Risk Factors for Thromboembolic Events in Patients With Dialysis-Dependent CKD: Pooled Analysis of Phase 3 Roxadustat Trials in Japan.

INTRODUCTION: Thromboembolic events have occurred in clinical trials of roxadustat. This post hoc analysis explored potential factors related to thromboembolic events in dialysis-dependent patients treated with roxadustat in four phase 3 clinical trials in Japan. METHODS: Thromboembolic events with onset before and after week 12 were evaluated. Baseline risk factors for thromboembolic events were investigated by Cox regression analyses. Nested case-control analyses using conditional logistic models with matched pairs of case-control data explored relationships between thromboembolic events and laboratory parameters. RESULTS: Of the 444 patients, 56 thromboembolic events were observed in 44 patients during ≤ 52 weeks of treatment. The proportion of venous and arterial thromboembolic events gradually increased after week 12. Baseline risk factors included hemodialysis (vs peritoneal dialysis), advanced age (≥ 65 years), shorter dialysis vintage (< 4 months), and history of thromboembolism. The absence of concomitant intravenous or oral iron therapy (including ferric citrate) was associated with thromboembolic events before week 12 (hazard ratio 11.25; 95% confidence interval [CI] 3.36-37.71; vs presence). Case-control analysis revealed that low average transferrin saturation (< 10%; unadjusted odds ratio [OR] 6.25; 95% CI 1.52-25.62; vs ≥ 20%), high average transferrin level (≥ 2.5 g/L; unadjusted OR 4.36; 95% CI 1.23-15.39; vs < 2.0 g/L), and high average roxadustat dose (≥ 150 mg; unadjusted OR 5.95; 95% CI 1.07-33.16; vs < 50 mg) over the previous 8 weeks before the event onset were associated with thromboembolic events after week 12. However, adjustment for iron status extinguished the significant relationship between roxadustat dose and events. Multivariate case-control analysis showed that increased transferrin from baseline (≥ 1.0 g/L; adjusted OR 7.85; 95% CI 1.82-33.90; vs < 0.5 g/dL) and decreased mean corpuscular volume (< - 2 fL; adjusted OR 5.55; 95% CI 1.73-17.83; vs ≥ 0 fL) were associated with increased risk of thromboembolic events. CONCLUSION: In addition to established risk factors, iron deficiency may be related to thromboembolic events. Graphical Abstract available for this article. TRIAL REGISTRATION: NCT02780726, NCT02952092, NCT02780141, NCT02779764.

Roxadustat is an oral medicine that treats anemia in patients with chronic kidney disease (CKD). Thromboembolic events, or blood vessels blocked by a blood clot, have occurred in clinical trials of roxadustat. This study explored potential factors that may be related to thromboembolic events in roxadustat-treated patients with anemia of CKD on dialysis before and after week 12. This study found that hemodialysis (vs peritoneal dialysis), advanced age (older than 65 years), short amount of time on dialysis (less than 4 months), previous history of thromboembolic events, and not receiving iron therapy were risk factors for thromboembolic events before week 12. Iron deficiency and high roxadustat dose were risk factors for thromboembolic events after week 12. When iron status was also considered, we did not find that roxadustat dose was related to thromboembolic events. A different model found that increased levels of transferrin, a protein that transports iron, from baseline and decreased mean corpuscular volume, or smaller red blood cells, increased the risk of thromboembolic events. Patients with anemia of CKD on dialysis may benefit from more intentional monitoring and management of iron while receiving roxadustat.

Use of molidustat, a hypoxia-inducible factor prolyl hydroxylase inhibitor, in chronic kidney disease-associated anemia in cats.

