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ENDOCRINE AND DIABETES

A Potent Immunomodulatory Compound, (S,R)-3-Phenyl-4,5-dihydro-5-isoxasole Acetic Acid, Prevents Spontaneous and Accelerated Forms of Autoimmune Diabetes in NOD Mice and Inhibits the Immunoinflammatory Diabetes Induced by Multiple Low Doses of Streptozotocin in CBA/H Mice

Institute for Biological Research "Sinisa Stankovic," Belgrade, Serbia (S.S.-G., I.C., D.M.-I., L.H., M.M., D.M.); Department of Biomedical Sciences, University of Catania, Catania, Italy (K.M., F.N.); Department of Pharmacobiological Sciences, University Magna Graecia of Catanzaro, Catanzaro, Italy (M.F.); Institute of Microbiology and Immunology, School of Medicine, Belgrade University, Belgrade, Serbia (D.P.); VGX Pharmaceuticals, Blue Bell, Pennsylvania (J.K.); and Laboratory of Medicinal Chemistry, North Shore-Long Island Jewish Health System, Manhasset, New York (Y.A.A.)

Received June 13, 2006; accepted December 4, 2006.

	Abstract

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(S,R)-3-Phenyl-4,5-dihydro-5-isoxasole acetic acid (VGX-1027) is an isoxazole compound that exhibits various immunomodulatory properties. The capacity of VGX-1027 to prevent interleukin (IL)-1 plus interferon--induced pancreatic islet death in vitro prompted us to evaluate its effects on the development of autoimmune diabetes in preclinical models of human type 1 diabetes mellitus (T1D). Administration of VGX-1027 to NOD mice with spontaneous or accelerated forms of diabetes induced either by injection of cyclophosphamide or by transfer of spleen cells from acutely diabetic syngeneic donors markedly reduced the cumulative incidence of diabetes and insulitis. In addition, VGX-1027 given either i.p. or p.o. to CBA/H mice made diabetic with multiple low doses of streptozotocin successfully counteracted the development of destructive insulitis and hyperglycemia. The animals receiving VGX-1027 exhibited reduced production of the proinflammatory mediators tumor necrosis factor-, IL-1, macrophage migration inhibitory factor, and inducible nitric-oxide synthase-mediated nitric oxide generation in both pancreatic islets and peripheral compartments. These results indicate that VGX-1027 probably exerts its antidiabetogenic effects by limiting cytokine-mediated immunoinflammatory events, leading to inflammation and destruction of pancreatic islets. VGX-1027 seems worthy of being considered as a candidate drug in the development of new therapeutic strategies for the prevention and early treatment of T1D.

One common mechanism by which infiltrating macrophages and autoreactive T lymphocytes promote cell death may be related to their capacity to produce proinflammatory mediators, such as tumor necrosis factor (TNF)-, interferon (IFN)-, interleukin (IL)-1, IL-18, and nitric oxide (NO), which have all been implicated as critical players in the initiation and propagation of the disease process (reviewed by Rabinovitch and Suarez-Pinzon, 1998). Although negating the action of proinflammatory and type 1 cytokines such as IL-1, IFN-, IL-12, IL-18, and TNF- may prevent autoimmune diabetogenesis in most of these models (Rabinovitch and Suarez-Pinzon, 1998), the efficacy of similar approaches in influencing the natural course of human T1D is not known because of the lack of clinical studies with specific inhibitors of these cytokines in the human disease counterpart.

However, despite the promising data obtained in these rodent models, there are several drawbacks limiting the possible application of specific cytokine inhibitors in human T1D, including their potential immunogenicity, their short half-life in vivo, the nonoral route of administration, the side effects, and the high costs of treatment. For this reason, studies aimed to discover orally available small chemical compounds that target the synthesis and/or the action of endogenous proinflammatory cytokines are warranted. For example, a variety of small synthetic inhibitors of TNF- have been designed to specifically inhibit the action of the cytokine at various levels, including transcriptional and post-transcriptional inhibition of TNF- production, secretion, down-regulation of TNF--receptor-mediated signal transduction, and inhibition of TNF- bioactivity (Holstad and Sandler, 2001; Namazi, 2004; Abdul-Hai et al., 2005).

Because in vitro and in vivo data have proven a synergistic action of type 1 proinflammatory cytokines in determining cell destruction (Rabinovitch and Suarez-Pinzon, 1998), it seems likely that compounds capable of simultaneously inhibiting most of them would prove more effective than specific neutralization of a single cytokine with the specific inhibitor. Along this line of research, in vitro and in vivo studies aimed to screen test compounds capable of inhibiting both the synthesis and the in vivo activity of TNF- were performed. In a first line of screening, a series of test compounds were analyzed for their capacity to inhibit TNF- synthesis from murine splenic mononuclear cells (SMNC) and peritoneal macrophages stimulated with either concanavalin A or lipopolysaccharide (LPS), respectively. The test compounds achieving a significant inhibition of TNF- in these assays were additionally evaluated for their efficacy in counteracting TNF--dependent LPS-induced lethality in mice. We found that, among the compounds tested, (S,R)-3-phenyl-4,5-dihydro-5-isoxasole acetic acid (VGX-1027) exhibited the highest potency in inhibiting the synthesis of TNF- from macrophages and/or T cells and afforded the highest degree of protection against the lethal effects of LPS in the mice (S. Stosic-Grujicic, Y. Al Abed, and F. Nicoletti, unpublished data).

These immunopharmacological characteristics and the above-mentioned pathogenic importance of TNF- in autoimmune diabetogenesis make VGX-1027 a suitable candidate for testing in preclinical models of human T1D and prompted us to evaluate its effects both in the NOD mouse and the mouse made diabetic with MLD-STZ. VGX-1027 exhibited powerful antidiabetogenic effects under in vitro, ex vivo, and in vivo conditions. In fact, it prevented IL-1 plus IFN--induced pancreatic islet death in vitro, and it powerfully suppressed clinical and histological signs of the disease in both spontaneous and accelerated (Like and Rossini, 1976; Kolb, 1987; Lukic et al., 1998; Shoda et al., 2005) forms of autoimmune diabetes in NOD mice and in the mouse made diabetic with MLD-STZ. Simultaneously, ex vivo studies carried out in mice with MLD-STZ-induced diabetes indicated that VGX-1027 dampened the immunoinflammatory diabetogenic processes at multiple levels, including the production of proinflammatory and cytotoxic mediators from both macrophages and pancreatic cells.

