure to different concentrations of 5,6-dihydroxy-hexan-2-one.
- Figure 18 is a graphical representation of smooth muscle cell numbers measured after 48 hours exposure to different concentrations of 5,6-dihydroxy-hexan-2-one.
- Figure 19 is a graphical representation of viable smooth muscle cell numbers measured after 48 hours exposure to different concentrations of 5,6-dihydroxy-hexan-2-one.
- Figures 20(a)-(l) are pictures that visually display the migration of coronary artery smooth muscle cells (CASMC) ( Figures 20(a)-(f)) and normal human dermal fibroblast (NHDF) cells ( Figures 20(g)-(0) exposed to various concentrations of 5,6-dihydroxy-hexan-2-one after 48 hours exposure, then cultured an additional 72 hours after removal of the 5,6- dihydroxy-hexan-2-one as described in Example 5.
- CASMC coronary artery smooth muscle cells
- NHDF normal human dermal fibroblast
- Figures 20(a) and (b) represent the CASMC control at time zero and 72 hours, respectively;
- Figures 20(c) and (d) represent CASMC (10 mg/ml) at time zero and 72 hours, respectively; and
- Figures 20(e) and (f) represent CASMC (1 mg/ml) at time zero and 72 hours, respectively.
- Figures 20(g) and (h) represent the NHDF cell control at time zero and 72 hours, respectively;
- Figures 20(i) and (j) represent NHDF cells (10 mg/ml) at time zero and 72 hours, respectively;
- Figures 20(k) and (I) represent NHDF cells (1 mg/ml) at time zero and 72 hours, respectively.
- EXAMPLE 6 Evaluation of the effects of 5,6-dihydroxy-hexan-2-one on human umbilical vein endothelial cells (HUVEC) and Glioblastoma/Astrocytoma cells U87 cellular proliferation and migration during 120 hours following 48 hour exposure to 5,6-dihydroxy-hexan-2- one at concentration of 10 mg/ml, 1 mg/ml, 100 micrograms/ml. Controls were cells not exposed to the agent.
- HUVEC human umbilical vein endothelial cells
- Glioblastoma/Astrocytoma cells U87 cellular proliferation and migration during 120 hours following 48 hour exposure to 5,6-dihydroxy-hexan-2- one at concentration of 10 mg/ml, 1 mg/ml, 100 micrograms/ml. Controls were cells not exposed to the agent.
- CyQuant from Invitrogen Corporation determines the density of cells in culture by using a green fluorescent dye which fluoresces when bound to cellular nucleic acids.
- Figure 21 is a graphical representation of human Glioblastoma/
- Time 0 refers to the replacement of the test agent with supplemented growth media.
- Figure 22 is a graphical representation of human umbilical vein endothelial cell (HUVEC) numbers measured after being exposed to various concentrations of 5,6-dihydroxy-hexan-2-one for 48 hours. Time 0 refers to replacement of the test agent with supplemented growth media.
- HUVEC human umbilical vein endothelial cell
- Figures 23(a)-(l) are pictures that visually display the migration over time of human umbilical vein endothelial cells (HUVEC) after 48 hours exposure to various concentrations of 5,6-dihydroxy-hexan-2-one. Specifically, Figures 23(a)-(d) represent the control, Figures 23(e)-(h) represent 10 mg/ml, and Figures 23(i)-(l) represent 1 mg/ml 5,6- dihydroxy-hexan-2-one.
- HUVEC human umbilical vein endothelial cells
- Figures 23 (a), (e), and (i) represent migration after 2 hours
- Figures 23 (b), (f), and (j) represent migration after 14 hours
- Figures 23 (c), (g), and (k) represent migration after 20 hours
- Figures 23 (d), (h), and (I) represent migration after 48 hours.
- Figures 24(a)-(l) are pictures that visually display the migration over time of human Glioblastoma/Astrocytoma U87 cells after 48 hours exposure to various concentrations of 5,6-dihydroxy-hexan-2-one. Specifically, Figures 24(a)-(d) represent the control, Figures 24(e)-(h) represent 10 mg/ml, and Figures 24(i)-(l) represent 1 mg/ml 5,6- dihydroxy-hexan-2-one.
