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
  • C08G63/68—Polyesters containing atoms other than carbon, hydrogen and oxygen
  • C08G63/692—Polyesters containing atoms other than carbon, hydrogen and oxygen containing phosphorus
• • 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
  • C08G63/68—Polyesters containing atoms other than carbon, hydrogen and oxygen
  • C08G63/692—Polyesters containing atoms other than carbon, hydrogen and oxygen containing phosphorus
  • C08G63/6924—Polyesters containing atoms other than carbon, hydrogen and oxygen containing phosphorus derived from polycarboxylic acids and polyhydroxy compounds
  • C08G63/6926—Dicarboxylic acids and dihydroxy compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
  • A61K9/1605—Excipients; Inactive ingredients
  • A61K9/1629—Organic macromolecular compounds
  • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
  • A61K9/1647—Polyesters, e.g. poly(lactide-co-glycolide)
• • 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
  • A61L17/12—Homopolymers or copolymers of glycolic acid or lactic acid
• • 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
  • 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
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
  • C08L85/00—Compositions of macromolecular compounds obtained by reactions forming a linkage in the main chain of the macromolecule containing atoms other than silicon, sulfur, nitrogen, oxygen and carbon; Compositions of derivatives of such polymers
  • C08L85/02—Compositions of macromolecular compounds obtained by reactions forming a linkage in the main chain of the macromolecule containing atoms other than silicon, sulfur, nitrogen, oxygen and carbon; Compositions of derivatives of such polymers containing phosphorus
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T428/00—Stock material or miscellaneous articles
  • Y10T428/29—Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
  • Y10T428/2913—Rod, strand, filament or fiber
  • Y10T428/2933—Coated or with bond, impregnation or core
  • Y10T428/2938—Coating on discrete and individual rods, strands or filaments
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T442/00—Fabric [woven, knitted, or nonwoven textile or cloth, etc.]
  • Y10T442/30—Woven fabric [i.e., woven strand or strip material]
  • Y10T442/3472—Woven fabric including an additional woven fabric layer
  • Y10T442/3528—Three or more fabric layers
  • Y10T442/3545—Woven fabric layers impregnated with a blend of thermosetting and thermoplastic resins

Definitions

• the present invention relates to biodegradable homopolymer and block copolymer compositions, in particular those containing both phosphate and terephthalate ester linkages in the polymer backbone, which degrade in vivo into non-toxic residues.
• the polymers of the invention are particularly useful as implantable medical devices and drug delivery systems.
• Biocompatible polymeric materials have been used extensively in therapeutic drug delivery and medical implant device applications. Sometimes, it is also desirable for such polymers to be, not only biocompatible, but also biodegradable to obviate the need for removing the polymer once its therapeutic value has been exhausted.
• a biodegradable medical device is intended for use as a drug delivery or other controlled-release system
• using a polymeric carrier is one effective means to deliver the therapeutic agent locally and in a controlled fashion, see Langer et al . , "Chemical and Physical Structures of Polymers as Carriers for Controlled Release of Bioactive Agents", J. Macro . Science, Rev. Macro . Chem . Phys . , C23 -. 1 , 61-126 (1983) .
• Polymers have been used as carriers of therapeutic agents to effect a localized and sustained release. See Leong et al .
• the steps leading to release of the therapeutic agent are water diffusion into the matrix, dissolution of the therapeutic agent, and diffusion of the therapeutic agent out through the channels of the matrix.
• the mean residence time of the therapeutic agent existing in the soluble state is longer for a non-biodegradable matrix than for a biodegradable matrix, for which passage through the channels of the matrix, while it may occur, is no longer required.
• therapeutic agents can decompose or become inactivated within the non-biodegradable matrix before they are released. This issue is particularly significant for many bio-macromolecules and smaller polypeptides, since these molecules are generally hydrolytically unstable and have low permeability through a polymer matrix. In fact, in a non- biodegradable matrix, many bio-macromolecules aggregate and precipitate, blocking the channels necessary for diffusion out of the carrier matrix.
• biodegradable matrix that, in addition to some diffusion release, also allows controlled release of the therapeutic agent by degradation of the polymer matrix.
• polyesters Pant et al . , "Biodegradable Drug Delivery Systems Based on Aliphatic Polyesters: Application to contraceptives and Narcotic Antagonists", Controlled Release of Bioactive Materials, 19- 44 (Richard Baker ed. , 1980); poly(amino acids) and pseudo- poly(amino acids) (Pulapura et al . , "Trends in the Development of Bioresorbable Polymers for Medical Applications", J. of Biomaterials Appl . , 6 : 1 , 216-50 (1992); polyurethanes (Bruin et al . , "Biodegradable Lysine Diisocyanate-based Poly (Glycolide-co- e Caprolactone) -
• biodegradable materials that are used as medical implant materials are polylactide, polyglycolide, polydioxanone, poly (lactide-co-glycolide) , poly (glycolide-co- polydioxanone), polyanhydrides, poly (glycolide-co- trimethylene carbonate), and poly (glycolide-co- caprolactone) .
• Polymers having phosphate linkages called poly (phosphates) , poly (phosphonates) and poly (phosphites) , are known. See Penczek et al . , Handbook of Polymer
• the versatility of these polymers comes from the versatility of the phosphorus atom, which is known for a multiplicity of reactions. Its bonding can involve the 3p orbitals or various 3s-3p hybrids; spd hybrids are also possible because of the accessible d orbitals. Thus, the physico-chemical properties of the poly (phosphoesters) can be readily changed by varying either the R or R' group.
• the biodegradability of the polymer is due primarily to the physiologically labile phosphoester bond in the backbone of the polymer. By manipulating the backbone or the sidechain, a wide range of biodegradation rates are attainable.
• phosphoesters An additional feature of poly (phosphoesters) is the availability of functional side groups. Because phosphorus can be pentavalent, drug molecules or other biologically active substances can be chemically linked to the polymer. For example, drugs with -O-carboxy groups may be coupled to the phosphorus via an ester bond, which is hydrolyzable .
