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Stimulatory Effect of a Sulfonylurea Analogue and Its Polymer Conjugate on Insulin Secretion from Rat Islets Akihiko KikuchiJ You Han Bae,*and Sung Wan Kim Department of Pharmaceutics and Pharmaceutical Chemistry, Center for Controlled Chemical Delivery, University of Utah, 421 Wakara Way, Suite 318, Salt Lake City, Utah 84108
A sulfonylurea with a polymerizable end group (a glyburide analogue) was synthesized. This monomer was copolymerized with NJV-dimethylacrylamide (DMAAm).In vitro bioactivity was evaluated by adding these sulfonylurea monomers and copolymers into normal rat islet cultures and measuring the insulin concentration in the supernatant. The sulfonylurea monomer showed a stimulatory effect on rat islets at glucose concentrations of 50 and 100 mg/dL, with equivalent stimulation indices for glyburide, the potent sulfonylurea drug, while no effect was observed at a glucose concentration of 200 mg/dL. The sulfonylurea polymer stimulated Langerhans islets to secrete insulin at a low glucose concentration of 50 mg/dL, with 80% bioactivity of its monomeric compound. However, at 100 and 200 mg/dL glucose concentrations, there was no significant stimulation in insulin secretion. Because it is suggested that the sulfonylurea receptors are located in the lipid phase on the ATP-sensitive K+ channels, the solubilization of sulfonylurea into, and its diffusion through, the lipid phase in the cell membrane may affect the interactions of sulfonylurea with its receptors. In the case of the sulfonylurea polymer, the sulfonylurea unit in the polymer can interact with its receptor as can be seen by the increased insulin secretion at a glucose concentration of 50 mg/dL, but its interaction would be interfered with to some extent by a large hydrophilic polymer backbone. Thus, less change in insulin secretion was observed at higher glucose concentrations by the addition of this particular sulfonylurea polymer.
Introduction Many efforts have been devoted to developing a biohybrid artificial pancreas for the treatment of insulindependent diabetes mellitus (IDDMl) [for reviews, see Colton and Avgoustiniatos (1991) and Reach (1990, 199211. Islet macroencapsulation into a chamber with vascular grafts (Maki et al., 1991; Sullivan et al., 1991) and microencapsulation of islets within permselective membranes [for example, Lim and Sun (1980), O’Shea et al. (1984), and Sun et al. (198l)l have been major approaches to the development of a biohybrid artificial pancreas. Until now, there appeared to be several unsolved problems with developing a bioartifical pancreas, such as the biocompatibility of immunoprotecting membranes, the necrosis or malhnction of the implanted islets due to local hypoxia and reduced nutrient supply, which is caused by the lack of a microvascular system around the islets, a large implant volume, and removal of implant or reseeding islets. Assuming that one will be able to replenish implanted islets after a certain implant period as research advances, it would be ben-
* Author to whom correspondence should be addressed. Current address: Institute of Biomedical Engineering, Tokyo Women’s Medical College, 8-1 Kawadacho, Shinjuku-ku, Tokyo 162, Japan. Abbreviations: DCC, 1,3-dicyclohexylcarbodiimide;DMAAm,
NJV-dimethylacrylamide;Su-OH,N-hydroxysuccinimide;DMSO, dimethyl sulfoxide; DMF, NJV-dimethylformamide;DCU, 1,3-dicyclohexylurea; BPO, tert-butylperoxy octanoate; PBS, phosphate-buffered saline solution; HBSS; Hanks’ balanced salt solution; HEPES, N-(2-hydroxyethyl)piperazine-N ’-2ethanesulfonic acid; FBS, fetal bovine serum; IDDM, insulindependent diabetes mellitus, NIDDM, non-insulin-dependent diabetes mellitus.
