WO2013049161A1 - Controlled hydrolysis of poly-4-hydroxybutyrate and copolymers - Google Patents
Controlled hydrolysis of poly-4-hydroxybutyrate and copolymers Download PDFInfo
- Publication number
- WO2013049161A1 WO2013049161A1 PCT/US2012/057277 US2012057277W WO2013049161A1 WO 2013049161 A1 WO2013049161 A1 WO 2013049161A1 US 2012057277 W US2012057277 W US 2012057277W WO 2013049161 A1 WO2013049161 A1 WO 2013049161A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- polymer
- copolymer
- p4hb
- molecular weight
- copolymers
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
- C08G63/91—Polymers modified by chemical after-treatment
- C08G63/912—Polymers modified by chemical after-treatment derived from hydroxycarboxylic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/26—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/42—Use of materials characterised by their function or physical properties
- A61L15/44—Medicaments
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L17/00—Materials for surgical sutures or for ligaturing blood vessels ; Materials for prostheses or catheters
- A61L17/005—Materials for surgical sutures or for ligaturing blood vessels ; Materials for prostheses or catheters containing a biologically active substance, e.g. a medicament or a biocide
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L17/00—Materials for surgical sutures or for ligaturing blood vessels ; Materials for prostheses or catheters
- A61L17/06—At least partially resorbable materials
- A61L17/10—At least partially resorbable materials containing macromolecular materials
- A61L17/105—Polyesters not covered by A61L17/12
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/001—Use of materials characterised by their function or physical properties
- A61L24/0015—Medicaments; Biocides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
- A61L24/046—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
- A61L26/0019—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/0066—Medicaments; Biocides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
- A61L29/04—Macromolecular materials
- A61L29/06—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
- A61L29/14—Materials characterised by their function or physical properties, e.g. lubricating compositions
- A61L29/16—Biologically active materials, e.g. therapeutic substances
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/04—Macromolecular materials
- A61L31/06—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L31/16—Biologically active materials, e.g. therapeutic substances
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
- C08G63/02—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds
- C08G63/06—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds derived from hydroxycarboxylic acids
Definitions
- the present invention generally relates to methods for controlling the hydrolysis of poly-4-hydroxybutyrate and copolymers thereof, and to compositions of poly-4-hydroxybutyrate and copolymers thereof with specific weight average molecular weights.
- the compositions can be used in implantable medical devices, controlled release applications, such as drug delivery, and certain compositions may be used for therapeutic delivery of 4- hydroxybutyrate.
- the compositions may also be used in blends, and as intermediates in synthesis.
- Poly-4-hydroxybutyrate (P4HB) and copolymers thereof can be produced using transgenic fermentation methods, see, for example, U.S. Patent No. 6,548,569 to Williams et al, and are produced commercially, for example, by Tepha, Inc. (Lexington, MA).
- Poly-4-hydroxybutyrate (P4HB, TephaFLEX ® biomaterial) is a strong, pliable thermoplastic polyester that, despite its biosynthetic route, has a relatively simple structure.
- the polymer belongs to a larger class of materials called
- PHAs polyhydroxyalkanoates
- polyesters are produced as storage granules inside cells, and serve to regulate energy metabolism. They are also of commercial interest because of their thermoplastic properties, and relative ease of production.
- biosynthetic routes are currently known to produce P4HB as shown in Figure 1.
- U.S. Patent Nos 6,245,537, 6,623,748 and 7,244,442 describe methods of making PHAs with little to no endotoxin, which is suitable for medical applications.
- U.S. Patent Nos. 6,548,569, 6,838,493, 6,867,247, 7,268,205, and 7, 179,883 describe use of PHAs to make medical devices.
- Copolymers of P4HB include 4-hydroxybutyrate copolymerized with 3- hydroxybutyrate or glycolic acid (U.S. patent application No. 20030211 131 by Martin and Skraly, U.S. Patent No. 6,316,262 to Huisman et al., and U.S. Patent No. 6,323,010 to Skraly et al.).
- Methods to control molecular weight of PHA polymers by biosynthetic methods have been disclosed by U.S. Patent No. 5,81 1,272 to Snell et al.
- compositions can be developed that allow the controlled release of a wide range of bioactive agents.
- Particularly desired compositions include polymers with weight average molecular weights less than 250,000, and more preferably less than 100,000.
- compositions should have a polydispersity of less than 3, and more preferably less than 2.5.
- lower molecular weight compositions of P4HB and copolymers thereof can be useful in the preparation of other chemicals, polymers, copolymers, and for blending with other materials.
- low molecular weight P4HB and copolymers thereof can be used in the preparation of polyurethanes or as nucleants or fillers in blends.
- US Patent No. 6,623,730 to Williams and Martin discloses the use of oligomers of P4HB as prodrugs to modulate endogenous levels of 4- hydroxybutyrate, and methods to prepare these oligomers by hydrolysis of P4HB.
- the method disclosed uses aliquots of sodium methoxide to hydrolyze P4HB dissolved in a solvent (anhydrous THF) to provide the oligomers of P4HB of the desired molecular weight. Starting with a polymer of molecular weight 430,000 g/mol, sodium methoxide is used to produce P4HB molecular weights of 320,000, 82,000 and 25,000.
- P4HB and copolymers thereof by hydrolysis of high molecular weight P4HB and copolymers without the use of solvents.
- compositions of P4HB and copolymers thereof that can be used in medical applications, for example, in implantable medical devices and or for the controlled release of bioactive substances, including the controlled release of 4-hydroxybutyrate.
- P4HB polymers and copolymers thereof that are useful for preparing controlled release systems, and as intermediates in chemical synthesis, have been developed. These methods avoid the use of organic solvents, and basic conditions that can cause transesterification reactions with polymer terminal end groups or elimination reactions.
- a preferred embodiment is a method for producing polymers of P4HB with weight average molecular weight less than 250,000, and more preferably, less than 100,000, which are useful in controlled release.
- a particularly preferred embodiment utilizes aqueous acetic acid to hydrolyze pellets of P4HB polymers and copolymers while in suspension.
- Figure 1 is a diagram of pathways leading to the biosynthesis of poly- 4-hydroxybutyrate.
- Figure 2 is a 1H NMR spectrum of hydrolysis product of P4HB in acetic acid for 27.5 hours.
- Poly-4-hydroxybutyrate as generally used herein means a homopolymer comprising 4-hydroxybutyrate units. It may be referred to herein as P4HB or TephaFLEX ® biomaterial (manufactured by Tepha, Inc., Lexington, MA).
- TephaFLEX ® biomaterial manufactured by Tepha, Inc., Lexington, MA.
- Copolymers of poly-4-hydroxybutyrate as generally used herein means any polymer comprising 4-hydroxybutyrate with one or more different hydroxy acid units.
- Controlled release or modified release refers to a release profile in which the drug release characteristics of time course and/or location are chosen to accomplish therapeutic or convenience objectives not offered by conventional dosage forms such as solutions, suspensions, or promptly dissolving dosage forms. Delayed release, extended release, and pulsatile release and their combinations are examples of modified release.
- Bioactive agent is used herein to refer to therapeutic, prophylactic, and/or diagnostic agents. It includes without limitation physiologically or pharmacologically active substances that act locally or systemically in the body.
- a biologically active agent is a substance used for, for example, the treatment, prevention, diagnosis, cure, or mitigation of disease or disorder, a substance which affects the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a predetermined physiological environment.
- Bioactive agents include biologically, physiologically, or pharmacologically active substances that act locally or systemically in the human or animal body.
- Bioactive agent includes a single such agent and is also intended to include a plurality
- Secondend as generally used herein means a physical combination of different polymers, as opposed to a copolymer comprised of two or more different monomers.
