Delivery system comprising lipid or polymeric nanoparticles comprising thyroid hormones

Abstract

The present invention relates to a delivery system of thyroid hormones comprising lipid or polymeric nanoparticles, preferably comprising a targeting agent for the TRCP6 receptor. Such a delivery system can be used in the treatment of diabetic cardiomyopathy or diabetic nephropathy.

Description

[0001] "DELIVERY SYSTEM COMPRISING LIPID OR POLYMERIC NANOPARTICLES

[0002] COMPRISING THYROID HORMONES"

[0003] Cross-Reference to Related Applications

[0004] This Patent Application claims priority from Italian Patent Application No. 102023000015291 filed on July 20, 2023, the entire disclosure of which is incorporated herein by reference.

[0005] Technical Field

[0006] The present invention relates to a delivery system comprising lipid or polymeric nanoparticles containing thyroid hormones, in particular triiodothyronine (T3) and / or thyroxine (T4), designed to specifically target stressed tissues in diabetes and to be used in the treatment of diabetic cardiomyopathy (DC) and diabetic nephropathy (DN).

[0007] Background of the Invention

[0008] Diabetes is one of the major health emergencies of the 21stcentury, affecting hundreds of millions of people worldwide. In Europe, there are tens of millions of people affected from diabetes; about 10.3% of men and 9.6% of women aged 25 years old and older. The incidence of diabetes is increasing among all age groups, especially due to an increase in overweight and obesity, unhealthy diets and physical inactivity, and adults affected by the disease are expected to increase in the future.

[0009] Diabetes can damage the heart, blood vessels, kidneys, eyes, and nerves over time. About seven people die every minute due to diabetes complications (WHO, Diabetes, 2018). Diabetic nephropathy (DN) and diabetic cardiomyopathy (DC) are the main complications and causes of death. DN develops in about 40% of diabetic patients, 25% to 45% of which progress to end-stage renal disease (ESRD). Cardiovascular diseases, on the other hand, are the most common cause of mortality and morbidity in diabetic populations (Matheus AS et al., 2013). Diabetes increases the risk of heart failure (HF), coronary artery disease, and myocardial infarction (MI). After a myocardial infarction, diabetic patients have higher rates of morbidity, mortality, and re-infarction than non-diabetics, with one-year mortality rates of almost 50%.

[0010] Standard treatments for diabetic patients are glucose and blood pressure control, lipid reduction, and blockade of the renin-angiotensin system; however, an effective treatment for DN and DC remains an unmet need in contemporary medicine. The development of an approach capable of stopping or even reversing the cardiac and renal complications of diabetes would save millions of lives, significantly improve the quality of life of patients with HF and ESRD, and save a significant amount of public and private funds currently spent on the treatment of diabetes complications.

[0011] Diabetes mellitus (DM) causes chronic stress on cells due to high glucose levels, oxygen free radicals, hypoxia, and glycation end-products. As a result, the diabetic organ undergoes a series of structural, metabolic and functional changes that often lead to diabetic cardiomyopathy and diabetic nephropathy. Evidence suggests that the phenotypic and morphological alterations that are observed in cardiomyocytes and podocytes in response to chronic damage are indications of an imperfect / maladaptive recapitulation of early developmental (i.e. embryonic) stages, which are controlled by the thyroid hormone (TH) / thyroid hormone receptor alpha 1 (TRal) axis. During foetal life, when L- triiodothyronine (T3) levels are low, the highly expressed TRal acts as an apo-receptor (unbound state), repressing and activating adult and foetal genes, respectively. After birth, there is a significant increase in T3 levels, and TRal switches to the holo-receptor state (bound state), inducing and promoting the expression of adult genes and structural and functional maturation of the organs. Interestingly, under various stress conditions and disease states, the foetal profile of the T3-TRal axis (low T3 and high TRal) is again adopted, suggesting that TRal plays a crucial role in reactivating the foetal gene program.

[0012] In particular, stressful stimuli, such as adrenergic stimulation, induce the upregulation of TRal in cardiomyocytes. Moreover, TRal has increased in vivo in surviving / undamaged myocardium following infarction, while its inhibition drastically restrains post-ischemic cardiac function and further deteriorates calcium handling. Similarly, diabetes induces decreased T3 levels, overexpression of TRal in podocytes, and re-expression of foetal genes in vivo, while high glucose levels induce the overexpression of TRal, downregulation of adult markers, and upregulation of embryonic markers in human podocytes in vitro.

[0013] Consistently with what mentioned above, epidemiological studies show that there is a strong association between stress-induced reduction of T3 levels and adverse clinical outcomes in patients with cardiac and renal diseases. It is therefore not surprising that exogenous administration of thyroid hormones (TH) during stress has shown considerable therapeutic potential. Treatment with TH after myocardial infarction increases viable cardiac mass with a physiological adult phenotype and improves cardiac function in diabetic hearts with MI.

[0014] Furthermore, TH treatment improves renal function in patients with chronic renal disease, while in experimental models of diabetes, T3 promotes re-differentiation and reduces pathological hypertrophy, improves renal structure and significantly reduces glomerulosclerosis.

[0015] However, translating this strategy into clinical practice is not problem-free. In order to obtain an efficient re-differentiation of stressed cells (overexpressing TRal), high doses of T3 are required; but this can cause various adverse effects, such as tachycardia, arrhythmias and hyperfiltration, thyroid gland dysfunctions and even death in case of long-term treatment.

[0016] Nanotechnological applications for the treatment of cardiac and renal diseases are under investigation and constantly developing, yet, no pharmaceuticals have been marketed in these areas so far. As for cardiovascular diseases, research has focused on the creation of nanoparticle-based (NP) drug delivery systems for the treatment of acute myocardial infarction by administrating a p38 inhibitor (Gray WD et al., 2011), anti-inflammatory drug pitavastatin (Nagaoka K et al., 2015), or insulin-like growth factor (IGF)-l (Chang MY et al., 2013) and VEGF (Scott RC et al., 2009). In addition, in order to treat DC, the effects of curcumin / PBLG-PEG-PBLG NPs (Tong F et al., 2018), or basic FGF nanoliposomes (Zhao YZ et al., 2016) have been investigated. Cerium oxide NPs (Jahani M et al., 2016), quercetin-loaded NPs (Tong F et al., 2017), gold NPs to deliver pomegranate peel extract (Manna K et al., 2019), and a core-shell NP to load dual-target drugs, such as AGEs inhibitors and RAGE inhibitors, have been tested to treat DN (Zhou X et al., 2012). Finally, US2011 / 0142947 describes poly (lactic-co-glycolic) acid (PLGA) and chitosan NPs encapsulating T3 for increased and sustained T3 administration for the treatment of cardiac arrest.

[0017] However, none of these strategies were able to selectively deliver the drug directly to stressed cells such as cardiomyocytes and podocytes.

[0018] In particular, there are currently no nanotechnological applications for the treatment of diabetic cardiomyopathy and diabetic nephropathy, and no strategy has yet been developed to specifically deliver a drug with regenerating and pro-differentiating properties such as T3 and / or T4 to damaged cardiomyocytes, podocytes and / or renal tubules in vivo.

[0019] In order to minimise adverse effects and maximise the repairing and regenerating abilities of thyroid hormones, there is thus a need in the art for drug delivery systems which can (i) target stressed cells, (ii) transport and deliver thyroid hormones into the cells of interest, (iii) deliver thyroid hormones intracellularly to act on the thyroid hormone receptors of stressed cells, and (iv) avoid or limit adverse reactions of thyroid hormones in normal cells and tissues.

[0020] Therefore, the object of the present invention is to provide a delivery system of thyroid hormones, in particular T3 and T4, which allows to maximise their repairing and regenerating abilities, that is selective towards stressed cells, and avoid the side effects associated with the administration of high doses of thyroid hormones.

[0021] Summary of the Invention

[0022] This object is achieved by a delivery system according to claims 1, 5 and 8, uses thereof according to claim 15, and pharmaceutical formulations comprising the same according to claim 16.

[0023] In particular, the present invention describes delivery systems comprising lipid or polymeric nanoparticles containing thyroid hormones, in particular triiodothyronine (T3) and / or thyroxine (T4), designed to specifically target stressed tissues in diabetes and to be utilised in the treatment of diabetic cardiomyopathy (DC) and diabetic nephropathy (DN). This is made possible thanks to a drug delivery system targeting the TRPC6 receptor, which is overexpressed in stressed podocytes and cardiomyocytes.

[0024] Brief Description of the Figures

[0025] The present invention will now be described in detail with reference to the accompanying drawings, in which:

[0026] - Figure 1 illustrates (A) the morphology and structure integrity analysis by CryoTEM and (B) size distribution stability one year after formulation, stored at 4 °C, of the empty (SAR-NPs) or T3-loaded (SAR-T3-NPs) lipid nanoparticles of the invention.

[0027] - Figure 2A illustrates (A-D) the quantification of apoptotic podocytes expressed as annexin V-positive cells by cell number in the field after 24 (A) and 72 (B) hours of treatment with the lipid nanoparticles of the invention compared to control cells (CTR), and the quantification of podocyte necrosis expressed as LDH delivery in the culture medium after 24 (C) and 72 (D) hours of treatment compared to control cells (CTR).

[0028] - Figure 2B illustrates (E-H) the quantification of apoptotic podocytes expressed as annexin V-positive cells by cell number in the field after 24 (E) and 72 (F) hours of treatment with the pH-sensitive polymeric nanoparticles of the invention compared to control cells (CTR), and the quantification of podocyte necrosis expressed as LDH delivery in the culture medium after 24 (G) and 72 (H) hours of treatment compared to control cells (CTR). Data are expressed as mean ± SEM. * = p<0.05, one-way ANOVA with Tukey's post hoc test. To quantify the number of positive cells, representative pictures were taken randomly in different areas of the cell cultures. Number of fields analysed: n = 20-25 per well (A, B). n = 2-4 wells per group.

[0029] - Figure 3A illustrates (A-D) the quantification of apoptotic cardiomyocytes expressed as annexin V-positive cells by cell number in the field after 24 (A) and 72 (B) hours of treatment with the lipid nanoparticles of the invention compared to control cells (CTR), and the quantification of cardiomyocyte necrosis expressed as LDH delivery in the culture medium after 24 (C) and 72 (D) hours of treatment compared to control cells (CTR).

[0030] - Figure 3B illustrates (E-H) the quantification of apoptotic cardiomyocytes expressed as annexin V-positive cells by cell number in the field after 24 (E) and 72 (F) hours of treatment with the pH-sensitive polymeric nanoparticles of the invention compared to control cells (CTR) and the quantification of cardiomyocyte necrosis after 24 (G) or 72 (H) hours. Data are expressed as mean ± SEM, ** = p<0.01 vs. control, *** = p<0.001 vs. control, one-way ANOVA with Tukey's post-hoc test. To quantify the number of positive cells, representative pictures were randomly taken in different areas of the cell cultures. Number of fields analysed: n = 20-25 per well (A, B). n = 2-4 wells per group.

[0031] - Figure 4 illustrates the concentration of lipid (A) and pH-sensitive polymeric (B) nanoparticles in serum after intravenous and intraperitoneal injections (10 pg / kg of T3).

