Troglitazone exhibits immunomodulatory activity on the cytokine production of activated human lymphocytes.

Troglitazone (TGL), a thiazolidinedione compound that improves the response of peripheral target tissue to insulin, also has anti-inflammatory properties, a potential means of protection from Type 1 (insulin dependent) diabetes. In order to test the ability of TGL to affect cytokine production, peripheral blood mononuclear cells from healthy donors were exposed to TGL in the presence or absence of a polyclonal activator (PHA) and the production of cytokines assayed. TGL enhanced PHA response, promoted secretion of the cytokines granulocyte and macrophage colony-stimulating factor and leukaemia inhibitory factor and inhibited tumour necrosis factor-alpha secretion, consistent with causing Th-2 differentiation in T-cells. These results suggest that TGL is capable of modulating cytokine production and could therefore influence Th1/Th2 differentiation.

Fas and Fas ligand-mediated apoptosis and its role in autoimmune diabetes.

The relationship between Fas-mediated apoptosis and Type 1 diabetes is currently under investigation. Fas/Fas ligand interaction could be involved both in the insulitis process and in beta-cell death. Nevertheless, different mechanisms appear to be involved in human Type 1 diabetes and in NOD mice. In the present work, we review recent evidence of the role of the Fas/Fas ligand system in human and NOD mouse diabetes, describing possible hypotheses for its involvement in the pathogenesis of the disease, with possible implications for therapy and islet transplantation.

Pharmacokinetic and immunogenic behavior of three recombinant human GM-CSF-EPO hybrid proteins in cynomolgus monkeys.

MEN 11,300, MEN 11,301, and MEN 11,303 are three recombinant human hybrid proteins that, as has recently been described, induce in vitro erythroid differentiation. This article provides data on their pharmacokinetic and immunogenic behavior after repeated i.v. administration to cynomolgus monkeys at 0.8 or 1.6 micrograms/kg doses. Pharmacokinetic data, obtained after the first administration, showed that the half-life (t1/2) and clearance (CL) values are dose dependent, with no significant differences among the three hybrid proteins. After the tenth administration, MEN 11,300 and MEN 11,301, both a high and low dose, and MEN 11,303 at high dose were undetectable in plasma, whereas MEN 11,303 at the lower dose showed no alteration in its pharmacokinetic profile. Immunologic analyses of plasma provided an explanation for this different pharmacokinetic behavior. In fact, plasma samples from animals treated repeatedly with MEN 11,300 and MEN 11,301 showed specific antibody formation in response to both the high- and the low-dose regimens. These antibodies exerted in vitro a strong neutralizing activity of the hybrid proteins, with a predominant specificity for the erythropoietin (EPO) portion. By contrast, MEN 11,303 at the lower dose did not induce a detectable antibody response whereas the antibodies observed on the high-dose regimen did not exert neutralizing activity against the hybrid proteins nor against granulocyte-macrophage colony-stimulating factor (GM-CSF) or EPO. Hematologic parameters were not affected by the treatments, thus indicating that the anti-EPO neutralizing antibody response does not cross react with the endogenous monkey cytokine. The overall immunogenicity data suggest that among the three fusion proteins, MEN 11,303 could have a lower immunogenic potential.

Troglitazone prevents insulin dependent diabetes in the non-obese diabetic mouse.

Troglitazone has recently been introduced in the treatment of Type 2 diabetes. In addition to its anti-diabetic effects it acts as a perixosome proliferator activated receptor-gamma (PPAR-gamma) agonist and has anti-inflammatory properties by inhibiting macrophage tumour necrosis factor-alpha (TNF-alpha) secretion. It also inhibits the production of endothelial selectin (e-selectin). Troglitazone also reduces interleukin-1alpha induced nitric oxide production in pancreatic beta-cells, which may be relevant in preventing nitric oxide mediated damage to these cells in the Type 1 diabetes process. We tested troglitazone in the spontaneous model of autoimmune diabetes, the non-obese diabetic (NOD) mouse, to determine its effect on the disease process. When administered by gavage from weaning at a dose of 400 mg/kg body weight (n = 32), troglitazone reduced the incidence of diabetes by 16 weeks compared to controls (n = 32) in a pattern that was maintained up to the conclusion of the experiment at 31 weeks of age (p < 0.05). Insulitis was unaltered (index = 1.05 +/- 0.71 vs. 1.13 +/- 0.82, treated vs. controls, p = 0.78). The study was repeated using troglitazone in the diet of NOD mice (n = 24) to give a dose of approximately 200 mg/kg body weight in order to provide a more consistent level of troglitazone during the time course of the experiment. There was a reduction of diabetes incidence in this group but it did not reach significance. Insulin levels were reduced in gavage treated mice although such reduction did not reach significance (p < 0.07). We conclude that, in view of its effect on this model of autoimmune diabetes and because of its known function as an insulin sensitiser, troglitazone might be considered for potential use in those patients with Type 1 masquerading as Type 2 diabetes.

The rhGM-CSF-EPO hybrid protein MEN 11300 induces anti-EPO antibodies and severe anaemia in rhesus monkeys.

A recombinant human GM-CSF-EPO hybrid protein named MEN 11300 was administered biweekly for a total of 6 weeks to rhesus monkeys in order to evaluate its pharmacokinetic behaviour, tolerability and immunogenicity. In this primate species a strong antibody response was induced which neutralized the in vitro biological activity of human EPO while no antibody response could be detected against human GM-CSF. A severe drop in reticulocyte counts at approximately 2 weeks after initiation of treatment was followed by a dramatic decrease in the number of erythrocytes. No effects were observed on GM-CSF-dependent hematopoietic lineages and the clinical chemistry analyses did not reveal signs of general toxicity. Reticulocyte and erythrocyte counts started to recover 3-4 weeks after discontinuation of treatment in concert with a decline in anti-EPO antibody titres. Nevertheless, cell numbers remained below basal levels up to 50 days after the last MEN 11300 administration. Haematological impairment indicates that the administration to non-human primate of human EPO fused to human GM-CSF, induces neutralizing autoantibodies to the self EPO. Present data do not allow prediction of the immunogenic potential of the fusion protein in humans and a dose-escalating phase I study should be addressed to investigate the safety of the product.

Production of recombinant human GM-CSF-EPO hybrid proteins: in vitro biological characterization.

Selective lineage differentiation depends upon the combined action of several colony-stimulating factors. Here we describe 3 human granulocyte-macrophage colony-stimulating factor-erythropoietin (GM-CSF-EPO) hybrid proteins generated by recombination of the relevant cDNAs. The expression vector containing the murine cytomegalovirus (mCMV) promoter and dihydrofolate reductase (DHFR) gene was used for the expression of the hybrid genes in Chinese hamster ovary (CHO) cells. Purified hybrid proteins from CHO transfectant cultures induced proliferation of both EPO and GM-CSF dependent cell lines. The clonogenic test, performed on purified human hematopoietic precursor cells, indicates that the hybrid proteins are more efficient at inducing erythroid differentiation compared with the equimolar mixture of GM-CSF and EPO.