Inorganic nitrate or nitrite for preventing contrast-induced nephropathy (CIN)

Abstract

The present invention provides inorganic nitrate for use in a method of preventing contrast-induced nephropathy (CIN) in a patient receiving contrast agent, said method comprising intravenously administering to said patient an inorganic nitrate at a dosage which results in a circulating concentration of nitrate in the patient of 300-400 umol / L up to 12 hours after administration, wherein the inorganic nitrate is administered concomitantly with said contrast agent. The present invention also provides inorganic nitrite for use in a method of preventing CIN in a patient receiving contrast agent, said method comprising intravenously administering to said patient an inorganic nitrite at a dosage which results in a circulating concentration of nitrite in the patient of 0.7-2.0 umol / L up to 6 hours after administration, wherein the inorganic nitrite is administered concomitantly with said contrast agent.

Description

[0001] USE FIELD OF THE INVENTION The present invention relates to the use of inorganic nitrate in a method of preventing contrast-induced nephropathy (CIN) in a patient receiving contrast agent. The present invention also relates to the use of inorganic nitrite in a method of preventing contrast-induced nephropathy (CIN) in a patient receiving contrast agent. BACKGROUND TO THE INVENTIONContrast induced nephropathy (CIN), also termed contrast-associated kidney injury, refers to a form ofacute kidney injury (AKI) that occurs after parenteral administration of radiopaque contrast agent. It is the 3rdmost common cause of hospital-acquired renal failure, after decreased renal perfusion and use of nephrotoxic medications and remains a major unmet clinical need. CIN is associated with a prolonged hospital stay, higher hospitalization costs, and a significant increase in morbidity and mortality. CIN is commonly seen following procedures for acute coronary syndromes (ACS) with rates 3-5 times higher compared to elective procedures, with reported incidence ranging from 5-55%, dependent upon the criteria used for diagnosis, the clinical setting and the investigated population. Additionally, emerging evidence suggests that 15–20% of patients who do not fulfill the current serum-creatinine-based consensus criteria for AKI are nevertheless likely to have acute tubular damage (that can be identified by raised serum biomarkers of renal injury including neutrophil gelatinase-associated lipocalin (NGAL), cystatin-C, and interleukin-18 (IL-18)) and may also benefit from therapies targeting CIN. Whilst the pathophysiology of CIN is not yet fully understood, an important mechanism thought to underlie the condition is the production of reactive oxygen species (ROS) and consequent vasoconstrictive renal hypoxic injury. One of the likely mechanisms of this is decreased levels of nitric oxide (NO) as a consequence of NO synthase dysfunction and NO scavenging (Devrim E, Cetin M, Namuslu M, Erguder IB, Cetin R, Durak I. Indian J Med Res.2009;130(4):433-436; Corwin S, Reiffel JA. Arch Intern Med.1985;145(3):538-543. doi:10.1001 / archinte.145.3.538). Limited data exists assessing the effect of NO on CIN, especially in patients undergoing percutaneous coronary intervention (PCI). In one small study (see Peguero JG, et al. J Cardiovasc Pharmacol Ther. 2014;19(3):310-314. doi:10.1177 / 1074248413515077), post-procedural renal function was compared between 112 patients who received an organic nitrate prior to PCI and 87 who did not. The authors concluded that the use of intravenous nitroglycerin infusion may be associated with a decreased incidence of CIN. In the NITRITE-AMI study (Jones DA, et al. Circ Res. 2015;116(3):437-447. doi:10.1161 / CIRCRESAHA.116.305082) the authors showed that a single bolus of intracoronary inorganic nitrite delivered as NaNO2 given during primary PCI for ST-elevation MI led to a significant rise in circulating NO2- levels. Additionally, during the first 48 hours after reperfusion, 5 patients (12.5%) in the control group developed CIN, which contrasted with only 1 patient (2.5%) in the NO2- treated group. Whilst these studies are encouraging there are limitations with either approach. It is well accepted that continuous / repeated administration of an organic nitrate is of limited benefit due to the development of tolerance (Münzel T, Sayegh H, Freeman BA, Tarpey MM, Harrison DG. J Clin Invest.1995;95(1):187- 194. doi:10.1172 / JCI117637) and that raising circulating levels of inorganic NO2- with a NO2- (nitrite) salt is impacted by delivery method due to its half short-life (i.e. needs to be administered parenterally) and issues of toxicity that occur due to the need to deliver very high doses to achieve circulating concentrations with activity (Pluta RM, Dejam A, Grimes G, Gladwin MT, Oldfield EH. JAMA. 2005;293(12):1477-1484. doi:10.1001 / jama.293.12.1477). In summary, contrast-induced nephropathy (CIN), an acute kidney injury resulting from the administration of intravascular iodinated contrast media, is a significant cause of morbidity / mortality following coronary angiographic procedures in high-risk patients. Despite preventative measures intended to mitigate the risk of CIN, there remains a need for novel effective treatments. SUMMARY OF THE INVENTION The present inventors have surprisingly found that certain circulating levels of nitrate and nitrite achieved by orally administering inorganic nitrate in the form of a nitrate salt, are effective in preventing CIN in patients undergoing invasive coronary procedures that involve the administration of a contrast agent. In particular, it has been found that the administration of inorganic nitrate has a surprising long-term effect on reducing major adverse kidney events (MAKE) and major adverse cardiac events (MACE) in such patients. Similar circulating levels of nitrate and nitrite can be achieved by intravenously administering inorganic nitrate or inorganic nitrite concomitantly with contrast agent. Accordingly, in a first aspect the present invention provides inorganic nitrate for use in a method of preventing contrast-induced nephropathy (CIN) in a patient receiving contrast agent, said method comprising intravenously administering to said patient an inorganic nitrate at a dosage which results in a circulating concentration of nitrate in the patient of 300-400 ^mol / L up to 12 hours after administration, wherein the inorganic nitrate is administered concomitantly with said contrast agent. In a second aspect the present invention provides inorganic nitrite for use in a method of preventing contrast-induced nephropathy (CIN) in a patient receiving contrast agent, said method comprising intravenously administering to said patient an inorganic nitrite at a dosage which results in a circulating concentration of nitrite in the patient of 0.7-2.0 ^mol / L up to 6 hours after administration, wherein the inorganic nitrite is administered concomitantly with said contrast agent. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to inorganic nitrate (NO3-) and inorganic nitrite (NO2-) for use in a method of preventing contrast-induced nephropathy (CIN) in a patient receiving contrast agent. UK Patent Application No. 2301355.0 filed on 31 January 2023 (PCT Application No. PCT / EP2024 / 052425 published as WO 2024 / 160924) is incorporated by reference in its entirety herein. Contrast-induced nephropathy (CIN), also termed contrast-associated kidney injury, is an acute kidney injury resulting from the administration of contrast media. By “contrast agent”, “contrast medium” or “contrast media” is meant a substance that is used to increase the contrast of structures or fluids within the body during medical imaging e.g. e.g. CT scans, X-ray fluoroscopy, MRI scans. For the purposes of the present invention, the contrast agent is typically iodinated. The contrast agent may be a radionuclide agent. The contrast agent may be a radiocontrast agent (or radiopaque contrast agent), i.e. one suitable for use in radiographic techniques using X-rays. Radiocontrast agents include iodine (typically used for intravenous administration) and barium sulphate (typically used for imaging of the digestive system and administered directly into the gastrointestinal tract). By “receiving contrast agent” is meant that a contrast agent is administered to a patient. This is typically done intravenously. The contrast agent is typically administered in the form of a solution and is also referred to herein as “contrast solution”. By “concomitantly” is meant at the same time, for example when the inorganic nitrate or inorganic nitrite is present in the contrast medium, or at approximately the same time, for example when the inorganic nitrate or inorganic nitrite is administered as a separate infusion, when the two solutions are administered as close together in time as practically possible, for example within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes of each other. By “circulating concentration” is meant the concentration of a particular compound or agent (in the present invention, nitrite or nitrate) in the blood, more particularly in the plasma. Circulating concentration can be measured by taking blood samples from a patient and determining the level of a particular compound or agent in the blood or plasma. This can be done using any suitable method, for example liquid phase ozone chemiluminescence (as described, for example, in Kapil et al. Hypertension 2015; 65: 320-327). By “preventing contrast-induced nephropathy (CIN)” is meant at least partially avoiding the incidence of CIN. “Preventing” in the context of the present invention means either that CIN is completely prevented or the incidence of CIN is lower than would otherwise have been the case (without the use of inorganic nitrate). The patient is typically a human patient, but the present invention also finds use in the veterinary field. The patient may therefore be a mammalian patient, such as a cat, dog or other companion animal. The present invention finds use in patients having any medical condition that requires them to undergo a medical procedure that involves administration of a contrast agent. For example, the invention finds use in patients undergoing coronary angiography or percutaneous coronary intervention (PCI). Coronary angiography or arteriography is an invasive diagnostic procedure that usually involves taking X-rays of the coronary arteries. During the procedure, a catheter is inserted into a blood vessel, typically in the groin or arm, and using X-ray images of the guide, the tip of the catheter is passed up to the heart and coronary arteries. A contrast agent is injected through the catheter and X-ray images (angiograms) are taken. Percutaneous coronary intervention (PCI) refers to a family of minimally invasive procedures used to open clogged coronary arteries (those that deliver blood to the heart). PCI may involvestent / scaffold / device implantation. PCI was formerly