Lipid nanoformulations of bet inhibitors for the treatment of kidney damage

Abstract

In cases of acute and chronic kidney damage, BET (Bromodomain and Extra-Terminal domain) inhibitors, such as the JQ1 molecule, can be used to inhibit the UPR (Unfolded Protein Response) pathway and other inflammatory and pro-fibrotic pathways, thus improving tissue damage and restoring kidney function. Nevertheless, JQ1 exhibits toxicity and low specificity, so that in order to achieve a suitable therapeutic effect, high doses that are incompatible with a low degree of toxicity must be used. The present invention proposes a solution to the poor pharmacokinetic profile of JQ1 and its low oral availability that comprises designing and developing lipid-based nanoparticles that make it possible to encapsulate and selectively administer the JQ1 inhibitor in sufficient quantity to achieve the desired therapeutic effect and at the same time reduce its toxicity.

Description

[0001]

[0002] Lipid nanoformulations of BET inhibitors for the treatment of kidney damage

[0003] TECHNICAL SECTOR

[0004] The present invention falls within the field of liposome formulations for medical use. More specifically, the invention describes lipid nanoformulations for use in the treatment of kidney damage.

[0005] BACKGROUND OF THE INVENTION

[0006] Acute kidney injury (AKI) is defined as a pathological condition characterized by an abrupt decline in glomerular filtration rate (GFR) and is associated with an increased risk of progression to chronic kidney disease (CKD) in surviving patients, due to the generation of maladaptive mechanisms in the kidney. Maladaptive repair is thought to be driven by a combination of factors, including persistent inflammation, oxidative stress, and dysregulated signaling pathways, which may contribute to the progressive decline in kidney function observed in CKD.

[0007] Although there are still no approved therapies to treat AKI or reduce its sequelae, epigenetics has been found to be key to regulating its pathogenesis. Epigenetics refers to reversible, heritable changes in gene expression that do not involve alterations in the original DNA sequence itself. In this sense, the bromodomain (BRD) and extraterminal domain (BET) family of proteins, which includes BRD2, BRD3, and BDR4 proteins and the testis-specific bromodomain protein (BRDT), are epigenetic "readers," whose function is to detect acetylated histones through their bromodomain and recruit the transcription elongation machinery to induce transcription initiation and extension.

[0008] The development of small-molecule bromodomain inhibitors has shed light on their role in acetylome alterations in various diseases. This has positioned them as important agents in cancer biology and inflammation. In the specific case of acute and chronic kidney damage, as a consequence of kidney failure or kidney transplantation, the unfolded protein response (UPR) pathway is deregulated, leading to increased expression of inflammatory and apoptotic genes. To prevent this situation, the molecule JQ1 can be used. This is a specific inhibitor of BET proteins that acts by inhibiting the UPR pathway and other inflammatory pathways, improving tissue damage and recovering kidney function (Morgado Pascual, JL et al. Bromodomain and Extraterminal Proteins as Novel Epigenetic Targets for Renal Diseases. Front. Pharmacol. 2019; 10: 1315).

[0009] However, JQ1 is toxic, so achieving an adequate therapeutic effect requires high doses that are incompatible with a low level of toxicity. To overcome this drawback, studies have been carried out in which JQ1 is combined with other inhibitors and administered via liposomes (WO2016061144) for a variety of applications, such as tumor treatment (CN114053223A) or to reduce, inhibit, and / or prevent acute cellular responses that lead to future cartilage degradation and osteoarthritis.

[0010] The present invention proposes a solution to the insufficient pharmacokinetic profile of JQ1 and its low oral bioavailability, which consists of the design and development of a series of lipid-based nanoparticles that allow the JQ1 inhibitor to be encapsulated and administered selectively, in a quantity sufficient to achieve the desired therapeutic effect and, at the same time, reduce its toxicity.

[0011] EXPLANATION OF THE INVENTION

[0012] The invention describes a novel approach to the design and development of lipid-based nanoparticles containing the JQ1 inhibitor, based on the application of nanobiotechnology techniques. Due to their translational potential, synthetic versatility, and bioavailability, a series of lipid nanoparticles are designed and validated in vitro and in vivo for the encapsulation of the drug JQ1. A pharmaceutical formulation based on these lipid nanoparticles is useful in the treatment of renal pathology (acute and chronic), generated by damage or after a kidney transplant, and in the imaging monitoring of their biodistribution. For each type of synthesized nanoparticle, an extensive characterization of the physicochemical properties is carried out, and the therapeutic effect is evaluated in vitro and in vivo, as well as their capacity to promote molecular imaging in vivo by fluorescence after systemic administration.

[0013] The liposomal nanoparticles containing JQ1 obtained (JQ1-NPs) and object of this invention present a notable specificity for the area to be treated (in this case, kidneys subjected to ischemia damage). Furthermore, these nanoparticles allow for dual molecular imaging using fluorescence and magnetic resonance techniques. Consequently, by combining therapeutic and potentially diagnostic activity in a single entity, these nanoparticles can allow for theragnosis (therapy + diagnosis) of kidney damage caused by ischemia.

