Polymeric Drugs and Drug Administration - American Chemical Society

Chapter 4

Protein Device for Glucose-Sensitive Release of Insulin Y. Ito and Y. Imanishi

Downloaded by IOWA STATE UNIV on February 12, 2017 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/bk-1994-0545.ch004

Department of Polymer Chemistry, Kyoto University, Kyoto 606-01, Japan

A glucose-sensitive protein device for insulin release was synthesized by coupling insulin to glucose oxidase via a disulfide bond. Glucose oxidation with glucose oxidase produces an electron that cleaves the disulfide bond. Several sophisticated devices are discussed.

The glucose-sensitive insulin-releasing systems that have been developed for the treatment of diabetes include encapsulation of viable pancreatic cells, electromechanical systems (using a glucose sensor and an insulin injection pump), and chemical systems. There are two major types of the chemical systems being used (1). One is a signal exchange system, such as a mixture of Concanavalin A and glycosylated insulin, or a mixture of boron- and diol-containing polymers. These chemical combinations utilize the specific interactions between lectin and glucose, or between boron and diol, respectively. The other system is a signal transduction which uses glucose-sensitive enzymes. The system transduces the glucose signal to other physicochemical signals, such as p H or redox. The enzymes are coupled to stimuli-responsive chemical systems. We have designed and synthesized p H sensitive and redox-sensitive membrane systems. The former is a porous cellulose membrane on which poly(acrylic acid) and glucose oxidase ( G O D ) were immobilized (2). When glucose is present, G O D catalyzes the conversion of glucose to gluconic acid, thus leading to a lower p H . The decrease in p H results in conformational modifications in the graft polymer chain which, in turn, alters the size of the membrane pores (3,4). The redox sensitive system is a composite membrane on which insulin is immobilized via a disulfide linkage. When glucose is oxidized by glucose dehydrogenase ( G D H ) in solution, the disulfide bonds are cleaved and the insulin is released (5,6). Since G D H has no redox site, the coupling with F A D and N A D enhances the sensitivity. In this paper a protein device was developed, in which G O D was directly coupled with insulin through a disulfide bond. The principal mechanism for the

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Ottenbrite; Polymeric Drugs and Drug Administration ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

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Figure 1. The principle of glucose-sensitive insulin-releasing protein device. release of insulin is shown in Figure 1. When G O D was used, the enzyme had two active sites containing F A D . Therefore, an S-S bond was incorporated directly onto the protein. According to Degani and Heller (7), although the active site is isolated from the protein surface, the electron transfer from the sites to the surface can be performed by coupling ferrocene derivatives in the presence of urea. In this study, the reversibly-cleavable S-S bonds are incorporated into G O D instead of the ferrocene derivatives. Experimental Materials. The glucose oxidase enzyme ( G O D ) from Aspergillus niger (G-8135) and insulin (1-5500) were purchased from Sigma Co. S^'-Dithiobis^-nitrobenzoic acid) (DTNB) and l-ethyl-3-(3-dimethylaniinopropyl)carbodiiniide hydrochloride, which is a water soluble carbodiimide (WSC), were purchased from Tokyo Kasei (Tokyo, Japan). Preparation of Protein Devices. The protein device was synthesized according to the scheme shown in Figure 2. The insulin carboxylic groups were blocked by a methyl ester to prevent inter- and intramolecular cross-linking reactions of proteins in the WSC-activated reaction between insulin and D T N B , or the DTNB-insulin and G O D . The protected insulin was purified by G P C (Sephadex G-15). The D T N B was activated with WSC (10 wt%) and reacted with the insulin methyl ester at 4°C for 20 h. The reaction product was purified on the same G P C column. The insulin methyl ester modified with D T N B (DTNB-insulin) was reactivated with WSC (10 wt%) and reacted with G O D , which was pretreated with urea (2M) at 0°C for 2 h. The reaction product was purified by G P C to collect the insulin/GOD hybrid. Insulin Release Assay. After the aqueous saccharide solution was added to the insulin-GOD hybrid solution, a portion of solution was removed for analysis. The amount of the insulin released was determined by the 276 nm absorption intensity of the solution eluted from a reverse-phase column packed with a Biofine R P C - P O (JASCO, Tokyo, Japan). A calibration curve based on the known amounts of native insulin was used in the insulin release issay.


4. ΠΌ & IMANISHI

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Figure 2. Scheme for the preparation of the protein device. Biological Activity Assay of the Released Insulin. The biological activity of the released insulin was determined by measuring the amount of glucose taken up by the adipocytes. Albumin-containing Krebs-Ringer bicarbonate buffer solution (1 mL) was added to an adipocyte culture (mouse origin). The mixture was incubated for 2 h in the presence of glucose/ C-glucose and insulin derivatives. The culture was terminated by adding sulfuric acid. Fat containing C-glucose, which was taken up by the cells, was extracted with a toluene scintillation solution. To assess the relative biological activity of the insulin derivative, the amount of C was determined by the scintillation counting and compared with that for the incubation with the native insulin. 14

14

1 4

Results The elemental analysis of the insulin product after the D T N B reaction (molar ratio 1/5) yielded 3.36% sulfur, indicating that two or three amino groups of the insulin molecule were used in the reaction. Summarized in Table 1 are the results for the reaction of DTNB-insulin and G O D . The increasing feed concentration of DTNB-insulin in the reaction with G O D resulted in the increase of DTNB-insulin incorporated into GOD.


