Ferroportin-Inhibitors For The Use In The Prevention And Treatment Of Kidney Injuries

Abstract

The invention relates to the use of ferroportin inhibitor compounds of the general formula (I) for preventing and treating kidney injuries, such as in particular acute kidney injuries, and the symptoms and pathological conditions associated therewith.

##STR00001##

Claims

1-16. (canceled)

17. A method of prevention and/or treatment of kidney injuries comprising administering to a patient in need thereof, compounds according to formula (I): ##STR00050## wherein X.sup.1 is N or O; and X.sup.2 is N, S or O; with the proviso that X.sup.1 and X.sup.2 are different; R.sup.1 is selected from the group consisting of hydrogen and optionally substituted alkyl; n is an integer of 1 to 3; A.sup.1 and A.sup.2 are independently selected from the group of alkanediyl R.sup.2 is hydrogen, or optionally substituted alkyl; or A.sup.1 and R.sup.2 together with the nitrogen atom to which they are bonded form an optionally substituted 4- to 6-membered ring; R.sup.3 indicates 1, 2 or 3 optional substituents, which may independently be selected from the group consisting of halogen, cyano, optionally substituted alkyl, optionally substituted alkoxy, and a carboxyl group; R.sup.4 is selected from the group consisting of hydrogen, halogen, C.sub.1-C.sub.3-alkyl, and halogen substituted alkyl; and pharmaceutically acceptable salts, solvates, hydrates and polymorphs of any of the foregoing.

18. The method of claim 17, wherein the kidney injuries are selected from kidney injuries induced by catalytic free iron.

19. The method of claim 17, wherein the kidney injuries are selected from acute kidney injury (AKI), renal ischemia-reperfusion injury (IRI) and AKI caused by ischemic injury, AKI following surgery or surgical intervention, and kidney injury associated with red blood cell (RBC) transfusion.

20. The method of claim 17, wherein the kidney injuries are selected from renal ischemia-reperfusion injury (IRI), ischemic injury and acute kidney injuries.

21. The method of claim 17, wherein the patient in need thereof is suffering from at least one symptom selected from the group consisting of (i) increased plasma creatinine levels, (ii) increased urine albumin excretion, (iii) decreased estimated glomerular filtration rate (eGFR), wherein each, of (i), (ii) and (iii) are measured as compared to normal physiological levels, and (iv) AKI.

22. The method of claim 17, wherein the patient in need thereof is at risk of suffering from AKI by any of the stages defined by the KDIGO or RIFLE/AKIN classification or by a CSA-NGAL score >0, or by an EGTI histology score >0.

23. The method of claim 17, wherein the prevention and/or treatment comprises at least one of a) decrease, accelerated decrease or prevention of increase of serum creatinine, b) increase or prevention of decrease of eGFR, c) decrease or prevention of increase of renal ferroportin, d) increase or prevention of decrease of H-ferritin levels, e) decrease or prevention of increase of renal neutrophil infiltration, and f) decrease or prevention of increase of serum IL-6 levels.

24. The method of claim 17, comprising administering to a patient in need thereof, wherein the patient in need thereof is at risk of at least one of IRI and AKI, compounds according to formula (I) one or more times within a time period of >0 to 48 hours, >0 to 36 hours, >0 to 24 hours, >0 to 20 hours, >0 to 18 hours, >0 to 16 hours, >0 to 12 hours, >0 to 10 hours, >0 to 8 hours, >0 to 6 hours, >0 to 5 hours, >0 to 4 hours, >0 to 3 hours, >0 to 2 hours, >0 to 1 hour, or >0 to 0.5 hours, prior to reperfusion, prior to red blood cell transfusion, prior to surgery or surgical intervention.

25. The method of claim 17, comprising administering to a patient in need thereof, compounds according to formula (I), one or more times within a time period between immediately after and up to 48 hours after a surgical intervention, RBC transfusion or an ischemic reperfusion event.