• the P-O-C group in the backbone also lowers the glass transition temperature of the polymer and, importantly, confers solubility in common organic solvents, which is desirable for easy characterization and processing.
• a number of other patents disclose flame retardants having a polyester-linked terephthalate recurring unit and may also have a poly (phosphonate) recurring unit having a -P-R' side chain in which an R' group has replaced the hydrogen atom of a poly (phosphite) , but lacking the intervening oxygen of a poly (phosphate) . See, for example, Desitter et al . , U.S. Patent No. 3,927,231 and Reader, U.S. Patent No. 3,932,566. Starck et al . , U.S. Patent No.
• biodegradable terephthalate polymers of the invention comprise the recurring monomeric units shown in formula I :
• R is a divalent organic moiety
• R' is an aliphatic, aromatic or heterocyclic residue
• x is ⁇ 1
• n is 0-5, 000, where the biodegradable polymer is sufficiently pure to be biocompatible and degrades to biocompatible residues upon biodegradation.
• the invention contemplates a process for preparing a biodegradable terephthalate homopolymer comprising the step of polymerizing p moles of a diol compound having formula II:
• R, R' and x are as defined above.
• the invention also contemplates a process for preparing a biodegradable block copolymer comprising the steps of :
• the invention comprises a biodegradable terephthalate polymer composition comprising r
• an article useful for implantation, injection, or otherwise being placed totally or partially within the body comprises the biodegradable terephthalate polymer of formula I or the above-described polymer composition.
• a method for the controlled release of a biologically active substance comprising the steps of:
• Figure 1A shows the DSC curve of P (BHET-EOP/TC, 80/20; and Figure IB shows the DSC curve of P (BHET-EOP/TC, 50/50)
• Figure 2A shows the 1 H-NMR spectrum
• Figure 2B shows the 31 P-NMR spectrum for P (BHET-EOP/TC, 80/20)
• Figure 3 shows the FT-IR spectrum for P (BHET-EOP/TC, 80/20) .
• Figure 4 shows the GPC chromatogram for P (BHET-EOP/TC, 80/20) .
• Figure 5 shows the molecular weights and elemental analyses for P (BHET-EOP/TC, 80/20) and P (BHET-HOP/TC, 90/10) .
• Figure 6 shows the storage stability of P (BHET-EOP/TC, 80/20) and P (BHET-EOP/TC, 85/15).
• Figures 7A and 7B show the in vi tro degradation data for P (BHET-EOP/TC, 80/20) and P (BHET-EOP/TC, 85/15).
• Figure 8 shows the change in molecular weight of P(BHDPT-EOP) and P (BHDPT-EOP/TC) poly (phosphoesters) during in vi tro degradation.
• Figure 9 shows the in vivo degradation of P(BHET-EOP)
• Figure 10 shows an electron microscopic photograph of P (BHET-EOP/TC, 80/20) microspheres containing FITC-BSA.
• Figure 11 shows the effect of loading level on the release kinetics of FITC-BSA from microspheres.
• Figure 12 shows the lidocaine release from polymer BHDPT-EOP and BHDPT-HOP microspheres .
• Figure 13 shows the release of lidocaine from copolymer P (BHDPT-EOP/TC) microspheres.
• Figure 14 shows the cytotoxicity of P (BHET-EOP/TC,
• Figure 15 shows a toxicity assay plot of relative cell growth (%) vs. concentration of degraded polymer in a tissue-culture well (mg/ml) for four separate polymers.
• Figure 16 shows a cell toxicity assay plot for two microspheres and their respective monomers.
• aliphatic refers to a linear, branched, or cyclic alkane, alkene, or alkyne.
• Preferred aliphatic groups in the poly (phosphate) polymer of the invention are linear or branched alkane having from 1 to 10 carbons, preferably being linear alkane groups of 1 to 7 carbon atoms .
• aromatic refers to an unsaturated cyclic carbon compound with 4n+2 T ⁇ electrons.
• heterocyclic refers to a saturated or unsaturated ring compound having one or more atoms other than carbon in the ring, for example, nitrogen, oxygen or sulfur.
• the biodegradable terephthalate polymer of the invention comprises the recurring monomeric units shown in formula I :
• R is a divalent organic moiety.
• R can be any divalent organic moiety so long as it does not interfere with the polymerization, copolymerization, or biodegradation reactions of the polymer.
• R can be an aliphatic group, for example, alkylene, such as ethylene, 1, 2-dimethylethylene, n-propylene, isopropylene, 2-methylpropylene, 2 , 2 ' -dimethyl -propylene or tert-butylene, tert-pentylene, n-hexylene, n-heptylene and the like; alkenylene, such as ethenylene, propenylene, dodecenylene, and the like; alkynylene, such as propynylene, hexynylene, octadecenynylene, and the like; an aliphatic group substituted with a non-interfering substituent, for example, hydroxy-, halogen- or
• R can also be a divalent aromatic group, such as phenylene, benzylene, naphthalene, phenanthrenylene, and the like, or a divalent aromatic group substituted with a non- interfering substituent.
• R can be a divalent heterocyclic group, such as pyrrolylene, furanylene, thiophenylene, alkylene-pyrrolylene-alkylene, pyridylene, pyridinylene, pyrimidinylene and the like, or may be any of these substituted with a non-interfering substituent.
• R is an alkylene group, a cycloaliphatic group, a phenylene group, or a divalent group having the formula:
• R is an alkylene group having from 1 to 7 carbon atoms and, most preferably, R is an ethylene group, a 2 -methyl -propylene group, or a 2 , 2 ' -dimethylpropylene group.
• R' in the polymer of the invention is an aliphatic, aromatic or heterocyclic residue.
