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Injectable Polyanhydride Granules Provide Controlled Release of Water-Soluble Drugs with a Reduced Initial Burst
Injectable Polyanhydride Granules Provide Controlled Release of Water-Soluble Drugs with a Reduced Initial Burst YASUHIKO TABATA'$,
ABRAHAM DOMB'*9, AND ROBERT
Received February 27, 1992, from the Department of Chemical Engineering, Massachusetts Institute of Technology, Cambrklge, MA 02739. Accepted for publication April 1, 1993". tPresent address: Research Center for Biomedical Engineering, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606, Japan. §Present address: School of Pharmacy, Hebrew University, Jerusalem, Israel.
Abstract 0 A method for preparing polyanhydride granules of an injectable size was developed. The resulting granules permitted a nearly constant release of low-molecular-weight, water-soluble drugs without an initial burst. The polyanhydrides used were poly(fatty acid dimer), poly(sebacicacid), and their copolymers. The dyes acid orange 63 and pnitroanilinewere usedas modelcompoundsfor drugs. Polymer degradation and drug release for disks and variously sized granules of copolymers containing drugs, prepared by a water-in-oil (W/O) emulsion method, were compared with those for devices prepared by the usual compressionmethod. I nthe W/O emulsion method, a mixture of aqueous drug solution and polymer-chloroform solution was emulsified by probe sonication to prepare a very flne W/O emusion. The powder obtainedby freez-ing of the W/O emulsion was pressed into circular disks. I n the compression method, the drug was mechanically mixedwith the polymer, and the mixture was compressed into circular disks. The resultingdisks were groundto prepare granules of different sizes. The granules encapsulated more than 95% of the drug, irrespective of the preparation method. Both methods were effective in preparing polymer disks capable of controlleddrug release without any initial burst. However, as the granule size decreased to an injectable size (diameter, <150 pm), a large difference in the drug release profile was observed between the two preparation methods. The injectable granules obtained by the W/O emulsion method showed nearly constant drug release without any large initial burst, in contrast to those prepared by the compression method, irrespective of the drug type. Degradation studies of the granules demonstrated no difference Inthedegradation profile of the granule matrixitself between the two methods. Light microscopic observations of polymer disk preparedbythe cornpressionmethodindicateda nonuniformdistribution of dye islands throughoutthe matrix. I ncontrast, a highly homogeneous mixing of dye and polymer was achieved for devices prepared by the W/O emulsion method. It is therefore possible that this highly uniform distribution of drug throughout the polymer matrix leads to a reduced iiiitial burst in drug release from the injectable granules obtained by the W/O emulsion method.
Controlled release of a variety of therapeutic agents has been studied through the use of biodegradable polymeric delivery systems.l-8 For the design of a bioerodible polymer system that may display surface erosion, we have suggested polyanhydrides as promising candidates. The anhydride linkage is water labile, and by rational selection of monomer units, the polymer can be made sufficientlyhydrophobicto discourage water penetration. We have studied the drug release and polymer degradation characteristics of various polyanhydride drug formulations.3.9-16 In particular,the copolymerpoly[bis@-carb0xyphenoxy)propane sebacic acid anhydride] has been used for the treatment of neurological disorders and brain tumors experimentally as well as clinically.17J8 For disk-type devices obtained by the compression method, the drug release profile closely matches the polymer degradation profile, a fact indicating a release mechanism that is dominantly degradation controlled. Degradation and release periods from several weeks to several years are possible by changing the type of polymer that constitutes the 0
Abstract publishedin Advance ACS Abstracts, November 15,1993.
0 1994, American Chemical Society and A d c a n Pharmaceutical Association
devices.9 However, the usual method of incorporating a hydrophilic drug into a hydrophobic polymer involves mixing particles of drug with particles of polymer and then compressing the mixture. The result is islands composed of small drug particles heterogeneously distributed throughout the polymer matrix. Therefore, a burst effect is seen when the drug islands located close to the matrix surface quickly dissolve after being immersed in solution. In most cases, this burst effect is undesirable because it releases an uncontrollable significant portion of the drug immediately at the beginning of the release period. This burst leaves smaller amounts of drug to be released over the entire release period. In addition, this initial burst may be toxic to the body. If the drug can be homogeneouslydispersed in the polymer matrix, this initial burst effect should be reduced greatly, irrespective of the shape of the drug formulations. In this study, a new method, namely, a water-in-oil(W/O) emulsion method, for preparing drug formulations that have a homogeneous distribution of drug throughout the polymer matrix, was developed. This method permitted the preparation of granules of an injectable size that could release water-soluble drugs at a nearly constant rate without any large initial burst. The granules could be a promising injectable controlled-release system that is an alternative to microspheres (19). In this study, granules of copolymers [P(FAD-SA)Iof poly(fatty aciddimer) (PFAD)and poly(sebacicacid) (PSA),prepared by a W/Oemulsion method, were compared with those prepared by the usual compression method with respect to polymer degradation and drug release. Granule degradation was examined by high-pressure liquid chromatography (HPLC), gel permeation chromatography (GPC),and differential scanning calorimetry (DSC). Morphology was observed by scanning electron microscopy (SEM). The effect of gelatin incorporation on the prolongation of the release of drug from granules is also described.
Experimental Section Materials-PSA, various copolymers of fatty acid dimer (FAD) derived from the naturally occurring oleic acid and sebacic acid (SA), and PFAD were kindly supplied by Nova Pharmaceutical Corp., Baltimore, MD. Gelatin (type A, from porcine skin) was purchased from Sigma Chemical Co.,St. Louis, MO. Acid orange 63 [AO;molecular weight, 832.801andp-nitroaniline(p-NA;molecular weight, 138.80)were purchased from Aldrich Chemical Co., Inc., Milwaukee, WI. Other chemical reagents were obtained from Sigma and used as obtained. Instrumentation-The molecular weights and dispersion characteristics of polyanhydrides were determined on a Perkin-Elmer GPC system consistingof the series 10 pump and the model 3600Data Station with refractive index detection (the LC-25 RI detector). Samples were eluted in chloroform through a Phenogel5-pm column (Phenomenex, Torrance, CA) at a flow rate of 0.90 mL/min. The molecular weights were determined relativeto those of polystyrene standards (Polysciences, Warrington, PA; molecular weight range, 25 000-500 OOO) by use of the CHROM I1 and GPC 5 computer programs (Perkin-Elmer). Thermal analysis of the polymers was conducted on a Perkin-Elmer DSC-2 differential scanning calorimeterby use of a heating rate of 10°C/ min to determine their melting temperature, heat of fusion, and
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crystallinity. Infrared (IR) spectroscopy was performed on a PerkinElmer model 1430spectrophotometer. Polymer samples were film cast onto NaCl plates from solutions of polymers in chloroform. P r e p a r a t i o n of P o l y a n h y d r i d e D i s k s a n d G r a n u l e s Drug-incorporated polyanhydride devices were formulated either by a W/O emusion method or by a compression method. For the standard W/O emulsion method, 100jtL of double-distilled water with or without 10 mg of gelatin was poured into 1mL of chloroform containing 235 mg of polyanhydride. The mixture was mixed for 20 s a t room temperature by use of a Vortex mixer at maximum speed (Vortex Genie; Scientific Inc.) and then subjected to probe sonication (model VC-250 apparatus; Sonic & Materials Inc.) at output 4 (50 W) for 30 s to form a very fine W/O emulsion. The W/O emulsion was frozen in liquid nitrogen and freeze-dried (Lab Conc. Inc.) for 24 h. No thawing of the emulsion sample was observed during the freeze-drying procedure. The powder obtained was pressed into circular disk in a Carver Test Cylinder Outfit (model C; Fred S. Carver Inc., Menomenee Falls, WI) at 27 000 psi and room temperature for 10min. For the compressionmethod, the polymers were ground in a very small mill grinder and filtered through sieves into sizes smaller than 50 pm. Then, model drugs, filtered through sieves into the same size range, were mixed with the polymers manually, and the mixtures were pressed into circular disks under the same conditions as for the W/O emulsion method. The disks obtained by the abovedescribed methods were 12 mm in diameter and 0.9-1.1 mm thick and weighed 150mg. The disks were ground with a mortar and pestle and sized by use of sieves with apertures of 150,425,600,840, and 1500 pm. Granules with different dyes and gelatin a t different loadings were prepared similarly, but the total amount of dye, gelatin, and polymer was always adjusted to 250 mg. For examination of the homogeneity of the drug dispersion in polymer formulations, cross sections were observed with a scanning electron microscope. Degradation Studies-Degradation experiments were carried out on a shaker a t 37 "C in an air gravity incubator (Imperial incubator; Lab Line Instrument Inc.). Polyanhydride granules (5mg) were suspended in 2.5 mL of 0.1 M phosphate-buffered solution (pH 7.4), and the buffer was changed periodically to approximate perfect sink conditions. The degradation of polyanhydride disks was studied similarly under the following conditions. A 150-mg disk was placed in 75 mL of buffer solution. The frequency of changing of the buffer solution was adjusted during the degradation studies to ensure that the concentrations of the drug and degradation producb were below 10% of their saturation values a t all times. Degradation kinetics were monitored by measuring the UV of periodically changed buffer solutions, by reverse-phase ion-pair HPLC with a polymeric C-18 column (Rainin Instrument Co., Inc., Woburn, MA). The mobile phase consisted of acetonitrile in aqueous tetrabutylammonium phosphate (0.05 mol/mL), with a gradient of 1530% acetonitrile. Molecular weights of polyanhydrides before and after granule preparation and during degradation studies were measured on a PerkinElmer GPC system. Morphology and degradation of the granules were observed with a scanning electron microscope (250 Mk; Cambridge Instrument) a t 3-10 kV. These examinations were done on granules before and immediately after preparation and after different periods of degradation. Granule samples for SEM were freeze-dried, mounted on metal stubs with double-sided tape, and coated with gold to a thickness of 200-500 A. Release Studies-Studies of release of A 0 and p-NA from polyanhydride disks and granules of different sizes were conducted under the same conditions as the degradation studies. The absorbance8 of the buffer solutions were measured on a Perkin-Elmer model 553 UV spectrophotometer to determine the amounts of the different dyes released (forAO, 424nm; for p-NA, 308 nm). Every dye absorbed strongly in the visible range and provided minimum interference with the UV analysis of matrix degradation products. A Micro BCA protein assay reagent (Pierce Chemical Co., Rockford, IL) that measures protein was used to assess gelatin release kinetics. Because the Micro BCA reagent reacts with very high concentrations of the polymer degradation products, care was taken to dilute the buffer solutions to a point a t which reaction of the degraded products with the Micro BCA reagent was negligible. However, for further correction of the Micro BCA assay procedure, a standard curve for the reaction of polymer products with the Micro BCA reagent was prepared. With the absorption values obtained from this curve, a calculated absorbance was subtracted to account for the presence of degraded products. The degradation and release experiments were done independently in triplicate.