26. The method of claim 17, comprising administering to a patient in need thereof, compounds according to formula (I), one or more times within a time period between immediately after and up to 12 hours after a surgical intervention or an ischemic reperfusion event.

27. The method of claim 17, wherein the compounds of the formula (I) are administered in a dose between 0.5 to 500 mg, or between 1 to 300 mg, or between 1 to 250 mg, or between 0.001 to 35 mg/kg body weight.

28. The method of claim 17, wherein in formula (I) n=1; R.sup.3=hydrogen; R.sup.4=hydrogen; A.sup.1=methylene or ethane-1,2-diyl; A.sup.2=methylene, ethane-1,2-diyl or propane-1,3-diyl; such that compounds according to formula (II) are defined: ##STR00051## or A.sup.1 and R.sup.2 together with the nitrogen atom to which they are bonded form an optionally substituted 4-membered ring, such that compounds according to formula (III) are defined: ##STR00052## wherein in formula (II) and (III) I is 0 or 1; and m is an integer of 1, 2 or 3.

29. The method of claim 17, wherein the compounds of formula (I) are in the form of a pharmaceutically acceptable salt with at least one acid selected from the group consisting of benzoic acid, citric acid, fumaric acid, hydrochloric acid, lactic acid, malic acid, maleic acid, methanesulfonic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid and toluenesulfonic acid, and solvates, hydrates and polymorphs of any of the foregoing.

30. The method of claim 28, wherein the compounds of formula (II) or (III) are in the form of a pharmaceutically acceptable salt with at least one acid selected from the group consisting of benzoic acid, citric acid, fumaric acid, hydrochloric acid, lactic acid, malic acid, maleic acid, methanesulfonic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid and toluenesulfonic acid, and solvates, hydrates and polymorphs of any of the foregoing.

31. The method of claim 17, wherein the compounds of the formula (I) are selected from the group consisting of: TABLE-US-00010 Exp No. Structure 1 2 4 40 94 118 126 127 193 206 208 233 and pharmaceutically acceptable salts, solvates, hydrates and polymorphs of any of the foregoing.

32. The method of claim 17, wherein the compounds of the formula (I) are selected from the group consisting of: TABLE-US-00011 Exp. No. Structure 1 40 94 127 208 and pharmaceutically acceptable salts, solvates, hydrates and polymorphs of any of the foregoing.

33. The method of claim 17, wherein the compounds of the formula (I) are selected from the group consisting of: ##STR00070## and pharmaceutically acceptable salts, solvates, hydrates and polymorphs of any of the foregoing; (b) a 1:1 sulfate salt having the formula ##STR00071## (c) a 1:1 phosphate salt having the formula ##STR00072## (d) a 1:3 HCl salt having the formula ##STR00073## and polymorphs of any of (b), (c) and (d).

34. The method of claim 17, wherein the compounds according to formula (I) are contained in a medicament, the medicament further comprising one or more pharmaceutical carriers and/or auxiliaries and/or solvents, and/or one or more additional pharmaceutically active compounds.

35. The method of claim 28, wherein the compounds according to formula (II) or (III) are contained in a medicament, the medicament further comprising one or more pharmaceutical carriers and/or auxiliaries and/or solvents, and/or one or more additional pharmaceutically active compounds.

36. The method of claim 17, wherein the method forms part of a combination therapy, wherein the combination therapy further comprises co-administration to the patient in need, one or more other pharmaceutically active compounds, wherein the co-administration of the combination therapy is carried out in a fixed dose combination therapy by co-administration of the compounds according to formula (I) together with one or more other pharmaceutically active compounds in a fixed-dose formulation, or wherein the co-administration of the combination therapy is carried out in a free dose combination therapy by co-administration of the compounds according to formula (I) together with one or more other pharmaceutically active compounds in free doses of the respective compounds, either by simultaneous administration of the individual compounds or by sequential use of the individual compounds administered over a time period.