[0014] Different encapsulation strategies are combined with multiple synthesis conditions (lipid type, proportion, lipid:drug ratio, or addition of specific additives, for example). The encapsulation strategies used are: (a) encapsulation of JQ1 in the liposome membrane, (b) encapsulation of JQ1 in the hydrophilic core of the liposomes, and (c) double encapsulation (JQ1 present in both the membrane and the core of the liposomes).

[0015] Encapsulation can be carried out, for example, by the thin lipid film hydration method, which comprises:

[0016] Prepare a solution containing lipids

[0017] Prepare a solution of JQ1

[0018] Mix the above solutions

[0019] Evaporate the solvents to obtain a thin film

[0020] Hydrate the thin film with distilled water

[0021] Promote the lifting of the thin film with the hydration solution and encourage the formation of lipid nanoparticles.

[0022] The lipids used in the present invention are dissolved in conventional volatile organic solvents according to their chemical nature. The JQ1 inhibitor is dissolved in an organic solvent such as acetonitrile, which must be miscible in both the organic phase and the aqueous phase, depending on whether it is encapsulated within the liposome core or its phospholipid membrane. Additives (such as cholesterol, for example) can be added to improve the encapsulation efficiency.

[0023] In the case of encapsulation in the core (type II liposomes) or in dual encapsulation, in the core and membrane of the liposomes (type III liposomes), a solution of JQ1 is also prepared to create a complex with complexing agents to increase the aqueous solubility of active substances that are poorly soluble in water, in order to increase their bioavailability and improve stability. In this invention, to allow the drug to be solvated within the aqueous core of the liposome, beta-cyclodextrin has been used, for example, although the use of other types of cyclodextrins is possible.

[0024] The lipid-based nanoparticles containing the JQ1 inhibitor obtained are characterized in that the JQ1 inhibitor is encapsulated in liposomes with a mixture of C-chain lipids. 14 to C 18 , in a JQ1:lipid ratio between 1:30 and 1:100 (w / w).

[0025] For each type of synthesized particle, an extensive characterization of the physical-chemical properties is carried out and the therapeutic effect is evaluated, in vitro and in vivo.

[0026] The proposed synthetic strategy can be applied to other inhibitor drugs from the same BET family, as well as to other drugs that act on different molecular targets but with the same purpose (treatment of acute kidney injury).

[0027] The presence of gadolinium in the lipid composition gives rise to nanoparticles whose relaxivity suggests their potential application as molecular contrast agents in magnetic resonance imaging. The simultaneous presence of a fluorophore in the lipid composition gives rise to nanoparticles that can be used in fluorescence imaging. Thus, they can be used as dual agents in multimodal MRI / OI molecular imaging.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1. Schematic representation of the preparation of liposomal nanoparticles containing JQ1: (A) Type I liposomes, (B) Type II liposomes and (C) Type III liposomes. Figure 2. Physicochemical characterization of JQ1 dual liposomes: a) Hydrodynamic radius; b) My orography TEM; c) Isoelectric point.

[0030] Figure 3. Analysis of molecular imaging properties and relaxivity. a) Ti map of different concentrations of JQ1-NPs in nuclear magnetic resonance imaging; b) Fluorescence image of different concentrations of JQ1-NPs. c) Analysis of Ti and T2 relaxation times based on gadolinium concentration.

[0031] Figure 4. Determination of HK-2 cell viability using the MTT assay.

[0032] Figure 5. Analysis of HK-2 cell viability using the Annexin V / 7AAD assay. Early apoptosis (Annexin V + and 7AAD −) and late apoptosis or necrosis (Annexin V + and 7AAD +).

[0033] Figure 6. In vitro efficacy of JQ1-loaded nanoparticles: (A) Representative flow cytometry histograms; (B) Mean fluorescence intensity (MFI) of RdR-NP-treated and untreated cells along with the percentage of positive cells, at different time points; and (C, D) IL6 and CCL2 gene expression levels analyzed by qPCR in HK-2 cells treated with empty NPs (JQ1-free NPs), JQ1-NPs, JQ1 (+), or JQ1 (-) for 24 h and TNF-α (10 ng / ml) stimulation for the last 3 h.

[0034] Figure 7. In vivo biodistribution assessment of fluorescently labeled NPs: a) Fluorescence signal captured in whole mice from the dorsal and ventral positions for sham-operated (SIM) and ischemic (l / R) mice; b) ex vivo images of isolated organs obtained from ol / R SIMs at 24 h post-NP injection; c) quantitative analysis of accumulation in the isolated organs; and d) average percentage efficiency (photons emitted by the samples per unit time and surface area).

[0035] Figure 8. Treatment of mice with NPs-JQ1 after bIRI: a) NGAL protein levels by Western blotting (WB); b) NGAL gene expression levels by qPCR; c) blood urea nitrogen (BUN, mg / dL) levels; and d) serum creatinine (mg / dL) assessed in mice at 24 h post-ischemic injury in mice administered 20, 30, and 40 mg / kg of NPs-JQ1.