known as angioplasty with stent and is typically anon-surgical procedure that uses a catheter to place a stent in blood vessels in order to open them up. By restoring blood flow, the treatment can improve symptoms of blocked arteries, such as chest pain or shortness of breath. As another example, the present invention finds use in patients undergoing a computerised tomography (CT) scan. A CT scan combines a series of X-ray images taken from different angles around the body and uses a computer to create detailed cross-sectional images of the inside of the body (for example the blood vessels, bones and soft tissue). CT scans are used to diagnose and monitor response to treatment in many conditions including orthopaedics and cancer. In a first aspect the present invention provides inorganic nitrate for use in a method of preventing contrast-induced nephropathy (CIN) in a patient receiving contrast agent, said method comprising intravenously administering to said patient an inorganic nitrate at a dosage which results in a circulating concentration of nitrate in the patient of 300-400 ^mol / L up to 12 hours after administration, wherein the inorganic nitrate is administered concomitantly with said contrast agent. Inorganic nitrate is used in the first aspect of the present invention. Inorganic nitrate is a polyatomic ion naturally found in a range of foods such as beetroot and green leafy vegetables and is also added in the form of nitrate salts (such as potassium nitrate and sodium nitrate) as a preservative to processed meat products such as ham and bacon. In the first aspect of the invention, an inorganic nitrate is intravenously administered to said patient concomitantly with said contrast agent, at a dosage which results in a circulating concentration of nitrate in the patient of 300-400 ^mol / L up to 12 hours after administration. The dosage of inorganic nitrate results in a circulating concentration of nitrate in the patient of 300-400 ^mol / L, for example 310-390 ^mol / L, 320-380 ^mol / L, 325-375 ^mol / L, 330-370 ^mol / L, 335-365 ^mol / L, 340-360 ^mol / L or 350-355 ^mol / L, for example approximately 300 ^mol / L, 325 ^mol / L, 350 ^mol / L, 375 ^mol / L or 400 ^mol / L,. This circulating concentration of nitrate will be achieved immediately after intravenous administration of the inorganic nitrate. The half-life of nitrate is 6-8 hours and so the circulating concentration of nitrate in the patient of 300-400 ^mol / L is achieved up to 12 hours (from 0-12 hours) after administration, for example from 0.5-11.5 hours, 1-11 hours, 2-10 hours, 3-9 hours, 4-8 hours or 5-6 hours after administration. The inorganic nitrate may be administered in the form of a nitrate salt, for example potassium nitrate (KNO3), sodium nitrate (NaNO3), calcium nitrate (Ca(NO3)2), zinc nitrate (Zn(NO3)2) or ammonium nitrate (NH4NO3). The inorganic nitrate is typically administered in the form of a nitrate salt solution. In a second aspect the invention provides inorganic nitrite for use in a method of preventing contrast- induced nephropathy (CIN) in a patient receiving contrast agent, said method comprising intravenously administering to said patient an inorganic nitrite at a dosage which results in a circulating concentration of nitrite in the patient of 0.7-2.0 ^mol / L up to 6 hours after administration, wherein the inorganic nitrite is administered concomitantly with said contrast agent. Inorganic nitrite is used in the second aspect of the present invention. In the second aspect of the invention, an inorganic nitrite is intravenously administered to said patient concomitantly with said contrast agent, at a dosage which results in a circulating concentration of nitrite in the patient of 0.7-2.0 ^mol / L up to 6 hours after administration. The dosage of inorganic nitrite results in a circulating concentration of nitrite in the patient of 0.7-2.0 ^mol / L, for example 0.8-1.9 ^mol / L, 0.9- 1.8 ^mol / L, 1.0-1.7 ^mol / L, 1.1-1.6 ^mol / L, 1.2-1.5 ^mol / L or 1.3-1.4 ^mol / L approximately 0.7^^mol / L, 0.8 ^mol / L, 0.9 ^mol / L, 1.0 ^mol / L, 1.1 ^mol / L, 1.2 ^mol / L, 1.3 ^mol / L, 1.4 ^mol / L 1.5 ^mol / L, 1.6 ^mol / L, 1.7 ^mol / L, 18. ^mol / L, 1.9 ^mol / L or 2.0 ^mol / L. This circulating concentration of nitrite will be achieved immediately after intravenous administration of the inorganic nitrite. The half-life of nitrite is 30 minutes but due to recycling of nitrate it will be in circulation for longer and so the circulating concentration of nitrite in the patient of 0.7-2.0 ^mol / L is achieved up to 6 hours (from 0-6 hours) after administration, for example from 0.1-6 hours, 0.5-5.5 hours, 1-5 hours, 1.5-4.5 hours, 2-4 hours, 2.5-3.5 hours or 3 hours after administration. The inorganic nitrite may be administered in the form of a nitrite salt, for example potassium nitrite (KNO2), sodium nitrite (NaNO2), calcium nitrite (Ca(NO2)2), zinc nitrite (Zn(NO2)2) or ammonium nitrite (NH4NO2). The inorganic nitrate is typically administered in the form of a nitrite salt solution. The inorganic nitrate or inorganic nitrite for use in the first and second aspects of the invention may be present in the contrast solution or administered as a separate infusion. The inorganic nitrate or inorganic nitrite may be administered in the form of a preparation or pharmaceutical composition, including any pharmaceutically acceptable excipient, diluent or binder that is suitable for intravenous administration. The inorganic nitrate or inorganic nitrite may be administered in combination with an antioxidant. The patient receiving contrast agent is typically suspected of suffering from one or more medical conditions in which contrast is used to enhance anatomical and functional imaging e.g. CT scans, X-ray fluoroscopy, MRI scans. For example, the patient may have angina, for example stable (chronic) angina. Angina is chest pain caused by reduced blood flow to the heart muscles. Stable angina is more common and less serious than unstable angina. Attacks typically have a trigger, for example stress or exercise, and usually stop within a few minutes of resting. Alternatively, the patient may have an acute coronary syndrome (ACS). Acute coronary syndromes include a range of conditions associated with sudden, reduced blood flow to the heart, for example unstable angina or myocardial infarction. Unstable angina is less common and more serious than stable angina. Attacks are more unpredictable and represent a medical emergency. Myocardial infarction ((MI), acute myocardial infarction (AMI) or heart attack) is a serious medical emergency and occurs when blood flow to the heart is suddenly blocked. The patient may in addition be suffering from diabetes, either type I or type II. The patient will undergo either a contrast CT scan and or x-ray fluoroscopy to obtain diagnostic images of the coronary arteries in order to make a diagnosis. X-ray fluoroscopy utilising contrast is for example used in guiding treatment of patients with acute coronary syndromes (e.g. with balloon angioplasty and stent insertion). In some embodiments of the first and second aspects of the invention, the method further comprises administering to said patient 8-20 mmol of an inorganic nitrate prior to the patient receiving said contrast agent and once per day for up to 7 days after the patient receives said contrast agent. In these embodiments, the inorganic nitrate is typically administered orally. For oral administration, the inorganic nitrate may be administered in the form of a capsule, pill, lozenge, spray, toothpaste or chewing gum. In these embodiments, inorganic nitrate may be administered in the form of dietary nitrate. Dietary nitrate may be in the form of any nitrate-rich vegetable or fruit, for example beetroot, green leafy vegetables (such as spinach, cabbage, Chinese cabbage and lettuce), fennel, rocket, radish and parsley, or a juice or dried concentrate thereof . The dietary nitrate may be administered in the form of beetroot juice or the juice of any nitrate-rich vegetable. In these embodiments, the dosage of inorganic nitrate of 8-20 mmol is administered to the patient prior to receiving (being administered) said contrast agent and then once per day for up to 7 days after the patient receives (is administered) said contrast agent. For example, from 9-19 mmol, 10-18 mmol, 11- 17 mmol, 12-16 mmol or 13-15 mmol of inorganic nitrate may be administered, for example 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mmol. Typically, 12 mmol of inorganic nitrate is administered. The dosage of inorganic nitrate administered to the patient prior to the patient receiving said contrast agent may be the same as or different from the dosage of inorganic nitrate administered after the patient receives said contrast agent, as long as both dosages of inorganic nitrate are from 8-20 mmol. An orally administered dosage of inorganic nitrate of 8-20 mmol typically results in a circulating concentration of nitrate in the patient of 300-400 ^mol / L, for example 350 ^mol / L, up to 72 hours after administration. For example, this dosage of inorganic nitrate may result in a circulating concentration of nitrate in the patient of 300 ^mol / L, 350 ^mol / L or 400 ^mol / L, from 1-72 hours, for example from 1-3 hours, 4-6 hours, 6-12 hours, 12-24 hours, 24-48 hours or 48-72 hours after oral administration. For example, this dosage of inorganic nitrate may result in a circulating concentration of nitrate in the patient of approximately 350 ^mol / L, 4-6 hours after oral administration and / or a circulating concentration of nitrate in the patient of approximately 325 ^mol / L, 48-72 hours after oral administration. An orally administered dosage of inorganic nitrate of 8-20 mmol typically results in a circulating concentration of nitrite in the patient of 1.0-1.5 ^mol / L up to 72 hours after administration. For example, this dosage of inorganic nitrite may result in a circulating concentration of nitrite in the patient of 1.0 ^mol / L or 1.5 ^mol / L, from 1-72 hours, for example from 1-3 hours, 4-6 hours, 6-12 hours, 12-24 hours, 24-48 hours or 48-72 hours after oral administration. For example, this dosage of inorganic nitrate may result in a circulating concentration of nitrite in the patient of approximately 1.5 ^mol / L, 4-6 hours after oral administration and / or a circulating concentration of nitrite in the patient of approximately 1.0 ^mol / L, 48-72 hours after oral administration. In these embodiments, the dosage of inorganic nitrate of 8-20 mmol is administered to the patient prior to the patient receiving (being administered) said contrast agent and then once per day for up to 7 days after the patient receives (is administered) said contrast agent. For example, the inorganic nitrate may be administered at least 2 hours prior to the patient receiving said contrast agent. Typically, the inorganic nitrate is administered no more than 24 hours prior to the patient receiving said contrast agent. Accordingly, the inorganic nitrate may be