Artemisinin and its derivatives for use in the treatment of kidney disease

Abstract

The present invention relates compounds according to Formula (I) wherein R.sup.1 and R.sup.2 are independently H or an optionally substituted group selected from an alkyl, a heteroalkyl, an aryl, a heteroaryl, an arylalkyl, and a heteroarylalkyl, and R.sup.3 and R.sup.4 taken together form a carbonyl (O); or wherein R.sup.1 and R.sup.2 are independently H or an optionally substituted group selected from an alkyl, a heteroalkyl, an aryl, a heteroaryl, an arylalkyl, and a heteroarylalkyl, R.sup.3 is H and R.sup.4 is H or OR.sup.5, wherein R.sup.5 is H or an optionally substituted group selected from an alkyl, a heteroalkyl, an aryl, a heteroaryl, an arylalkyl, and a heteroarylalkyl; or pharmaceutically acceptable salts or esters thereof, for use in the treatment of kidney disease, in particular in the treatment of acute kidney injury. The present invention also relates to methods of treatment of the same and methods of kidney transplant surgery and coronary artery bypass graft surgery using the compounds of Formula (I). ##STR00001##

Claims

1. A method of treating acute kidney injury or a method of treating acute kidney injury in a patient undergoing kidney dialysis or surgery that results in ischaemia-reperfusion of the whole or part of the kidney, kidney transplantation surgery, kidney and pancreas transplantation surgery, or coronary artery bypass surgery comprising administering a compound according to Formula I ##STR00004## or a pharmaceutically acceptable salt or ester thereof, or a pharmaceutical composition comprising the compound according to Formula I or a pharmaceutically acceptable salt or ester thereof wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, to a patient in need thereof, wherein: R.sup.1 and R.sup.2 are independently H or an optionally substituted group selected from an alkyl, a heteroalkyl, an aryl, a heteroaryl, an arylalkyl, and a heteroarylalkyl; and R.sup.3 and R.sup.4 taken together form a carbonyl (O); or R.sup.3 is H and R.sup.4 is H or OR.sup.5, wherein R.sup.5 is H or an optionally substituted group selected from an alkyl, a heteroalkyl, an aryl, a heteroaryl, an arylalkyl, and a heteroarylalkyl; and further wherein: a) the compound or pharmaceutical composition is administered to the patient at least 12 hours after ischaemia-reperfusion of the whole or part of the kidney or at least 12 hours after kidney transplantation surgery, kidney and pancreas transplantation surgery, or coronary artery bypass surgery; b) the compound or pharmaceutical composition is administered to the patient after diagnosis of acute kidney injury or after diagnosis of a risk of acute kidney injury; c) the method further comprises measuring serum creatinine levels after surgery and administering the compound or pharmaceutical composition only if acute kidney injury is diagnosed or if the patient is deemed at risk of developing acute kidney injury; or d) the patient is undergoing kidney transplantation, wherein the composition comprising the compound of Formula I, or pharmaceutically acceptable salt or ester thereof, is a reperfusion solution, wherein the reperfusion solution further comprises one or more volume expanders, and wherein the method further comprises bathing the donor kidney in or reperfusing the donor kidney with the reperfusion solution.

2. The method according to claim 1, wherein the compound or pharmaceutical composition is administered at least 12 hours after acute kidney injury.

3. The method according to claim 1, wherein: R.sup.1 and R.sup.2 are independently H or an optionally substituted C.sub.1-C.sub.10 alkyl; and R.sup.3 and R.sup.4 taken together form a carbonyl (O) group; or R.sup.3 is H and R.sup.4 is H or OR.sup.5, wherein R.sup.5 is H or an optionally substituted group selected from an alkyl, a heteroalkyl, an aryl, a heteroaryl, an arylalkyl, and a heteroarylalkyl; or a pharmaceutically acceptable salt or ester thereof.

4. The method according to claim 1, wherein: R.sup.1 and R.sup.2 are independently H or an optionally substituted C.sub.1-C.sub.3 alkyl; and R.sup.3 and R.sup.4 taken together form a carbonyl (O) group; or R.sup.3 is H and R.sup.4 is H or OR.sup.5, wherein R.sup.5 is H or an optionally substituted group selected from an alkyl, a heteroalkyl, an aryl, a heteroaryl, an arylalkyl, and a heteroarylalkyl; or a pharmaceutically acceptable salt or ester thereof.

5. The method according to claim 1, wherein: R.sup.1 and R.sup.2 are independently H or an optionally substituted methyl; and R.sup.3 and R.sup.4 taken together form a carbonyl (O) group; or R.sup.3 is H and R.sup.4 is OR.sup.5, wherein R.sup.5 is H or an optionally substituted group selected from an alkyl, a heteroalkyl, an aryl, a heteroaryl, an arylalkyl, and a heteroarylalkyl; or a pharmaceutically acceptable salt or ester thereof.

6. The method according to claim 1, wherein: R.sup.1 and R.sup.2 are both independently methyl; and R.sup.3 and R.sup.4 taken together form a carbonyl (O) group; or R.sup.3 is H and R.sup.4 is OR.sup.5, wherein R.sup.5 is H or an optionally substituted group selected from an alkyl, a heteroalkyl, an aryl, a heteroaryl, an arylalkyl, and a heteroarylalkyl; or a pharmaceutically acceptable salt or ester thereof.

7. The method according to claim 1, wherein R.sup.5 is H, an alkyl, or an arylalkyl, wherein the alkyl and/or arylalkyl is/are optionally substituted with one more or more of halo, O, COOR.sup.6, OR.sup.6 and OCOR.sup.6, wherein R.sup.6 is H or a C.sub.1-C.sub.6 alkyl.

8. The method according to claim 1, wherein R.sup.5 is selected from the group consisting of H, CH.sub.3, CH.sub.2CH.sub.3, CO(CH.sub.2).sub.2COOH and CH.sub.2C.sub.6H.sub.4COOH.

