Synthesis and Biological Properties of Insulin−Deoxycholic Acid

Bioconjugate Chem. 2005, 16, 615−620

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Synthesis and Biological Properties of Insulin-Deoxycholic Acid Chemical Conjugates Seulki Lee, Kwangmeyung Kim, Tadiparthi Suresh Kumar, Jisook Lee, Sang Kyoon Kim, Dong Yun Lee, Yong-kyu Lee, and Youngro Byun* Department of Materials Science and Engineering, Gwangju Institute of Science and Technology, 1 Oryong-dong, Buk-gu, Gwangju, 500-712, Korea. Received June 2, 2004; Revised Manuscript Received March 23, 2005

Bile acids have been considered very useful in the preparation of new pharmaceuticals, and more recently in the preparation of peptide and protein drugs because of their natural chemical and biological properties. In this study, we modified recombinant human insulin by covalently attaching deoxycholic acid (DOCA) derivatives in order to synthesize orally active insulin analogues. DOCA derivatives, namely succinimido deoxycholate and succinimido bisdeoxycholyl-L-lysine were prepared and site specifically conjugated at LysB29 of insulin. The resultant insulin conjugates, [NB29-deoxycholyl] insulin (Ins-DOCA) and [NB29-bisdeoxycholyl-L-lysil] insulin (Ins-bisDOCA), were studied for their chemical, structural, and biological properties. Their chemical properties were determined by HPLC, MALDITOF mass spectroscopy, and dynamic light scattering. Lipophilicity and self-aggregation behavior of insulin conjugates were enhanced with increasing number of labeled bile acid. The far-ultraviolet region of circular dichroism spectra showed no significant change of the tertiary structure of insulin in aqueous solution due to conjugation. Competitive insulin binding assay with HepG2 cells revealed that monosubstituted insulin conjugates still retained high binding affinity to the insulin receptor. When the insulin conjugates were intravenously administered (0.33 IU/kg) to streptozotocin (STZ)induced diabetic rats, the conjugates showed sustained biological activity for a longer period with the similar lowest blood glucose level (glucose nadir), compared to native insulin. In further studies, the resulting new insulin conjugates will be investigated for their oral efficiency as a long-acting insulin formulation for the treatment of diabetic patients.

INTRODUCTION

Insulin is the primary hormone utilized for treatment of virtually all types of diabetic patients. Insulin can be administered only by parenteral injection; however, conventional injectable insulin replacement therapy has several therapeutic risk factors, such as poor compliance and unsatisfactory metabolic regulation in diabetic patients (1, 2). Many attempts have therefore been made to enhance therapeutic performance of insulin using various delivery systems (3-5). However, these alternative delivery methods of insulin still have many problems mainly due to natural characteristics of insulin such as physicochemical instability, increased susceptibility to proteolysis, solubility, large molecular weight, and limited ability to traverse biological barriers (6, 7). To overcome these limitations, several kinds of chemical modifications of insulin have been proposed. For instance, lipid modification of insulin could improve solubility and showed extended-action via binding to an existing serum protein (8-10). In addition, poly(ethylene glycol), 9-fluorenylmethoxycarbonyl, transferrin, polysialic acid, and poly(ethylene glycol)-alkyl linker chemically conjugated insulin derivatives showed improved therapeutic profiles, such as solubility, enzymatic stability, circulation half-life, or permeability against mucosal membrane (11-16). * To whom correspondence should be addressed: Youngro Byun, Ph.D., Department of Materials Science and Engineering, Gwangju Institute of Science and Technology, 1 Oryong-dong, Buk-gu, Gwangju, 500-712, Korea. Phone, 82-62-970-2312; fax, 82-62-970-2354; e-mail, [email protected].

Among all the different alternative route of insulin delivery, peroral formulation is the most preferable for its significantly pronounced therapeutic and patient acceptability. In our previous study, deoxycholic acid (DOCA)-conjugated heparin was highly absorbed in the intestine without damaging the tissue structure of the mucous membrane (17, 18). More recently, we have prepared a new oral delivery carrier based on bile acid which improves the oral bioavailability of human insulin (19). Based on substantiated observations that associated bile acids could enhance the absorption of poorly permeable macromolecules in the intestine, we have designed site-specific DOCA-conjugated insulin analogues. DOCA is one of the bile acids which are natural compounds consisting of a facially amphiphilic steroid nucleus with a hydrophobic β-side and a hydrophilic R-side. They are synthesized from cholesterol in the liver, released in the small intestine, and returned to the liver by absorption through the bile acid transporter (20, 21). Bile acids have been considered in the preparation of new drugs because bile acid-conjugated materials can retain the properties of bile acids, such as amphiphilicity, capacity of selfassembling, high chemical stability, and binding ability to bile acid transporters in the intestine (22, 23). Taken together, this spectrum of properties gives the therapeutic peptide a unique pharmacological profile that is attractive for developing new orally active formulations. In this respect, we hypothesized that covalent coupling of bile acid to a specific site of insulin may improve the therapeutic profiles of insulin while maintaining its biological activity. We synthesized, in this study, site specifically labeled insulin-DOCA conjugates and char-