administered from 2-24 hours prior to the patient receiving said contrast agent, for example from 3-22 hours, 4-21 hours, 5-20 hours, 6-19 hours, 7-18 hours, 8-17 hours, 9-16 hours, 10-15 hours, 11-14 hours, 12-13 hours prior to the patient receiving said contrast agent. This dosage of inorganic nitrate is then administered to the patient once per day for up to 7 days after the patient receives said contrast agent, for example once per day for 1, 2, 3, 4, 5, 6 or 7 days after receiving said contrast agent, for example for 5 days after receiving said contrast agent. For example, the inorganic nitrate may be administered to the patient once per day for 2-4 days after the patient receives said contrast agent, corresponding to the typical length of a hospital stay for example for an elective or emergency PCI of 3 days. After cessation of treatment of the patient with 8-20 mmol of inorganic nitrate, inorganic nitrate may be administered at a lower dose for a longer period of time, for example, 2-12 mmol once per day, typically from 2.5-8 mmol once per day. For example, 2-11 mmol, 2.5-10 mmol, 2.5-9 mmol, 3-7 mmol, 4-6 mmol or 4-5 mmol, for example 4 mmol or 5 mmol of nitrate may be administered once per day. Administration of the inorganic nitrate at this lower dose may continue on a once daily basis for 1-5 months after cessation of treatment of the patient with 8-20 mmol of inorganic nitrate. For example, administration of the inorganic nitrate at this lower dose may continue on a once daily basis for 1 month, 2 months, 3 months, 4 months or 5 months or up to 6 months, either after the patient has received a contrast agent or after cessation of treatment of the patient with 8-20 mmol of inorganic nitrate. Alternatively, administration of the inorganic nitrate at this lower dose may continue on a once daily basis for at least 6 months, either after the patient has received a contrast agent or after cessation of treatment of the patient with 8-20 mmol of inorganic nitrate. For example, administration of the inorganic nitrate at this lower dose may continue on a once daily basis for at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 14 months, at least 16 months, at least 18 months or at least 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years or 10 years. The inorganic nitrate may be administered at this lower dose on a once daily basis indefinitely, for the remainder of the patient’s life. The present inventors have surprisingly found that administration of inorganic nitrate or inorganic nitrite reduces the incidence of major adverse cardiac events (MACE) and major adverse kidney events (MAKE) in patients undergoing procedures that require administration of a contrast agent. Accordingly, the first aspect of the present invention may alternatively be worded as inorganic nitrate for use in a method of reducing the incidence of or preventing MACE and / or MAKE in a patient undergoing a procedure requiring administration of a contrast agent, said method comprising intravenously administering to said patient an inorganic nitrate at a dosage which results in a circulating concentration of nitrate in the patient of 300-400 ^mol / L up to 12 hours after administration, wherein the inorganic nitrate is administered concomitantly with said contrast agent. The second aspect of the present invention may alternatively be worded as inorganic nitrite for use in a method of reducing the incidence of or preventing MACE and / or MAKE in a patient undergoing a procedure requiring administration of a contrast agent, said method comprising intravenously administering to said patient an inorganic nitrite at a dosage which results in a circulating concentration of nitrite in the patient of 0.7-2.0 ^mol / L up to 6 hours after administration, wherein the inorganic nitrite is administered concomitantly with said contrast agent. The present invention can also be worded as: ^ A method of preventing contrast-induced nephropathy (CIN) in a patient receiving contrast agent, said method comprising intravenously administering to said patient an inorganic nitrate at a dosage which results in a circulating concentration of nitrate in the patient of 300-400 ^mol / L up to 12 hours after administration, wherein the inorganic nitrate is administered concomitantly with said contrast agent. ^ A method of preventing contrast-induced nephropathy (CIN) in a patient receiving contrast agent, said method comprising intravenously administering to said patient an inorganic nitrite at a dosage which results in a circulating concentration of nitrite in the patient of 0.7-2.0 ^mol / L up to 6 hours after administration, wherein the inorganic nitrite is administered concomitantly with said contrast agent. ^ A method of reducing the incidence of or preventing MACE and / or MAKE in a patient undergoing a procedure requiring administration of a contrast agent, said method comprising intravenously administering to said patient an inorganic nitrate at a dosage which results in a circulating concentration of nitrate in the patient of 300-400 ^mol / L up to 12 hours after administration, wherein the inorganic nitrate is administered concomitantly with said contrast agent. ^ A method of reducing the incidence of or preventing MACE and / or MAKE in a patient undergoing a procedure requiring administration of a contrast agent, said method comprising intravenously administering to said patient an inorganic nitrite at a dosage which results in a circulating concentration of nitrite in the patient of 0.7-2.0 ^mol / L up to 6 hours after administration, wherein the inorganic nitrite is administered concomitantly with said contrast agent. Preferred features for the various aspects of the invention are as for the first aspect of the invention mutatis mutandis. It will be appreciated that all embodiments described herein are considered to be broadly applicable and combinable with any and all other consistent embodiments, as appropriate. Such combinations are considered to fall within the scope of the present invention. BRIEF DESCRIPTION OF FIGURES Figure 1 – Schematic of study flowchart. NSTEACS: Non-ST segment elevation acute coronary syndrome; PCI: percutaneous intervention; CIN: contrast induced nephropathy; ESRF: end stage renal failure; STEMI: ST segment elevation myocardial infarction; U&E: urea and electrolytes; NOx: nitric oxide levels (NO2- / NO3-); MACE: major adverse cardiac events; EQ5D: quality of life questionnaire. Figure 2 – Recruitment and follow-up. Accounting for pandemic restrictions. Figure 3 – Calculation of MEHRAN score Figure 4 – Cumulative incidence of major adverse cardiac events (MACE). Top line represents placebo and bottom line represents active nitrate. Figure 5 – Cumulative incidence of major adverse cardiac events (MACE) with removal of procedural-related myocardial infarctions (MI). Top line represents placebo and bottom line represents active nitrate. Figure 6 – Plasma nitrate / nitrate levels over time in the NITRATE-CIN cohort. Figure 6A shows the concentration of plasma nitrate at 4-6h and 48-72h after IMP administration in the inorganic nitrate (KNO3, 12mmol daily for five days) group compared to placebo (KCl 12mmol daily for five days) with no difference in the groups at 3 months. Figure 6B demonstrates changes in plasma nitrite concentrations at 4-6 h and 48-72h after IMP administration. Values shown are mean^ SEM of n=431 at 4-6hrs, 94 at 48-72hrs and 105 at 3 months. Two-way ANOVA shown as a number within the graph with Sidak’s post-test comparing groups at each timepoint shown as ** for P<0.01 or **** for P<0.0001 Figure 7 – Blood pressure and heart rate haemodynamics in the NITRATE-CIN cohort Figure 7 shows systolic blood pressure (A), diastolic blood pressure (B) and heart rate (C) at baseline, 4-6h after the first IMP administration and then at 3 months in the inorganic nitrate treated (KNO3, 12mmol daily for five days; N=316, 262 and 111 respectively)) group compared to placebo (KCl 12mmol daily for five days; N=319, 263, 106 respectively). Values shown as mean^ SEM. Two-way ANOVA shown as a number within the graph with Sidak’s post-test comparing groups at each timepoint shown as *** for P<0.001. Figure 8. Adverse Events by System Organ Class and Treatment allocation. The numbers of events in each applicable system organ class are shown stacked by severity: orange=mild, red=moderate, brown=severe. The most frequent adverse event was acute kidney injury (renal and urinary disorders) followed by procedure-related myocardial infarctions (injury, poisoning and procedural). EXAMPLES Example 1 – Nitrate CIN study to assess the effect of inorganic nitrate treatment on CIN in patients with ACS This study was designed to investigate the hypothesis that inorganic nitrate treatment decreases the rate of CIN as part of semi-emergent coronary angiography for ACS. Inorganic nitrate is a simple and easy to administer intervention that may prove useful in prevention of CIN in at-risk patients undergoing coronary angiographic procedures. The NITRATE-CIN trial is a single-centre, randomised double-blinded placebo-controlled trial, with 640 patients recruited presenting with acute coronary syndromes (ACS) who were at risk of contrast-induced nephropathy (CIN). Patients were randomised to either inorganic nitrate therapy (capsules containing12 mmol KNO3) or placebo capsules containing potassium chloride (KCl 12 mmol) daily for 5 days. Theprimary endpoint was development of CIN using the Kidney Disease Improving Global Outcomes (KDIGO) criteria. A key secondary endpoint was renal function over a 3-month follow-up period. Additional secondary endpoints include serum renal biomarkers (e.g. neutrophil gelatinase-associated lipocalin) at 6 h, 48 h and 3 months following administration of contrast. Cost-effectiveness of inorganic nitrate therapy was also evaluated. Study Population Patients presenting with non-ST-elevation acute coronary syndrome (NSTE-ACS) undergoing invasive coronary procedures that involve the administration of intracoronary iodinated contrast media (angiography + / -PCI) that require CIN prophylaxis were recruited to the study. These patients were recruited at The Barts Heart Centre, based at St Bartholomew’s Hospital. This is the largest cardiac centre in the UK, serving a population of approximately 6 million people from North East and Central London and is a 24 / 7 centre performing approximately 6000 angiograms and 2000 non-primary angioplasties a year. A total of 640 patients (male and female, aged ≥18) undergoing angiography for NSTE-ACS who require CIN prophylaxis as per local guidelines were recruited. See Table 1 for full inclusion and exclusion criteria. Table 1. Inclusion and Exclusion Criteria of NITRATE-CIN study Inclusion Criteria 1. Patients undergoing coronary angiography+ / -PCI for NSTE-ACS 2. Requirement for CIN prophylaxis as per Barts Heart Centre Criteria for CIN prophylaxis: o eGFR<60ml / min OR o 2 of the following: diabetes, liver failure (cirrhosis), age > 70yr, exposure to contrast in last 7 days, heart failure (or LVEF<40%), concomitant renally active drugs (ACEi, ARB, NSAIDs, aminoglycosides, diuretics) 3. Aged ≥18 4. Patients able and willing to give their written informed consent. Exclusion Criteria 1. ST segment myocardial infarction undergoing Primary PCI. 2. Patients with eGFR<20ml / min or on renal replacement therapy 3. Subjects presenting with cardiogenic shock (systolic blood pressure <80 mmHg for >30 minutes, or requiring inotropes or emergency intra aortic balloon pump for hypotension treatment) or cardiopulmonary resuscitation 4. Current life-threatening condition other than vascular disease that may prevent a subject completing the study. 