9. The method according to claim 1, wherein the compound is selected from the group consisting of artesunate, artemisinin, artemether, dihydroartemisinin, artelinic acid and artemotil.

10. The method according to claim 1, wherein the pharmaceutical composition further comprises an additional pharmaceutically active agent.

11. The method according to claim 1, wherein the compound or composition is administered simultaneously with, separately from, or sequentially to the administration of one or more further pharmaceutically active agents.

12. The method according to claim 1, wherein the compound or pharmaceutical composition is administered by the oral, parenteral, intravenous, intramuscular, intrathecal or intraperitoneal route, or is administered by inhalation.

13. The method according to claim 1, wherein the compound or pharmaceutical composition is administered to the patient at least 12 hours after ischaemia-reperfusion of the whole or part of the kidney or at least 12 hours after kidney transplantation surgery, kidney and pancreas transplantation surgery, or coronary artery bypass surgery.

14. The method according to claim 1, wherein the compound or pharmaceutical composition is administered to the patient after diagnosis of acute kidney injury or after diagnosis of a risk of acute kidney injury.

15. The method according to claim 1, wherein the method further comprises measuring serum creatinine levels after surgery and administering the compound or pharmaceutical composition only if acute kidney injury is diagnosed or if the patient is deemed at risk of developing acute kidney injury.

16. The method of claim 15, wherein the serum creatinine levels are measured at least 12 hours after surgery.

17. The method of claim 15, wherein the acute kidney injury is diagnosed or the patient is deemed at risk of developing acute kidney injury when the serum concentration of creatinine is greater than 1.5 mg/dl.

18. The method according to claim 1 in a patient undergoing kidney transplantation, wherein the composition comprising the compound of Formula I, or pharmaceutically acceptable salt or ester thereof, is a reperfusion solution, wherein the reperfusion solution further comprises one or more volume expanders, and wherein the method further comprises bathing the donor kidney in or reperfusing the donor kidney with the reperfusion solution.

19. The method of claim 18, wherein the volume expander is a colloid or a crystalloid.

20. The method of claim 19, wherein the colloid comprises an aqueous solution comprising at least one component selected from the group consisting of gelatin, succinylated gelatin, albumin, dextran, blood and etherified starch.

21. The method of claim 19, wherein the crystalloid is an aqueous solution comprising at least three ions selected from the group consisting of sodium ions, chloride ions, lactate ions, potassium ions and calcium ions.

22. A method of perfusing a kidney comprising bathing the kidney in a reperfusion solution comprising a compound according to Formula I ##STR00005## or a pharmaceutically acceptable salt or ester thereof, and one or more volume expanders, wherein: R.sup.1 and R.sup.2 are independently H or an optionally substituted group selected from an alkyl, a heteroalkyl, an aryl, a heteroaryl, an arylalkyl, and a heteroarylalkyl; and R.sup.3 and R.sup.4 taken together form a carbonyl (O); or R.sup.3 is H and R.sup.4 is H or OR.sup.5, wherein R.sup.5 is H or an optionally substituted group selected from an alkyl, a heteroalkyl, an aryl, a heteroaryl, an arylalkyl, and a heteroarylalkyl.

23. The method according to claim 22 further comprising implanting the kidney into a patient.

24. The method of claim 1, wherein the compound is administered by or during kidney dialysis.

Description

(1) The invention will now be described with reference to the following Examples, which are presented for the purposes of reference only and are not intended to be limiting on the scope of the invention. In the Examples, references are made to a number of Figures, in which:

(2) FIG. 1 shows alterations in MAP in rats subjected to (i) surgical procedure alone (Sham, n=4), or surgical procedure and haemorrhagic shock then treated with (ii) vehicle (10% DMSO, 1 ml/kg i.v., HS Control, n=10) or (iii) artesunate (1, 3 or 10 mg/kg i.v., HS+Artesunate 1 mg/kg; n=6, HS+Artesunate 3 mg/kg; n=7 and HS+Artesunate 10 mg/kg; n=8, respectively) on resuscitation. Data is expressed as meanSEM. .star-solid. P<0.05 sham vs. HS Control.

(3) FIGS. 2A, 2B, and 2C show alterations in serum levels of (a) urea and (b) creatinine; and (c) creatinine clearance, in rats subjected to (i) surgical procedure alone (Sham, n=4), or surgical procedure and haemorrhagic shock then treated with (ii) vehicle (10% DMSO, 1 ml/kg i.v., HS Control, n=10) or (iii) artesunate (1, 3 or 10 mg/kg i.v., HS+Artesunate 1 mg/kg; n=6, HS+Artesunate 3 mg/kg; n=7 and HS+Artesunate 10 mg/kg; n=8, respectively) on resuscitation. Data is expressed as meanSEM. .star-solid. P<0.05 vs. HS Control.

(4) FIGS. 3A, 3B, and 3C show alterations in serum levels of (a) AST, (b) ALT and (c) CK in rats subjected to (i) surgical procedure alone (Sham, n=4), or surgical procedure and haemorrhagic shock then treated with (ii) vehicle (10% DMSO, 1 ml/kg i.v., HS Control, n=10) or (iii) artesunate (1, 3 or 10 mg/kg i.v., HS+Artesunate 1 mg/kg; n=6, HS+Artesunate 3 mg/kg; n=7 and HS+Artesunate 10 mg/kg; n=8, respectively) on resuscitation. Data is expressed as meanSEM. .star-solid. P<0.05 vs. HS Control.

(5) FIGS. 4A and 4B show alterations in serum levels of (a) urea and (b) creatinine, in rats subjected to (i) surgical procedure alone and treated with vehicle (Sham+10% DMSO, n=4), burn injury and treated with (ii) vehicle (Burn+10% DMSO, n=10) or (iii) artesunate (Burn+Artesunate, n=9). Data is expressed as meanSEM, *P<0.05 when compared to Burn+10% DMSO.

