Synthesis of Poly (Carboxyphenoxypropane- Sebacic Anhydride) for the Delivery of Drugs to the Brain ABRAHAM J. DOMB I and MICHAEL KUBEK 2 27 JDepartment of Medicinal Chemistry and Natural Products School of Pharmacy - Faculty of Medicine The Hebrew University of Jerusalem, Jerusalem 91120, Israel; 2Departments of Anatomy and Cell Biology, and Psychiatry Indiana University School of Medicine, Indianapolis, IN, 46202 USA, 1. INTRODUCTION Bioerodible polymeric implants have been used for the delivery of a range of anticancer drugs [1-5]. These devices are favorable for clinical use because the device is eliminated from the brain after the drug has been depleted. Most studies have been conducted by Brem and co-workers using poly(1,3-p-carboxyphenoxypropane-sebacic acid) [P(CPP-SA)] as drug carrier [4-11]. The biocompatibility and elimination processes of this polymer carrier have been extensively investigated and found to be biocompatible and biodegradable in the brain [12-14]. The inflammatory reaction elicited by these polymers when implanted in the brain of a rat, rabbit and monkey was compared with the clinically used implants of Surgicel (oxidized regenerated cellulose) and Gelfoam (absorbable gelatin sponge) [12-14]. The metabolic disposition and elimination processes of radiolabelled P(CPP:SA)20:80 implanted in the rat brain was studied [15]. Nearly all the radioactivity in the polymers was excreted by the four week time point and the device was completely eliminated from the implant site, four weeks after implantation. This biodegradable polymer was used clinically in the brain. Poly(CPP-SA) wafers loaded with 1,3-bis-(2-chloroethyl)-I-nitrosourea Blood-Brain Barrier Edited by Kobiler et al., Kluwer Academic/Plenum Publishers, New York, 2001 351
Transcript
Synthesis of Poly (CarboxyphenoxypropaneSebacic Anhydride) for the Delivery of Drugs to the Brain
ABRAHAM J. DOMBI and MICHAEL KUBEK2
27
JDepartment of Medicinal Chemistry and Natural Products School of Pharmacy - Faculty of Medicine The Hebrew University of Jerusalem, Jerusalem 91120, Israel; 2Departments of Anatomy and Cell Biology, and Psychiatry Indiana University School of Medicine, Indianapolis, IN, 46202 USA,
1. INTRODUCTION
Bioerodible polymeric implants have been used for the delivery of a range of anticancer drugs [1-5]. These devices are favorable for clinical use because the device is eliminated from the brain after the drug has been depleted. Most studies have been conducted by Brem and co-workers using poly(1,3-p-carboxyphenoxypropane-sebacic acid) [P(CPP-SA)] as drug carrier [4-11].
The biocompatibility and elimination processes of this polymer carrier have been extensively investigated and found to be biocompatible and biodegradable in the brain [12-14]. The inflammatory reaction elicited by these polymers when implanted in the brain of a rat, rabbit and monkey was compared with the clinically used implants of Surgicel (oxidized regenerated cellulose) and Gelfoam (absorbable gelatin sponge) [12-14].
The metabolic disposition and elimination processes of radio labelled P(CPP:SA)20:80 implanted in the rat brain was studied [15]. Nearly all the radioactivity in the polymers was excreted by the four week time point and the device was completely eliminated from the implant site, four weeks after implantation.
This biodegradable polymer was used clinically in the brain. Poly(CPP-SA) wafers loaded with 1,3-bis-(2-chloroethyl)-I-nitrosourea
Blood-Brain Barrier
Edited by Kobiler et al., Kluwer Academic/Plenum Publishers, New York, 2001 351
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(BCNU) were studied for the treatment of glioma multifonna [16]. A number of review articles have been published describing the developmental processes of this implant [4-8,17]. The synthesis and characterization of poly(CPP-SA)20:80 and other biodegradable polyanhydrides has been investigated for over 10 years and enonnous data is available [18-23]. However, the effect on the polymer composition and preparation of monomers on the physical properties was not published.
This report describes the controlled preparation of CPP and SA prepolymers and various compositions of CPP-SA copolymers, and the effect of the preparation and composition on the polymer physical properties.