BACKGROUND: Erythropoietic effects of molidustat, a hypoxia-inducible factor prolyl hydroxylase (HIF-PH) inhibitor, were previously demonstrated in healthy cats. OBJECTIVE: To evaluate the safety and erythropoietic effects of daily PO administration of molidustat in anemic cats with chronic kidney disease (CKD). ANIMALS: Twenty-one client-owned CKD cats (4-17 years old) with anemia. METHODS: Multicenter field study; randomized, masked, and placebo-controlled. Cats were treated PO once daily for 28 days with suspensions of control product (CP; n = 6) or 5 mg/kg of molidustat (n = 15). Hematocrit (HCT) was evaluated at weekly intervals. Individual cat treatment success was defined as a ≥4% point increase in HCT compared to baseline. RESULTS: Control group mean HCT remained low throughout the study (20.1%-23.4%). Mean HCT of molidustat-treated cats increased weekly, and a significant increase compared to baseline (23.6%) was first observed on Day 21 (27.3%; P < .001; 95% confidence interval [CI], 1.69-5.67). Compared to CP group, mean HCT was significantly higher on Day 21 (27.3% vs 20.1%; P < .001; 95% CI, 2.91-10.75) but not significantly higher on Day 28 (27.8% vs 23.4%; P = .06; 95% CI, -0.23 to 9.88). The number of individual treatment successes on Day 28 was higher among remaining molidustat-treated cats (7/14) compared to remaining control cats (1/5), but there was no significant difference between groups. CONCLUSIONS AND CLINICAL IMPORTANCE: Daily PO molidustat administration may stimulate a clinically relevant erythropoietic response in anemic cats with CKD. This HIF-PH inhibitor may be an alternative for managing anemia in cats compared to recombinant EPO treatment.

[Hypoxia-inducible factor-prolyl hydroxylase inhibitors: the "alternative" for EPO?] / Hypoxie-induceerbare-factor-prolylhydroxylaseremmers.

Hypoxia-inducible factor-prolyl hydroxylase inhibitors (HIF-PHI) are a new drug class for the treatment of renal anemia. HIF-PHI increase the expression of genes such as erythropoietin and genes involved in iron homeostasis. HIF-PHI were found to be superior to placebo in increasing hemoglobin levels and non-inferior to erythropoiesis stimulating agents (ESA). Furthermore, HIF-PHI appeared to positively influence iron parameters and also appeared to be effective in patients with elevated inflammatory values. The cardiovascular safety of HIF-PHI was found to be similar to ESA in most studies. However, a stronger risk of deep vein thrombosis and thrombosis of the shunt was found with treatment of HIF-PHI compared to ESA. HIF-PHI can be considered as an alternative to ESA, with the positive effect on iron homeostasis, the oral administration and the potential possibility to treat patients with ESA hyporesponsiveness as additional benefits, although effectiveness in this subgroup has yet to be demonstrated.

First evidence of the incorporation of daprodustat and other hypoxia-inducible factor stabilizers into equine hair by passive transfer based on segmental quantitative analysis.

Daprodustat is a hypoxia-inducible factor prolyl hydroxylase domain (HIF-PHD) inhibitor and is used as an erythropoiesis stimulant for the treatment of anemia in humans. In general, administering daprodustat to horses will result in a lifetime ban from both equestrian sports and horseracing by the International Federation of Horseracing Authorities and the Fédération Équestre Internationale, respectively. To control the misuse/abuse of daprodustat, we conducted nasoesophageal administration of daprodustat (100 mg/day for 3 days) to three thoroughbred mares and the post-administration hair samples collected from the three horses over 6 months were analyzed to demonstrate the potential longer-term detection of daprodustat and its metabolites in hair compared with the detection times of daprodustat of 1 and 2 weeks in plasma and urine respectively. The results of the quantitative 2-cm segmental analysis showed that daprodustat was primarily localized in the proximal region (0-2 cm) at 0.375-0.463 pg/mg at 1 month post-administration. These drug bands were gradually spread out along the hair shaft at a rate consistent with the reported growth rate of horse mane hair (approximately 2.5 cm/month) over the following 6 months. In addition, to attain deeper insight into the mechanism of drug incorporation into hair, a total of 11 relevant parameters, including the actual PK parameters and simulated physicochemical and biopharmaceutical parameters for three HIF stabilizers (i.e., daprodustat, vadadustat, and IOX4), were investigated after normalization of the z-scores of all these parameters. Multiple regression analysis indicated that the major factors contributing to the incorporation of the three drugs into hair were their maximum plasma concentrations and lipophilicities, strongly suggesting that the three HIF stabilizers permeated from the bloodstream into the hair bulb via passive transfer with concentration gradients. This work is the first reported evidence showing the incorporation of HIF stabilizers into hair via passive transfer. In addition, cross-species comparison of drug incorporations into hair between daprodustat in horse and roxadustat in human was made in order to have a better understanding of the interactive interpretations about the analysis results obtained from different species. The above findings are not only useful and beneficial for the purpose of doping control but also provide a better understanding of the mechanism of drug incorporation into horse hair.