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6 / Journal of Pharmaceutical Sciences Vol. 83, No. 1, January 1994
Table 1-Physical
Properties of Various Polyanhydrldes' DSC Results molecular weightb
Polymer (FAD/SA Ratio)
Mw
PSA 29 400 P(FADSA)(8:92) 23 700 P(FAD-SA)(25~75) 42 900 P(FAD-SA)(25~75) 29 000 P(FAD-SA)(25~75) 19 700 P(FAD-SA)(25~75) 12 300 P(FAD-SA) (44~56) 18 000 PFAD
16 100
Mn
%
Melting
Tempera- AH Crystalture ('(2) (cal/g)= linity
16 500 10 100 17 200 13 400 9400 7000 9800 7500
80.5 73.7 63.0 61.0 60.5 59.8 34.7
28.69 24.48 14.01 13.86 13.27 13.52 4.21
57.0 52.9 37.1 36.7 35.2 35.8 14.9
0.0
a T h e IR spectra showed doublets at 1740 and 1800 cm-'.
* Determined by GPC.
Mw, weight-average molecular weight; Mn, number-average molecular weight. Determined by DSC.
Results Polymer Characterization-Table 1summarizes the physical properties of the polyanhydrides used in this study. P(FAD-SA)samples of approximately the same molecular weights were used to investigate the effect of FAD content on the degradation and release profiles of the disk and granules. The identity of each polyanhydridewas confirmed by IR spectroscopy. The IR spectra of PSA, PFAD, and P(FAD-SA) copolymers showed doublets at 1740 and 1800cm-I, which are characteristic of the carboxylic anhydrides. The melting point of a polymer decreased with increasing amounts of FAD in the copolymer. The SA homopolymer became more flexible through its copolymerization with FAD. Heat of fusion values for the polymers demonstrated a decrease as FAD was added to SA. However, no major difference in the heat of fusion was observed for P(FADSA) of different molecular weights. When two monomers (SA forming a crystalline homopolymer and FAD forming an amorphous homopolymer) were copolymerized, the degree of crystallinity decreased as FAD was added to the SA homopolymer. If we assume that the heat of fusion described only the change from a crystalline to an amorphous polymer, then there was a large decrease in crystallinity when the amount of FAD in the copolymer was increased. The relative degree of copolymer crystallinity decreased with increasing amounts of FAD in the copolymer, as calculated from the crystallinity of the SA homopolymer (57%),the heat of fusion, and the molar fraction of the monomers in each copolymer, by use of formulas reported by Mathiowitz et al. (19). Comparison of Release Characteristics of P(FAD-SA) Devices Obtained by t h e W/OEmulsion Method and t h e Compression Method-Characteristic release curves for the P(FAD-SA) disks and granules of different sizes are shown in Figure 1. The gelatin-free disks and granules were prepared by the W/O emulsion method and the compression method. A 0 was released from both types of disks at a nearly constant rate (Figure 1A). However, as the granule size became smaller, the difference in the profiles of release of A 0 from granules prepared by the W/O emulsion method and the compression method became larger. Granules of an injectable size (diameter, <150 Mm) prepared by the compression method exhibited a large initial burst in release and a fast release in comparison with those prepared by the W/O emulsion method (Figure 1B). In addition, a similar influence of the preparation method and the device size on the profile of drug release was observed when p-NA was used as a model drug (data not shown). It is possible that the difference in release profiles between injectable granules prepared by the two different methods may be due to the difference in dye distribution throughout the
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Flgure 1-A) Release of A 0 (2% loading) from gelatin-free disks of P(FAD-SA) (2575) with a molecular weight of 42 900 in 0.1 M phosphate buffer at37 O C . Key: (0)W/Oemulsion method; (0)compressionmethod. (B) Release of A 0 (2% loading) from gelatin-free granules [P(FAD-SA), 2575; molecular weight, 42 9001 of different sizes, prepared by the W/O emulsion method (0and A)and the compression method (0 and A),in 0.1 M phosphate buffer at 37 OC. Key: (0and .)granules with diameters of <150 pm; (Aand A)granules with diameters of >600 but
polyanhydride matrix. Cross sections of P(FAD-SA) disks containing A 0 were analyzed on an optical microscope to examine the homogeneityof A 0 distribution in the matrix. As is apparent from Figure 2, A 0 was dispersed much more homogeneously in the P(FAD-SA) disk prepared by the W/O emulsion method than in that prepared by the compression method. A nonuniform distribution of dye islands throughout the polymer matrix was observed for the disk prepared by the compression method. In contrast, a highlyhomogeneous mixing of the dye and the polymer was achieved for the disk prepared by the W/O emulsion method. Degradation Characteristics of P(FAD-SA)GranulesThe effect of granule size on the release of SA from P(FAD-SA) granules with loadings of 2% A 0 and 4 % gelatin is shown in Figure 3; the smaller the granules, the faster the release. The same trend for SA release was observed for gelatin-free granules, a result indicating no effect of gelatin on polymer degradation itself. Moreover, no difference in the SA release profile was observed between granules prepared by the W/O emulsion method and those prepared by the compression method (data not shown). This result indicates no effect of dye dispersion conditions on polymer degradation itself. Figure 4 shows a typical curve of molecular weight loss for P(FAD-SA) granules with loadings of 2% A 0 and 4% gelatin. The molecular weight decreased rapidly within the initial 72 h for the granules prepared by the W/O emulsion method and the compression method. This trend was observed irrespective of the type of polyanhydride and dye and gelatin loadings (data not shown). The molecular weight distribution at all times was unimodal and relatively narrow, with no evidence of shoulders corresponding to low- or intermediate-molecular-weight fragments (data not shown). After 125 h in buffer, the polymer molecular weight decreased from 42 900 to about 600. This molecular weight corresponded to that of the FAD monomer, a result suggesting that the granules were completely degraded to form chloroform-solubleFAD monomers in addition to water-
Flgure2-Fhotomicrographs of cross section of 4 % gelatin-loadedP(FADSA) (2575; molecular weight, 42 900; 2 % A 0 loading) disks prepared by the WIO emulsion method (A) and the compression method (B).