[0036] Figure 9. Efficacy of JQ1 delivery via liposomes compared to its unencapsulated state: a) NGAL protein levels measured by WB after administration of p-cyclodextrin vehicle and encapsulated JQ1 using p-cyclodextrin as vehicle (p-actin as control protein); b) NGAL expression levels by qPCR; c) PAS-stained sections for sham and ischemic animals treated with NPs without JQ1, with JQ1-NPs and unencapsulated JQ1 at a dose of 40 mg / kg; d) blood urea nitrogen (BUN, mg / dL); and e) serum creatinine (mg / dL).

[0037] Figure 10. Decreased liver toxicity after encapsulation of JQ1 in nanoparticles: (A) analysis of liver serum markers and (B) histological evaluation of the liver.

[0038] Figure 11. Change in body weight of rats treated with NPs-JQ1 in the first 14 days after administration of 40 mg / kg.

[0039] Figure 12. Suppression of the inflammatory response in the kidney after treatment with NPs-JQ1: a) expression of proinflammatory cytokine genes measured by qPCR in kidney samples from different groups of mice at 24 h post-reperfusion; b and c) representative flow cytometry dot plot of neutrophil (left) and monocyte (right) cell infiltration into the mouse kidneys; d) quantification of the total number of neutrophil and monocyte cells per kidney in the CD45+ population.

[0040] Figure 13. Prevention of renal fibrosis with JQ1-NPs after bIRI: a) quantitative qPCR analysis of the expression of fibrosis-related genes; b) protein levels measured by WB of α-SMA and Fibronectin-1 (β-actin as a control protein) and by qPCR; c) Representative images of Masson's trichrome staining on day 21 of ischemic kidneys treated as indicated and quantification (%) of the stained area; d) levels of the kidney damage marker NGAL by qPCR; and e) blood urea nitrogen (BUN, mg / dl). PREFERRED EMBODIMENT OF THE INVENTION

[0041] The present invention is illustrated by the following examples, which are not intended to be limiting of its scope.

[0042] Example 1. Synthesis of lipid-based nanoparticles.

[0043] Three liposome formulations are optimized, the scheme of which is represented in Figure 1: liposomes with membrane-encapsulated JQ1 (type I); liposomes with core-encapsulated JQ1 (type II); and liposomes with dual encapsulation, both in the membrane and the core (type III).

[0044] In all cases, liposomes are synthesized by the thin film lipid hydration method.

[0045] In general, to do this, first, lipid stock solutions are prepared by dissolving DMPC at 10 mg / ml in chloroform and dissolving MHPC and Gd-DTPA-BSA at 10 mg / ml in a chloroform:methanol mixture (3:1 v / v).

[0046] On the other hand, JQ1 is dissolved in acetonitrile at a concentration of 5 mg / ml.

[0047] These solutions are then mixed in the appropriate proportion to achieve the desired liposome composition. In this case, the lipids DMPC, MHPC, and Gd-DTPA-BSA are mixed in a molar ratio of 7.6:1, 1:1, and JQ1 in acetonitrile is added in a 1:30 weight ratio to the total amount of lipids.

[0048] For the encapsulation of JQ1 in the liposome core (type II liposomes), a 1% CD-JQ1 complex is also produced in water with 5 mg / ml of JQ1 in acetonitrile, in a CD:JQ1 volume ratio of 9:1, allowing the drug to solvate within the aqueous core of the liposome. An encapsulation yield of 50% is achieved.

[0049] The encapsulation of JQ1 in both the membrane and the core of the liposome (type III liposomes) is carried out by encapsulating a total of 50% of JQ1 in both the membrane and the core. The aqueous hydration solution is added in a proportion of 1 ml of water per 2.5 mg of lipids. The process of obtaining each type of liposomal nanoparticle is schematized in Figure 1. In the case of type I liposomes (Figure 1A), the solvent of the lipid mixture and JQ1 (1) is evaporated under reduced pressure to form the thin film (2), then hydrated with distilled water (3) and subsequently, the solution is sonicated for 1 hour at 60 ° C. For type II liposomes (Figure 1 B), the solvent of the lipid mixture (1) is evaporated under reduced pressure to form the thin film (3); It is then hydrated with distilled water containing the CD-JQ1 complex (3) and subsequently subjected to ultrasound for 1 hour at 60°C.For type III liposomes (Figure 1 C), the solvent from the lipid mixture and JQ1 (1) is evaporated under reduced pressure to form the thin film (2), then hydrated with distilled water containing the CD-JQ1 complex (3) and sonicated for 1 h at 60°C. Finally, all samples are centrifuged in Amicon 100 kDa molecular weight cut-off (MWCO) concentrators at 2900 g for 10 min and washed three times with distilled water to remove uncomplexed lipids and unencapsulated drug.