Effects of hypoxia-inducible factor-prolyl hydroxylase inhibitors vs. erythropoiesis-stimulating agents on iron metabolism in non-dialysis-dependent anemic patients with CKD: A network meta-analysis.

Objective: To compare the effects of five hypoxia-inducible factor-prolyl hydroxylase domain inhibitors (HIF-PHIs), two erythropoiesis-stimulating agents (ESAs), and placebo on iron metabolism in renal anemia patients with non-dialysis-dependent chronic kidney disease (NDD-CKD). Method: Five electronic databases were searched for studies. Randomized controlled clinical trials comparing HIF-PHIs, ESAs, and placebo in NDD-CKD patients were selected. The statistical program used for network meta-analysis was Stata/SE 15.1. The main outcomes were the change in hepcidin and hemoglobin (Hb) levels. The merits of intervention measures were predicted by the surface under the cumulative ranking curve method. Results: Of 1,589 original titles screened, data were extracted from 15 trials (3,228 participants). All HIF-PHIs and ESAs showed greater Hb level-raising ability than placebo. Among them, desidustat demonstrated the highest probability of increasing Hb (95.6%). Hepcidin [mean deviation (MD) = -43.42, 95%CI: -47.08 to -39.76], ferritin (MD= -48.56, 95%CI: -55.21 to -41.96), and transferrin saturation (MD = -4.73, 95%CI: -5.52 to -3.94) were decreased, while transferrin (MD = 0.09, 95%CI: 0.01 to 0.18) and total iron-binding capacity (MD = 6.34, 95%CI: 5.71 to 6.96) was increased in HIF-PHIs versus those in ESAs. In addition, this study observed heterogeneity in the ability of HIF-PHIs to decrease hepcidin. Compared with darbepoetin, only daprodustat (MD = -49.09, 95% CI: -98.13 to -0.05) could significantly reduce hepcidin levels. Meanwhile, daprodustat also showed the highest hepcidin-lowering efficacy (84.0%), while placebo was the lowest (8.2%). Conclusion: For NDD-CKD patients, HIF-PHIs could ameliorate functional iron deficiency by promoting iron transport and utilization, which may be achieved by decreasing hepcidin levels. Interestingly, HIF-PHIs had heterogeneous effects on iron metabolism. Systematic review registration: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=242777, Identifier CRD42021242777.

Correlation between blood concentration of roxadustat and clinical efficacy in patients with anemia of chronic kidney disease.

Roxadustat has been associated with the efficacy and safety in patients with chronic kidney disease-related anemia. However, the relationship between roxadustat blood concentration and clinical efficacy is lacking. To explore of the correlation between clinical efficacy and blood concentration of roxadustat in patients with renal anemia of chronic kidney diseases, so as to provide reference for rational clinical drug use. A total of 46 patients were selected with a diagnosis of renal anemia who were prescribed roxadustat at the department of nephrology of the First Hospital of Hebei Medical University from December 2019 to March 2020. The roxadustat blood concentration was determined at 12 weeks of treatment, according to the cumulative response rate, patients were divided into the response group and the nonresponse group, and the relationship between roxadustat blood concentration and treatment effect was analyzed. We also explored the correlation between various factors and the blood concentration. The patients in the response group had higher roxadustat blood concentrations than the nonresponse group (P < .05), and there was no correlation between blood concentration and clinical characteristics such as age, gender, and dosage (P > .05). The blood concentration of roxadustat was correlated with clinical efficacy. The higher the blood concentration, the better the clinical efficacy, meaning it might be a predictor of efficacy.

Roxadustat: Do we know all the answers?