[0032] - Figure 5 illustrates the variations in body weight in healthy rats after 2 weeks of treatment with the lipid (A) and pH-sensitive polymeric (B) nanoparticles of the invention.

[0033] - Figure 6 illustrates the thyroid hormone levels in serum of healthy rats after 2 weeks of treatment with the lipid (A-C) and pH-sensitive polymeric (D-F) nanoparticles of the invention.

[0034] - Figure 7 illustrates the variation of body temperature and heart rate after 2 weeks of treatment with lipid (A-B) and pH-sensitive polymeric (C-D) nanoparticles in healthy rats.

[0035] - Figure 8 illustrates the liver function of healthy rats after 2 weeks of treatment with the lipid (A-B) and pH- sensitive polymeric (C-D) nanoparticles of the invention.

[0036] - Figure 9 illustrates the renal function of healthy rats after 2 weeks of treatment with the lipid (A-B) and pH- sensitive polymeric (C-D) nanoparticles of the invention.

[0037] - Figure 10 illustrates the cardiac function, assessed by cardiac ultrasound, in healthy rats after 2 weeks of treatment with the lipid nanoparticles of the invention. - Figure 11 illustrates the heart function, assessed by echocardiography, in healthy rats after 2 weeks of treatment with the pH-sensitive polymeric nanoparticles of the invention.

[0038] - Figure 12 illustrates serum lipid concentrations in healthy rats after 2 weeks of treatment with the lipid (A) and pH-sensitive polymeric (B) nanoparticles of the invention.

[0039] - Figure 13A illustrates the evaluation of the intracellular T3 content (ng / dl) in both control and glucose- stressed podocytes (A) and cardiomyocytes (B), treated with T3 dissolved in the culture medium or with the lipid nanoparticles of the invention by ELISA tests.

[0040] - Figure 13B illustrates representative confocal images of both control and glucose-stressed podocytes (C) and cardiomyocytes (D), treated with FITC-labelled lipid nanoparticles of the invention (T3-FITC-NPs(L) or SAR-T3- FITC-NPs (L)). In the left panels, the cells are highlighted by staining the nuclei with DAPI, while in the right panels the cells are visible in bright-field images. Data are expressed as mean ± SEM, n=2-3 independent wells per group. Magnification 40x, scale bars 20 pm (C, D).

[0041] - Figure 14A illustrates the differentiation of C2C12 myoblasts following the administration of the lipid nanoparticles of the present invention. Control= no treatment, T3= free triiodothyronine in a dose of 10 nM in the culture medium, SAR-T3-NPs = SAR-T3-NPs in a dose equivalent to 10 nM T3 in the culture medium, SAR-NPs = SAR- NPs in the same dose.

[0042] - Figure 14B illustrates the differentiation of C2C12 myoblasts following the administration of the pH-sensitive polymeric nanoparticles of the present invention. Control= no treatment, T3= free triiodothyronine in a dose of 10 nM in the culture medium, SAR-T3-NPs = SAR-T3-NPs in a dose equivalent to 10 nM T3 in the culture medium, SAR-NPs = SAR- NPs in the same dose.

[0043] - Figure 15 illustrates the differentiation of C2C12 myoblasts following the administration of the lipid nanoparticles of the present invention following conjugation with SAR7334. Control= no treatment, T3= free triiodothyronine in a dose of 10 nM in the culture medium, T3-NPs (AB199) = T3-NPs without SAR in a dose equivalent to 10 nM T3 in the culture medium, SAR-T3-NPs (AB201) = SAR-T3- NPs in a dose equivalent to 10 nM T3 in the culture medium.

[0044] - Figure 16A illustrates the molecular indices of C2C12 myoblast differentiation following the treatment with lipid nanoparticles. Data are expressed as mean ± SEM. * p<0.05 vs SAR-NPs, ** p<0.05 vs SAR-NPs and T3 10 nM, non-parametric Mann-Whitney test.

[0045] - Figure 16B illustrates the molecular indices of C2C12 myoblasts differentiation following the treatment with pH- sensitive polymeric nanoparticles. Data are expressed as mean ± SEM. * p<0.05 vs SAR-NPs, ** p<0.05 vs SAR-NPs and T3 10 nM, non-parametric Mann-Whitney test.

[0046] - Figure 17 illustrates the differentiation of C2C12 myoblasts, pre-treated or not with H2O2, and subsequently exposed to treatment with ROS-sensitive polymeric nanoparticles (SAR-T3-NPs(P)) in a dose equivalent to 0.5 nM T3 in the culture medium, SAR-NPs(P)= ROS-sensitive SAR- NPs (P) in the same dose. - Figure 18 illustrates left ventricular (LV) systolic function assessed by echocardiography after treatment with pH-sensitive lipid and polymeric nanoparticles of the present invention for 3 months in healthy and diabetic rats. Lean SAR-NPs= SAR-NP lipid and pH-sensitive polymeric nanoparticles administered to healthy rats; DM SAR-NPs= SAR- NP lipid and pH-sensitive polymeric nanoparticles administered to ZDF diabetic rats; DM SAR-T3-NPs(P)= SAR-T3- NP pH-sensitive polymeric nanoparticles administered to ZDF diabetic rats; DM SAR-T3-NPs(L)= SAR-T3-NP lipid nanoparticles administered to ZDF diabetic rats. Comparisons between groups were performed with one-way ANOVA and post hoc tests (Bonferroni or Dunnett correction).

[0047] - Figure 19 illustrates left ventricular (LV) diastolic function after the treatment with the lipid and pH-sensitive polymeric nanoparticles of the present invention for 3 months in healthy and diabetic rats. Lean SAR-NPs= SAR-NP lipid and pH-sensitive polymeric nanoparticles administered to healthy rats; DM SAR-NPs= SAR-NP lipid and pH-sensitive polymeric nanoparticles administered to ZDF diabetic rats; DM SAR-T3- NPs(P)= SAR-T3-NP pH-sensitive polymeric nanoparticles administered to ZDF diabetic rats; DM SAR-T3-NPs(L)= SAR-T3- NP lipid nanoparticles administered to ZDF diabetic rats. Comparisons between groups were performed with one-way ANOVA and post hoc tests (Bonferroni or Dunnett correction).

[0048] - Figure 20 illustrates the heart rate assessed during echocardiography after 3 months of treatment with the lipid and pH-sensitive polymeric nanoparticles of the present invention in healthy and diabetic rats. Lean SAR-NPs= SAR- NP lipid and pH-sensitive polymeric nanoparticles administered to healthy rats; DM SAR-NPs= SAR-NP lipid and pH-sensitive polymeric nanoparticles administered to ZDF diabetic rats; DM SAR-T3-NPs(P)= SAR-T3-NP pH-sensitive polymeric nanoparticles administered to ZDF diabetic rats; DM SAR-T3-NPs(L)= SAR-T3-NP lipid nanoparticles administered to ZDF diabetic rats.

[0049] - Figure 21 illustrates serum creatinine levels in healthy and diabetic rats after 3 months of treatment with the lipid and pH-sensitive polymeric nanoparticles of the present invention. Lean SAR-NPs= SAR-NP lipid and pH- sensitive polymeric nanoparticles administered to healthy rats; DM SAR-NPs= SAR-NP lipid and pH-sensitive polymeric nanoparticles administered to ZDF diabetic rats; DM SAR-T3- NPs(P)= SAR-T3-NP pH-sensitive polymeric nanoparticles administered to ZDF diabetic rats; DM SAR-T3-NPs(L)= SAR-T3- NP lipid-sensitive nanoparticles administered to ZDF diabetic rats.

[0050] - Figure 22 illustrates fasting blood glucose in healthy and diabetic rats after 3 months of treatment with the lipid and pH-sensitive polymeric nanoparticles of the present invention. Lean SAR-NPs= SAR-NP lipid and pH-sensitive polymeric nanoparticles administered to healthy rats; DM SAR-NPs= SAR-NP lipid and pH-sensitive polymeric nanoparticles administered to ZDF diabetic rats; DM SAR-T3- NPs(P)= SAR-T3-NP pH-sensitive polymeric nanoparticles administered to ZDF diabetic rats; DM SAR-T3-NPs(L)= SAR-T3- NP lipid nanoparticles administered to ZDF diabetic rats.

[0051] - Figure 23 illustrates total cholesterol and triglycerides in healthy and diabetic rats after 3 months of treatment with the lipid and pH-sensitive polymeric nanoparticles of the present invention. Lean SAR-NPs= SAR- NP lipid and pH-sensitive polymeric nanoparticles administered to healthy rats; DM SAR-NPs= SAR-NP lipid and pH-sensitive polymeric nanoparticles administered to ZDF diabetic rats; DM SAR-T3-NPs(P)= SAR-T3-NP pH-sensitive polymeric nanoparticles administered to ZDF diabetic rats; DM SAR-T3-NPs(L)= SAR-T3-NP lipid nanoparticles administered to ZDF diabetic rats.

[0052] - Figure 24 illustrates the effect of treatment with the lipid and pH-sensitive polymeric nanoparticles of the present invention on renal function. Data are expressed as mean ± SEM. The data were analysed by one-way ANOVA and comparisons between groups (comparison groups 1= Lean, DM SAR-Nps and DM SAR-Peg / T3; comparison groups 2= Lean, DM SAR-Nps and DM SAR-Lp / T3) were performed with post hoc tests (Tukey's correction). Lean SAR-NPs= SAR-NP lipid and pH- sensitive polymeric nanoparticles administered to healthy rats; DM SAR-NPs= SAR-NP lipid and pH-sensitive polymeric nanoparticles administered to ZDF diabetic rats; DM SAR-T3- NPs(P)= SAR-T3-NP pH-sensitive polymeric nanoparticles administered to ZDF diabetic rats; DM SAR-T3-NPs(L)= SAR-T3- NP lipid nanoparticles administered to ZDF diabetic rats.

[0053] - Figure 25 illustrates left ventricular (LV) systolic and diastolic function assessed by echocardiography after administration of doxorubicin (Dox) and treatment with the ROS-sensitive polymeric nanoparticles of the present invention for 4 weeks in C57 / bl6 mice. Data are presented as mean ± SEM. Comparisons between groups were performed with the independent-sample t-test.

[0054] Description of Embodiments LIPID NANOPARTICLES

[0055] According to a first aspect of the invention, a thyroid hormones delivery system comprising lipid nanoparticles (also called liposomes) is provided, comprising:

[0056] (1) at least one lipid

[0057] (2) at least one lipid with stealth properties

[0058] (3) at least one functionalised lipid having structure (I)

[0059] LIFID I- LINKER |- 1 TARGETING

[0060] :. j i 1 AGENT wherein said targeting agent is a targeting agent for the TRCP6 receptor, and in which said thyroid hormones are encapsulated in said nanoparticles or immobilised on said nanoparticles .

[0061] Preferably, thyroid hormones are triiodothyronine (T3) and thyroxine (T4).

