METHODS AND PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT OF CHRONIC KIDNEY DISEASE

Abstract

The present invention relates to methods and pharmaceutical compositions for the treatment of chronic kidney disease. The inventors used the CMap resources to identify the sesquiterpene lactone parthenolide that was subsequently analyzed for its in vivo capacity to reduce the development of DKD in the type I diabetes mouse model. In particular, the invention relates to a method of treating chronic kidney disease (CKD) in patient in need thereof comprising administering to the patient a therapeutically effective amount of Dimethylaminoparthenolide (DMAPT).

Claims

1. A method of treating chronic kidney disease (CKD) in patient in need thereof comprising administering to the patient a therapeutically effective amount of Dimethylaminoparthenolide (DMAPT).

2. The method according to claim 1 wherein the CKD is a disease selected from the group consisting of nephropathy, glomerulonephritis, interstitial nephritis, lupus nephritis, idiopathic nephrotic syndrome (INS), obstructive uropathy, polycystic kidney disease, cardiovascular diseases, hypertension, diabetes, and kidney graft rejection.

3. The method according to claim 1 wherein the CKD is a diabetic kidney disease (DKD).

4. The method according to claim 3 wherein the patient with DKD has type 1 diabetes.

5. The method according to claim 3 wherein the patient with DKD has type 2 diabetes.

6. The method according to claim 1 wherein the CKD is a progressive CKD after a partial nephrectomy.

7. The method according to claim 1 wherein the CKD is a focal segmental glomerulosclerosis (FSGS).

8. The method according to claim 1 wherein the patient has a vascular calcification.

9. The method according to claim 8 wherein the vascular calcification is an intimal calcification or a medial calcification.

10. The method according to claim 1 wherein the DMAPT is combined with a nephroprotective treatment.

11. The method according to claim 10 wherein the nephroprotective treatment is sodium-glucose cotransporter 2 inhibitor (SGLT2i).

12. The method according to claim 10 wherein the nephroprotective treatment is a renin angiotensin aldosterone system inhibitor (RAASi).

13. The method according to claim 12 wherein the RAASi is an angio-converting enzyme inhibitors (ACEi).

14. The method according to claim 13 wherein the ACEi is ramipril.

15. The method according to claim 12 wherein the RAASi is an angiotensin-receptor blockers (ARBs).

16. The method according to claim 10 wherein the nephroprotective treatment is an anti-endothelin-1 receptor.

17. The method according to claim 2 wherein the nephropathy is membranous nephropathy (MN), diabetic nephropathy or hypertensive nephropathy, the glomerulonephritis is membranous glomerulonephritis or membranoproliferative glomerulonephritis (MPGN), the INS is minimal change nephrotic syndrome (MCNS) or focal segmental glomerulosclerosis (FSGS), the polycystic kidney disease is Autosomal Dominant Polycystic Kidney Disease (ADPKD) or Autosomal Recessive Polycystic Kidney Disease (ARPKD), the diabetes is diabetic kidney disease, and the kidney graft rejection is acute or chronic kidney rejection.

18. The method of claim 17, wherein the MPGN is rapidly progressive glomerulonephritis (RPGN).

Description

FIGURE

[0072] FIG. 1: Comparative influence of DMAPT and Ramipril-treatment on Ins2Akita mice. 4 month old diabetic Ins2Akita (DKD) that had been treated or not (WT) with Ramipril (DKD+R) or DMAPT (DKD+D) for 2 months before sacrifice (scale bar=50 μm). (A-F) Quantification of ACR (A), glomerular injury (B), glomerular area (C), fibrosis (D), glycemia (E), and body weight (F) in WT (n=10), DKD (n=9), DKD+R (n=9) and DKD+D (n=9). Values are mean±SEM and One-way ANOVA test for multiple comparisons. Comparison to WT: *: P<0.05; **:P<0.01; ***: P<0.001; ****: P<0.0001. Comparison of DKD+R or DKD+D to DKD: #: P<0.05; ##:P<0.01; ###: P<0.001; ####: P<0.0001. Comparison of DKD+R to DKD+D: @@:P<0.01; @@@@: P<0.0001.