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Time ( h r ) Figure3-Degradation profiles for the P(FADSA)(25:75: molecular weight 42 900)disk and granules of different sizes prepared by the W/O emulsion method in 0.1 M phosphate buffer at 37 O C ; granules had diameters of <150 pm (0), >150 but C425 p m (O),and >600 but <840 pm (A);and disk (A).
soluble SA monomers. The profile of molecular weight loss for the original polymer was quite similar to that for the granules, a result indicating no effect of dye or gelatin incorporation on polymer degradation itself. SEM observation of P(FAD-SA) Granules-Figure 5 shows typical scanning electron micrographs of P(FAD-SA) granules with loadings of 2% A 0 and 4% gelatin. The granules were prepared from P(FAD-SA) with a molecular weight of 42 900 and a 25:75 FAD/SA molar ratio. Immediately after preparation, the surface of the granules appeared smooth,without Journal of Pharmaceutical Sciences / 7 Vol. 83, No. 1, January 1994
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Flgure 4-Molecular weight loss for P(FADSA) (2575) granules (<150 Fm In diameter) in 0.1 M phosphate buffer at 37 ' C . Granules were prepared by the WIO ernulslon method (0)and the compression method (0). (0) Original polymer.
any visible pores; no traces of dye were found on the outer surface (Figure 5A). A SEM photograph of a granule after 25 h of degradation showed that the surface possessed small pores and had an irregular structure (Figure 5B). The same granule but after 135 h of degradation is shown in Figure 5C. The granules displayed a deformed shape, and some oily substances were left behind. A similar profile of granule degradation was observed for gelatin-freegranules. Again, the presence of dye in the granule matrix and the method of granule preparation had no effect on the degradation profile €or the granules, irrespective of gelatin incorporation. Release Characteristics of Gelatin-Free P(FAD-SA) Granules-Characteristic release curves for the gelatin-free disks and granules prepared by the W/O emulsion method are shown in Figure 6. A 0 was released from all devices without any large initial burst. For pure surface erosion, we expect release rates to depend on granule size, while for pure bulk erosion, no dependence on size should exist. In Figure 6, one can see the influence of granule size on the release rate: the smaller granules showed the faster release. The same rule applied for polymer erosion (Figure 3), a result suggesting that pure bulk erosion did not occur in this system. There was a good correlation between A 0 release and SA release. In addition, this was true for granules loaded with p-NA (data not shown). The result suggests that diffusional escape of dye from the granule matrix is minimal and that release is controlled by matrix degradation caused by polymer surface erosion. Release Characteristics of Gelatin-Loaded P(FAD-SA) Granules-Characteristic release curves for 4 % gelatin-loaded granules prepared from P(FAD-SA) with different molecular weights are shown in Figure 7A. A 0 was released from the injectable granules without any large initial burst. However, no effect of polymer molecular weight on A 0 release was observed. Figure7B showstheeffectof loading on A 0 releasefromgranules. A 0 loading had no effect on the release profile over the range of dye loading studied. Figure 7C shows A 0 release from various polyanhydride granules with 4% gelatin loading. The rate of release from the granules decreased with increasing amounts of FAD in the copolymer. This result may be due to the more hydrophobic nature of the FAD monomer. The profile of release of A 0 and p-NA from granules with loadings of 2 % dye and 4 % gelatin are shown in Figure 8. Both dyes were released at a nearly constant rate, without any large initial burst, and no difference in the release profiles was observed for the two dyes. Figure 9 shows profiles of release of A 0 and SA from P(FADSA) granules prepared with or without gelatin. For the gelatinfree granules, the A 0 release pattern cloeely matched that of polymer degradation, and both processes were almost complete within 90 h. This result suggests a release mechanism that is dominantlydegradation controlled. However,the incorporation of gelatin in thegranules permitted the prolonged release of AO, 8 1 Journal of Pharmaceutical Sciences vol. 83, No. 1, January 1994
Figure I-SEM of ~(FAD-SA)granules at different degradation stages: immediately after preparation (A), after 25 h (B),after 135 h (c)in 0.1 M phosphate buffer at 37 'C.
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Figure 8-Release of A 0 from the gelatin-free disk (0)and granules of different sires in 0.1 M phosphate buffer at 37 O C ; granules had dlameters of <150 pm (0),>150 but <425 pm (O),>600 but <840 pm (A),and >840 but <1500 pm (A).P(FADSA) was used at 25:75, had a molecular weight of 42 900, and contained 2% A 0 loading.
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Flgure 8-Release of various dyes from P(FAD-SA) (2575; molecular weight, 42 900) granules with dlameters of < 150 pm In 0.1 M phosphate AO; (0)p-NA. buffer at 37 O C . Key: (0)
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Tlme (hr) Flgure9-Profiles of releaseof A 0 (circles)and SA (triangles)from P(FAD SA) (2575) granules with diameters of <150 pm In 0.1 M phosphate buffer at 37 O C . Key: (0 and A)4% gelatin-loaded granules; (0 and A) gelatin-free granules. P(FAPSA) molecular weight. 42 900 A 0 loading, 2%.
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Time (hr) Flgure 7-A) Release of A 0 (2% loading) from granules of P(FADSA) (25:75) with different molecular weights (<150 pm in diameter) in 0.1 M phosphate buffer at 37 O C ; molecular weights were 12 300 (0),29 000 (O), 42 900 (A). (6)Release of A 0 from P(FAPSA) (2575) granules (<150 pm in diameter) with different A 0 loadings in 0.1 M phosphate buffer at 37 O C ; A 0 loadings were 1% (0),2% (O),and 4% (A). P(FADSA) molecular weight, 42 900. Gelatin loading, 4 % (C) Release of A 0 (2% loading) from granules of various polyanhydrkfes in 0.1 M phosphate buffer at 37 O C . Key: (0)PSA; (0)V F A P S A ) (8:92); (A) P(FADSA) (2575); (A)P(FADSA) (4456). Qranules had a diameter of <150 pm.
Flgure 10-A) Release of A 0 (2% loadlng) (0) and gelatin (0)from 4% gelatin-loaded granules wlth diameters of <150 pm in 0.1 M pho@-ate buffer at 37 OC. (B)Release of A 0 (2% loading) (0) and gelailn (0)from 8 qC, gelatin-loadedgranules wlthdlametersof <150pm in 0.1 M phosphate buffer at 37 O C . P(FADSA) molecular weight, 42 900.
althoughthe granules exhibited an SA release profile similar to that of gelatin-free granules. A 0 release from P(FAD-SA) granules (25:75) containing gelatin in differentamounta is shownFigure 10. A 0 was released from the granules faster than gelatin, irrespective of the amount
of gelatin incorporated in the granule matrix. The period of A 0 release was significantly prolonged by increasing the amounta of gelatin in the granule matrix. Althoughgelatinreleaselagged behind A 0 release, the correlationbetween the two profiles was clear.
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JounRl of m m w l sdences / 9 Vol. 83, No. 1, January 1984
for gelatin, AO, and FAD monomers over a temperature range of -25-150 "C (data not shown). Degradation studies demonstrated that after 135 h in buffer, the granules were completely degraded to remnants composed of FAD monomers and gelatin in addition of AO. These DSC data suggest that a crystalline structure is formed from the remnants produced by granule degradation.