- Molecular weight refers to the weight average molecular weight (Mw), not the number average molecular weight (Mn), and is measured by gel permeation chromatography (GPC) relative to polystyrene.
- Biocompatible as generally used herein means the biological response to the material or device being appropriate for the device's intended application in vivo. Any metabolites of these materials should also be biocompatible.
- Methods have been developed to produce low molecular weight (Mw) compositions of P4HB and copolymers thereof in high yield. These methods may be used to prepare P4HB and copolymers thereof with weight average molecular weights (Mw) ranging from 1 to 250 kDa, and more preferably with Mw ranging from 5 to 100 kDa. The methods also yield P4HB and copolymers with narrow polydispersity (PDI), less than 3.0 and preferably a PDI of less than 2.5.
- the P4HB and copolymers thereof are prepared by hydrolysis with aqueous acids, and preferably with acetic acid.
- pellets of the polymers may be suspended in acid, and hydro lyzed while in suspension to yield polymers of predictable weight average molecular weight and narrow polydispersity.
- purification is straightforward, side reactions are essentially absent, and yields are very high.
- P4HB poly-4- hydroxybutyrate
- Copolymers include P4HB with another hydroxyacid, such as 3-hydroxybutyrate, and P4HB with glycolic acid or lactic acid monomer.
- P4HB and copolymers thereof can be obtained from Tepha, Inc. of Lexington, MA.
- the starting P4HB homopolymer and copolymers thereof are extruded into pellets.
- An extruder can be used for this purpose.
- the pellets are then suspended in acid for hydrolysis. This facilitates isolation of the product, and provides a high yield of lower molecular weight product.
- P4HB and copolymers thereof can be prepared with a weight average molecular weight less than 250,000, and more preferably, less than 100,000. It has been discovered that low molecular weight (M w ) polymers and copolymers of P4HB can be produced with unexpectedly high yields, without solvent, and in high purity with side reactions essentially absent. Notably, the hydrolysis can be controlled so that lower molecular weight (M w ) polymers and copolymers can be predictably produced with high purity, high yield, and with narrow polydispersity.
- the polymers can be hydrolyzed by suspending the polymer in an acid or an acid solution. Therefore, in one embodiment, the acid or acid solution is one in which the polymer pellet is insoluble or partially insoluble.
- the acid can be an organic acid or an inorganic acid. For those
- the acid can be a
- Suitable organic acids include, but are not limited to, acetic acid, halogenated acetic acid, such as mono-, di-, trihalogenated acetic acid, lactic acid, glycolic acid, propanoic acid, butanoic acid, pentanoic acid, hexanoic acid, benzoic acid, fumaric acid, maleic acid, tartaric acid, citric acid, malic acid, and pyruvic acid.
- Suitable inorganic acids include, but are not limited to, hydrochloric acid, sulfuric acid, hydrobromic acid, and hydroiodic acid.
- a particularly preferred embodiment utilizes aqueous acetic acid to hydrolyze P4HB polymers and copolymers. More preferably, the polymers and copolymers are first extruded into pellets, and then suspended in aqueous acid. It has been discovered that the pellets will remain suspended in the acid (with shaking or stirring) during hydrolysis as the molecular weights of the polymers decrease in a predictable manner. Even though the polymers and copolymers remain suspended, and the polymer does not dissolve, the polydispersity of the lower molecular weight (M w ) product remains narrow. Because the pellets do not dissolve in the acid, the product may be easily isolated at the end of the hydrolysis, for example, by decanting, washing with water, and drying. There is no need to evaporate acid or solvent, or use a solvent extraction process to recover the low molecular weight polymers.
- Granules or other non-pelletized forms of the polymer or copolymers can be hydrolyzed with the method, however, these forms are more likely to become sticky during hydrolysis, making isolation of the product more difficult.
- the polymer or copolymer pellets are suspended in aqueous acetic acid, and either stirred or shaken.
- the hydrolysis may be performed at ambient or elevated temperature.
- Preferably the hydrolysis is undertaken at constant temperature in the range of 37 to 50°C.
- the strength of the acid may be adjusted to speed up or slow down the rate of hydrolysis of the polymers and copolymers.
- acetic acid is used with a concentration in the range of 5M to 12M. However, lower concentrations may be used to slow down the rate of hydrolysis, and higher concentrations may be used to speed up the rate of hydrolysis.
- the length of the hydrolysis reaction will be determined by the molecular weight (M w ) desired, form of the polymer or copolymer, temperature of the reaction, type of acid, and concentration of the acid.
- M w molecular weight
- the molecular weights (Mw) of the polymer with 10M and 12M acetic acid are 85kDa and 40kDa, respectively.
- Low molecular weight polymers and copolymers of P4HB have properties that are substantially improved for many medical applications relative to higher molecular weight versions. These materials have lower melt viscosities, and higher solubilities in solvents that can make them easier to process.
- the lower molecular weight P4HB polymer and copolymers thereof can also provide more desirable controlled release profiles for delivery of therapeutic, prophylactic or diagnostic agents or other bioactive agent, such as a small-molecule drug, peptide, protein, antibody, sugar, polysaccharide, nucleotide, oligonucleotide, aptamer, siRNA, nucleic acid, and combinations thereof.
- the lower molecular weight P4HB may also be used to make medical devices, such as, but not limited to, sutures, stents, stent grafts, stent coatings, devices for temporary wound or tissue support, devices for soft or hard tissue repair, repair patches, tissue engineering scaffolds, retention membranes, anti-adhesion membranes tissue separation membranes, hernia repair devices, device coatings, cardiovascular patches, catheter balloons, vascular closure devices, slings, biocompatible coatings, rotator cuff repair devices, meniscus repair devices, adhesion barriers, guided tissue repair/regeneration devices, articular cartilage repair devices, nerve guides, tendon repair devices, intracardiac septal defect repair devices, including, but not limited to, atrial septal defect repair devices and PFO (patent foramen ovale) closure devices, left atrial appendage (LAA) closure devices, pericardial patches, bulking and filling agents, plastic surgery devices (including facial and breast cosmetic and reconstructive devices), vein valves, heart valves, bone
- Lower molecular weight polymers and copolymers of P4HB and copolymers thereof can also be used to modulate endogenous levels of 4- hydroxybutyrate as disclosed by US Patent No. 6,623,730 to Williams and Martin.
- Lower molecular weight polymers and copolymers of P4HB and copolymers thereof can also be used in implantable applications that require a more rapid rate of degradation or absorption than the higher Mw polymers.
- the implanted device will degrade and the low Mw polymer will be absorbed within six months
- P4HB polymers and copolymers with low molecular weights may also be used in blends with other materials to improve polymer properties.
- the low molecular polymers may also be used in the synthesis of other materials, for example, in reactive polymerization. II. Methods of Manufacturing Low Molecular Weight P4HB and Copolymers.
- a low molecular weight (M w ) P4HB polymer or copolymer may be prepared as follows. Pellets of a P4HB polymer are suspended in aqueous acetic acid, and placed in a shaker incubator. A number of parameters can be varied to control the hydrolysis of the polymer or copolymer including, but not limited to, the concentration of the acid, the type of acid, the temperature of the reaction, the speed (rpm) of the shaker (or stirrer speed), and the reaction time.
- the reaction is quenched by decanting the acid, and rinsing the polymer or copolymer with water. Residual acid and water may be removed by drying in vacuo.