EXAMPLE

[0073] Material & Methods

[0074] Animal

[0075] Male C57BL/6-Ins2Akita/J (Ins2Akita) and C57BL/6J (WT) mice (Charles River) were housed with unrestricted access to water and were maintained on a 12-h light-dark cycle in a pathogen-free environment on standard mouse chow. All experiments were conducted in accordance with the Guide for the care and use of laboratory animals of the National Institute of Health, eighth edition and the French Institute of Health guidelines for the care and use of laboratory animals. The project was approved by the local (Inserm/UPS US006 CREFRE) and national ethics committees (ethics committee for animal experiment, CEEA122; Toulouse, France; approval 02867.01).

[0076] Treatments and Urine Collection

[0077] In a first series of experiments, 2 months old mice were treated with or without ACEi ramipril (10 mg/kg/d in drinking water) for 2 months. In a second series of experiments 2 months old mice were treated with or without ACEi ramipril as in the first series or with dimethylaminoparthenolide monofumarate (DMAPT, 10 mg/kg/d by gavage) for 2 months. In each series of experiments, urine was collected in metabolic cages for 12 h, few days before sacrifice.

[0078] Isolation of Glomeruli

[0079] Glomeruli were isolated as previously described.sup.31 with minor modifications. Mice were anesthetized by ip injection of a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg) in phosphate buffered saline (PBS). A catheter was placed into the abdominal aorta after ligation of the cava vein, the upper abdominal aorta, the mesenteric and the celiac arteries. The lower part of the abdominal aorta was perfused with 40 ml of Dynabeads M-450 Tosylactivated (4.5 μm diameter, Dynal A.S., Oslo, Norway) at a concentration of 2.106 beads/ml followed by 15 ml of cold PBS. This procedures allows accumulation of beads in glomeruli.sup.32. Next, the left and right kidneys were collected and decapsulated. The animal was sacrificed by cervical dislocation and blood was collected by intra-cardiac puncture in heparinized tubes and plasma was prepared by centrifugation at 1500×g at 4° C. for 5 min and stored at −20° C. for further use. One portion of the right kidney was snap-frozen in liquid nitrogen and stored at −80° C. Another portion of the right kidney cortex was fixed in Carnoy's solution (ethanol/chloroform/glacial acetic acid: 60/30/10, v/v/v) for further histological analysis.

[0080] The left kidney was gently pressed manually through a 70 μm cell strainer using a flattened pestle followed by washing of the cell strainer with 20 ml of cold PBS. The filtrate was centrifuged at 200×g for 5 minutes at 4° C., and the glomerular pellet was adjusted to 2 ml with PBS and transferred to an Eppendorf tube that was placed in a magnetic particle concentrator (Dynal A.S., Oslo, Norway) to concentrate the glomeruli into a pellet. The supernatant was discarded and the pellet was washed 5×with 1 ml of PBS. The final pellet was resuspended in 100 μl PBS. This procedure allows the isolation of ˜4000 glomeruli per kidney.

[0081] Based on light microscopy survey, our glomerular suspensions were highly enriched for glomeruli. Moreover, mRNA quantification showed that isolated glomeruli were enriched in glomerular-specific genes (nphs2, podx1 and cldn5) while proximal (aqp1, slc22a13), distal tubules (wnk), and of loop of Henle (aqp1, slcl2a3) specific genes were not, or poorly enriched compared to total kidney. Moreover, among the 2422 proteins detected by LC-MS/MS, several glomerular-specific proteins were ranked within the first upper quartile of relative abundance including NphS1 (rank 196), Tjpl/ZO-1 (rank 221) Nphs2 (rank 339), Synpo (rank 388), Actn4 (rank 7), cd2ap (rank 543), while several tubular-specific proteins Aqp1 (rank 591), Umod (rank 1634) were ranked as less abundant proteins, or were not detected (slc22a13, Wnk). These observations support the relative high purity of our glomerular preparations.

[0082] Renal Histology

[0083] Right kidney cortexes were fixed in Carnoy's solution for 24 h, dehydrated in successive baths with increasing alcohol concentrations, embedded in paraffin and 2 μm sections were cut and mounted, and stained with either periodic acid-Schiff (PAS) or Masson trichrome. Stained sections were scanned using a Nanozoomer 2.0 RS (Hamamatsu Photonics SARL) and treated with the Morpho-expert image-analysis software (version 1.00, Explora Nova) for renal corpuscle surface quantification. At least 50 glomeruli including superficial and juxtaglomerular cortical area, were examined for each animal. The extent of glomerular injury was expressed as the percentage of glomerular area fraction occupied by PAS positive matrix.sup.3.