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Temperature ( O C ) Figure 11-DSC thermograms of P(FAD-SA) (2575) granules with diameters of < 150 pm at different degradation stages: immediatelyafter preparation (A), after 27 h (B) and after 135 h (C) for 4% gelatin-loaded granules and immediately after preparation (D) after 27 h (E), after 135 h (F) for gelatin-free granules in 0.1 M phosphate buffer at 37 OC. A 0 loading, 2 YO. The physical state of P(FAD-SA)granules during degradation was characterized by DSC measurements of the granules (Figure 11). P(FAD-SA) granules displayed a sharp endotherm around 60 "C before degradation, irrespective of the incorporation of gelatin in the granule matrix. This endotherm corresponds to the melting of the crystalline regions of the polymers in the granule matrix. The heat of fusion values were 13.45 and 13.10 cal/g for gelatin-free granules and 4 %-loaded granules, respectively. These values are quite similar to the heat of fusion value for the origianal polymer, 13.27 cal/g, a result indicating that the crystalline state of the polymer remains unchanged when A 0 and gelatin are loaded into the granule matrix. The endotherm became broader and the heat of fusion for the granules decreased with degradation time for both types of granules. However, a new, sharp endotherm at 120 "C was observed 135 h after degradation of the granules with 4 % gelatin loading, in contrast to gelatin-freegranules. In addition, no endotherm was observed 10 /Journal of Pharmaceutical Sciences Vol. 83, No. 1, January 1994
The present study demonstrates the differences in release profiles for water-soluble drugs in polyanhydride devices prepared by the W/O emulsion method and the usual compression method. When the two types of disk were compared, no significant difference in release profiles was observed, although the disk prepared by the latter method exhibited somewhat faster release than that prepared by the former method (Figure 1A). For devices of a smaller size (granules), however, the release profiles were quite different; the granules obtained by the compression method tended to release A 0 faster, with an initial burst, than those prepared by the W/O emulsion method (Figure 1B). A similar result was obtained for granules with diameters of <150 pm prepared from different polyanhydrides and loaded with different dyes. The difference in release pattern may be explained in terms of the distribution of the dye throughout the polymer matrix. Light microscopic observations of P(FAD-SA) disks demonstrated that the dye was dispersed much more homogeneouslyin the disk obtained by the W/O emulsion method than in that obtained by the compression method (Figure 2). The heterogeneous distribution of the dye and the large size of the dye islands throughout the polymer matrix did not lead to a significant initial burst in dye release from polyanhydride devices of a large size, such as a disk. For granules of an injectable size (<150 pm), however, the uniformity of dye distribution and the size of the dye islands became important contributing factors in the regulation of dye release profiles. It is possible that the dye islands located close to the surface of granules obtained by the compression method were responsible for a burst effect. In contrast, granules prepared by the W/O emulsion method showed a nearly constant release without any large initial burst (Figure 1B). The dispersion homogeneity must depend on the preparation method, because water-soluble drugs are poorly miscible with hydrophobic polyanhydrides. This W/O emulsion method is more effective in preparing polyanhydride-drug formulations with a highly uniform drug distribution than the compression method. The W/O method permitted the preparation of polyanhydride granules that had diameters of <150 pm and that could achieve a nearly constant release of water-soluble drugs. The preparation procedure was reproducible with respect to release profiles and yield; the yield was very high, -95%. Water-soluble A 0 was released at a nearly constant rate without any large initial burst, and the release profile was determined by the type of matrix polymer. Polyanhydrides possess a water-labile linkage. Thus, we had to try to minimize the contact time between the polymer and water to delay polymer degradation. However, the contact time in the granule preparation was very short, and a GPC study demonstrated no loss of polymer molecular weight before or after granule preparation (Figure 4). In addition, the granule preparation could be carried out at a low temperature, a fact that may be important in preventing the inactivation of some drugs. Polyanhydride erosion takes place because of the hydrolysis of water-labile anhydride linkages. The complete loss of SA after a short period (-90 h) indicated that water had completely penetrated the polymer matrix. Then, dissolution and/or diffusion through the polymer matrix of the degradation products, at least those connected with water-soluble SA,
occurred after the complete degradation of anhydride bonds. GPC and SEM studies showed that after 135 h in buffer, the remnants were oily substances soluble in chloroform, and their molecular weights were similar to those of FAD monomers (Figures4 and 5). Again, no difference in the degradation profile of the granule matrix itself was observed between granules prepared by the W/O emulsion method and those prepared by the compression method when judged on the basis of SA release and polymer molecular weight. In addition, the complete disappearance of anhydride bonds was observed near this time, as evaluated by IR spectroscopy (data not shown). Similar degradation profiles were observed for P(FAD-SA) microspheres.15 At 135h, complete cleavage of anhydride bonds in P(FAD-SA) took place and left oily, pooly water-soluble FAD monomers behind, irrespective of the type of devices. The incorporationof gelatin in the granule matrix was effective in prolonging the release period for water-soluble drugs. The profile of release of SA for granules containing gelatin was quite similar to that for gelatin-free granules (Figure 9). GPC and SEM studies demonstrated that no difference in the degration profile of the granule matrix itself was observed between gelatinloaded granules and gelatin-free granules (Figures 4 and 5). The DSC study of the granules at different degradation stages showed that after 135 h in buffer, a sharp endotherm at -120 "C was observed for granules containing gelatin but not for gelatin-free granules (Figure 11). Similar results were obtained for granules containingdyes other than AO. In addition, the same endotherm pattern could be seen when A 0 was mixed with FAD monomers, irrespective of the amount of A 0 (data not shown). At this stage of degradation, gelatin and FAD monomers in addition to dye remained in the granule matrix. A similar gelatin effect on the profile of release of water-soluble drugs has been found for P(FAD-SA) microspheres.15 These results demonstrated the formation of a crystalline structure between gelatin and FAD monomers produced via the degradation of P(FAD-SA). Thus, it is possible that an interaction between FAD monomers and gelatin molecules causes the continued release of drugs even after the polymer matrix has been completely degraded. The crystalline structure formed may function as a reservior for watersolubledrugs and result in a prolonged release period, irrespective of device type.
-
References and Notes 1. Sidman, K. R.; Schwope, A. D.; Steber, W. D.; Rudolph, S.E.; Poulin, S. B. J. Membr. Sci. 1990, 7, 227-291. 2. Lewis, D. H. In BiodegradablePolymers as Drug Delivery Systems; Chasin, M.; Langer, R., Eds.; Dekker: New York, 1990; pp 1-42. 3. Chasin,M.; Domb,A.; Ron, E.; Mathiowitz,E.; Leong,K.; Laurencin, C.; Brem, H.; Grossman, S.; Langer, R. In BiodegradablePolymers as Drug Delivery Systems; Chasin, M.; Langer, R., Eds.; Dekker: New York, 1990; pp 43-70. 4. Pitt, C. G. In Biodegradable Polymers as Drug Delivery Systems; Chasin, M.; Langer, R., Ed.; Dekker: New York, 1990; pp 71-120. 5. Heller, J.; S arer, R. V.; Zentner, G. M. In BiodegradablePolymers as Drug Degvery Systems; Chasin, M.; Langer, R., Eds.; Dekker: New York, 1990; pp 121-162. 6. Allcock, H. R. In Biodegradable Polymers as Drug Delivery Systems; Chasin, M.; Langer, R., Eds.; Dekker: New York, 1990; DD 163-194. 7. Kohn, J. In Biodegradable Polymers as Drug Delivery Systems; Chasin, M.; Langer, R., Eds.; Dekker: New York, 1990; pp 195230. 8. Bogdansky, S. In Biodegradable Polymers as Drug Delivery Systems; Chasin, M.; Langer, R., Eds.; Dekker: New York, 1990,
.-
DD 231-260. r r ---
9. Leong, K. W.; Brott, B. C.; Langer, R. J.Biomed. Mater. Res. 1985, 19,941-955. 10. Mathiowitz, E.; Langer, R. J. Controlled Release 1987, 5, 13-22. 11. Bindschaedler, C.; Leong, K.; Mathiowitz, E.; Langer, R. J. Pharm. Sci. 1988, 77, 696-698. 12. Mathiowitz, E.; Saltzman, W. E.; Domb, A.; Dor, P.; Langer, R. J. A d . Polvm. Sci. 1988. 35. 755-774. 13. Mkhiowhz, E.; Amato,'C.;'Dor, P.; Langer, R. Polymer 1990,31, 547-555. 14. Mathiowitz, E.; Bernstein, H.; Giannos, S.; Dor, P.; Turek, T.; Langer, R. J. Appl. Polym. Sci. 1992,45, 125-134. 15. Tabata, T.; Langer, R. Pharm. Res. 1993,10, 391-399. 16. Tamada, J.; Langer, R. J. Biomater. Sci. Polym. Ed. 1992,3,315353. 17. Brem, H.; Mahaley, M. S., Jr.; Vick, N. A.; Black, K. L.; Schold, S. C., Jr.; Burger, P. C.; Friedman, A. H.; Ciric, J. S.; Eller, T. W.; Cozzens, J. F.; Kenealy, J. N. J. Neurosurg. 1991, 74, 441-446. 18. Langer, R. J. Controlled Release 1991, 16, 53-60. 19. Mathiowitz, E.; Ron, E.; Mathiowitz, G.; Amato, C.; Langer, R. Macromolecules 1990,23, 3212-3218.
Acknowledgments This study was supported by NIH grant lUOlCA48508. We thank Dr. Janet Tamada for assistance and helpful discussions.