- P4HB pellets Tepha, Inc., Lexington, MA
- M w 367 kDa 30 grams of P4HB pellets (Tepha, Inc., Lexington, MA) (M w 367 kDa) were suspended in 100 mL of 10 M aqueous acetic acid, and incubated at 37°C at 50 rpm in a shaker incubator. A sample was removed after 4 hours, and the weight average molecular weight of the polymer was determined by GPC (relative to polystyrene) to be 224 kDa. After 27.5 hours, the reaction was quenched by decanting the acetic acid, and rinsing the polymer with distilled water.
- the pellets did not clump or aggregate during the reaction time, as was seen for the similar reaction performed in 12 M acetic acid.
- the polymer pellets were then dried in vacuo to remove residual acetic acid and water.
- the weight average molecular weight of the polymer was determined to be 85 kDa after 27.5 hours, and the polymer was recovered in high yield and without loss of material.
- a 20 gram sample of P4HB pellets (Tepha, Inc., Lexington, MA) (M w 347 kDa) was suspended in 200 mL of 8 M aqueous acetic acid, and shaken at 50 rpm at 37°C in a shaker incubator. Samples were removed periodically, and the weight average molecular weight of the polymer and polydispersity were determined by GPC (relative to polystyrene).
- the hydrolysis of P4HB pellets or granules suspended in aqueous solution of acetic acid has been performed successfully under mild conditions with reasonable reaction times.
- the hydrolysis product, low Mw P4HB was prepared in a high yield, close to 100%, and its Mw could also be predicted based on the Mw versus reaction time curve under fixed reaction conditions, i.e., temperature and concentration of AcOH.
- the polydispersity of the hydrolysis products are low and similar to that of the starting polymer, about 2, showing that the hydrolysis reaction occurs uniformly throughout the bulk of the material, rather than being localized to the surface, even though the pellets/granules remain undissolved and suspended in the acetic acid solution.
- the kinetics of the hydrolysis reaction could be affected by a couple of factors such as the reaction temperature (room temperature to 50 °C) and the concentration of AcOH solution (5— 10 M). Because the P4HB pellets/granules were simply suspended in the AcOH solution at relative low temperatures instead of forming a homogeneous solution and the ratio of P4HB/AcOH could be 3g/10mL, the hydrolysis reaction described herein should be scalable for production of P4HB oligomer with controlled molecular weight.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Materials Engineering (AREA)
- Surgery (AREA)
- Vascular Medicine (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Hematology (AREA)
- Dermatology (AREA)
- Heart & Thoracic Surgery (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Transplantation (AREA)
- General Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Inorganic Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Polyesters Or Polycarbonates (AREA)
- Materials For Medical Uses (AREA)
- Medicinal Preparation (AREA)
Abstract
Methods for making P4HB polymers and copolymers thereof that are useful for preparing controlled release systems, medical devices and as intermediates in chemical synthesis, have been developed. These methods avoid the use of organic solvents, and basic conditions that can cause transesterification reactions with polymer terminal end groups or elimination reactions. A preferred embodiment is a method for producing polymers of P4HB with weight average molecular weight less than 250,000, and more preferably, less than 100,000, and a Pd of less than 3, which are useful in controlled release. A particularly preferred embodiment utilizes aqueous acetic acid to hydrolyze pellets of P4HB polymers and copolymers while in suspension.
Description
CONTROLLED HYDROLYSIS OF
POLY-4-HYDROXYBUTYRATE AND COPOLYMERS
Field of the Invention
The present invention generally relates to methods for controlling the hydrolysis of poly-4-hydroxybutyrate and copolymers thereof, and to compositions of poly-4-hydroxybutyrate and copolymers thereof with specific weight average molecular weights. The compositions can be used in implantable medical devices, controlled release applications, such as drug delivery, and certain compositions may be used for therapeutic delivery of 4- hydroxybutyrate. The compositions may also be used in blends, and as intermediates in synthesis.
Background of the Invention
Poly-4-hydroxybutyrate (P4HB) and copolymers thereof can be produced using transgenic fermentation methods, see, for example, U.S. Patent No. 6,548,569 to Williams et al, and are produced commercially, for example, by Tepha, Inc. (Lexington, MA). Poly-4-hydroxybutyrate (P4HB, TephaFLEX® biomaterial) is a strong, pliable thermoplastic polyester that, despite its biosynthetic route, has a relatively simple structure.
The polymer belongs to a larger class of materials called
polyhydroxyalkanoates (PHAs) that are produced by numerous
microorganisms (see, for example, Steinbuchel A., et al. Diversity of Bacterial Polyhydroxyalkanoic Acids, FEMS Microbial. Lett. 128:219-228 (1995)). In nature these polyesters are produced as storage granules inside cells, and serve to regulate energy metabolism. They are also of commercial interest because of their thermoplastic properties, and relative ease of production. Several biosynthetic routes are currently known to produce P4HB as shown in Figure 1.
Chemical synthesis of P4HB has been attempted, but it has been impossible to produce the polymer with a sufficiently high molecular weight that is necessary for most applications (see Hori, Y., et al, Polymer 36:4703- 4705 (1995); Houk, K.N., et al, J. Org. Chem., 2008, 73 (7), 2674-2678; and Moore, T., et al, Biomaterials 26:3771-3782 (2005)). In fact, it has been calculated to be thermodynamically impossible to chemically synthesize a high molecular weight homopolymer under normal conditions (Moore, T., et al, Biomaterials 26:3771-3782 (2005)).
U.S. Patent Nos 6,245,537, 6,623,748 and 7,244,442 describe methods of making PHAs with little to no endotoxin, which is suitable for medical applications. U.S. Patent Nos. 6,548,569, 6,838,493, 6,867,247, 7,268,205, and 7, 179,883 describe use of PHAs to make medical devices. Copolymers of P4HB include 4-hydroxybutyrate copolymerized with 3- hydroxybutyrate or glycolic acid (U.S. patent application No. 20030211 131 by Martin and Skraly, U.S. Patent No. 6,316,262 to Huisman et al., and U.S. Patent No. 6,323,010 to Skraly et al.). Methods to control molecular weight of PHA polymers by biosynthetic methods have been disclosed by U.S. Patent No. 5,81 1,272 to Snell et al.
PHAs with controlled degradation and degradation in vivo of less than one year are disclosed by U.S. Patent No. 6,548,569, 6,610,764,
6,828,357, 6,867,248, and 6,878,758 to Williams et al. and WO 99/32536 to Martin et al. Applications of P4HB have been reviewed in Williams, S.F., et al, Polyesters, III, 4:91-127 (2002), and by Martin, D. et al. Medical Applications of Poly-4-hydroxybutyrate: A Strong Flexible Absorbable Biomaterial, Biochem. Eng. J. 16:97-105 (2003). Medical devices and applications of P4HB have also been disclosed by WO 00/56376 to Williams et al. Several patents including U.S. Patent Nos. 6,555, 123, 6,585,994, and 7,025,980 describe the use of PHAs in tissue repair and engineering.
In controlled release there currently exists a need for P4HB and copolymers thereof with narrowly defined weight average molecular weight ranges. By controlling these ranges, compositions can be developed that allow the controlled release of a wide range of bioactive agents. Particularly
desired compositions include polymers with weight average molecular weights less than 250,000, and more preferably less than 100,000.
Preferably, the compositions should have a polydispersity of less than 3, and more preferably less than 2.5.
In addition to being useful in controlled release, lower molecular weight compositions of P4HB and copolymers thereof can be useful in the preparation of other chemicals, polymers, copolymers, and for blending with other materials. For example, low molecular weight P4HB and copolymers thereof can be used in the preparation of polyurethanes or as nucleants or fillers in blends.