eficial if one could reduce the number of implant islets by maximizing the insulin secretion function of the islets because of limited islet resources, low isolation yields, and preservation problems, as well as implant recipient comfort. It was reported that the minimal requirement of islets implanted for the correction of hyperglycemia is more than 5000 isletskg of body weight (human islet size equivalent), indicating that a large number (i.e,, volume) of islets would be required for a biohybrid artificial pancreas (Warnock and Rajotte, 1988). Sulfonylureas have been used for the treatment of noninsulin-dependent diabetes mellitus (NIDDM) for decades (Groop, 1992). An NIDDM patient is characterized by a low response in insulin secretion toward increased blood glucose levels. Sulfonylureas directly interact with the P-cells of Langerhans islets which results in increased insulin secretion (Gorus et al., 1988). Sulfonylureas interact with sulfonylurea receptors, which exist on the ATP-sensitive K+ channel on the pancreatic P-cell membrane surface (Hellman et al., 1971; Schmid-Antomarchi et al., 1987; Henquin, 1990; Sturgess et al., 1985; Schwanstecher et al., 1992a-e; Boyd, 1988; Gaines et al., 1988; Misler et al., 1989; Panten et al., 1989, 1992; Ziinkler et al., 1989). This interaction inhibits K+ efflux, causing membrane depolarization followed by an increase in Ca2+influx through voltage-dependent Ca2+channels (Boyd, 1988; Cook et al., 1984; Henquin, 1990; Nelson et al., 1987; Panten et al., 1992). The increase in cytoplasmic Ca2+concentration eventually triggers insulin secretion from pancreatic islets (Boyd, 1988; Henquin, 1990; Nelson et al., 1987; Turk et al., 1993). Since sulfonylureas react with cell surface membrane receptors (Hellman et al., 1971; Schwanstecher et al., 1992a,e), these
8756-7938/94/3010-0630$04.50/0 0 1994 American Chemical Society and American Institute of Chemical Engineers
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bCH,
Figure 1. Structural formula of glyburide.
may not necessarily be internalized into cells for insulin secretion (Hellman et al., 1971; Schwanstecher et al., 1992a). Sulfonylureas taken orally may, although this is not confirmed, interact with implanted islets for enhanced insulin secretion. It would be more beneficial for the implant recipient to avoid taking oral sulfonylureas. Sulfonylureas covalently attached to soluble polymer chains may interact with islets and enhance insulin secretion from the islets when these conjugates are entrapped with islets within the immunoprotective membranes, thus residing inside the membranes until the whole content is replaced. It is assumed that this may reduce the required number of islets for glucose homeostasis. In addition, this conjugate concept may offer more means for detailed investigations of drug unit interactions with corresponding receptor cells. Pioneering work on sulfonylurea conjugation to water soluble polymers was performed by Obereigner et al. (1979). They synthesized several types of polymerizable derivatives from carbutamide and then copolymerized them with (2-hydroxypropyl) methacrylate. These polymers exhibited the lowering effect of blood glucose level by 70% of basal glucose concentration in vivo after administration of polymer solution (20 m&g body weight corresponding to sulfonylurea unit) to rat intravenously. Although they showed the reduced blood glucose level in vivo, there is no detailed information on enhanced insulin secretion from Langerhans islets. Furthermore, carbutamide is a less potent sulfonylurea drug compared to the second-generation sulfonylurea drug, glyburide (see Figure 1 for its structure), which has a 2 orders of magnitude higher potency (70 pgkg in humans) than carbutamide. The primary aims of this study are to synthesize a sulfonylurea compound (a glyburide analogue) with a polymerizable functional group, since glyburide has no functional group, for conjugation to a polymer chain and to test the bioactivities of the compound and its copolymers with Nfl-dimethylmethacrylamide.Nfl-Dimethylmethacrylamide, one of the most hydrophilic monomers, has been selected for the increased water solubility of sulfonylurea copolymers.