Anemia is a common complication of chronic kidney disease (CKD), and its prevalence rises as the disease progresses. Intravenous or subcutaneous erythropoiesis-stimulating agents (ESAs) are advised to treat CKD-associated anemia, since shortage of erythropoietin (EPO) and iron are the main cause of anemia. However, ESA resistance and safety have spurred a lot of interest in the development of alternate anemia therapies. Roxadustat, an orally administered hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI) that increases erythropoiesis and may modulate iron metabolism, was recently licensed in China, Chile, South Korea, Japan and the European Union for the treatment of CKD-related anemia. Despite this, clinical trials have shown a number of adverse effects, including cardiovascular disease, hyperkalemia, and infections. In addition, of concern is roxadustat's possible effects on other organs and systems. In this review, based on clinical evidence, we discuss the potentially detrimental effects of roxadustat to the known biology on systems other than kidney, and the need for long-term follow-up in order for roxadustat to be approved in more countries in the future.

Future perspectives of anemia management in chronic kidney disease using hypoxia-inducible factor-prolyl hydroxylase inhibitors.

For the past 3 decades, erythropoiesis-stimulating agents (ESA) in conjunction with iron supplementation has been the mainstay of treatment for anemia in chronic kidney disease (CKD). Although ESAs are well-established and highly efficacious treatment, clinical trials demonstrated that the use of ESAs with a high hemoglobin (Hb) target was associated with increased risk of cardiovascular events. This safety concern raised considerable interest in developing an alternative therapeutic strategy. Hypoxia-inducible factor-prolyl hydroxylase inhibitors (HIF-PHIs) are such novel agents to treat anemia in CKD. They stimulate endogenous erythropoietin production via HIF activation and thereby induce erythropoiesis. At least 6 small-molecule HIF-PHIs have been developed to date. The phase 3 clinical trials demonstrated that their effects were noninferior to ESAs. HIF-PHIs may have several advantages over the conventional treatment, such as oral route of administration and their ability to raise Hb levels in patients with chronic inflammation. Although many of the phase 3 clinical trials demonstrated that HIF-PHIs were noninferior to placebo or ESAs with respect to cardiovascular safety, one of the compounds failed to meet the prespecified noninferiority criterion in non-dialysis-dependent CKD patients, and some studies of another HIF-PHI indicated potential risks for thromboembolic events. While the regulatory agencies of some countries including Japan and the European Union concluded that roxadustat, one of the HIF-PHIs, had a favorable benefit-risk profile, the U.S. Food and Drug Administration decided not to approve the drug because of safety reasons. In order to establish the optimal anemia management in CKD, further studies are needed to evaluate important aspects of HIF-PHIs, such as long-term safety, appropriate Hb target, and the types of patients who would gain benefits from these new drugs.

Treatment of Renal Anemia in Patients With Hemodialysis Using Hypoxia-inducible Factor (HIF) Stabilizer, Roxadustat: A Short-term Clinical Study.

BACKGROUND/AIM: Renal anemia is a major complication in patients with chronic kidney disease (CKD) and hemodialysis, increasing morbidity and mortality. Roxadustat is a novel oral hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor (PHI), which is administrated for renal anemia. Different from erythropoiesis-stimulating agents (ESAs), Roxadustat could increase erythropoietin physiologically, improving the therapeutic effects. It has not been so long since Roxadustat was approved by the European Commission (EC). Thus, only a few studies have reported on the treatment of renal anemia using Roxadustat. PATIENTS AND METHODS: In this study, we evaluated the efficacy of Roxadustat in patients undergoing hemodialysis (HD). Nine patients under HD (72±10 years old) were enrolled in this study. Patients received Roxadustat first time or changed from ESAs (5-10 mg, 3 times a week after HD). Observation period was 5.3±2.9 months. RESULTS: Roxadustat treatment effectively increased and maintained hemoglobin levels. Levels of ferritin and C-reactive protein tended to decrease, but the difference was not statistically significant. No significant adverse effects were observed in all patients during the study. CONCLUSION: Roxadustat is effective and relatively tolerant for treating renal anemia in patients subjected to hemodialysis.

Are there advantages of daprodustat over erythropoiesis-stimulating agents (ESAs) in treating anemia associated with chronic kidney disease (CKD)?