[0062] The present invention is advantageous compared to existing approaches because the nanoparticles are able to deliver the drug specifically only to stressed cells (podocytes and cardiomyocytes) thanks to overexpression of the TRCP6 receptor, reducing the potential adverse effects while maintaining the repairing and regenerating properties of thyroid hormones and simultaneously treating two important complications of diabetes.

[0063] In addition, the delivery system of the invention minimises systemic distribution, allows lower doses of required thyroid hormones and reduces toxicity.

[0064] The lipid (1)

[0065] The delivery system comprises a lipid (1) responsible for the integrity and stability of the nanoparticle. At the same time, it allows the encapsulation of hydrophobic compounds, such as the small molecules T3 and T4, through hydrophobic interactions within the lipid bilayer.

[0066] Preferably, lipid (1) is a phospholipid.

[0067] In the present invention, lipids (1) can be any lipid species present in a charged, uncharged or neutral zwitterionic form. Lipids (1) can be chosen, by way of example and without limitation, from the group consisting of l-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1,2- distearoyl-glycero-3-phosphocholine (DSPC), 1,2- dipalmitoyl-glycero-3-phosphocholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE) dilauroylphosphatidylethanolamine (DLPE), diacylphosphatidylethanolamine, monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, dielaidoylphosphatidylethanolamine (DEPE), stearoyloleoylphosphatidylethanolamine (SOPE) palmitoyloleoyl-phosphatidylethanolamine (POPE), 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), distearoylphosphatidylcholine (DSPC), dipalmitoyl-phosphatidylcholine (DPPC), dimyristoyl-phosphatidylcholine (DMPC), dilauroylphosphatidylcholine (DLPC), diacyl-phosphatidylcholine, palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleol- phosphatidylglycerol (POPG), dioleoyl-phosphatidylglycerol (DOPG), distearoyl-phosphatidylglycerol (DSPG), dipalmitoyl-phosphatidylglycerol (DPPG), dimyristoyl- phosphatidylglycerol (DMPG), dilauroyl-phosphatidylglycerol (DLPG), glycosyldiacylglycerols, phosphoinositides, phosphatidylserine, ceramide, cerebrosides (such as in particular galactocerebrosides) , cephalin, sphingolipids (such as in particular sphingomyelin and dihydrosphingomyelin), phosphatidic acids and lysophosphatidic acid, asialogangliosides (such as in particular asialo GM1 and GM2), cardiolipin, bis(monoacylglycerol)phosphate, prostaglandins, eicosanoids, glycerides, ether lipids, oxidised lipids, sterol-modified phospholipids. Lipids comprising a single fat chain can also be utilised, including lysophosphatides, lys©phosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol, lys©phosphatidylserine, lysophosphatidylglycerols, lysophosphatidylserines or even lysophosphatidic acids and their mixtures. Lipids (1) may contain saturated or unsaturated fatty acids. Other examples of lipids (1) include non-phosphorus- containing lipids such as, e.g. stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulphate, alkylaryl sulphate, polyethyl-amides of fatty acids, dioctyldimethylammonium bromide, ceramide and sphingomyelin.

[0068] The lipid with stealth properties (2)

[0069] The lipid with stealth properties (2) plays the role of increasing colloidal stability, resistance to opsonisation and reticuloendothelial clearance and thus the biodistribution lifetime after nanoparticle administration. The term "lipid with stealth properties" means a lipid bound to a large inert polymeric material so as to create steric hindrance and thus reducing the aggregation of nanoparticles with each other. The term can also refer to lipids that increase the circulating stability of liposomes, reduce harmful non-specific interactions, reduce their aggregation and thus improve drug delivery efficiency.

[0070] Examples of lipids with stealth properties (2) include e.g. polyethylene glycol (PEG)-modified lipids (pegylated lipids such as PEG-DMG and PEG-DMA), monosialoganglioside (Gml) and polyamide oligomers ("PAO"). Lipids modified with polyethylene glycol are preferred.

[0071] Suitable pegylated lipids include, but are not limited to, PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG- CerC20), PEG-modified dialkylamines, PEG-modified 1,2- diacyloxypropan-3-amines, PEG-modified diacylglycerols and dialkylglycerols, mPEG (PM=2000)- distearoylphosphatidylethanolamine (PEG-DSPE), 1,2- distearoylglycerol, 2-distearoyl-glycero-3- phosphoethanolamine-N- [amino (polyethylene glycol)] (DSPE- PEG) 1,2-dimyristoyl-glycero-3-methoxy [polyethylene glycol] (DMG-PEG), 1,2-dioleoyl-glycero-3-phosphoethanolamine-N-

[0072] [amino (polyethylene glycol) (DOPE-PEG). Other examples of PEG-conjugated lipids may include 1- (monomethoxy-polyethylene glycol) -2,3-dimyristoylglycerol (PEG-DMG), pegylated phosphatidylethanolamine (PEG-PE), dimyristoyl-rac-glycero-3-methoxy-polyethylene glycol (PEG- S-DMG), a pegylated ceramide (PEG-cer), or a pegylated dialkoxy-propyl carbamate such as methoxy (polyethoxy)ethyl- N- (2,3-di(tetradecanoxy)propyl)carbamate or 2,3- di (tetradecanoxy)propyl-N- (c)- methoxy (polyethoxy)ethyl)carbamate.

[0073] According to the present invention, polyethylene glycol (PEG) is a linear, water-soluble polymer consisting of repeating ethylene units with two terminal hydroxyl groups. PEGs are classified according to their molecular weight and include the following: monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol-succinate (MePEG-S), monomethoxypolyethylene glycol- succinimidylsuccinate (MePEG-SNHS), monomethoxypolyethylene glycolamine (MePEG-NH), monomethoxypolyethylene glycol tresylate (MePEG-TRES), monomethoxypolyethylene glycolimidazolylcarbonyl (MePEG-IM), as well as compounds containing a terminal hydroxyl group instead of a terminal methoxyl group (e.g., HO-PEG-S, HO-PEG-S NHS, HO-PEG-NH).

[0074] In the lipids with stealth properties described herein, the PEG group can have an average molecular weight between 550 Daltons and 10,000 Daltons. In some cases, the PEG group has an average molecular weight between 750 Daltons and 5,000 Daltons (e.g. 1,000 Daltons to 5,000 Daltons, 1,500 Daltons to 3,000 Daltons, 750 Daltons to 3,000 Daltons, 750 Daltons to 2,000 Daltons). In the preferred examples, the PEG group has an average molecular weight of 2,000 Daltons or 750 Daltons.

[0075] In some cases, PEG may optionally be replaced by an alkyl, alkoxy, acyl or aryl group.

[0076] The PEG can be conjugated directly to the lipid or can be bound to the lipid via a bridge unit. Any suitable bridge unit may be used to couple PEG to a lipid, comprising, for example, bridge units not containing esters and bridge units containing esters. In a preferred embodiment, the bridge unit is a bridge unit containing no esters. Bridge units containing no esters include, but are not limited to, starch (-C(O)NH-), amino (-NR-), carbonyl (-C (0)-), carbamate (- NHC(O)O-), urea (-NHC (0)NH-), disulphide (-S-S-), ether (- 0-), succinyl (- (0)CCHC (0)-), succinamidyl (- NHC (0)CHCHC (0)NH-), as well as combinations thereof (such as, for example, a bridge unit containing both a carbamate bridge unit and a starch bridge unit). In a preferred embodiment, a carbamate bridge unit is used for coupling the PEG and the lipid together.

[0077] In other cases, a bridge unit containing an ester is used for coupling the PEG and the lipid together. Among the suitable bridge units containing esters, for example, carbonate (-OC(O)O-), succinyl-phosphate (-O(O)POHO-) esters, sulphonate esters and their combinations can be found. Phosphatidylethanolamines having a variety of acyl chain groups of varying length and saturation degree can be conjugated to PEG to form lipids with stealth properties. Such phosphatidylethanolamines are commercially available or can be isolated or synthesised by conventional techniques known to those skilled in the art. Phosphatidylethanolamines containing saturated or unsaturated fatty acids with carbon chain lengths between CIO and C20 are preferred. Phosphatidylethanolamines with mono- or di-unsaturated fatty acids and mixtures of saturated and unsaturated fatty acids can also be used. Suitable phosphatidylethanolamines include, but are not limited to, dimyristoylphosphatidylethanolamine (DMPE), dipalmitoyl- phosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE) and distearoylphosphatidylethanolamine (DSPE).

[0078] In other cases, PEG may be coupled to a diacylglycerol moiety. In this definition, "diacylglycerol" or "DAG" comprises a compound with 2 fatty acyl chains, both independent, with a number of carbons between 2 and 30, and bound at the 1- and 2-positions of the glycerol by ester bonds. The acyl groups can be saturated or have varying degrees of unsaturation. Suitable acyl groups include, but are not limited to, lauroyl (C12), myristoyl (C14), palmitoyl (C16), stearoyl (C18) and icosoyl (C20). In the preferred embodiments, the two acyl chains are the same, both being either myristoyl (i.e. dimyristoyl) or stearoyl (i.e. distearoyl) .

[0079] In other cases, PEG can be coupled to a "dialkylpropyl moiety" or "DAA", which comprises a compound with 2 alkyl chains having independently between 2 and 30 carbons. The alkyl groups may be saturated or have varying degrees of unsaturation. Preferably, the PEG-DAA conjugate is a PEG- didecyloxypropyl conjugate (CIO), a PEG-dilauryloxypropyl conjugate (C12), a PEG-dimethyloxypropyl conjugate (C14), a PEG-dipalmitoxypropyl conjugate (C16) or a PEG- distearyloxypropyl conjugate (C18).

[0080] In these cases, PEG preferably has an average molecular weight of 750 or 2,000 Daltons. In particular embodiments, the terminal hydroxyl group of PEG is replaced by a methyl group, thus creating a terminal methoxyl group.

[0081] In addition to the above, the lipid with stealth properties can have other hydrophilic polymers other than PEG as an inert polymeric material. Examples of suitable polymers that can be used instead of PEG include polyglycerol, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid and derivatised celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

[0082] The functionalised lipid having structure (I) (3)

[0083] The delivery system of the present invention further comprises at least one functionalised lipid having structure (I) (3) which allows the binding of the targeting agent on the surface of the nanoparticles by means of a covalent bond and thus to specifically deliver the nanoparticles to the receptors on the cell surface.

[0084] The lipid portion of the functionalised lipid having structure (I) (3) comprises, but is not limited to, lipids selected from the group consisting of those ones described for the abovementioned lipids in groups (1) or (2).

