A novel drug delivery system for type 1 diabetes: Insulin-mimetic vanadyl-poly(γ-glutamic acid) complex

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Inorganic Biochemistry Journal of Inorganic Biochemistry 100 (2006) 1535–1546 www.elsevier.com/locate/jinorgbio

A novel drug delivery system for type 1 diabetes: Insulin-mimetic vanadyl-poly(c-glutamic acid) complex Subarna Karmaker, Tapan K. Saha, Yutaka Yoshikawa, Hiroyuki Yasui, Hiromu Sakurai

*

Department of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan Received 29 December 2005; received in revised form 12 May 2006; accepted 12 May 2006 Available online 24 May 2006

Abstract Insulin-mimetic vanadyl-poly(c-glutamic acid) complex, VO-c-PGA, is proposed as a novel drug delivery system for treating type 1 diabetic animals. The structure of VO-c-PGA in solution as well as in solid state was analyzed by electronic absorption, infra-red, and electron spin resonance spectra, and proposed that the equatorial coordination mode of VO2+ is in either carboxylate(O)–VO– (OH2)3 or 2 carboxylate(O2)–VO–(OH2)2. In vitro insulin-mimetic activity, metallokinetic feature in the blood of healthy rats, and in vivo normoglycemic effect of the complex prepared in solution were evaluated in streptozotocin(STZ)-induced type 1 diabetic mice, and these effects were compared with those of a solution containing only VOSO4 as a positive control. The in vitro insulin-mimetic activity of VO-c-PGA was examined by determining both inhibition of free fatty acid (FFA) release and glucose uptake in isolated rat adipocytes, in which the concentration of VO-c-PGA for 50% inhibition of FFA release was significantly lower than that of VOSO4. Metallokinetic study suggested that the bioavailability of VO-c-PGA complex was much higher than that of VOSO4. The complex showed a significant hypoglycemic activity within at least 4 h after a single oral administration, the effect being sustained for at least 24 h. Furthermore, VO-c-PGA normalized the hyperglycemia in STZ-mice within 3 days when it was given orally at doses of 5–10 mg V kg1 body mass for 16 days. The improvement in diabetes was also supported by the results on oral glucose tolerance test, HbA1c levels, and blood pressure.  2006 Elsevier Inc. All rights reserved. Keywords: Vanadyl-poly(c-glutamic acid) complex; Drug delivery system; Diabetes; Hyperglycemia; Pharmacokinetics

1. Introduction Diabetes mellitus (DM) is a serious disease characterized by abnormally high blood sugar levels. The two main types of DM are insulin dependent type 1 DM and non-insulin dependent type 2 DM. Type 1 DM usually manifests in childhood or adolescence, and the patients require exogenous insulin because of the destruction of insulin-producing b-cells in the pancreas by autoimmune reaction [1]. The prevalence of type 2 DM begins to rise in early middle age and increases along with age. Exogenous insulin is not always a necessity for these patients because insulin pro*

Corresponding author. Tel.: +81 75 595 4629; fax: +81 75 595 4753. E-mail address: [email protected] (H. Sakurai).

0162-0134/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2006.05.005

duction is frequently high compared to that of type 1 DM. Currently, type 1 DM is treated with daily insulin injections following careful monitoring of blood glucose levels. Type 2 DM is treated by oral administration of hypoglycemic drugs such as sulfonylureas [2], metformin [3], a-glucosidase inhibitors, thiazolidinediones and meglitinides [4], and in some cases a combination therapy (insulin plus hypoglycemic drug) [5] together with regular exercise and maintaining a low glycemic index diet to control blood sugar level. As of 2005, there is no other clinically available form of pharmaceutics other than insulin injection for the treatment of type 1 DM. Vanadyl sulfate, VOSO4, which is +4 oxidative form of vanadium has been shown to reduce hyperglycemia and insulin resistance not only in