[0050] When a fluorescent probe is needed, Liss Rhod PE or Cy 5.5 (at 1 mg / ml in chloroform) is prepared and added to the lipid mixture at a rate of 0.2% of the total lipid molar amount.

[0051] Example 2. Influence of the chain length of mixed lipids.

[0052] Due to the hydrophobic nature of JQ1, encapsulation of JQ1 in liposome membranes (a) (type I liposomes) is carried out by comparing the effect of using a mixture of 18-carbon chain lipids (DSP:PC:Gd-DTPA-BSA) with a mixture of 14-carbon chain lipids (DMPC:MHPC:Gd-DTPA-BSA), in both cases the JQ1 lipid weight ratio being 1:50. As shown in Table 1 , encapsulation with shorter chain lipids (A) results in better yields than encapsulation with longer chain lipids (B). For both types of lipids, the encapsulation yield achieved by adding other adjuvants such as cholesterol is compared but, as also observed in Table 1 , said yield is not significantly improved. Table 1. Different combinations of lipids in different molar ratios with a JQ1 Hypoids weight ratio of 1:50.

[0053] Example 3. Influence of the JQ1 Hypoids relationship

[0054] To determine the influence of the JQ1:Hypodos ratio in the case of the cholesterol-free short-chain lipid mixture (A), JQ1:Hypodos ratios of 1:100 (E) and 1:30 (F) were tested. Table 2 shows no significant changes in terms of encapsulation efficiency. Therefore, the final JQ1:Hypodos ratio was set at 1:30 to utilize a lower amount of lipids per unit of drug.

[0055] Table 2. Different weight ratios of JQ1 Hipidos for the combination of short-chain lipids

[0056] Example 4. Encapsulation effectiveness.

[0057] The encapsulation efficiency of JQ1 within liposomes was measured by high-performance liquid chromatography (HPLC). For each preparation, a 50 pL sample volume was evaporated at 50 °C and then rehydrated in the same volume with the mobile phase (80% acetonitrile and 20% milliQ H2O). The sample was analyzed using an RP C18 column (250 x 4.6 mm, 5 pm, Gemini) with a flow rate of 1 ml / min and a column temperature of 25 °C. The mobile phase contained 80% acetonitrile and 20% milliQ H2O, the wavelength was set at 254 nm, and the elution time of JQ1 was approximately 5.4 min. The peak of the compound was identified using a commercial standard reference.

[0058] For the long-chain lipids shown in Table 1, 7.2 pg of JQ1 were encapsulated per mg of lipid; and for the short-chain lipids, 10 pg of JQ1 were encapsulated per mg of lipid.

[0059] In the case of encapsulation of JQ1 in the liposome core (type II liposomes) with a JQ1 lipid ratio of 1:30 using short chain lipids and adding 1% cyclodextrin to form a complex with JQ1, the encapsulation yield turns out to be 17.8 pg of encapsulated JQ1 per mg of lipids.

[0060] Finally, for type III liposomes, a total encapsulation of 36 pg of JQ1 per mg of lipid is achieved.

[0061] Example 5. Physicochemical characterization.

[0062] The type III liposome formulation of example 4 was characterized by dynamic light scattering (DLS), measuring its hydrodynamic radius and isoelectric point (Figure 2).

[0063] Regarding size, liposomes with an average hydrodynamic radius of 66 nm and a polydispersity index of 0.225 were obtained (Figure 2a). The size and morphology of the synthesized dual liposomes were also studied by TEM (Figure 2b), showing a spherical morphology without aggregates, and with an average size of approximately 60 nm. These data are in agreement with the results obtained with DLS. The isoelectric point (pH at which the net charge of the nanoparticles is 0) was studied, obtaining a value of pH 6 (Figure 2c). Furthermore, the surface charge was found to be negative, -5.53 mV at a physiological pH of 7.

[0064] These results are optimal for obtaining a stable, small-sized lipid formulation with a negative surface charge at physiological pH. In fact, under these conditions, the nanoparticles are not expected to aggregate, thus maintaining their optimal colloidal properties. Example 6. Characterization of the ability of NPs to act as a dual contrast agent in promoting molecular imaging by magnetic resonance imaging and fluorescence.

[0065] Since a liposome model has been designed with different components in its structure, regardless of the drug they contain, the different molecular imaging possibilities they can provide have been evaluated. The overall potential of JQ1 liposomes as contrast agents for use in multimodal molecular imaging was evaluated in vitro using model samples at different concentrations, or "phantoms." On the one hand, due to the possible presence of a fluorophore in their structure, using optical imaging. On the other hand, given the incorporation of a contrast agent such as gadolinium, using magnetic resonance imaging (Figure 3).

[0066] To this end, the properties of liposomes for fluorescent imaging were first evaluated. The fluorescence of different concentrations of liposomes was measured, and it was found that the higher the concentration of nanoparticles, the greater the fluorescence intensity. Thus, the concentration-dependent fluorescence makes these liposomes good candidates for use in optical imaging both in vitro and in vivo (Figure 3a).