(6) FIGS. 5A and 5B show alterations in serum levels of (a) AST and (b) ALT, in rats subjected to (i) surgical procedure alone and treated with vehicle (Sham+10% DMSO, n=4), burn injury and treated with (ii) vehicle (Burn+10% DMSO, n=10) or (iii) artesunate (Burn+Artesunate, n=9). Data is expressed as meanSEM, *P<0.05 when compared to Burn+10% DMSO.

(7) FIGS. 6A, 6B, 6C, and 6D show the effect of 30 min ischemia followed by different lengths of reperfusion (24, 48 or 72 h) on glomerular and tubular function. Serum urea (A), serum creatinine (B) and estimated creatinine clearance (C) were measured as indicators of glomerular function, and fractional excretion of sodium (D) as an indicator of tubular function (in different sets of animals for each time point). The peak of dysfunction in all parameters occurs 24 h after the onset of reperfusion. Pre-ischemia: n=4; 24 h reperfusion: n=4; 48 h reperfusion: n=8; 72 h reperfusion: n=4. Data are presented as means.e.m. of n observations, .star-solid..star-solid. P<0.0001 vs. pre-ischemia, .star-solid. P<0.001 vs. pre-ischemia.

(8) FIGS. 7A, 7B, 7C, 7D, and 7E show the effect of 30 min ischemia followed by different lengths of reperfusion (24, 48 or 72 h) on renal injury. Representative histological H&E images of rat renal tissue were taken from groups without renal ischemia (pre-ischemia, A), or from rats subjected to 30 min of renal ischaemia followed by reperfusion for 24 h (B), 48 h (C) or 72 h (D). Ten randomly selected fields from three individual kidneys (n=3) per group were selected and analysed (total fields=30) for the determination of percentage background white space using ImageJ software and represented as tissue surface area per field (E). Data are presented as means.e.m. of n observations, .star-solid. P<0.05 vs. pre-ischemia.

(9) FIGS. 8A, 8B, and 8C show the effect of 30 min ischemia followed by different length of reperfusion (24, 48 or 72 h) on the activation of IB, NF-B and endothelial nitric oxide synthase (eNOS). Activation of IB (A) was measured as phosphorylation of Ser32/36 on IB during the course of reperfusion. The activation of IB results in nuclear translocation of the p65 subunit of NF-B (B). The activation of eNOS (C) was measured as phosphorylation of Ser1177 during the course of reperfusion. Data are presented as means.e.m. of n observations, .star-solid. P<0.05 vs. pre-ischemia.

(10) FIGS. 9A, 9B, 9C, and 9D show serum urea (A) and creatinine levels (B) measurements, from rats, as biochemical markers of renal dysfunction over the course of reperfusion (n=4). Serum urea (C) and creatinine levels (D) were measured at the end of 48 h of reperfusion subsequent to either sham-operation (Sham+Vehicle, n=8) or renal ischaemia/reperfusion (IRI+Vehicle, n=8; IRI+Artesunate 0.3 mg/kg i.v., n=7). Vehicle or artesunate were administered 24 h after the onset of reperfusion, i.e. the peak of dysfunction (see A & B). Data represent meanSEM for n observations. Data were analysed by a one-way ANOVA followed by a Dunnett's test for comparison of the sham/treated groups with IRI+Vehicle group; .star-solid. P<0.05 vs. IRI+Vehicle.

(11) FIGS. 10A and 10B show creatinine clearance (A) measurements, from rats, as a biochemical marker of glomerular dysfunction over the course of reperfusion (n=4). Estimated creatinine clearance (B) was measured at the end of 48 h of reperfusion subsequent to either sham-operation (Sham+Vehicle, n=8) or renal ischaemia/reperfusion (IRI+Vehicle, n=8; IRI+Artesunate 0.3 mg/kg i.v., n=7). Vehicle or artesunate were administered 24 h after the onset of reperfusion, i.e. the peak of dysfunction (see A). Data represent meanSEM for n observations. Data were analysed by a one-way ANOVA followed by a Dunnett's test for comparison of the sham/treated groups with IRI+Vehicle group; .star-solid. P<0.05 vs. IRI+Vehicle.

(12) FIGS. 11A and 11B show fractional excretion of sodium (A), which was repeatedly measured, from rats, as a biochemical marker of tubular dysfunction over the course of reperfusion (n=4). Fractional excretion of sodium (B) was measured at the end of 48 h of reperfusion subsequent to either sham-operation (Sham+Vehicle, n=8) or renal ischaemia/reperfusion (IRI+Vehicle, n=8; or IRI+Artesunate 0.3 mg/kg i.v., n=7). Vehicle or artesunate were administered 24 h after the onset of reperfusion, i.e. the peak of dysfunction (see A). Data represent meanSEM for n observations. Data were analysed by a one-way ANOVA followed by a Dunnett's test for comparison of the sham/treated groups with IRI+Vehicle group; .star-solid. P<0.05 vs. IRI+Vehicle.

EXAMPLES

(13) The animal protocols followed in this study were approved by the local Animal Use and Care Committee in accordance with the derivatives of both the Home Office Guidance on the Operation of Animals (Scientific Procedures) Act 1986 published by Her Majesty's Stationary Office and the Guide for the Care and Use of Laboratory Animals of the National Research Council.