Journal of Pharmaceutical Sciences / 11 Vol. 83,No. 1, January 1994
ABRAHAM DOMB'*9, AND ROBERT
Received February 27, 1992, from the Department of Chemical Engineering, Massachusetts Institute of Technology, Cambrklge, MA 02739. Accepted for publication April 1, 1993". tPresent address: Research Center for Biomedical Engineering, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606, Japan. §Present address: School of Pharmacy, Hebrew University, Jerusalem, Israel.
Abstract 0 A method for preparing polyanhydride granules of an injectable size was developed. The resulting granules permitted a nearly constant release of low-molecular-weight, water-soluble drugs without an initial burst. The polyanhydrides used were poly(fatty acid dimer), poly(sebacicacid), and their copolymers. The dyes acid orange 63 and pnitroanilinewere usedas modelcompoundsfor drugs. Polymer degradation and drug release for disks and variously sized granules of copolymers containing drugs, prepared by a water-in-oil (W/O) emulsion method, were compared with those for devices prepared by the usual compressionmethod. I nthe W/O emulsion method, a mixture of aqueous drug solution and polymer-chloroform solution was emulsified by probe sonication to prepare a very flne W/O emusion. The powder obtainedby freez-ing of the W/O emulsion was pressed into circular disks. I n the compression method, the drug was mechanically mixedwith the polymer, and the mixture was compressed into circular disks. The resultingdisks were groundto prepare granules of different sizes. The granules encapsulated more than 95% of the drug, irrespective of the preparation method. Both methods were effective in preparing polymer disks capable of controlleddrug release without any initial burst. However, as the granule size decreased to an injectable size (diameter, <150 pm), a large difference in the drug release profile was observed between the two preparation methods. The injectable granules obtained by the W/O emulsion method showed nearly constant drug release without any large initial burst, in contrast to those prepared by the compression method, irrespective of the drug type. Degradation studies of the granules demonstrated no difference Inthedegradation profile of the granule matrixitself between the two methods. Light microscopic observations of polymer disk preparedbythe cornpressionmethodindicateda nonuniformdistribution of dye islands throughoutthe matrix. I ncontrast, a highly homogeneous mixing of dye and polymer was achieved for devices prepared by the W/O emulsion method. It is therefore possible that this highly uniform distribution of drug throughout the polymer matrix leads to a reduced iiiitial burst in drug release from the injectable granules obtained by the W/O emulsion method.
Controlled release of a variety of therapeutic agents has been studied through the use of biodegradable polymeric delivery systems.l-8 For the design of a bioerodible polymer system that may display surface erosion, we have suggested polyanhydrides as promising candidates. The anhydride linkage is water labile, and by rational selection of monomer units, the polymer can be made sufficientlyhydrophobicto discourage water penetration. We have studied the drug release and polymer degradation characteristics of various polyanhydride drug formulations.3.9-16 In particular,the copolymerpoly[bis@-carb0xyphenoxy)propane sebacic acid anhydride] has been used for the treatment of neurological disorders and brain tumors experimentally as well as clinically.17J8 For disk-type devices obtained by the compression method, the drug release profile closely matches the polymer degradation profile, a fact indicating a release mechanism that is dominantly degradation controlled. Degradation and release periods from several weeks to several years are possible by changing the type of polymer that constitutes the 0
Abstract publishedin Advance ACS Abstracts, November 15,1993.
0 1994, American Chemical Society and A d c a n Pharmaceutical Association
devices.9 However, the usual method of incorporating a hydrophilic drug into a hydrophobic polymer involves mixing particles of drug with particles of polymer and then compressing the mixture. The result is islands composed of small drug particles heterogeneously distributed throughout the polymer matrix. Therefore, a burst effect is seen when the drug islands located close to the matrix surface quickly dissolve after being immersed in solution. In most cases, this burst effect is undesirable because it releases an uncontrollable significant portion of the drug immediately at the beginning of the release period. This burst leaves smaller amounts of drug to be released over the entire release period. In addition, this initial burst may be toxic to the body. If the drug can be homogeneouslydispersed in the polymer matrix, this initial burst effect should be reduced greatly, irrespective of the shape of the drug formulations. In this study, a new method, namely, a water-in-oil(W/O) emulsion method, for preparing drug formulations that have a homogeneous distribution of drug throughout the polymer matrix, was developed. This method permitted the preparation of granules of an injectable size that could release water-soluble drugs at a nearly constant rate without any large initial burst. The granules could be a promising injectable controlled-release system that is an alternative to microspheres (19). In this study, granules of copolymers [P(FAD-SA)Iof poly(fatty aciddimer) (PFAD)and poly(sebacicacid) (PSA),prepared by a W/Oemulsion method, were compared with those prepared by the usual compression method with respect to polymer degradation and drug release. Granule degradation was examined by high-pressure liquid chromatography (HPLC), gel permeation chromatography (GPC),and differential scanning calorimetry (DSC). Morphology was observed by scanning electron microscopy (SEM). The effect of gelatin incorporation on the prolongation of the release of drug from granules is also described.
Experimental Section Materials-PSA, various copolymers of fatty acid dimer (FAD) derived from the naturally occurring oleic acid and sebacic acid (SA), and PFAD were kindly supplied by Nova Pharmaceutical Corp., Baltimore, MD. Gelatin (type A, from porcine skin) was purchased from Sigma Chemical Co.,St. Louis, MO. Acid orange 63 [AO;molecular weight, 832.801andp-nitroaniline(p-NA;molecular weight, 138.80)were purchased from Aldrich Chemical Co., Inc., Milwaukee, WI. Other chemical reagents were obtained from Sigma and used as obtained. Instrumentation-The molecular weights and dispersion characteristics of polyanhydrides were determined on a Perkin-Elmer GPC system consistingof the series 10 pump and the model 3600Data Station with refractive index detection (the LC-25 RI detector). Samples were eluted in chloroform through a Phenogel5-pm column (Phenomenex, Torrance, CA) at a flow rate of 0.90 mL/min. The molecular weights were determined relativeto those of polystyrene standards (Polysciences, Warrington, PA; molecular weight range, 25 000-500 OOO) by use of the CHROM I1 and GPC 5 computer programs (Perkin-Elmer). Thermal analysis of the polymers was conducted on a Perkin-Elmer DSC-2 differential scanning calorimeterby use of a heating rate of 10°C/ min to determine their melting temperature, heat of fusion, and
0022-3549/94/120&5$04.50/0
Journal of Pharmaceutical Sciences / 5 Vol. 83, No. 1, January 1994
crystallinity. Infrared (IR) spectroscopy was performed on a PerkinElmer model 1430spectrophotometer. Polymer samples were film cast onto NaCl plates from solutions of polymers in chloroform. P r e p a r a t i o n of P o l y a n h y d r i d e D i s k s a n d G r a n u l e s Drug-incorporated polyanhydride devices were formulated either by a W/O emusion method or by a compression method. For the standard W/O emulsion method, 100jtL of double-distilled water with or without 10 mg of gelatin was poured into 1mL of chloroform containing 235 mg of polyanhydride. The mixture was mixed for 20 s a t room temperature by use of a Vortex mixer at maximum speed (Vortex Genie; Scientific Inc.) and then subjected to probe sonication (model VC-250 apparatus; Sonic & Materials Inc.) at output 4 (50 W) for 30 s to form a very fine W/O emulsion. The W/O emulsion was frozen in liquid nitrogen and freeze-dried (Lab Conc. Inc.) for 24 h. No thawing of the emulsion sample was observed during the freeze-drying procedure. The powder obtained was pressed into circular disk in a Carver Test Cylinder Outfit (model C; Fred S. Carver Inc., Menomenee Falls, WI) at 27 000 psi and room temperature for 10min. For the compressionmethod, the polymers were ground in a very small mill grinder and filtered through sieves into sizes smaller than 50 pm. Then, model drugs, filtered through sieves into the same size range, were mixed with the polymers manually, and the mixtures were pressed into circular disks under the same conditions as for the W/O emulsion method. The disks obtained by the abovedescribed methods were 12 mm in diameter and 0.9-1.1 mm thick and weighed 150mg. The disks were ground with a mortar and pestle and sized by use of sieves with apertures of 150,425,600,840, and 1500 pm. Granules with different dyes and gelatin a t different loadings were prepared similarly, but the total amount of dye, gelatin, and polymer was always adjusted to 250 mg. For examination of the homogeneity of the drug dispersion in polymer formulations, cross sections were observed with a scanning electron microscope. Degradation Studies-Degradation experiments were carried out on a shaker a t 37 "C in an air gravity incubator (Imperial incubator; Lab Line Instrument Inc.). Polyanhydride granules (5mg) were suspended in 2.5 mL of 0.1 M phosphate-buffered solution (pH 7.4), and the buffer was changed periodically to approximate perfect sink conditions. The degradation of polyanhydride disks was studied similarly under the following conditions. A 150-mg disk was placed in 75 mL of buffer solution. The frequency of changing of the buffer solution was adjusted during the degradation studies to ensure that the concentrations of the drug and degradation producb were below 10% of their saturation values a t all times. Degradation kinetics were monitored by measuring the UV of periodically changed buffer solutions, by reverse-phase ion-pair HPLC with a polymeric C-18 column (Rainin Instrument Co., Inc., Woburn, MA). The mobile phase consisted of acetonitrile in aqueous tetrabutylammonium phosphate (0.05 mol/mL), with a gradient of 1530% acetonitrile. Molecular weights of polyanhydrides before and after granule preparation and during degradation studies were measured on a PerkinElmer GPC system. Morphology and degradation of the granules were observed with a scanning electron microscope (250 Mk; Cambridge Instrument) a t 3-10 kV. These examinations were done on granules before and immediately after preparation and after different periods of degradation. Granule samples for SEM were freeze-dried, mounted on metal stubs with double-sided tape, and coated with gold to a thickness of 200-500 A. Release Studies-Studies of release of A 0 and p-NA from polyanhydride disks and granules of different sizes were conducted under the same conditions as the degradation studies. The absorbance8 of the buffer solutions were measured on a Perkin-Elmer model 553 UV spectrophotometer to determine the amounts of the different dyes released (forAO, 424nm; for p-NA, 308 nm). Every dye absorbed strongly in the visible range and provided minimum interference with the UV analysis of matrix degradation products. A Micro BCA protein assay reagent (Pierce Chemical Co., Rockford, IL) that measures protein was used to assess gelatin release kinetics. Because the Micro BCA reagent reacts with very high concentrations of the polymer degradation products, care was taken to dilute the buffer solutions to a point a t which reaction of the degraded products with the Micro BCA reagent was negligible. However, for further correction of the Micro BCA assay procedure, a standard curve for the reaction of polymer products with the Micro BCA reagent was prepared. With the absorption values obtained from this curve, a calculated absorbance was subtracted to account for the presence of degraded products. The degradation and release experiments were done independently in triplicate.