[0067] The characterization was also carried out to evaluate the use of these liposomes as contrast agents in magnetic resonance imaging, using different sample concentrations (Figure 3b). To this end, relaxation times (T1 and T2) were measured for different dilutions of the initial concentration of gadolinium-based lipids and, subsequently, the corresponding relaxation rates Ri = 1 / T1 and R2 = 1 / T2 were calculated and plotted against the total lipid concentration in each sample. These graphs were fitted to linear regressions to obtain the slopes r1 and r2, which correspond to the ionic relaxivity of the sample under study. Thus, values ​​of 13.96 mM' were obtained. 1 s' 1 and 19.43 mM' 1 s' 1for r1 and <2, respectively, thus obtaining an r2Íri value of 1.39 (Figure 3c). This ratio indicates the tendency of the contrast agent to promote a perturbation of the longitudinal or transverse relaxivity of water during the generation of the magnetic resonance image. The closer this ratio is to the value of 1, the more its tendency will be to generate a perturbation of the longitudinal relaxivity. In this case, the contrast is said to be of the positive type (T1), generating a whiter and brighter image where the molecular agent is most concentrated. As expected, in magnetic resonance phantoms of a series of dilutions in a T1-weighted sequence, a positive white contrast was generated in a concentration-dependent manner (Figure 3b).

[0068] Consequently, due to the presence of Gd-based and fluorescence-based lipids in their membrane, these liposomes are expected to efficiently act as dual molecular contrast agents in T1-weighted MRI and in vivo fluorescence imaging (IVIS), respectively.

[0069] Example 7. Cell viability.

[0070] To evaluate the cell viability of the three types of liposomes obtained, their effect on the renal tubular epithelial cell line HK-2 is evaluated by MTT (3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium) assay after treatment with nanoencapsulated JQ1 (NPs-JQ1) for 24 hours.

[0071] Cells are treated with unencapsulated JQ1 (+) or its inactive enantiomer JQ1 (-), or with JQ1 (+) encapsulated in liposomal NP types I, II, and III. Empty NPs (JQ1-free nanoparticles) are used to assess NP- and JQ1-independent toxicities. The results (Figure 4) are expressed as percentages relative to control cells (untreated cells, black bars).

[0072] It was observed that all three types of liposomes behaved equally in terms of cytotoxicity, with each reducing cell viability by only about 20% at the highest dose of JQ1 (500 nM). Furthermore, this reduction was the same as that observed after treating cells with unencapsulated JQ1 for the same time and dose.

[0073] Since the MTT assay only measures the metabolic activity of cells, the annexin V / 7AAD assay was also performed to evaluate cell death and apoptosis after treatment of cells with JQ1 type III NPs alone, and comparing them with unencapsulated JQ1 (+) or its inactive enantiomer JQ1 (-), obtaining similar results (Figure 5). The data show that, in all cases, the percentage of apoptotic and necrotic cells was less than 12%. Example 8. In vitro efficacy of JQ1-loaded nanoparticles

[0074] Cellular uptake of the designed nanoparticles (NPs-JQ1 type III) by the renal tubular epithelial cell line HK-2 (target cell type) is evaluated since internalization into the target cell is a prerequisite for proper activity of the encapsulated drug. Cells are cultured in Gibco™ RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% penicillin / streptomycin, 1% Gibco™ insulin-transferrin-selenium (ITS-G) and 5 ng / ml hydrocortisone (Sigma-Aldrich®).

[0075] Rhodamine red (RdR) fluorophore-labeled nanoparticles are added to HK-2 cell cultures for 1, 3, 6, 18, and 24 h at a fixed concentration of 500 nM JQ1 (equivalent to a lipid dose of 25 μM) and analyzed by flow cytometry. Figure 6A shows the fluorescent intensity of RdR nanoparticle uptake in HK-2 cells compared to untreated control cells at 1, 3, 6, 18, and 24 h. It is observed that the cells efficiently incorporated JQ1 nanoparticles and a maximum fluorescence intensity index (MIF) was detected at 24 h, where almost 100% of the cells were positive for Rhodamine red (Figure 6B).

[0076] Functional experiments were then performed at 24 h, analyzing the gene expression of IL6 and CCL2 (known targets of the BET inhibitor JQ1) by qPCR in HK-2 cells after stimulation with TNF-α. The effect of encapsulated JQ1 (JQ1-NPs type III) was compared with its non-encapsulated counterpart at the same doses and at 24 h. Empty nanoparticles and the inactive negative enantiomer JQ1 (-) were used as their respective negative controls. The results are shown in Figures 6C and D and it is deduced that JQ1-NPs exert the same effect in decreasing the expression of IL6 and CCL2 compared to free JQ1 and in a dose-dependent manner (10 - 250 nM), which means that all the JQ1 encapsulated in the nanoparticle is functional and is efficiently released into the target cell; while empty nanoparticles and JQ1 (-) did not affect gene expression, as expected.