2. EXPERIMENTAL
Instrumentation: Infrared spectroscopy (Ane1ect Instruments FT-IR model fx-6160) was perfonned on solutions of monomers and oligomer samples in CH2C12 cast on NaCI plates. Ultraviolet (UV) spectroscopy was perfonned using a Kontron Instruments Uvikon model 930. Thennal analysis was detennined on a Mattler TA4000 differential scanning calorimeter, calibrated with Zn and In standards, at a heating rate of 100C/min and a temperature range between -1000C and 2500C under nitrogen. Molecular weights of the polyanhydrides were estimated on a gel penneation chromatography (GPC) system consisting of a Spectra Physics (Darmstadt, Gennany) P1000 pump with UV detection (Applied Bioscience 759A Absorbance UV detector) at 254 nm, a Rheodyne (Coatati, CA) injection valve with a 20 !J.L loop, and a Spectra Physics Data Jet integrator. Samples were eluted with CHCl3 through a linear Styrogel column (lOA pore size) at a flow rate of 1 mllmin. The molecular weights were detennined relative to polystyrene standards (Polyscience, Warrington, PA) with a molecular weight range of 400 to 10,000 using a WINner1286 computer program. 1H NMR spectra (CDCI3/TMS/B/ppm) were obtained on a Varian 300 MHz spectrometer.
Materials: All materials and solvents were of high purity, the sources are listed in the CPP and SA preparation protocols (enclosed). Prepolymers of CPP and SA were synthesized according to the GMP production protocols unless otherwise mentioned. SA prepolymers with Dp=7.4 and 12, were prepared from the reaction of SA monomer with excess acetic anhydride (1: 10 w/v) for 5 and 20 min, following evaporation to dryness using a vacuum pump and precipitation in ether:petroleum ether from a 10% solution in dichloromethane. SA of Dp=4.3 was prepared from the reaction of sebacic acid with acetyl chloride at room temperature in dichloromethane in the presence of crosslinked poly(vinyl pyridine) (PVP) as an acid acceptor. After removal of the PVP:HCI by filtration, the solvent
Synthesis of poly(carboxyphenoxypropane-sebacic anhydride) 353
was evaporated to yield 70% of SA prepolymer of Dp=4.3 (by GPC). SA prepolymers with Dp of 22 and 44 were prepared by melt condensation of SA prepolymer ofDp=12 at 1800C for 5 and 20 min under vacuum (0.3 mm Hg), respectively. The number average molecular weight (Mn) of the prepolymers was determined by GPC using 500A crosslinked polystyrene column. The Dp of SA prepolymers was calculated from the Mn using: Dp=(Mn-l02)/184.
Preparation of CPP prepolymers: The procedure for the synthesis was similar for all preparations. CPP diacid monomer was added to preheated acetic anhydride solution in a 1: 10 w/v ratio and allowed to react at a predetermined temperature and time. After heating the reaction mixture was left to cool to room temperature, filtered through a red ribbon filter paper, concentrated to about 20% of the starting volume, and left to crystallize over night at OoC. The precipitate was isolated by filtration and reprecipitated into a petroleum ether:ether 1: 1 v/v ratio from a 10% dichloromethane solution. The filtrate was farther concentrated and treated as the starting solution to yield additional amount of CPP prepolymer. The product was analyzed to meet the prepolymer specifications.
Preparation of CPP-SA mixed prepolymer: CPP monomer was mixed with SA monomer at a 5, 10, 30, and 80 mole %. The mixed powder was added to refulxing acetic anhydride (1:10 w/v) and left to react for 30 minutes. Acetic anhydride was evaporated to dryness and reprecipitated in access petroleum ether:ether 1: 1 mixture from a 10% dichloromethane solution to yield a white powder.