• R' is aliphatic, it is preferably alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, -C 8 H 17 , and the like; alkyl substituted with a non-interfering substituent, such as halogen, alkoxy or nitro; or alkyl conjugated to a biologically active substance to form a pendant drug delivery system.
• R' is aromatic, it typically contains from about 5 to about 14 carbon atoms, preferably about 5 to 12 carbon atoms and, optionally, can contain one or more rings that are fused to each other.
• R' is heterocyclic, it typically contains from about 5 to 14 ring atoms, preferably from about 5 to 12 ring atoms, and one or more heteroatoms.
• heterocyclic groups include furan, thiophene, pyrrole, isopyrrole, 3 -isopyrrole, pyrazole, 2-isoimidazole, 1 , 2 , 3-triazole, 1 , 2 , 4-triazole, oxazole, thiazole, isothiazole, 1 , 2 , 3 -oxadiazole, 1 , 2 , 4 -oxadiazole, 1 , 2 , 5-oxadiazole, 1 , 3 , 4-oxadiazole, 1, 2 , 3 , 4-oxatriazole, 1 , 2 , 3 , 5-oxatriazole, 1 , 2 , 3 -dioxazole, 1, 2 , 4-dioxazole, 1, 3 , 2-dioxazole, 1, 3 , 4-dioxazole, 1, 2 , 5-oxatriazole, 1,2-pyran,
• R' is heterocyclic, it is selected from the group consisting of furan, pyridine, N-alkylpyridine, 1,2,3- and 1, 2 , 4-triazoles, indene, anthracene and purine .
• R' is an alkyl group or a phenyl group and, even more preferably, an alkyl group having from 1 to 7 carbon atoms. Most preferably, R' is an ethyl group.
• x can vary greatly depending on the desired solubility of the polymer, the desired Tg, the desired stability of the polymer, the desired stiffness of the final polymers, and the biodegradability and the release characteristics desired in the polymer.
• x generally is ⁇ 1 and, typically, varies between about 1 and 40.
• x is from about 1 to about 30, more preferably, from about 1 to about 20 and, most preferably, from about 2 to about 20.
• the most common way of controlling the value of x is to vary the feed ratio of the "x" portion relative to the other monomer. For example, in the case of making the polymer:
• EOP ethyl phosphoro- dichloridate
• TC terephthaloyl chloride reactant
• Feed ratios of EOP to TC can easily vary from 99:1 to 1:99, for example, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 50:50, 45:55, 20:80, 15:85, and the like.
• the EOP/TC feed ratio varies from about 90:10 to about 50:50; even more preferably, from about 85:15 to about 50:50; and, most preferably from about 80:20 to about 50:50.
• n can vary greatly depending on the biodegradability and the release characteristics desired in the polymer, but typically varies between about 0 to 5,000, preferably between about 2 and 500. More preferably, n is from about 5 to about 300 and, most preferably, from about 5 to about 200.
• Biodegradable polymers differ from non-biodegradable polymers in that they can be degraded during in vivo therapy. This generally involves breaking down the polymer into its monomeric subunits . In principle, the ultimate hydrolytic breakdown products of a poly (phosphate) are phosphate, alcohol, and diol, all of which are potentially non-toxic.
• the intermediate oligomeric products of the hydrolysis may have different properties, but the toxicology of a biodegradable polymer intended for implantation or injection, even one synthesized from apparently innocuous monomeric structures, is typically determined after one or more in vi tro toxicity analyses.
• biodegradable polymer of the invention is preferably sufficiently pure to be biocompatible itself and remains biocompatible upon biodegradation.
• biocompatible is meant that the biodegradation products or the polymer are non-toxic and result in only minimal tissue irritation when implanted or injected into vasculated tissue.
• the polymer of the invention is preferably soluble in one or more common organic solvents for ease of fabrication and processing.
• Common organic solvents include such solvents as chloroform, dichloromethane, acetone, ethyl acetate, DMAC, N-methyl pyrrolidone, dimethylformamide, and dimethylsulfoxide .
• the polymer is preferably soluble in at least one of the above solvents.
• the glass transition temperature (Tg) of the polymer of the invention can vary widely depending upon the degree of branching of the diols used to prepare the polymer, the relative proportion of phosphorous-containing monomer used to make the polymer, and the like. However, preferably, the Tg is within the range of from about -10°C to about 80°C and, even more preferably, between about 0 and 50°C.
• a Friedel-Crafts reaction can also be used to synthesize poly (phosphates) .
• Polymerization typically is effected by reacting either bis (chloromethyl) compounds with aromatic hydrocarbons or chloromethylat-ed diphenyl ether with triaryl phosphates.
• Poly (phosphates) can also be obtained by bulk condensation between phosphorus diimidazolides and aromatic diols, such as resorcinol and quinoline, usually under nitrogen or some other inert gas.
• An advantage of bulk polycondensation is that it avoids the use of solvents and large amounts of other additives, thus making purification more straightforward. It can also provide polymers of reasonably high molecular weight . Somewhat rigorous conditions, however, are often required and can lead to chain acidolysis (or hydrolysis if water is present). Unwanted, thermally- induced side reactions, such as cross-linking reactions, can also occur if the polymer backbone is susceptible to hydrogen atom abstraction or oxidation with subsequent macroradical recombination. To minimize these side reactions, the polymerization is preferably carried out in solution. Solution polycondensation requires that both the diol and the phosphorus component be soluble in a common solvent .
• a chlorinated organic solvent such as chloroform, dichloromethane, or dichloroethane .
• the solution polymerization is preferably run in the presence of equimolar amounts of the reactants and a stoichiometric amount of an acid acceptor, usually a tertiary amine such as pyridine or triethylamine .
• the product is then typically isolated from the solution by precipitation with a non- solvent and purified to remove the hydrochloride salt by conventional techniques known to those of ordinary skill in the art, such as by washing with an aqueous acidic solution, e.g. , dilute HCl .