5. Use of an investigational device or investigational drug within 30 days or 5 half-lives (whichever is the longer) preceding the first dose of study medication. 6. Patients considered unsuitable to participate by the research team (e.g., due to medical reasons, laboratory abnormalities, or subject’s unwillingness to comply with all study related procedures). 7. Severe acute infection 8. Pregnancy. This was tested by urine HcG measurement 9. Any Inclusion Criteria not met Table 1 – Inclusion and Exclusion Criteria of NITRATE-CIN study Intervention Patients were randomised to receive KNO3 capsules (12 mmol giving 744 mg of NO3-) or an equivalent dose (12 mmol) of KCl as placebo control. The capsules were taken by the patient prior to their coronary angiogram procedure and they received a daily dose for four days post procedure (five doses in total). A previous study (Kapil V, Milsom AB, Okorie M, et al. Hypertens (Dallas, Tex 1979).2010;56(2):274- 281. doi:10.1161 / HYPERTENSIONAHA.110.153536) confirms tolerability of KNO3 with minimal side effects. In that study it was also demonstrated that the levels of circulating NO2- achieved with a 12mmol KNO3 dose were approximately 1.0 µmol / L; a level that persisted for up to 3 hours following ingestion. In contrast, a lower dose of 4 mmol did not achieve these levels (rising to no more than 0.5 µmol / L). In the NITRITE AMI trial (Jones DA, et al, supra) where a reduction of CIN events appeared to occur, the levels of circulating NO2- were 0.67±0.18 µmol / L. Thus, a 12mmol dose for this study was used to ensure sufficient efficacious levels of the anion were achieved. The KNO3 and matching KCl placebo capsules were supplied by the Pharmacy Manufacturing Unit based at Guy’s and St Thomas’ NHS Foundation Trust (GSTT). GSTT Pharmaceuticals hold a license with the MHRA, which allows the manufacture, storage and distribution of a range of Sterile and Non Sterile Investigational Medical Products (IMPs) for Phase I, II, III and IV clinical trials. Randomisation and Blinding Process Patients were block randomised on a 1:1 basis to receive either KNO3 capsules or placebo KCl capsules using an algorithm written in a statistical package with a reliable pseudorandom number generator, and stratified by diabetes status. Block lengths were varied randomly. Treatment assignment in both the intervention and placebo groups remained blinded until data lock and statistical analysis at the end of the study. Experimental Protocol Patients were approached on their arrival to Barts Heart Centre. Following consent, and prior to angiography, up to 40ml of venous blood was taken. This sample was used to measure baseline renal function, inflammatory markers (including hs-CRP), renal biomarkers, and baseline plasma NO3- / NO2- (collectively referred to as NOx) levels. Where possible, urine and saliva samples were also taken for analysis of NOx levels, renal biomarkers and NO3- reducing bacteria. The participant was then randomised to daily ingestion of either KNO3 or KCl capsules (placebo). This was commenced prior to their scheduled angiogram and continued daily (two capsules) for five days. A quality of life assessment (EQ5D) was performed prior to PCI (and study drug administration). EQ5D is an instrument, which evaluates the generic quality of life that was developed in Europe and is widely used. The EQ5D descriptive system is a preference-based HRQL measure with one question for each of the five dimensions that include mobility, self-care, usual activities, pain / discomfort, and anxiety / depression. Following the angiogram + / - PCI procedure a blood sample was collected 4-6 hours later for renal biomarkers, inflammatory markers and troponin (if PCI performed) assessment, along with urine and saliva sample collection. At 48-72 hours a blood sample for renal function was taken, along with urine and saliva sample collection where possible. The participant was asked to return to the clinical centre at 3 months for review. At this stage venous blood and urine samples were collected as well as EQ5D and assessment for MACE. After 1 year, the participant was contacted by telephone for assessment of MACE and EQ5D. The whole duration of the study was 1 year for each participant (see Figure 1). Participants had no obligation to complete the whole study and were free to withdraw at any point Blood, saliva and urine analysis U&E and hs-CRP sample analysis were performed at the local Barts Health Biochemistry laboratory. Blood samples were taken from the venous side of the circulation from a hand vein using a yellow butterfly needle (19 gauge). Blood samples were centrifuged immediately for plasma, and samples stored at -80oC for the purposes of making biochemical measurements (e.g. NOx / cGMP). All samples were discarded once used as per local procedures. Saliva and urine was collected in a falcon tube at the time of visits and stored for analysis. Saliva was centrifuged and a pellet generated. This pellet contained oral bacteria that had dislodged from the oral cavity. This pellet was frozen (-80oC) until identification of the oral microbiota by second-generation genome sequencing. NOx concentrations in saliva, blood and urine were determined using the technique of chemiluminescence (as described, for example, in Kapil et al. Hypertension 2015; 65: 320-327), which was performed at The William Harvey Research Institute (WHRI), Queen Mary University of London (QMUL) and measured at baseline, 4-6 hours, 48-72 hours, and at 3 months after IMP administration. Measures of renal biomarkers (serum and urine) were conducted at WHRI (QMUL). These were also measured at baseline, 4-6 hours, and at 48-72 hours. Statistical Analysis The reported incidence of ACS-associated AKI is extremely heterogeneous, ranging from 5% to 55%, and it varies with the criteria used for diagnosing AKI, the clinical setting and the investigated population. The most recent data from the Acute Coronary Treatment and Intervention Outcomes Network (ACTION) registry demonstrated that AKI occurred in 16% of 59970 patients with acute myocardial infarction (AMI) (Fox CS, Muntner P, Chen AY, Alexander KP, Roe MT, Wiviott SD. Circulation. 2012;125(3):497-504. doi:10.1161 / CIRCULATIONAHA.111.039909), with similar studies showing rates of 15% according to RIFLE criteria and 13% according to AKIN criteria in patients with AMI (Marenzi G, Cabiati A, Bertoli S V, et al. Am J Cardiol. 2013;111(6):816-822. doi:10.1016 / j.amjcard.2012.11.046). These data were recently confirmed in the large-scale Harmonizing Outcomes With Revascularization and Stents in Acute Myocardial Infarction (HORIZONS-AMI) trial, which reported AKI in 16% of the patients (Narula A, Mehran R, Weisz G, et al. Eur Heart J.2014;35(23):1533-1540. doi:10.1093 / eurheartj / ehu063). For our study calculations, we thereforepropose a conservative CIN incidence of 12%. Preliminary data demonstrated an 80%-related reduction in CIN in AMI patients (Jones DA, et al, supra). For our power calculations, we have proposed an effect at the lower end of this range and have powered the study to determine a difference of 60%. For a power of 80% and a significance level of 0.05, 232 patients needed to be recruited into each trial arm, 464 patients in total, allowing for 27.5% drop out (primary endpoint) this meant 320 patients in each arm and a total study population of 640 ACS patients. Sub-group analysis of the primary endpoint was undertaken. Unadjusted effect sizes were shown by subgroup and for all patients using a modified forest plot, along with a test for heterogeneity. Subgroups provided insight into the following mechanistic and exploratory questions: 1). Pre-existing nitrate use versus no prior use: Do patients already established on an organic nitrate derive similar benefits from inorganic nitrate therapy after an ACS? Previous studies have suggested less effect in patients established on organic nitrates. 2). Diabetic versus non-diabetic patients: Is dietary nitrate equally effective in diabetic and non- diabetic patients? Previous studies have suggested less benefit of nitrate in patients with diabetes. 3). Troponin positive versus troponin negative: Is the risk of CIN higher and dietary nitrate more effective in patients with evidence of myocardial infarct (troponin positive (NSTEMI) rather than troponin negative (unstable angina)). 4). Mehran risk score group (low, moderate etc): Is the benefit of inorganic nitrate seen throughout patients of varying CIN risk or is it only beneficial in those at the highest risk (Mehran score 11-15 / ≥16)? Study Endpoints 1) To determine if inorganic NO3- reduces renal adverse events including long-term renal impairment caused by CIN, need for haemofiltration or haemodialysis. 2) To determine whether levels of serum biomarkers released secondary to renal injury can predict CIN. 3) To determine if inorganic NO3- ingestion decreases renal injury as assessed by serum levels of renal biomarkers. 4) To determine if inorganic NO3- ingestion decreases rates of post-procedural MI (Society for Cardiovascular Angiography and Intervention definition). 5) Assessment of cost effectiveness of inorganic NO3- (Quality-adjusted Life Years [QALYs]) measured over 12 months of follow-up. 6) Assessment of cardiovascular endpoints (Major Adverse Cardiac Events [MACE]) including death, non-fatal MI, and unscheduled revascularization. Recruitment and follow-up 640 patients were recruited over 2 years and 5 months.319 patients were allocated active treatment, whilst 321 were allocated placebo.625 patients completed the 3-month follow up. 580 patients completed the study to 1 year.299 of these patients had received the active treatment, whilst 281 of these patients had received the placebo. 6 subjects withdrew, 2 subjects were withdrawn by the investigator and 52 subjects died. Baseline characteristics and demographics Characteristics and demographics were collated, see Table 2 below. The mean age was 71 years, 73% of the subjects were male.16% of the subjects were Asian, 8% were black and 75% were white.76% of the subjects had some history of hypertension, and 85% presented with non-ST-elevation myocardial infarction (NSTEMI), involving partial blockage of one of the coronary arteries, causing reduced flow of oxygen-rich blood to the heart muscle.