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animal model for diabetes [6] but also in diabetic patients [7]. However, the absorption and incorporation of this inorganic salt was generally very low [8,9]. Therefore, in 1990, we first noticed that vanadyl-cysteinate methyl ester complex normalized the high blood glucose level of streptozotocine(STZ)-induced type 1 diabetic rats by daily oral administrations [10]. Since then, our research group and others have been developed many types of vanadyl complexes with different coordination modes around vanadyl ion such as, VO(S4), VO(N2O2), VO(O4), VO(S2O2), and VO(N4) [11–24]. These complexes are more effective than VOSO4 in reducing circulating glucose levels in type 1 DM as well as in type 2 DM animals. In the wake of these findings we have continued our research to find more effective vanadyl complexes using various kind of ligands as well as to prepare sustained drug delivery systems. Recently, we found that the entericcoated encapsulation of VOSO4 improved bioavailability of vanadyl species compared with that associated with either gelatin-coated capsules containing VOSO4 or the solution of VOSO4 [25]. On the other hand, we focused on the use of a polymer that is both biodegradable and biocompatible and that would be effective in enhancing drug targeting specificity, lowering systemic drug toxicity, improving treatment absorption rates and providing protection for pharmaceuticals against biochemical degradation. Polyc-glutamic acid, c-PGA, is a naturally occurring biodegradable and biocompatible polymer, which is non-toxic toward humans and the environment [26]. Therefore, the potential applications of this biopolymer have been of interest in a broad range of industrial fields such as food, medicine and water treatment [27–29]. It has carboxyl groups on the side-chain that offer attachment points for the conjugation of compounds, thereby rendering the compound more soluble and easier to administer. It was reported that the c-PGA-paclitaxel conjugate exhibited markedly greater anti-tumor activity against murine tumors and human tumor xenografts than that of paclitaxel [28,29]. In order to achieve a safer and more effective treatment for DM, a controlled dosing regimen and dosage standard for the oral administration of VOSO4 or vanadyl complexes need to be established. Therefore, the high biological importance of c-PGA promoted us to use this polymer as a carrier of vanadyl ion (VO2+) to make vanadyl-c-PGA (VO-c-PGA) complex for a sustained drug delivery system. In this study, we have prepared and characterized VO-c-PGA complex in solution as well as in solid state. VO-c-PGA complex in solution was used to evaluate its in vitro insulin-mimetic activity, metallokinetic feature in the blood of healthy rats using blood circulation monitoring-ESR (BCMESR) and in vivo hypoglycemic effect in STZ-induced type 1 diabetic mice (STZ-mice), comparing with those of VOSO4 as a positive control.

H COOH N α H

β

γ O n

Structure of γ-PGA

2. Experimental 2.1. Materials The sample of poly-c-glutamic acid, c-PGA, with a D:L enantiomeric mixture having the average molecular weight 5.0 · 105 Da was kindly provided from BioLeaders Japan Corporation (Osaka, Japan). The polymer was used without further purification. VOSO4 Æ nH2O (Wako Pure Chemical Industries, Osaka, Japan) was standardized by complexometric titration with EDTA (ethylenediamine-N,N,N,N-tetraacetic acid) and 2.3H2O was determined as an adduct with VOSO4. Bovine serum albumine (BSA; fraction V), (±)-epinephrine monohydrochloride (Epi), and collagenase were purchased from Sigma Chemical (Sigma, St. Louis, MO, USA). All other reagents were commercially available in the highest grade of purity and were used without further purification. The solution of c-PGA was prepared in aqueous solution by adding microliters amount of 5 M NaOH. 2.2. Preparation of VO-c-PGA complex in solid state VO-c-PGA complex was prepared by mixing various amounts of VOSO4 (1 mM) and 4 mL of c-PGA (1%) solutions under magnetic stirring overnight at room temperature. Acetone was added to the solution to complete precipitation of VO-c-PGA complex. The resulting precipitate was washed with distilled water and acetone, and dried on silica gel under a vacuum condition at room temperature. To determine vanadium content, VO-c-PGA complex (10 mg) was decomposed in concentrated nitric acid (HNO3) and evaporated to dry, and then the ash was dissolved in 5 mL of 6% HNO3. The vanadium concentration in the solution was measured by inductively coupled plasma mass spectrometer (ICP-MS) (Shimadzu ICP-MS8500, Kyoto, Japan). The concentration of vanadium was calculated using a calibration curve at a concentration range of 5–100 ppb for the standard vanadium solutions (Wako Pure Chemical Industries Ltd., Osaka, Japan). The detection limit of vanadium concentration was approximately 0.01 ppb. The correlation coefficient of linear regression was r = 0.999 for a total