Polymerization: Copolymers at various ratios of CPP to SA were prepared by melt condensation of the appropriate prepolymer ratios at 1800C for 60 min under a vacuum of 300±50 mm Hg. The polymers were purified by precipitating the polymer in ether:petroleum ether from a 10% solution in dichloromethane and evaporation to dryness. In a typical experiment, CPP prepolymer (5g) was mixed with increasing amounts of SA prepolymer (Tables 1 and 2) and polymerized at 1800C / 300±50 mm Hg for 60 min. (>90% yield). The copolymers were purified by precipitation in a 1:1 mixture of petroleum ether:ether (100 ml) from a 10% solution in dichloromethane (10 ml). The precipitated polymer was analyzed by IH NMR (CPP:SA ratio), GPC (Mn and Mw), DSC (Tmax and % crystallinity), IR, and degradation in buffer.
Determination of CPP in polymer by UV spectroscopy in chloroform: Solutions of 10 to 2 mg/ml of CPP prepolymer in chloroform (HPLC grade) were monitored by UV at 271 and 248 nm. Solutions of polymers at a concentration of 10 mg/ml were used to determine the CPP content in the copolymer. A correction to the acetate anhydride end groups of the CPP prepolymer was made by dividing the OD data by 0.85 (for CPP prepolymer of Mn=700g/mol, 102g/mol belong to the acetate end groups. The net CPP is 598/700=85% of the obtained Mn.
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Determination of CPP in polymer by UV spectroscopy in IN NaOH solution: 10 mg of CPP was dissolved in IN NaOH solution (1 ml) and the UV absorption was determined at 248 and 271 nm.
Determination of acetic anhydride in the CPP and SA prepolymers. Acetic anhydride in the prepolymer samples was determined by GC using an Alltech packed 10% FF AP CAW A 80/20 and FID detractor. Samples of 1 microliter acetic anhydride were used as standards. Samples of prepolymers (100 mg) were dissolved in 0.5 ml dichloromethane and precipitated in 1.5 ml of ether: petrol ether mixture. Samples (1 l)[jJ)ffilheO precipitation solution was injected to GC for analysis. A typical Rt for acetic anhydride was 4.95 min. Both prepo1ymers had additional peaks attributed to short chain prepolymers.
3. RESULTS AND DISCUSSION:
Two series of CPP and SA copolymers were synthesized from two batches of CPP prepolymer (Dp=1.8) and SA prepolymer (Dp=10.3) (EO-38-1 t07 series and EO-28-1a to 6a and 1b-6b series). The physical properties of these polymers are summarized in Tables 1 and 2.
All polymers had a molecular weight in the range ofMw=25,000:"80,000 and Mn=4,000-8,000. The polymers showed anhydride peaks at 1804 and 1742 cm-1. The crystallinity of the polymers is correlated with the dH of the polymers [6]. Both the melting point (Tmax) and the crystallinity decreased with the increase ofCPP content. A large variability in the dH (crystallinity) is related to the variability in the polymer handling conditions (cooling rate, storage in freezer, etc.).
A good correlation was found between the 'H NMR analysis and the monomer entry. Purification of the polymers by precipitation from dichloromethane solution had a slide effect (inconsistent) on the polymer properties which is attributed to the precipitation process that may remove a low MW fraction but in the other hand a decrease in MW may occur during solubilization in dichloromethane [5].
The degradation rate of these polymers in O.lM phosphate buffer pH7.4 at 37 oC was studied. The degradation was determined by monitoring the CPP released to the buffer using UV analysis. All polymers released the CPP content for about 20 days at a constant rate in a similar rate within a range of about ±6% throughout the degradation period. After 20 days of degradation about 80% of the tablet mass was degraded and eliminated with
Synthesis of poly(carboxyphenoxypropane-sebacic anhydride) 355
the remaining mass were composed of pure CPP monomer as determined by 'H NMR and IR (Table 3). The similar CPP release rate from CPP-SA copolymers of 16 to 25% CPP content can be explained by the fact that most of the sebacic acid component is degraded and released within 5 to 7 days leaving the water insoluble CPP monomer to slowly dissolve and eliminate [4].
EO-40-71 :4.08 12 13.54 0.18 68.3 72.5 72,400 4,600 Polymers were prepared by melt condensation of CPP and SA prepolymers at a 2.3 weight ratio. Polymerization at 1800C for 60 min. under a vacuum of 300±50 mm Hg. Dp was determined from the Mn obtained by OPC using the equation: (Mn-I02)/184. % CPP was determined from the integration ratio of the singlet at 1.33 ppm (8H of SA) and the triplet at 4.25 ppm (4H ofCPP). SA distribution in the copolymer is represented as either the ratio of the aromatic doublet at 8.1 and 7.9 ppm or the aliphatic triplets at 2.6 and 2.43 ppm.