[0084] Biochemical Analysis

[0085] Urinary albumin concentration was measured by ELISA using the AlbuWell kit (WAK-Chemie Medical GmbH, Steinbach, Germany). Urinary creatinine concentration was measured by the colorimetric method of Jaffe according to the protocol Creatinine Assay Kit (Bio Assay Systems). Blood glucose levels were measured in caudal blood from fasted awake mice using a glucometer (Glucometer Elite XL; Bayer Healthcare, Elkhart, Ind.).

[0086] Quantitative Proteomics of Glomerular Samples

[0087] Glomerular sample preparation for proteomics—Isolated glomeruli were homogenized in RIPA buffer under agitation for 3 min and centrifuged 15 min at 13000×g to pellet the beads together with cell debris. The supernatant was collected and stored at −80° C. at a protein concentration of 1-2 mg/ml before being processed for mass spectrometry (MS) analysis. Protein samples were air-dried in a SpeedVac concentrator and then reconstituted in 1× final Laemmli buffer containing 25 mM dithiothreitol and heated at 95° C. for 5 min. Cysteines were alkylated for 30 min at room temperature by the addition of a solution of 75 mM chloroacetamid. Proteins were loaded onto a 12% acrylamide SDS-PAGE gel and concentrated in a single band visualized by Coomassie staining (Instant Blue—Expedeon). The gel band containing the whole sample was cut and washed several times in 50 mM ammonium bicarbonate:acetonitrile (1:1) for 15 min at 37° C. Proteins were in-gel digested using 0.6 μg of modified sequencing-grade trypsin (Promega) in 50 mM ammonium bicarbonate overnight at 37° C. Peptides were extracted from the gel by two incubations in 10% formic acid:acetonitrile (1:1) for 15 min at 37° C. The extracted fractions were pooled with the initial digestion supernatant and dried under speed-vaccum. The resulting peptides were resuspended with 14 μL of 5% acetonitrile, 0.05% trifluoroacetic acid for nanoLC-MS/MS analysis.

[0088] NanoLC-MSMS analysis—Peptides were analyzed by nanoLC-MS/MS using an UltiMate 3000 system (Dionex) coupled to an LTQ Orbitrap Velos ETD mass spectrometer (Thermo Fisher Scientific). Five microliters of each sample were loaded onto a C18 precolumn (300 μm inner diameter×5 mm; Dionex) at 20 μl/min in 5% acetonitrile, 0.05% trifluoroacetic acid. After 5 min of desalting, the precolumn was switched online with the analytical C18 column ((75 m inner diameter×50 cm; in-house packed with Reprosil C18) equilibrated in 95% solvent A (5% acetonitrile, 0.2% formic acid) and 5% solvent B (80% acetonitrile, 0.2% formic acid). Peptides were eluted using a 5 to 50% gradient of solvent B over 110 min at a flow rate of 300 nl/min. The mass spectrometer was operated in a data-dependent acquisition mode with Xcalibur software. Survey MS scans were acquired in the Orbitrap on the 300 to 2000 m/z range with the resolution set at 60,000. The 20 most intense ions per survey scan were selected for CID fragmentation and the resulting fragments were analyzed in the linear ion trap (LTQ). A dynamic exclusion of 60 s was used to prevent repetitive selection of the same peptide. Each sample was injected once for MS analysis.

[0089] Protein identification and quantifcationfrom raw nanoLC-MS/MS data—Raw nanoLC-MS/MS files were processed with the MaxQuant software (version 1.5.2.8) for database search with the Andromeda search engine and for quantitative analysis. Data were searched against “Mus musculus” entries in the Swiss-Prot protein database (UniProtKB/Swiss-Prot protein knowledgebase release 2015_01; 16,695 entries). Carbamidomethylation of cysteine was set as a fixed modification whereas oxidation of methionine and protein N-terminal acetylation were set as variable modifications. Specificity of trypsin digestion was set for cleavage after K or R and two missed trypsin cleavage sites were allowed. The precursor mass tolerance was set to 20 ppm for the first search and 4.5 ppm for the main Andromeda database search. The mass tolerance in MS/MS mode was set to 0.8 Da. Minimum peptide length was set to 7 amino acids and minimum number of unique peptides was set to 1. Andromeda results were validated by the target-decoy approach using a reverse database at both a peptide and protein FDR of 1%. For label-free relative quantification of the samples, the “match between runs” option of MaxQuant was enabled with a time window of 3 min to allow cross-assignment of MS features detected in the different runs.