	Materials and Methods

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Reagents and Drugs. Streptozotocin (STZ, S-0130), [³H]thymidine, sulfanilamide, naphthylethylenediamine dihydrochloride, and cyclophosphamide (CY) were purchased from Sigma-Aldrich (St. Louis, MO). RPMI 1640 medium was supplemented with 1 mM HEPES buffer, 5 to 10% fetal calf serum, as indicated, 1% sodium pyruvate, 2 mM l-glutamine (all from Flow Laboratories, Irvine, Scotland, UK), penicillin/streptomycin, and 5 x 10^-5 M 2-mercapto-ethanol (Sigma). Recombinant mouse cytokines IFN-, TNF-, and IL-1 were from Sigma. VGX-1027 (Fig. 1) was synthesized as described previously (Eichenger et al., 1997).

View larger version (16K): [in this window] [in a new window]   Fig. 1. Synthesis and structure of (S,R)-3-phenyl-4,5-dihydro-5-isoxasole acetic acid (VGX-1027).

The in vivo doses of VGX-1027 were chosen on the basis of preliminary studies carried out in the TNF--dependent (Mohler et al., 1993) mouse model of LPS-induced lethality. In these studies, the doses of 20 mg/kg b.wt. i.p. and 100 mg/kg b.wt. p.o. were the most effective doses of those tested (5, 10, and 20 mg/kg b.wt. i.p. and 60, 100, and 120 mg/kg b.wt. p.o.) in significantly reducing the cumulative incidence of lethality compared with vehicle-treated controls (F. Nicoletti, data not shown).

Mice. Inbred CBA/H male mice that are genetically susceptible to development of immunoinflammatory diabetes after MLD-STZ were originally obtained from the Jackson Laboratory (Bar Harbor, ME) and have then been bred at the Institute for Biological Research (Belgrade, Serbia) for 20 years. Adult male mice, at 6 to 8 weeks of age with body weight ranging from 25 to 30 g, were used in all experiments, and each experimental group consisted of seven to eight mice. Female NOD mice were obtained from Charles River (Calco, Italy) and were kept at the animal house of the Department of Biomedical Science of the University of Catania (Catania, Italy).

Both strains of mice were kept under standard laboratory conditions (nonspecific pathogen-free) with free access to food and water. The handling of animals and the study protocol were in accordance with international guidelines and approved by the local Institutional Animal Care and Use Committee.

Pharmacokinetic Analysis of VGX-1027 in Mice. Single doses of VGX-1027 (20 mg/kg b.wt.) in vehicle (500 mM Na₂HPO₄) were administered i.p. Blood samples (0.25–0.3 ml) were collected before dose administration and 0.30, 1, 1.30, 2, 3, 4, 6, and 8 h after dosing via vena cava puncture and processed for plasma. Each determination consists of plasma samples pooled from 2 mice. There were 10 mice for each time point considered.

Sample Preparation. Before the extraction procedure, clear and homogeneous plasma samples obtained for pharmacokinetic evaluation were thawed in a water bath at 30°C and then centrifuged at 2500g for 10 min. Plasma samples (500 µl) pooled from different normal (VGX-1027-untreated) mice containing VGX-1027 were mixed with a 0.1 M pH 7 phosphate buffer (500 µl). Then samples were purified by solid-phase extraction. The extraction procedure was performed by using conditioned Oasis HLB solid-phase extraction cartridges (1 ml, 30 mg) from Waters (Milford, MA) that were connected to a LiChrolut extraction unit (Merck, Darmstadt, Germany). The stationary phase the cartridges was activated by elution under vacuum with 2 ml of a methanol-water (HPLC grade) mixture. Samples were charged on extraction cartridges, washed under vacuum with a 5% (v/v) methanol aqueous solution (1 ml), and then eluted twice with 1 ml of methanol acidified (pH 2.4) with trifluoroacetic acid. The elutate was collected in a glass tube and evaporated under a nitrogen stream at 40°C by using a sample concentrator (Techne Dri-Block-3D). The residue was dissolved with acetonitrile (200 µl), centrifuged by a Mini Spin Eppendorf centrifuge, filtered by Whatman Anotop 10 liquid chromatography filters (0.2 µl/10 mm), and then injected onto the chromatographic apparatus. Control samples were prepared by adding 100 µl of an acetonitrile VGX-1027 solution at scalar concentrations. Control samples were frozen at -20°C and then submitted to the procedure reported above to validate both the analytical method and the extraction procedure.

Extraction Efficiency. The mean recovery of VGX-1027 from spiked mouse plasma samples (control samples) was evaluated to test the efficiency and reproducibility of the extraction procedure. The determination was carried out in replicate (n = 3) in all samples. The extraction was performed as described above. The responses of these standards obtained by the extraction procedures have been compared with those of standard solution at the same concentration injected directly into the liquid chromatographic apparatus. The peak areas were compared with that of standard aqueous samples without extraction.

The extraction efficiency was expressed as the percentage of the amount of VGX-1027 that was able to be extracted from control plasma samples spiked with known amounts of the drug. The extraction efficiency of the method, as a function of the drug amount, is reported in Table 1.

HPLC Experiments. The chromatographic system was a Jasco PU 1580 intelligent HPLC pump (Jasco, Tokyo, Japan) equipped with a Jasco MD 1510 multiwavelength detector set at VGX-1027 _(max) 260 nm. A 100-µl loop was used for analytical determinations.

The HPLC apparatus was connected to a computer running Jasco Borwin software, version 1.5, for both acquisition and data analysis. The chromatographic separation was performed with an Agilent Nucleosil C₁₈ reversed-phase column (250 x 4.6 mm, 5 µm) with an Agilent column guard. The analytical column was thermostated to 25°C with a Gastorr GF 103 block heater (Jones Chromatography, Lakewood, CO). The mobile phase was an acetonitrile-pH 2.01 water (acidified with trifluoroacetic acid) mixture (35:65, v/v). The mobile phase was delivered at a flow rate of 1.0 ml/min.