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6 / Journal of Pharmaceutical Sciences Vol. 83, No. 1, January 1994
Table 1-Physical
Properties of Various Polyanhydrldes' DSC Results molecular weightb
Polymer (FAD/SA Ratio)
Mw
PSA 29 400 P(FADSA)(8:92) 23 700 P(FAD-SA)(25~75) 42 900 P(FAD-SA)(25~75) 29 000 P(FAD-SA)(25~75) 19 700 P(FAD-SA)(25~75) 12 300 P(FAD-SA) (44~56) 18 000 PFAD
16 100
Mn
%
Melting
Tempera- AH Crystalture ('(2) (cal/g)= linity
16 500 10 100 17 200 13 400 9400 7000 9800 7500
80.5 73.7 63.0 61.0 60.5 59.8 34.7
28.69 24.48 14.01 13.86 13.27 13.52 4.21
57.0 52.9 37.1 36.7 35.2 35.8 14.9
0.0
a T h e IR spectra showed doublets at 1740 and 1800 cm-'.
* Determined by GPC.
Mw, weight-average molecular weight; Mn, number-average molecular weight. Determined by DSC.
Results Polymer Characterization-Table 1summarizes the physical properties of the polyanhydrides used in this study. P(FAD-SA)samples of approximately the same molecular weights were used to investigate the effect of FAD content on the degradation and release profiles of the disk and granules. The identity of each polyanhydridewas confirmed by IR spectroscopy. The IR spectra of PSA, PFAD, and P(FAD-SA) copolymers showed doublets at 1740 and 1800cm-I, which are characteristic of the carboxylic anhydrides. The melting point of a polymer decreased with increasing amounts of FAD in the copolymer. The SA homopolymer became more flexible through its copolymerization with FAD. Heat of fusion values for the polymers demonstrated a decrease as FAD was added to SA. However, no major difference in the heat of fusion was observed for P(FADSA) of different molecular weights. When two monomers (SA forming a crystalline homopolymer and FAD forming an amorphous homopolymer) were copolymerized, the degree of crystallinity decreased as FAD was added to the SA homopolymer. If we assume that the heat of fusion described only the change from a crystalline to an amorphous polymer, then there was a large decrease in crystallinity when the amount of FAD in the copolymer was increased. The relative degree of copolymer crystallinity decreased with increasing amounts of FAD in the copolymer, as calculated from the crystallinity of the SA homopolymer (57%),the heat of fusion, and the molar fraction of the monomers in each copolymer, by use of formulas reported by Mathiowitz et al. (19). Comparison of Release Characteristics of P(FAD-SA) Devices Obtained by t h e W/OEmulsion Method and t h e Compression Method-Characteristic release curves for the P(FAD-SA) disks and granules of different sizes are shown in Figure 1. The gelatin-free disks and granules were prepared by the W/O emulsion method and the compression method. A 0 was released from both types of disks at a nearly constant rate (Figure 1A). However, as the granule size became smaller, the difference in the profiles of release of A 0 from granules prepared by the W/O emulsion method and the compression method became larger. Granules of an injectable size (diameter, <150 Mm) prepared by the compression method exhibited a large initial burst in release and a fast release in comparison with those prepared by the W/O emulsion method (Figure 1B). In addition, a similar influence of the preparation method and the device size on the profile of drug release was observed when p-NA was used as a model drug (data not shown). It is possible that the difference in release profiles between injectable granules prepared by the two different methods may be due to the difference in dye distribution throughout the
'0
120. (A)
0
$ 100m
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-8
01
0
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0
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0
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O O 0
0.O
50
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2 10
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Time (hr)
2 0 b 0
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Flgure 1-A) Release of A 0 (2% loading) from gelatin-free disks of P(FAD-SA) (2575) with a molecular weight of 42 900 in 0.1 M phosphate buffer at37 O C . Key: (0)W/Oemulsion method; (0)compressionmethod. (B) Release of A 0 (2% loading) from gelatin-free granules [P(FAD-SA), 2575; molecular weight, 42 9001 of different sizes, prepared by the W/O emulsion method (0and A)and the compression method (0 and A),in 0.1 M phosphate buffer at 37 OC. Key: (0and .)granules with diameters of <150 pm; (Aand A)granules with diameters of >600 but
polyanhydride matrix. Cross sections of P(FAD-SA) disks containing A 0 were analyzed on an optical microscope to examine the homogeneityof A 0 distribution in the matrix. As is apparent from Figure 2, A 0 was dispersed much more homogeneously in the P(FAD-SA) disk prepared by the W/O emulsion method than in that prepared by the compression method. A nonuniform distribution of dye islands throughout the polymer matrix was observed for the disk prepared by the compression method. In contrast, a highlyhomogeneous mixing of the dye and the polymer was achieved for the disk prepared by the W/O emulsion method. Degradation Characteristics of P(FAD-SA)GranulesThe effect of granule size on the release of SA from P(FAD-SA) granules with loadings of 2% A 0 and 4 % gelatin is shown in Figure 3; the smaller the granules, the faster the release. The same trend for SA release was observed for gelatin-free granules, a result indicating no effect of gelatin on polymer degradation itself. Moreover, no difference in the SA release profile was observed between granules prepared by the W/O emulsion method and those prepared by the compression method (data not shown). This result indicates no effect of dye dispersion conditions on polymer degradation itself. Figure 4 shows a typical curve of molecular weight loss for P(FAD-SA) granules with loadings of 2% A 0 and 4% gelatin. The molecular weight decreased rapidly within the initial 72 h for the granules prepared by the W/O emulsion method and the compression method. This trend was observed irrespective of the type of polyanhydride and dye and gelatin loadings (data not shown). The molecular weight distribution at all times was unimodal and relatively narrow, with no evidence of shoulders corresponding to low- or intermediate-molecular-weight fragments (data not shown). After 125 h in buffer, the polymer molecular weight decreased from 42 900 to about 600. This molecular weight corresponded to that of the FAD monomer, a result suggesting that the granules were completely degraded to form chloroform-solubleFAD monomers in addition to water-
Flgure2-Fhotomicrographs of cross section of 4 % gelatin-loadedP(FADSA) (2575; molecular weight, 42 900; 2 % A 0 loading) disks prepared by the WIO emulsion method (A) and the compression method (B).