[0090] Data processing and statistical analysis—Protein entries identified as potential contaminants from the ‘proteinGroups.txt’ files generated by MaxQuant were eliminated from the analysis, as were proteins identified by fewer than two peptides. Protein relative quantification was performed by comparisons of different groups of eight samples each (8 biological replicates per group: WT, DKD, DKD+R, WT+R) (Table S1). Protein intensities were normalized across all conditions by the median intensity. For each comparison, only proteins which were quantified in at least 4 biological replicates (4 intensities values retrieved by MaxQuant) in at least one of the groups were considered for further processing and statistical analysis (Filter 1, columns AR to AU, Table S1). Remaining missing values were then replaced by a constant noise value determined independently for each analytical run as the 1% percentile of the total protein population. The mean proportion of missing values over the whole analytic run was 2.1% (line 2435, column S, Table S1). Proteins with a p-value of less than 0.05 were considered as significantly varying between two groups.

[0091] Proteomic Data Availability

[0092] Because of the confinement resulting COVID-19 pandemic, access to our MS facility was not possible before submission and the proteomic raw data can therefore not be deposited at the time of manuscript submission. Once the pandemic is over, the deposit will be made immediately on ProteomeXchange and the accession number will be communicated.

[0093] Pathway Enrichment Analysis

[0094] Pathway enrichment analysis of up- and down-regulated proteins was using the Gene Set Enrichement Analysis (GSEA) software package (https://www.gsea-msigdb.org).sup.10,34 using “Hallmarks gene sets” and “Canonical Pathways” as Compute Overlaps.

[0095] Connectivity Map Analysis

[0096] The initial version of CMap (CMap1: https://portals.broadinstitute.org/cmap) consists of 6,100 differential expression profiles obtained after treatment of 3 cultured human cells (MCF7, PC3, and HL60) with varying concentrations of 1309 compounds.sup.11. More recently, a new CMap version was released.sup.35 (CMap2: https://clue.io/) encompassing 8549 differential expression profiles obtained after treatment of 9 cultured human cells (VCAP, A375, A549, HAE1, HCC515, HEPG2, HT29, MCF7, PC3) with varying concentrations of 2428 compounds. For our experiments, each mouse protein ID was first converted to its human ortholog and then converted into human gene ID. Up- and down-gene IDs were then queried to CMap1 and CMap2 to retrieve compounds with best negative enrichment as recently recommended.sup.17.

[0097] DMAPT Synthesis

[0098] Dimethylaminoparthenolide monofumarate [(13-(N,N-dimethyl)-amino-4a,5b-epoxy-4,10-dimethyl-6a-hydroxy-12-oic acid-c-lactonegermacra-1(10)-ene monofumarate)] was synthesized by reaction of parthenolide (Sigma-Aldrich) with dimethylamine (Sigma-Aldrich) and isolated as the fumarate salt as previously described.sup.19. Analytical data (1H and 13C NMR, mass spectrometry and melting point) are consistent to those previously reported.sup.16. DMAPT fumarate purity was checked by elemental analysis.

[0099] Statistics

[0100] Comparison between 2 groups of values was implemented using a two-tailed unpaired Welch's t-tests. Comparison between more than 2 groups, was implemented using an ordinary one-way ANOVA followed by Homl-Sidak's multiple comparisons test was used. P<0.05 was considered statistically significant. For the proteomic data the P values were adjusted for the false discovery rate (Benjamini-Hochberg).

[0101] Results

[0102] Influence of ramipril on diabetic nephropathy.