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Table 2. ProEerties ofEoly(CPP-SA) at various CPP content
EO-28-6a 1:2.8 17.0 15.4 14.0 68.3 67.8 55,300 8,300 EO-28-6b 1:2.8 17.0 17.0 17.5 71.1 69.3 47,000 8,000 Copolymers were prepared under similar conditions described for Table 1 but with different batches of CPP and SA prepolymers.
Table 3. ProEerties of Degraded Eol~(CPP-SA) After 20 Days in Buffer
EO-28-5a 19.4 6.6 79.2 EO-28-6a 17.0 4.8 85.9 Compressed tablets (200 mg) were degraded in O.1M phosphate buffer pH7.4 at 37oC. CPP release was determined by UV absorption at 248 nm. The solid remaining after 20 days was pure CPP monomer as determined by H NMR.
The effect of the chain length of SA prepolymer (Dp), added to the polymerization with CPP prepolymer, on the properties of poly(CPPSA)20:80 was detennined. SA prepolymers of DP = 4.3, 7.6, 10.3, 27, and 44 were copolymerized with CPP (Dp= 1.8) at a 20:80 molar ratio. The
Synthesis of poly(carboxyphenoxypropane-sebacic anhydride) 357
copolymerization was carried out at 1800C for 90 minutes and the resulted polymer was analyzed by IH NMR for polymer composition and SA diad length. The SA prepolymer chain length did not affect the polymer composition (CPP-SA ratio), SA block length in the polymer, the polymer molecular weight and its melting point (Table 4).
Table 4. Poly(CPP-SA)20:80 proEerties as a function of the Dp of SA plepol~er
Polymers were prepared by melt condensation of CPP and SA prepolymers at a 2.3 weight ratio. Polymerization at 1800C for 60 min. under a vacuum of 300±50 mm Hg. Dp was determined from the Mn obtained by OPC using the equation: (Mn-I02)/184. % CPP was determined from the integration ratio of the singlet at 1.33 ppm (8H of SA) and the triplet at 4.25 ppm (4H of CPP). SA distribution in the copolymer is represented as either the ratio of the aromatic doublet at 8.1 and 7.9 ppm or the aliphatic triplets at 2.6 and 2.43 ppm. % CPP determined by UV at 271nm using a calibration curve. Data was converted from weight % to mole %.
In an attempt to fmd simpler methods for the determination ofpoly(CPPSA) composition (i.e. CPP:SA ratio), we have analyzed chloroformic
solutions of the above polymers by IH NMR and UV. A standard curve for CPP was obtained from chloroformic solutions of CPP prepolymer. Calibration curves for CPP prepolymer at 271 and 248 nm were obtained
(r2=0.97). UV determination at 248 or 271 nm of the series of polymers listed in Tables 1 and 2 did not show a good correlation with the entry amounts. The determination of the CPP content in poly(CPP-SA) by hydrolysis of the polymer in IN NaOH solution and monitoring the UV absorption at 248 and 271 nm resulted in data with an error as high as 10%. For comparison, 'H NMR analysis provided a good correlation with the
35,200
55,000
26,400
48,200
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monomer entry. The 'H NMR is recommended for the detennination of polymer composition.
In an attempt to improve the yield of the preparation of the CPP prepolymer the following experiments were conducted: 1. Reaction of CPP monomer in 1: 1 mixtures of acetic anhydride and one of
the solvents, dichloromethane, chlorofonn, toluene, and xylene at a 1: 10 w/v CPP to solution ratio.
2. Reaction of CPP monomer in acetic anhydride at lower temperatures (100 to 1400C) for a few hours.
3. Reaction of CPP monomer in acetic anhydride at 1000C in the presence of catalytic amounts ofH3P04 or ZnCl2. 4. Reaction of CPP monomer in acetic anhydride using azeotrope collection of acetic acid fonned during carboxylic acid acetylation. A different approach is the preparation of CPP-SA mixed prepolymer. The results of these experiments are summarized in Table 5.