[0002] Table 2 – Baseline characteristics and demographics of study recruits. Note: missing date: 3 for SBP, 3 for DBP, 3 for HR, 1 for creatinine, 6 for urea, 1 for eGFR. *First degree relative < 55 male, < 65 female. Previous studies have suggested less benefit of nitrate in patients with diabetes, therefore subjects were stratified by diabetes status. Diabetes status was balanced.46% of subjects receiving active treatment were diabetic, whilst 54% of subjects receiving active treatment were non-diabetic. Also 46% of subjects receiving placebo were diabetic, whilst 54% of subjects receiving placebo were non-diabetic. The majority of type II diabetic subjects were on drug therapy. See Table 3 below. Table 3 – Diabetes status. Diabetes stratification was balanced. Mehran score is a risk score for CIN. Mehran scores were calculated post procedure. Mehran scores comprise 8 components (see below). The 8 components are scored and together sum to an overall risk score (see schematic in Figure 3). 1. Risk factors 2. Intra-aortic balloon pump (IABP) 3. Congestive heart failure (CHF) 4. Age >75 years 5. Anemia 6. Diabets 7. Contrast media volume 8. Serum creatine > 1.5 mg / dl OR eGFR < 60 ml / min / 1.7m2 Mehran score outputs are shown in Table 4 below. Approximately 83-84% of subjects were categorised in the medium or high risk groups, whilst approximately 8-9% of subjects were categorised as very high risk. There was an overall balance between the randomised active versus placebo group. Table 4 – Mehran Score calculations. Primary Endpoints The primary endpoint is development of Contrast induced nephropathy (CIN) using the Kidney Disease Improving Global Outcomes (KDIGO) criteria (see Table 5). Table 5 shows the odds ratio (OR) which is a measure of association between exposure and an outcome, and p-value. The data was covariate adjusted for baseline creatine and diabetes status. A covariate adjustment refers to use of information measured on a subject before the time of randomisation (see Tables 2 & 3) for estimating and testing treatment effects between randomised groups. Table 5 – Contrast induced nephropathy (CIN) determination. Covariate adjusted for baseline creatine and diabetes status. In subgroup analysis it was explored whether the effect on CIN would differ by: 1. Pre-existing organic nitrate use 2. Diabetic status 3. Presentation 4. Mehran score The effects were consistent for most subgroups, with a minor reduced effect in those on organic nitrate at baseline (the p-value for the interaction between those on organic nitrate at baseline vs. those not on organic nitrate at baseline was 0.04). Rates of CIN were higher among diabetics, however with no evidence for a differing effect. As expected, those with higher Mehran risk scores (a risk score for CIN) displayed higher rates of CIN, however the treatment effect was similar no matter the risk level. See Table 6. Table 6 – Contrast induced nephropathy (CIN) determination – subgroup analysis. Covariate adjusted for baseline creatine and diabetes status. Secondary Endpoints A key secondary endpoint is renal function over a 3-month follow-up period. Additional secondary endpoints include serum renal biomarkers (e.g. neutrophil gelatinase-associated lipocalin) at 6 h, 48 h and 3 months following administration of contrast. Cost-effectiveness of inorganic nitrate therapy was also evaluated. Renal function at 3 months Renal function was measured at 3 months in subjects. In the placebo treatment arm, creatine levels rose more than creatine levels in the active treatment arm, however the higher starting point of creatine levels in the active treatment arm meant that the crude estimated difference in creatine between treatment and placebo groups at 3 months was small. Upon adjustment for baseline values of creatine (accounting for the difference in the change), it was determined that creatine levels were more than 10 units lower in the active treatment arm of the study compared to the placebo arm of the study, and this difference was statistically significant. A similar pattern was demonstrated for eGFR. eGFR was found at lower baseline levels in the active treatment arm of the study. Whilst eGFR declined in the placebo group, it remained in and around baseline in the active treatment group. This resulted in an adjusted difference of a 5.17 improvement in the active treatment subjects. See Table 7.