(14) 1. Evaluation of the Effects of Artesunate on Organ Injury and Dysfunction Induced by Trauma Haemorrhage in the Rat

(15) 1.1 Surgical Procedure

(16) Thirty-five male Wistar rats (2715 g) were anaesthetised with sodium thiopentone (120 mg/kg i.p., LINK Pharmaceuticals Ltd., West Sussex, UK) and anaesthesia was maintained by supplementary injections (10 mg/kg i.v.) of sodium thiopentone as and when required. The animals were placed onto a thermostatically controlled heating mat (Harvard Apparatus Ltd., Kent, UK) and body temperature was maintained at 371 C. by means of a rectal probe attached to a homeothermic blanket. A tracheotomy was performed by inserting into the lumen of the trachea a small length of polyethylene tubing [Internal Diameter (ID) 1.67 mm, Portex, Kent, UK] to maintain airway patency and facilitate spontaneous respiration. The left femoral artery was cannulated (ID 0.40 mm, Portex) and connected to a pressure transducer (SP844 blood pressure sensor, Memscap, U.S.A.) for the measurement of mean arterial blood pressure (MAP) and derivation of heart rate (HR) from the pulse waveform, which were both displayed on a data acquisition system (Powerlab 8SP, Chart v5.5.3, AD Instruments, Hastings, U.K.) installed on an Intel-based computer running Windows XP for the duration of the experiment. The right carotid artery was cannulated (ID 0.58 mm, Portex) to facilitate the withdrawal of blood using a heparinised syringe. The right jugular vein was cannulated (ID 0.40 mm, Portex) for the administration of Ringer's Lactate (RL), shed blood, test compounds and/or vehicle. The bladder was also cannulated (ID 0.76 mm, Portex) for the collection of urine. Upon completion of the surgical procedure, cardiovascular parameters were allowed to stabilise for a period of 15 min.

(17) 1.2 Haemorrhage and Resuscitation

(18) After the stabilisation period, blood was withdrawn via the cannula inserted in the right carotid artery in order to achieve a fall in MAP to 355 mmHg within 10 min. From this point onwards, MAP was maintained at 355 mmHg for a period of 90 min either by further withdrawal of blood during the compensation phase (MAP rises following blood withdrawal due to sympathetic response) or administration of Ringer's Lactate i.v. during the decompensation phase (animals are unable to increase and maintain high MAP). The average volume of blood withdrawn during haemorrhage was 9.80.2 ml (n=31, across all haemorrhaged groups). At 90 min after initiation of haemorrhage, resuscitation was performed with 20 ml/kg Ringer's Lactate i.v. over a period of 10 min and then half the shed blood mixed with 100 u/ml heparinised saline i.v. over a period of 50 min. At the end of 1 h resuscitation, an i.v. infusion of Ringer's Lactate (1.5 ml/kg/h) was started as fluid replacement and maintained for a further 3 h.

(19) 1.3 Quantification of Organ Injury/Dysfunction

(20) Four hours after the onset of resuscitation, 1.2 ml blood was collected from the right carotid artery and decanted into serum gel tubes (Sarstedt, Numbrecht, Germany), after which the heart was removed to terminate the experiment. The samples were centrifuged (9900 rpm for 3 min) to separate serum from which urea, creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and creatinine kinase (CK) were measured within 24 hours (Idexx Laboratories Ltd., West Yorkshire, UK). Urine collected during the last 3 h of the experiment was analysed for creatinine levels in order to estimate creatinine clearance as an indicator of glomerular dysfunction and was calculated as follows:

(21) $\underset{\left(\mathrm{ml}\text{/}\min \right)}{\mathrm{Creatinine}\mathrm{Clearance}}=\frac{\mathrm{urine}\mathrm{creatinine}\left(\mathrm{.Math.mol}\text{/}I\right)\mathrm{urine}\mathrm{flow}\left(\mathrm{ml}\text{/}\min \right)}{\mathrm{serum}\mathrm{creatinine}\left(\mathrm{.Math.mol}\text{/}I\right)}$
1.4 Experimental Design

(22) Rats were randomly allocated into the following groups: (i) Sham (n=4) (ii) HS Control (n=10) (iii) HS+Artesunate 1 mg/kg (n=6) (iv) HS+Artesunate 3 mg/kg (n=7) (v) HS+Artesunate 10 mg/kg (n=8)

(23) Sham-operated rats underwent identical surgical procedures but without haemorrhage or resuscitation. Animals received either 10% DMSO (1 ml/kg i.v.) or artesunate (1, 3 or 10 mg/kg i.v.) on resuscitation.

(24) 1.5 Materials

(25) Unless otherwise stated, all compounds were obtained from Sigma-Aldrich Company Ltd (Poole, Dorset, U.K.). All stock solutions were prepared in non-pyrogenic saline [0.9% (w/v) NaCl: Baxter Healthcare Ltd., Thetford, Norfolk, U.K.]. Ringer's Lactate was also obtained from Baxter Healthcare Ltd. Sodium thiopentone (Thiovet) was obtained from Link Pharmaceuticals, Horsham, U.K. Multiparin (Heparin injection B.P., 5,000 iu/ml) was obtained from National Veterinary Services, Stoke-on-Trent, U.K., 0.1 ml Multiparin added to 4.9 ml 0.9% (w/v) sodium chloride to give concentration of 100 u/ml and 5 ml Multiparin added to 1 liter 0.9% (w/v) sodium chloride to give concentration of 25 u/ml. Artesunate was also obtained from Sigma-Aldrich Company Ltd (Poole, Dorset, U.K.).

(26) 1.6 Statistical Analysis

(27) All values described in the text and figures are expressed as meanstandard error of the mean (SEM) for n observations. Each data point represents biochemical measurements obtained from up to 10 separate animals. Statistical analysis was carried out using GraphPad Prism 5.03 (GraphPad Software, San Diego, Calif., USA). Data without repeated measurements was assessed by one-way ANOVA followed by Dunnett's post hoc test. Data with repeated measurements was assessed by two-way ANOVA followed by Bonferroni's post hoc test. A P value of less than 0.05 was considered to be significant.

(28) 1.7 Effect of Artesunate on the Circulatory Failure Caused by Haemorrhagic Shock

(29) When compared to sham-operated rats, HS-rats treated with vehicle demonstrated a significant reduction in MAP during the resuscitation period (P<0.05, FIG. 1). The administration of artesunate (1, 3 or 10 mg/kg) on resuscitation failed to attenuate the decline in MAP caused by haemorrhage during the resuscitation phase (P>0.05, FIG. 1).

