Abstract
The present invention relates to peroxiredoxin 6 or a synthetic analogue thereof for use as a hypoglycaemic agent, for example in the treatment of diabetes, such as type 1 diabetes mellitus and type 2 diabetes mellitus.
Claims
1-17. (canceled)
18. A method of treating diabetes mellitus, the method comprising administering to a patient peroxiredoxin 6 or a synthetic analogue thereof.
19. The method of claim 18, wherein said diabetes mellitus is type 2 diabetes mellitus or type 1 diabetes mellitus.
20. The method of claim 18, wherein the patient is affected by obesity.
21. The method of claim 18, wherein said synthetic analogue of peroxiredoxin 6 is a synthetic analogue modified in one or more catalytic domains selected from the peroxidase site, phospholipase-A.sub.2 site, lysophosphatidylcholine acyltransferase site, dimerization site and sumoylation site.
22. The method of claim 18, wherein said synthetic analogue of peroxiredoxin 6 is selected from the following mutant Prdx6s: mutant with the dimerization site inhibited, mutant with the phospholipase-A.sub.2 site inhibited, mutant with the peroxidase site inhibited, and mutant with the sumoylation site inhibited.
23. The method of claim 22, wherein said mutant with the dimerization site inhibited is selected from L145E, L148E and L145E/L148E.
24. The method of claim 22, wherein said mutant with the phospholipase-A.sub.2 site inhibited is selected from S32A or S32T.
25. The method of claim 22, wherein said mutant with the peroxidase site inhibited is C47S.
26. The method of claim 22, wherein said mutant with the sumoylation site inhibited is selected from K122R, K142R and K122R/K142R.
27. The method of claim 18, wherein said administering is of a pharmaceutical composition that comprises peroxiredoxin 6 and/or a synthetic analogue thereof as an active ingredient, together with one or more excipients and/or adjuvants.
28. The method of claim 27, wherein said composition further comprises a hypoglycaemic agent other than peroxiredoxin 6 or said synthetic analogue thereof.
29. The method of claim 28, wherein said hypoglycaemic agent is selected frommetformin, GLP-1 agonists, DPP-4 inhibitors, SGLT-2 inhibitors, human insulin and slow, rapid or ultra-rapid analogue insulin.
30. The method of claim 27, wherein said composition is in a form for subcutaneous administration.
31. The method of claim 18, wherein said administering is by subcutaneous administration.
32. The method of claim 18, wherein said peroxiredoxin 6 or said synthetic analogue thereof is administered in combination with at least one hypoglycaemic agent other than peroxiredoxin 6 or said synthetic analogue thereof.
33. The method of claim 32, wherein said at least one hypoglycaemic agent is selected from metformin, GLP-1 agonists, DPP-4 inhibitors, SGLT-2 inhibitors, human insulin and slow, rapid or ultra-rapid insulin analogue.
34. The method of claim 33, wherein said at least one hypoglycemic agent and said peroxiredoxin 6 or said synthetic analogue thereof are administered separately or sequentially.
35. The method of claim 32, wherein the patient is affected by obesity.
36. A method of treating obesity, the method comprising administering to a patient peroxiredoxin 6, a synthetic analogue thereof or a pharmaceutical composition comprising peroxiredoxin 6 and/or a synthetic analogue thereof as an active ingredient, together with one or more excipients and/or adjuvants, for use in therapy for obesity.
37. The method of claim 36, wherein said synthetic analogue thereof is a synthetic analogue modified in one or more catalytic domains selected from the peroxidase site, the phospholipase-A.sub.2 site, the lysophosphatidylcholine acyltransferase site, the dimerization site and the sumoylation site.
38. The method of claim 37, wherein said synthetic analogue of peroxiredoxin 6 is selected from the following mutant Prdx6s: mutant with the dimerization site inhibited, mutant with the phospholipase-A.sub.2 site inhibited, mutant with the peroxidase site inhibited, and mutant with the sumoylation site inhibited.
39. The method of claim 38, wherein said mutant with the dimerization site inhibited is selected from L145E, L148E and L145E/L148E.
40. The method of claim 38, wherein said mutant with the phospholipase-A.sub.2 site inhibited is selected from S32A or S32T.
41. The method of claim 38, wherein said mutant with the peroxidase site inhibited is C47S.
42. The method of claim 38, wherein said mutant with the sumoylation site inhibited is selected from K122R, K142R and K122R/K142R.