Description

[0345] FIG. 1: Illustration of the dosing regimen in Example II

[0346] FIG. 2: Serum iron levels in naïve C57BL/6 mice treated with Fpn127 at 120 mg/kg or 300 mg/kg for 4 h or 8 h. Vehicle was 0.5% methylcellulose in water

EXAMPLES

[0347] The invention is illustrated in more detail by the following examples. The examples are merely explanatory, and the person skilled in the art can extend the specific examples to further ferroportin inhibitor compounds according to the present invention.

I. Ferroportin Inhibitor Example Compounds

[0348] Regarding the preparation of the specific Ferroportin Inhibitor Example Compounds Nos. 1, 2, 4, 40, 94, 118, 126, 127, 193, 206, 208 and 233 as described herein and the preparation of pharmaceutically acceptable salts thereof reference is made to the international applications WO2017/068089, WO2017/068090 and WO2018/192973.

[0349] Regarding the preparation of the specific Ferroportin Inhibitor Compounds described in WO2020/123850 A1 reference is made to the preparation methods described in said international application WO2020/123850 A1.

II. Pharmacological Assays

II. 1 Reduction of Serum Iron by Fpn127 in C57BL/6 Mice

[0350] To determine the dose of Fpn127 that causes sustained serum iron reduction, C57BL/6 mice received either 120 mg/kg or 300 mg/kg of Fpn127 po for 4 h or 8 h. Serum iron was reduced significantly by both doses at 4 h post dosing. However, only the dose of 300 mg/kg sustained the hypoferremia for 8 h (FIG. 2). These data in naïve C57BL/6 mice suggested to use the dose of 300 mg/kg po in the bilateral ureteral obstruction model of AKI.

II. 2 In Vivo Efficacy of the Ferroportin Inhibitor Fpn127 in the Bilateral Ischemic Acute Kidney Injury Mouse Model

[0351] Renal ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury (AKI) and iron-mediated oxidative stress by non-transferrin bound iron (NTBI) is implicated in IRI pathogenesis (Baliga R, Ueda N, Shah S V: Biochem J 291: 901-905, 1993). Hepcidin, the key regulator or iron homeostasis preventing iron export from cells via ferroportin, has been shown to mediate protection in renal IRI (Scindia Y et al, JASN, 2015).

[0352] The efficacy of the ferroportin inhibitor compounds of the present invention in treating kidney injuries, such as IRI and AKI, can be determined in a model of bilateral ischemic kidney injury. As an exemplary ferroportin inhibitor compound according to formula (I) Example Compound No. 127 (Fpn127) can be used.