- Figures 24 (a), (e), and (i) represent migration after 2 hours
- Figures 24 (b), (f), and (j) represent migration after 14 hours
- Figures 24 (c), (g), and (k) represent migration after 20 hours
- Figures 23 (d), (h), and (I) represent migration after 48 hours.
- EXAMPLE 7 Evaluation of the effects upon 24 and 48 hour exposure of human coronary artery endothelial cells (HCAEC) to six compounds.
- [133] Cells were exposed to the test compounds for 24 to 48 hours and then allowed to grow for 96 hours in regular media (without test compound). The final concentrations for cell treatment were as follows: compounds 13358-18-1 , 13358-18-2, 13358-18-4, and 13358-18-6 were evaluated at 10 mg/ml, 1 mg/ml, 100 micrograms/ml, and 10 micrograms/ml. Compounds 13358-18-3 and 13358-18-5 were evaluated at 50 mg/ml, 10 mg/ml, 1 mg/ml, and 100 micrograms/ml.
- a non-radioactive cytotoxicity assay available under the trade designation CytoTox 96 from Promega Corporation (Madison, Wl) is designed to determine the cytotoxicity of a substance by quantitatively measuring the presence of lactate dehydrogenase (LDH) in cell culture supernatant. LDH is released in cells that are undergoing cell death and the assay converts it to a red formazan product that can allows for the proportional amount of LDH absorbance to be read.
- LDH lactate dehydrogenase
- a cell proliferation assay available under the trade designation CyQuant from Invitrogen Corporation determines the density of cells in culture by eluding a green fluorescent dye which fluoresces when bound to cellular nucleic acids. The amount of nucleic acid present is proportional to the amount of living cells in culture, therefore an accurate cell count can be obtained.
- a luminescent cell viability assay available under the trade designation CellTiter-Glo from Promega Corporation uses the amount ATP present in the cell cytoplasm as an indicator for metabolic activity. The amount of ATP present is proportional to the measured amount of a luminescent signal. This assay was used to confirm if the cells are still viable after exposure to the test compounds.
- Figures 25(a) and (b) are graphical illustrations of cell counts and cell viability, respectively, for human coronary artery endothelial cells (HCAEC) exposed to 5,6-dihydroxy-6-phenyl-hexan-2-one (13358-18-1 ) for 24 hours and 48 hours, then allowed to grow for 96 hours after removal of 5,6-dihydroxy-6-phenyl-hexan-2-one.
- HCAEC human coronary artery endothelial cells
- Figures 26(a) and (b) are graphical illustrations of cell counts and cell viability, respectively, for human coronary artery endothelial cells (HCAEC) exposed to 5,6-dihydroxy-heptan-2-one (13358-18-2) for 24 hours and 48 hours, then allowed to grow for 96 hours after removal of 5,6-dihydroxy-heptan-2-one.
- HCAEC human coronary artery endothelial cells
- Figures 27(a) and (b) are graphical illustrations of cell counts and cell viability, respectively, for human coronary artery endothelial cells (HCAEC) exposed to 5,6-dihydroxy-5-methyl-hexan-2-one (13358-18-3 for 24 hours and 48 hours, then allowed to grow for 96 hours after removal of 5,6-dihydroxy-5-methyl-hexan-2-one.
- HCAEC human coronary artery endothelial cells
- Figures 28(a) and (b) are graphical illustrations of cell counts and cell viability, respectively, for human coronary artery endothelial cells (HCAEC) exposed to 2-(2',3'-dihydroxypropyl)-cyclohexanone (13358-18- 4) for 24 hours and 48 hours, then allowed to grow for 96 hours after removal of 2-(2',3'-dihydroxypropyl)-cyclohexanone.
- HCAEC human coronary artery endothelial cells
- Figures 29(a) and (b) are graphical illustrations of cell counts and cell viability, respectively, for human coronary artery endothelial cells (HCAEC) exposed to 2, 3-d i hydroxy propyl acetate (13358-18-5) for 24 hours and 48 hours, then allowed to grow for 96 hours after removal of 2,3-dihydroxypropyl acetate.