(30) 1.8 Effect of Artesunate on the Organ Injury and Dysfunction Induced by Haemorrhagic Shock

(31) When compared to sham-operated rats, HS-rats treated with vehicle developed significant increases in serum urea (P<0.001, FIG. 2A) and creatinine (P<0.001, FIG. 2B); creatinine clearance was significantly reduced when compared to sham-operated rats (P<0.005, FIG. 2C) indicating the development of renal and glomerular dysfunction. Treatment of HS-rats with 3 mg/kg artesunate significantly attenuated the rises in serum urea (P<0.05, FIG. 2A) and creatinine (P<0.005, FIG. 2B) when compared to HS-rats; whereas treatment with 10 mg/kg artesunate significantly attenuated the rise in serum creatinine (P<0.005, FIG. 2B) and the fall in creatinine clearance (P<0.005, FIG. 2C). Treatment of HS-rats with 1 mg/kg artesunate had no significant effect on the rises in serum urea (P>0.05, FIG. 2A) and creatinine (P>0.05, FIG. 2B) or on the fall in creatinine clearance (P>0.05, FIG. 2C).

(32) When compared to sham-operated rats, HS-rats treated with vehicle developed significant increases in serum AST (P<0.005, FIG. 3A), ALT (P<0.005, FIG. 3B) and creatinine kinase (P<0.05, FIG. 3C) indicating the development of liver injury and skeletal-muscle injury. Treatment of HS-rats with artesunate (both 3 and 10 mg/kg) significantly attenuated the rises in serum AST (P<0.05, FIG. 3A), ALT (P<0.005, FIG. 3B) and creatinine kinase (P<0.05, FIG. 3C). Treatment with 1 mg/kg artesunate had no significant effect on the rises in serum AST (P>0.05, FIG. 3A), ALT (P>0.05, FIG. 3B) or creatinine kinase (P>0.05, FIG. 3C).

(33) 2. Evaluation of the Effects of Artesunate on the Organ Injury and Dysfunction Induced by Burn Injury in the Rat

(34) 2.1. Surgical Procedure

(35) Twenty-two male Wistar rats (Harlan, Udine, Italy) weighing 300 to 350 g were anaesthetised with sodium pentobarbital (Eutasil, 60 mg/kg i.p.; Sanofi Veterinria, Algs, Portugal), which was supplemented as required. Anaesthetised rats were shaved (dorsum and abdomen) and placed onto a thermostatically controlled heating mat (Harvard Apparatus Ltd, Kent, U.K.) and body temperature maintained at 371 C. by means of a rectal probe attached to a homeothermic blanket. A tracheotomy was performed to maintain airway patency and to facilitate spontaneous respiration. Thirty minutes prior to burn injury, rats were treated with vehicle or drug, as described in section 3.2. To induce burn injury, a 60% third degree skin burn was induced by immersing dorsal shaved skin in 99 C. water for 10 s using a synthetic foam template. The rats were then dried and placed over the heating mat. Rats were sacrificed at 6 hours after burn injury by overdose of the anaesthetic and serum samples obtained for analysis of organ injury and dysfunction.

(36) 2.2 Experimental Design

(37) Rats were randomly allocated into the following groups: (i) Sham (n=4) (ii) Burn+10% DMSO (n=10) (iii) Burn+Artesunate (n=9)

(38) Sham-operated rats underwent identical surgical procedures but without burn injury (immersed in room temperature water). Animals received either 10% DMSO (1 ml/kg i.v.) or artesunate (3 mg/kg i.v.) 30 min prior to burn injury and 30 min after burn injury

(39) 2.3 Materials

(40) Unless otherwise stated, all compounds were obtained from Sigma-Aldrich Qumica S.A. (Sintra, Portugal). Pentobarbital sodium (Eutasil) was obtained from Sanofi Veterinria (Miraflores, Algs, Portugal). All stock solutions were prepared in non-pyrogenic saline (0.9% NaCl; B. Braun Medical Lda, Queluz, Portugal).

(41) 2.4 Statistical Analysis

(42) Each data point represents measurements obtained from up to 10 separate animals. Data was assessed using Mann-Whitney U test.

(43) 2.5 Effect of Artesunate on the Renal Dysfunction Induced by Burn Injury

(44) When compared to sham-operated rats, rats subjected to burn injury and treated with vehicle developed significant increases in serum urea (P<0.05, FIG. 4A) and creatinine (P<0.05, FIG. 4B) indicating the development of renal dysfunction. Treatment of burn injury rats with 3 mg/kg artesunate significantly attenuated the rises in serum urea (P<0.05, FIG. 4A) and creatinine (P<0.05, FIG. 4B) when compared to burns injury rats.

(45) 2.6 Effect of Artesunate on the Hepatic Injury Induced by Burn Injury

(46) When compared to sham-operated rats, rats subjected to burn injury and treated with vehicle developed significant increases in serum aspartate aminotransferase, AST (P<0.05, FIG. 5A) and alanine aminotransferase, ALT (P<0.05, FIG. 5B) indicating the development of hepatic injury. Treatment of burn injury rats with 3 mg/kg artesunate had no significant effect on the rises in serum AST (P>0.05, FIG. 5A) and ALT (P>0.05, FIG. 5B) induced by burn injury.

(47) 2.7 Summary

(48) Treatment of rats subjected burn injury with vehicle resulted in significant renal dysfunction (as indicated by rises in serum urea and creatinine) and significant hepatic injury (as indicated by rises in serum AST and ALT).

(49) Treatment of rats subjected to burn injury with 3 mg/kg artesunate resulted in a significant reduction in the renal dysfunction (measured using serum urea and creatinine) caused by burn injury. However, treatment of rats subjected to burn injury with 3 mg/kg artesunate had no significant effect on the hepatic injury (measured using serum AST and ALT) caused by burn injury.