[0085] The linker may be selected from, but is not limited to, the group consisting of azide, carboxy-N-hydroxy succinimide (carboxy-NHS), isothiocyanates, isocyanates, acyl azides, N- hydroxy succinimide esters (NHS esters), sulphonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, arylating agents, imidoesters, carbodiimides, anhydrides, haloacetyl derivatives, alkyl halide derivatives, maleimides, aziridines, acryloyl derivatives, thiol disulphide, carbonyl diimidazole, diazoalkane compounds, diazoacetyl compounds, N,N'-Disuccinimidyl carbonate, hydrazine derivatives, homofunctional linkers, heterobifunctional linkers, trifunctional crosslinkers. The functionalised lipid with structure (I) (3) also comprises a targeting agent for TRCP6 (transient receptor potential cation channel, subfamily C, member 6). This receptor is overexpressed in cardiomyocytes in response to stress (Onohara N et al., 2006; Kuwahara K et al., 2006) and is increased in damaged podocytes (Ilatovskaya DV et al., 2015). Interestingly, TRPC6 is highly expressed in podocytes of Zucker Diabetic Fatty (ZDF) rats - an animal model of type II diabetes - whereas it is almost completely absent in healthy control rats. Although TRPC6 is also expressed in healthy control animals, there was a strong increase in protein levels in the heart in response to diabetes. TRPC6 is also highly expressed in immortalised human podocytes (Saleem MA et al., 2002) and in cardiomyocytes (Davidson MM et al., 2005) under normal culture conditions. These cell lines were therefore used to assess the toxicity of the delivery system according to the present invention and to optimise its specificity.

[0086] Preferably, the targeting agent chosen in the group consisting of 4- [[(1R,2R)-2- [(3R)-3-amino-l-piperidinyl]- 2,3-dihydro-lH-inden-l-yl]oxy]-3-chlorobenzonitrile dichlorhydrate (SAR7334) of formula

[0087] Larixyl acetate, 1,3-dihydro-l- [1- [(5,6,7,8- tetrahydro-4H-cyclohepta [b]thien-2-yl)carbonyl]-4- piperidinyl]-2H-benzimidazol-2-one (GSK-2934A), 1— (2— (3— (4— methoxyphenyl)propoxy)-4-methoxyphenylethyl) -lH-imidazole

[0088] (SKF96365) [4- (6-aminopiridazin-3-yl)piperidin-l-yl]- [4- [4- (trifluoromethyl)fenoxy]phenyl]metanone (BI749327), 4-

[0089] [ [(1R,2R,3aR,7aS)-2- [(3R)-3-aminopiperidin-l-yl]-7a-methyl- 5-oxo-2,3,3a,4,6,7-hexahydro-lH-inden-l-yl] oxy]benzonitrile (DS88790512), (IS,4S,4aR,8aS)-4- [(3S)-3-hydroxy-3- methylpent-4-en-l-yl]-4a,8,8-trimethyl-3-methyliden- decahydronaphthalene-l-yl N-methylcarbamate (SH045), [4- (6- aminopyridazin-3-yl)piperidin-l-yl]- [4- [4-

[0090] (trifluoromethyl)phenoxy]phenyl]methanone (BI749327), ethyl 4- [7-hydroxy-2-methyl-3- [4-

[0091] (trifluoromethyl)phenyl]pyrazol [1,5-a]pyrimidin-5- yl]piperidin-l-carboxylate (HDM), ethyl 4- [7-hydroxy-2,5- dimethyl-3- [4- (trifluoromethyl)phenyl]pyrazol[1,5— a]pyrimidin-5-yl]piperidin-l-carboxylate (HQR).

[0092] Other components

[0093] The delivery system according to the present invention may also comprise:

[0094] (4) at least one sterol and / or

[0095] (5) at least one cationic lipid or

[0096] (6) at least one ionisable lipid

[0097] Group 5) and group 6) lipids can facilitate the interactions between nanoparticles and the cell membrane, promote cellular uptake and improve endosomal drug delivery within cells.

[0098] The sterol (4)

[0099] According to an embodiment, the delivery system according to the present invention may also comprise a sterol (4) such as cholesterol or a derivative thereof. Non-limiting examples of cholesterol derivatives include polar analogues such as 5C-cholestanol, 5C-coprostanol, cholesteryl-(2- hydroxy)-ethyl ether, cholesteryl- (4'-hydroxy)-butyl ether, 6-chetocolestanol; non-polar analogues such as 50- cholestane, cholestenone, 5C-cholestanone, cholesteryl decanoate; and mixtures thereof. In other cases, the cholesterol derivative is a polar analogue such as cholesteryl- (4'-hydroxy)-butyl ether.

[0100] The cationic lipid (5)

[0101] According to a further embodiment, the delivery system according to the present invention may also comprise (5) a cationic lipid such as monovalent cationic lipids in the form of quaternary ammonium salts such as DOTMA (N-[l-(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP (1,2-dioleyloxy-3-trimethylammonium propane) or DDAB (dioctadecyldimethylammonium bromide); monovalent cationic lipids in the form of pyridinium salts, such as SAINT-2 (N- methyl-4- (dioleyl)methylpyridinium chloride); multivalent cationic lipids in the form of lipospermines such as DOGS (dioctadecylamidoglycyl spermine) and DOSPA (2,3- dioleoyloxy-N- [2- (spermine-carboxamido)ethyl]-N,N-dimethyl- 1-propanaminium); multivariate cationic lipids in the form of lipopolysins, cationic derivatives of cholesterol such as DC-Chol (3p[N- (N '—N ',-dimethylaminomethane)- carbamoyl]cholesterol), amino acid-based lipids such as dioleoyl-glycero-succinyl-cystamido-ornithine, dioleoyl- glycero-succinyl-cystamido-arginine, dioleoyl-glycero- succinyl-glutamido-bis- (cystamido-ornithine), dioleoyl- glycero-succinyl-cystamido-bis-lysine and all other related lipids as described in EP2285772. The ionisable lipid (6)

[0102] As an alternative to the cationic lipid, the deliverysystem of the present invention may comprise (6) an ionisable lipid.

[0103] By "ionisable lipid" is preferably meant an amphiphilic organic molecule including in its structure a hydrophobic non-polar and hydrophilic polar carbonaceous region bound together by a chemical spacer. It is defined as ionisable due to the propensity of its hydrophilic region to include one or more positive charges depending on the pH of its direct environment, allowing the user to adjust its chemical properties by choosing the right pH. Consequently, it has no permanent positive charges at physiological pH (7.0-7.4), while it becomes cationic when the pH of its physiological environment decreases. Its amphiphilic nature makes it generally water-insoluble and soluble to some extent in an organic solvent. This also implies that such molecules can form self-assembled structures in an aqueous environment if handled correctly.

[0104] The ionisable lipid may also be an ionisable cholesterol derivative, designed to be positively charged at acidic and neutral pH under physiological conditions, in order to allow efficient drug encapsulation and intracytoplasmic delivery upon protonation under acidic conditions.

[0105] Any type of ionisable lipid or ionisable cholesterol can be considered, such as, but not limited to, (10Z,13Z)- 1- (9Z,12Z)-9,12-octadecadien-l-yl-10,13-nonadecadien-l-yl ester of 4- (dimethylamino)-butanoic acid (D-Lin-MC3-DMA), 2,2-dilinoleyl-4- (2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1 ,2-dioleyloxy-3-dimethylaminopropane (DODMA), ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis (2-hexyldecanoate) (ALC-0315), 1-octylnononyl ester of 8- [ (2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl]amino]-octanoic acid (SM-102), l-methyl-4,4-bis[(9Z,12Z)-9,12-octadecadien- 1-yloxy]-piperidine (YSK05), 1,1'- ((2- (4- (2- (2- (bis(2- hydroxydodecyl)amino)ethyl) (2- hydroxydodecyl)amino)ethyl)piperazin-1- yl)ethyl)azanediyl)bis (dodecan-2-ol) (C12-200), decyl (2- (dioctylammonium)ethyl) phosphate (9A1P9) and those listed in the following publications: Martijn J. W. Evers et al, "State-of-the-Art Design and Rapid-Mixing Production Techniques of Lipid Nanoparticles for Nucleic Acid Delivery", Small Methods, 2018, 2, 1700375; Yulia Eygeris et al., "Chemistry of Lipid Nanoparticles for RNA Delivery", Acc. Chem. Res. 2022, 55, 1, 2-12; Xucheng Hou et al., "Lipid nanoparticles for mRNA delivery", Nature Reviews Materials volume 6, pages 1078-1094, (2021); Sean A. Dilliard et al, "Passive, active and endogenous organ-targeted lipid and polymer nanoparticles for delivery of genetic drugs", Nature Reviews Materials volume 8, pages 282-300, (2023); Da Sun et al., "Structure and Function of Cationic and Ionizable Lipids for Nucleic Acid Delivery", Pharmaceutical Research volume 40, pages 27-46 (2023).

[0106] The molar ratio of lipids of groups 1-6 and the amount of targeting agents attached to the nanoparticle surface can be modulated to obtain optimal delivery in specific organs.

[0107] Group (1) lipids in the nanoparticle formulation may preferably be between 5 and 96 mole percent, group (2) lipids between 0.1 and 10 mole percent, group (3) functionalised lipids between 0.1 and 10 mole percent, optional sterol compounds may be present between 0 and 50 mole percent, and optional cationic and ionisable lipids may be present between 0 and 50 mole percent.

[0108] The lipid ratio of groups 1-6 can also be adjusted to obtain maximum encapsulation and stability of the drug within the nanoparticles.

[0109] Lipids of groups 1-6 can also be labelled with fluorophores or biotin or radio markers, which are used in some formulations to easily monitor the biodistribution of nanoparticles (e.g. DSPE-Rodamine, DSPE-NBD, DSPE-PEG-N- Cy5).

[0110] The delivery system according to the present invention is used in the treatment of a disorder selected from the group consisting of diabetic cardiomyopathy and diabetic nephropathy .

[0111] The nanoparticles of the delivery system according to the present invention can be produced by means of methods known in the art.

[0112] For instance, nanoparticles can be prepared through microfluidic technology with pressure-driven flow control. First, all the lipid mixtures defined above, supplemented with T3 or T4 drug, are dissolved in an organic solvent (e.g. CHCI3, CHC13:MeOH, MeOH) before being evaporated under high vacuum to form a dried thin film., the lipid thin film is then redissolved in a solvent (e.g. i-PrOH, EtOH, MeOH), before is made to flow by a rapid mixing with an antisolvent (e.g. an aqueous buffer). At the junction of the microfluidic chip, the compounds self-assemble into nanoparticles by a nucleation-growth process before being collected at the end of the microfluidic system. The resulting nanoparticles are further purified (e.g. by dialysis, ultra-centrifugation, tangential flow filtration) and stored at 4 °C.

[0113] Alternatively, nanoparticles can be prepared using the ethanol injection technique. The lipid mixture and the T3 or T4 drug in EtOH solution are rapidly injected into an aqueous solution in which small unilamellar vesicles spontaneously form. Depending on the concentration of the lipid formulation, liposomes of different sizes can be obtained. The resulting nanoparticles are further purified (e.g. by dialysis, ultra-centrifugation, tangential flow filtration) and stored at 4 °C.

[0114] Lipid nanoparticles can also be obtained by means of the thin film rehydration method. The lipid thin film containing the T3 or T4 drug is rehydrated (e.g. with an aqueous buffer) to form a suspension of large multilamellar vesicles. The suspension is extruded under high pressure (10-40 bar) through a polycarbonate membrane with selected pore sizes (e.g. 80 nm, 100 nm or 200 nm). The extrusion process forces the nanoassembly of materials and generates monodisperse nanoparticles with a well-defined size distribution. Once collected, the lipid nanoparticles are purified (e.g. by dialysis, ultra-centrifugation, tangential flow filtration) and stored at 4 °C.