[0003] Table 7 – Secondary endpoint – renal function at 3 months. Covariate adjusted for baseline level and diabetes status at baseline. Assessment of cardiovascular endpoints Major adverse cardiac events (MACE) including death, non-fatal myocardial infarction and unscheduled revascularisation were assessed starting at the 3-month timepoint (see Table 8). Events were categorised as MACE (overall composite of MACE), then individual components (1) All-cause mortality; (2) cardiovascular mortality; (3) non-fatal myocardial infarction (MI); and (4) unscheduled revascularisation. 12 MACE events in the active treatment group were identified, compared to 52 MACE events in the placebo group, with an adjusted odds ratio (OR) of 0.2 and a very small p value of <0.001. In each of the individual components of MACE, fewer events were found in the active treatment group compared to the placebo group, with significant reductions for non-fatal myocardial infarction and unscheduled revascularisation.

[0004] Table 8 – Secondary endpoint – major adverse cardiac events (MACE) at 3 months. Covariate adjusted for diabetes status at baseline. MACE was further assessed at 12 months (see Table 9). At 12 months there were 34 MACE events in the active group compared to 81 MACE events in the placebo group, with an adjusted odds ratio (OR) of 0.35 in favour of the active treatment group. At 12 months a significant difference was observed for all-cause mortality, as well as non-fatal MI and unscheduled revascularisation. 17 deaths occurred in the active treatment group versus 35 deaths in the placebo group, with a p-value of 0.011. Table 9 – Secondary endpoint – major adverse cardiac events (MACE) at 12 months. Covariate adjusted for diabetes status at baseline. The cumulative incidence of MACE was also assessed (see Figure 4) comparing the active treatment group (active nitrate) with the placebo group. At each time-point from 3 months onwards there was a higher number of MACE events in the placebo group than the active treatment group. The large number of MACE events in the placebo group at 0 months was due to a number of procedural-related myocardial infarction events. Data in Table 10 below and Figure 5 accounts for removal of said procedural-related MI events, however a significant reduction in MACE events remained in the active treatment group. Table 10 – Cumulative incidence of major adverse cardiac events (MACE) with removal of procedural-related myocardial infarction vents. Covariate adjusted for diabetes status at baseline. Assessment of kidney endpoints Major adverse kidney events (MAKE) were assessed starting at the 3-month timepoint (see Table 11). Events were categorised as MAKE (overall composite of MAKE), then individual components (1) All- cause mortality; (2) new-onset renal replacement therapy; and (3) persistent renal dysfunction. 15 MAKE events in the active treatment group were identified, compared to 62 MAKE events in the placebo group, with an adjusted odds ratio (OR) of 0.21 and a very small p-value of <0.001. In each of the individual components of MAKE, fewer events were found in the active treatment group compared to the placebo group, with a significant reduction in persistent renal dysfunction. Table 11 – Secondary endpoint – major adverse kidney events (MAKE) at 3 months. Covariate adjusted for diabetes status at baseline. MAKE was further assessed at 12 months (see Table 12). At 12 months there were 35 MAKE events in the active group compared to 94 MAKE events in the placebo group, with an adjusted odds ratio (OR) of 0.30 in favour of the active treatment group and a p-value of <0.001. At 12 months a significant