To determine the amount of VGX-1027 in mouse plasma samples, a calibration curve was determined by preparing control samples spiked with known amounts of drug added in plasma samples and plotted as a function of the chromatographic peak area (Fig. 2). The calibration curve presented a r² value of 0.99984. The curve was constructed from five replicate measurements of five different drug concentrations over an interval of 0.1 to 50 µg/ml.

View larger version (15K): [in this window] [in a new window]   Fig. 2. HPLC calibration curve of mouse plasma samples spiked with known amounts of VGX-1027. The curve was constructed from five replicate measurements of five different drug concentrations over an interval of 0.1 to 50 µg/ml

Clinical Evaluation of Diabetes. NOD mice were examined for diabetes development by twice weekly determination of glycosuria followed, when positive, by measurement of glycemia. The NOD mice were defined as diabetic when fasting blood glucose levels exceeded 11.8 mM for 2 consecutive days. The development of the disease in MLD-STZ-induced diabetes was evaluated by measuring blood glucose level and body weight loss on a weekly basis. For determination of blood glucose levels in MLD-STZ diabetes, nonfasting mice were bled at the indicated time points from the retro-orbital venous plexus with heparinized capillary tubes. The plasma glucose concentration was determined by a glucose oxidase method, using a blood glucose meter with electrode (Sensimac; IMACO GmbH, Lüdersdorf, Germany).

Spontaneous and Accelerated Diabetes in NOD Mice and in Vivo Treatments. To evaluate the impact of VGX-1027 on the development of spontaneous type 1 diabetes in the NOD mouse, euglycemic female 12-week-old NOD mice were treated i.p. with either 20 mg/kg b.wt. VGX-1027 or its vehicle six times a week from the 12th until the 25th week of age. An additional control group of mice was left untreated.

For induction of diabetes with CY, the drug was dissolved in water for injection and injected i.p. at the dose of 200 mg/kg b.wt. into 12- to 14-week-old euglycemic NOD mice. For adoptively transferred diabetes, 1 x 10⁷ spleen cells from acutely diabetic mice were injected i.v. to euglycemic 4-week-old female NOD mice.

Both in CY-induced and adoptively transferred diabetes, the mice were treated i.p. with either 20 mg/kg b.wt. VGX-1027 or its vehicle daily, six times a week, starting 1 day after the diabetogenic challenge. Treatment was continued until the end of the studies, 2 weeks after CY injection or 40 days after cell transfer. At the end of the experiment, these latter mice were sacrificed, and pancreata specimens were collected for histological analyses.

Histological Evaluation of Insulitis in NOD Mice. Mononuclear cell infiltration of the pancreatic islets was graded in a blind fashion as described elsewhere (Nicoletti et al., 1996): 0, no infiltrate; 1, periductular infiltrate; 2, peri-islet infiltrate; 3, intraislet infiltrate; and 4, intraislet infiltrate associated with cell destruction. At least 15 islets were counted for each mouse. A mean score for each pancreas was calculated by dividing the total score by the number of islets examined. Insulitis scores are expressed as mean values ± S.D.

MLD-STZ-Induced Diabetes in CBA/H Mice and in Vivo Treatments. Immunoinflammatory diabetes was induced with MLD-STZ as described earlier (Lukic et al., 1991). In brief, STZ was dissolved in citrate buffer, pH 4.5, and injected i.p. at doses of 40 mg/kg (low dose) daily for 5 consecutive days. Day 0 was defined as the first injection of STZ.

To evaluate the effect of VGX-1027 on disease development, the drug was administered as a continuous 12-day treatment, as either an "early" or "late" prophylactic regimen: for the former, treatment of the mice started 1 day before MLD-STZ, and for the latter, it started the day after the last STZ dose. VGX-1027 was dissolved in 500 mM Na₂HPO₄ (pH 8.5), further diluted in H₂O, and administered to mice either i.p. or p.o., as indicated under Results. A daily i.p. dose of VGX-1027 was 10 or 20 mg/kg b.wt., whereas 100 mg/kg b.wt. was used for p.o. treatment. The control mice were treated under similar experimental conditions with an equivalent amount of vehicle.

Histology and Immunohistochemical Analysis of Pancreas in MLD-STZ Diabetic Mice. In selected experiments in MLD-STZ-induced diabetes, the pancreata from individual mice (n = 7–8 per group) were fixed in 10% formalin, embedded in paraffin, sectioned, and routinely stained with hematoxylin and eosin for histological examination by light microscopy. Multiple nonconsecutive sections (4) randomly selected from each pancreas (a total of 20–40 islets/animal) were analyzed.

Immunohistochemical analyses were performed on frozen 9-µm-thick pancreata sections obtained from mice with MLD-STZ-induced diabetes as described elsewhere (Lukic et al., 1991) or 5-µm-thick paraffin sections. To optimize immunohistological staining of paraffin sections, the microwave antigen retrieval technique was used (Penkowa and Hidlago, 2000). In brief, deparaffinized sections were immersed in 0.01 M sodium citrate buffer (pH 6.0), boiled in a 750-W microwave oven for 10 min, and cooled to room temperature. Staining was performed using primary monoclonal Abs: guinea pig antiswine insulin Ab, strongly cross-reactive with insulin from several mammalian species, including mouse (Tian et al., 2004) (A0564; Dako, Hamburg, Germany), and rabbit anti-mouse iNOS (N 7782; Sigma). For detection, we then used the ExtrAvidin peroxidase staining kit (Sigma) with 3,3'-diaminobenzidine as substrate. Sections were counterstained with Mayer's hematoxylin and mounted in Canada balsam.

Apoptosis was assessed with a basic terminal deoxynucleotidyl transferase (TUNEL) assay a using TACS TdT in situ apoptosis detection kit (R&D Systems, Oxford, UK) according to the manufacturer's guidelines. The slides were then counterstained in methyl green and examined on a Zeiss Axiolab microscope. At least 4 mice/condition and 20 to 40 islets/mouse were examined.