[0115] Finally, other classic and commonly known methods can be used for the production of liposomes, such as solvent injection, mechanical dispersion, reversed-phase evaporation, double emulsification, water in the organic phase or solubilisation with detergents.

[0116] The nanoparticles obtained were evaluated with additional analytical techniques (e.g., dynamic light scattering, electron microscopy (EM), UV-vis spectroscopy, fluorescence, HPLC) to assess, inter alia, their size distribution, polydispersity index, charge surface area, morphology, encapsulation efficiency (e.e.) and stability, SAR active agent grafting rate and sterility.

[0117] Figure 1 shows the morphology, size, stability and structure of SAR-T3-NPs (lipid nanoparticles encapsulating the T3 hormone functionalised with SAR7334) and SAR-NPs, (empty lipid nanoparticles functionalised with SAR7334) over time. The lipid nanoparticles range from 80 to 100 nm in size during the first month and the size slightly increases with time, until reaching a maximum of 160 nm after one year of storage at 4 °C.

[0118] The nanoparticles showed an encapsulation efficiency rate of 70-90% of T3 and T4 molecules.

[0119] When preparing the functionalised lipids, the covalent bond of SAR7334 molecules to the lipid was optimal when 2.5 molar percentage of lipids was used.

[0120] This molar percentage has proven to be optimal for a better internalisation within targeted cell lines (e.g., human cardiomyocytes and podocytes). Molar percentages of functionalised lipids containing SAR7334 can be between 1% and 25%. Furthermore, the nanoparticles size is approximately 80-160 nm with a high monodispersion for a period of up to 1 year when stored at 4 °C. Electron microscopy (EM) images confirmed the morphology and structural integrity of the delivery systems of the invention containing lipid nanoparticles. pH—SENSITIVE POLYMERIC NANOPARTICLES

[0121] According to a second aspect of the invention, a thyroid hormones delivery system comprising polymeric nanoparticles comprising an amphiphilic block copolymer comprising a hydrophobic block of poly (y-benzyl-L-glutamate) and a hydrophilic block of amino-polyethyleneglycol or amino-N3- polyethyleneglycol wherein said thyroid hormones are immobilised on said nanoparticles by means of a pH-sensitive bridge covalently binding said hormones to said hydrophobic block is provided.

[0122] Advantageously, such nanoparticles are sensitive to acid pH and are therefore able to deliver thyroid hormones in ischaemic areas, damaged tissues or within the cell at the level of endosomes.

[0123] In particular, the pH-sensitive bridge can be a bridge of formula (III)

[0124] In an embodiment, the delivery system further comprises a targeting agent, in particular chosen from the group consisting of Larixyl acetate, 1,3-dihydro-l- [1- [(5,6,7,8- tetrahydro-4H-cyclohepta [b]thien-2-yl)carbonyl]-4- piperidinyl]-2H-benzimidazol-2-one (GSK-2934A), 1— (2— (3— (4— methoxyphenyl)propoxy)-4-methoxyphenylethyl) -lH-imidazole (SKF96365), [4- (6-aminopyridazin-3-yl)piperidin-l-yl]- [4-

[0125] [4- (trifluoromethyl)phenoxy]phenyl]methanone (BI749327), 4- [ [(1R,2R,3aR,7aS)-2- [(3R)-3-aminopiperidin-l-yl]-7a-methyl- 5-oxo-2,3,3a,4,6,7-hesahydro-lH-inden-l-yl] oxy]benzonitrile (DS88790512), (IS,4S,4aR,8aS)-4- [(3S)-3-hydroxy-3- methylpent-4-en-l-yl]-4a,8,8-trimethyl-3-methyliden- decahydronaphtalen-l-yl N-methylcarbamate (SH045), [4— (6— aminopyridazin-3-yl)piperidin-l-yl]- [4- [4-

[0126] (trifluoromethyl)phenoxy]phenyl]methanone (BI749327), ethyl 4- [7-hydroxy-2-methyl-3- [4-

[0127] (trifluoromethyl)phenyl]pyrazol [1,5-a]pyrimidin-5- yl]piperidin-l-carboxylate (HDM), ethyl 4- [7-hydroxy-2,5- dimethyl-3- [4- (trifluoromethyl)phenyl]pyrazol[1,5- a]pyrimidin-5-yl]piperidin-l-carboxylate (HQR), more specifically 4- [[(1R,2R)-2- [(3R)-3-amino-l-piperidinyl]- 2,3-dihydro-lH-inden-l-yl]oxy]-3-chlorobenzonitrile dichlorhydrate (SAR7334).

[0128] The presence of such a targeting agent allows selective delivery of nanoparticles to stressed cells (podocytes and cardiomyocytes) only, thanks to the overexpression of the TRCP6 receptor, reducing potential adverse effects while maintaining the repairing and regenerating properties of thyroid hormones and simultaneously treating two important complications of diabetes.

[0129] The nanoparticles are synthesised from modified PEG-b- PBLG and N3-PEG-b-PBLG copolymers. The copolymers consist of a PEG block that increases the nanoparticle stability and half-life in the bloodstream and minimises the opsonisation after administration.

[0130] In one embodiment, the block copolymer comprises one PEG block and one PBLG block and the PEG / PBLG weight ratio is between 30 / 70 and 70 / 30, preferably 45 / 55.

[0131] POLYMERIC NANOPARTICLES SENSITIVE TO REACTIVE OXYGEN SPECIES (ROS)

[0132] Finally, according to a third aspect of the invention, a thyroid hormones delivery system comprising polymeric nanoparticles comprising an amino-polyethylene glycol residue or amino-N3-polyethylene glycol residue and a thioketalic bridge having formula (II) is provided wherein said thyroid hormones are immobilised on said nanoparticles .

[0133] Advantageously, this delivery system is able to deliver thyroid hormones in a controlled manner in a microenvironment rich in reactive oxygen species (ROS). Reactive oxygen species are produced under stressful conditions and play a key role in the progression of cardiovascular diseases. High levels of ROS can be found in stressed or injured tissues, especially in diabetic patients, and ROS can also be present in myocardial infarction. In cardiovascular diseases, the organs which are the most affected by ROS are the heart and kidneys, and a drug administration system responsive to ROS has a competitive advantage in treating stress-damaged tissues.

[0134] Nanoparticles according to the third aspect of the invention consist of PEG-NH2 or N3-PEG-NH2, a ROS-reactive, biocompatible and biodegradable thioketalic bridge, and thyroid hormones.

[0135] In one embodiment, the ROS-sensitive nanoparticles can be derivatised to bind a Larixyl acetate targeting agent, 1,3-dihydro-l- [1- [(5,6,7,8-tetrahydro-4H- cyclohepta [b]thien-2-yl)carbonyl]-4-piperidinyl]-2H- benzimidazol-2-one (GSK-2934A), 1— (2— (3— (4— methoxyphenyl)propoxy)-4-methoxyphenylethyl) -lH-imidazole (SKF96365), [4- (6-aminopyridazin-3-yl)piperidin-l-yl]- [4- [4- (trifluoromethyl)phenoxy]phenyl]methanone (BI749327), 4- [ [(1R,2R,3aR,7aS)-2- [(3R)-3-aminopiperidin-l-yl]-7a-methyl- 5-oxo-2,3,3a,4,6,7-hesahydro-lH-inden-l-yl] oxy]benzonitrile (DS88790512), (IS,4S,4aR,8aS)-4- [(3S)-3-hydroxy-3- methylpent-4-en-l-yl]-4a,8,8-trimethyl-3-methyliden- decahydronaftalen-l-yl N-methylcarbamate (SH045), [4- (6- aminopyridazin-3-yl)piperidin-l-yl]- [4- [4-

[0136] (trifluoromethyl)phenoxy]phenyl]methanone (BI749327), ethyl 4- [7-hydroxy-2-methyl-3- [4-

[0137] (trifluoromethyl)phenyl]pyrazol [1,5-a]pyrimidin-5- yl]piperidin-l-carboxylate (HDM), ethyl 4- [7-hydroxy-2,5- dimethyl-3- [4- (trifluoromethyl)phenyl]pyrazol[1,5— a]pyrimidin-5-yl]piperidin-l-carboxylate (HQR), more specifically 4- [[(1R,2R)-2- [(3R)-3-amino-l-piperidinyl]- 2,3-dihydro-lH-inden-l-yl]oxy]-3-chlorobenzonitrile dichlorhydrate (SAR7334).

[0138] Both types of polymeric nanoparticle-based delivery systems exhibit excellent stability, particularly when stored at 4 °C.

[0139] The amount of targeting agent bound to the surface of the nanoparticles can be modulated to obtain optimal delivery to specific organs. Polymeric nanoparticles can also be conjugated with fluorophores (e.g., rhodamine B) to easily monitor biodistribution.

[0140] The delivery systems of the present invention are all suitable for delivering thyroid hormones, in particular triiodothyronine or thyroxine. They are also useful for use in treating a selected disorder in the group consisting of diabetic cardiomyopathy and diabetic nephropathy.

[0141] Hereinafter, the present invention will be illustrated by means of some examples, which are not intended to limit the scope of the invention.

[0142] In the following examples, the following abbreviations are used: empty lipid nanoparticles (free-NPs(L)); T3-loaded lipid nanoparticles (T3-NPs(L)); SAR7334-derivatised empty lipid nanoparticles (SAR-NPs(L)); SAR7334-derivatised T3- loaded lipid nanoparticles (SAR-T3-NPs(L)); empty polymeric nanoparticles (free-NPs(P)); T3-loaded polymeric nanoparticles (T3-NPs(P)); SAR7334-derivatised empty polymeric nanoparticles (SAR-NPs(P)); SAR7334-derivatised T3-loaded polymeric nanoparticles (SAR-T3-NPs(P)).

[0143] EXAMPLES

[0144] Example 1

[0145] Preparation method of pH-sensitive nanoparticles

[0146] The copolymers were synthesised by polymerisation via ring-opening of N-carboxylic anhydride of benzyl-L- glutamate. Subsequently, the PBLG block was modified with hydrazine [PEG-b-P (LG-HYD) or N3- PEG-b-P (LG-HYD)] and levulinic acid [PEG-b-P (LG-HYD-LA) or N3-PEG~b-P (LG-HYD-LA)] to form a pH-sensitive hydrazone bond. To this modified copolymer block, thyroid hormones are covalently bound through a chemical bond [PEG-b-P (LG-HYD-LA)-T3 / T4 or N3-PEG- b-P (LG-HYD-LA)-T3 / T4]. The synthesis scheme for preparing the block polymer to obtain nanoparticles according to the second aspect of the invention is shown below in Synthesis Scheme 1.

[0147]

[0148] The terminal azide group of N3-polyethylene glycol can be employed to attach the targeting agent SAR7334. SAR7334 is attached on the PEG side and must be on the outer surface of the nanoparticles in order to bind easily to the receptors. Specifically, the conjugation between SAR7334 and PEG is obtained through a selective alkyne-azide reaction. SAR7334 was modified with a molecule (DBCO) offering a triple bond that reacts with the azide of PEG. As demonstrated by in vitro and in vivo results, SAR7334 retains the ability to bind to the receptor, even after the conjugation with PEG.