10.1021/bc049871e CCC: $30.25 © 2005 American Chemical Society Published on Web 04/19/2005

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acterized their biological activity in vitro and in vivo, respectively. EXPERIMENTAL PROCEDURES

Materials. Human crystalline zinc insulin (Zn2+ insulin) was purchased from Serologicals Corp. (Norcross, GA) and used without further purification. (3-[125I]IodotyrosylA14)-insulin (human recombinant) was obtained from Amersham Biosciences Corp. (Piscataway, NJ). Deoxycholic acid (DOCA), L-lysine ethyl ester, N,N′-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), tetrahydrofuran (THF), anhydrous N,N-dimethylformamide (DMF), triethylamine (TEA), ethyl acetate (EtOAc), absolute ethanol (EtOH), ammonium bicarbonate (NH4HCO3), magnesium sulfate anhydrous (MgSO4), and streptozotocin (STZ) were purchased from Sigma-Aldrich Co. (St. Louis, MO). Before use, THF was distilled from sodium benzophenone. All reagents and organic solvents used were at least ACS grade. Preparation of DOCA Derivatives. Succinimido Deoxycholate. DOCA (200 mg, 0.5 mmol) and NHS (76 mg, 0.67 mmol) were dissolved in anhydrous THF (20 mL). DCC (136 mg, 0.67 mmol) was added and stirred at 4 °C for 6 h. After urea derivatives were filtered, the filtrate was poured into cold n-hexane (120 mL) and the precipitate was dried in a vacuum. The synthesized succinimido deoxycholate was obtained as white powder. 1 H NMR, 350 MHz, CDCl3: 0.59 (s, 3H, 18-Me); 0.86 (s, 3H, 19-Me); 0.91 (d, 3H, 21-Me); 1.03-2.50 (25H, steroidal H); 2.81 (s, 4H, CH2CH2); 3.78 (m, 1H, 3β-H); 4.06 (br s, 1H, 12β-H). MALDI-MS: calcd MW, 489.6; found, 490. Succinimido Bisdeoxycholyl-L-lysine. A suspension of L-lysine ethyl ester HCl (50 mg, 0.24 mmol) in anhydrous DMF (15 mL) containing TEA (140 µL, 1 mmol) was stirred at room temperature for 30 min, followed by addition of DOCA (190 mg, 0.48 mmol) and NHS (71 mg, 0.62 mmol). DCC (129 mg, 0.62 mmol), dissolved in anhydrous DMF (5 mL), was added dropwise to the mixture and then stirred at room temperature for 12 h. The mixture was filtered, and the filtrate was diluted with EtOAc (40 mL). The organic mixture was successively washed with 15 mL of 0.5 N HCl, distilled water, 0.5 N NaOH, and distilled water. The dried products were dissolved in EtOH (10 mL) containing saturated NaOH and allowed reaction followed by refluxing on a stream bath at 60 °C for 6 h. After filtration at room temperature, the filtrate was added dropwise to a cold acid solution (pH 3). The precipitates were filtered, washed, and dried in a vacuum. Finally, bisdeoxycholyl-L-lysine was obtained as white powder. The carboxylic group of bisdeoxycholyl-L-lysine was activated by NHS as described above to obtain succinimido bisdeoxycholyl-Llysine. 1H NMR, 350 MHz, CDCl3-CD3OD: 0.58 (s, 6H, 2 × 18-Me); 0.84 (s, 6H, 2 × 19-Me); 0.92 (d, 6H, 2 × 21-Me); 1.05-2.40 (56H, steroidal 50H, 3 × CH2); 2.75 (s, 4H, CH2CH2); 2.98 (m, 2H, CH2-NHCO); 3.61 (m, 2H, 2 × 3β-H); 4.16 (br s, 1H, 2 × 12β-H); 4.47 (s, 1H, CH-NHCO); 7.73 (br s, 1H, CH2-NHCO); 7.96 (br s, 1H, CH-NHCO). MALDI-MS: calcd MW, 992.4; found, 991. Preparation of Insulin Conjugates. [NB29-Deoxycholyl] Insulin. This conjugate was synthesized according to a modified method used for the acylation of insulin with fatty acid (9). Briefly, Zn2+ insulin (100 mg, 17 µmol) was dissolved in DMF/H2O (6 mL, 3/2 v/v, pH 10) at room temperature. Succinimido deoxycholate (12.6 mg, 25.8 µmol) was dissolved in anhydrous DMF (1 mL) and