(50) 3.0 Effect of Time on Acute Kidney Injury and the Activation of Intracellular Proteins

(51) 3.1 Acute Kidney InjurySurgical Procedure and Quantification of Organ Injury/Dysfunction

(52) This study was carried out on 63 male Wistar rats (Charles River Ltd, Margate, UK) weighing between 240-290 g and receiving a standard diet and water ad libitum. Animals were anesthetized using a ketamine (150 mg/kg) and xylazine (15 mg/kg) mixture i.p. (1.5 ml/kg). The hair was shaved and the skin cleaned with 70% alcohol (v/v). The animals were then placed on a homoeothermic blanket set at 37 C. Animals received 0.1 mg/kg s.c. buprenorphine (0.1 ml/kg) prior to commencement of surgery. A mid-line laparotomy was then performed. The right renal pedicle (consisting of the renal artery, vein and nerve) was isolated and tied off using sterile 4-0 silk-braided suture (Pearsalls Ltd., Taunton, UK). The right kidney was then surgically removed. The left renal pedicle was isolated and clamped using a non-traumatic microvascular clamp at time 0. After 30 min of unilateral renal ischemia, the clamp was removed to allow reperfusion. For reperfusion, the kidneys were observed for a further 5 min to ensure reflow, following which 8 ml/kg saline at 37 C. was injected into the abdomen and all incisions were sutured in two layers (Ethicon Prolene 4-0). Animals were then allowed to recover on the homeothermic blanket and placed into cages upon recovery. Twenty-four hours prior to the end of the experiment, rats were placed in metabolic cages for the collection of urine and the subsequent determination of both estimated creatinine clearance and fractional excretion of sodium. At the end of the experiment, blood was taken by cardiac puncture into non-heparinized syringes and immediately decanted into 1.3 ml serum gel tubes (Sarstedt, Germany). The blood was centrifuged at 9900 g for 5 min to separate serum. All biochemical markers in serum and urine were measured in a blinded fashion by a commercial veterinary testing laboratory (IDEXX Ltd, West Sussex, UK). The left kidney was removed following removal of the heart. Half of the left kidney was snap frozen and stored at 80 C., and the other half was stored in 10% neutral buffered formalin.

(53) 3.2 Experimental Design (Time Course)

(54) Rats were randomly allocated into the following groups: (i) pre-ischemia (n=4); (ii) 24 h reperfusion (n=4); (iii) 48 h reperfusion (n=8); and (v) 72 h reperfusion (n=4).

(55) 3.3 Histological Evaluation and Scoring

(56) Kidneys were fixed in 10% neutral buffered formalin for 48 h before being dehydrated with 70% ethanol. Tissues were embedded in paraffin and sections were cut at 4 m by a single technician in order to minimize variations in section thickness. The slides were deparaffinised with xylene, stained with haematoxylins and eosin and viewed with a Keyence Biozero BZ-8000 microscope (Ontario, Canada). Histological features such as glomerular shrinkage, tubular dilatation, basophilia, necrosis and luminal congestion were noted. Ten random images were taken per slide and quantified for total tissue surface area using ImageJ as a marker of renal injury.

(57) 3.4 Western Blot Analysis

(58) Western blots were carried out as previously described[22]. Three separate experiments of western blot analysis were performed for each marker and tissues were done separately for each western blot experiment. Briefly, previously snap frozen rat kidney samples were homogenized and centrifuged at 4,000 g for 5 min at 4 C. Supernatants were removed and centrifuged at 15,000 g at 4 C. for 40 min to obtain the cytosolic fraction. The pelleted nuclei were re-suspended in extraction buffer. The suspensions were centrifuged at 15,000 g for 20 min at 4 C. The resulting supernatants containing nuclear proteins were carefully removed, and protein content was determined using a bicinchoninic acid (BCA) protein assay following the manufacturer's directions. Proteins were separated by 8% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinyldenedifluoride (PVDF) membrane, which was then incubated with a primary antibody (mouse anti-total IB dilution 1:1000; mouse anti-pIB Ser.sup.32/36 dilution 1:1000; rabbit anti-total eNOS dilution 1:200; goat anti-peNOS Ser.sup.1177 dilution 1:200; rabbit anti-NF-B p65 dilution 1:1000). Blots were then incubated with a secondary antibody conjugated with horseradish peroxidase (dilution 1:10000) and developed using the ECL detection system. The immunoreactive bands were visualized by autoradiography. The membranes were stripped and incubated with -actin monoclonal antibody (dilution 1:5000) and subsequently with an anti-mouse antibody (dilution 1:10000) to assess gel-loading homogeneity. Densitometric analysis of the bands was performed using Gel ProAnalyzer 4.5, 2000 software (Media Cybernetics, Silver Spring, Md., USA) and optical density analysis was expressed as fold-increase versus the sham group. In the sham group, the immunoreactive bands of the gel were respectively measured and normalized against the first immunoreactive band (standard sham sample) and the results of all the bands belonging to the same group were expressed as meanSEM. This provides SEM for the sham group where a value of 1 is relative to the first immunoreactive band. The membranes were stripped and incubated with -actin monoclonal antibody and subsequently with an anti-mouse antibody to assess gel-loading homogeneity. Relative band intensity was assessed and normalized against parallel -actin expression. Each group was then adjusted against corresponding Sham data to establish relative protein expression when compared to Sham animals.

(59) 3.5 Materials

(60) Unless otherwise stated, all compounds used in this study were purchased from Sigma-Aldrich Company Ltd. (Poole, Dorset, U.K.). All stock solutions were prepared using non-pyrogenic saline (0.9% [w/v] NaCl; Baxter Healthcare Ltd., Thetford, Norfolk, U.K.). Ringer's Lactate was purchased from Baxter Healthcare Ltd. Antibodies for western blot analysis were purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany).

(61) 3.6 Statistical Analysis

(62) All values described in the text and figures are expressed as meanstandard error of the mean (SEM) for n observations. Each data point represents biochemical measurements obtained from up to 11 separate animals. Statistical analysis was carried out using GraphPad Prism 6.0b (GraphPad Software, San Diego, Calif., USA). Data without repeated measurements was assessed by one-way ANOVA followed by Bonferroni's multiple-comparison post hoc test. A P value of less than 0.05 was considered to be significant.