• Reaction times tend to be longer with solution polymerization than with bulk polymerization. However, because overall milder reaction conditions can be used, side reactions are minimized, and more sensitive functional groups can be incorporated into the polymer.
• the disadvantages of solution polymerization are that the attainment of high molecular weights, such as a Mw greater than 20,000, is less likely.
• Interfacial polycondensation can be used when high molecular weight polymers are desired at high reaction rates. Mild conditions minimize side reactions. Also the dependence of high molecular weight on stoichiometric equivalence between diol and dichloridate inherent in solution methods is removed. However, hydrolysis of the acid chloride may occur in the alkaline aqueous phase. Sensitive dichloridates that have some solubility in water are generally subject to hydrolysis rather than polymerization. Phase transfer catalysts, such as crown ethers or tertiary ammonium chloride, can be used to bring the ionized diol to the interface to facilitate the polycondensation reaction.
• Phase transfer catalysts such as crown ethers or tertiary ammonium chloride
• the yield and molecular weight of the resulting polymer after interfacial polycondensation are affected by reaction time, molar ratio of the monomers, volume ratio of the immiscible solvents, the type of acid acceptor, and the type and concentration of the phase transfer catalyst.
• the process of making a biodegradable terephthalate homopolymer of formula I comprises the step of polymerizing p moles of a diol compound having formula II :
• R' is defined as above, and p>q, to form q moles of a homopolymer of formula IV, shown below: wherein R, R' and x are as defined above.
• the homopolymer so formed can be isolated, purified and used as is. Alternatively, the homopolymer, isolated or not, can be used to prepare a block copolymer of the invention by:
• the function of the polymerization reaction of step (a) is to phosphorylate the di-ester starting material and then to polymerize it to form the homopolymer.
• the polymerization step (a) can take place at widely varying temperatures, depending upon the solvent used, the molecular weight desired, the solubility desired, and the susceptibility of the reactants to form side reactions.
• the polymerization step (a) takes place at a temperature from about -40 to about +160°C for solution polymerization, preferably from about 0 to 65 °C; in bulk, temperatures in the range of about +150 °C are generally used.
• the time required for the polymerization step (a) also can vary widely, depending on the type of polymerization being used and the molecular weight desired. Preferably, however, the polymerization step (a) takes place during a time between about 30 minutes and 24 hours.
• the polymerization step (a) may be in bulk, in solution, by interfacial polycondensation, or any other convenient method of polymerization
• the polymerization step (a) is a solution polymerization reaction.
• an acid acceptor is advantageously present during the polymerization step (a) .
• a particularly suitable class of acid acceptor comprises tertiary amines, such as pyridine, trimethylamine, triethylamine, substituted anilines and substituted aminopyridines .
• the most preferred acid acceptor is the substituted aminopyridine 4-dimethylaminopyridine ("DMAP") .
• the addition sequence for the polymerization step (a) can vary significantly depending upon the relative reactivities of the diol of formula II, the phosphorodichloridate of formula III, and the homopolymer of formula IV; the purity of these reactants; the temperature at which the polymerization reaction is performed; the degree of agitation used in the polymerization reaction; and the like.
• the diol of formula II is combined with a solvent and an acid acceptor, and then the phosphorodichloridate is added slowly.
• a solution of the phosphorodichloridate in a solvent may be trickled in or added dropwise to the chilled reaction mixture of diol, solvent and acid acceptor, to control the rate of the polymerization reaction.
• step (b) The purpose of the copolymerization of step (b) is to form a block copolymer comprising (i) the phosphorylated homopolymer chains produced as a result of polymerization step (a) and (ii) interconnecting polyester units.
• the result is a block copolymer having a microcrystalline structure particularly well-suited to use as a controlled release medium.
• the copolymerization step (b) of the invention usually takes place at a slightly higher temperature than the temperature of the polymerization step (a) , but also may vary widely, depending upon the type of copolymerization reaction used, the presence of one or more catalysts, the molecular weight desired, the solubility desired, and the susceptibility of the reactants to undesirable side reaction.
• the copolymerization step (b) when carried out as a solution polymerization reaction, it typically takes place at a temperature between about -40 and 100°C.
• Typical solvents include methylene chloride, chloroform, or any of a wide variety of inert organic solvents .
• step (b) The time required for the copolymerization of step (b) can also vary widely, depending on the molecular weight of the material desired and, in general, the need to use more or less rigorous conditions for the reaction to proceed to the desired degree of completion. Typically, however, the copolymerization step (b) takes place during a time of about 30 minutes to 24 hours.
• the addition sequence for the copolymerization step (b) can vary significantly depending upon the relative reactivities of the homopolymer of formula IV and the terephthaloyl chloride of formula V; the purity of these reactants; the temperature at which the copolymerization reaction is performed; the degree of agitation used in the copolymerization reaction; and the like.
• the terephthaloyl chloride of formula V is added slowly to the reaction mixture, rather than vice versa.
• a solution of the terephthaloyl chloride in a solvent may be trickled in or added dropwise to the chilled or room temperature reaction, to control the rate of the copolymerization reaction.
• the polymer of formula I is isolated from the reaction mixture by conventional techniques, such as by precipitating out, extraction with an immiscible solvent, evaporation, filtration, crystallization and the like.
• the polymer of formula I is both isolated and purified by quenching a solution of said polymer with a non-solvent or a partial solvent, such as diethyl ether or petroleum ether.
• the addition sequence of the reactive chlorides and the reaction temperatures in each step are preferably optimized to obtain the combination of molecular weight desired with good solubility in common organic solvents.
• the additive sequence comprises dissolving the bis-terephthalate starting material with an acid acceptor in a solvent in which both are soluble, chilling the solution with stirring, slowly adding an equal molar amount of the phosphorodichloridate (dissolved in the same solvent) to the solution, allowing the reaction to proceed at room temperature for a period of time, slowly adding an appropriate amount of terephthaloyl chloride, which is also dissolved in the same solvent, and increasing the temperature to about 50°C before refluxing overnight.