[0005] difference was observed for all-cause mortality, as well as persistent renal dysfunction. Table 12 – Secondary endpoint – major adverse kidney events (MAKE) at 12 months. Covariate adjusted for diabetes status at baseline. Assessment of Adverse Events There were 184 AEs in 156 patients. More than twice as many patients in the placebo arm experienced AEs (38.1% vs.16.4%, p<0.001). Most patients experienced 1 AE while 24 patients experienced two AEs (15 patients in the placebo arm vs.5 in the active arm) and 2 patients experienced three AEs (both on the placebo arm). Of these AEs 75.5% (139 / 184) were procedure-related events occurring in 116 patients.16 patients experienced 2 procedural AEs: 14 in the placebo arm vs.2 in the active arm. Only 31 procedure-related AEs were myocardial infarctions. Most AEs were of a mild intensity (83.2%, 153 / 184).15.8% of AEs were graded moderate intensity (29 / 184) while 2 AES were graded as severe (1.09%). Tolerance and effect of intervention on plasma nitrate / nitrite levels Inorganic nitrate intervention and placebo were both tolerated well, as shown herein. There was a significant increase in the plasma nitrate concentration at both 4-6 hours (349.5 ± 193.2 μmol / L vs 34.0 ± 22.4 μmol / L, P<0.001) and 48-72 hours (376.5 ± 260.7 μmol / L vs 32.2 ± 19.5 μmol / L, P<0.001) after angiography in those receiving inorganic nitrate when compared to placebo (Figure 6). There were also increased levels in plasma nitrite concentrations seen at both 4-6hours (1.40±5.3 μmol / L vs 0.5 ± 0.37μmol / L, P=0.038) and 48-72 hours (0.9±1.2 μmol / L vs 0.4±0.3 μmol / L, P=0.036) in those receiving inorganic nitrate compared to placebo indicating that the enterosalivary circuit of nitrate was intact in these patients. In contrast there were no differences in the plasma nitrate (31.5 ±16.2 μmol / L vs 33.6±18.6 μmol / L, P=0.9982) or nitrite concentrations (0.3 ±0.2 μmol / L vs 0.3±0.2 μmol / L, P=0.999) at 3 months between the two groups indicating washout of nitrate in those receiving inorganic nitrate. In addition, assessment of blood pressure confirmed both delivery and efficacy of inorganic nitrate with a reduction in systolic blood pressure and no significant difference between the groups in diastolic blood pressure or heart rate (Figure 7). Results NITRATE-CIN enrolled 640 patients who were recruited over 2 years and 5 months.319 patients were allocated active treatment, whilst 321 were allocated placebo.625 patients completed the 3-month follow up. The baseline demographic data of the two groups is similar indicating effective randomisation (Table 2). The primary endpoint of CIN was substantially reduced in the patients receiving the inorganic nitrate compared to those receiving the placebo. Importantly, this effect was observed whether the patients had diabetes or not and this finding is very important since previous studies have suggested that inorganic nitrate is ineffective in patients with this condition. Our results disagree with this view and show benefit whether one has diabetes or not. Whilst these effects mentioned above suggest positive influence over the damaging effects of contrast the observations that demonstrated the longer-term benefits upon both renal function in the form of MAKE and cardiovascular events in the form of MACE are very surprising. These results suggest that treatment with the relatively high dose of 12mmol (the ADI is ~4mmol) for just prior and then up to 5 days post contrast prevents the renal damage caused by said contrast and that this in the long term reduces risk of further serious renal or cardiac events. This treatment leads to the circulating levels of nitrate and nitrite shown in Figure 6A and B respectively. Interpretation: In patients who receive contrast to aid with a surgical intervention, treatment with dietary nitrate, prevents the acute damage caused by the contrast and also reduces the long term risk of further renal and cardiac events. These findings may also apply to patients receiving contrast as part of diagnostic procedures, and can be replicated by administering inorganic nitrate or inorganic nitrite to the patient concomitantly with contrast agent