US Patent No. 6,623,730 to Williams and Martin discloses the use of oligomers of P4HB as prodrugs to modulate endogenous levels of 4- hydroxybutyrate, and methods to prepare these oligomers by hydrolysis of P4HB. The method disclosed uses aliquots of sodium methoxide to hydrolyze P4HB dissolved in a solvent (anhydrous THF) to provide the oligomers of P4HB of the desired molecular weight. Starting with a polymer of molecular weight 430,000 g/mol, sodium methoxide is used to produce P4HB molecular weights of 320,000, 82,000 and 25,000. The disadvantages of this procedure are as follows: (i) the use of methoxide can result in terminal esterification of the polyester chains (producing methyl esters); (ii) the relatively low solubility of P4HB in THF (and other solvents) requires large volumes of solvent to dissolve the polymer which is difficult to handle, expensive, and costly to remove; and (iii) basic conditions could potentially lead to elimination reactions in the polymer causing unwanted side reactions.
It is an object of the present invention to provide methods to produce
P4HB and copolymers thereof by hydrolysis of high molecular weight P4HB and copolymers without the use of solvents.
It is a further object of the present invention to provide methods to produce P4HB and copolymers thereof by hydrolysis of high molecular weight P4HB and copolymers without the use of basic conditions that can esterify polymer terminal end groups or cause basic elimination in the polymer chains.
It is another object of the present invention to provide methods for hydrolysis of P4HB and copolymers thereof that maintain the polymers in solid state during hydrolysis resulting in easy isolation of the hydro lyzed products.
It is still another object of the present invention to provide compositions of P4HB and copolymers thereof that can be used in medical applications, for example, in implantable medical devices and or for the controlled release of bioactive substances, including the controlled release of 4-hydroxybutyrate.
Summary of the Invention
Methods for making P4HB polymers and copolymers thereof that are useful for preparing controlled release systems, and as intermediates in chemical synthesis, have been developed. These methods avoid the use of organic solvents, and basic conditions that can cause transesterification reactions with polymer terminal end groups or elimination reactions. A preferred embodiment is a method for producing polymers of P4HB with weight average molecular weight less than 250,000, and more preferably, less than 100,000, which are useful in controlled release. A particularly preferred embodiment utilizes aqueous acetic acid to hydrolyze pellets of P4HB polymers and copolymers while in suspension.
Brief Description of the Drawings
Figure 1 is a diagram of pathways leading to the biosynthesis of poly- 4-hydroxybutyrate.
Figure 2 is a 1H NMR spectrum of hydrolysis product of P4HB in acetic acid for 27.5 hours.
Detailed Description of the Invention Definitions
"Poly-4-hydroxybutyrate" as generally used herein means a homopolymer comprising 4-hydroxybutyrate units. It may be referred to herein as P4HB or TephaFLEX® biomaterial (manufactured by Tepha, Inc., Lexington, MA).
"Copolymers of poly-4-hydroxybutyrate" as generally used herein means any polymer comprising 4-hydroxybutyrate with one or more different hydroxy acid units.
"Controlled release" or "modified release, as used herein, refers to a release profile in which the drug release characteristics of time course and/or location are chosen to accomplish therapeutic or convenience objectives not offered by conventional dosage forms such as solutions, suspensions, or promptly dissolving dosage forms. Delayed release, extended release, and pulsatile release and their combinations are examples of modified release.
"Bioactive agent" is used herein to refer to therapeutic, prophylactic, and/or diagnostic agents. It includes without limitation physiologically or pharmacologically active substances that act locally or systemically in the body. A biologically active agent is a substance used for, for example, the treatment, prevention, diagnosis, cure, or mitigation of disease or disorder, a substance which affects the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a predetermined physiological environment. Bioactive agents include biologically, physiologically, or pharmacologically active substances that act locally or systemically in the human or animal body. Examples can include, but are not limited to, small-molecule drugs, peptides, proteins, antibodies, sugars, polysaccharides, nucleotides, oligonucleotides, aptamers, siRNA, nucleic acids, and combinations thereof. "Bioactive agent" includes a single such agent and is also intended to include a plurality
"Blend" as generally used herein means a physical combination of different polymers, as opposed to a copolymer comprised of two or more different monomers.
"Molecular weight" as used herein, unless otherwise specified, refers to the weight average molecular weight (Mw), not the number average molecular weight (Mn), and is measured by gel permeation chromatography (GPC) relative to polystyrene.
"Absorbable" as generally used herein means the material is broken down in the body and eventually eliminated from the body within 5 years.
"Biocompatible" as generally used herein means the biological response to the material or device being appropriate for the device's intended application in vivo. Any metabolites of these materials should also be biocompatible.
I. Composition
Methods have been developed to produce low molecular weight (Mw) compositions of P4HB and copolymers thereof in high yield. These methods may be used to prepare P4HB and copolymers thereof with weight average molecular weights (Mw) ranging from 1 to 250 kDa, and more preferably with Mw ranging from 5 to 100 kDa. The methods also yield P4HB and copolymers with narrow polydispersity (PDI), less than 3.0 and preferably a PDI of less than 2.5. The P4HB and copolymers thereof are prepared by hydrolysis with aqueous acids, and preferably with acetic acid. A major advantage of the method is that pellets of the polymers may be suspended in acid, and hydro lyzed while in suspension to yield polymers of predictable weight average molecular weight and narrow polydispersity. In addition, purification is straightforward, side reactions are essentially absent, and yields are very high.
A. Polymers
The processes described herein can typically be used with poly-4- hydroxybutyrate (P4HB) or a copolymer thereof. Copolymers include P4HB with another hydroxyacid, such as 3-hydroxybutyrate, and P4HB with glycolic acid or lactic acid monomer. P4HB and copolymers thereof can be obtained from Tepha, Inc. of Lexington, MA.
In a preferred embodiment, the starting P4HB homopolymer and copolymers thereof are extruded into pellets. An extruder can be used for this purpose. The pellets are then suspended in acid for hydrolysis. This facilitates isolation of the product, and provides a high yield of lower molecular weight product.
B. Hydrolysis
In a preferred embodiment, P4HB and copolymers thereof can be prepared with a weight average molecular weight less than 250,000, and
more preferably, less than 100,000. It has been discovered that low molecular weight (Mw) polymers and copolymers of P4HB can be produced with unexpectedly high yields, without solvent, and in high purity with side reactions essentially absent. Notably, the hydrolysis can be controlled so that lower molecular weight (Mw) polymers and copolymers can be predictably produced with high purity, high yield, and with narrow polydispersity.
The polymers can be hydrolyzed by suspending the polymer in an acid or an acid solution. Therefore, in one embodiment, the acid or acid solution is one in which the polymer pellet is insoluble or partially insoluble. The acid can be an organic acid or an inorganic acid. For those
embodiments where the acid is an organic acid, the acid can be a
monocarboxylic acid or a polycarboxylic acid, such as a di- or tricarboxylic acid. Suitable organic acids include, but are not limited to, acetic acid, halogenated acetic acid, such as mono-, di-, trihalogenated acetic acid, lactic acid, glycolic acid, propanoic acid, butanoic acid, pentanoic acid, hexanoic acid, benzoic acid, fumaric acid, maleic acid, tartaric acid, citric acid, malic acid, and pyruvic acid. Suitable inorganic acids include, but are not limited to, hydrochloric acid, sulfuric acid, hydrobromic acid, and hydroiodic acid.