Table 5. Synthesis ofCPP Er9!oly!!!er at various conditions No. Solution Temp. Time Solubility" Unreactedb Yield (%y
(oc) (Hrs) ofCPPpp (%) I total (mg/ml)
1. CHC13:AcAn 1:1 60 (ref) 24 150 95 <5
2. (CH2hC12:AcAn 1: 1 82 (ref) 24 55 90 <5
3. Toluene:AcAn 1: 1 110 (ref) 24 40 80 10
4. Xylene:AcAn 1: 1 140 (ref) 24 40 75 10
5. Acetic anhydride 91 24 30 90 <5
6. Acetic anhydride 110 6 30 70 17
7. Acetic anhydride 122 6 30 40 38
8. Acetic anhydride 135 6 30 20 50
9. Acetic anhyd+H3P04 reflux 0.5 30 70 <5
10. Acetic anhydride reflux 0.5 30 50 28
CPP monomer (GMP grade) was mixed (I: 10 w/v) with preheated acetic anhydride mixture and allowed to react for the time listed in the Table. AcAn-acetic anhydride; ref-reflux. a. The solubility was determined for the CPP prepolymer at room temperature. b. Unreacted CPP was isolated from the reaction mixture at room temperature at the end of the reaction. c. The yield was of the first 'crop' and the total is with the second 'crop' after concentrating the reaction solution.
The synthesis of CPP prepolymer by reflux in 1: 1 mixtures of acetic anhydride with good solvents for CPP prepolymer (chlorinated hydrocarbons and aromatics) at reflux for 24 hours resulted in very low yields «5%). The mixture with xylene yielded about 20% of pure CPP prepolymer. In all experiments about 50 to 80% of the added CPP monomer did not dissolve and separated by filtration.
Reaction at 950 C acetic anhydride (1: 10 w/v) for 24 hours resulted in a low yield (5-10%) with most CPP monomer remain unreacted. Addition of either phosphoric acid or ZnCl2 did not improve the yield but rather formed a colored (yellow to brown) reaction mixture. The use of azeotrope to remove the acetic acid formed during the reaction also did not improve the yield (although Azeotrope was effective in removing acetic anhydride from the reaction mixture).
The processes that resulted in better yields were the reaction of CPP with acetic anhydride at temperatures between 125 and 1400 C for 5 hours.
A different approach is the preparation of mixed prepolymer of CPPSA. The preparation of CPP-SA mixed prepolymer at a 20:80 ratio is possible and yielded a good quality mixed prepolymer that can be directly polymerized into the final polymer. The success of this approach is dependent on the purity of the prepolymers obtained. Because high quality CPP and SA monomers are currently produced, a mixed prepolymer should be considered. Alternatively, the CPP prepolymer can be prepared in the presence of a small amount of SA monomer (5 to 20 mole %). The addition of SA monomer reduces the risk of formation of insoluble large CPP oligomers. The acetic anhydride solution was clear and uncolored and the CPP-SA mixed prepolymer was white which indicated a pure product.
The acetic anhydride content in the CPP and SA prepolymers was determined by GC using an Alltech column packed with 10% FF Ap CW A W 80/100. These experiments indicate that the prepolymers contain between about 0.5 to 2% acetic anhydride.
4. CONCLUSIONS
I. 1 H NMR is the preferred method for the determination of CPP content is P(CPP-SA).
2. Copolymers of CPP-SA at a 16 to 25% CPP content melt at temperature range (Tmax) between 60 and noc. This copolymers degrade in vitro in a similar rate (about 10%/day during the first week).
3. The chain length of SA prepolymer does not affect the polymer properties.
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4. The preferred method for the synthesis of CPP prepolymer is heating at 13SoC for 5 hours and purification by chloroform extraction.
5. The prepolymers contain between 0.5 and 2.0 wt % acetic anhydride.
ACKNOWLEDGMENTS
This work was supported in part by a Binational Science Foundation grant (BSF) to M.J. Kubek and 1. Ringel. A.J. Domb is affiliated with The David R. Bloom Center for Pharmacy, and the Alex Grass Center for Drug Design, The Hebrew University.
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