[0103] Our investigations were performed in the Ins2Akita mouse since this model at the timepoints investigated is recognized as a useful model of early to moderately advanced renal morphological changes and renal dysfunction in type I DKD 14. Moreover, it was shown that ACEi reduces albuminuria in this model.sup.15,16 Ins2Akita mice (DKD) became significantly hyperglycemic at 1 month of age (Data not shown) and exhibited significant increased ACR at 2 months (Data not shown) compared to WT mice. Ramipril treatment, starting at 2 months of age, of Ins2Akita mice (DKD+R) significantly reduced ACR that reached a level close to that of WT mice (Data not shown) Ramipril also slightly but significantly reduced glycemia (Data not shown) but had no influence on body weight (Data not shown). These data confirmed the ability of ramipril to reduce albuminuria in Ins2Akita mice.

[0104] Identification of Ramipril-Sensitive and -Insensitive Glomerular Proteins.

[0105] Since our objective was to identify a drug able to inhibit DKD acting through a RAAS-independent mechanism, and since, glomerular injury is considered as the initial key event in DKD progression, we aimed to identify glomerular proteins (GPs) modified in DKD but not counter regulated by ramipril treatment. We hypothesized that such proteins could be potential valuable targets for new pharmacological treatments of DKD on top of ACEi. High quality glomeruli were isolated (see Methods) from each mouse of 4 different groups: WT, DKD, DKD+R and WT+R (8 mice per group) and GPs of each animal were separately analyzed by large-scale quantitative MS-based proteomics.

[0106] Based on the analysis of 32 glomerular samples (4 groups of 8 animals), a total of 2422 GPs were identified and quantified (Data not shown). Three sets of comparison (Set #1, Set #2, Set #3) were then performed (Data not shown). Proteins were considered significantly different in those comparisons based on a p<0.05. In Set #1, comparison of DKD vs WT mice identified 666 GPs with significantly varying abundances [329up with increased and 337down with decreased abundance] that we considered as DKD-associated and that we entitled DKD-GPs (DKD-associated glomerular proteins) (Data not shown). In Set #2, comparison of DKD+R vs DKD mice and identified 543 significant proteins (316 up, 227 down) corresponding to ramipril-sensitive GPs (RS-GPs), and 1879 not significant proteins corresponding to ramipril-insensitive GPs (RI-GPs) in a DKD context (Data not shown). Finally, in Set #3 comparison of WT+R vs WT mice identified 500 (252 up, 248 down) significant RS-GPs, and 1922 not significant RI-GPs in a non-DKD context (Data not shown).

[0107] We then classified the 666 DKD-GPs identified in Set #1 in Set #2 and Set #3 (Data not shown). 86 of the 666 DKD-GPs in Set #1 were significant in Set #2 with a ratio opposite to Set #1 (Data not shown) and were classified as RS-DKD-GPs indicating their ability to be counter regulated by ramipril. Nevertheless, in Set #3, 3 of these 86 DKD-GPs showed a regulation opposite to Set #1 (Data not shown) indicating a sensitivity to ramipril not specific to DKD. These 3 proteins were therefore removed from the definitive RS-DKD-GPs list that finally included 83 proteins (35 up, 48 down) (Data not shown). Pathway analysis of these RS-DKD-GPs showed a highly significant enrichment in proteins involved in small molecules transport and in folding of actin and tubulin by the CCT-TRIC complex (Data not shown).

[0108] In contrast, 518 DKD-GPs were not significant in Set #2 (Data not shown) and were classified as RI-DKD-GPs indicating their insensitivity to ramipril in a DKD context. Nevertheless, 168 of them were significant in Set #3 (Data not shown), indicating their sensitivity to ramipril in a non-diabetic context. Since our objective was to identify a ramipril-insensitive signature specific to DKD, these 168 proteins were removed from the RI-DKD-GPs list that finally included 346 proteins (173 up, 173 down) (Data not shown). RI-DKD-GPs included 48 proteins with a more than 2-fold increase abudance (Data not shown), and 36 proteins with a more than 2 fold decreased abundance (Data not shown). RI-DKD-GPs represented 52% of all DKD-GPs indicating ample space for improvement of DKD treatment. Pathway analysis of RI-DKD-GPs showed a highly significant enrichment in proteins involved in the metabolism of the amino acids, protein localization and peroxisomal protein import (Data not shown) suggesting that RI-DKD-GPs are involved in quite different molecular pathways than RS-DKD-GPs. Overall these data suggested that RI-DKD-GPs are potential targets for new pharmacological treatments of DKD.