A particularly preferred embodiment utilizes aqueous acetic acid to hydrolyze P4HB polymers and copolymers. More preferably, the polymers and copolymers are first extruded into pellets, and then suspended in aqueous acid. It has been discovered that the pellets will remain suspended in the acid (with shaking or stirring) during hydrolysis as the molecular weights of the polymers decrease in a predictable manner. Even though the polymers and copolymers remain suspended, and the polymer does not dissolve, the polydispersity of the lower molecular weight (Mw) product remains narrow. Because the pellets do not dissolve in the acid, the product may be easily isolated at the end of the hydrolysis, for example, by decanting, washing with water, and drying. There is no need to evaporate acid or solvent, or use a solvent extraction process to recover the low molecular weight polymers.
Granules or other non-pelletized forms of the polymer or copolymers can be hydrolyzed with the method, however, these forms are more likely to
become sticky during hydrolysis, making isolation of the product more difficult.
In a preferred method, the polymer or copolymer pellets are suspended in aqueous acetic acid, and either stirred or shaken. The hydrolysis may be performed at ambient or elevated temperature. Preferably the hydrolysis is undertaken at constant temperature in the range of 37 to 50°C.
The strength of the acid may be adjusted to speed up or slow down the rate of hydrolysis of the polymers and copolymers. In a preferred method, acetic acid is used with a concentration in the range of 5M to 12M. However, lower concentrations may be used to slow down the rate of hydrolysis, and higher concentrations may be used to speed up the rate of hydrolysis.
The length of the hydrolysis reaction will be determined by the molecular weight (Mw) desired, form of the polymer or copolymer, temperature of the reaction, type of acid, and concentration of the acid. For example, pellets of P4HB (Mw = 346kDa) suspended in aqueous acetic acid at 37°C for 4 hours will have a Mw = 224kDa if the concentration of the acid is 10M, and Mw =1 15kDA if the concentration of the acid is 12M. Under the same conditions, at 27.5 hours, the molecular weights (Mw) of the polymer with 10M and 12M acetic acid are 85kDa and 40kDa, respectively.
C. Uses of Low Molecular Weight P4HB and P4HB
Copolymers
Low molecular weight polymers and copolymers of P4HB have properties that are substantially improved for many medical applications relative to higher molecular weight versions. These materials have lower melt viscosities, and higher solubilities in solvents that can make them easier to process. The lower molecular weight P4HB polymer and copolymers thereof can also provide more desirable controlled release profiles for delivery of therapeutic, prophylactic or diagnostic agents or other bioactive agent, such as a small-molecule drug, peptide, protein, antibody, sugar, polysaccharide, nucleotide, oligonucleotide, aptamer, siRNA, nucleic acid,
and combinations thereof. The lower molecular weight P4HB may also be used to make medical devices, such as, but not limited to, sutures, stents, stent grafts, stent coatings, devices for temporary wound or tissue support, devices for soft or hard tissue repair, repair patches, tissue engineering scaffolds, retention membranes, anti-adhesion membranes tissue separation membranes, hernia repair devices, device coatings, cardiovascular patches, catheter balloons, vascular closure devices, slings, biocompatible coatings, rotator cuff repair devices, meniscus repair devices, adhesion barriers, guided tissue repair/regeneration devices, articular cartilage repair devices, nerve guides, tendon repair devices, intracardiac septal defect repair devices, including, but not limited to, atrial septal defect repair devices and PFO (patent foramen ovale) closure devices, left atrial appendage (LAA) closure devices, pericardial patches, bulking and filling agents, plastic surgery devices (including facial and breast cosmetic and reconstructive devices), vein valves, heart valves, bone marrow scaffolds, meniscus regeneration devices, ligament and tendon grafts, ocular cell implants, spinal fusion devices, imaging devices, skin substitutes, dural substitutes, bone graft substitutes, wound dressings, and hemostats.
Lower molecular weight polymers and copolymers of P4HB and copolymers thereof can also be used to modulate endogenous levels of 4- hydroxybutyrate as disclosed by US Patent No. 6,623,730 to Williams and Martin.
Lower molecular weight polymers and copolymers of P4HB and copolymers thereof can also be used in implantable applications that require a more rapid rate of degradation or absorption than the higher Mw polymers. Preferrably, the implanted device will degrade and the low Mw polymer will be absorbed within six months
P4HB polymers and copolymers with low molecular weights may also be used in blends with other materials to improve polymer properties. The low molecular polymers may also be used in the synthesis of other materials, for example, in reactive polymerization.
II. Methods of Manufacturing Low Molecular Weight P4HB and Copolymers.
A. Method of Making Low Molecular Weight P4HB Polymer
In a preferred method, a low molecular weight (Mw) P4HB polymer or copolymer may be prepared as follows. Pellets of a P4HB polymer are suspended in aqueous acetic acid, and placed in a shaker incubator. A number of parameters can be varied to control the hydrolysis of the polymer or copolymer including, but not limited to, the concentration of the acid, the type of acid, the temperature of the reaction, the speed (rpm) of the shaker (or stirrer speed), and the reaction time.
When the desired molecular weight (Mw) is achieved, the reaction is quenched by decanting the acid, and rinsing the polymer or copolymer with water. Residual acid and water may be removed by drying in vacuo.
The present invention will be further understood by reference to the following non-limiting examples.
Example 1: Hydrolysis of P4HB with 12M Aqueous Acetic Acid
Materials and Methods
30 grams of P4HB pellets (Tepha, Inc., Lexington, MA) (Mw 367 kDa) were suspended in 100 mL of 12 M aqueous acetic acid (AcOH), and incubated at 37°C at 50 rpm in a shaker incubator. After 4 and 27.5 hours, the Mw of polymer samples taken from the reaction were 115 and 40 kDa, respectively.
Results
After 4 hours the P4HB pellets became sticky and the AcOH solution turned cloudy. After 30 hours, the whole reaction mixture appeared milky, and when cooled down, it gelled into a semi-solid. It was therefore determined that 12 M AcOH is too strong to be used as the hydrolysis medium for P4HB pellets since this concentration may partially dissolve the polymer. As a result, the collection of the hydrolysis products would less practical and the reaction conditions would be less homogeneous with some
hydrolysis occurring in solution and some in the solid phase (i.e. within the pellets).
Example 2: Hydrolysis of P4HB with 10 M Aqueous Acetic Acid
Materials and Methods
30 grams of P4HB pellets (Tepha, Inc., Lexington, MA) (Mw 367 kDa) were suspended in 100 mL of 10 M aqueous acetic acid, and incubated at 37°C at 50 rpm in a shaker incubator. A sample was removed after 4 hours, and the weight average molecular weight of the polymer was determined by GPC (relative to polystyrene) to be 224 kDa. After 27.5 hours, the reaction was quenched by decanting the acetic acid, and rinsing the polymer with distilled water.
Results
The pellets did not clump or aggregate during the reaction time, as was seen for the similar reaction performed in 12 M acetic acid. The polymer pellets were then dried in vacuo to remove residual acetic acid and water.
The weight average molecular weight of the polymer was determined to be 85 kDa after 27.5 hours, and the polymer was recovered in high yield and without loss of material.
Example 3: Hydrolysis of P4HB with 8M Aqueous Acetic Acid
Materials and Methods
A 20 gram sample of P4HB pellets (Tepha, Inc., Lexington, MA) (Mw 347 kDa) was suspended in 200 mL of 8 M aqueous acetic acid, and shaken at 50 rpm at 37°C in a shaker incubator. Samples were removed periodically, and the weight average molecular weight of the polymer and polydispersity were determined by GPC (relative to polystyrene).
Results
The results are tabulated below (time in hours).
TABLE 1 : Molecular Weight and PDI of P4HB over Time
Example 4: Hydrolysis of P4HB with 5M Aqueous Acetic Acid
Materials and Methods
A 20 gram sample of P4HB granules (Tepha, Inc., Lexington, MA)
(Mw 347 kDa) was suspended in 200 mL of 5M aqueous acetic acid, and stirred at 45°C. Samples were removed periodically, and the weight average molecular weight of the polymer was determined by GPC (relative to polystyrene).