[0353] To determine the optimal level of kidney injury in this model, a pilot study comparing 25 min and 30 min of bi-lateral renal ischemia is conducted, as described in Wei Q and Dong Z. “Mouse model of ischemic acute kidney injury: technical notes and tricks”, Am J Physiol Renal Physiol 303: F1487-F1494, 2012. The mouse is anesthetized with 50-60 mg/kg of pentobarbital sodium by intraperitoneal injection. Pentobarbital solution is diluted with sterile saline to have a concentration of 5 mg/ml for injection. Shortly after pentobarbital injection, 50 μg/kg of buprenorphine is administered subcutaneously for relief from pain and distress. After pentobarbital and buprenorphine injections, the hair on both sides of the mouse is removed with the hair clipper. The skin in the surgical area is then wiped clean with 70% alcohol swab. Immediately after the skin preparation, the mouse is placed on the homeothermic blanket of a homeothermic monitor system and covered by sterile gauze. The body temperature is monitored through a rectal probe and controlled in the range of 36.5-37° C. (our routine set-point is 36.7° C. and temperature varies in 0.1° C. range). Surgery will not be started until 1) the body temperature is stabilized at the set-point, and 2) the mouse is in deep anesthesia and thus does not respond to pain induced by toe pinch. It usually takes 30 min after pentobarbital injection to achieve deep anesthesia. The mouse is placed on the thermostatic station laying on the right side. The skin and muscle on the left flank side are cut open along the back to expose the left kidney. The incision is positioned at ⅓ of the body from the back of the mouse and the incision size is 1-1.5 cm along the back. The kidney is then pushed out from the cut with sterile cotton swabs to expose the renal pedicle. Dissection of the pedicle tissue is done with ultra-fine-point tweezers to remove the tissue around the renal pedicle to expose the blood vessels for renal pedicle clamping. After the preparation, the left kidney is returned to the abdomen cavity. The right renal pedicle is prepared by a similar surgical procedure, but the incision is closer to the rib due to the different position of the right kidney. After the pedicle preparation, both kidneys are returned back to their original positions in the abdomen cavity. The mouse is then covered with sterile gauze on the thermostatic station for its body temperature to stabilize again, which usually takes 5-10 min. The right kidney is gently pushed out of the body cavity with cotton swabs to expose the pedicle. A microaneurysm is used to clamp the pedicle to block the blood flow to the kidney to induce renal ischemia. The duration of right kidney ischemia starts from the time of clamping. Complete ischemia is indicated by color change of the kidney from red to dark purple in a few seconds. After verification of the kidney color changes, the kidney is returned to the abdomen cavity. The mouse is then laid on its right side for the left renal pedicle clamping and ischemia. There is around 1-1.5 min time latency between the right and left kidney clamping. However, the ischemic time of each side is recorded separately to ensure both kidneys receive the same durations of ischemia. After the ischemia, the micro-aneurysm clips are released at desired times for each kidney to start the reperfusion, which is indicated by the change of kidney color to red. A Vicryl suture is used to close the muscle layer of the incision followed by the closure of the skin wound with Michel wound clips. Immediately after the wound closure, 0.5 ml warm sterile saline is given intraperitoneally to each mouse. The animal is then kept on a heating pad until it gains full consciousness before being returned to its housing cage. Kidneys are exposed to reperfusion for 24 hours. Sham-operated mice undergo bilateral flank incisions without clamping of renal pedicles. 24 hours after ischemia, mice are euthanized and kidney and blood are collected. Serum creatinine levels are measured and serve as marker for severity of injury. The following groups of 8- to 10-week-old C578L/6J male mice; n=4/group are used in the pilot study: [0354] 1. Sham operated [0355] 2. IRI— 25 min [0356] IRI— 30 min

Main Study:

[0357] Based on the results from the pilot study, the ischemic duration with plasma creatinine levels between 2.5-3 mg/dL is selected for the main study. Mice are pre-treated with Fpn127 (300 mg/kg, per os, p.o), Fpn127 (100 mg/kg, intravenous, i.v.), Hepcidin (50 μg/mouse, intraperitoneal, i.p.), or vehicle (0.5% methylcellulose, p.o.) for 24 hours before IRI. Then, both renal pedicles are exposed and cross-clamped either for 25 min or 30 min in anaesthetized mice. Clamps are removed, and kidneys are allowed to reperfuse for 24 hours. Sham-operated mice undergo bilateral flank incisions without clamping of renal pedicles. 24 hours after ischemia, mice are euthanized and kidneys and blood are collected.

[0358] The following groups of mice are included: 8- to 10-week-old C57BL/6J male mice n=8/group. [0359] 4. Sham operated—vehicle (0.5% Methylcellulose, 10 ml/kg, p.o.) [0360] 5. IRI— vehicle, (0.5% Methylcellulose, 10 ml/kg, p.o.) [0361] 6. IRI— Fpn127 (300 mg/kg, 10 ml/kg, 24 h before IRI, p.o.) [0362] 7. IRI—Fpn127 (100 mg/kg, 5 ml/kg, 24 h before IRI, i.v.) [0363] 8. IRI— Hepcidin (50 μg/mouse, 5 ml/kg, 24 h before IRI), i.p.