Anemia is common in CKD and increases the risk of developing heart disease. Although ESAs relieve the symptoms of anemia, they have adverse effects and do not reduce the adverse outcomes associated with anemia. This evaluation is of the phase 3 ASCEND clinical trials of the hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor daprodustat versus ESAs in subjects with CKD undergoing dialysis or not. Daprodustat was non-inferior to ESAs in increasing hemoglobin, and in the incidence of cardiovascular events and adverse effects. Daprodustat is effective in subjects who are hyporesponsive to ESAs, and this is one circumstance when daprodustat may be preferred to ESAs. However, to become a widely used medicine in subjects with CKD responsive to ESAs, daprodustat needs to be well tolerated, used by a high percentage of subjects over a long time, and be superior to ESAs in improving clinical outcomes. As this may not be the case, there is not a strong basis for recommending daprodustat over ESAs. The other 'dustats' (roxadustat, vadadustat) have also not been shown to be superior to the ESAs, and none have been approved by the FDA to date.

Therapeutic Effect of Roxadustat on Patients With Posttransplant Anemia.

BACKGROUND: Roxadustat, a hypoxia-inducible factor prolyl hydroxylase inhibitor, has been reported to be effective in treating conservative renal failure and renal anemia in patients undergoing dialysis. Nonetheless, its effect on posttransplant anemia (PTA) has not yet been analyzed. METHODS: This study was conducted in accordance with the 1975 Declaration of Helsinki, as revised in 2013. Roxadustat was administered in 31 patients with a hemoglobin level ≤11 g/dL after renal transplant. The mean hemoglobin, serum iron, ferritin, and low-density lipoprotein (LDL) cholesterol levels and the estimated glomerular filtration rate at 4, 8, 12, 16, and 20 weeks after administration were compared with those before administration. RESULTS: The average (standard deviation) hemoglobin level in 25 patients (6 patients dropped out) increased from 9.8 (0.78) g/dL before administration to 12.1 (1.44) g/dL (P < .001) after 12 weeks of roxadustat administration. The mean ferritin level in patients decreased from 107.6 (84.95) ng/mL before administration to 51.7 (44.04) ng/mL (P = .022) after 8 weeks of roxadustat administration. The mean LDL cholesterol level decreased from 114.1 (31.67) mg/dL before administration to 78.7 (18.26) mg/dL (P = .0012) after 8 weeks of roxadustat administration. Complications observed in patients after roxadustat administration included reduced hemoglobin levels in 3 patients, gastrointestinal symptoms in 2 patients, and myocardial infarction in 1 patient. CONCLUSIONS: Hemoglobin levels significantly increased, whereas ferritin and LDL cholesterol levels significantly decreased in patients with PTA after roxadustat administration. Roxadustat seems to be an effective treatment for patients with PTA; however, the blood clotting tendency due to iron deficiency should be monitored in patients.

Roxadustat prevents Ang II hypertension by targeting angiotensin receptors and eNOS.

The prevalence of hypertension is increasing globally, while strategies for prevention and treatment of hypertension remain limited. FG-4592 (Roxadustat) is a potentially novel, orally active small-molecule hypoxia-inducible factor (HIF) stabilizer and is being used clinically to treat chronic kidney disease (CKD) anemia. In the present study, we evaluate the effects of FG-4592 on hypertension. In an angiotensin II (Ang II) hypertension model, FG-4592 abolished hypertensive responses; prevented vascular thickening, cardiac hypertrophy, and kidney injury; downregulated AGTR1 expression; and enhanced AGTR2, endothelial NO synthase (eNOS), and HIF1α protein levels in the aortas of mice. Additionally, the levels of thiobarbituric acid reactive substances (TBARs) in blood and urine were diminished by FG-4592 treatment. In vascular smooth muscle cells, FG-4592 treatment reduced angiotensin receptor type 1 (AGTR1) and increased AGTR2 levels, while preventing Ang II-induced oxidative stress. In vascular endothelial cells, FG-4592 upregulated total and phosphorylated eNOS. Moreover, FG-4592 treatment was hypotensive in L-NAME-induced hypertension. In summary, FG-4592 treatment remarkably ameliorated hypertension and organ injury, possibly through stabilizing HIF1α and subsequently targeting eNOS, AGTR1, AGTR2, and oxidative stress. Therefore, in addition to its role in treating CKD anemia, FG-4592 could be explored as a treatment for hypertension associated with high renin angiotensin system (RAS) activity or eNOS defects.

Hypoxia-Inducible Factor-Prolyl Hydroxylase Domain Inhibitors to Treat Anemia in Chronic Kidney Disease.