(63) 3.7 Results

(64) When compared to baseline (pre-ischemia), rats that underwent 30 min of unilateral renal ischemia developed significant renal (as measured by rises in serum urea and creatinine), glomerular (as measured by a fall in estimated creatinine clearance) and tubular dysfunction (as measured by a rise in fractional excretion of sodium) at 24 h of reperfusion, followed by a progressive recovery of renal, glomerular and tubular function without intervention (FIGS. 6a, 6B, 6C, and 6D). When compared to baseline (pre-ischemia), rats that underwent 30 min of unilateral renal ischemia developed histological signs of significant renal injury (see methods) at 48 h of reperfusion (FIGS. 7A, 7B, 7C, 7D, and 7E). These findings indicate the development of acute kidney injury. When compared to baseline (pre-ischemia), rats that underwent 30 min of unilateral renal ischemia developed significant phosphorylation of Ser32/36 on IB and, hence, activation of the IKK complex, at 48 h of reperfusion (FIG. 8A). Subsequently, activation of the IKK complex resulted in significant nuclear translocation of the NF-B subunit p65 at 48 h of reperfusion (FIG. 8B). In addition, at 48 h of reperfusion the phosphorylation of Ser1177 on eNOS was significantly reduced when compared to baseline (FIG. 8C).

(65) 4.0 Effect of Late Administration of Artesunate in a Recovery Rat Model of Unilateral Renal Ischaemia/Reperfusion Injury

(66) 4.1 Surgical Procedure

(67) Thirty-one male Wistar rats (240-270 g, Charles River, Margate, U.K.) were used in this study. Rats were anaesthetised with a ketamine (100 mg/ml) and xylazine (20 mg/ml) mixture (2:1; 1.5 ml/kg, i.p.) and anaesthesia was maintained by supplementary injections (200 g/kg i.p.) of ketamine/xylazine. Buprenorphine was administered at a dose of 0.1 mg/kg s.c. (0.5 ml/kg). Rats were then, placed onto a thermostatically controlled heating mat (Harvard Apparatus Ltd., Kent, U.K.) set at 37 C. A midline laparotomy was then performed. The anatomical right kidney was removed following permanent ligation of the renal artery and vein. The left renal pedicle (consisting of the renal artery, vein and nerve) was isolated and clamped using a non-traumatic microvascular clamp at time 0 for 30 min. Body temperature was maintained at 351 C. during ischaemia by means of measuring rectal temperature with a thermometer. After 30 min of unilateral renal ischaemia, the clamp was removed to allow reperfusion for 48 h. After the renal clamp were removed, the kidney were observed for a further 5 min to ensure reflow after which 2 ml saline at 37 C. was injected into the abdomen and all incisions were sutured in two layers. Rats were then allowed to recover from anaesthesia on the homoeothermic blanket and placed back into cages. Twenty-four hours prior to sacrifice rats were individually placed into metabolic cages for the collection of urine. Rats were re-anaesthetised with sodium thiopentone (120 mg/kg i.p., LINK Pharmaceuticals Ltd., West Sussex, UK) at the end of 48 h reperfusion.

(68) 4.2 Sample Collection

(69) For the time-course experiments serial blood samples were collected from rats over the course of the reperfusion phase. Following the reperfusion period, 3.5 ml of blood was taken from the right ventricle of the heart via cardiac puncture into non-heparinised 5 ml syringes and immediately decanted into 1.3 ml serum gel tubes (Sarstedt, Germany). The blood was centrifuged at 9900 g for 3 min to separate serum, which was subsequently stored at 80 C. until analysis. The lungs, liver and kidney were excised of which one section of each organ was snap frozen in liquid nitrogen and stored at 80 C. and the other was placed in 10% formalin until analysis (if required). All biochemical markers in serum and urine were measured in a blinded fashion by a commercial veterinary testing laboratory (IDEXX Ltd, West Sussex, UK). Serum urea and creatinine are used as indicators of renal dysfunction. Creatinine clearance is used as an indicator of glomerular dysfunction and is calculated as follows

(70) $\underset{\left(\mathrm{ml}\text{/}\min \right)}{\mathrm{Creatinine}\mathrm{Clearance}}=\frac{\mathrm{urine}\mathrm{creatinine}\left(\mathrm{.Math.mol}\text{/}I\right)\mathrm{urine}\mathrm{flow}\left(\mathrm{ml}\text{/}\min \right)}{\mathrm{serum}\mathrm{creatinine}\left(\mathrm{.Math.mol}\text{/}I\right)}$

(71) Fractional excretion of sodium is used as an indicator of tubular dysfunction and is calculated as follows:

(72) $\underset{\mathrm{of}\mathrm{Sodium}\left(\%\right)}{\mathrm{Fractional}\mathrm{Excretion}}=\frac{\mathrm{urin}e\mathrm{sodium}\left(\mathrm{mmol}\text{/}I\right)\mathrm{serum}\mathrm{sodium}\left(\mathrm{mmol}\text{/}I\right)}{\mathrm{urine}\mathrm{creatinine}\left(\mathrm{.Math.mol}/I\right)\mathrm{serum}\mathrm{creatinine}\left(\mathrm{.Math.mol}\text{/}I\right)}$
4.3 Experimental Design

(73) Animals were randomised into seven experimental groups and treated with either sodium bicarbonate as a 1 ml/kg i.v. bolus 24 h into reperfusion or artesunate at 0.3 mg/kg as an i.v. bolus 24 h into reperfusion (see Tables 2 and 3, where n represents the number of animals studied).