Lee et al.

added to the insulin solution. Apparent pH was adjusted constantly by the addition of 1 N NaOH. After 30 min, the reaction was quenched by addition of distilled water, and the pH was adjusted to 2 with 1 N HCl. The reaction mixture was immediately filtered, dialyzed against 0.01% of NH4HCO3, and lyophilized. The lyophilized sample was further purified by preparative reversed-phase HPLC (Shimadzu, Tokyo, Japan) with a Shim-pack Prep-ODS column (20 mm × 250 mm). The mobile phase consisted of 0.1% TFA in distilled water (eluent A) and acetonitrile containing 0.1% TFA (eluent B). The mobile phase was run with a linear gradient from 35 to 45% eluent B for 15 min at a flow rate of 10 mL/min, and the UV absorbance of the eluent was monitored at 280 nm. [NB29-Bisdeoxycholyl-L-lysil] Insulin. Zn2+ insulin (100 mg, 17 µmol) and succinimido bisdeoxycholyl-Llysine (25.7 mg, 25.8 µmol) were treated and purified as described above. The mobile phase of preparative reversedphase HPLC was run with a linear gradient from 45 to 55% eluent B for 15 min at a flow rate of 10 mL/min. Characterization. MALDI-TOF Mass Spectroscopy. Molecular weight was obtained from MALDI-TOF mass spectrometry using a Voyager Biospectrometry Workstation (PerSeptive Biosystem, Framingham, MA). Samples were prepared by mixing 2 µL of aliquot with 2 µL of the matrix solution, a saturated solution of R-CHCA in 50% of water/acetonitrile with 0.3% TFA. One microliter of the sample mixture was spotted into a well of the sample plate and dried by vacuum prior to mass spectrometry. Data for 2 ns pulses of the 337 nm nitrogen laser were averaged for each spectrum in a linear mode, and positive ion TOF detection was performed using an accelerating voltage of 25 kV. Structural Verification. Insulin and the conjugates were incubated with trypsin (1 mg/mL) at 37 °C for 2 h. This treatment was expected to cleave the Arg-Gly bond in the β-chain of insulin. After incubation, the mixture was acidified with 0.1% TFA to quench the reaction and the mass of resulting proteolytic fragment was determined by using MALDI-TOF mass spectrometry. Circular Dichroism. All samples were prepared in 10 mmol HCl at concentrations of 0.1 mmol and filtered through a 0.2 µm syringe filter. A Jasco J-720 spectropolarimeter (Tokyo, Japan) was used, and three to five scans were averaged before the final spectrum was acquired. The data (ellipticity in mdeg) was then transformed to mean residue ellipticity (θm) using the expression, θm ) (θM)/(Cl), where θ is the observed ellipticity (mdeg), M is the mean residue molecular weight (g/mol), C is the protein concentration (g/mL), and l is the optical path length (cm). Dynamic Light Scattering. The characteristics of selfaggregates of insulin analogues in PBS (pH 7.4) were investigated by dynamic light scattering at a wavelength of 488 nm with an Ar ion laser (Cyonics, San Jose, CA). The scattered light was measured at an angle of 90° and was collected with Multi-8 correlator (Malvern Instruments Ltd., U.K.). The hydrodynamic radius was analyzed by using the PCS software provided by the same company. Competitive Insulin Receptor-Binding Assay. The biological activities of the insulin conjugates were determined using a competitive receptor-binding assay at steady state (24). HepG2 human hepatoma cell line (ATCC, Rockville, MD) were cultured at 37 °C in a 5% CO2-humidified atmosphere in culture flasks with Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum, 2 mM glutamine, 50 µg/mL streptomycin, and 50 U/mL penicillin. For binding stud-