[0077] Example 9. In vivo distribution of nanoparticles on an animal model of bilateral ischemia-reperfusion injury (bIRI) To carry out the study of the in vivo efficacy of type III JQ1-NPs, wild-type C57BL / 6N mice (Janvier Labs.), which is a congenic strain (genetically homogeneous), are used. More specifically, animals aged 8 to 10 weeks old are used and are acclimatized for 1 week before the study. This study was previously approved by the Animal Experimentation Committee of the University of Oviedo (Spain) and is carried out in accordance with European and Spanish legislative and regulatory guidelines (European convention ETS 123, on the use and protection of vertebrate mammals for experimental and other scientific purposes; Law 6 / 2013 and Royal Decree RD 53 / 2013, on the protection and use of animals in scientific research), making every effort to minimize the discomfort caused to the mice.

[0078] First, the in vivo distribution of type III JQ1-NPs conjugated to the fluorophore Cy5.5 and administered intraperitoneally to mice after 45 min of ischemia or in sham-operated mice is analyzed. For this purpose, images are taken using the IVIS in vivo imaging system at 1, 3, 6, and 24 h after nanoparticle injection. Epifluorescence detected (Figure 7a) from the dorsal position of the mice indicates that nanoparticles reach the kidneys as early as 1 h after injection and remain there after 24 h. Moreover, ventral images show that nanoparticles disseminate throughout the peritoneum in short times (1–3 h) but eventually relocate to the upper-middle zone, corresponding to the liver, at longer times (Figure 7b).

[0079] These results were quantitatively corroborated at 24 hours by ex vivo epifluorescence detection of organs, where the greatest accumulation of nanoparticles was detected in the liver, followed by the pancreas and kidneys (Figures 7b and 7c). Within a short time after administration, a significant uptake by the kidneys was detected. These results were confirmed by ex vivo analysis, determining that the nanoparticles reached the kidneys.

[0080] Example 10. In vivo efficacy of JQ1-loaded nanoparticles on an animal model of bilateral ischemia-reperfusion injury (bIRI)

[0081] Once the distribution of the nanoparticles in the organs of the mice was verified (example 9), different doses (10, 20, 30 and 40 mg / kg) of type III JQ1-NPs were evaluated in vivo for the bilateral ischemic reperfusion-induced kidney injury (bIRI) model. JQ1-NPs were administered intraperitoneally 1 hour after ischemia, and their effects were analyzed after 24 hours of reperfusion.

[0082] The renal injury marker NGAL was analyzed by Western Blot (WB) (Figure 8a) and qPCR (Figure 8b), and compared with the levels in sham and ischemic mice treated with empty NPs (nanoparticles without JQ1). The results show a significant and dose-dependent reduction in NGAL in mice treated with 30 and 40 mg / kg of JQI-NPs.

[0083] Kidney function parameters, blood urea nitrogen (BUN) (Figure 8c) and creatinine (Figure 8d), were then measured in blood samples collected by cardiac puncture. Serum creatinine and urea were determined using a creatinine assay kit (ab204537, Abeam) and a blood urea nitrogen calorimetric detection kit K024-H5, Arbor Assays, respectively. A significant reduction in both parameters was detected only at the highest dose of 40 mg / kg.

[0084] Example 11. Efficacy and toxicity of liposome-mediated delivery of JQ1.

[0085] In order to verify that type III NPs-JQ1 nanoparticles contribute to reducing the effective dose of JQ1 through their formulation in liposome-based NPs in the bIRI kidney model, markers of kidney damage and functional parameters are analyzed in mice treated with a JQ1 dose of 40 mg / kg and the results are compared between its encapsulated and non-encapsulated formulation (free JQ1). To this end, the same experimental design (example 10) is carried out and free JQ1 is administered at 40 mg / kg 1 hour after reperfusion and markers of kidney damage and function are analyzed at 24 hours (Figure 9).

[0086] The results show that unencapsulated JQ1 has no effect on decreasing the tissue injury marker NGAL, assessed by WB and qPCR (Figure 9a) and (Figure 9b). Furthermore, PAS (Phenodic Acid-Schiff) staining used to assess pathological damage (Figure 9c) showed that the cortico-medullary zone, which is the most affected area in the kidney after ischemic kidney injury, showed a reduction in tubular necrosis after administration of JQ1-NPs type III, detected by the presence of shed tubular cells and loss of the brush border. Additionally, there was a reduction in the number of intratubular deposits, or “casts,” composed of shed cells, proteins, or other materials that obstruct the tubule and prevent normal kidney function. Overall, less kidney damage was detected in mice treated with JQ1-NPs than in those treated with unencapsulated JQ1 (+).

[0087] Regarding blood urea nitrogen (Figure 9d) and serum creatinine (Figure 9e) levels, the results show that the free JQ1 formulation does not significantly reduce either of these 2 parameters, contrary to the reduction observed with encapsulated JQ1 (Figure 8d and 8e).