Cell Preparations and Cultures. Pancreas, spleen, and resident peritoneal cells (PC) were collected from individual mice given MLD-STZ and treated with either VGX-1027 or its vehicle, on day 15 after the first injection of STZ, as well as from normal untreated animals. The pancreatic islets, SMNC, and PC were prepared as described previously (Stosic-Grujicic et al., 2004; Cvetkovic et al., 2005). Cell culture supernatants, used for ex vivo detection of cytokines and NO, were obtained by culturing the cells for 48 h in 24-well Limbro culture plates in 1 ml of a standard medium (1 x 10⁶ PC, 5 x 10⁶ SMNC, or 1 x 10⁵ islets/well).

Culture of Murine Insulinoma Cell Lines. Mouse insulinoma MIN6 cells (with permission of Dr. J.-I. Miyazaki, Osaka University, Osaka, Japan) (Miyazaki et al., 1990) and the rat insulinoma RIN-m5F cell line were kindly donated by Dr. Karsten Buschard (Bartholin Instituttet, Copenhagen, Denmark). Cells were cultured in tissue culture flasks (Sarsted, Numbrecht, Germany) in RPMI 1640 medium containing 10% fetal calf serum, until reaching 80% confluence, when they were detached by standard trypsinization procedure. Cells were washed and seeded for cytokine and NO production into 96-well flat-bottomed cell culture plates (6 x 10⁴/well) in the presence of various combinations of recombinant mouse cytokines (IL-1, IFN-, and/or TNF-), with or without VGX-1027. Culture supernatants were collected after 48 h. In some experiments the assessment of cell viability after cytokine treatment was performed. The cytotoxic action of cytokines was evaluated by fluorescein diacetate (FDA) staining and by crystal violet colorimetric assay (Kaludjerovic et al., 2005) after 24 and 48 h of cell cultivation, respectively, at the same conditions as described above. The nonfluorescent molecule FDA is hydrolyzed inside the viable cells to fluorescein by the intracellular esterases, the activity of which is reduced in the cells undergoing apoptosis. To discriminate live and dead cells, FDA dissolved in RPMI 1640 medium was added in cell cultures (100 ng/ml) and after 20 min at 37°C, cells were trypsinized, washed twice, and analyzed on FACSCalibur flow cytometer using CellQuestPro software (Becton Dickinson, Heidelberg, Germany). FDA-positive cells were considered viable. Staining with crystal violet has been used as a test for the viability of adherent cells (Kaludjerovic et al., 2005). At the end of incubation, cell cultures were washed with phosphate-buffered saline to remove nonadherent dead cells; the remaining adherent cells were fixed with methanol and stained with 1% crystal violet. After thorough washing, the stain was extracted by the addition of 33% acetic acid, and the absorbance of the dissolved dye, corresponding to the number of live adherent cells, was measured at 570 nm in a microplate reader.

Nitric Oxide Production. Nitrite accumulation, an indicator of NO release in the supernatant, was determined using the Griess reaction as described previously (Cvetkovic et al., 2005). In brief, samples of cell-free culture supernatants were mixed with an equal volume of Griess reagent (a 1:1 mixture of 0.1% naphthylethylenediamine dihydrochloride and 1% sulfanilamide in 5% H₃PO₄). After 10 min, the optical density was measured at 570 nm in a microplate reader and compared with a standard curve of NaNO₂.

Measuring of Cytokines in Culture Supernatants. Cell culture supernatant samples were analyzed in duplicate for murine TNF-, IL-1, IFN-, and IL-10 by ELISA using anti-mouse paired antibodies (R&D Systems) according to manufacturer's instructions.

Cell-Based ELISA. The ex vivo expression of MIF in PC and islets of Langerhans was determined by a cell-based ELISA according to a previously described protocol (Cvetkovic et al., 2005). Rabbit anti-mouse MIF antibody (at 1:2000 dilution) was used as a primary Ab, whereas the detecting Ab was horseradish peroxidase-conjugated anti-rabbit IgG (U.S. Biochemical Corp., Cleveland, OH) at 1:2000 dilution. The data obtained by measuring the light absorbance at 492 nm were corrected for differences in cell number by staining the cells with crystal violet after the ELISA procedure.

Semiquantitative Reverse Transcriptase-PCR. Total RNA was isolated from pancreatic islets with TRIzol reagent (Genosys, Woodlands, TX) according to the manufacturer's instructions. RNA was reverse-transcribed using Moloney leukemia virus reverse transcriptase and random primers (Pharmacia, Uppsala, Sweden). PCR amplification of cDNA with primers specific for TNF- and -actin as a housekeeping gene was carried out in a Mastercycler Gradient thermal cycler (Eppendorf, Hamburg, Germany) as follows: 30 s of denaturation at 95°C, 30 s of annealing at 58°C, and 30 s of extension at 72°C. For each gene, preliminary experiments were conducted to ascertain that amplification of cDNA was in the linear range under the respective cycling conditions. For TNF-, the primers were sense, 5'-ACGCTCTTCTGTCTACTGAAC-3', and antisense, 5'-CTTGTCCCTTGAAGAGAACC-3', and the PCR product was 304 base pairs. The primers for -actin were sense, 5'-TCCTTCTTGGGTATGG-3', and antisense, 5'-ACGCAGCTCAGTAACAG-3', and the PCR product was 358 base pairs. The PCR products were visualized by electrophoresis through 2.5% agarose gels containing ethidium bromide, gels were photographed, and results were analyzed by densitometry using Kodak 1D 3.6 software.

Statistical Analysis. Results are shown as mean values ± S.D. Unless otherwise specified, the statistical significance of differences between groups was evaluated using analysis of variance, followed by a Student-Newman-Keuls test for multiple comparisons between treatment groups, an unpaired Student's t test for means between two groups, or a ² test as indicated. In the spontaneous model of diabetes of NOD mice, differences in the kinetic and cumulative incidence of the diabetes were assessed by the log-rank test (Mantel-Cox). A p < 0.05 was considered to be significant.