• the polymer of formula I is usually characterized by a release rate of the biologically active substance in vivo that is controlled, at least in part, as a function of hydrolysis of the phosphoester bond of the polymer during biodegradation. Additionally, the biologically active substance to be released may be conjugated to the phosphorus side chain R' to form a pendant drug delivery system.
• the structure of the side chain can influence the release behavior of the polymer. For example, it is expected that conversion of the phosphorous side chain to a more lipophilic, more hydrophobic or bulky group would slow down the degradation process. Thus, for example, release is usually faster from polymer compositions with a small aliphatic group side chain than with a bulky aromatic side chain.
• the lifetime of a biodegradable polymer in vivo also depends upon its molecular weight, crystallinity, biostability, and the degree of cross-linking. In general, the greater the molecular weight, the higher the degree of crystallinity, and the greater the biostability, the slower biodegradation will be. Accordingly, degradation times can vary widely, preferably from less than a day to several months .
• the polymer of formula I can be used either alone or as a composition containing, in addition, a biologically active substance to form a variety of useful biodegradable materials.
• the polymer of formula I can be used to produce a biosorbable suture, an orthopedic appliance or bone cement for repairing injuries to bone or connective tissue, a laminate for degradable or non- degradable fabrics, or a coating for an irnplantable device, even without the presence of a biologically active substance.
• the biodegradable terephthalate polymer composition comprises both:
• the biologically active substance of the invention can vary widely with the purpose for the composition.
• the active substance (s) may be described as a single entity or a combination of entities.
• the delivery system is designed to be used with biologically active substances having high water-solubility as well as with those having low water- solubility to produce a delivery system that has controlled release rates.
• biologically active substance includes without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of disease or illness-; or substances which affect 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.
• Non-limiting examples of broad categories of useful biologically active substances include the following therapeutic categories: anabolic agents, antacids, anti- asthmatic agents, anti-cholesterolemic and anti-lipid agents, anti-coagulants, anti-convulsants, anti-diarrheals, anti-emetics, anti-infective agents, anti-inflammatory agents, anti-manic agents, anti-nauseants, anti-neoplastic agents, anti-obesity agents, anti-pyretic and analgesic agents, anti-spasmodic agents, anti-thrombotic agents, anti- uricemic agents, anti-anginal agents, antihistamines, anti- tussives, appetite suppressants, biologicals, cerebral dilators, coronary dilators, decongestants, diuretics, diagnostic agents, erythropoietic agents, expectorants, gastrointestinal sedatives, hyperglycemic agents, hypnotics, hypoglycemic agents, ion exchange resins, laxative
• non-limiting examples of useful biologically active substances include the following therapeutic categories: analgesics, such as nonsteroidal anti- inflammatory drugs, opiate agonists and salicylates; antihistamines, such as H ⁇ blockers and H 2 -blockers; anti- infective agents, such as anthelmintics, antianaerobics , antibiotics, aminoglycoside antibiotics, antifungal antibiotics, cephalosporin antibiotics, macrolide antibiotics, miscellaneous ⁇ -lactam antibiotics, penicillin antibiotics, quinolone antibiotics, sulfonamide antibiotics, tetracycline antibiotics, antimycobacterials, antituberculosis antimycobacterials, antiprotozoals, antimalarial antiprotozoals, antiviral agents, anti- retroviral agents, scabicides, and urinary anti-infectives ; antineoplastic agents, such as alkylating agents, nitrogen mustard aklylating agents, nitros,
• NSAIDs nonsteroidal anti-inflammatory drugs
• analgesics such as diclofenac, ibuprofen, ketoprofen, and naproxen
• opiate agonist analgesics such as codeine, fentanyl, hydromorphone, and morphine
• salicylate analgesics such as aspirin (ASA) (enteric coated ASA)
• ⁇ -blocker antihistamines such as clemastine and terfenadine
• H 2 -blocker antihistamines such as cimetidine, famotidine, nizadine, and ranitidine; (6) anti-infective agents-, such as mupirocin; (7) antianaerobic anti-infectives, such as chloramphenicol and clindamycin; (8) antifungal antibiotic anti-infectives, such as amphotericin b, clotrimazole, fluconazole, and ketoconazole; (9) macrolide antibiotic anti-infectives, such as azithromycin and erythromycin; (10) miscellaneous ⁇ -lactam antibiotic anti-infectives, such as aztreonam and imipenem; (11) penicillin antibiotic anti- infectives, such as nafcillin, oxacillin, penicillin G, and penicillin V; (12) quinolone antibiotic anti-infectives, such as ciprofloxacin and norfloxacin; (13) tetracycline antibiotic anti
• hormones and hormone modifiers such as bromocriptine
• abortifacients such as methotrexate; (83) antidiabetic agents, such as insulin; (84) oral contraceptives, such as estrogen and progestin; (85) progestin contraceptives, such as levonorgestrel and norgestrel; (86) estrogens such as conjugated estrogens, diethylstilbestrol (DES), estrogen (estradiol, estrone, and estropipate) ; (87) fertility agents, such as clomiphene, human chorionic gonadatropin (HCG) , and menotropins; (88) parathyroid agents such as calcitonin; (89) pituitary hormones, such as desmopressin, goserelin, oxytocin, and vasopressin (ADH) ; (90) progestins, such as medroxyprogesterone, norethindrone , and progesterone; (91) thyroid hormones, such as levothyroxine; (91) thyroid
• the following less common drugs may also be used: chlorhexidine; estradiol cypionate in oil; estradiol valerate in oil; flurbiprofen; flurbiprofen sodium; ivermectin; levodopa; nafarelin; and somatropin.