Materials and Methods Materials. Terephthalic acid, 442-aminoethy1)benzenesulfonamide, N-hydroxysuccinimide, 1,3-dicyclohexylcarbodiimide, cyclohexyl isocyanate, allylamine, Nfldimethylacrylamide, and glybenzcyclamide (glyburide) were purchased from Aldrich Chemical Co. (Milwaukee, WI). Hanks' balanced salts, RPMI 1640 powder medium, sodium pyruvate, L-glutamine, penicillin-streptomycin solution (10000 IU/mL penicillin and 10 000 pglmL streptomycin), amphotericin B, collagenase type XI, Ficoll-DL400, and N42-hydroxyethy1)piperazine-N'Sethanesulfonic acid (HEPES) were obtained from Sigma Chemical Co. (St. Louis, MO). Fetal bovine serum (FBS, No. 11152102)was purchased from Hyclone Laboratories, Inc. (Logan, UT). Islet culture medium was RPMI 1640 supplemented with 11.1 mM glucose (unless otherwise noted), 23.8 mM sodium bicarbonate, 2 mM sodium pyruvate, 2 mM L-glutamine, 6 mM HEPES, antibiotics (100 IU/mL
631
penicillin, 100 pg/mL streptomycin, and 2.5 pg/mL amphotericin B),and 10% FBS. Methods. I, Syntheeie of Sulfonylurea Compound- 4~[2-(4-Carboxyphenyl)amidoethyllbenzenesulfonamide (XI) (Atherton and Sheppard, 1989;Bodanszky and Bodanszky, 1984;Jones, 1992). nYenty millimoles of terephthalic acid (I) and 20 mmol of N-hydroxysuccinimide (Su-OH) were dissolved in 40 mL of dimethyl sulfoxide (DMSO) a t room temperature. To this solution was added 20 mmol of 1,3-dicyclohexylcarbodiimide (DCC) solution in DMSO (20 mL), and the mixture was stirred to form the active ester of I for 1 h at 20-23 "C. Then, 20 mmol of 4-(2-aminoethyl)benzenesulfonamide (11) in DMSO solution (20 mL) was added and stirred for overnight to prepare III. The dicyclohexylurea (DCU) formed was removed by filtration, and the filtrate was poured dropwise into an excess amount of distilled water. The precipitate was collected by filtration and then dissolved in 400 mL of dilute NaOH (25 mmol) aqueous solution. The insoluble part was removed by filtration, and the filtrate was acidified by adding 4 M HC1 dropwise while stirring. The precipitate was recovered by filtration and dried under vacuum (yield, 95%). N-[4-[2-[(4-Carboxyphenyl)amido/ethyl]phenyl]sulfonyllN'-cyclohexylurea (V)(Takla, 1981). Ten millimoles of I11 was mixed with 40 mL of acetone and 10 mL of 2 mol/dm3 NaOH aqueous solution, Then, approximately 40 mL of water was added to this mixture while stirring. The solution was cooled on an ice-water bath to 0-5 "C, and cyclohexyl isocyanate (IV,12 mmol) was added dropwise to this solution. After 3 h of stirring at 0-5 "C, the reaction mixture was diluted with 20 mL of methanol and 60 mL of distilled water followed by filtration. The filtrate was acidified with 4 M HC1. Precipitate (V) was collected by filtration and washed with distilled water, followed by thorough drying under vacuum (yield, 71.6%). 'H NMR (DMSO-dd: 13.2 ppm, HOOC; 10.4 ppm, S02NECONH; 8.7 ppm, CON-HCH2CH2; 8.1-7.2 ppm, phenylene; 6.4 ppm, S02NHCONB; 3.6 ppm, CONHCH2C&; 3.2 ppm, C1 proton of cyclohexyl; 2.9 ppm, CONHCbCH2; 1.8-1.0 ppm, cyclohexyl. N-[[4-[2-[[4-(Allylcarbamido)phenyllamidolethyllphenyllsulfonyll-N-cyclohexylurea (VII). To 7 mmol of V and 7 mmol of Su-OH in DMSO solution (25 mL) was added DCC (7 mmol) in DMSO solution (15 mL), and the mixture was stirred for 1.5h at 22 "C to form the active ester of V. Allylamine (7 mmol) was then added to this solution, which was allowed to stir for 1 day at 22 "C. DCU formed was removed by filtration, and the filtrate was poured into an excess amount of distilled water to precipitate the product MI). The precipitate was collected by filtration, followed by dissolution into NaOH (10 mmol) aqueous solution. After removal of the insoluble part, the filtrate was acidified with HC1 aqueous solution. The product was filtered and washed with distilled water, followed by vacuum drying (yield, 70.8%). IH NMR (DMSO-dd: 10.4ppm, S02NHCONH; 8.8 ppm, allyl-NBCO, CONBCH2CH2; 8.1-7.1 ppm, phenylene; 6.4 ppm, S0,NHCONB; 5.9 ppm, CH2=CHCH2; 5.1 ppm, C13,=CHCH2; 3.9 ppm, CHz=CHCH,; 3.6 ppm, CONHCCB,; 3.2 ppm, C1 proton of cyclohexyl; 2.9 ppm, CONHCI12CH2;1.8-1.0 ppm, cyclohexyl. Copolymerization of Sulfonylurea Monomer (VIn with N,N-Dimethylacrylamide (DMAAm). Predetermined amounts of DMAAm and sulfonylurea monomer M I ) were dissolved in DMSO at a monomer concentration of 10% (w/v). tert-Butylperoxy octanoate (BPO, Polysciences, Inc., Warrington, PA) was used as an initiator (10 mmol/dm3). After 15 min of bubbling dried N2 gas,