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Insulin−DOCA Conjugates

ies, cells were seeded on 24-well plates at a density of 4 × 105 cells/well. Cells were used after 3 days when confluence was reached. Cells were washed twice with cell-binding media (DMEM containing 20 mM Hepes and 0.1% human albumin, pH 7.4), and competitive binding of unlabeled insulin and conjugates at steady state was measured by 2 h incubation at 4 °C with 1 mL binding media containing 25 pM [125I]-insulin as the radio ligand in the presence of various concentrations of unlabeled insulin and the conjugates. Incubation was performed at 4 °C to inhibit endocytosis of receptor-bound insulin and the conjugates. After incubation, cells were washed twice with cold PBS, extracted with 1 mL of 1 N NaOH, and an aliquot of 100 µL was transferred into a glass tube for γ-radiation counting. All assays were made in triplicate. Nonspecific binding was determined in the presence of 10-6 M unlabeled insulin. The results were analyzed according to the four-parameter logistic model, and the affinity of the each conjugate was expressed relative to that of native insulin. The concentration giving half-maximal inhibition of receptor-bound tracer (IC50) was also estimated. In Vivo Hypoglycemic Effect. We followed the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals (NIH publication 85-23 Rev. 1985). After an initial 3-day acclimation period, male Sprague-Dawley rats weighing from 230 to 250 g were fasted for 12 h before inducing diabetes mellitus. Diabetes was induced by a single intraperitoneal injection of STZ (in citrate buffer, pH 4.5) at 70 mg/kg. Five days after the STZ treatment, the test solutions (insulin, 0.33 IU/kg, 0.4 mL/kg and the equivalent amount of the conjugates) were administered intravenously into the tail vain. Blood samples for the analysis of blood glucose were taken from the tail veins and determined immediately on fresh samples by using a one touch blood glucose monitoring system (Glucocard II, Arkray, Kyoto, Japan). Binding of Insulin Conjugates to Serum Albumin. Binding of insulin conjugates to serum albumin was assessed using size exclusion chromatography (7.8 mm × 300 mm Ultrahydrogel 250, Waters). Elution profiles were obtained for insulin and the conjugates (both 5 µmol) in the absence and presence of bovine serum albumin (BSA; 25 µmol; Sigma). The mobile phase was run with distilled water for 20 min at a flow rate of 0.5 mL/min, and the UV absorbance of the eluent was monitored at 280 nm. Statistical Analysis. Data are expressed as mean ( SE. Student’s t-test was performed, and a value of p < 0.05 was considered to be statistically significant. RESULTS AND DISCUSSION

Synthesis and Characterization of Insulin Conjugates. The succinimidyl ester of two kinds of DOCA derivatives, succinimido deoxycholate and succinimido bisdeoxycholyl-L-lysine, were synthesized and conjugated to the -amino group of LysB29 insulin. Since the pKa’s of two N-terminal R-amino groups of insulin are reported as 8.4 and 7.1 for GlyA1 and PheB1, respectively, and above 9.8 for -amino group of LysB29, insulin could be selectively labeled to LysB29 in a base condition (9, 11, 25). Conjugates were prepared in a basic (pH 10) 60% DMF solution, where the amino groups of LysB29 are most reactive. The conjugate was obtained with above 99% purity (Figure 2) with above 45% product yield. The molecular weight of conjugates analyzed by MALDI-TOF mass

Figure 1. Chemical structures of succinimido deoxycholate 1 and succinimido bisdeoxycholyl-L-lysine 2 and insulin conjugates. R′ ) deoxycholyl and bisdeoxycholyl-L-lysil, respectively.

Figure 2. Reversed-phase HPLC chromatograms (detection at 280 nm): (A) human insulin, (B) [NB29-deoxycholyl] insulin, and (C) [NB29-bisdeoxycholyl-L-lysil] insulin. Table 1. HPLC Retention Times (the Last Three Columns) and Molecular Mass of Insulin and Their Conjugate Fragments after Trypsin Protease Digestion of the HPLC-Purified Peaka proteolytic fragment

insulin standard

Ins-DOCA

A(1-21)-B(1-22) 22.0 min 21.96 min (4865.5/4864.5) (4865.5/4864.8) B(23-29) 19.39 min (859.0/863.4) B(23-30) 31.1 min (1334.7/1336.5)

Ins-bisDOCA 21.96 min (4865.5/4865.0)