[0088] Furthermore, in vivo toxicity is determined by analyzing liver serum markers (Figure 10A) and by histological evaluation of the liver (Figure 10B), finding that mice treated with NPs-JQ1 have better liver function compared to free JQ1. Animals treated with NPs-JQ1 have reduced levels of the liver enzymes ALT, AST and AKP and at the histological level, the hydropic degeneration (swelling / dilation) of hepatocytes observed in the livers of mice treated with unencapsulated JQ1 (+) is much greater than in those that were administered NPs-JQ1.

[0089] On the other hand, non-ischemic mice (control mice) were administered a dose of 40 mg / kg of encapsulated JQ1 (NPs-JQ1 type III) and their changes in body weight were measured, finding no differences from day 1 to day 14 (Figure 11 ). Finally, the data from the hemogram and blood cell count (Table 3) confirm that NPs-JQ1 are not toxic, since no significant differences were detected with respect to empty NPs at the different times evaluated.

[0090] These results demonstrate that encapsulation of JQ1 in liposomal NPs offers a significant improvement by increasing drug efficacy and reducing its toxicity in the renal bIRI model. Table 3. Complete blood count and blood cell count of mice treated with JQ1-NPs. Mice were administered IP JQ1-NPs at a dose of 40 mg / kg and blood samples were analyzed on days 1, 7, and 14 thereafter. Results are means and ± SD is indicated from 5 animals per group.

[0091] Neu: Neutrophils; Lym: Lymphocytes; Mo: Monocytes; Eos: Eosinophils; Bas: Basophils; RBC: Red Blood Cells; HGB: Hemoglobin; HOT: Hematocrit; MCV: Mean Corpuscular Volume (average size of red blood cells); MCH: Mean Corpuscular Hemoglobin (average amount of hemoglobin in red blood cells); MCHC: Mean Corpuscular Hemoglobin Concentration (average amount of hemoglobin per unit volume in a single RBC); RDW CV: RBC Distribution Width (range of variation of RBC volume); PLT: Platelets; MPV: Mean Platelet Volume.

[0092] Example 12. Decreased expression of proinflammatory cytokines and chemokines and infiltration of myeloid cells in the kidney by administration of NPs-JQ1.

[0093] In IRI, inflammation becomes excessive, with innate immune cells playing a pivotal role in mediating the response to injury. Following an ischemic event, there is a marked increase in proinflammatory cytokines and chemokines within the tissue, and innate immune cells, neutrophils, and monocytes, are massively recruited following reperfusion, further exacerbating tissue damage. Therefore, early suppression of the inflammatory response is key to improving renal damage after ischemia. For this reason, this model is considered appropriate for analyzing the enhanced efficacy of JQ1 caused by its encapsulation in liposomal NPs.

[0094] The anti-inflammatory effects of encapsulated versus unencapsulated JQ1 are compared at 24 h post-bIRI using a dose of 40 mg / kg and an administration time of 1 h post-ischemia. Figure 12a shows that only JQ1 contained within NPs could decrease the expression level of cytokines such as I116 and chemokines such as Csf2, Ccl2, Ccl5, in contrast to free JQ1 which shows no effect. Consequently, a decrease in neutrophil infiltration (CD45 + CD11 + Ly6G ft / Ly6C / OW ) and monocytes (CD45 + CD11 + Ly6G' Ly6C hi F4 / 80 / OW ) within the kidney, by flow cytometry, in the group of mice administered NPs-JQ1. In contrast, no significant differences were detected when free JQ1 was administered to the mice (Figure 12b-d).

[0095] Example 13. Effect of NPs-JQ1 on the progression of post-ischemic renal fibrosis.

[0096] We examined whether inhibition of the inflammatory burden in the acute phase of renal bIRI may be sufficient to preserve long-term organ function, since inflammatory cell infiltration is a key factor in the development of subsequent fibrosis.

[0097] For this purpose, a 30-minute bIRI model was used, since the 45-minute ischemia model is lethal after 3 or 4 days, and therefore, the animals' progression could not be assessed. Following the experimental model described in Example 10, 1 hour after ischemia, mice were administered a dose of 40 mg / kg of type III JQ1-NPs, and the damage was analyzed after 21 days.

[0098] Figure 13a shows a notable decrease in the expression of pro-fibrotic genes, such as Fibronectin 1 (Fib-1), fibroblast-specific protein (Fsp-1), α-smooth muscle actin (α-Sma), and collagen 1α1 (Col1α1), in the group of mice injected with JQ1-NPs in contrast to the group of control mice to which empty NPs were administered. The decrease in some of these pro-fibrotic molecules is also validated by Western Blot technique and a notable decrease is detected in the group of mice treated with JQ1-NPs (Figure 13b). By Masson's trichrome staining, it is determined that the administration of a single dose of 40 mg / kg of JQ1-NPs is sufficient to improve interstitial fibrosis on day 21 (Figure 13c). The levels of the kidney damage marker NGAL, analyzed by pPCR, are not as high as those observed in the acute phase, but a significant decrease is observed in the group of mice treated with NPs-JQ1 (Figure 13d).Similarly, the disruption of renal malfunction in the group of mice treated with NPs-JQ1 is evidenced by the decrease in blood urea nitrogen (BUN) levels in the serum of treated mice compared to those administered empty NPs (Figure 13e).