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Table 1. Copolymerization of Sulfonylurea Monomer VI1 with N,"Dimethylacrylamide (DMAAm) copolymer feed composition VI1 feed ratio composition DMAAm VI1 mol wt yield mol wt run (mmol) (mmol) o/o % (%) % % 1 23.97 0.24 1.0 5.0 81 1.2 5.8 2 23.38 1.2 5.9 0.36 1.5 7.3 93 2.5 11.7 93 2.1 10.0 3 22.27 0.57 4 19.82 5.0 21.4 88 4.5 19.6 1.04
polymerization proceeded a t 80 "C for 1h with vigorous stirring. Polymers were precipitated in an excess amount of diethyl ether to separate them from unreacted chemicals and dried under vacuum. The yield of the copolymers ranged from 81% to 93%. The amount of sulfonylurea unit in the copolymer was determined from lH NMR spectra using DMSO-& as a solvent. The results of copolymerization are summarized in Table 1. II. In Vitro Bioactivity of Sulfonylurea Compounds. Langerhans islets were obtained from the pancreases of Sprague-Dawley male rats (Sasco, Omaha, NB, 250-300 g body weight) by a collagenase digestion technique (Lacy and Kostianovsky, 1967), followed by density gradient centrifugation (Linda11et al., 1969).Two hundred to four hundred islets per rat pancreas were collected for each isolation. The islets were cultured in RPMI 1640 medium supplemented with 10% FBS and antibiotics at 37 "C in a humidified atmosphere of 7% COz. The day after islet isolation, round-shaped islets were hand-picked under a microscope and subcultured in RPMI 1640 supplemented with 10% FBS. The subcultured islets were suspended in three different RPMI 1640 media with glucose concentrations of 50, 100, and 200 mg/dL, respectively, without serum and then placed into a 24-well tissue culture plate (Falcon 3047, Becton and Dickinson, Lincoln Park, NJ) at an islet concentration of 40-50 islets/mL in each well. Sulfonylsulfonylurea copolymer (sulfonylurea monomer (MI), urea content, 19.6 wt %), and glyburide (commercially available second-generation compound) were dissolved in DMSO at a definite concentration of sulfonylurea. Ten microliters of these solutions were added to each well and incubated in a humidified atmosphere at 37 "C for 2 h. The final DMSO concentration in the medium was less than 1%(v/v), and at this concentration, no apparent DMSO toxicity to islets was observed by islet viability. Before and after incubation, the number of islets was counted. Islet viability after 2 h of incubation was above 90%. The islet suspension was spun down, and then supernatant was collected and kept frozen until insulin radio-immunoassay was performed. Scheme 1. Synthesis of Sulfonylurea Compounds
W N C ) IV
V
WOOC 1 \
NaOH, Acetone, 0-5 "C, 3 h
Insulin concentration in the supernatant was determined by INSULIN RIA kit (ICN Biomedicals, Costa Mesa, CAI. Data were expressed as a mean with the standard error of the mean (SEM) (pIUlisletl2 h). The stimulation index (SI) is defined as the ratio of insulin secreted from rat islets in the presence of sulfonylurea compounds to that in the absence of sulfonylurea compounds at each experimental condition:
SI = (insulin secreted with sulfonylurea)/ (insulin secreted without sulfonylurea)
Results and Discussion Synthesis of Sulfonylurea Compounds. A sulfonylurea compound with a polymerizable vinyl group was synthesized following Scheme 1. This compound has a structure similar to that of glyburide, which is one of the most effective second-generation drugs used for the treatment of non-insulin-dependent diabetes mellitus (NIDDM). The structure of this monomer was confirmed by lH NMR according to the assignment of glyburide (Takla, 1981). The sulfonylurea monomer (vn)was then copolymerized with a hydrophilic monomer, DMAAm in DMSO, by using a radical initiator. Copolymer compositions were determined by 'H NMR. In the following in vitro experiments, the sulfonylurea copolymer with a sulfonylurea content of 19.6 wt % was used. Solubility of the Sulfonylurea Monomer and Copolymer. The solubility of glyburide in aqueous milieu was limited because of its high hydrophobicity. The solubility of this drug was increased in alkaline aqueous solution due to the formation of salts (4 pg/mL a t pH 4 and 600 pg/mL at pH 9) (AHFS, 1993; Takla, 1981). Due to structural similarity, the solubility