Description
[0055] The present invention will now be described by way of non-limiting illustration according to a preferred embodiment thereof, with particular reference to the examples and the figures in the appended drawings, wherein:
[0056] - FIG. 1 shows the deletion of Prdx6 which alters insulin secretion by modulating the synthesis of ATP and the intracellular content of Ca.sup.2+; A) Stable silencing of Prdx6 (Prdx6.sup.KD) and respective control cells silenced for the gene encoding GFP (green fluorescent protein) (Scramble, Scr), in murine insulinoma cells βTC6; B) Evaluation of insulin secretion after stimulation with 20 mM of glucose at different time intervals (0-5-10-15-20-25-30 min.); C) the production of ATP was measured under the same experimental conditions as described in point B; D) the intracellular calcium content was analysed with a cytofluorometric assay under the same experimental conditions as used for the evaluation of insulin secretion; (*p<0.05; ***p<0.0005);
[0057] - FIG. 2 shows the structural and functional alterations of mitochondria in Prdx6.sup.KD cells; A-B) Evaluation of the mitochondrial ultrastructure in Scr and Prdx6.sup.KD cells through an analysis with electron microscopy; C) the mitochondrial mass was measured by flow cytofluorometry analysis using the marker MitoTracker green; D) Ratio between mitochondrial area and cytoplasmic area. E-F) Evaluation of mitochondrial functionality with an analysis of the levels of the membrane potential and oxygen consumption; (*p<0.05; **p<0.005);
[0058] - FIG. 3 shows the evaluation of insulin secretion and calcium (Ca.sup.2+) levels: A-B) insulin secretion was evaluated in murine insulinoma cells βTC6) first following stimulation with different concentrations of recombinant Prdx6 for 15 minutes, and then with Prdx6 400 nM at different time intervals; C) The analysis of calcium levels used during stimulation with Prdx6 400 nM for 15 min., in association with increased levels of insulin secretion, reveals a calcium-mediated Prdx6 secretion mechanism; D) Analysis of the insulin secretion in βTC6 cells and in βTC6 cells silenced for Prdx6 (Prdx6.sup.KD); the Prdx6.sup.KD cells that show morphological-structural alterations of the mitochondria, responsible for the insulin secretion process, are not capable of responding to stimulation with Prdx6; (**p<0.005; ***p<0.0005);
[0059] - FIG. 4 shows the localisation of the Prdx6-biotin complex; for the purpose of localising Prdx6, the latter was associated with biotin by cross-linking and the Prdx6-biotin complex was used for treatment of the cells A-B) Western blot analysis of the membrane fractionation reveals a localisation of the complex in question mainly at the level of the cytoplasmic membrane; C-D) Flow cytofluorometry analysis for the evaluation and validation of the localisation of the Prdx6-biotin complex through the use of streptavidin, which recognises and binds biotin; (*p<0.05);
[0060] - FIG. 5 shows the Prdx6-mediated insulin secretion in human islets; Islets of healthy donors were treated with Prdx6 400 nM for 15 min.; the insulin secretion was subsequently evaluated, (**p<0.005; ***p<0.0005) (N .sub.= 6);
[0061] - FIG. 6 shows the Prdx6-mediated incretin secretion in human colorectal adenocarcinoma cells; colorectal adenocarcinoma cells were treated with glucose (A), Prdx6 (B), and Prdx6 + glucose (C); stimulation with Prdx6 is capable of inducing the release of GLP-1; (*p<0.05; **p<0.005);
[0062] - FIG. 7 shows the incretin secretion in Prdx6.sup.-/- mouse models; A-B) Prdx6.sup.-/- mouse models were fed regularly and in the postprandial phases both the secretion of GLP-1 and the glycaemic levels were evaluated; subsequently, to validate the data, the animals were subjected to oral administration of a glucose load (OGTT) C). The secretion of GLP-1 is decreased in the absence of Prdx6. *p<0.05; **p<0.005; ***p<0.0005; (N = 5).
EXAMPLE1 EXPERIMENTAL DESIGN AND RESULTS: PRDX6 AND INSULIN
[0063] The mechanism whereby Prdx6 modulates glucose-dependent insulin secretion was studied in vitro using murine insulinoma cells (βTC6) stably silenced for Prdx6 (Prdx6.sup.KD) ([3TC6: ATCC-CRL-11506, Manassas, Virginia, USA) (FIG. 1, Panel A).