BACKGROUND: Hypoxia-inducible factor (HIF) stabilizers, also known as inhibitors of HIF prolyl hydroxylase domain (PHD) inhibitors enzymes, are novel small-molecule agents to treat renal anemia. They increase endogenous erythropoietin (EPO) production by stabilizing HIF. This review focuses on the mechanisms by which PHD inhibitors ameliorate anemia in chronic kidney disease (CKD) and summarizes the current clinical experience with and prospects for these drugs. SUMMARY: Anemia is a serious complication of CKD and is an independent risk factor for congestive heart failure. Appropriate treatment of anemia is important in the management of advanced stage CKD, as it might help to extend life expectancy and improve the physical function of patients with CKD. However, at present, adverse effects of treatment, such as thromboembolic events, as well as high therapeutic cost have a negative impact on society. PHD inhibitors stabilize the transcription factor HIF, increasing the expression of downstream target genes, including EPO and enzymes involved in iron metabolism, resulting in increased EPO production and improved iron utilization. Key Messages: The potential advantages of PHD inhibitors over conventional EPO-based therapies include a more physiologic response to renal anemia, noninvasive oral administration, and lower cost. Phase III trials of more than 5 PHD inhibitors are ongoing, with overall demonstration of success in increasing hemoglobin levels. In this review, we focus on the mechanisms of PHD inhibitors in improving renal anemia in CKD and summarize the current clinical findings regarding these drugs.

Anemia in conventional hemodialysis: Finding the optimal treatment balance.

Renal anemia is a serious and common complication in hemodialysis (HD) patients. The introduction of erythropoiesis-stimulating agents (ESAs) has dramatically improved hemoglobin levels and outcomes. Several interventional studies reported that excessive correction of anemia and the massive use of ESA can trigger cardiovascular disease (CVD), and consequently may worsen the prognosis of patients undergoing HD. Therefore, it has been widely recognized that large doses of ESA should be used with caution. An effective use of iron preparations is required to yield the optimal effect of ESA. It is well-known that iron utilization is inhibited under pathological conditions, such as chronic inflammation, resulting in ESA resistance. It is postulated that a new class of therapeutic agents for renal anemia, hypoxia inducible factor prolyl hydroxylase (HIF-PH) inhibitors, will have beneficial treatment effects in patients on HD. HIF is induced by hypoxia and promotes erythropoietin production. In the absence of a hypoxic state, HIF is decomposed by the HIF catabolic enzyme. HIF-PH inhibitors inhibit this degrading enzyme and stimulate endogenous erythropoietin production via HIF induction. Additionally, HIF-PH inhibitors promote effective utilization of iron and raise erythropoietin to physiological concentrations. Accordingly, HIF-PH inhibitors improve anemia and iron metabolism. It appears that this effect persists irrespective of chronic inflammatory conditions. HIF-PH inhibitors do not overshoot erythropoietin above physiological concentrations like ESAs. Therefore, it is hypothesized that HIF-PH inhibitors would not increase the risk of CVD in patients undergoing HD.

HIF prolyl hydroxylase inhibitors for the treatment of renal anaemia and beyond.

Small-molecule stabilizers of hypoxia inducible factor (HIF) are being developed for the treatment of renal anaemia. These molecules inhibit prolyl hydroxylase domain-containing (PHD) enzymes, resulting in HIF activation and increased production of erythropoietin. Currently, renal anaemia is treated with recombinant human erythropoietin or related analogues, referred to as conventional erythropoiesis stimulating agents (ESAs). Advantages of PHD enzyme inhibitors over conventional ESAs include their oral administration and their simpler - and potentially cheaper - production. Importantly, inhibition of PHD enzymes is likely to have a range of consequences other than increasing levels of erythropoietin, and these effects could be beneficial - for instance by reducing the need for parenteral iron - but might in some instances be harmful. Several companies are currently testing PHD enzyme inhibitors in patients with renal anaemia and have reported clear evidence of efficacy without serious safety concerns. A central question that current studies are beginning to address is whether using PHD enzyme inhibitors will influence hard end points, including mortality and the rate of cardiovascular events. In terms of approaches to therapy, the exquisite specificity of conventional ESAs is a striking contrast to the pleiotropic effects of activating HIF. Excitingly, PHD inhibitors could also be useful for conditions besides renal anaemia, such as protection from ischaemic injury.