of the sulfonylurea monomer (VII) was close to that of glyburide. MI was soluble in DMSO and alkaline aqueous solutions (pH > 10) and partly soluble in DMF and dioxane. The solubility of VI1 in phosphate-buffered saline (isotonic PBS, pH 7.4) was 25 pg/mL. Copolymers of the sulfonylurea monomer with DMAAm were also soluble in alkaline aqueous solution (pH > 101, THF, DMF, and DMSO. Copolymers were soluble in PBS (isotonic pH 7.4) at 50 pg/mL based on the amount of sulfonylurea monomer unit. Although the solubility of sulfonylurea compounds in aqueous media is increased after copolymerization with the hydrophilic comonomer, DMAAm, the extent of the solubility increase in aqueous media is lower than expected. This may be due to the high hydrophobicity of the sulfonylurea moiety, where two benzene rings and one cyclohexyl ring exist. Bioactivity of Sulfonylurea Compounds in Vitro. Since glyburide has no functional groups in its structure
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633 Table 2. Effects of Glyburide, Sulfonylurea Monomer
and Sulfonylurea Polymer on Insulin Secretion from 3W, Rat Ialeta
la
glucose concentration (mg/dL)
50 control (without
C
1 -
3Q)
-
8
C
6,
sulfonylurea)(n = 2-5) glyburide (n = 2) 1 Pcgl" 10 &mL sulfonylurea monomer VI1 (n = 3) 1 PcglmL 10 pg/mL sulfonylurea in copolymer (run 4)(n = 5) 1 pg/mLb 10 pg/mLb 50 pglmLb
1.0
100 1.0
200 1.0
1.13 f 0.07 1.43 f 0.18 1.21f 0.02 1.30 f.0.12 1.42f 0.05 1.13f 0.22 1.35f 0.29 1.50f 0.19 1.14 f 0.14 1.65 f.0.48 1.61 f 0.15 1.09f 0.10 1.04 f 0.05 0.99f 0.06 0.94f 0.04 1.44f 0.22 1.12 f 0.03 0.97f 0.02 1.27f 0.11 1.07 f 0.08 0.89 f 0.05
Data are presented as mean f.SEM. * Sulfonylurea concentrations were calculated based on sulfonylurea unit (VII)in the copolymer.
for coupling to a polymer, a sulfonylurea monomer (VI11 with a structure similar to that of glyburide was synthesized. To evaluate the bioactivity of the sulfonylurea monomer as an islet stimulant, a sulfonylurea monomer solution in DMSO was added to the islet culture and incubated for 2 h at 37 "C. The glucose concentration of the culture medium was varied from 50 to 200 mg/dL. Figure 2 shows the amount of insulin secreted from rat islets stimulated by VI1 plotted against glucose concentration. As expected, insulin secretion from Langerhans islets increased with an increasing glucose concentration of the medium in the absence of sulfonylurea monomer. At low glucose concentrations, i.e., 50 and 100 mg/dL, the addition of VII to islet culture resulted in the increase in insulin secretion from islets compared to the control. This demonstrates the same trend as the results obtained when glyburide was added to islet culture (Table 2). At these glucose concentrations, insulin secretion from sulfonylurea-stimulated islets was significant compared to insulin secretion from unstimulated islets. However, the difference in insulin secretion was not significant at a glucose concentration of 200 mg/dL, regardless of the sulfonylurea Concentration. It has been reported that the difference between insulin secretion from normal islets in the absence and the presence of sulfonylureas was relatively smaller at high glucose concentrations than the difference at low glucose concentrations (Kiekens et al., 1992; Pipeleers, 1992; Schuit et al., 1988). Glucose itself is the stimulant for Langerhans islets to secrete insulin, and thus, there was little effect of sulfonylurea a t 200 mg/dL glucose. It is obvious from the results, as summarized in Table 2, that rat islets were stimulated to secrete insulin with VI1 at 50 and 100 mg/dL glucose concentrations. These values are equivalent to or greater than the glyburide effect. No significant difference in the stimulation index is seen at a glucose concentration of 200 mg/dL, regardless of the sulfonylurea concentration. At glucose concentrations of 50 and 100 mg/dL, the amount of insulin secreted from islets was increased by the addition of a small amount of a sulfonylurea compound (1pg/mL, ca. 2 p M ) . At a higher concentration (20 p M ) of either glyburide or VII, the stimulation effect was not improved. Consequently, the newly synthesized