	Results

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In Vitro Effects of VGX-1027 on the Production of TNF- and NO by Islet Cells and on Their Survival. The exposure of islet cells to IL-1 in combination with IFN- and/or TNF- is known to induce severe functional suppression and death (Rabinovitch and Suarez-Pinzon, 1998; Eizirik and Mandroup-Poulsen, 2001). Therefore, experiments were carried out to ascertain whether VGX-1027 could modulate production of proinflammatory mediators as well as survival of cytokine-induced pancreatic islet death in vitro. Because the sensitivity of the islets to the action of cytokines is known to vary among different species, we carried out the experiments in freshly isolated pancreatic islets and rat (RIN-m5F) and mouse (MIN6) insulinoma cell lines as cell models. Exposure of MIN6 or RIN-m5F cells to 5 ng/ml of recombinant IL-1 + IFN- for 48 h resulted in the production of TNF- (Fig. 3A). Likewise, overnight cytokine exposure of freshly isolated islets and RIN-m5F cells resulted in high levels of nitrite (Fig. 3B). VGX-1027 (10 µg/ml) significantly inhibited both IL-1/IFN--induced TNF- and nitrite accumulation (Fig. 3, A and B). We also assessed the capacity of VGX-1027 to interfere with the cytotoxic effects of the cytokines by examining the death and survival of cells. Results from FDA staining (Fig. 3C) and crystal violet assay (Fig. 3D) of MIN6 cells showed that a significant increase in cell survival was observed in the presence of VGX-1027. In contrast, the protective effect of VGX-1027 was annulled by exposure to exogenous TNF-. These in vitro results urged us to proceed with testing of VGX-1027 in various preclinical models of T1D.

View larger version (21K): [in this window] [in a new window]   Fig. 3. In vitro effects of VGX-1027 on the production of TNF- and NO and survival of islet cells. TNF- production was determined in MIN6 or RIN-m5F cell culture supernatants (A) and NO accumulation in fresh pancreatic islets or MIN6 cells (B). Viability of MIN6 cells was determined by colorimetric assay with FDA staining (C) or crystal violet staining (D). *, p < 0.05 refers to treatment with IFN-/IL-1.

Pharmacokinetic Analysis of VGX-1027 in Mice. A highly sensitive and selective analytical method was developed to allow the determination of VGX-1027 in the microgram range in plasma samples. This procedure consisted of the use of solid-phase extraction and reversed-phase high-performance liquid chromatography with ultraviolet detection.

Figure 4A shows a retention time for VGX-1027 of 5.027 ± 0.3 min. The lower limit of detection was 0.1 µg/ml, whereas the lower limit of quantification was 0.5 µg/ml. The high selectivity of the chromatographic method is proven by the absence of an interfering peak at the retention time of VGX-1027 (Fig. 4, A and B).

View larger version (11K): [in this window] [in a new window]   Fig. 4. Typical HPLC chromatograms of plasma samples of untreated mice (A), plasma samples of mice spiked with the drug (10 µg/ml) (B), and plasma samples of mice treated with VGX-1027 2 h after i.p. administration (C). The black arrow in A shows the time at which the peak of VGX-1027 has to appear.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Plasma concentration of VGX-1027 as a function of time after i.p. administration in mice.

VGX-1027 Prevents Development of Spontaneous Type 1 Diabetes in NOD Mice and Also Counteracts Accelerated Diabetogenesis Induced by CY Challenge or Adoptive Transfer of Diabetogenic Spleen Cells in NOD Mice. Most of the female NOD mice that were either treated with the vehicle of VGX-1027 (13/16, 81.3%) or left untreated (12/16, 75%) developed diabetes by the end of the study at the age of 25 weeks. In contrast, prolonged treatment with VGX-1027 markedly and significantly reduced the cumulative incidence of the disease in these mice with only 5 of 16 mice (31.3%) becoming diabetic by this age (Fig. 6A) [p < 0.0001 versus both vehicle-treated controls and untreated NOD mice by the log-rank test (Mantel-Cox)]. Importantly, prolonged treatment with the drug appeared to be well tolerated by the mice as judged from their behavior and general appearance. No difference in food consumption was observed between either the mice treated with VGX-1027 and the mice treated with the vehicle or left untreated (data not shown). Accordingly, there were no significant differences in body weight gain between the mice treated with VGX-1027 or its vehicle or the mice that were left untreated and remained euglycemic throughout the study period [the diabetic mice were not considered in the analysis as they characteristically lose body weight after disease onset (data not shown)].

View larger version (19K): [in this window] [in a new window]   Fig. 6. Reduction of spontaneous and adoptively transferred type 1 diabetes in NOD mice by VGX-1027. A, euglycemic female 12-week-old NOD mice were treated i.p. with either 20 mg/kg b.wt. VGX-1027 or its vehicle six times a week from the 12th until the 25th week of age. An additional control group of mice was left untreated. Each group consisted of 16 mice. By the 25th week of age, the incidence of diabetes was 81.3% (13/16) in the mice treated with the vehicle of VGX-1027, 75% (12/16) in the mice left untreated, and 31.3% (5/16) in the mice treated with VGX-1027. The data are representative of two independent experiments that were merged because of interstudy variability <10%. Statistical analysis was performed by a log-rank (Mantel-Cox). B, diabetes was induced by i.v. transfer of 1 x 107 spleen cells from acutely diabetic mice to euglycemic 4-week-old female NOD mice. The mice were treated i.p. with either 20 mg/kg b.wt. VGX-1027 or its vehicle daily, six times a week starting 1 day after the diabetogenic challenge. Each group consisted of 16 mice. On day 40 post-transfer, the incidence of diabetes was 81.2% (13/16) in the vehicle-treated mice and 25% (4/16) in the mice receiving VGX-1027. The data are representative of two independent experiments that were merged because of interstudy variability <10%.

Concurring with the antidiabetogenic effects of VGX-1027 observed in the spontaneous model of diabetes, the drug turned out to markedly reduce the development of the disease in accelerated models of autoimmune diabetogenesis. VGX-1027 was first tested in the model of CY-induced diabetes. As expected, within 2 weeks after the challenge, most of the control NOD mice that were treated with the vehicle of VGX-1027 (15/20, 75%) developed classic signs of diabetes with two or more consecutive days of glycosuria and fasting hyperglycemia. In contrast, the incidence of diabetes was significantly lower in the mice that were treated with VGX-1027, with only 6/20 (30%) having developed diabetes during the same period of time (p = 0.01 versus vehicle-treated controls by ²) (not shown in Fig. 6).