0
50
100
150
200
Time ( h r ) Figure3-Degradation profiles for the P(FADSA)(25:75: molecular weight 42 900)disk and granules of different sizes prepared by the W/O emulsion method in 0.1 M phosphate buffer at 37 O C ; granules had diameters of <150 pm (0), >150 but C425 p m (O),and >600 but <840 pm (A);and disk (A).
soluble SA monomers. The profile of molecular weight loss for the original polymer was quite similar to that for the granules, a result indicating no effect of dye or gelatin incorporation on polymer degradation itself. SEM observation of P(FAD-SA) Granules-Figure 5 shows typical scanning electron micrographs of P(FAD-SA) granules with loadings of 2% A 0 and 4% gelatin. The granules were prepared from P(FAD-SA) with a molecular weight of 42 900 and a 25:75 FAD/SA molar ratio. Immediately after preparation, the surface of the granules appeared smooth,without Journal of Pharmaceutical Sciences / 7 Vol. 83, No. 1, January 1994
1
50000 1 40000
30000 20000
0
50
100 150 Time (hr)
200
Flgure 4-Molecular weight loss for P(FADSA) (2575) granules (<150 Fm In diameter) in 0.1 M phosphate buffer at 37 ' C . Granules were prepared by the WIO ernulslon method (0)and the compression method (0). (0) Original polymer.
any visible pores; no traces of dye were found on the outer surface (Figure 5A). A SEM photograph of a granule after 25 h of degradation showed that the surface possessed small pores and had an irregular structure (Figure 5B). The same granule but after 135 h of degradation is shown in Figure 5C. The granules displayed a deformed shape, and some oily substances were left behind. A similar profile of granule degradation was observed for gelatin-freegranules. Again, the presence of dye in the granule matrix and the method of granule preparation had no effect on the degradation profile €or the granules, irrespective of gelatin incorporation. Release Characteristics of Gelatin-Free P(FAD-SA) Granules-Characteristic release curves for the gelatin-free disks and granules prepared by the W/O emulsion method are shown in Figure 6. A 0 was released from all devices without any large initial burst. For pure surface erosion, we expect release rates to depend on granule size, while for pure bulk erosion, no dependence on size should exist. In Figure 6, one can see the influence of granule size on the release rate: the smaller granules showed the faster release. The same rule applied for polymer erosion (Figure 3), a result suggesting that pure bulk erosion did not occur in this system. There was a good correlation between A 0 release and SA release. In addition, this was true for granules loaded with p-NA (data not shown). The result suggests that diffusional escape of dye from the granule matrix is minimal and that release is controlled by matrix degradation caused by polymer surface erosion. Release Characteristics of Gelatin-Loaded P(FAD-SA) Granules-Characteristic release curves for 4 % gelatin-loaded granules prepared from P(FAD-SA) with different molecular weights are shown in Figure 7A. A 0 was released from the injectable granules without any large initial burst. However, no effect of polymer molecular weight on A 0 release was observed. Figure7B showstheeffectof loading on A 0 releasefromgranules. A 0 loading had no effect on the release profile over the range of dye loading studied. Figure 7C shows A 0 release from various polyanhydride granules with 4% gelatin loading. The rate of release from the granules decreased with increasing amounts of FAD in the copolymer. This result may be due to the more hydrophobic nature of the FAD monomer. The profile of release of A 0 and p-NA from granules with loadings of 2 % dye and 4 % gelatin are shown in Figure 8. Both dyes were released at a nearly constant rate, without any large initial burst, and no difference in the release profiles was observed for the two dyes. Figure 9 shows profiles of release of A 0 and SA from P(FADSA) granules prepared with or without gelatin. For the gelatinfree granules, the A 0 release pattern cloeely matched that of polymer degradation, and both processes were almost complete within 90 h. This result suggests a release mechanism that is dominantlydegradation controlled. However,the incorporation of gelatin in thegranules permitted the prolonged release of AO, 8 1 Journal of Pharmaceutical Sciences vol. 83, No. 1, January 1994
Figure I-SEM of ~(FAD-SA)granules at different degradation stages: immediately after preparation (A), after 25 h (B),after 135 h (c)in 0.1 M phosphate buffer at 37 'C.
I
120 1
0 0
4 7
0
50
'1
150
100
Time (hr)
200
Figure 8-Release of A 0 from the gelatin-free disk (0)and granules of different sires in 0.1 M phosphate buffer at 37 O C ; granules had dlameters of <150 pm (0),>150 but <425 pm (O),>600 but <840 pm (A),and >840 but <1500 pm (A).P(FADSA) was used at 25:75, had a molecular weight of 42 900, and contained 2% A 0 loading.
0
200
400
Flgure 8-Release of various dyes from P(FAD-SA) (2575; molecular weight, 42 900) granules with dlameters of < 150 pm In 0.1 M phosphate AO; (0)p-NA. buffer at 37 O C . Key: (0)
I
" I
0 0 Y . 0 200 1
0
200
.
I
'
1
.
I
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Tlme (hr) Flgure9-Profiles of releaseof A 0 (circles)and SA (triangles)from P(FAD SA) (2575) granules with diameters of <150 pm In 0.1 M phosphate buffer at 37 O C . Key: (0 and A)4% gelatin-loaded granules; (0 and A) gelatin-free granules. P(FAPSA) molecular weight. 42 900 A 0 loading, 2%.
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Time (hr) Flgure 7-A) Release of A 0 (2% loading) from granules of P(FADSA) (25:75) with different molecular weights (<150 pm in diameter) in 0.1 M phosphate buffer at 37 O C ; molecular weights were 12 300 (0),29 000 (O), 42 900 (A). (6)Release of A 0 from P(FAPSA) (2575) granules (<150 pm in diameter) with different A 0 loadings in 0.1 M phosphate buffer at 37 O C ; A 0 loadings were 1% (0),2% (O),and 4% (A). P(FADSA) molecular weight, 42 900. Gelatin loading, 4 % (C) Release of A 0 (2% loading) from granules of various polyanhydrkfes in 0.1 M phosphate buffer at 37 O C . Key: (0)PSA; (0)V F A P S A ) (8:92); (A) P(FADSA) (2575); (A)P(FADSA) (4456). Qranules had a diameter of <150 pm.
Flgure 10-A) Release of A 0 (2% loadlng) (0) and gelatin (0)from 4% gelatin-loaded granules wlth diameters of <150 pm in 0.1 M pho@-ate buffer at 37 OC. (B)Release of A 0 (2% loading) (0) and gelailn (0)from 8 qC, gelatin-loadedgranules wlthdlametersof <150pm in 0.1 M phosphate buffer at 37 O C . P(FADSA) molecular weight, 42 900.
althoughthe granules exhibited an SA release profile similar to that of gelatin-free granules. A 0 release from P(FAD-SA) granules (25:75) containing gelatin in differentamounta is shownFigure 10. A 0 was released from the granules faster than gelatin, irrespective of the amount
of gelatin incorporated in the granule matrix. The period of A 0 release was significantly prolonged by increasing the amounta of gelatin in the granule matrix. Althoughgelatinreleaselagged behind A 0 release, the correlationbetween the two profiles was clear.
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JounRl of m m w l sdences / 9 Vol. 83, No. 1, January 1984
for gelatin, AO, and FAD monomers over a temperature range of -25-150 "C (data not shown). Degradation studies demonstrated that after 135 h in buffer, the granules were completely degraded to remnants composed of FAD monomers and gelatin in addition of AO. These DSC data suggest that a crystalline structure is formed from the remnants produced by granule degradation.