S. Karmaker et al. / Journal of Inorganic Biochemistry 100 (2006) 1535–1546

of four metal concentrations. Plasma conditions for ICPMS were as follows: coolant gas-flow, plasma gas-flow and carrier gas-flow were 7.0, 1.5, and 0.61 L min1, respectively, and the sampling depth was 6 mm. Three repeated measurements of metal concentration were performed. 2.3. Preparation of VO-c-PGA complex in solution Typically, a sample solution at the appropriate pH was prepared by mixing VOSO4 (1 M) and c-PGA (1–40% w/v) solutions under magnetic stirring. The final concentration of VOSO4 was 1–20 mM and that of c-PGA was 0.1– 40% (w/v) depending on the measurement conditions. The pH of the reaction mixtures was determined by using HORIBA pH meter M-12 (Japan). The pH of the samples was adjusted by adding microliter quantities of 5 M NaOH or 5 M HCl. The VO-c-PGA complex in solution was used in vitro and in vivo experiments. 2.4. Spectroscopic measurements Visible absorption spectra were measured on an Agilent 8453 UV–vis spectrometer (Agilent, Germany) in the wavelength region of 400–900 nm at room temperature. The diffuse reflectance absorption spectra of the solid complex and VOSO4 mixed with KBr was recorded by a MCPD-1000 spectrometer (Otsuka Electronics, Osaka, Japan) in the wavelength region of 400–800 nm. IR spectra of the VO-c-PGA complex, c-PGA and VOSO4 were measured with the samples in compressed KBr discs by a Shimadzu FTIR-8100A spectrophotometer (Shimadzu, Kyoto, Japan). Electron spin resonance (ESR) spectra were recorded both at room temperature and liquid nitrogen (77 K) temperature by means of an X-band ESR spectrometer (JES-RE1X, Jeol, Tokyo, Japan) under the following conditions: frequency, 9.4 GHz; microwave power, 5.0 mW; modulation frequency, 100 kHz; modulation amplitude width, 0.63 mT; response, 0.03 s; scanning time 4 min; magnetic field 340 ± 100 mT; standards, tetracynoquinodi-methane lithium salt (TCNQ-Li) (g = 2.00252) and Mn(II) in MgO (magnetic field between the third and fourth signals due to Mn(II), 8.69 mT). The hyperfine coupling constant (A0 value) and g0 value were obtained from the spectra at room temperature, where the A0 value was estimated as the magnetic field between MI = 1/2 and 1/2 hyperfine components. Ai and gi values were obtained from the spectra at 77 K, where Ai value was estimated as the mean on the basis of the spectral regions for the MI = 5/2 and 7/2 (A1) and MI = 5/2 and 7/2 (A2) hyperfine components. The g^ and A^ values were obtained using the following equations: g? ¼ 1=2ð3g0  gk Þ

ð1Þ

A? ¼ 1=2ð3A0  Ak Þ

ð2Þ

1537

2.5. Evaluation of in vitro insulin-mimetic activity Insulin-mimetic activity of the VO-c-PGA complex was evaluated by in vitro experiments, in which the inhibitory activity of complex on release of free fatty acid (FFA) [30] and enhancement of glucose-uptake ability [31] in isolated rat adipocytes treated with epinephrine (Epi) were compared with those of VOSO4 as a positive control. Male Wistar rats (weighing 200 g) were sacrificed under anesthesia with ether, and the adipose tissues were removed. The adipose tissues were chopped up with scissors and digested with collagenase for 1 h at 37 C in Krebs-Ringer bicarbonate (KRB) buffer, pH 7.4, containing 2% BSA. The obtained adipocytes were then separated from undigested tissues by filtration through nylon mesh (250 lm) and washed three times with the buffer without collagenase. The complex (30 lL) at various concentrations prepared in saline solution and 10 lL of glucose (final concentration: 5 mM) were added to 240 lL of the isolated adipocytes (1 · 106 cells mL1), and the resulting suspensions were incubated at 37 C for 30 min. Finally, 15 lL of Epi (final concentration: 10 lM) was added to the suspensions and the resulting mixture was incubated at 37 C for 3 h. The reactions were stopped by cooling as well as by centrifuging at 3000 rpm at 4 C for 10 min. The FFA concentration in the outer solution of the cells was determined with an FFA kit (NEFA C-test Wako, Wako Pure Chemical Industries, Osaka, Japan) [30]. The IC50 value of the complex (concentration required to inhibit 50% of the FFA release) was determined from the curve drawn for the complex concentration-dependent inhibitory effect of FFA release from isolated rat adipocytes treated with epinephrine. In addition, glucose concentration in the outer solution of the cells was estimated using a Fuji Dry Chem analyzer (Fuji Medical Co. Ltd., Tokyo, Japan) [31]. The glucose-uptake ability of the compounds was evaluated with the apparent EC50 values, the 50% enhancing concentration of the compound with respect to the maximal glucose-uptake concentration in epinephrine-treated adipocytes. 2.6. Metallokinetic analysis of VO-c-PGA complex by blood circulation monitoring-ESR (BCM-ESR) The metallokinetic features of vanadyl species in the blood of healthy rats receiving VOSO4 and VO-c-PGA complex at a dose of 0.5 mg V kg1 body mass were analyzed by in vivo BCM-ESR [32,33]. Briefly, rats were anesthetized by i.p. injection of pentobarbital and kept at 35 C on a Deltaphase Isothermal Pad (Model 39 DP, Braintree Scientific, MA, USA). Heparinized polyethylene tubes were cannulated into the left femoral artery and vein. The free ends of cannula were joined with heparinized silicon tubes to make a blood circuit outside the body, which was directly connected to an ESR cell (a quartz