Results
The results (average of three runs) are tabulated in Table 2.
TABLE 2: Molecular Weight and PDI of P4HB over Time
The XH NMR spectrum of the hydrolysis product from Example 2 after 27.5 hours is shown in Figure 2. As expected, this low Mw hydrolysis product shows a small ΧΗ peak of the polymer end group -CH2OH at a chemical shift of about 3.69 ppm. The integration area of this peak is 0.50 compared to an integration area of about 100 for the polymer methylene group at chemical shift of about 4.12 ppm. As such, the end group ratio of this sample is 0.5/100, or 0.005, which would suggest the product is a 200 mer (i.e. approx. Mn 17 kDa). This value of Mn from end group analysis is reasonable considering the polymer's GPC Mw of 85 kDa and Mn of 44 kDa. (Note: The GPC values relative to PS are about double the absolute values for the polymer from LLS).
The hydrolysis of P4HB pellets or granules suspended in aqueous solution of acetic acid has been performed successfully under mild conditions with reasonable reaction times. The hydrolysis product, low Mw P4HB, was prepared in a high yield, close to 100%, and its Mw could also be predicted based on the Mw versus reaction time curve under fixed reaction conditions, i.e., temperature and concentration of AcOH. Surprisingly for a biphasic reaction, the polydispersity of the hydrolysis products are low and similar to that of the starting polymer, about 2, showing that the hydrolysis reaction occurs uniformly throughout the bulk of the material, rather than being localized to the surface, even though the pellets/granules remain undissolved and suspended in the acetic acid solution.
From the NMR analysis, it appears that the end groups are hydroxyl groups just like the starting polymer. This shows that no new functional groups (i.e. acetate groups) have been added to the polymer and confirms the reaction as a true hydrolysis with no derivatization of the terminal end groups.
The kinetics of the hydrolysis reaction could be affected by a couple of factors such as the reaction temperature (room temperature to 50 °C) and the concentration of AcOH solution (5— 10 M). Because the P4HB
pellets/granules were simply suspended in the AcOH solution at relative low temperatures instead of forming a homogeneous solution and the ratio of P4HB/AcOH could be 3g/10mL, the hydrolysis reaction described herein should be scalable for production of P4HB oligomer with controlled molecular weight.
Claims
1. A poly-4-hydroxybutyrate polymer or copolymer thereof, wherein the polymer or copolymer is derived by acidic hydrolysis of a higher molecular weight poly-4-hydroxybutyrate polymer or copolymer, and wherein the polymer or copolymer has a weight average molecular weight between 1,000 and 250,000 Da.
2. The polymer or copolymer of claim 1 wherein the polymer or copolymer is suspended as pellets or particles in an aqueous acidic solution for a period of time and at a temperature to hydrolyse the polymer.
3. The polymer or copolymer of claim 2 wherein the higher molecular weight poly-4-hydroxybutyrate polymer or copolymer are in pellet form during hydrolysis.
4. The polymer or copolymer of claim 1 wherein aqueous acetic acid is used for the acidic hydrolysis of the polymer or copolymer.
5. The polymer of copolymer of claim 1 wherein the
polydispersity after acidic hydrolysis is between 3 and 1.
6. A medical device comprising the polymer or copolymer of any of claims 1-5.
7. The device of claim 6 selected from the group consisting of a suture, stent, stent graft, stent coating, drug delivery device, device for temporary wound or tissue support, device for soft or hard tissue repair, repair patch, tissue engineering scaffold, retention membrane, anti-adhesion membrane, tissue separation membrane, hernia repair device, device coating, cardiovascular patch, catheter balloon, vascular closure device, sling, biocompatible coating, rotator cuff repair device, meniscus repair device, adhesion barrier, guided tissue repair/regeneration device, articular cartilage repair device, nerve guide, tendon repair device, intracardiac septal defect repair device, including but not limited to atrial septal defect repair devices and PFO (patent foramen ovale) closure devices, left atrial appendage (LAA) closure device, pericardial patch, bulking and filling agent, plastic surgery devices (including facial and breast cosmetic and reconstructive devices), vein valve, heart valve, bone marrow scaffold, meniscus regeneration device, ligament and tendon graft, ocular cell implant, spinal fusion device, imaging device, skin substitute, dural substitute, bone graft substitute, wound dressing, and hemostat.
8. The device of claim 6 wherein the device is used for controlled release of a bioactive agent.
9. The device of claim 8 wherein the bioactive agent is a therapeutic, prophylactic, and/or diagnostic agent.
10. The device of claim 9 wherein the bioactive agent is a small- molecule drug, peptide, protein, antibody, sugar, polysaccharide, nucleotide, oligonucleotide, aptamer, siRNA, nucleic acid, and combinations thereof.
1 1. The device of claim 9 wherein the device is used to raise endogenous levels of 4-hydroxybutyrate in a patient.
12. The polymer or copolymer of claim 1 used as an additive or in a polymer blend.
13. A method of using a device of any of claims 6-11, comprising administering to or implanting in an individual in need thereof a device comprising poly-4-hydroxybutyrate polymers or copolymers with weight average molecular weight below 250,000 Da, wherein the polymers or copolymers are derived by acidic hydrolysis of higher molecular weight poly -4-hydroxybutyrate polymers or copolymers, and wherein the acidic hydrolysis takes place in suspension.