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Temperature ( O C ) Figure 11-DSC thermograms of P(FAD-SA) (2575) granules with diameters of < 150 pm at different degradation stages: immediatelyafter preparation (A), after 27 h (B) and after 135 h (C) for 4% gelatin-loaded granules and immediately after preparation (D) after 27 h (E), after 135 h (F) for gelatin-free granules in 0.1 M phosphate buffer at 37 OC. A 0 loading, 2 YO. The physical state of P(FAD-SA)granules during degradation was characterized by DSC measurements of the granules (Figure 11). P(FAD-SA) granules displayed a sharp endotherm around 60 "C before degradation, irrespective of the incorporation of gelatin in the granule matrix. This endotherm corresponds to the melting of the crystalline regions of the polymers in the granule matrix. The heat of fusion values were 13.45 and 13.10 cal/g for gelatin-free granules and 4 %-loaded granules, respectively. These values are quite similar to the heat of fusion value for the origianal polymer, 13.27 cal/g, a result indicating that the crystalline state of the polymer remains unchanged when A 0 and gelatin are loaded into the granule matrix. The endotherm became broader and the heat of fusion for the granules decreased with degradation time for both types of granules. However, a new, sharp endotherm at 120 "C was observed 135 h after degradation of the granules with 4 % gelatin loading, in contrast to gelatin-freegranules. In addition, no endotherm was observed 10 /Journal of Pharmaceutical Sciences Vol. 83, No. 1, January 1994
The present study demonstrates the differences in release profiles for water-soluble drugs in polyanhydride devices prepared by the W/O emulsion method and the usual compression method. When the two types of disk were compared, no significant difference in release profiles was observed, although the disk prepared by the latter method exhibited somewhat faster release than that prepared by the former method (Figure 1A). For devices of a smaller size (granules), however, the release profiles were quite different; the granules obtained by the compression method tended to release A 0 faster, with an initial burst, than those prepared by the W/O emulsion method (Figure 1B). A similar result was obtained for granules with diameters of <150 pm prepared from different polyanhydrides and loaded with different dyes. The difference in release pattern may be explained in terms of the distribution of the dye throughout the polymer matrix. Light microscopic observations of P(FAD-SA) disks demonstrated that the dye was dispersed much more homogeneouslyin the disk obtained by the W/O emulsion method than in that obtained by the compression method (Figure 2). The heterogeneous distribution of the dye and the large size of the dye islands throughout the polymer matrix did not lead to a significant initial burst in dye release from polyanhydride devices of a large size, such as a disk. For granules of an injectable size (<150 pm), however, the uniformity of dye distribution and the size of the dye islands became important contributing factors in the regulation of dye release profiles. It is possible that the dye islands located close to the surface of granules obtained by the compression method were responsible for a burst effect. In contrast, granules prepared by the W/O emulsion method showed a nearly constant release without any large initial burst (Figure 1B). The dispersion homogeneity must depend on the preparation method, because water-soluble drugs are poorly miscible with hydrophobic polyanhydrides. This W/O emulsion method is more effective in preparing polyanhydride-drug formulations with a highly uniform drug distribution than the compression method. The W/O method permitted the preparation of polyanhydride granules that had diameters of <150 pm and that could achieve a nearly constant release of water-soluble drugs. The preparation procedure was reproducible with respect to release profiles and yield; the yield was very high, -95%. Water-soluble A 0 was released at a nearly constant rate without any large initial burst, and the release profile was determined by the type of matrix polymer. Polyanhydrides possess a water-labile linkage. Thus, we had to try to minimize the contact time between the polymer and water to delay polymer degradation. However, the contact time in the granule preparation was very short, and a GPC study demonstrated no loss of polymer molecular weight before or after granule preparation (Figure 4). In addition, the granule preparation could be carried out at a low temperature, a fact that may be important in preventing the inactivation of some drugs. Polyanhydride erosion takes place because of the hydrolysis of water-labile anhydride linkages. The complete loss of SA after a short period (-90 h) indicated that water had completely penetrated the polymer matrix. Then, dissolution and/or diffusion through the polymer matrix of the degradation products, at least those connected with water-soluble SA,
occurred after the complete degradation of anhydride bonds. GPC and SEM studies showed that after 135 h in buffer, the remnants were oily substances soluble in chloroform, and their molecular weights were similar to those of FAD monomers (Figures4 and 5). Again, no difference in the degradation profile of the granule matrix itself was observed between granules prepared by the W/O emulsion method and those prepared by the compression method when judged on the basis of SA release and polymer molecular weight. In addition, the complete disappearance of anhydride bonds was observed near this time, as evaluated by IR spectroscopy (data not shown). Similar degradation profiles were observed for P(FAD-SA) microspheres.15 At 135h, complete cleavage of anhydride bonds in P(FAD-SA) took place and left oily, pooly water-soluble FAD monomers behind, irrespective of the type of devices. The incorporationof gelatin in the granule matrix was effective in prolonging the release period for water-soluble drugs. The profile of release of SA for granules containing gelatin was quite similar to that for gelatin-free granules (Figure 9). GPC and SEM studies demonstrated that no difference in the degration profile of the granule matrix itself was observed between gelatinloaded granules and gelatin-free granules (Figures 4 and 5). The DSC study of the granules at different degradation stages showed that after 135 h in buffer, a sharp endotherm at -120 "C was observed for granules containing gelatin but not for gelatin-free granules (Figure 11). Similar results were obtained for granules containingdyes other than AO. In addition, the same endotherm pattern could be seen when A 0 was mixed with FAD monomers, irrespective of the amount of A 0 (data not shown). At this stage of degradation, gelatin and FAD monomers in addition to dye remained in the granule matrix. A similar gelatin effect on the profile of release of water-soluble drugs has been found for P(FAD-SA) microspheres.15 These results demonstrated the formation of a crystalline structure between gelatin and FAD monomers produced via the degradation of P(FAD-SA). Thus, it is possible that an interaction between FAD monomers and gelatin molecules causes the continued release of drugs even after the polymer matrix has been completely degraded. The crystalline structure formed may function as a reservior for watersolubledrugs and result in a prolonged release period, irrespective of device type.
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References and Notes 1. Sidman, K. R.; Schwope, A. D.; Steber, W. D.; Rudolph, S.E.; Poulin, S. B. J. Membr. Sci. 1990, 7, 227-291. 2. Lewis, D. H. In BiodegradablePolymers as Drug Delivery Systems; Chasin, M.; Langer, R., Eds.; Dekker: New York, 1990; pp 1-42. 3. Chasin,M.; Domb,A.; Ron, E.; Mathiowitz,E.; Leong,K.; Laurencin, C.; Brem, H.; Grossman, S.; Langer, R. In BiodegradablePolymers as Drug Delivery Systems; Chasin, M.; Langer, R., Eds.; Dekker: New York, 1990; pp 43-70. 4. Pitt, C. G. In Biodegradable Polymers as Drug Delivery Systems; Chasin, M.; Langer, R., Ed.; Dekker: New York, 1990; pp 71-120. 5. Heller, J.; S arer, R. V.; Zentner, G. M. In BiodegradablePolymers as Drug Degvery Systems; Chasin, M.; Langer, R., Eds.; Dekker: New York, 1990; pp 121-162. 6. Allcock, H. R. In Biodegradable Polymers as Drug Delivery Systems; Chasin, M.; Langer, R., Eds.; Dekker: New York, 1990; DD 163-194. 7. Kohn, J. In Biodegradable Polymers as Drug Delivery Systems; Chasin, M.; Langer, R., Eds.; Dekker: New York, 1990; pp 195230. 8. Bogdansky, S. In Biodegradable Polymers as Drug Delivery Systems; Chasin, M.; Langer, R., Eds.; Dekker: New York, 1990,
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9. Leong, K. W.; Brott, B. C.; Langer, R. J.Biomed. Mater. Res. 1985, 19,941-955. 10. Mathiowitz, E.; Langer, R. J. Controlled Release 1987, 5, 13-22. 11. Bindschaedler, C.; Leong, K.; Mathiowitz, E.; Langer, R. J. Pharm. Sci. 1988, 77, 696-698. 12. Mathiowitz, E.; Saltzman, W. E.; Domb, A.; Dor, P.; Langer, R. J. A d . Polvm. Sci. 1988. 35. 755-774. 13. Mkhiowhz, E.; Amato,'C.;'Dor, P.; Langer, R. Polymer 1990,31, 547-555. 14. Mathiowitz, E.; Bernstein, H.; Giannos, S.; Dor, P.; Turek, T.; Langer, R. J. Appl. Polym. Sci. 1992,45, 125-134. 15. Tabata, T.; Langer, R. Pharm. Res. 1993,10, 391-399. 16. Tamada, J.; Langer, R. J. Biomater. Sci. Polym. Ed. 1992,3,315353. 17. Brem, H.; Mahaley, M. S., Jr.; Vick, N. A.; Black, K. L.; Schold, S. C., Jr.; Burger, P. C.; Friedman, A. H.; Ciric, J. S.; Eller, T. W.; Cozzens, J. F.; Kenealy, J. N. J. Neurosurg. 1991, 74, 441-446. 18. Langer, R. J. Controlled Release 1991, 16, 53-60. 19. Mathiowitz, E.; Ron, E.; Mathiowitz, G.; Amato, C.; Langer, R. Macromolecules 1990,23, 3212-3218.
Acknowledgments This study was supported by NIH grant lUOlCA48508. We thank Dr. Janet Tamada for assistance and helpful discussions.
Journal of Pharmaceutical Sciences / 11 Vol. 83,No. 1, January 1994