• the following new drugs may also be used: recombinant beta-glucan; bovine immunoglobulin concentrate; bovine superoxide dismutase; the formulation comprising fluorouracil, epinephrine, and bovine collagen; recombinant hirudin (r-Hir) , HIV-1 immunogen; human anti-TAC antibody; recombinant human growth hormone (r-hGH) ; recombinant human hemoglobin (r-Hb) ; recombinant human mecasermin (r-IGF-1) ; recombinant interferon beta-la; lenograstim (G-CSF) ; olanzapine; recombinant thyroid stimulating hormone (r-TSH) ; and topotecan.
• recombinant beta-glucan bovine immunoglobulin concentrate
• bovine superoxide dismutase the formulation comprising fluorouracil, epinephrine, and bovine collagen
• intravenous products may be used: acyclovir sodium; aldesleukin; atenolol; bleomycin sulfate, human calcitonin; salmon calcitonin; carboplatin; carmustine; dactinomycin, daunorubicin HCl; docetaxel; doxorubicin HCl; epoetin alfa; etoposide (VP-16) ; fluorouracil (5-FU) ; ganciclovir sodium; gentamicin sulfate; interferon alfa; leuprolide acetate; meperidine HCl; methadone HCl; methotrexate sodium; paclitaxel; ranitidine HCl; vinblastin sulfate; and zidovudine (AZT) .
• useful biologically active substances from the above categories include: (a) antineoplastics such as androgen inhibitors, antimetabolites, cytotoxic agents, and immunomodulators ; (b) anti-tussives such as dextromethorphan, dextromethorphan hydrobromide, noscapine, carbetapentane citrate, and chlorphedianol hydrochloride; (c) antihistamines such as chlorpheniramine maleate, pheninda ine tartrate, pyrilamine maleate, doxylamine succinate, and phenyltoloxamine citrate; (d) decongestants such as phenylephrine hydrochloride, phenylpropanolamine hydrochloride, pseudoephedrine hydrochloride, and ephedrine; (e) various alkaloids such as codeine phosphate, codeine sulfate and morphine; (f) mineral supplements such as potassium chloride, zinc chloride, calcium carbon
• TGF- /. fibroblast growth factor
• FGF tumor necrosis factor- ⁇ & /.
• TNF-c ⁇ & /_ nerve growth factor
• NGF nerve growth factor
• GRF growth hormone releasing factor
• EGF epidermal growth factor
• FGFHF fibroblast growth factor homologous factor
• HGF hepatocyte growth factor
• IGF insulin growth factor
• IIF-2 invasion inhibiting factor-2
• BMP 1--7 bone morphogenetic proteins 1-7
• somatostatin thymosin- ⁇ -1, ⁇ -globulin, superoxide dismutase (SOD)
• complement factors hGH, tPA, calcitonin, ANF, EPO and insulin
• anti- infective agents such as antifungals, anti-virals, antiseptics and antibiotics.
• the biologically active substance may be a radiosensitizer, such as metoclopramide, sensamide or neusensamide (manufactured by Oxigene) ; profiromycin (made by Vion) ; RSR13 (made by Allos) ; Thymitaq (made by Agouron) , etanidazole or lobenguane (manufactured by Nycomed) ; gadolinium texaphrin (made by Pharmacyclics) ; BuDR/Broxine (made by NeoPharm) ; IPdR (made by Sparta) ; CR2412 (made by Cell Therapeutic) ; L1X (made by Terrapin) ; or the like.
• a radiosensitizer such as metoclopramide, sensamide or neusensamide (manufactured by Oxigene) ; profiromycin (made by Vion) ; RSR13 (made by Allos) ; Thymit
• the biologically active substance is selected from the group consisting of peptides, polypeptides, proteins, amino acids, polysaccharides, growth factors, hormones, anti-angiogenesis factors, interferons or cytokines, and pro-drugs.
• the biologically active substance is a therapeutic drug or pro-drug, most preferably a drug selected from the group consisting of chemotherapeutic agents and other anti-neoplasties such as paclitaxel, antibiotics, anti-virals, antifungals, anti-inflammatories, and anticoagulants.
• the biologically active substances are used in amounts that are therapeutically effective. While the effective amount of a biologically active substance will depend on the particular material being used, amounts of the biologically active substance from about 1% to about 65% have been easily incorporated into the present delivery systems while achieving controlled release. Lesser amounts may be used to achieve efficacious levels of treatment for certain biologically active substances.
• Pharmaceutically acceptable carriers may be prepared from a wide range of materials. Without being limited thereto, such materials include diluents, binders and adhesives, lubricants, disintegrants , colorants, bulking agents, flavorings, sweeteners and miscellaneous materials such as buffers and adsorbents in order to prepare a particular medicated composition.
• a biodegradable therapeutic agent delivery system consists of a dispersion of the therapeutic agent in a polymer matrix.
• the therapeutic agent is typically released as the polymeric matrix biodegrades in vivo into soluble products that can be absorbed by and eventually excreted from the body.
• an article is used for implantation, injection, or otherwise placed totally or partially within the body, the article comprising the biodegradable terephthalate polymer composition of the invention.
• the biologically active substance of the composition and the polymer of the invention may form a homogeneous matrix, or the biologically active substance may be encapsulated in some way within the polymer.
• the biologically active substance may be first encapsulated in a microsphere and then combined with the polymer in such a way that at least a portion of the microsphere structure is maintained.
• the biologically active substance may be sufficiently immiscible in the polymer of the invention that it is dispersed as small droplets, rather than being dissolved, in the polymer. Either form is acceptable, but it is preferred that, regardless of the homogeneity of the composition, the release rate of the biologically active substance in vivo remain controlled, at least partially as a function of hydrolysis of the phosphoester bond of the polymer upon biodegradation.
• the article of the invention is designed for implantation or injection into the body of an animal. It is particularly important that such an article result in minimal tissue irritation when implanted or injected into vasculated tissue.
• the polymer compositions of the invention provide a physical form having specific chemical, physical, and mechanical properties sufficient for the application, in addition to being a composition that degrades in vivo into non-toxic residues.