[0364] The dosing time interval is illustrated in FIG. 1.

[0365] The following markers are measured in the end of the study: Plasma creatinine, blood urea nitrogen (BUN), total plasma iron, NTBI, plasma hepcidin, spleen, kidney and liver iron.

[0366] Hematoxylin/eosin (HE) staining of kidney sections is performed to evaluate the extent of kidney tissue injury with tubular injury score as readout. Immunohistochemistry using caspase-3 staining on kidney sections are performed to assess the level of kidney damage.

[0367] ROS-mediated oxidative stress in kidney is assessed by detection of 4-HNE in kidney sections. Ferroportin gene expression in liver, spleen and kidney is measured by qPCR.

[0368] H-Ferritin expression in organs is measured by western blot and qPCR.

[0369] Infiltration of leukocytes in kidney is detected by staining with anti-CD45 antibody using flow cytometry.

[0370] The neutrophils are identified by anti-Ly6G and Ly6C labeling of CD11b+ cells and flow cytometry analysis.

[0371] In patients, the onset of acute kidney injury is unforeseen and ideally drug dosing closer to the ischemia event would be preferable. To optimize the dosing regimen of the ferroportin inhibitor mice are treated with Fpn127 for 1, 3, 6, 9, 12 h and 15 h before the IRI. The following groups of mice are included: 8- to 10-week-old C57BL/6J male mice n=8/group. [0372] 1. Sham operated—vehicle (0.5% Methylcellulose, 10 ml/kg, p.o.) [0373] 2. IRI— vehicle, (0.5% Methylcellulose, 10 ml/kg, p.o.) [0374] 3. IRI— Fpn127 (300 mg/kg, 10 ml/kg, 2 h before IRI, p.o.) [0375] 4. IRI— Fpn127 (300 mg/kg, 10 ml/kg, 4 h before IRI, p.o.) [0376] 5. IRI Fpn127 (300 mg/kg, 10 ml/kg, 6 h before IRI, p.o.) [0377] 6. IRI Fpn127 (300 mg/kg, 10 ml/kg, 8 h before URI, p.o.) [0378] 7. IRI— Fpn127 (300 mg/kg, 10 ml/kg, 12 h before IRI, p.o.) [0379] 8. IRI— Fpn127 (300 mg/kg, 10 ml/kg, 16 h before IRI, p.o.)

[0380] The parameters of kidney function measured in the main study are used as efficacy readouts. To further optimize the dosing regimen mice are administered with Fpn127 via i.v. route for 0.5 h, 1 h and 3 h before IRI or 1 h after IRI. The following groups of mice are included: 8- to 10-week-old C57BL/6J male mice n=8/group.

[0381] Sham operated—vehicle (saline, 5 ml/kg, i.v.) [0382] 1. IRI vehicle, (saline, 5 ml/kg, i.v.)) [0383] 2. IRI— Fpn127 (100 mg/kg, 5 ml/kg, 0.5 h before IRI, i.v.) [0384] 3. IRI— Fpn127 (100 mg/kg, 5 ml/kg, 1 h before IRI, i.v.) [0385] 4. IRI Fpn127 (100 mg/kg, 5 ml/kg, 3 h before IRI, i.v.) [0386] 5. IRI— Fpn127 (100 mg/kg, 5 ml/kg, 1 h after IRI, i.v.)

[0387] The parameters of kidney function measured in the main study are used as efficacy readouts.

II. 3 Reduction of the Proportion of ROS in Kidney Tissue

[0388] The effect of the ferroportin inhibitor, e.g. of Fpn127, on ROS levels in kidney tissue can be monitored by commercially available far-red emitting ROS-sensitive sensor.

[0389] In particular, ROS determination can be used as an efficiency marker, similar as described in Scindia et al., 2015 (cited above).