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20 lL capillary tube). Blood from the femoral artery was returned and recirculated to the femoral vein after flowing through the ESR cell using the blood pressure provided by the rat’s own heartbeat, which was always maintained at a constant rate. VOSO4 and VO-c-PGA complex in saline solutions were given by a single i.v. injection to the rats, and ESR spectra were measured at room temperature every 30 s using an X-band EPR spectrometer. Data were collected and analyzed by a Windows computer using WINRAD Data Analyzer (Radical Research, Tokyo, Japan). Disappearance of ESR signal due to vanadyl species in the blood was plotted against time following the administration of the VOSO4 and VO-c-PGA complex. To determine the concentrations of vanadyl species, 20 lL of VOSO4 and VO-c-PGA complex dissolved in the blood of untreated rats was applied to the ESR cell. Each calibration curve obtained by monitoring the signal intensities of the central peak due to the corresponding vanadyl complex was freshly prepared by using the fresh blood spike obtained with each complex. Metallokinetic parameters for vanadyl complexes were obtained on the basis of a two-compartment model. The equation [Cb = A exp(at) + B exp(bt)] was fitted to each individual profile of the determined concentrations of vanadyl complexes using a nonlinear least-squares regression program MULTI [34] which is newly rewritten with Visual Basic, where Cb is the blood concentration, a and b are the apparent rate constants, A and B are the corresponding zero time intercepts, respectively, t is time. The metallokinetic parameters such as area under the concentration curve (AUC), mean residence time (MRT), total body clearance (CLtot), and steady-state distribution volume in the body (Vdss) were calculated from the fitted results as follows: AUC = A/a + B/b, MRT = (A/a2 + B/b2)/(A/a + B/b), CLtot = Dose/(A/a + B/b), and Vdss = Dose Æ (A/a2 + B/ b2)/(A/a + B/b), where Dose is the amount of vanadyl complexes intravenously administrated. 2.7. Evaluation of in vivo antidiabetic activity Experimental type 1 DM was induced in male Std: ddY mice, weighing about 30 g, by a single i.p. injection of freshly prepared streptozotocin (STZ) (100 mg kg1 body mass) in 0.1 M citrate buffer (pH 5). Blood samples for the analysis of glucose level were obtained from tail vein of STZ-mice, and the levels were measured using glucose oxidase method with a Glucocard (Arkray, Kyoto, Japan). STZ-mice with a blood glucose level of 450–550 mg dL1 (25–30.6 mM) at 4 weeks after STZ administration were used for the experiments. The in vivo anti-diabetic activity of VO-c-PGA complex was assessed in STZ-mice by both a single oral gavage and daily oral administrations of the complex for 16 days. The change of blood glucose level of STZ-mice after a single oral gavage of VOSO4 (10 mg kg1 body mass) and VO-c-PGA complex at doses 5 mg and 10 mg V kg1 body mass were monitored. For daily oral administrations, the STZ-mice were treated with

VOSO4 and VO-c-PGA complex at doses 5–10 mg V kg1 body mass, and equivalent volume of 2% c-PGA for 16 days. VO-c-PGA complex was freshly prepared by mixing adequate amount of VOSO4 (1 M) with c-PGA (2% w/v) in saline solution at pH 3.2. The final concentration of vanadyl ion in complex was 19.96 mM. The blood sample for the analyses of glucose levels was obtained from the tail vein of each mouse and the blood glucose level was measured with a Glucocard as described. Body mass of STZmice, which were allowed free access to solid food (MF, Oriental Yeast, Tokyo, Japan) and tap water, were measured daily during the administration of saline, VOSO4, VO-c-PGA, and c-PGA. In addition, intakes of solid food and drinking water in each mouse were checked daily throughout the experiment. After daily oral administration of VOSO4, VO-c-PGA, and c-PGA for 16 days, blood samples collected from orbital exsanguinations of the mice under anesthesia with ether were centrifuged at 5000 rpm for 10 min at 4 C, and serum samples for the analysis of urea nitrogen (UN), glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), triglycerides (TG), total cholesterol (TCHO), FFA and insulin levels were separated. The serum UN, GPT, GOT, TG and TCHO levels were estimated by using a Fuji Dry Chem analyzer (Fuji Medical Co., Tokyo, Japan). A NEFA C-test and Glazyme insulin-EIA test (Wako Pure Chemical Industries, Osaka, Japan) were used to determine the serum FFA and insulin levels, respectively. In addition, HbA1c levels in the blood obtained from the tail vein of the mice after the administration of VOSO4, VO-c-PGA, and c-PGA were measured by using a DCA 2000 system (Bayer, Tokyo, Japan). 2.8. Oral glucose tolerance test After administrations of the saline, VOSO4, VO-c-PGA, and c-PGA for 16 days, an oral glucose tolerance test (OGTT) was performed. The STZ-mice were fasted for 12 h and glucose at a dose 1 g kg1 body mass was given orally. Blood samples were obtained from the tail vein at 0, 15, 30, 45, 60, 90, 120 and 180 min after glucose administration. Blood glucose levels were measured with the Glucocards. 2.9. Measurement of systolic blood pressure Systolic blood pressure in the conscious state was measured after administrations of the saline, VOSO4, VO-cPGA, and c-PGA for 16 days by the indirect tail cuff method using a Model MK-2000 BP monitor for rats and mice (Muromachi Kikai, Tokyo, Japan) according to the manufacturer’s instructions. The measurement was carried out under room temperature condition (25 C), because the instrument does not require pre-warming of the animals. Five readings were obtained from each mouse and mean value was considered.