[0064] The cells were plated in a 24-well multiwell plate for 24 hours and were then incubated in a Krebs solution (20% Solution A: 610 mM NaCI, 24 mM KCI, 6 mM KH.sub.2PO.sub.4, 6 mM MgSO.sub.4-7H.sub.2O, 5 mM CaCl.sub.2; 25% Solution B: 10 mM HEPES, 20 mM NaHCO.sub.3, 20 mM NaCI, 55% H.sub.2O and 2 mM Glucose) for 45 minutes (starvation). At the end of starvation, the culture medium was removed and a Krebs solution containing 20 mM glucose was added for 0, 5′, 15′ and 30′ minutes (FIG. 1, Panel B). The data obtained confirmed what had previously been observed in vivo: 15 minutes after stimulation with glucose, the secretion of insulin showed to be significantly reduced in the Prdx6.sup.KD cells (FIG. 1, Panel B). Furthermore, under the same experimental conditions, a significant reduction in the synthesis of ATP was present in the Prdx6.sup.KD cells (FIG. 1, Panel C) and, consequently, a reduced intracellular flow of Ca.sup.2+ (FIG. 1, Panel D).
[0065] These data thus showed a direct involvement of Prdx6 in the ‘classic’ insulin secretion process. The morphology and structure of the mitochondria were subsequently analysed by electron microscopy (FIG. 2, Panels A and B) and a significant morphological-structural alteration in Prdx6.sup.KD cells was documented. Furthermore, both the mass and the dimensions of the mitochondria were evaluated, revealing a reduction in Prdx6.sup.KD cells (FIG. 2, Panels C and D respectively). In association with the morphological analysis, mitochondrial functionality was also evaluated. In particular, a reduction was observed both in the mitochondrial membrane potential and in oxygen consumption in the absence of Prdx6 (FIG. 2, Panels E and F respectively). These data reveal, at least in part, the cause that determines the reduced insulin secretion in response to glucose. Subsequently, the effect of Prdx6 in stimulating insulin secretion was evaluated hypothesising a hormone-like action of Prdx6, on the basis of the results obtained. For this purpose, the murine insulinoma-βTC6 control cells and the Prdx6.sup.KD cells were treated with Krebs as previously described and, after the starvation process, Krebs containing increasing concentrations of recombinant Prxd6 (1, 10, 100, 400, 1000 nM) was added for 15 minutes. The supernatant was then recovered and used to measure the insulin by means of the ELISA assay (Mercodia). The largest increase in insulin secretion, which was comparable to the one obtained after stimulation with 20 mM glucose, occurred after the treatment with 400 nM Prdx6 (p<0.0064), whereas at higher concentrations (1000 nM) it was not possible to observe a further increase in insulin secretion (FIG. 3, Panel A). Subsequently, a time response curve (0, 2, 10, 15, 30, 60 minutes) was derived by using 400 nM of Prdx6 with the aim of validating the previously selected timing, following the same experimental procedure as described above. The data showed an insulin secretion peak after 15 minutes (p<0.0019), which, despite decreasing, confirmed to be significant also after 60 minutes (p<0.03) (FIG. 3, Panel B).