III. Effect of Fpn127 on NTBI and LPI Levels in the IRI/AKI Mouse Model Described Above

[0390] As described above, elevated plasma NTBI levels as a result of ferroportin-mediated export of iron from macrophages recycling damaged cells, such as RBCs and other types of damaged cells during AKI are considered to induce tissue injury. Dosing of ferroportin inhibitors of the present invention, such as Fpn127 has the potential to reduce the levels of plasma NTBI (and LPI) and the associated adverse effects.

[0391] The levels of NTBI in the mouse model of IRI/MAKI described above are investigated in mice treated with either vehicle or ferroportin inhibitors of the present invention, such as Fpn127, as indicated above. The nitrilotriacetate-NTBI method (NTA-NTBI) previously described (Singh S, Hider R C, Porter J B. “A direct method for quantification of non-transferrin-bound iron.“Anal Biochem. 1990 May 1; 186(2):320-3) is used with minor modifications.

[0392] Briefly, 0.02 mL of 800-mM NTA (at pH 5.7) is added to 0.18-mL mouse serum pool and allowed to stand for 30 minutes at 22° C. The solution is ultrafiltered using Whatman Vectaspin ultracentrifugation devices (30 kDa) at 12320 g and the ultrafiltrate (0.02 mL) injected directly onto an high-performance liquid chromatography column (ChromSpher-ODS, 5 μM, 100×3 mm, glass column fitted with an appropriate guard column) equilibrated with 5% acetonitrile and 3-mM deferiprone (DFP) in 5-mM MOPS (pH 7.8). The NTA-iron complex then exchanges to form the DFP-iron complex detected at 460 nm by a Waters 996 photodiode array. Injecting standard concentrations of iron prepared in 80-mM NTA is used to generate a standard curve. The 800-mM NTA solution used to treat the samples and prepare the standards is treated with 2-μM iron to normalize the background iron that contaminates reagents. This means that the zero standard gives a positive signal because it contains the added background iron as an NTA-complex. When unsaturated transferrin is present in sera, this additional background iron can be donated to vacant transferrin sites resulting in a loss of the background signal and yielding a negative NTBI value.

[0393] NTBI is also measured using an alternative method (CP851 bead-NTBI) assay as described in Garbowski M W, Ma Y, Fucharoen S, Srichairatanakool S, Hider R, Porter J B. “Clinical and methodological factors affecting non-transferrin-bound iron values using a novel fluorescent bead assay.” Transl Res. 2016). The standards for this assay are prepared as follows: 1-mM iron-NTA complex (1:2.5 molar ratio), prepared from 100-mM NTA and 18-mM atomic absorption standard iron solution, is diluted with MilliQ water to a final concentration between 0 and 100 μM. For the standard curve, 120 μL of probe-labeled bead suspensions are incubated with 20 μL of buffered NTA-iron solutions of known concentration for 20 minutes at room temperature, with subsequent addition of 20 μL control serum from wild type mice (without free iron) and 40-μL paraformaldehyde (10% in MOPS) at a final concentration of 2%. The suspensions in sealed 96-well plates are incubated at 37° C. for 16 hours with shaking before fluorescence measurement by flow-cytometry. For serum samples of unknown iron concentrations, 140 μL quantities of beads are incubated with 20 μL of serum samples for 20 minutes, with subsequent addition of 40-μL paraformaldehyde at a final concentration of 2%. The chelatable fluorescent beads are mixed with serum from wild type mice as a control to set up the fluorescence at 100% and the relative fluorescence of chelatable fluorescent beads with serum from mice tested in the IRI/AKI model described above was calculated accordingly. Measurements are performed on Beckman Coulter F C500 flow-cytometer and analysis on FlowJo software. Gates were based on dot-plots of untreated bead populations. Median fluorescence of 10,000 events was recorded and corrected for bead auto-fluorescence. A standard curve was fitted with variable-slope sigmoidal dose response function.