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2.10. Statistical analysis

0.3 (c)

3. Results and discussion

(b)

Absorbance

All experimental results are expressed as the mean values ± standard deviations (SD). Statistical analysis was performed by analysis of variance (ANOVA) or Student’s t-test at 5% (p < 0.05), 1% (p < 0.01), or 0.1% (p < 0.001) significance level of the difference.

0.2 (d) (e)

0.1

3.1. Preparation and characterization of VO-c-PGA complex

(a)

The VO-c-PGA complex was prepared in aqueous saline solution (pH 3.2) by mixing VOSO4 (1 M) and c-PGA (1– 40% w/v) solutions. The structure of the new complex VO-c-PGA was characterized by visible absorption, IR, and ESR spectra. The same complex was prepared in solid state to confirm the structure in aqueous solution. The physico-chemical characters of VO-c-PGA and VOSO4 are summarized in Table 1. In solution, the absorption maxima of VO-c-PGA complex were found in the visible range at around 780 nm with a shoulder at 600 nm, whereas free c-PGA absorbed only the UV-region and VOSO4 showed an absorption band at 765 nm with a shoulder at 620 nm (Fig. 1). Thus the red shift (15 nm) of the absorption band at around 780 nm and the blue shift (20 nm) of the absorption band at 600 nm in the visible region must be ascribed to transition of electrons associated with the complex formation. The spectrum of the solid complex also presented two bands in the visible range at around 600 nm and 780 nm. The shift of the two typical bands of free VO2+ [35,36] to red (15 nm) and blue (20 nm), respectively, in the complex suggests coordination of this metal ion through the carboxylate groups of the c-PGA. Similar shift of the absorption bands by the complexation has been proposed in vanadyl-ciprofloxacin, vanadyl-quinic acid and vanadyl-suprofen complexes [37–39]. As the position of the electronic absorption bands either in solution or in solid state was found to be quite similar, the structure of the complex is almost analogues in both the solution and solid states.

0 500

600

700

800

900

Wavelength (nm) Fig. 1. Visible spectra of c-PGA (a), VOSO4 (b), VO-c-PGA (c) in aqueous solution at pH 3.2 and diffuse reflectance electronic spectra of VOSO4 (d) and VO-c-PGA (e) complex in KBr.

When 2% c-PGA was titrated with 5–20 mM VOSO4 solutions at pH 3.2 (Fig. 2A), the absorbance intensities at 780 nm and 600 nm were linearly increased with increasing the initial concentration of VOSO4 solution (Fig. 2B), however, the precipitation of the complex was observed at 25 mM of VOSO4. The results indicated that the concentration of VO-c-PGA complex was increased with increasing of VOSO4, and the complex was remained in solution up to 20 mM of VOSO4 for 2% c-PGA. The FT-IR spectrum of c-PGA exhibited the following absorption bands: the strong NAH stretching band at 3295 cm1, the strong stretching vibration band due to the [email protected] in the COOH group at 1734 cm1, the bands at the region 1620–1660 cm1 for amide I ([email protected] stretch) and 1540–1560 cm1 for amide II (NAH bend). On the other hand, the new band due to [email protected] stretching frequency was found at 980 cm1 in the spectrum of VO-c-PGA complex as observed in the case of bis(6-hydroxypicolinato) oxovanadium(IV) complex at 978 cm1 [33]. Moreover, the frequency at 1734 cm1 of c-PGA was shifted to the lower frequency at 1726 cm1 in complex with a greatly

Table 1 Physico-chemical characters of VOSO4 and VO-c-PGA complex prepared in aqueous solution Sample

Solvent

pH

Visible absorption maxima (nm)

IR in KBr [email protected] (cm1)a

ESR parameters A-value (104 cm1)

g-value

VOSO4 VO-c-PGA

H2O H2O

3.00 1.72 2.16 3.02 4.00 5.00

Sh.  = absorption shoulder. a Data from solid complex.