[0149] The synthesis scheme for the conjugation of the block polymer with SAR7334 is shown below in Synthesis Scheme 2.

[0150] Synthesis Scheme 2 Example 2

[0151] Method of preparing ROS-sensitive nanoparticles

[0152] In this formulation, the thioketalic bridge is first bound to PEG-NH2 (or N3-PEG-NH2) amine group. Then, the thyroid hormones are covalently bound to the thioketal bridge (PEG-TK-T3 / T4) . The entire process is simple and has high drug encapsulation yields. Again, the azide group of PEG can be used to bind a targeting agent, in particular SAR7334, using the same procedure as described above (SAR7334-PEG-TK- T3 / T4).

[0153] The thioketalic bridge can be synthesised from 3- mercaptopropionic acid and acetone as depicted in Synthesis Scheme 3.

[0154] Synthesis Scheme 3

[0155] Polymeric nanoparticles according to the third aspect of the invention can be prepared as illustrated in Synthesis scheme 4.

[0156]

[0157] Example 3

[0158] Liposome formulations were prepared according to the film hydration method combined with the extrusion method. Unmodified liposomes (Unmod-LIPO) were obtained by dissolving a mixture of POPC (l-palmitoyl-2- oleylphosphatidylcholine) (9.35 pmol) or DOPC (1,2-dioleoyl- sn-glycero-3-phosphocholine) (9.35 pmol) in chloroform / methanol (1 / 1, v / v), + / - DDAB (Dimethyldioctadecylammonium) (0.75 pmol) or dioleoyl- glycero-succinyl-cistammido-arginine (0.75 pmol) + / - cholesterol and T3 (10 pmol) (as a methanol solution). DSPE- PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol)-2000]) (0.3 pmol) was added to the solution to obtain stealth liposomes (Stealth-LIPO). The DSPE-PEG-maleimide or DSPE-PEG-azide (0.25 pmol) lipid linker was added to the solution to obtain selective stealth liposomes (SAR-stealth-LIPO and T3 SAR-stealth-LIPO, respectively) .

[0159] Lipid mixtures in chloroform / methanol were added to a round-bottomed flask and evaporated in vacuo by rotoevaporation (Rotavapor Biichi R110, Flawil, Switzerland) to form a thin film. The thin lipid films were hydrated at 65 °C with 1 mL of PBS solution (pH 7.4) by mechanical stirring for 2 hours. The resulting liposome dispersions were sonicated with a Soniprep 150 ultrasonic disintegrator (MSE Crowley, London, UK) until a clear, opalescent dispersion was obtained (5 seconds on and 2 seconds off, 60 cycles). The liposomes were then extruded through polycarbonate membranes with 100 nm pores using the Lipex® extruder. At this stage, SAR7334 was covalently conjugated to the distal end of the DSPE-PEG-azide by click chemistry (0.75 pmol of SAR-DBCO were used) or the DSPE-PEG-maleimide lipid by thioether bond (0.75 pmol of SAR-Tiol were used), and then bound to the stealth liposomes surface as a targeting function. To bind the linker with a thioether bond, SAR7334 was thiolated using a molar ratio of 1:40 SAR / iminothiolane (Gijsens et al., 2002), while for binding the azide group, SAR7334 was coupled to DBCO (dibenzocycloctyl) by means of a coupling reaction with N- hydroxysuccinimide . The liposomes were finally purified on a Sepharose CL-4B gel filtration column using phosphate buffer as eluent, or by centrifugation with an Amicon filter (lOOkMW), and sterilised by filtration with a 0.22 pm filter.

[0160] Example 4

[0161] Effects of nanoparticles of the invention on the viability of human cell lines

[0162] For the cytotoxicity studies, human cardiomyocytes and podocytes were exposed for 24 and 72 hours to different delivery systems according to the invention containing rhodamine-labelled lipid and polymeric nanoparticles, specifically (i) free-NPs; (ii) T3-NPs or SAR-NPs; and (iii) SAR-T3-NPs, at concentrations equivalent to 100 nM, 10 nM and 0.1 nM T3. At the end of treatment, cell viability was analysed by assessing apoptosis and necrosis by annexin V staining and lactate dehydrogenase (LDH) assay, respectively.

[0163] - The effect of the delivery system of the invention on the viability of human podocytes

[0164] The results of these cytotoxicity studies indicated that the delivery system of the invention did not lead to a substantial cell death in human podocytes under all conditions and time points tested using both the lipid and pH-sensitive polymeric nanoparticles of the invention (Figure 2A panels A-D and 2B panels E-H, respectively). Specifically, none of the delivery system formulations according to the invention produced signs of apoptosis in human podocytes after 24 (panel A) and 72 (panel B) hours of treatment compared to control cells (CTR). Only the highest dose of SAR-T3-NPs(L) led to a significant necrosis increase in podocytes compared to control cells after 24 hours of treatment (panel C), whereas no apparent changes in podocyte viability were observed among the other groups after 24 (panel C) or 72 (panel D) hours of treatment. No signs of cytotoxicity were also observed for the pH-sensitive polymeric nanoparticles (Figure 2B panels E-H).

[0165] - Effect of the delivery system of the invention on the viability of human cardiomyocytes

[0166] When tested on human cardiomyocytes, only the highest dose of SAR-T3-NPs(L) formulation led to a significant increase in number of the apoptotic cells compared to the control group after 24 hours of treatment (Figure 3A panel A). Likewise, after a 72-hour exposure to the systems of the invention, only the highest dose of SAR-T3-NPs(L) and T3- NPs (L) led to a significant apoptosis increase compared to control cells (Figure 3A panel B). The results of the necrosis test showed no change in the viability of cardiomyocytes treated with the delivery systems of the invention compared to the control group, both after 24 and 72 hours of treatment (Figure 3A panels C and D and Figure 3B panels G and H), suggesting that the delivery systems of the invention do not have a remarkable effect on cardiomyocyte viability.

[0167] - Properties and acute effects of in vivo delivery systems of the invention in healthy rats

[0168] Acute toxicity and pharmacokinetics were determined for lipid and pH-sensitive polymeric nanoparticles (SAR-NPs and SAR-T3-NPs) labelled with rhodamine in healthy animals. Two different doses corresponding to 1 pg / kg of T3 and 10 pg / kg of T3 were studied. Intraperitoneal (i.p.) administration was compared to intravenous administration (Figure 4). After a single injection, the concentration of nanoparticles in serum was analysed at different time intervals by fluorescence ELISA. The biodistribution in different tissues was determined by fluorescence microscopy. The results show that lipid nanoparticles have a serum peak 6 hours after i.p. injection, they remain in the bloodstream after 24 hours and their concentration is very low after 48 hours (Figure 4A). The pH-sensitive polymeric nanoparticles have a serum peak 6 hours after i.p. injection, they remain in the bloodstream at stable levels after 24 hours, and their concentration had been detected for at least 72 hours (Figure 4B).

[0169] Both lipid and polymeric nanoparticles did not build up only in the heart and kidney, but also in the spleen of normal animals. A very low concentration of lipid and polymeric nanoparticles was detected in the liver and lungs of normal rats. No presence was detected in the brain.

[0170] - Chronic effects of in vivo delivery systems according to the invention in healthy rats

[0171] The chronic effects of the delivery systems according to the invention were determined for lipid and pH-sensitive polymeric nanoparticles (SAR-NPs and SAR-T3-NPs) in healthy animals. For the lipid nanoparticles, two different doses corresponding to 1 pg / kg of T3 and 10 pg / kg of T3 were used, 3 times a week for a total of 2 weeks. For the polymeric nanoparticles, two different doses corresponding to 2.3 pg / kg of T3 and 23 pg / kg of T3 were used, once every 5 days for a total of 2 weeks.

[0172] Hyperthyroidism is known to cause tachycardia, increased body temperature, increased cardiac function, and cardiac hypertrophy and reduced serum lipids. No mortality or toxic effects similar to hyperthyroidism were observed after chronic administration of SAR-T3-NPs. No changes were found in heart rate (Figures 7B, 7D), core temperature (Figures 7A, 7C) and body weight (Figures 5A and 5B). Liver function (Figures 8A-D), renal function (Figures 9A-D), cholesterol and triglycerides (Figures 12A-D) assessed by serum biochemical tests were similar among groups. After 2 weeks of treatment, cardiac function in normal rats did not change (Figures 10A-D and Figures 11A-D). As for serum thyroid hormones measurements, a small "delivery" of T3 into the bloodstream was observed after chronic treatment only in the group of animals treated with a high dose of lipid nanoparticles (10 pg / kg) (Figures 6A-F). This delivery caused a 10-15% increase in serum T3 levels and explains why no hyperthyroid-like effects were observed. No changes in thyroid hormones levels were observed with either low-dose or high-dose polymeric nanoparticles.

[0173] - The delivery system of the invention increases the concentration of intracellular T3 and enhances the effects of T3 in in vitro cell cultures

[0174] (a) Functionalisation with SAR7334 strongly improved the ability of cells to absorb the delivery system of the invention

[0175] As described above, human cardiomyocytes and podocytes were exposed to different rhodamine-labelled delivery systems of the invention for 24 and 72 hours. Rhodamine labelling allowed the intracellular uptake of the different formulations to be assessed. After treating with lipid or pH-sensitive polymeric nanoparticles, a marked increase in the intracellular rhodamine signal was observed in both cell lines treated with SAR-functionalised nanoparticles compared to other formulations, indicating that SAR functionalisation strongly increases the uptake of the systems of the invention.

[0176] (b) The delivery system of the invention can direct and deliver T3 into glucose-damaged cells

[0177] To assess the selectivity of the delivery systems of the invention for damaged cells, podocytes and cardiomyocytes were first exposed to normal (CTR) or elevated glucose concentrations to simulate the diabetic insult. The cells were then treated with lipid nanoparticles according to the invention (T3-NPs(L) and SAR-T3-NPs(L)) to assess T3 intracellular uptake by ELISA. Other groups of both control (CTR T3) and glucose-damaged (Glu T3) cells were treated with the same concentration of T3 (10 nM) dissolved in culture medium to compare its uptake with that of the lipid nanoparticle systems according to the invention. In another set of experiments, cells were exposed to lipid nanoparticles loaded with T3 conjugated with fluorescein isothiocyanate (FITC) as a marker. Following the same experimental design as described above, both control and glucose-stressed cells were treated with different labelled nanoparticle formulations, and T3 intracellular uptake was determined by confocal microscopy techniques. The results of the ELISA experiments indicated that T3 intracellular uptake strongly increased in podocytes treated with the inventive systems compared to control and glucose-stressed cells treated with T3 dissolved in the culture medium (Figure 13A). Furthermore, functionalisation of the nanoparticles with SAR7334 increased the T3 intracellular uptake in glucose-stressed podocytes compared to control cells, although glucose treatment positively influenced the uptake of T3 delivered with lipid nanoparticles, even in the absence of SAR7334 (Figure 13A) . Similarly, treatment with the inventive systems significantly increased T3 uptake compared to cells treated with T3 powder dissolved in culture medium also in cardiomyocytes (Figure 13B). A slight increase in T3 intracellular uptake was also observed in glucose-stressed cardiomyocytes treated with SAR-T3-NPs(L) compared to other groups (Figure 13B). Testing by fluorescence and bright- field microscopy showed an increased uptake of FITC- conjugated T3 (in green) in glucose-damaged podocytes treated with SAR-T3-FITC-NPs(L) compared to those exposed to T3-FITC-NPs (L) and both control groups, indicating that functionalisation with SAR7334 of the inventive systems increased T3 uptake in damaged podocytes (Figure 13B panel C). On the other hand, immunofluorescence analysis of the cardiomyocytes treated with the inventive systems showed that FITC signal was higher in control and glucose-damaged cells exposed to SAR-T3-NPs(L) than in the T3-NPs(L)-treated groups, suggesting that T3 uptake increased because of the functionalisation with SAR7334, although in this cell line it did not appear to be affected by glucose treatment (Figure 13 panel D). Similar results were obtained by treating podocytes with rhodamine-labelled polymeric nanoparticles. Indeed, the uptake of the polymeric nanoparticles was clearly higher in glucose-stressed cells treated with SAR- functionalised polymeric nanoparticles than in non-stressed control cells.