Claims

CLAIMS 1. Nanoformulations of BET inhibitors comprising lipid-based nanoparticles containing the JQ1 inhibitor characterized in that the JQ1 inhibitor is encapsulated in liposomes with a mixture of C-chain lipids. 14 to C 18 , in a JQ1:lipid ratio between 1:30 and 1:100 (w / w).

2. Nanoformulations, according to claim 1, where the lipid mixture is DSP:PC:Gd-DTPA-BSA.

3. Nanoformulations, according to claim 1, where the lipid mixture is DMPC:MHPC:Gd-DTPA-BSA.

4. Nanoformulations, according to claims 2 and 3, where the proportion of lipids in the mixture is 7.6:1, 1:1, 0.

5. Nanoformulations, according to claim 1, where cholesterol is added as an additive.

6. Nanoformulations, according to claim 1, where the lipid mixture contains Liss Rhod PE.

7. Nanoformulations, according to claim 1, where the lipid mixture contains Cy 5.

5.

8. Nanoformulations, according to claim 1, where the JQ1 inhibitor is encapsulated in the liposome membrane.

9. Nanoformulations, according to claim 1, where the JQ1 inhibitor is encapsulated in the core of the liposomes.

10. Nanoformulations, according to claim 1, where the JQ1 inhibitor is encapsulated in both the membrane and the nucleus of the liposomes.

11. Method for obtaining liposomal nanoparticles containing JQ1, according to claim 8, comprising: Prepare a solution containing C^ to Cis chain lipids (DMPC, MHPC, Gd-DTPA-BSA) by dissolving in chloroform (for DMPC) or chloroform-methanol 3:1 (v / v) (for MHPC and Gd-DTPA-BSA) Prepare a solution of JQ1 in acetonitrile. Mix the above solutions Evaporate the solvents to obtain a thin film Hydrate the thin film with distilled water Promote the lifting of the thin film with the hydration solution and encourage the formation of lipid nanoparticles.

12. Method for obtaining liposomal nanoparticles containing JQ1, according to claim 9, comprising: Prepare a solution containing C-chain lipids 14 to C 18 (DMPC, MHPC, Gd-DTPA-BSA) by dissolving in chloroform (for DMPC) or chloroform-methanol 3:1 (v / v) (for MHPC and Gd-DTPA-BSA) Evaporate the solvents from the lipid mixture to obtain a thin film Prepare a solution of JQ1 in acetonitrile Prepare a complex of cyclodextrin and JQ1 (CD-JQ1) in distilled water from the solution of JQ1 in acetonitrile Hydrate the thin film with distilled water containing the cyclodextrin complex and JQ1 Growing the thin film 13. Method for obtaining liposomal nanoparticles containing JQ1, according to claim 10, comprising: Prepare a solution containing C-chain lipids 14 to C 18 (DMPC, MHPC, Gd-DTPA-BSA) by dissolving in chloroform (for DMPC) or chloroform-methanol 3:1 (v / v) (for MHPC and Gd-DTPA-BSA) Prepare a solution of JQ1 in acetonitrile. Mix the above solutions Evaporate the solvents from the lipid mixture and JQ1 to obtain a thin film Prepare a complex of cyclodextrin and JQ1 in distilled water from the dissolution of JQ1 in acetonitrile. Hydrate the thin film with distilled water containing the cyclodextrin complex and JQ1 Growing the thin film 14. Method according to any one of claims 11 to 13, wherein the lipid mixture is obtained by dissolving DMPC in chloroform and MHPC and Gd-DTPA-BSA in a mixture of chloroform and methanol (ratio 3:1 (v / v)).

15. Method according to claim 14, wherein the solution of JQ1 is prepared by dissolving JQ1 in acetonitrile.

16. Method according to claim 15, where the solvents are evaporated under reduced pressure.

17. Method according to claim 14, where the growth of the thin film is carried out by ultrasound and at a temperature of 60°C.

18. Method according to claims 12 and 13, wherein the ratio of cyclodextrin and JQ1 in the complex is 9:1 by volume.

19. Method according to any of claims 11 to 13, wherein Liss Rhod PE or Cy 5.5 is added to the lipid mixture to obtain a fluorescent probe.

20. Method according to claim 18, wherein Liss Rhod PE or Cy 5.5 are added in a proportion of 0.2% of the total amount of lipids in moles.

21. Use of the claimed nanoformulations in the diagnosis of acute and chronic renal pathology.

22. Use of the claimed nanoformulations in the therapy and monitoring of acute and chronic renal pathology.

23. Use of the claimed nanoformulations in the theragnosis of acute and chronic renal pathology.

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