- HCAEC human coronary artery endothelial cells
- Figures 30(a) and (b) are graphical illustrations of cell counts and cell viability, respectively, for human coronary artery endothelial cells (HCAEC) exposed to 5,6-dihydroxy-hexan-2-one (13358-18-6) for 24 hours and 48 hours, then allowed to grow for 96 hours after removal of 5,6-dihydroxy-hexan-2-one.
- HCAEC human coronary artery endothelial cells
Abstract
Description
Claims
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/US2007/018972 WO2009029069A1 (en) | 2007-08-29 | 2007-08-29 | Methods of inhibiting or suppressing cellular proliferation |
Publications (1)
Publication Number | Publication Date |
---|---|
EP2180890A1 true EP2180890A1 (en) | 2010-05-05 |
Family
ID=39308034
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP07837460A Withdrawn EP2180890A1 (en) | 2007-08-29 | 2007-08-29 | Methods of inhibiting or suppressing cellular proliferation |
Country Status (2)
Country | Link |
---|---|
EP (1) | EP2180890A1 (en) |
WO (1) | WO2009029069A1 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE102017010898A1 (en) * | 2017-11-24 | 2019-05-29 | Eberhard Karls Universität Tübingen | New inhibitors of shikimic acid pathway |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007098041A1 (en) * | 2006-02-17 | 2007-08-30 | Medtronic, Inc. | Polyketal polymers, and methods of making and using same |
-
2007
- 2007-08-29 EP EP07837460A patent/EP2180890A1/en not_active Withdrawn
- 2007-08-29 WO PCT/US2007/018972 patent/WO2009029069A1/en active Application Filing
Non-Patent Citations (1)
Title |
---|
See references of WO2009029069A1 * |
Also Published As
Publication number | Publication date |
---|---|
WO2009029069A1 (en) | 2009-03-05 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US8106146B2 (en) | Therapeutic polymers and methods of generation | |
WO2008115694A2 (en) | Polymerization of multifunctional azides, and polymers therefrom | |
ES2451653T3 (en) | Implantable medical device with surface erosion polyester drug supply coating | |
US9603976B2 (en) | Biodegradable coatings for implantable medical devices | |
US7442721B2 (en) | Durable biocompatible controlled drug release polymeric coatings for medical devices | |
US7741375B2 (en) | Polyketal polymers, and methods of making and using same | |
JP2007532197A (en) | Coating composition for bioactive substances | |
US20110052503A1 (en) | Biodegradable contrast agents | |
US20090043378A1 (en) | Biocompatible Polymer System for Extended Drug Release | |
JP2008526371A (en) | Biodegradable coating composition comprising a blend | |
JP2008526322A (en) | Biodegradable coating composition comprising multiple layers | |
JP6351617B2 (en) | Coating comprising polyesteramide copolymer for drug delivery | |
EP2634198B1 (en) | Copolymers containing phosphorylcholine groups and methods of preparing and using the same | |
JP2005530561A (en) | Silicone mixtures and composites for drug delivery | |
EP2716307A1 (en) | Drug eluting stent with a biodegradable release layer attached with an electro-grafted primer coating | |
US20110065809A1 (en) | Polymerization of Multifunctional Azides, and Polymers Therefrom | |
US20090269390A1 (en) | Medical devices, polymers, compositions, and methods for delivering a haloacetate | |
US20090061515A1 (en) | Methods of inhibiting or suppressing cellular proliferation | |
WO2009029069A1 (en) | Methods of inhibiting or suppressing cellular proliferation | |
CN111603614B (en) | Catheter and preparation method thereof | |
CN109010931B (en) | Interventional medical device and application of aphidicolin | |
US20100240852A1 (en) | Heteroatom-containing polymers and metathesis polymerization methods for making same |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20100218 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE SI SK TR |
|
AX | Request for extension of the european patent |
Extension state: AL BA HR MK RS |
|
RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: KING, KRYSTAL Inventor name: DONOVAN, MAURA Inventor name: CASAS-BEJAR, JESUS Inventor name: LUO, LIAN, LEON Inventor name: ROBINSON, TIMOTHY, H. Inventor name: BENZ, MICHAEL, ERIC |
|
DAX | Request for extension of the european patent (deleted) | ||
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20130301 |