In another model of accelerated diabetes in NOD mice, starting from 23 days after the transfer of spleen cells from acutely diabetic donors, the control mice treated with the vehicle of VGX-1027 progressively started to develop diabetes with a cumulative incidence of 81.2% (13/16) on day 40 post-transfer (Fig. 6B). In agreement with the preventive effects of VGX-1027 in CY-induced diabetes, the drug was also capable of significantly reducing the development of adoptively transferred diabetes in NOD mice as only 25% (4/16; p = 0.005 versus vehicle-treated controls by ²) of the VGX-1027-treated mice developed diabetes within the same period of time. In the VGX-1027-treated mice that developed disease, there was no significant difference in the kinetics of disease development between VGX-1027 and vehicle-treated diabetic mice (30.3 ± 3.0 versus 31.6 ± 4.1 days, respectively). Concurring with the clinical findings, histological analyses carried out in pancreatic specimens obtained in these two groups of mice at the end of the study showed that VGX-1027-treated mice had a significantly milder form of insulitis than the vehicle-treated controls (insulitis score = 1.94 ± 0.91 versus 2.91 ± 0.8, respectively; p = 0.003 by one-way analysis of variance).

VGX-1027 Treatment Reduces Clinical Signs of MLD-STZ-Induced Diabetes and Suppresses Pathohistological Changes of Pancreas. To evaluate the ability of VGX-1027 to interfere with immunoinflammatory diabetogenic pathways induced by MLD-STZ, CBA/H mice were treated i.p. daily for 12 consecutive days with either 10 or 20 mg/kg b.wt. of the drug. The group of control mice that were challenged with MLD-STZ and received the vehicle from day–1 to day 10 developed persistent hyperglycemia that started from 2 weeks after the first injection of the STZ. In contrast, mice treated under the same experimental conditions with VGX-1027 exhibited a dose-dependent reduction in blood glucose levels (Fig. 7A). This protection did not depend on continuous application of the drug, because none of the mice developed hyperglycemia throughout the entire follow-up period after treatment withdrawal. Moreover, VGX-1027 (100 mg/kg b.wt./day) showed a similar effect when given p.o. (Fig. 7B).

View larger version (60K): [in this window] [in a new window]   Fig. 7. Effect of VGX-1027 treatment on the MLD-STZ-induced hyperglycemia, body weight loss, and histopathology of pancreatic islets. A, B, and C, plasma glucose levels in control CBA/H mice receiving STZ (40 mg/kg/day, for 5 consecutive days) in conjunction with 12 daily i.p. injections of vehicle (STZ) or in mice treated with STZ and VGX-1027 given as a continuous 12-day treatment, applied according to the following treatment protocols: A, different doses of VGX-1027, 20 and 10 mg/kg b.wt., administered i.p. from day -1 to day +10; B, 100 mg/kg b.wt. of VGX-1027, administered p.o. from day -1 to day +10; C, 20 mg/kg b.wt. VGX-1027 administered i.p. as an early (STZ plus VGX-1027 early) or late (STZ plus VGX-1027 late) prophylactic treatment (as described under Materials and Methods). D, percent change in body weight from the start of the experiment, determined in mice treated according to protocol C. Results from a representative experiment are presented as the means ± S.D. for seven to eight mice per group. *, p < 0.05 refers to treatment with MLD-STZ. E and F, light micrographs showing morphological profiles of pancreatic islets by day 62 after disease induction (hematoxylin and eosin staining). E, control MLD-STZ-treated animals; note atrophy and loss of islet margins. F, MLD-STZ-treated animals after early prophylactic i.p. treatment with VGX-1027; note the well-preserved morphology. G and H, immunocytochemical detection of insulin expression in pancreatic islets by day 27 after disease induction (immunoperoxidase staining). G, control MLD-STZ-treated animals; note the paucity of insulin-containing cells. H, MLD-STZ-treated animals after late prophylactic i.p. treatment with VGX-1027; note the homogeneous pattern of well-preserved insulin-containing cell mass. I and J, detection of apoptotic cells in pancreatic islets by day 15 post-MLD-STZ treatment (TUNEL staining); note the increased number of apoptotic cells in control MLD-STZ-treated animals (I) compared with VGX-1027-treated mice (J). E through J, images are representative of four to eight animals per group. Original magnification, 400x.

We next evaluated the impact of VGX-1027 on histopathological events taking place at the level of pancreatic cells during development of diabetes in MLD-STZ-induced diabetes. Representative examples of the light microscopic evaluation of the islets are presented in Fig. 7, E to J. We have previously reported the appearance of a limited but significant insulitis and subsequent depletion of cells in mice by 10 to 20 days after MLD-STZ administration (Lukic et al., 1991). Consistent with our earlier studies, as early as 15 days after the injections of MLD-STZ, mild mononuclear cell infiltrates of endocrine pancreas accompanied by initial necrotic changes could be observed (not shown). From this period up to the end of the serial measurement of blood glucose levels (8 weeks), the destructive process progressed, and most of the islets lost clear margins (Fig. 7E). By contrast, the prophylactic administration of VGX-1027 abrogated the development of these changes. The pancreatic sections from VGX-1027-treated mice did not harbor a substantial infiltrate, and the majority of pancreatic islets appeared normal, with no signs of inflammation (Fig. 7F). Concordant with the clinical status of these mice, the distribution of insulin-positive cells (Fig. 7H) was normal long after treatment cessation. Moreover, histological evidence for apoptosis was consistently found in the control diabetic mice, as evidenced by TUNEL staining (Fig. 7I), whereas in the islets of VGX-1027-treated mice no such evidence for apoptosis was found throughout the entire follow-up period (Fig. 7J), indicating that treatment with VGX-1027 preserved islets from autoimmune attack, thus enabling appropriate regulation of plasma glucose levels. These data confirmed that the antidiabetogenic effect of VGX-1027 was maintained at both the clinical and histological level.

VGX-1027 Treatment Down-Regulates the Production of Proinflammatory Mediators. To elucidate the mechanisms by which VGX-1027 treatment prevents disease onset, functional studies were performed on both pancreas and peripheral compartments during early progression of the disease. Hence, pancreatic islets, splenocytes, and peritoneal cells were harvested on day 15 after MLD-STZ injection from mice that had been treated i.p. daily for 12 consecutive days with either VGX-1027 (20 mg/kg) or its vehicle, and the levels of the diabetogenic mediators TNF-, IL-1, MIF, and NO were examined ex vivo.