40.95 min (1836.8/1837.9)

a Parentheses indicate mass; calculated molecular weight/ measured molecular weight.

spectroscopy (calculated molecular weight/measured molecular weight; for insulin, Ins-DOCA and Ins-bisDOCA were 5807/5805.2, 6181.6/6176.3 and 6679.4/6679.7, respectively) clearly indicated that both conjugates were purely monosubstituted. Native insulin and purified insulin conjugates were subjected to the trypsin digestion to determine the positional substitution of DOCA derivatives. It was expected that proteolytic fragment with a DOCA derivative attached to it should result in a shift in retention time compared to the respective unlabeled fragment. By peptide mapping using HPLC and MALDI-TOF, it was confirmed that both bile acid derivatives were labeled at LysB29 of insulin (Table 1). Retention time in HPLC profiles of insulin, Ins-DOCA, and Ins-bisDOCA was 22.87, 28.13, and 33.83 min, respectively. Calculated lipophilic index (lipophilic index ) log((tR - t0)/t0); t0, retention time of solvent) of insulin,

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Figure 3. Far-UV-CD spectra of Zn2+ insulin (O), Ins-DOCA (b), and Ins-bisDOCA (1). Solutions were prepared in 10 mmol HCl to a protein concentration of 0.1 mmol.

Ins-DOCA, and Ins-bisDOCA was 0.701, 0.806, and 0.898, respectively. The lipophilic indices of conjugates were higher than that of native insulin, indicating that these derivatives were more lipophilic compared to native insulin. In addition, the lipophilicity of the conjugates increased with increasing number of DOCA molecules that were chemically attached. Circular dichroism (CD) spectroscopy has been applied to evaluate the conformational change of insulin conjugates (Figure 3). Because insulin conjugates showed higher self-aggregation behavior mainly due to the labeled bulky hydrophobic DOCA derivatives, insulin conjugate samples for CD analysis were prepared in acid condition to maintain a monomeric form and clear solution. The far-UV-CD band at 208 nm primarily arises from R-helix structure, and 223 nm is for component of the β-structure. The ratio between both bands ([φ]208/[φ]223] can be used to inform a qualitative measure of overall conformation structure of insulin and conjugates. The [φ]208/[φ]223 ratio value for insulin, Ins-DOCA, and Ins-bisDOCA was 1.32, 1.49, and 1.38, respectively. The spectral characteristics of the conjugates indicated that conjugation of DOCA derivatives to insulin does not alter the overall tertiary structure of native peptide. Competitive Insulin Receptor-Binding Assay. Human hepatoma cell line HepG2 has been chosen as a model for the in vitro studies of insulin-receptor binding of insulin conjugates. The HepG2 cells maintain the function of differentiated liver cells, and the relative binding affinities for the insulin receptor in HepG2 cells are consistent with the relative metabolic potencies shown by fat cells (26, 27). The competition curves of insulin or the conjugates to insulin receptors on intact HepG2 cells are shown in Figure 4. The absolute mean value of IC50 for the insulin receptor was 1.28 × 10-9 M for insulin, 3.37 × 10-9 M for Ins-DOCA, and 2.24 × 10-9 M for Ins-bisDOCA. Therefore, monosubstituted insulin conjugates still retained high binding affinity to the insulin receptor. In Vivo Hypoglycemic Effect. The pharmacodynamic activity of insulin conjugates was evaluated in STZ-induced diabetic rats. Figure 5 illustrates the blood glucose level following intravenous injection of insulin and the conjugates. Reduction in glucose level for insulin, Ins-DOCA, and Ins-bisDOCA was 46.1 ( 7.9%, 41.1 ( 8.7%, 39.0 ( 11.1%, respectively. Compared to native insulin, insulin conjugates did not show any differences in glucose nadir. Although the glucose nadir was similar

Lee et al.

Figure 4. Inhibition of 125I-insulin binding to HepG2 cells by native insulin and synthetic insulin conjugates. Insulin and conjugates were incubated with HepG2 cells as described under Experimental Procedures. Zn2+ insulin (b), Ins-DOCA (O), and Ins-bisDOCA (1). Results are mean ( SE (n ) 3).