14. A medical device comprising a poly-4-hydroxybutrate polymer or copolymer, wherein the polymer or copolymer is derived by acidic hydrolysis, and wherein the polymer or copolymer has a weight average molecular weight less than 250,000 Da, and wherein the polymer or copolymer has a polydispersity between 3 and 1.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP12769859.5A EP2760911B1 (en) | 2011-09-27 | 2012-09-26 | Controlled hydrolysis of poly-4-hydroxybutyrate and copolymers |
ES12769859.5T ES2659525T3 (en) | 2011-09-27 | 2012-09-26 | Controlled hydrolysis of poly-4-hydroxybutyrate and copolymers |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161539695P | 2011-09-27 | 2011-09-27 | |
US61/539,695 | 2011-09-27 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2013049161A1 true WO2013049161A1 (en) | 2013-04-04 |
Family
ID=47003276
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2012/057277 WO2013049161A1 (en) | 2011-09-27 | 2012-09-26 | Controlled hydrolysis of poly-4-hydroxybutyrate and copolymers |
Country Status (4)
Country | Link |
---|---|
US (1) | US8680228B2 (en) |
EP (2) | EP2760911B1 (en) |
ES (1) | ES2659525T3 (en) |
WO (1) | WO2013049161A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2019112925A1 (en) | 2017-12-04 | 2019-06-13 | Tepha, Inc. | Vacuum membrane thermoformed poly-4-hydroxybutyrate medical implants |
Families Citing this family (20)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011119839A1 (en) | 2010-03-24 | 2011-09-29 | Jazz Pharmaceuticals, Inc. | Controlled release dosage forms for high dose, water soluble and hygroscopic drug substances |
US9505927B2 (en) | 2012-08-17 | 2016-11-29 | Metabolix, Inc. | Biobased modifiers for polyvinylchloride blends |
US9464187B2 (en) | 2012-08-17 | 2016-10-11 | Metabolix, Inc. | Biobased modifiers for polyvinylchloride blends |
CN104755538B (en) | 2012-08-17 | 2018-08-31 | Cj 第一制糖株式会社 | Bio-rubber modifying agent for blend polymer |
EP3004225A1 (en) | 2013-05-30 | 2016-04-13 | Metabolix, Inc. | Recyclate blends |
US9302029B2 (en) | 2013-10-31 | 2016-04-05 | Tepha, Inc. | Pultrusion of poly-4-hydroxybutyrate and copolymers thereof |
EP3065789B1 (en) | 2013-11-05 | 2018-10-24 | Tepha, Inc. | Compositions and devices of poly-4-hydroxybutyrate |
EP3122817B1 (en) | 2014-03-27 | 2020-07-29 | CJ CheilJedang Corporation | Highly filled polymer systems |
US10398662B1 (en) | 2015-02-18 | 2019-09-03 | Jazz Pharma Ireland Limited | GHB formulation and method for its manufacture |
US11986451B1 (en) | 2016-07-22 | 2024-05-21 | Flamel Ireland Limited | Modified release gamma-hydroxybutyrate formulations having improved pharmacokinetics |
US11602513B1 (en) | 2016-07-22 | 2023-03-14 | Flamel Ireland Limited | Modified release gamma-hydroxybutyrate formulations having improved pharmacokinetics |
UY37341A (en) | 2016-07-22 | 2017-11-30 | Flamel Ireland Ltd | FORMULATIONS OF GAMMA-MODIFIED RELEASE HYDROXIBUTIRATE WITH IMPROVED PHARMACOCINETICS |
US11504347B1 (en) | 2016-07-22 | 2022-11-22 | Flamel Ireland Limited | Modified release gamma-hydroxybutyrate formulations having improved pharmacokinetics |
US11602512B1 (en) | 2016-07-22 | 2023-03-14 | Flamel Ireland Limited | Modified release gamma-hydroxybutyrate formulations having improved pharmacokinetics |
US20180263936A1 (en) | 2017-03-17 | 2018-09-20 | Jazz Pharmaceuticals Ireland Limited | Gamma-hydroxybutyrate compositions and their use for the treatment of disorders |
KR20210094513A (en) | 2018-11-19 | 2021-07-29 | 재즈 파마슈티칼즈 아일랜드 리미티드 | Alcohol-Resistant Drug Formulations |
BR112021013766A2 (en) | 2019-03-01 | 2021-09-21 | Flamel Ireland Limited | GAMMA-HYDROXYBUTYRATE COMPOSITIONS WITH IMPROVED PHARMACOKINETICS IN THE FED STATE |
CN113292713B (en) * | 2021-06-08 | 2022-10-25 | 中山大学附属第六医院 | Molecular weight grading and purifying method of polyhydroxyalkanoate |
US11779557B1 (en) | 2022-02-07 | 2023-10-10 | Flamel Ireland Limited | Modified release gamma-hydroxybutyrate formulations having improved pharmacokinetics |
US11583510B1 (en) | 2022-02-07 | 2023-02-21 | Flamel Ireland Limited | Methods of administering gamma hydroxybutyrate formulations after a high-fat meal |
Citations (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5191016A (en) * | 1990-07-19 | 1993-03-02 | Manssur Yalpani | Functionalized poly(hydroxyalkanoates) and method of manufacturing same |
US5811272A (en) | 1996-07-26 | 1998-09-22 | Massachusetts Institute Of Technology | Method for controlling molecular weight of polyhydroxyalkanoates |
WO1999032536A1 (en) | 1997-12-22 | 1999-07-01 | Metabolix, Inc. | Polyhydroxyalkanoate compositions having controlled degradation rates |
WO2000056376A1 (en) | 1999-03-25 | 2000-09-28 | Metabolix, Inc. | Medical devices and applications of polyhydroxyalkanoate polymers |
US6245537B1 (en) | 1997-05-12 | 2001-06-12 | Metabolix, Inc. | Removing endotoxin with an oxdizing agent from polyhydroxyalkanoates produced by fermentation |
US6316262B1 (en) | 1997-09-19 | 2001-11-13 | Metabolix, Inc. | Biological systems for manufacture of polyhydroxyalkanoate polymers containing 4-hydroxyacids |
US6323010B1 (en) | 1998-05-22 | 2001-11-27 | Metabolix, Inc. | Polyhydroxyalkanoate biopolymer compositions |
US20020141967A1 (en) * | 1999-09-14 | 2002-10-03 | Tepha, Inc. | Polyhydroxyalkanoate compositions for soft tissue repair, augmentation, and viscosupplementation |
US6610764B1 (en) | 1997-05-12 | 2003-08-26 | Metabolix, Inc. | Polyhydroxyalkanoate compositions having controlled degradation rates |
US6623748B2 (en) | 1997-09-02 | 2003-09-23 | Gensci Orthobiologics, Inc. | Reverse phase connective tissue repair composition |
US6623730B1 (en) | 1999-09-14 | 2003-09-23 | Tepha, Inc. | Therapeutic uses of polymers and oligomers comprising gamma-hydroxybutyrate |
US20030211131A1 (en) | 2002-05-10 | 2003-11-13 | Metabolix, Inc. | Bioabsorbable polymer containing 2-hydroxyacid monomers |
US6828357B1 (en) | 1997-07-31 | 2004-12-07 | Metabolix, Inc. | Polyhydroxyalkanoate compositions having controlled degradation rates |
US6867248B1 (en) | 1997-05-12 | 2005-03-15 | Metabolix, Inc. | Polyhydroxyalkanoate compositions having controlled degradation rates |
US20060177513A1 (en) * | 2005-01-28 | 2006-08-10 | Tepha, Inc. | Embolization using poly-4-hydroxybutyrate particles |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1991287B1 (en) * | 2006-02-07 | 2012-07-04 | Tepha, Inc. | Polymeric, degradable drug-eluting stents and coatings |
WO2009085823A1 (en) * | 2007-12-19 | 2009-07-09 | Tepha, Inc. | Medical devices containing melt-blown non-wovens of poly-r-hydroxybutyrate and copolymers |
ES2426774T3 (en) * | 2008-06-25 | 2013-10-25 | Metabolix, Inc. | Compositions of branched PHA, procedures for its production and use in applications |
-
2012
- 2012-09-26 US US13/627,545 patent/US8680228B2/en active Active
- 2012-09-26 ES ES12769859.5T patent/ES2659525T3/en active Active
- 2012-09-26 WO PCT/US2012/057277 patent/WO2013049161A1/en active Application Filing
- 2012-09-26 EP EP12769859.5A patent/EP2760911B1/en active Active
- 2012-09-26 EP EP17188627.8A patent/EP3266806A1/en not_active Withdrawn
Patent Citations (25)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5191016A (en) * | 1990-07-19 | 1993-03-02 | Manssur Yalpani | Functionalized poly(hydroxyalkanoates) and method of manufacturing same |