[0394] NTBI, which encompasses all forms of serum iron that are not tightly associated with transferrin, is chemically and functionally heterogeneous. LPI represents a component of NTBI that is both redox active and chelatable, capable of permeating into organs and inducing tissue iron overload. LPI assay (Esposito BP1, Breuer W, Sirankapracha P, Pootrakul P, Hershko C, Cabantchik Z I. “Labile plasma iron in iron overload: redox activity and susceptibility to chelation.” Blood. 2003) measures the iron-specific capacity of a given sample to produce ROS and is considered one of the most relevant reactive iron species involved in tissue injury, such as AKI.

[0395] FeROS™ LPI kit (Aferrix Ltd.) is used to measure LPI in sera of mice treated with either vehicle or ferroportin inhibitors of the present invention, such as Fpn127.

[0396] NTBI and LPI levels in mice tested in the IRI/AKI model have been found to serve as translational markers allowing to evaluate the efficiency of the ferroportin inhibitor therapy.

[0397] This model can also be used to optimally design the dosing regimen of ferroportin inhibitors (such as e.g. Fpn127) for treating IRI and AKI. Therewith an optimal combination therapy for AKI can be established using the ferroportin inhibitors of the present invention.

[0398] With the models and examples described above, it is possible to demonstrate the capacity of the ferroportin inhibitors of the present invention in preventing and improving IRI and AKI.

IV. Serum Creatinine, Urine Albumin Excretion, BUN, NGAL, Hemoglobin (Hb), Kidney H-Ferritin, Total Plasma Iron, RBC Hemolysis, Renal Ferroportin and KIM-1

[0399] These parameters can be determined using conventional methods.

[0400] For example, iron levels in plasma can be determined by the MULTIGENT Iron assay (Abbott Diagnostics). Total organ iron and is determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES) in rodent models or by magnetic resonance imaging in patients.

V. Serum Hepcidin, IL-6, Nonheme Iron, Renal Neutrophil Infiltration

[0401] These parameters can be determined as described by Scindia et al., 2015 (cited above).

VI. Tissue/Organ Iron Levels

[0402] Iron levels, such as, e.g., liver, spleen or kidney iron levels can be determined using conventional assay(s). For example, iron levels can be determined by magnetic resonance imaging.

VII. Tissue Morphology and Histology/Tubular Necrosis and Apoptosis

[0403] Tissue morphology and histopathology, such as tubular necrosis and apoptosis, can be performed as described by Scindia et al., 2015 (cited above).

VIII. Efficacy of the Ferroportin Inhibitor VIT-2653 (Example Compound No. 40) to Attenuate Renal Injury Following Red Blood Cell Transfusion in Guinea Pigs

[0404] The efficacy of the ferroportin inhibitor compounds of the present invention in the prevention and treatment of acute kidney injuries in accordance with the present invention has further been confirmed by the results of J. H. Baek et al. “Ferroportin inhibition attenuates plasma iron, oxidant stress, and renal injury following red blood cell transfusion in guinea pigs”; Transfusion 2020 March; 60(3):513-523.

[0405] Said experiments have been carried out by intravenously administering the small-molecule ferroportin inhibitor VIT-2653, corresponding to Example Compound No. 40 of the present invention and further confirm the findings of the present invention.

[0406] The NTBI and Hb levels following exchange transfusion were significantly improved by dosing of the ferroportin inhibitor.

[0407] Total iron in kidneys following transfusion can be reduced by dosing of the ferroportin inhibitor. The contribution of circulating Hb on renal iron loading and the subsequent effects on oxidative stress and cellular injury was evaluated revealing that dosing of the ferroportin inhibitor to transfused mice significantly reduced the occurrence of changes in plasma creatinine >0.3 mg/dL, which is used as indicator of early acute kidney injury (AKI).

[0408] The experimental details and study conditions and the concrete study results can be derived from the mentioned paper.