[0178] (c) In vitro efficacy of the inventive system functionalised with SAR7334 to enhance the effects of T3

[0179] In order to demonstrate the efficacy of the inventive systems in increasing intracellular T3 concentrations and enhancing their effects, a cell culture model of skeletal myoblasts (C2C12) was exposed to lipid or pH-sensitive polymeric nanoparticles according to the invention (free- NPs, T3-NPs, and SAR-T3-NPs) with a concentration corresponding to 10 nM T3. As above, for these experiments other groups of cells were treated with the same concentration of T3 dissolved in the culture medium. The results of these experiments showed that the treatment with the system of the invention functionalised with SAR7334 increased the expression of differentiation markers (e.g., SERCA1 and actin, Figures 16A and B) compared to free T3 or the delivery system of the invention not functionalised with SAR7334. Most importantly, SAR-T3-NPs significantly enhanced the differentiation of C2C12 myoblasts into myofibers - which is known to be directly dependent on T3 concentrations - compared to the other groups (Figures 14 and 15).

[0180] To demonstrate the efficacy of the ROS-sensitive polymeric nanoparticle system in enhancing the effects of T3 under normal and oxidative conditions, a cell culture model of skeletal myoblasts (C2C12) was exposed to this system (SAR-NPs(P) and SAR-T3-NPs(P)) with a concentration corresponding to 0.5 nM T3 with or without 5 pM H2O2. The results of these experiments showed that treatment with ROS- sensitive SAR-T3-NPs(P) significantly enhanced C2C12 myoblast differentiation into myofibers, even at the low dose of 0.5 nM. The presence of hydrogen peroxide further improved the effect of SAR-T3-NPs(P) (Figure 17).

[0181] - The selective delivery system of the invention specifically delivers T3 into the heart and kidney damaged by diabetes

[0182] In order to assess the selectivity of the inventive systems for in vivo diabetes-damaged cardiomyocytes and podocytes, tissues from control (healthy) and diabetic (ZDF) rats treated with rhodamine-labelled nanoparticles were collected at the end of the pharmacokinetic studies (see above for further details). Animals (n=2 per group) were sacrificed 2 hours after a single i.p. administration of rhodamine-labelled lipid nanoparticles (T3-NPs(L) or SAR-T3- NPs (L), 10 pg / kg of T3), and heart and kidneys were collected to assess nanoparticle specificity by means of a confocal microscopy analysis. It should be noted that ZDF rats treated with SAR-T3-NPs(L) showed a remarkable increase in rhodamine signal at both the cardiac and glomerular level compared to ZDF rats treated with T3-NPs(L) and healthy rats treated with SAR-T3-NPs(L), indicating that functionalisation with SAR7334 confers a greater specificity to cardiac and renal tissues damaged by diabetes. Interestingly, only diabetic animals show a rhodamine signal in the glomerulus, suggesting that functionalisation with SAR7334 increases nanoparticle uptake at glomerular level.

[0183] To further determine the distribution of the drug in the body and, more importantly, whether the inventive systems effectively deliver T3 to damaged cardiomyocytes and podocytes, an in vivo specificity study was performed. For this purpose, healthy and diabetic ZDF rats (n=3 per group) were treated with a single i.v. administration of systems functionalised with SAR7334. In order to follow the distribution in the different tissues, lipid nanoparticles were loaded with fluorescein isothiocyanate (FITC)- conjugated T3 (T3-FITC-NPs(L) and SAR-T3-FITC-NPs(L)), while polymeric nanoparticles were labelled with rhodamine. The inventive systems were administered with a dose corresponding to 10 pg / kg of T3 and rats were sacrificed 2 or 24 hours after the injection of lipid or polymeric nanoparticles (depending on the respective pharmacokinetics) to collect kidney, heart, spleen, liver, brain and lung and perform the subsequent confocal microscopy analyses. As previously described for the biodistribution of nanoparticles, a clear increase in FITC-T3 signal was found in cardiomyocytes and glomeruli of diabetic rats compared to the same regions of healthy control rats in these studies. In particular, in immunofluorescence experiments, the FITC- T3 signal was found to colocalise with a podocyte-specific marker (WT1). Furthermore, in this study as well, renal tubules and spleen were the main accumulation sites of the FITC-T3 signal, whereas almost no FITC signal was found in liver, lungs and brain. Taken together, these results suggest that the system of the invention (i) reaches organs without drug leakage; (ii) crosses biological barriers and delivers T3 into glomeruli, in podocytes, before being reabsorbed by renal tubules; and (iii) when it is functionalised with SAR7334, it can specifically target and deliver T3 to podocytes and cardiomyocytes damaged by diabetes.

[0184] Similar results were obtained by confocal analysis of tissue sections from healthy and diabetic rats treated with pH-sensitive polymeric nanoparticles. Indeed, the delivery system comprising polymeric nanoparticles also reached and accumulated efficiently in the damaged cardiomyocytes and podocytes of diabetic animals. As above, the results of the immunofluorescence experiment showed that the rhodamine- labelled nanoparticles co-localised with a podocyte-specific marker (WT1). Finally, no apparent accumulation of nanoparticles was found in other organs but spleen.

[0185] - Efficacy of the delivery system of the invention in treating diabetic nephropathy and cardiomyopathy

[0186] The efficacy of the lipid and pH-sensitive polymeric nanoparticles of the present invention was determined in ZDF diabetic rats. Nanoparticles with (SAR-T3-NPs) and without T3 (SAR-NPs) were administered intraperitoneally with doses corresponding to 3 pg / kg of T3, 3 times a week for the lipid nanoparticles, or 6 pg / kg once every 5 days for the polymeric nanoparticles for a total of 3 months.

[0187] Echocardiography was used to assess left ventricular systolic and diastolic function. Ejection fraction is an index of global systolic function, while left ventricular wall systolic velocity, assessed by tissue Doppler imaging, is an index of regional myocardial function. Increased deceleration time indicates increased left ventricular filling pressures due to diastolic dysfunction. As it can be seen from Figures 18 and 19, chronic treatment with SAR-T3-NPs in diabetic rats significantly improved systolic and diastolic function as assessed by echocardiography .

[0188] After treatment with both systems of the present invention, no effect on serum lipids (cholesterol and triglycerides, Figure 23), blood glucose (Figure 22), and creatinine clearance (Figure 21) was observed, which indicates the selectivity of the nanoparticles for diabetic hearts and kidneys. Furthermore, nanoparticle treatment did not result into changes in heart rate (Figure 20).

[0189] The therapeutic efficacy of treatment with the lipid and polymeric nanoparticles of the present invention on renal function was assessed by analysing the proteinuria / creatininuria ratio. In particular, Figure 24 illustrates the effect of treatment with the nanoparticles of the present invention on renal function at the beginning of the study (A, day 0), after 30 days of treatment (B) and at the end of the study (C, day 90). Treatment with both nanoparticles of the present invention significantly reduces protein loss in urine in diabetic rats (C) treated with nanoparticles compared to diabetic animals treated with vehicle.

[0190] - Efficacy of ROS-sensitive polymeric nanoparticles in the treatment of doxorubicin-induced cardiomyopathy

[0191] The efficacy of ROS-sensitive polymeric nanoparticles was determined in C57 / B16 mice treated with doxorubicin (6 intraperitoneal injections of 2.5 mg / kg per dose, for a period of 2 weeks). This model has been shown to cause cardiomyopathy and heart failure due to altered calcium homeostasis and increased oxidative stress. SAR-NPs and SAR- T3-NPs were administered intraperitoneally with doses corresponding to 20 pg / kg of T3, twice a week for a total of 4 weeks. Echocardiography was used to assess left ventricular systolic and diastolic functions. Chronic treatment with SAR-T3-NPs significantly improved systolic and diastolic functions as assessed by echocardiography. No effect on heart rate was observed (Figure 25).

Claims

CLAIMS1.- A delivery system of thyroid hormones comprising polymeric nanoparticles comprising an amphiphilic block copolymer comprising a hydrophobic block of poly (y_benzyl- L-glutamate) and a hydrophilic block of amino-polyethylene glycol or amino-N3-polyethylene glycol, wherein said thyroid hormones are immobilized on said nanoparticles by means of a pH-sensitive bridge covalently binding said hormones to said hydrophobic block.2.- The delivery system according to claim 1, characterized in that said pH-sensitive bridge is a bridge of formula (III).3.- The delivery system according to claim 1, characterized in that it further comprises a targeting agent.4.- The delivery system according to claim 3, characterized in that said targeting agent is chosen from the group consisting of 4- [[(1R,2R)-2- [(3R)-3-amino-l- piperidinyl]-2,3-dihydro-lH-inden-l-yl] oxy]-3- chlorobenzonitrile dihydrochloride (SAR7334), Larixyl acetate, 1,3-dihydro-l- [1- [(5,6,7,8-tetrahydro-4H- cyclohepta [b]thien-2-yl)carbonyl]-4-piperidinyl]-2H- benzimidazol-2-one (GSK-2934A), 1— (2— (3— (4— methoxyphenyl)propoxy)-4-methoxyphenylethyl) -lH-imidazole (SKF96365), [4- (6-aminopiridazin-3-yl)piperidin-l-yl]- [4-[4- (trifluoromethyl)phenoxy]phenyl]methanone (BI749327), 4- [ [(1R,2R,3aR,7aS)-2- [(3R)-3-aminopiperidin-l-yl]-7a-methyl- 5-oxo-2,3,3a,4,6,7-hexahydro-lH-inden-l-yl] oxy]benzonitrile(DS88790512), (IS,4S,4aR,8aS)-4- [(3S)-3-hidroxy-3- methylpent-4-en-l-yl]-4a,8,8-trimethyl-3-methyliden- decahydronaphthalene-l-yl N-methylcarbamate (SH045), [4- (6- aminopyridazin-3-yl)piperidin-l-yl]- [4- [4-(trifluoromethyl)phenoxy]phenyl]methanone (BI749327), ethyl 4- [7-hydroxy-2-methyl-3- [4-(trifluoromethyl)phenyl]pyrazol [1,5-a]pyrimidin-5- yl]piperidin-l-carboxylate (HDM), ethyl 4- [7-hydroxy-2,5- dimethyl-3- [4- (trifluoromethyl)phenyl]pyrazol[1,5— a]pyrimidin-5-yl]piperidin-l-carboxylate (HQR).5.- The delivery system of thyroid hormones comprising polymeric nanoparticles comprising an amino-polyethylene glycol or amino-N3-polyethylene glycol residue and a thioketalic bridge having formula (II)wherein said thyroid hormones are immobilized on said nanoparticles .6.- The delivery system according to claim 5, characterized in that it further comprises a targeting agent.