US5811272A (en) | 1996-07-26 | 1998-09-22 | Massachusetts Institute Of Technology | Method for controlling molecular weight of polyhydroxyalkanoates |
US6610764B1 (en) | 1997-05-12 | 2003-08-26 | Metabolix, Inc. | Polyhydroxyalkanoate compositions having controlled degradation rates |
US7244442B2 (en) | 1997-05-12 | 2007-07-17 | Metabolix, Inc. | Method for making devices using polyhydroxyalkanoate having pyrogen removed |
US6878758B2 (en) | 1997-05-12 | 2005-04-12 | Metabolix, Inc. | Polyhydroxyalkanoate compositions having controlled degradation rates |
US6245537B1 (en) | 1997-05-12 | 2001-06-12 | Metabolix, Inc. | Removing endotoxin with an oxdizing agent from polyhydroxyalkanoates produced by fermentation |
US6867248B1 (en) | 1997-05-12 | 2005-03-15 | Metabolix, Inc. | Polyhydroxyalkanoate compositions having controlled degradation rates |
US6828357B1 (en) | 1997-07-31 | 2004-12-07 | Metabolix, Inc. | Polyhydroxyalkanoate compositions having controlled degradation rates |
US6623748B2 (en) | 1997-09-02 | 2003-09-23 | Gensci Orthobiologics, Inc. | Reverse phase connective tissue repair composition |
US6316262B1 (en) | 1997-09-19 | 2001-11-13 | Metabolix, Inc. | Biological systems for manufacture of polyhydroxyalkanoate polymers containing 4-hydroxyacids |
WO1999032536A1 (en) | 1997-12-22 | 1999-07-01 | Metabolix, Inc. | Polyhydroxyalkanoate compositions having controlled degradation rates |
US6323010B1 (en) | 1998-05-22 | 2001-11-27 | Metabolix, Inc. | Polyhydroxyalkanoate biopolymer compositions |
US7268205B2 (en) | 1999-03-25 | 2007-09-11 | Metabolix, Inc. | Medical devices and applications of polyhydroxyalkanoate polymers |
US6548569B1 (en) | 1999-03-25 | 2003-04-15 | Metabolix, Inc. | Medical devices and applications of polyhydroxyalkanoate polymers |
US7179883B2 (en) | 1999-03-25 | 2007-02-20 | Metabolix, Inc. | Medical devices and applications of polyhydroxyalkanoate polymers |
WO2000056376A1 (en) | 1999-03-25 | 2000-09-28 | Metabolix, Inc. | Medical devices and applications of polyhydroxyalkanoate polymers |
US6838493B2 (en) | 1999-03-25 | 2005-01-04 | Metabolix, Inc. | Medical devices and applications of polyhydroxyalkanoate polymers |
US6867247B2 (en) | 1999-03-25 | 2005-03-15 | Metabolix, Inc. | Medical devices and applications of polyhydroxyalkanoate polymers |
US6623730B1 (en) | 1999-09-14 | 2003-09-23 | Tepha, Inc. | Therapeutic uses of polymers and oligomers comprising gamma-hydroxybutyrate |
US7025980B1 (en) | 1999-09-14 | 2006-04-11 | Tepha, Inc. | Polyhydroxyalkanoate compositions for soft tissue repair, augmentation, and viscosupplementation |
US6555123B2 (en) | 1999-09-14 | 2003-04-29 | Tepha, Inc. | Polyhydroxyalkanoate compositions for soft tissue repair, augmentation, and viscosupplementation |
US20020141967A1 (en) * | 1999-09-14 | 2002-10-03 | Tepha, Inc. | Polyhydroxyalkanoate compositions for soft tissue repair, augmentation, and viscosupplementation |
US6585994B2 (en) | 1999-09-14 | 2003-07-01 | Tepha, Inc. | Polyhydroxyalkanoate compositions for soft tissue repair, augmentation, and viscosupplementation |
US20030211131A1 (en) | 2002-05-10 | 2003-11-13 | Metabolix, Inc. | Bioabsorbable polymer containing 2-hydroxyacid monomers |
US20060177513A1 (en) * | 2005-01-28 | 2006-08-10 | Tepha, Inc. | Embolization using poly-4-hydroxybutyrate particles |
Non-Patent Citations (8)
Title |
---|
HORI Y ET AL: "Chemical synthesis of high molecular weight poly(3-hydroxybutyrate-co-4-hydroxybutyrate)", POLYMER, ELSEVIER SCIENCE PUBLISHERS B.V, GB, vol. 36, no. 24, 1 November 1995 (1995-11-01), pages 4703 - 4705, XP004025461, ISSN: 0032-3861, DOI: 10.1016/0032-3861(95)96838-Y * |
HORI, Y. ET AL., POLYMER, vol. 36, 1995, pages 4703 - 4705 |
HOUK, K.N. ET AL., J. ORG. CHEM., vol. 73, no. 7, 2008, pages 2674 - 2678 |
MARTIN, D. ET AL.: "Medical Applications of Poly-4-hydroxybutyrate: A Strong Flexible Absorbable Biomaterial", BIOCHEM. ENG. J., vol. 16, 2003, pages 97 - 105 |
MOORE T ET AL: "Chemosynthesis of bioresorbable poly(gamma-butyrolactone) by ring-opening polymerisation: a review", BIOMATERIALS, ELSEVIER SCIENCE PUBLISHERS BV., BARKING, GB, vol. 26, no. 18, 1 June 2005 (2005-06-01), pages 3771 - 3782, XP027767944, ISSN: 0142-9612, [retrieved on 20050601] * |
MOORE, T. ET AL., BIOMATERIALS, vol. 26, 2005, pages 3771 - 3782 |
STEINBÜCHEL A. ET AL.: "Diversity of Bacterial Polyhydroxyalkanoic Acids", FEMS MICROBIAL. LETT., vol. 128, 1995, pages 219 - 228 |
WILLIAMS, S.F. ET AL., POLYESTERS, III, vol. 4, 2002, pages 91 - 127 |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2019112925A1 (en) | 2017-12-04 | 2019-06-13 | Tepha, Inc. | Vacuum membrane thermoformed poly-4-hydroxybutyrate medical implants |
US11160898B2 (en) | 2017-12-04 | 2021-11-02 | Tepha, Inc. | Vacuum membrane thermoformed poly-4-hydroxybutyrate medical implants |
Also Published As
Publication number | Publication date |
---|---|
US8680228B2 (en) | 2014-03-25 |
EP2760911A1 (en) | 2014-08-06 |
EP3266806A1 (en) | 2018-01-10 |
US20130085185A1 (en) | 2013-04-04 |
EP2760911B1 (en) | 2017-11-22 |
ES2659525T3 (en) | 2018-03-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP2760911B1 (en) | Controlled hydrolysis of poly-4-hydroxybutyrate and copolymers | |
JP3126637B2 (en) | Biocompatible block copolymer | |
CN101611063B (en) | Cross-linked hyaluronic acid and method for producing the same | |
DE60036863T2 (en) | Medical devices and uses of polyhydroxyalkanoate polymers | |
JP4376455B2 (en) | Polyhydroxyalkanoate composition having controlled degradation rate | |
CA2905765C (en) | Compositions and devices of poly-4-hydroxybutyrate | |
CA2928731A1 (en) | Compositions and devices of poly-4-hydroxybutyrate | |
CA2800679A1 (en) | Compositions and methods for enhancement of nucleic acid delivery | |
Cipurković et al. | Biodegradable Polymers: Production, properties and application in medicine | |
Lipsa et al. | Poly (α-hydroxyacids) in biomedical applications: synthesis and properties of lactic acid polymers | |
WO2003042277A1 (en) | The polyester containing active drugs and having amino acids in the main chain, and its preparation method | |
US8962008B2 (en) | Hybrid polymeric materials for medical applications and preparation thereof | |
CA2682291C (en) | Device made at least partially of n-acetylchitosan with controlled biodissolution | |
CN115970051B (en) | Degradable tissue engineering filling material and preparation method and application thereof | |
CN115785403B (en) | Malic acid group biological material and preparation method and application thereof | |
CN109970957B (en) | Controllable biodegradable copolymer with alkaline copolymerization center | |
Bhilare et al. | Biodegradability and Biodegradable Polymers: An Overview | |
JP2002167428A (en) | Vital material |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 12769859 Country of ref document: EP Kind code of ref document: A1 |
|
DPE1 | Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101) | ||
NENP | Non-entry into the national phase |
Ref country code: DE |
|
REEP | Request for entry into the european phase |
Ref document number: 2012769859 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2012769859 Country of ref document: EP |