620 sh. ; 765

600 sh. ; 780

980 980

g0

gi

g^

A0

Ai

A^

1.969 1.969 1.967 1.965 1.962 1.966

1.935 1.935 1.937 1.941 1.940 1.942

1.986 1.986 1.982 1.978 1.974 1.977

106 106 106 106 105 106

186 186 187 180 179 176

66 69 65 68 69 70

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0.8

A

(d)

Absorbance

0.6

(c) (b)

0.4

(a)

0.2 0 500

B

600

900

0.8 y = 0.0249x + 0.1664 (b) R = 0.9996

0.6 Absorbance at 600 and 780 nm

700 800 Wavelength (nm)

(a) 0.4 y = 0.0133x + 0.2352 R = 0.9955

0.2

0

0

5

10 15 [VOSO4]int. (mM)

20

25

Fig. 2. Titration curves of 2% c-PGA with VOSO4 (5–20 mM) in aqueous solution at pH 3.2 (A) and the changes in absorbance at 600 (a) and 780 nm (b) (B). Concentrations of VOSO4 were (a) 5 mM, (b) 10 mM, (c) 15 mM, and (d) 20 mM.

reduced intensity. The shift of this band to lower frequency may be due to the [email protected] stretching of the coordinated carboxylic groups to the metal center and the partially remaining protonated carboxylic acid groups. The strong absorption bands of the asymmetrical and symmetrical valency vibrations due to the COO group were not clearly observed at around 1600 cm1 and 1400 cm1, respectively, however, the broad absorption bands were observed at around 1640 cm1 and 1404 cm1, respectively. The broad band centered at 1640 cm1 might be the convoluted one due to the amide I stretching at 1620–1660 cm1 and the asymmetrical valency vibration due to the COO group at around 1600 cm1. The relatively large value of D[mas(COO)  ms(COO)] = 236 cm1 [40] was indicative of monodentate carboxylate coordination to VO2+. ESR spectra of VO-c-PGA complex in aqueous solution (pH 3.02) exhibited eight lines at room temperature and an anisotropic spectrum at liquid nitrogen temperature (Fig. 3), when the complex was prepared at the ratio of 1:0.02 of VOSO4 and c-PGA (final concentration of VOSO4: 1 mM and c-PGA: 1%), indicating the existence of vanadyl species upon the complexation of VO2+ with c-PGA. The anisotropic spectrum of the complex was not observed at room temperature under the experimental conditions probably be due to the random structure of VO-cPGA complex. It was reported that the c-PGA (COOH type) has a parallel b structure, while the sodium salt of c-PGA (COONa type) has a random structure [41,42]. In this study, the aqueous solution of c-PGA was prepared by adding microliters amount of 5 M NaOH. When the concentration of c-PGA was increased to that of VOSO4 at the ratio of 1:0.8 of VOSO4 and c-PGA (final

A

B

C

D

240

340 Magnetic field (mT)

440

240

340 Magnetic field (mT)

440

Fig. 3. ESR spectra of VOSO4 at room temperature (A) and at liquid nitrogen temperature (LNT) (B); and VO-c-PGA at room temperature (C) and at LNT (D) at pH 3.02. VO-c-PGA complex was prepared by mixing at the ratio of 1:0.02 of VOSO4 and c-PGA (final concentration of VOSO4: 1 mM and c-PGA: 1%).

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A

250 ESR signal intensity at 290 mT

concentration of VOSO4: 1 mM and c-PGA: 40%), the ESR spectrum of VO-c-PGA became anisotropic at room temperature (Fig. 4A). Similar phenomenon was observed in the ESR spectrum of 1:1 solution of VOSO4 and human serum albumin (HSA) at room temperature [43]. Moreover, the spectrum of the complex (Fig. 4B) at liquid nitrogen temperature exhibited anisotropy as observed in 1% c-PGA (Fig. 3D). These results indicate that the VOc-PGA complex is formed by the same coordination of VO2+ through the side chain carboxylic groups of the c-PGA at both low and high concentrations, and the structure of the complex is same in both liquid and solid states as expected. In order to determine the coordination mode of VO-cPGA complex and the preferable pH value for the complexation, ESR spectra of the complex were taken at room and liquid nitrogen temperatures at different pH, where the final concentrations of VOSO4 (1 mM) and cPGA (1%) were kept constant. The estimated ESR parameters are tabulated in Table 1. The values of gi and Ai were found to be almost constant at pH 1.7–5.0 (Table 1) indicating the coordination mode around vanadyl ion is VO(O4) as judged from the reference values reported previously [10,16,33]. However, the highest signal intensity of VO-c-PGA complex at liquid nitrogen temperature was observed at pH 3 among the observed pH at 1.7–5.0 (Fig. 5), suggesting that the most suitable pH was 3 for the VO-c-PGA complexation. The hyperfine coupling constants, A0 and Ai, of VO-c-PGA complex were also calculated with the application of the socalled additivity relationship [44],

200

150

100

50

0

0

1

2

3 pH

4

5

6

Fig. 5. pH dependent change of ESR signal intensity at 290 mT of VO-c-PGA complex at liquid nitrogen temperature (LNT).