(74) TABLE-US-00002 TABLE 2 Experimental Design for Characterisation of Acute Kidney Injury in a Rat Model of Unilateral Renal Ischaemia/Reperfusion Injury Protocol Group Treatment Ischaemia (min) Reperfusion (h) n A Baseline 4 B 24 h 30 24 4 C 48 h 30 48 8 D 72 h 30 72 4

(75) TABLE-US-00003 TABLE 3 Experimental Design for Treatment with Artesunate in a Rat Model of Unilateral Renal Ischaemia/Reperfusion Injury 24 h into reperfusion Bolus Dose Dose Group Treatment Conc. (mg/mL) (mg/kg) Volume (ml/kg) n E Sham + Sodium 1 8 Vehicle Bicarbonate F IRI + Sodium 1 8 Vehicle Bicarbonate G IRI + 0.3 0.3 1 7 Artesunate
4.4 Materials and Drug Preparation

(76) Sodium bicarbonate was purchased from Sigma-Aldrich Co Ltd and stored as 10 ml tubes at 20 C. Saline was obtained from Baxter Healthcare Ltd (UKF7124). Artesunate (Guilin Pharmaceuticals Co Ltd, China) was prepared prior to the start of the study in 3 mg/ml aliquots and stored at 20 C. for no longer than one month. On the day of injection one aliquot was removed from the freezer and diluted to the required concentration (see below).

(77) Artesunate 0.3 mg/kg: i.v. Bolus Dose 0.3 mg/kg

(78) Compound is administered in a volume of 1 ml/kg.

(79) Therefore, concentration of the compound assuming each rat weighs 250 g is:

(80) p.o. Bolus Concentration=0.3 mg/ml

(81) Concentration of stock=3 mg/ml in 1 ml aliquots.

(82) Dilution factor: 30.3=10

(83) Allow 1 ml per rat for a Bolus Dose, i.e. 100 l of 3 mg/ml artesunate stock required per 900 l of sodium bicarbonate.

(84) 4.5 Statistical Evaluation

(85) All data in the text and figures are presented as meanSEM of n observations, where n represents the number of animals studied. All statistical analysis was calculated using GraphPad Prism 6 (GraphPad Software, San Diego, Calif., USA). Biochemical data was analysed by one-way ANOVA for data representing more than two groups, followed by a Dunnett's post hoc test for comparison of sham/treated groups with the control vehicle. A P-value of less than 0.05 was considered to be statistically significant.

(86) 4.6 Effects of Time and Artesunate on the Renal Dysfunction Caused by Ischaemia and Reperfusion

(87) When compared to baseline (0 h of reperfusion), rats that underwent 30 min of unilateral renal ischaemia developed a peak of renal dysfunction at 24 h of reperfusion, followed by a progressive recovery of renal function without intervention (FIGS. 9A and 9B).

(88) When compared sham-operated rats, rats that underwent 30 min of unilateral renal ischaemia and 48 h of reperfusion exhibited a significant increase in serum urea from 5.750.28 to 34.132.23 mmol/L (P<0.05, FIG. 9C) and serum creatinine from 35.601.26 to 205.9022.23 mol/L (P<0.05, FIG. 9D). These findings indicate the development of acute kidney injury. Compared to rats subjected to ischaemia/reperfusion only, treatment with 0.3 mg/kg artesunate 24 h into reperfusion (the peak of dysfunction) significantly attenuated serum urea from 34.132.23 to 21.631.51 mmol/L (P<0.05, FIG. 9C) and serum creatinine from 205.9022.23 to 115.49.72 mol/L (P<0.05, FIG. 9D).

(89) 4.7 Effects of Time and Artesunate on the Glomerular Dysfunction Caused by Ischaemia and Reperfusion

(90) When compared to baseline (0 h of reperfusion), rats that underwent 30 min of unilateral renal ischaemia developed a peak of glomerular dysfunction at 24 h of reperfusion, followed by a progressive recovery of glomerular function without intervention (FIG. 10A).

(91) When compared sham-operated rats, rats that underwent 30 min of unilateral renal ischaemia and 48 h of reperfusion exhibited a significant increase in estimated creatinine clearance from 0.510.02 to 0.080.01 ml/min/100 g bw (P<0.05, FIG. 10B) These findings indicate the development of acute kidney injury. Compared to rats subjected to ischaemia/reperfusion only, treatment with 0.3 mg/kg artesunate 24 h into reperfusion (the peak of dysfunction) significantly attenuated estimated creatinine clearance from 0.080.01 to 0.140.02 ml/kg/100 g bw (P<0.05, FIG. 10B).

(92) 4.8 Effects of Time and Artesunate on the Tubular Dysfunction Caused by Ischaemia and Reperfusion

(93) When compared to baseline (0 h of reperfusion), rats that underwent 30 min of unilateral renal ischaemia developed a peak of tubular dysfunction at 24 h of reperfusion, followed by a progressive recovery of tubular function without intervention (FIG. 11A).

(94) When compared sham-operated rats, rats that underwent 30 min of unilateral renal ischaemia and 48 h of reperfusion exhibited a significant increase in fractional excretion of sodium from 0.590.07 to 4.450.65% (P<0.05, FIG. 11B) These findings indicate the development of acute kidney injury. Compared to rats subjected to ischaemia/reperfusion only, treatment with 0.3 mg/kg artesunate 24 h into reperfusion (the peak of dysfunction) significantly attenuated fractional excretion of sodium from 4.450.65 to 1.900.17% (P<0.05, FIG. 11B).

(95) 4.9 Summary and Conclusions

(96) Rats subjected to unilateral renal ischemia for 30 min, following right kidney nephrectomy, developed transient increases in renal dysfunction (as indicated by a rise in serum creatinine and urea), glomerular dysfunction (as indicated by a decline in estimated creatinine clearance) and tubular dysfunction (as indicated by a rise in fractional excretion of sodium). The peak of dysfunction occurred 24 h after the onset of reperfusion, which progressively declined and resulted in recovery of function 72 h after the onset of reperfusion.

(97) Treatment with artesunate at a dose of 0.3 mg/kg (i.v.) at the peak of renal, glomerular and tubular dysfunction (24 h after the onset of reperfusion) resulted within 24 h in a significant improvement in renal, glomerular and tubular function.