Figure 5. Blood glucose response following intravenous administration of insulin (0.33 IU/kg, b, n ) 5) and an equivalent amount of Ins-DOCA (O, n ) 8) and Ins-bisDOCA (1, n ) 8) prepared in 10 mmol PBS. Samples were injected into the tail vein of STZ-induced diabetic rats. Results are mean ( SE.

to that of native insulin, the conjugates induced a slower rate of the blood glucose levels for Ins-DOCA and InsbisDOCA, were slowly reduced, and were maintained at low level for about 6 h, while the blood glucose level of native insulin was rapidly decreased after insulin injection and increased again. Therefore, the duration of the blood glucose lowering effect of the conjugates seemed to be sustained compared to native insulin. To evaluate whether sustained biological action of the conjugates is responsible for existing serum protein (e.g., albumin) binding ability derived by conjugated DOCA, the binding affinity of insulin conjugates to serum albumin was evaluated using a size exclusion chromatography. Figure 6 illustrates the elution profiles of BSA, human insulin, and the conjugates in the absence (Figure 6A) and presence (Figure 6B) of BSA. It was hypothesized that, if insulin conjugates could bind to the BSA, the elution position of the conjugates would be identical to that of BSA. As shown in Figure 6, the elution position of insulin and the conjugates changed slightly; however, it was nearly unaffected by the presence of BSA. This result confirmed that the insulin conjugates did not bind

Insulin−DOCA Conjugates

Figure 6. Size exclusion chromatography assessment of binding of insulin and insulin conjugates to bovine serum albumin (BSA). (A) Insulin and insulin conjugates (5 µmol), run separately, and (B) insulin and insulin conjugates (5 µmol) + BSA (25 µmol), run separately.

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inactive. However, when the aggregate form slowly gets dissociated due to dilution in the blood stream, the conjugate’s biological activity would gradually appear. Therefore, different glucodynamic profiles of insulin conjugates in vivo could be explained by the aggregation property of the insulin conjugates. In conclusion, we have successfully conjugated two different kinds of DOCA derivatives to LysB29 of insulin. The lipophilicity and self-aggregation behavior of insulin were enhanced by increasing the number of conjugated bile acid. The overall tertiary structure of insulin was preserved after conjugation, and it still retained high binding strength to the insulin receptor despite the fact that a bulky group of bile acid derivatives was attached to it. The insulin conjugates showed a gradual biological response, increased duration of action, and increased residence time compared to native insulin due to the high order of aggregation properties. Beyond the development of orally active insulin formulation based on bile acid acylation techniques, our concern was to develop site-specific bile acid-conjugated insulin analogues which can maintain their biological activity while bearing the properties of bile acid. To this end, we have designed and prepared biologically active DOCA-conjugated insulin analogues. Furthermore, we have found that chemically bound bile acid could prolong the biological activity of native insulin in the physiological condition. The potential of our DOCA-insulin conjugates for oral delivery is under evaluation using in vivo models, and we anticipate that our new insulin conjugates would be applied to enhance the clinical efficiency of the present treatment for diabetic patients. ACKNOWLEDGMENT

This study was supported by National Research Laboratory (NRL) Project from the Ministry of Science and Technology in Korea. Figure 7. Size distribution profiles of insulin and conjugates at various concentrations determined by dynamic light scattering. Solutions were prepared in 10 mmol PBS at 25 °C for insulin (b), Ins-DOCA (O), and Ins-bisDOCA (1).

Supporting Information Available: The HPLC and mass spectrometry data of insulin and their conjugates after trypsin protease digestion. This material is available free of charge via the Internet at http://pubs.acs.org.

to serum albumin, and the sustained action of the conjugate was not related to the binding affinity with albumin. On the other hand, the sustained biological action of the insulin conjugates could be explained by the selfassociation profile of the conjugates. We introduced bulky hydrophobic region to the surface of the peptide. Labeled DOCA derivatives have high capacity of self-assembling (by combining an amphiphilic steroid nucleus with the reactivity of the side groups of DOCA), and these could increase hydrophobic aggregation behavior above a certain concentration. Consequently, different physical states of insulin in solution might impact its pharmacological profile. In view of the degree of aggregation of insulin conjugates in PBS, DLS measurement was selected to characterize self-association properties. As illustrated in Figure 7, insulin conjugates showed higher aggregation behavior compared to insulin and possessed mean hydrodynamic diameter of 130 and 240 nm for Ins-DOCA and Ins-bisDOCA, while native insulin showed negligible size distribution. The tendency of aggregation was reversible and decreased by dilution. As the mole ratio of the conjugated DOCA molecules to insulin was increased, the aggregate size of insulin conjugates increased. Self-aggregated states correspond to unusual micelle-like structures that result biologically

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