A0;calcd ¼

X

ni A0;i =4

ð3Þ

where i denotes the different types of donor atoms ligated to the equatorial positions of VO2+, ni(=1–4) is the number of donor atoms of type i, and A0,i is the observed coupling constant (from model studies) when all four equatorial donor atoms are of type i. The theoretical A0 and Ai values were estimated based on various (O4) equatorial coordination modes of VO2+ in VO-c-PGA complex. The calculated A0 and Ai values of 104 · 104 cm1 and 180 · 104 cm1 for carboxylate(O)–VO–(OH2)3 and 102 · 104 cm1 and 177 · 104 cm1 for 2 carboxylate(O2)–VO–(OH2)2 are very close to the observed values of A0 and Ai for the complex as shown in Table 1. Therefore, the equatorial coordination mode of VO2+ in VO-c-PGA complex was proposed to be in either carboxylate(O)–VO–(OH2)3 or 2 carboxylate(O2)–VO–(OH2)2. The proposed structures of VO-cPGA complex are depicted in Fig. 6. The average number of moles of vanadium ion bound with one mole of c-PGA in the solid VO-c-PGA complex was estimated to be (6.21 ± 1.5) · 104 using ICP-MS method. 3.2. Evaluation of in vitro insulin-mimetic activity of VO-c-PGA complex

B

250

300 350 Magnetic Field (mT)

400

Fig. 4. ESR spectra of VO-c-PGA at room temperature (A) and at LNT (B) at pH 3.02 The complex was prepared by mixing at the ratio of 1:0.8 of VOSO4 and c-PGA (final concentration of VOSO4: 1 mM and c-PGA: 40%).

To evaluate the in vitro insulin-mimetic activity of VO-cPGA complex, the inhibition of release of free fatty acid (FFA) from isolated rat adipocytes treated with Epi was examined in the presence of VOSO4, VO-c-PGA, and cPGA [30]. The concentration-dependent inhibitory effects of VOSO4 and VO-c-PGA complex on FFA release from isolated rat adipocytes treated with Epi are shown in Fig. 7A, from which the IC50 values of the VOSO4 and VO-c-PGA complex were estimated (Table 2). The inhibitory activity of VO-c-PGA complex (IC50 = 0.18 ± 0.05 mM) was significantly (p < 0.05) higher than that of VOSO4 (IC50 = 0.38 ± 0.15 mM).

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O

O C

O

C

OH2

O

OH2 O

O V

OH2

V

and C

OH2

OH2

O

O

Fig. 6. The proposed structure of VO-c-PGA.

A

Table 2 IC50 and EC50 values of VOSO4 and VO-c-PGA in isolated rat adipocytes treated with epinephrine

1.5

-1

FFA (mEq L )

Sample

1.0

VOSO4 VO-c-PGA a

Glucose-uptake assay

IC50 (mM)

EC50 (lM)

0.38 ± 0.15 0.17 ± 0.05a

21.2 ± 0.1 19.1 ± 3.2

p < 0.05 vs. VOSO4.

0.5

0.0

Blank Control 0.1 0.5 1.0 VOSO4 (mM)

0.1 0.5 1.0 VO-γ-PGA (mM)

B 120

Glucose uptake (%)

FFA assay

obtained from these data, as shown in Table 2. The EC50 value of VO-c-PGA complex (19.1 ± 3.2 lM) was found to be slightly lower than that of VOSO4 (21.2 ± 0.1 lM). However, the polymer ligand c-PGA alone did not show any in vitro insulin-mimetic activity under the same experimental conditions. These results indicated that the VO-cPGA complex has higher insulin-mimetic activity than that of VOSO4.

100

3.3. Metallokinetic feature of VO-c-PGA complex in the blood of healthy rats by blood circulation monitoring-ESR (BCM-ESR)

80 60 40 20 0

n.d. n.d. 0 10

25 50 75 VOSO4 (µΜ)

n.d. n.d. 0 10 25 50 75 VO-γ-PGA (µΜ)

Fig. 7. Inhibitory effects of VOSO4 and VO-c-PGA (0.1–1.0 mM) on FFA release (A) and enhancing effect of VOSO4 and VO-c-PGA (10–75 lM) on glucose-uptake ability (B) in rat isolated adipocytes (2.5 · 106 cells mL1) treated with 0.01 mM Epi in the presence of 1 mg mL1 glucose.