[0066] With the aim of better understanding the role of Prdx6 in the modulation of insulin secretion, the levels of cytoplasmic Ca.sup.2+ were measured to verify whether the secretion was Ca.sup.2+-dependent, similarly to what occurs after stimulation with glucose. The intracellular levels of calcium were measured both under basal conditions and in response to stimulation with 400 nM Prdx6 for 15 minutes; a significant reduction was observed in the intracellular levels of Ca.sup.2+ at the end of the treatment, indicating that Prdx6-mediated insulin secretion is a calcium-dependent process (FIG. 3, Panel C). In order to confirm that the mitochondrial defect observed in Prdx6.sup.KD cells was a key mechanism for modifying the insulin secretion, the knockdown cells were also stimulated with recombinant Prdx6 (FIG. 3, Panel D). As expected, there being a morphological-functional defect of the mitochondria, no increase was observed in insulin secretion in response to the treatment with Prdx6. Since no increase in insulin secretion was found after stimulation with exogenous Prdx6 in the knockdown cells, it can be hypothesised that the effect of Prdx6 in stimulating insulin secretion is receptor-dependent and not mediated by the transport of Prdx6 into the cell. With the aim of confirming the hypothesis, the localisation of Prdx6 was analysed after stimulation with biotinylated recombinant Prdx6. In particular, using the EZ-Link NHS-Biotin Reagents kit (Invitrogen) and following the protocol provided, recombinant Prdx6 was biotinylated and used at a concentration of 400 nM to stimulate the cells for 15 minutes, as previously described. The cells thus treated were lysed on ice for 30 minutes with a lysis buffer containing 1% NP-40 (137 mM NaCI, 20 mM Tris pH 7.6, 1 mM MgCl.sub.2, 1 mM CaCl.sub.2, 10% Glycerol, protease inhibitor cocktail, phosphatase inhibitor cocktail). The lysate was centrifuged at 14,000 rpm - 18,600 g for 20 minutes at +4° C. and the supernatant with the cytoplasmic proteins was recovered. The pellet containing the membrane was washed with PBS 3 times to eliminate the cytoplasmic contaminant.
[0067] Subsequently, the pellet was resuspended in a lysis buffer containing 1.5% NP-40 and kept on ice for 30 minutes. The lysate obtained, containing the membrane proteins, was then processed in an ultracentrifuge at 50,000 rpm - 150,600 g for 1 hour at +4° C. The supernatant was recovered and the membrane and cytoplasmic proteins were quantified by means of the Bradford assay. 20 .Math.g of proteins were separated by SDS-PAGE on a polyacrylamide gel at a 4-12% gradient and subsequently transferred onto a nitrocellulose membrane. The membrane was then incubated with an anti-biotin antibody. The results showed that the biotinylated protein is localised mainly at the level of the cytoplasmic membrane, supporting the hypothesis of a receptor-dependent mechanism of action (FIG. 4, Panel A). After the removal (stripping) of the previously used primary antibody, the same membrane was incubated with an anti-Prdx6 antibody capable of detecting both the endogenous protein and the biotinylated one. The results obtained confirmed an increase in Prdx6 in the cytoplasmic membrane (FIG. 4, Panel B). The data were validated by cytofluorometric analysis, in which the localisation of biotinylated Prdx6 was evaluated at both an extra- and intracellular level. For extracellular marking, the cells were plated and treated as previously described. Subsequently, the cells were detached using trypsin and washed with PBS and the pellet was incubated with a PBS solution containing streptavidin diluted 1:10 (which interacts directly with biotin) for 30 minutes in the dark and at room temperature. This was followed by washing with PBS and the subsequent cytofluorometric analysis. For intracellular marking, on the other hand, the cells were plated for 24 hours as described above, detached with trypsin, washed with PBS and subsequently permeabilised and fixed using the BD Fixation/Permeabilization Solution Kit (BD). Afterwards, they were stimulated with 400 nM of biotinylated Prdx6 for 15 minutes and at the end of the stimulation they were washed to eliminate the excess protein component, incubated with the solution of streptavidin and processed as described for the extracellular marking. The results confirmed that biotinylated Prdx6 is mainly localised in the cytoplasmic membrane (FIG. 4, Panel C) and only a small part is internalised (FIG. 4, Panel D), suggesting the presence of a receptor-dependent mechanism of action.
[0068] Finally, an assessment was made of the Prdx6-mediated insulin secretion in human pancreatic islets of healthy donors not suitable for transplantation (Approved by the Ethics and Scientific Committee of Niguarda Ca′ Granda Hospital in the meeting of 16.12.2009) obtained from Prof. Federico Bertuzzi, Niguarda Hospital, Milan, and through the purchase of material from the company Tebu-Bio (HUMAN ISLETS: Tebu-Bio SRL, Cat. 196HIR-IEQ-1000 Magenta, Milan, Italy).
[0069] The islets were maintained in a specific culture medium for 24 h after their arrival to enable their physiological recovery after transport. They were subsequently withdrawn using a stereomicroscope and transferred to a 24-well multiwell plate, where they were treated as previously described for the insulin secretion. The results (normalised for the number of islets in each well) showed that stimulation with Prdx6 (400 nM) was capable of inducing a significant increase in insulin secretion compared both to the control cells (p<0.0001) and to the cells treated with glucose (p<0.0033) (FIG. 5). Furthermore, although the data were not significant, an increasing trend in insulin secretion was observed following co-treatment with glucose and Prdx6, compared to the treatment with glucose alone, suggesting a synergic mechanism of secretion (FIG. 5).