• Typical structural medical articles include such implants as orthopedic fixation devices, ventricular shunts, laminates for degradable fabric, drug-carriers, biosorbable sutures, burn dressings, coatings to be placed on other implant devices, and the like.
• the composition of the invention may be useful for repairing bone and connective tissue injuries.
• a biodegradable porous material can be loaded with bone morphogenetic proteins to form a bone graft useful for even large segmental defects.
• a biodegradable material in the form of woven fabric can be used to promote tissue ingrowth.
• the polymer composition of the invention may be used as a temporary barrier for preventing tissue adhesion, e.g., following abdominal surgery.
• the presence of a biodegradable supporting matrix can be used to facilitate cell adhesion and proliferation.
• the tubular article can also serve as a geometric guide for axonal elongation in the direction of functional recovery.
• the polymer compositions of the invention provide a polymeric matrix capable of sequestering a biologically active substance and provide predictable, controlled delivery of the substance. The polymeric matrix then degrades to non-toxic residues.
• Biodegradable medical implant devices and drug delivery products can be prepared in several ways.
• the polymer can be melt processed using conventional extrusion or injection molding techniques, or these products can be prepared by dissolving in an appropriate solvent, followed by formation of the device, and subsequent removal of the solvent by evaporation or extraction.
• the polymers may be formed into drug delivery systems of almost any size or shape desired, for example, implantable solid discs or wafers or injectable rods, microspheres, or other microparticles .
• a medical implant article Once a medical implant article is in place, it should remain in at least partial contact with a biological fluid, such as blood, internal organ secretions, mucous membranes, cerebrospinal fluid and the like.
• a biological fluid such as blood, internal organ secretions, mucous membranes, cerebrospinal fluid and the like.
• BHET having excellent purity may be prepared according to the following reaction scheme:
• BHET is also commercially available.
• TC terephthaloyl chloride
• the structure of P (BHET-EOP/TC, 80/20) was ascertained by -NMR, 31 P-NMR and FT-IR spectra, as shown in Figures 2 and 3. The structure was also confirmed by elemental analysis, which correlated closely with theoretical ratios. The results of the elemental analysis are shown in Figure 5.
• the molecular weight of P (BHET-EOP/TC, 80/20) was first measured by gel permeation chromatography (GPC) with polystyrene as the calibration standard. The resulting graph established a weight average molecular weight (Mw) of about 6100 and a number average molecular weight (Mn) of about 2200, as shown in Figure 4. Vapor pressure osmometry (“VPO”) for this copolymer gave an Mn value of about 7900. The results of these molecular weight studies are also shown in Figure 5.
• a series of other P (BHET-EOP/TC) copolymers of the invention were prepared by following the procedure described above in Example 2 except that the feed ratio of the EOP to TC used during the initial polymerization step and copolymerization step respectively was varied. The results are shown below in Table 1. From the feed ratio of EOP/TC, the value of "x" from the formula shown below can be calculated. For example, in P (BHET-EOP/TC, 80/20) prepared above in Example 2, x is 8.
• the phosphoester copolymers P (BHET-HOP/TC, 80:20) and P (BHET-HOP/TC, 90:10) were prepared by the procedure described above in Example 2, except that hexyl phosphorodichloridate (“HOP") was substituted for the monomer ethyl phosphoro-dichloridate (EOP) during the initial polymerization step, and the feed ratio was varied.
• HOP hexyl phosphorodichloridate
• EOP monomer ethyl phosphoro-dichloridate
• BHDPT Bis ( 3 -hydroxy- 2 , 2 ' -dimethylpropyl ) terephthalate
• the BHDPT monomer prepared in Example 5 above and the acid acceptor 4-dimethylaminopyridine (DMAP) were dissolved in methylene chloride.
• the resulting solution was chilled to -70°C using a dry ice/acetone bath, and an equal molar amount of ethyl phosphorodichloridate (EOP) was slowly added.
• EOP ethyl phosphorodichloridate
• the reaction mixture was then heated and refluxed overnight.
• the salt formed in the polymerization was removed by filtration.
• the remaining polymer solution (filtrate) was washed with a saturated NaCl solution three times, and the homopolymer was precipitated in diethyl ether.
• Copolymers of P (BHDPT-EOP) with TC were synthesized by the two-step solution copolymerization shown above. After the reaction between BHDPT and EOP had proceeded at room temperature for one hour, the reaction flask was cooled in a dry ice/acetone bath. An appropriate amount of TC (the number of moles of TC and EOP combined equaled the number of moles of BHDPT) was slowly added to the flask. The reaction mixture was then heated and refluxed overnight. The salt formed in the polymerization was removed by filtration. The remaining copolymer solution (filtrate) was washed with a saturated NaCl solution three times, and the copolymer was precipitated out in diethyl ether.
• Example 8 Feed Ratio Variations for P (BHDPT-EOP/TC)
• a series of other P (BHDPT-EOP/TC) copolymers of the invention were prepared by following the procedure described above in Example 7, except that the feed ratio of EOP to TC, which were used during the initial polymerization step and the copolymerization step respectively, were varied. The results are shown below in Table 2. From the feed ratio of EOP/TC, the value of x from the formula shown below can be - calculated. For example, in P (BHDPT-EOP/TC, 80/20), the value of x is 8.
• the BHDPT monomer prepared in Example 5 above and the acid acceptor 4-dimethylaminopyridine (DMAP) were dissolved in methylene chloride.
• the resulting solution was chilled to -70°C using a dry ice/acetone bath, and an equal molar amount of hexyl phosphorodichloridate (HOP) was slowly added.
• the reaction mixture was then heated and refluxed overnight.
• the salt formed in the polymerization was removed by filtration.
• the remaining polymer solution (filtrate) was washed with a saturated NaCl solution three times, and the homopolymer was precipitated in diethyl ether.