[0109] Connectivity Mapping of RI-DKD-GPs Signature.

[0110] In an attempt to identify a pharmacological compound able to inhibit DKD through a ramipril-independent mechanism, UP and DOWN RI-DKD-GPs were analyzed in silico using both CMap1 and CMap2 as recommended by Lim and Pavlidis.sup.17 to select to most probable candidates using this in-silico strategy (Methods). Using CMap1, we found 2 top compounds (quizapine and parthenolide) that exhibited the highest negative enrichment score with best “percent non-nul” (100) (Data not shown). Quizapine is a serotonin receptor agonist. Parthenolide is a sesquiterpene lactone naturally present in a plant (Tanacetum parthenium).sup.18. When using CMap2, quizapine was not retrieved, but parthenolide was found within the top 20 compounds with highest negative enrichment (Data not shown). These observations suggested that parthenolide has the potential to inhibit the ramipril-insensitive glomerular DKD protein signature and therefore the DKD phenotype.

[0111] Influence of Parthenolide on DKD.

[0112] Following CMap prediction, we decided to verify the capacity of parthenolide to inhibit DKD. Nevertheless, parthenolide has a poor water-solubility that constitutes a major limitation for in vivo studies and for further development as a clinical therapeutic agent. To circumvent this issue, an orally bioavailable analog of parthenolide, DMAPT (dimethyamino-parthenolide, fumarate salt) was developed (supplementary materials and.sup.19,20) We therefore tested the influence of a DMAPT-treatment comparatively to ramipril on the development of DKD in Ins2Akita mice.

[0113] Treatment of Ins2Akita mice (DKD) with DMAPT (DKD+P) led to a significant reduction of ACR to the same extent to that of ramipril (DKD+R) (FIG. 1A). DMAPT also significantly reduced glomerular injury (PAS staining) (FIG. 1B) and fibrosis (Masson trichrome staining) (FIG. 1D), and tended to reduce glomerular area without reaching significance (FIG. 1C). In contrast, ramipril had no significant influence on these 3 parameters (FIG. 1A-FIG. 1D). Neither glycemia (FIG. 1E) nor body weight (FIG. 1F) were significantly influenced by DMAPT and ramipril. For ramipril this contrasts with the slight reduction in glycemia observed in the first experiment (FIG. 1A). In conclusion, these data indicated that DMAPT inhibits DKD-associated albuminuria and that, in contrast to ramipril, DMAPT is also able to inhibit DKD-associated kidney injuries.

[0114] Discussion

[0115] Because of their beneficial effect on high blood pressure, ACEi reduce cardiovascular risk and CKD progression in patients with advanced DKD with macroalbuminuria, but are poorly efficient in preventing DKD patients with microalbuminuria.sup.4. Here, we found that among the glomerular proteins that are modified during DKD in Ins2Akita mouse, a model of moderately advanced type I DKD, only a small proportion (12%) is counter regulated by the ACEi ramipril. The remaining insensitive to ramipril proteins are potential targets for new drug-treatment of DKD through a ramipril independent mechanism. By browsing the “drug repurposing” data base CMap, we found that the best negative enrichment of the ramipril-insensitive glomerular DKD protein signature is obtained with parthenolide predicting that this molecule could potentially inhibit DKD progression. Very interestingly, in vivo treatment with DMAPT, an orally bioavailable analog of parthenolide.sup.19,20 potently reduces urinary ACR in Ins2Akita mice demonstrating that the in silico prediction with CMap was transferable in vivo. Parthenolide was shown to have a beneficial impact on proteinuria and renal injury in immune glomerulonephritis in rat.sup.21, but to the best of our knowledge the beneficial impact of parthenolide on DKD has not been reported yet. Moreover, our results show that DMAPT is not only able to reduce urinary ACR but is also able to reduce kidney lesions associated with DKD in Ins2Akita mice. This is contrasting with the absence of effects of ramipril-treatment on kidney lesions seen in our and other studiesis.sup.15,16. Parthenolide is a sesquiterpene lactone naturally present in a plant (Tanacetum parthenium) that has anti-cancer and anti-inflammatory effects by inhibiting the activity of the NF kappa B transcription factor complex.sup.18. Therefore, the beneficial impact of DMAPT on kidney injuries could depend on the NF kappa B dependent pathways. The hypothesis is in agreement with previous reports showing that dysregulation of NF kappa B is involved in podocyte damage and proteinuria in DKD.sup.22-26. The hypothesis is also in agreement with a previous report showing that parthenolide is also able to alleviate renal inflammation and insulin resistance in type 2 diabetic db/db mice.sup.27. DMAPT was also reported to inhibit histone deacetylase (HDAC) activity and this effect is independent of NF kappa B.sup.28 and there are numerous evidences for renoprotective effects of HDAC inhibitors in experimental DKD.sup.29. Therefore, the protective effect of DMAPT in DKD could also result from HDAC inhibition.