49

50


Table 1. The Amount of Insulin Coupled with Glucose Oxidase


Molar r a t i o of DTNB-insulin/ GOD i n f e e d *

*

Content of S i n protein h y b r i d (%)

GOD a m i n o g r o u p s used f o r DTNB-insulin c o u p l i n g (mol%)

10

/

1

3.39

38

1

/

1

2.27

20

1

/

10

1 .67

1 1

D T N B - i n s u l i n was p r e p a r e d i n 5/1 m o l a r r a t i o .

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insulin

Insulin Release. When glucose was added to an aqueous solution containing the protein device, the H P L C pattern showed a new peak which was not distinguish able from native insulin, as shown in Figure 3. This result indicates that there was no disulfide bond cleavage in the insulin. As shown in Figure 4, sugars other than glucose did not induce the insulin release from the insulin/GOD hybrid, indicating that the insulin/GOD hybrid has glucose specificity. O n addition of a reducing agent, 2-mercaptoethanol, insulin was released in a manner similar to that for the glucose addition. This suggests that the release mechanism is based on the reduction cleavage of the disulfide bond between the insulin and the G O D . Shown in Figure 5 is that insulin was repeatedly released on further addition of glucose. The protein hybrid with a higher amount of the incorporated insulin released more insulin for the same amount of the glucose added. The amount of the insulin released per glucose molecule ranged from 0.12 to 0.28 χ 10" . The efficiency was higher for the insulin hybrid with the higher insulin content. 2

Biological Activity of the Released Insulin. The biological activity of insulin derivatives is demonstrated in Figure 6. The activity of the insulin methyl ester and DTNB-insulin are about 95% and 70% of the native insulin activity, respectively. The activity of the insulin released was about 80% of that for the native insulin. The H P L C pattern of the released insulin indicates no reduction cleavage of the disulfide bond in the insulin molecule. Discussion We have synthesized a prototype protein device which quickly releases insulin in response to glucose. However, the practical application of the device requires the


4. ΓΓΟ & IMANISHI

Protein Device for Glucose-Sensitive Release of Insulin

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Time (min) Figure 4. Insulin release from the protein device in the presence of 2-mercaptoethanol ( A ) , maltose ( Q ) , galactose ( Ο ) · A l l concentrations are 15 m M .


51

52



120

Time

(min)

Figure 5. Insulin release from various protein devices in response to 15 m M glucose at 37°C. Protein hybrid synthesized with a molar ratio of DTNB-insul i n / G O D in feed: 10/1 ( Q ) , 1/1 ( · ) , 1/10 ( Δ ) .

consideration of two other problems. The one is the immunogenicity of the device. The other is the lack of a need-response function for the device. The former can be overcome by encapsulation using semipermeable membranes, which are permeable to insulin but not to any immunoglobulin or covalent bonded bioinert polymers, such as poly(ethylene glycol). In the latter case, the device releases insulin in an almost linear manner in response to the glucose concentration, as shown in Figure 7. However, in the body insulin is only required at high levels of glucose in the blood, while at low glucose levels insulin release can cause serious problems. Normally, a healthy pancreas controls these conditions precisely. In general, the intelligent materials used under similar biological conditions should have three functions: sensing, processing, and reacting. However, the processing function, which is usually performed by microcomputers in an electromechanical artificial pancreas, has not been included in the chemical systems. We, therefore, have introduced a processing control function into this system. To reduce the insulin release at the low glucose level in the blood, an inhibitor was incorporated in the protein device, as shown in Figure 7. We believe that this approach will result in more sophisticated artificial pancreas designs.


4.

53 Protein Device for Glucose-Sensitive Release of Insulin

ITO&IMANISHI

Insulin

Relative biological activity

derivatives

0.5 Downloaded by IOWA STATE UNIV on February 12, 2017 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/bk-1994-0545.ch004

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Figure 6. Biological activity of native, esterified, DTNB-modified, and released insulin from the protein hybrid in response to 15 m M glucose added. The activity of native insulin is taken as the standard.



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Figure 7. Scheme of the design for sophisticated glucose-sensitive insulin-releasing system.

Literature Cited 1. Ito, Y. In Synthesis of Biocomposite Material: Chemical and Biological Modifications of Natural Polymers. Imanishi, Y., Ed., CRC Press, Boca Raton, 1992, 137-180. 2. Ito, Y., Casolaro, M., Kono., K., Imanishi, Y.J.Controlled Release 1989, 10, 195203. 3. Ito, Y., Kotera, S., Inaba, M., Kono., K., Imanishi, Y. Polymer 1990, 31, 21572161. 4. Ito, Y., Inaba, M., Chung, D.J., Imanishi, Y. Macromolecules 1992, 25, 7313-7316. 5. Ito, Y., Chung, D.J., Imanishi, Y. Artif. Organs 1990, 14, 234-236. 6. Chung, D.J., Ito, Y., Imanishi, Y. J. Controlled Release 1992, 18, 45-54. 7. Degani, Y., Heller, A.J.Am. Chem. Soc. 1988, 110, 2615-2620. RECEIVED September 30, 1993


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