• Copolymers of P(BHDPT-HOP) with TC were synthesized by a two-step solution polymerization. After the reaction between BHDPT and HOP had proceeded at room temperature for one hour, the reaction flask was cooled in a dry ice/acetone bath. An appropriate amount of TC (the number of moles of TC and HOP combined equaled the number of moles of BHDPT) was slowly added to the flask. The reaction mixture was then heated and refluxed overnight . The salt formed during the copolymerization was removed by filtration. The remaining copolymer solution (filtrate) was washed with a saturated NaCl solution three times, and the copolymer was precipitated out in diethyl ether.
• TC the number of moles of TC and HOP combined equaled the number of moles of BHDPT
• Example 11 Other Diol Variations Diol terephthalates that are structurally related to that of BHET and BHDPT were synthesized similarly to that in Example 5 by reacting TC with either n-propylenediol or 2- methylpropylenediol , the structures of which are shown below, to form the corresponding diol terephthalate.
• the Tg increased as the proportion of EOP decreased and the proportion of TC increased.
• Tg glass transition temperature
• R is -CH 2 CH 2 -; -CH 2 CH 2 CH 2 - ; -CH 2 CH (CH 3 ) CH 2 - ;
• Example 16 Solubilities of the Polymers of the Invention
• BHET-EOP/TC copolymers of the invention were placed in a desiccator at room temperature, and their stability was monitored by intrinsic viscosity and GPC. The copolymers were stable under these conditions without the need for storage under inert gas .
• the weight average molecular weight of P (BHET-EOP/TC, 80/20) decreased about 20% in three days. After 18 days, the P (BHET-EOP/TC, 85/15) and P (BHET-EOP/TC, 80/20) discs had lost about 40% and 20% in mass respectively.
• Figure 9 shows the in vivo degradation of P(BHET- EOP/TC, 80/20), as measured by weight loss.
• Example 21 In vi tro Biocompatability/
• the cytotoxicity of P (BHET-EOP/TC, 80/20) copolymer was assessed by culturing human embryonic kidney (HEK) cells on a cover slip that had been coated with the copolymer P (BHET- EOP/TC, 80/20) .
• HEK cells were also cultured on a coverslip coated with TCPS .
• the cells cultured on the copolymer-coated cover slip exhibited normal morphology at all times and proliferated significantly in 72 days, as compared to a considerably lower amount when identical HEK cells were cultured on TCPS.
• a 100 mg polymer wafer was formed from P (BHET-EOP/TC, 80/20) and, as a reference, a copolymer of lactic and glycolic acid (75/25, "PLGA") known to be biocompatible. These wafers were inserted between muscle layers of the right limb of adult SPF Sprague-Dawley rats under anesthesia. The wafers were retrieved at specific times, and the surrounding tissues were prepared for histopathological analysis by a certified pathologist using the following scoring:
• the phosphoester copolymer P (BHET-EOP/TC, 80/20) was shown to have an acceptable biocompatability similar to that exhibited by the PLGA reference wafer.
• Microspheres were prepared via a double- emulsion/solvent-extraction method using FITC-labeled bovine serum albumin (FITC-BSA) as a model protein drug.
• FITC-BSA FITC-labeled bovine serum albumin
• One hundred ⁇ L of an FITC-BSA solution (10 mg/mL) were added to a solution of 100 mg of P (BHET-EOP/TC, 80/20) in 1 mL of methylene chloride, and emulsified via sonication for 15 seconds on ice.
• the resulting emulsion was immediately poured into 5 mL of a vortexing aqueous solution of 1% polyvinyl alcohol (PVA) and 5% NaCl. The vortexing was maintained for one minute.
• PVA polyvinyl alcohol
• the resulting emulsion was poured into 20 mL of an aqueous solution of 0.3% PVA and 5% NaCl, which was being stirred vigorously. Twenty- five mL of a 2% isopropanol solution was added, and the mixture was kept stirring for one hour to ensure complete extraction. The resulting microspheres were collected via centrifugation at 3000 X g, washed three times with water, and lyophilized. Empty microspheres were prepared in the same way except that water was used as the inner aqueous phase.
• the loading level of FITC-BSA was determined by assaying for FITC after hydrolyzing the microspheres in a 0.5 N NaOH solution overnight. Loading levels were determined by comparison with a standard curve, which had been generated by making a series of FITC-BSA solutions in 0.5 N NaOH. Protein loading levels of 1.5, 14.1 and 22.8 wt . % were readily obtained.
• the encapsulation efficiency of FITC-BSA by the microspheres was determined at different loading levels by comparing the quantity of FITC-BSA entrapped with the initial amount in solution via fluorometry. As shown below in Table 9, encapsulation efficiencies of 84.6 and 99.6% were obtained. These results showed that encapsulation efficiencies of 70-90% would be readily obtainable.
• PVA polyvinyl alcohol
• a copolymer/drug solution was prepared by combining 900 mg of P (BHDPT-EOP/TC, 50/50) copolymer and 100 mg of lidocaine in 9 mL of methylene chloride and vortex-mixing.
• FITC-BSA was released within the first two days, with an additional amount of about 5% being released after 10 days in PBS at 37°C.
• the release kinetics of FITC-BSA from P (BHET-EOP/TC, 80/20) microspheres at different loading levels are shown in Figure 11.
• P BHET-EOP/TC, 80/20 microspheres were added to 96- well tissue culture plates at different concentrations. The wells were then seeded with human gastric carcinoma cells (GT3TKB) at a density of 10 4 cells/well. The cells were incubated with the microspheres for 48 hours at 37°C. The resulting cell proliferation rate was analyzed by MTT assay and plotted as % relative growth vs. concentration of copolymer microspheres in the tissue culture well . The results are shown in Figure 14.
• GT3TKB human gastric carcinoma cells
• GT3TKB (GT3TKB) at a density of 10 4 / ell.
• the degraded polymer products were incubated with the GT3TKB cells for 48 hours.

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