EXAMPLE 2. EXPERIMENT DESIGN AND RESULTS: PRDX6 AND GLP-1
[0070] In order to better characterise the hormone-like action of Prdx6, moreover, an assessment was made of its effect in modulating the secretion of GLP-1 (Glucagon-like Peptide-1), a peptide hormone of 37 amino acids synthesised and secreted in the gut by the L cells of the ileum and colon, with an increase in secretion in the postprandial period. GLP-1 increases insulin secretion in response to glucose and, in patients with DMT2, this action is altered due to a defect in the secretion of GLP-1. In particular, as the incretin release mechanism is similar to the glucose-mediated insulin release mechanism, it was verified whether Prdx6 may have a role in boosting insulin secretion also by acting on the secretion of GLP-1. Therefore, colorectal adenocarcinoma cells, NCI-H716, (ATCC-CCL-251 [H716], Manassas, Virginia, USA) were maintained in RPMI 1640 culture medium with 10% FBS and penicillin/streptomycin and subsequently stimulated with Prdx6. In particular, the cells were plated at a density of 1 x10.sup.6 overnight in 24-well multiwell plates, previously treated with basal membrane matrix (BMM) (BD Biosciences) to allow a better adhesion of the NCI-H716 and differentiation towards endocrine cell lines. The cells were then incubated with a specific buffer (138 mM NaCI, 4.5 mM KCI, 4.2 mM NaHCO.sub.3, 1.2 mM NaH.sub.2PO.sub.4, 2.5 mM CaCl.sub.2, 1.2 mM MgCl.sub.2, 10 mM HEPES and 0.1% (wt/vol) BSA (pH = 7)) and treated with 25 mM glucose and 400 nM Prdx6.
[0071] At the end of the treatment, GLP-1 (the active isoforms GLP-1 (7-36) GLP-1 (7-37)) was measured at time 0 and after 15, 30, 60 and 120 minutes by means of the ELISA assay (Millipore) (FIG. 6, Panel A). Surprisingly, the treatment with Prdx6 increased the secretion of GLP-1 with a peak after 15 minutes (218.46±34.2) (FIG. 6, Panel B) and the co-treatment with glucose further significantly increased the secretion of GLP-1 after 15 minutes (286.1±31.04) (FIG. 6, Panel C). These data highlight the potential action of Prdx6 as a ‘hormone’ that stimulates the secretion of GLP-1. In order to confirm the data obtained on GLP-1 secretion in vitro, it was evaluated whether Prdx6.sup.-/- mouse models, currently available for purchase from The Jackson Lab, (Sacramento, CA, USA, strain B6.129-Prdx6tm1 Abf/Mmjax, MMRRC (Stock No:43402-JAX-1-cysPrx KO) and kindly donated by Prof. Xiaosong Wang (The Jackson Laboratory), showed a defect in GLP-1 production in the postprandial period (fed state). As shown in FIG. 7, Panel A, the amount of secreted GLP-1 was lower in Prdx6.sup.-/- mice than in Wild Type (WT) mice (p<0.005), in association with an increase in glycaemia (p<0.005), suggesting an alteration in the GLP-1-mediated insulin secretion process in Prdx6.sup.-/- mice (FIG. 7, Panel B). In order to further confirm the data obtained, the Prdx6.sup.-/- mice were subjected to oral administration of a glucose load (OGTT) by gavage (2 g/kg glucose after 16 hours of fasting). As shown in FIG. 7, Panel C, 15 minutes after stimulation with glucose the Prdx6.sup.-/- mice showed a significant reduction in GLP-1 secretion compared to the WT animals (p<0.05). The data reported above point to a pharmacological role of Prdx6 as an insulin secretagogue agent, which would be able for the first time to increase insulin secretion with an anti-apoptotic effect (this effect is amply documented in the literature in relation to the role of Prdx6 as an antioxidant enzyme). Furthermore, it would be the first hypoglycaemic agent capable of simultaneously stimulating the secretion of insulin and of GLP-1, reducing the levels of oxidative stress.