sulfonylurea monomer is concluded to be active in stimulating Langerhans islets insulin secretion in uitro, and this result suggests that a sulfonylurea monomer incorporated into a polymer may have a stimulatory effect on Langerhans islets. VI1 is then incorporated into polymers by radical copolymerization with DMAAm. The bioactivity of this sulfonylurea polymer was explored by adding the copolymer to islet culture. The sulfonylurea concentration was calculated by the monomer unit in the copolymer (i.e., 19.6 w t % in the copolymer used). The glucose-dependent insulin secretion activity of the islets in the presence of the copolymer was normal. Insulin secretion was increased by the addition of the sulfonylurea copolymer a t a glucose concentration of 50 mg/dL. At a glucose concentration of 50 mg/dL and a sulfonylurea concentration of 10 pg/mL, the sulfonylurea copolymer exhibited approximately 80% bioactivity of VII. At glucose concentrations of 100 and 200 mg/dL, however, no significant effects of the sulfonylurea copolymer were observed. In other words, the effect of sulfonylurea in the polymer chain is considered to be lower when compared to VI1 a t the same sulfonylurea concentration. It is considered that the stimulatory effect of sulfonylurea on insulin secretion from Langerhans islets was concealed by the stimulatory effect of glucose at higher glucose concentrations. When sulfonylurea is added to islet cell culture, it is partitioned into the lipid phase of the cellular membrane or the proteins because of their high hydrophobicities (Groop, 1992; Panten et al., 1992; Pearson, 1985; Takla, 1981). It is suggested that sulfonylurea receptors on the ATP-sensitive K+ channels are located in the lipid phase of the cellular membrane (Schwanstecher et al., 1992a; Zunkler et al., 19891, and thus sulfonylureas are likely to access their receptors in the lipid phase of the P-cell membrane via lateral diffusion. Taking into account this consideration, the sulfonylurea moiety in the copolymer can be partitioned into the cell membrane lipid phase, but it is difficult to access the sulfonylurea receptors because they are conjugated to a relatively hydrophilic polymer backbone. The polymer backbone may interfere with the lateral diffusion of sulfonylurea within the p-cell membrane to its receptors on the ATP-sensitive K+ channels, which may lead to reduced binding of sulfonylurea to its receptor. In addition, it is plausible that the
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penetration of sulfonylurea polymer within islets is also limited due to its large size in a hydrated state, resulting in the reduced contact with /3-cells inside of Langerhans islets. Therefore, the stimulatory effect of the sulfonylurea copolymer cannot be seen at glucose concentrations of 100 and 200 mg1dL. This deficit may be improved by conjugating the sulfonylurea moiety to the polymer via a spacer.
Conclusion A sulfonylurea with a polymerizable end group (a glyburide analogue) and its copolymers with N,N-dimethylmethacrylate were synthesized. An in vitro bioactivity test demonstrated that the sulfonylurea monomer has a stimulatory effect on rat islets equivalent to that of glyburide. The sulfonylurea polymer stimulated Langerhans islets to secrete insulin a t a low glucose concentration of 50 mgIdL, with 80% bioactivity of its monomeric compound, and showed no significant stimulation at higher glucose concentrations (100 and 200 mgl dL). The sulfonylurea unit in the polymer can interact with its receptor, as can be seen by the increased insulin secretion at a low glucose concentration, but its interaction would be interfered with to some extent by a large hydrophilic polymer backbone. Although further molecular design of the sulfonylurea conjugate for enhanced interactions is necessary, it is proved that the synthesized monomer and its polymer conjugate have bioactivity to islets. This approach may find potential application to a bioartificial pancreas and as a tool for investigating drug-cell surface receptor interactions. Studies on long-term biocompatibility and a more detailed investigation for islet stimulation of the copolymers under various conditions are in progress.
Acknowledgment We are grateful to Dr. Moon Kyu Lee for his technical advice on rat islet isolation and cultivation. We also acknowledge Dr. Harvey A. Jacobs for his valuable discussions and comments throughout this work. This work was supported by NIH Grant DK 46458-01.
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