As shown in Fig. 8, both pancreatic and peripheral immune cells established from mice treated with 0.5 mg of VGX-1027 i.p. released significantly reduced amounts of TNF- compared with control diabetic animals. The inhibition by VGX-1027 treatment in PC (Fig. 8A) and SMNC (Fig. 8B) was 46.6 and 93.4%, respectively, and in the islets was 91.4% (Fig. 8C). Consistent with this finding, a marked reduction in mRNA expression was detected between pancreatic islets isolated from VGX-1027-treated versus diabetic mice (inhibition of 61.3%), indicating that the VGX-1027 effect occurs through down-regulation of TNF- gene expression (Fig. 8D).

View larger version (26K): [in this window] [in a new window]   Fig. 8. Effect of VGX-1027 treatment on TNF- expression. PC (A), SMNC (B), and pancreatic islets (C and D) were isolated from mice that were not challenged with STZ, (STZ-untreated) or treated with STZ and vehicle (STZ+vehicle) or STZ and VGX-1027 (STZ+VGX-1027) under the early prophylactic i.p. regimen by day 15 after disease induction. Production of TNF- was measured by ELISA in the 48-h culture supernatants of cells. Results are given as means ± S.D. for five mice per group, done in duplicate. D, expression of TNF- mRNA was assessed by RT-PCR. Results from the representative of three separate experiments with similar results are presented as relative expression of TNF- mRNA compared with -actin. *, p < 0.05 refers to the control group treated with vehicle.

View larger version (32K): [in this window] [in a new window]   Fig. 9. Effect of VGX-1027 treatment on the expression of MIF, secretion of IL-1, NO production, and iNOS expression. PC (A, C, and E) and pancreatic islets (B, D, and F) were isolated from the same group of mice as described in the legend to Fig. 5. A and B, MIF expression was determined by a cell-based ELISA as described under Materials and Methods. C and D, production of IL-1 was measured by ELISA in the 48-h culture supernatants of cells. E and F, nitrite accumulation was measured by the Griess reaction in the 48-h culture supernatants of cells. Results are given as means ± S.D. for five mice per group, done in triplicate. *, p < 0.05 refers to treatment with MLD-STZ. G and H, immunocytochemical detection of iNOS expression in pancreatic islets by day 15 after disease induction (immunoperoxidase staining). G, control MLD-STZ-treated animals; note the strong staining of iNOS-containing cells. H, MLD-STZ-treated animals after early prophylactic i.p. treatment with VGX-1027; note the absence of iNOS-containing cells. Original magnification 400x.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
VGX-1027 is an orally bioactive small isoxazole compound that is being developed for the treatment of immune-mediated pathological conditions. We have shown here for the first time that VGX-1027 exhibited multiple and powerful antidiabetogenic effects including prevention of cell death and suppression of clinical and histological signs of immunoinflammatory diabetes both in spontaneous and accelerated models of the disease in the NOD mouse as well as in mice with MLD-STZ-induced diabetes.

Although pathogenic and immunotherapeutic differences occur between MLD-STZ-induced diabetes and NOD mouse diabetes, it is believed that in both models the damaged pancreatic islets ultimately become targets for cell-mediated (auto)immune reactions mounted from T lymphocytes and macrophages, which migrate into the pancreatic islets. Cell destruction could result from the toxic effect of free radicals (, and NO·), cytokines (IL-1, TNF-, TNF-, and IFN-), and other inflammatory products released by activated macrophages and T cells, as well as from cells themselves (Like and Rossini, 1976; Kolb, 1987; Lukic et al., 1998; Rabinovitch, 1998; Rabinovitch and Suarez-Pinzon, 1998; Wachlin et al., 2003; Gurgul et al., 2004; Shoda et al., 2005). In addition, it has been shown that TNF- and IL-1 may increase vulnerability of pancreatic cells to autoimmune destruction by inducing the expression of Fas on the cell surface (Wachlin et al., 2003). Therefore, the capacity of VGX-1027 to counteract the generation of TNF- and NO by cytokine-stimulated islet cells in vitro implies that its antidiabetogenic property relies on the down-modulation of cytotoxic mediators within the islets. Although the mechanism by which VGX-1027 regulates the level of TNF- expression is not clear, it seems that VGX-1027-mediated rescue of islet cells is secondary to reduced synthesis of the cytokine rather than to antagonism of its bioactivity as the addition of exogenous TNF- reversed VGX-1027-mediated protection.

In agreement with these in vitro observations, VGX-1027 showed beneficial effects in both spontaneous and accelerated forms of diabetes in NOD mice. Likewise, in MLD-STZ-induced diabetes, VGX-1027 counteracted the diabetogenic effects of the toxin even when administered in a late prophylactic regimen after the last injection of STZ. Ex vivo studies carried out in this model showed that VGX-1027 acted in a bimodal manner, entailing both a direct protective effect on the cells as well as a profound impact on key immunopathogenic events associated with disease development, including diminished production of proinflammatory and cytotoxic mediators by macrophages and pancreatic cells.

An interesting immunopharmacological characteristic of VGX-1027 arising from these data is its impact on the macrophage secretory capacity of the type 1 proinflammatory cytokines TNF-, IL-1, and MIF and also of the pleiotropic soluble immunomodulatory mediator NO. Type 1 cytokines of the innate immune system have been proposed as key players in initiating, coordinating, and maintaining cell destruction during autoimmune diabetogenesis (Beyan et al., 2003). NO has repeatedly been reported to synergize with cytokines in inducing apoptosis of pancreatic cells possibly via potentiation of c-Jun NH₂-terminal kinase activity and inhibition of Akt (Storling et al., 2005). In agreement with these observations, we and others have shown that negating the actions of endogenous TNF-, IL-1, MIF, and NO either with specific inhibitors or by gene deletion prevents development of autoimmune diabetes in NOD mice and/or mice given MLD-STZ (Lukic et al., 1991; Nicoletti et al., 1994; Sandberg et al., 1994; Yang et al., 1994; Flodstrom et al., 1999; Holstad and Sandler, 2001; Suarez-Pinzon et al., 2001; Drage et al., 2002; Wang et al., 2002; Thomas et al., 2004). However, note that it is unlikely that prevention of diabetes by VGX-1027 is due solely to interference with priming and activation of lymphocytes, because the protective effect was also achieved by late prophylactic treatment, after the initial induction of the disease.