[0116] Although further studies should investigate a complementary protective effect of ACEi and DMAPT in DKD (in both type I and II diabetes), our data strongly suggested that parthenolide or its derivatives stand as potential new drug candidates for DKD treatment that would advantageously complement the use of ACEi. Phase I trial with standardized doses in patients with cancer showed that parthenolide was well tolerated without dose-limiting toxicity.sup.30. Whether parthenolide could be used in patients with DKD remains to be tested.

REFERENCES

[0117] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. [0118] 1. Tung, C.-W., Hsu, Y.-C., Shih, Y.-H., Chang, P.-J. & Lin, C.-L. Glomerular mesangial cell and podocyte injuries in diabetic nephropathy. Nephrology (Carlton) 23 Suppl 4, 32-37 (2018). [0119] 2. Zeni, L., Norden, A. G. W., Cancarini, G. & Unwin, R. J. A more tubulocentric view of diabetic kidney disease. J. Nephrol. 30, 701-717 (2017). [0120] 3. Lewis, E. J., Hunsicker, L. G., Bain, R. P. & Rohde, R. D. The effect of angiotensin-converting-enzyme inhibition on diabetic nephropathy. The Collaborative Study Group. N. Engl. J. Med. 329, 1456-1462 (1993). [0121] 4. Majewski, C. & Bakris, G. L. Has RAAS Blockade Reached Its Limits in the Treatment of Diabetic Nephropathy? Curr. Diab. Rep. 16, 24 (2016). [0122] 5. Yamout, H., Lazich, I. & Bakris, G. L. Blood pressure, hypertension, RAAS blockade, and drug therapy in diabetic kidney disease. Adv Chronic Kidney Dis 21, 281-286 (2014). [0123] 6. Ramos, A. M. et al. Design and optimization strategies for the development of new drugs that treat chronic kidney disease. Expert Opin Drug Discov 15, 101-115 (2020). [0124] 7. Ruegg, C., Tissot, J.-D., Farmer, P. & Mariotti, A. Omics meets hypothesis-driven research. Partnership for innovative discoveries in vascular biology and angiogenesis. Thromb. Haemost. 100, 738-746 (2008). [0125] 8. Colhoun, H. M. & Marcovecchio, M. L. Biomarkers of diabetic kidney disease. Diabetologia 61, 996-1011 (2018). [0126] 9. Mokou, M., Lygirou, V., Vlahou, A. & Mischak, H. Proteomics in cardiovascular disease: recent progress and clinical implication and implementation. Expert Rev Proteomics 14, 117-136 (2017). [0127] 10. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. U.S.A. 102, 15545-15550 (2005). [0128] 11. Lamb, J. et al. The Connectivity Map: using gene-expression signatures to connect small molecules, genes, and disease. Science 313, 1929-1935 (2006). [0129] 12. Musa, A. et al. A review of connectivity map and computational approaches in pharmacogenomics. Brief. Bioinformatics 19, 506-523 (2018). [0130] 13. Schanstra, J. P. et al. Systems biology identifies cytosolic PLA2 as a target in vascular calcification treatment. JCI Insight 4, (2019). [0131] 14. Kitada, M., Ogura, Y. & Koya, D. Rodent models of diabetic nephropathy: their utility and limitations. Int J Nephrol Renovasc Dis 9, 279-290 (2016). [0132] 15. Lo, C.-S. et al. Dual RAS blockade normalizes angiotensin-converting enzyme-2 expression and prevents hypertension and tubular apoptosis in Akita angiotensinogen-transgenic mice. Am. J. Physiol. Renal Physiol. 302, F840-852 (2012). [0133] 16. You, H., Gao, T., Cooper, T. K., Morris, S. M. & Awad, A. S. Arginase inhibition: a new treatment for preventing progression of established diabetic nephropathy. Am. J. Physiol. Renal Physiol. 