7. A delivery system according to claim 6, characterized in that said targeting agent is chosen from the group consisting of 4- [[(1R,2R)-2- [(3R)-3-amino-l-piperidinyl]- 2,3-dihydro-lH-inden-l-yl]oxy]-3-chlorobenzonitrile dihydrochloride (SAR7334), Larixyl acetate, 1,3-dihydro-l- [1- [(5,6,7,8-tetrahydro-4H-cyclohepta [b]thien-2- yl)carbonyl]-4-piperidinyl]-2H-benzimidazol-2-one (GSK- 2934A), 1- (2- (3- (4-methoxyphenyl)propoxy)-4- methoxyphenylethyl)-lH-imidazole (SKF96365), [4- (6-aminopyridazin-3-yl)piperidin-l-yl]- [4- [4-(trifluoromethyl)phenoxy]phenyl]methanone (BI749327), 4-[ [(1R,2R,3aR,7aS)-2- [(3R)-3-aminopiperidin-l-yl]-7a-methyl- 5-oxo-2,3,3a,4,6,7-hexahydro-lH-inden-l-yl] oxy]benzonitrile (DS88790512), (IS,4S,4aR,8aS)-4- [(3S)-3-hydroxy-3- methylpent-4-en-l-yl]-4a,8,8-trimethyl-3-methyliden- decahydronaphthalene-l-yl N-methylcarbamate (SH045), [4- (6- aminopyridazin-3-yl)piperidin-l-yl]- [4- [4-(trifluoromethyl)phenoxy]phenyl]methanone (BI749327), ethyl 4- [7-hydroxy-2-methyl-3- [4-(trifluoromethyl)phenyl]pyrazol [1,5-a]pyrimidin-5- yl]piperidin-l-carboxylate (HDM), ethyl 4- [7-hydroxy-2,5- dimethyl-3- [4- (trifluoromethyl)phenyl]pyrazol[1,5— a]pyrimidin-5-yl]piperidin-l-carboxylate (HQR).8.- The delivery system of thyroid hormones comprising lipid nanoparticles comprising:(1) at least one lipid(2) at least one lipid with stealth properties(3) at least one functionalised lipid having structure(I) wherein said targeting agent is a targeting agent for the TRCP6 receptor, and wherein said thyroid hormones are encapsulated in or immobilised on said nanoparticles.9.- The delivery system according to claim 8, characterised in that said lipid (1) is a phospholipid.10.- The delivery system according to claim 8, characterised in that said lipid (1) is selected from thegroup consisting of l-palmitoyl-2-oleoyl-glycero-3- phosphocholine (POPC), 1,2-distearoyl-glycero-3- phosphocholine (DSPC), 1,2-dipalmitoyl-glycero-3- phosphocholine (DPPC), dioleoyl-phosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE) dilauroylphosphatidylethanolamine (DLPE), diacylphosphatidylethanolamine, monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, dielaidoylphosphatidylethanolamine (DEPE), stearoyloleoylphosphatidylethanolamine (SOPE), palmitoyl- oleoyl-phosphatidylethanolamine (POPE), 1,2-dioleoyl-sn- glycero-3-phosphocholine (DOPC), distearoylphosphatidylcholine (DSPC), dipalmitoyl-phosphatidylcholine (DPPC), dimyristoyl-phosphatidylcholine (DMPC), dilauroylphosphatidylcholine (DLPC), diacyl-phosphatidylcholine, palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleol- phosphatidylglycerol (POPG), dioleoyl-phosphatidylglycerol (DOPG), distearoyl-phosphatidylglycerol (DSPG) dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidylglycerol (DMPG), dilauroyl-phosphatidylglycerol (DLPG), glycosyldiacylglycerols, phosphoinositides, phosphatidylserine, ceramide, cerebrosides, cephalin, sphingolipids, phosphatidic acids, lysophosphatidic acid, asialogangliosides, cardiolipin, bis (monoacylglycerol)phosphate, prostaglandins, eicosanoids, glycerides, ether lipids, oxidised lipids, sterol-modified phospholipids, lysophosphatides,lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol, lysophosphatidylserine, lysophosphatidylglycerols, lysophosphatidylserines, lysophosphatidic acids, and mixtures thereof.11.- A delivery system according to claim 8, characterised in that said lipid with stealth properties is selected from the group consisting of lipids modified with polyethylene glycol (PEG), monosialoganglioside (Gml) and polyamide oligomers.12.- The delivery system according to claim 8, characterised in that said functionalised lipid having structure (I) comprises:- a targeting agent chosen from the group consisting of4- [[(1R,2R)-2- [(3R)-3-amino-l-piperidinyl]-2,3-dihydro-lH- inden-l-yl]oxy]-3-chlorobenzonitrile dihydrochloride(SAR7334), Larixyl acetate, 1,3-dihydro-l- [1- [(5,6,7,8- tetrahydro-4H-cycloepta [b]thyen-2-yl)carbonyl]-4- piperidinyl]-2H-benzimidazol-2-one (GSK-2934A), l-(2-(4- methoxyphenyl)propoxy)-4-methoxyphenylethyl) -lH-imidazole (SKF96365), [4- (6-aminopyridazin-3-yl)piperidin-l-yl][4—(4— (trifluoromethyl)phenoxy)phenyl)methanone (BI749327), 4-[ [(1R,2R,3aR,7aS)-2- [(3R)-3-aminopiperidin-l-yl]-7a-methyl-5-oxo-2,3,3a,4,6,7-hexahydro-lH-inden-l-yl] oxy]benzonitrile(DS88790512), (IS,4S,4aR,8aS)-4- [(3S)-3-hydroxy-3- methylpent-4-en-l-yl]-4a,8,8-trimethyl-3-methyliden- decahydronaphthalene-l-yl N-methylcarbamate (SH045), [4—(6— aminopyridazin-3-yl)piperidin-l-yl]- [4- [4-(trifluoromethyl)phenoxy]phenyl]methanone (BI749327), ethyl 4- [7-hydroxy-2-methyl-3- [4- (trifluoromethyl)phenyl]pyrazol [1,5-a]pyrimidin-5-yl]piperidin-l-carboxylate (HDM), ethyl 4- [7-hydroxy-2,5- dimethyl-3- [4- (trifluoromethyl)phenyl]pyrazol[1,5- a]pyrimidin-5-yl]piperidin-l-carboxylate (HQR);- a linker chosen from the group consisting of azides, carboxy-N-hydroxy succinimide, isothiocyanates, isocyanates, acyl azides, N-hydroxy succinimide esters, sulphonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, arylating agents, imidoesters, carbodiimides, anhydrides, haloacetyl derivatives, alkyl halide derivatives, maleimide, aziridines, acryloyl derivatives, thiol disulphide, carbonyl diimidazole, diazoalkane compounds, diazoacetyl compounds, N,N'- Disuccinimidyl carbonate, hydrazine derivatives, homofunctional linkers, heterobifunctional linkers, trifunctional crosslinkers;- a lipid chosen from the group consisting of 1- palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1,2- distearoyl-glycero-3-phosphocholine (DSPC) and 1,2- dipalmitoyl-glycero-3-phosphocholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), diacylphosphatidylethanolamine, monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, dielaidoylphosphatidylethanolamine (DEPE), stearoyloleoylphosphatidylethanolamine (SOPE), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylcholine (DOPC), distearoyl-phosphatidylcholine (DSPC), dipalmitoyl-phosphatidylcholine (DPPC) dimyristoylphosphatidylcholine (DMPC), dilauroyl-phosphatidylcholine (DLPC), diacyl-phosphatidylcholine, palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleol- phosphatidylglycerol (POPG), dioleoyl-phosphatidylglycerol (DOPG), distearoyl-phosphatidylglycerol (DSPG), dipalmitoyl-phosphatidylglycerol (DPPG), dimyristoylphosphatidylglycerol (DMPG), dilauroyl-phosphatidylglycerol (DLPG) glycosyldiacylglycerols, phosphoinositides, phosphatidylserine, ceramide, cerebrosides, cephalin, sphingolipids, phosphatidic acids, lysophosphatidic acid, asialogangliosides, cardiolipin, bis (monoacylglycerol)phosphate, prostaglandins, eicosanoids, glycerides, ether lipids, oxidised lipids, sterol-modified phospholipids, lysophosphatides, lys©phosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol, lys©phosphatidylserine, lysophosphatidylglycerols, lys©phosphatidylserines, lysophosphatidic acids, PEG-modified phosphatidylethanolamine and PEG-modified phosphatidic acid, PEG-ceramide conjugates, PEG-modified dialkylamines, PEG- modified 1,2-diacyloxypropan-3-amines, PEG-modified diacylglycerols and dialkylglycerols, mPEG (PM=2000)- distearoylphosphatidylethanolamine (PEG-DSPE), 1,2- distearoylglycerol, 2-distearoyl-glycero-3- phosphoethanolamine-N- [amino (polyethylene glycol)] (DSPE- PEG), 1,2-dimyristoyl-glycero-3-methoxy [polyethylene glycol] (DMG-PEG), 1,2-dioleoyl-glycero-3- phosphoethanolamine-N- [amino (polyethylene glycol) (DOPE-PEG), 1- (monomethoxy-polyethylene glycol)-2,3- dimyristoylglycerol (PEG-DMG), pegylated phosphatidylethanolamine (PEG-PE), dimyristoyl-rac-glycero- 3-methoxy-polyethylene glycol (PEG-S-DMG), pegylated ceramide (PEG-cer), pegylated dialkoxypropyl carbamate, PEG- modified phosphatidic acid, PEG-ceramide conjugates, PEG- modified dialkylamines, PEG-modified 1,2-diacyloxypropan-3- amines, PEG-modified diacylglycerols and dialkylglycerols, and mixtures thereof.13.- The delivery system according to any one of claims 8 to 12, characterised in that it further comprises at least one of (4) at least one sterol, (5) at least one cationic lipid and (6) at least one ionisable lipid.14.- The delivery system according to any one of claims 1 to 13, characterised in that said thyroid hormones are chosen from triidothyronine and thyroxine.15.- The delivery system according to any one of claims 1 to 13 for use in treating a disorder selected from the group consisting of diabetic cardiomyopathy and diabetic nephropathy .16.- A pharmaceutical formulation comprising a delivery system according to any one of claims 1 to 13.

Images