The insulin-mimetic activity of VOSO4 and VO-c-PGA complex was also estimated using the glucose-uptake assay [31]. As shown in Fig. 7B, the concentration-dependent enhancing effects of glucose-uptake by VOSO4 as well as VO-c-PGA were observed at lower concentrations than those in the FFA-release assay, and EC50 values were

Metallokinetic profiles of paramagnetic vanadyl species in the blood of healthy rats receiving VOSO4 and VO-cPGA complex were analyzed using BCM-ESR method [32,33]. Fig. 8A shows a typical example for the ESR spectral change with a time-dependent decrease of its signal intensities produced by vanadyl species during BCM-ESR in rats administered the VO-c-PGA complex. The central peak intensity gradually decreased with time as expected. Fig. 8B shows the time courses for the determined vanadyl species in the circulating blood of rats given vanadyl compounds as well as the simulated curves, which represent the theoretical curves fitted to the mean data on the basis of the two-compartment model with nonlinear least-squares regression [34]. Detected vanadyl species in the blood of rats given vanadyl compounds decreased in a biphasic pattern, and then the clearance curves were fitted to the individual with the two-compartment model. The in vivo vanadyl species disappeared time-dependently in the circulating blood of rats, indicating that vanadyl species taken

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S. Karmaker et al. / Journal of Inorganic Biochemistry 100 (2006) 1535–1546

25

A 20 15 10 5 3

Time (min)

1.5 0.5 300

320

340 360 Magnetic Field (mT)

380

1000

Vanadyl concentration in the blood of rats

B

100

10 0

10

20

30

Time (min) Fig. 8. In vivo BCM-ESR feature of vanadyl species. A rat was administered VO-c-PGA intravenously at a dose of 0.5 mg of V kg1 body mass under anesthesia (A). ESR spectra were recorded at room temperature every 30 s following administration of the complex. Time courses of vanadyl concentration in the blood of rats treated intravenously with VOSO4 (s; n = 4), and VO-c-PGA (d; n = 3) as monitored by the BCM-ESR method (B). The corresponding theoretical curves (solid lines) were fitted to the mean values.

Table 3 Metallokinetic parameters of VOSO4 and VO-c-PGA complex Sample

AUC (lmol min mL1)

MRT (min)

CLtot (mL min1 kg1)

Vdss (mL kg1)

t1/2, a (min)

t1/2, b (min)

VOSO4 VO-c-PGA

0.26 ± 0.04 2.08 ± 0.03a

4.4 ± 0.7 22.3 ± 1.1a

38.9 ± 5.8 4.7 ± 0.1a

167 ± 10 105 ± 4a

0.66 ± 0.02 0.88 ± 0.29a

1.0 ± 0.2 17.2 ± 1.0a

Data are expressed as the means ±SDs for 3 or 4 rats. Rats were treated with vanadyl complexes VOSO4 (n = 4) and VO-c-PGA (n = 3). V: 0.5 mg kg1 body mass by i.v. injection under anesthesia. a Significantly different at the 1% level of ANOVA in comparison with VOSO4.

up into the blood were distributed to the peripheral tissues and eliminated from the whole body. Vanadyl concentrations in the blood of rats given VO-c-PGA complex were remained significantly higher and longer than those of VOSO4 as shown in Fig. 8B. The metallokinetic parameters of vanadyl species after administration of VOSO4 and VO-c-PGA complex are summarized in Table 3. AUC and MRT values for the rats treated with VO-c-PGA complex (2.08 ± 0.03 lmol min mL1, 22.3 ± 1.1 min, respectively) were significantly higher than those of VOSO4 (0.26 ± 0.04 lmol min mL1, 4.4 ± 0.7 min, respectively), which related with the clearance rates of vanadyl species from the blood of rats. In fact, the values of CLtot and Vdss for the rats treated with VO-c-PGA complex (4.7 ± 0.1 mL min1 kg1 and 105 ±

4 mL kg1, respectively) were significantly lower than those of VOSO4 (38.9 ± 5.8 mL min1 kg1 and 167 ± 10 mL kg1, respectively). These results suggested that the bioavailability of VO-c-PGA complex was much higher than that of VOSO4. Therefore, VO-c-PGA complex was expected to normalize the elevated blood glucose levels in STZ-induced diabetic mice. 3.4. Evaluation of in vivo anti-diabetic activities of VOSO4 and VO-c-PGA complex Based on the results of in vitro insuline-mimetic activity and metallokinetic features of vanadyl compounds, the antidiabetic activity of VOSO4, VO-c-PGA complex, and c-PGA was evaluated in STZ–mice.

1544

A

700 600

VOSO4: 10 mg V kg –1 body mass

35 -1

500

a

25

400

a

a

a

a

a

a a

300

a

a 200

20 15

a

10

Blood glucose level (mM)

30

-1

Blood glucose level (mg dL )

600

30 500

0

8

16

24

32

40

0 48

25 1

d b b

200 100 10

B

-2

0

5 2

20 15 10

(VO-γ-PGA) 10 mg V kg–1 body mass

4

6

8 10 Day

12

14

5 16

18

0

12 p
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