309, F447-455 (2015). [0134] 17. Lim, N. & Pavlidis, P. Evaluation of Connectivity Map shows limited reproducibility in drug repositioning. bioRxiv 845693 (2019) doi:10.1101/845693. [0135] 18. Ghantous, A., Sinjab, A., Herceg, Z. & Darwiche, N. Parthenolide: from plant shoots to cancer roots. Drug Discov. Today 18, 894-905 (2013). [0136] 19. Neelakantan, S., Nasim, S., Guzman, M. L., Jordan, C. T. & Crooks, P. A. Aminoparthenolides as novel anti-leukemic agents: Discovery of the NF-kappaB inhibitor, DMAPT (LC-1). Bioorg. Med. Chem. Lett. 19, 4346-4349 (2009). [0137] 20. Guzman, M. L. et al. An orally bioavailable parthenolide analog selectively eradicates acute myelogenous leukemia stem and progenitor cells. Blood 110, 4427-4435 (2007). [0138] 21. Löpez-Franco, O. et al. Nuclear factor-kappa B inhibitors as potential novel anti-inflammatory agents for the treatment of immune glomerulonephritis. Am. J. Pathol. 161, 1497-1505 (2002). [0139] 22. Wiggins, J. E. Aging in the glomerulus. J. Gerontol. A Biol. Sci. Med. Sci. 67, 1358-1364 (2012). [0140] 23. Brähler, S. et al. Intrinsic proinflammatory signaling in podocytes contributes to podocyte damage and prolonged proteinuria. Am. J. Physiol. Renal Physiol. 303, F1473-1485 (2012). [0141] 24. Zhao, X., Hsu, K.-S., Lim, J. H., Bruggeman, L. A. & Kao, H.-Y. α-Actinin 4 potentiates nuclear factor κ-light-chain-enhancer of activated B-cell (NF-κB) activity in podocytes independent of its cytoplasmic actin binding function. J. Biol. Chem. 290, 338-349 (2015). [0142] 25. Wei, M., Li, Z., Xiao, L. & Yang, Z. Effects of ROS-relative NF-κB signaling on high glucose-induced TLR4 and MCP-1 expression in podocyte injury. Mol. Immunol. 68, 261-271 (2015). [0143] 26. Bao, W. et al. Toll-like Receptor 9 Can be Activated by Endogenous Mitochondrial DNA to Induce Podocyte Apoptosis. Sci Rep 6, 22579 (2016). [0144] 27. Liu, Q. et al. Inhibition of NF-κB Reduces Renal Inflammation and Expression of PEPCK in Type 2 Diabetic Mice. Inflammation 41, 2018-2029 (2018). [0145] 28. Nakshatri, H. et al. NF-κB-dependent and -independent epigenetic modulation using the novel anti-cancer agent DMAPT. Cell Death Dis 6, e1608 (2015). [0146] 29. Hadden, M. J. & Advani, A. Histone Deacetylase Inhibitors and Diabetic Kidney Disease. Int J Mol Sci 19, (2018). [0147] 30. Curry, E. A. et al. Phase I dose escalation trial of feverfew with standardized doses of parthenolide in patients with cancer. Invest New Drugs 22, 299-305 (2004). [0148] 31. Liu, X. et al. Isolating glomeruli from mice: A practical approach for beginners. Exp Ther Med 5, 1322-1326 (2013). [0149] 32. Takemoto, M. et al. A new method for large scale isolation of kidney glomeruli from mice. Am. J. Pathol. 161, 799-805 (2002). [0150] 33. Klein, J. et al. Urinary peptidomics provides a noninvasive humanized readout of diabetic nephropathy in mice. Kidney Int. 90, 1045-1055 (2016). [0151] 34. Reimand, J. et al. Pathway enrichment analysis and visualization of omics data using g:Profiler, GSEA, Cytoscape and EnrichmentMap. Nat Protoc 14, 482-517 (2019). [0152] 35. Subramanian, A. et al. A Next Generation Connectivity Map: L1000 Platform and the First 1,000,000 Profiles. Cell 171, 1437-1452.e17 (2017).