Claims

CLAIMS 1. Inorganic nitrate for use in a method of preventing contrast-induced nephropathy (CIN) in a patient receiving contrast agent, said method comprising intravenously administering to said patient an inorganic nitrate at a dosage which results in a circulating concentration of nitrate in the patient of 300-400 ^mol / L up to 12 hours after administration, wherein the inorganic nitrate is administered concomitantly with said contrast agent.

2. Inorganic nitrate for use according to claim 1, wherein the dosage of inorganic nitrate results in a circulating concentration of nitrate in the patient of 310-390 ^mol / L, 320-380 ^mol / L, 325-375 ^mol / L, 330-370 ^mol / L, 335-365 ^mol / L, 340-360 ^mol / L or 350-355 ^mol / L.

3. Inorganic nitrate for use according to claim 2, wherein the inorganic nitrate is a nitrate salt.

4. Inorganic nitrate for use according to claim 3, wherein the nitrate salt is potassium nitrate (KNO3) or sodium nitrate (NaNO3).

5. Inorganic nitrite for use in a method of preventing contrast-induced nephropathy (CIN) in a patient receiving contrast agent, said method comprising intravenously administering to said patient an inorganic nitrite at a dosage which results in a circulating concentration of nitrite in the patient of 0.7-2.0 ^mol / L up to 6 hours after administration, wherein the inorganic nitrite is administered concomitantly with said contrast agent.

6. Inorganic nitrite for use according to claim 5, wherein the dosage of inorganic nitrite results in a circulating concentration of nitrite in the patient of 0.8-1.9 ^mol / L, 0.9-1.8 ^mol / L, 1.0-1.7 ^mol / L, 1.1-1.6 ^mol / L, 1.2-1.5 ^mol / L or 1.3-1.4 ^mol / L.

7. Inorganic nitrite for use according to claim 6, wherein the inorganic nitrite is a nitrite salt.

8. Inorganic nitrite for use according to claim 7, wherein the nitrite salt is potassium nitrite (KNO2) or sodium nitrite (NaNO2).

9. Inorganic nitrate or inorganic nitrite for use according to any one of the preceding claims, wherein the inorganic nitrate or inorganic nitrite is present in the contrast solution or administered as a separate infusion.

10. Inorganic nitrate or inorganic nitrite for use according to any one of the preceding claims, wherein the patient is undergoing coronary angiography, percutaneous coronary intervention (PCI) and / or a computerised tomography (CT) scan.

11. Inorganic nitrate or inorganic nitrite for use according to any one of the preceding claims, wherein the patient has an acute coronary syndrome (ACS) or stable angina or another medical condition requiring administration of a contrast agent.

12. Inorganic nitrate or inorganic nitrite for use according to any one of the preceding claims, wherein the contrast agent is a radiocontrast agent.

13. Inorganic nitrate or inorganic nitrite for use according to any one of the preceding claims, wherein the patient has diabetes.

14. Inorganic nitrate or inorganic nitrite for use according to any one of the preceding claims, wherein administration of the inorganic nitrate reduces major adverse cardiac events (MACE) or major adverse kidney events (MAKE).

15. Inorganic nitrate or inorganic nitrite for use according to any one of the preceding claims, said method further comprising administering to said patient 8-20 mmol of an inorganic nitrate prior to the patient receiving said contrast agent and once per day for up to 7 days after the patient receives said contrast agent.

16. Inorganic nitrate or inorganic nitrite for use according to claim 15, wherein the 8-20 mmol of inorganic nitrate is administered orally.

17. Inorganic nitrate or inorganic nitrite for use according to claim 16, wherein the 8-20 mmol of inorganic nitrate is administered in the form of a capsule, pill, lozenge, spray, toothpaste or chewing gum.

18. Inorganic nitrate for use according to any one of claims 15 to 17, wherein the inorganic nitrate is dietary nitrate.

19. Inorganic nitrate for use according to claim 18, wherein the dietary nitrate is administered in the form of beetroot, green leafy vegetables or any other nitrate-rich vegetable or fruit or the juice thereof.

20. Inorganic nitrate for use according to any one of claims 15 to 19, wherein 12 mmol of inorganic nitrate is administered.

21. Inorganic nitrate for use according to any one of claims 15 to 20, wherein the inorganic nitrate is administered at least 2 hours prior to the patient receiving said contrast agent.

22. Inorganic nitrate for use according to claim 21, wherein the inorganic nitrate is administered no more than 24 hours prior to the patient receiving said contrast agent.

23. Inorganic nitrate for use according to any one of claims 15 to 22, wherein the inorganic nitrate is administered once per day for up to 4 days after the patient receives said contrast agent.

24. Inorganic nitrate for use according to claim 23, wherein the inorganic nitrate is administered once per day for 2-4 days after the patient receives said contrast agent.

25. Inorganic nitrate for use according to any one of claims 15 to 24, wherein 2-12 mmol of an inorganic nitrate is administered once per day after cessation of said treatment with 8-20 mmol of an inorganic nitrate.

26. Inorganic nitrate for use according to claim 25, wherein 2.5-8 mmol of an inorganic nitrate is administered once per day after cessation of said treatment with 8-20 mmol of an inorganic nitrate.

27. Inorganic nitrate for use according to claim 25 or 26, wherein 2-12 mmol of an inorganic nitrate is administered once per day for 1-5 months or for at least 6 months.

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