The progression of T1D in mice is marked by two general "checkpoints": the first is associated with "benign" insulitis with limited cell destruction, when animals remain diabetes-free, and the second corresponds to the shift to "aggressive" insulitis when cells are destroyed to promote overt diabetes (Andre et al., 1996). The transition from benign to pernicious insulitis requires an islet cell response to TNF- (Pakala et al., 1999), which is consistent with the recent observation that TNF- plays a central role in the effector function of diabetogenic CD4⁺ Th1 T cell clones (Cantor and Haskins, 2005). The marked inhibitory effects of VGX-1027 on in vitro and in vivo production of TNF- at the pancreatic level as well as the fact that VGX-1027-induced reduction of TNF- synthesis correlated with reduced severity of pancreatic inflammation all suggest that inhibition of TNF- may represent an important mechanism by which VGX-1027 influences the transition to aggressive insulitis and hence to the clinical appearance of diabetes. Nonetheless, because the pathogenic effect of endogenous TNF- in NOD mice is age-dependent and occurs primarily in the early stages of the diabetogenic process (Yang et al., 1994; Pakala et al., 1999; Green and Flavell, 2000; Christen et al., 2001; Cantor and Haskins, 2005), it is possible that the antidiabetogenic action of VGX-1027 might have depended on its presently demonstrated capacity to simultaneously inhibit the production of other diabetogenic mediators such as IL-1, MIF, and NO.

In immunoinflammatory diabetes, the Th1 response has been connected with a cell-destructive insulitis, whereas a Th2/3-type response was associated with protection from the disease (Kolb, 1997; Rabinovitch and Suarez-Pinzon, 1998). Pharmacological compounds capable of converting the predominant Th1 state to a Th2/Th3 state have been considered suitable candidates for the prevention and early treatment of the disease. However, despite its in vitro and in vivo antidiabetogenic efficiency as well as its capacity to prevent pancreatic and/or macrophage production of diabetogenic type 1 proinflammatory cytokines and NO, VGX-1027 failed to influence the production of the two prototypical Th1 and Th2 cytokines, IFN- and IL-10. Knowing the powerful stimulatory effects of IFN- on macrophages, these data suggest that VGX-1027 inhibits macrophage functions in an IFN--independent manner.

Two observations of particular relevance from the clinical point of view are the apparent low toxicity of the drug that was demonstrated by the lack of characteristic body weight loss occurring in MLD-STZ-induced diabetes, which, unlike that in control mice, was not observed in VGX-1027-treated mice, and the efficacy of the drug to prevent MLD-STZ diabetes equally regardless of whether it was given i.p. or p.o. The proper evaluation of VGX-1027 in the treatment of patients with newly diagnosed T1D will require preclinical studies carried out under "therapeutic" dosing conditions to animals with established disease. Nonetheless, the capacity of the drug to counteract the diabetogenic effects of MLD-STZ even when administered as a late prophylactic regimen, started 1 day after the last of the five injections of the toxin had been given, is encouraging for the translation of these findings to the clinical setting. As this is a period of time when early diabetogenic pathways are fully activated in this model (Karabatas et al., 2005), the above findings indicate that VGX-1027 is capable of delaying/reversing an already initiated process of cell destruction and implies its possible prophylactic use in individuals at risk for developing T1D who can be selected on the basis of immunological markers associated with actively ongoing cell destruction. In addition, if the long-term persistence of the antidiabetogenic effects of VGX-1027 observed in MLD-STZ diabetic mice after treatment interruption also occur in humans, this would have the obvious advantage of avoiding the requirements for prolonged and possibly lifelong treatment of patients with both newly diagnosed T1D and individuals at risk for development of the disease.

Finally, the preliminary observation emerging on ex vivo analyses that VGX-1027 may preferentially inhibit immunoinflammatory events leading to inflammation and destruction of pancreatic islets without influencing IFN- and IL-10 production may also be important in the clinical setting as it might predict down-regulation of the immune system that spares its physiological functioning. Preservation of the IL-12-IFN- axis might indicate that VGX-1027 may be less likely than other immunosuppressants to provoke compromise of innate immunity with consequential reduced immunity to opportunistic pathogens (Ware, 2005).

Taken together, these findings all indicate that VGX-1027 has an immunopharmacological and toxicological profile. Thus, VGX-1027 is worthy of being further studied for its use in the prevention and early treatment of human T1D and other autoimmune diseases.

	Acknowledgements

 
We acknowledge the kind assistance of Drs. Jun-ichi Miyagaki (Osaka University, Osaka, Japan) and Karsten Buschard (Bartholin Instituttet Kommunehospitalet, Copenhagen, Denmark) for providing the MIN6 and RIN-m5F cell lines. We also thank Dr. Marija Mostarica-Stojkovic (Institute for Microbiology and Immunology, School of Medicine, University of Belgrade, Belgrade, Serbia) and Dr. C. Jo White (VGX Pharmaceuticals, Blue Bell, PA) for critical review of the manuscript.

	Footnotes

 
This work was partly supported by The Serbian Ministry of Science (Grants 143029 and 145066).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.109272.

ABBREVIATIONS: T1D, type 1 diabetes; MLD-STZ, multiple low doses of streptozotocin; TNF, tumor necrosis factor; IFN, interferon; IL, interleukin; NO, nitric oxide; SMNC, spleen mononuclear cells; LPS, lipopolysaccharide; VGX-1027, (S,R)-3-phenyl-4,5-dihydro-5-isoxasole acetic acid; STZ, streptozocin; CY, cyclophosphamide; HPLC, high-performance liquid chromatography; iNOS, inducible nitric-oxide synthase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; PC, peritoneal cells; PCR, polymerase chain reaction; FDA, fluorescein diacetate; ELISA, enzyme-linked immunosorbent assay; MIF, macrophage migration inhibitory factor; Th, T helper type.

Address correspondence to: Dr. Ferdinando Nicoletti, Department of Biomedical Sciences. Via Androne, 83, 95124, Catania, Italy. E-mail: ferdinic{at}unict.it

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