Randomised phase 2 trial of intra-coronary nitrite during acute myocardial infarction

Abstract

Rationale

Pre-clinical evidence demonstrates that inorganic nitrite, following its in situ conversion to nitric oxide, attenuates consequent myocardial reperfusion injury.

Objective

We investigated whether intra-coronary injection of nitrite during primary percutaneous coronary intervention (PCI) might improve infarct size in ST-elevated myocardial infarction (STEMI).

Methods and Results

Patients undergoing primary PCI (n=80) were randomised to receive intracoronary (10mL) sodium nitrite (1.8μmol) or NaCl (placebo) before balloon inflation. The primary endpoint was infarct size assessed by measuring creatine kinase (CK) release. Secondary outcomes included infarct size assessed by troponin T release and by cardiac magnetic resonance imaging (CMR) on day 2.

Baseline characteristics were similar between the groups. No evidence of differences in CK release (p=0.92), troponin T (p=0.85) or CMR-assessed infarct size (p=0.254) were evident. In contrast there was an improvement in myocardial salvage index (p=0.05) and reduction in MACE at 1 year (2.6% vs 15.8%, p=0.04) in the nitrite group. In a 66-patient sub-group with TIMI≤1 flow there was reduced serum CK (p=0.030) and a 19% reduction in CMR-determined infarct size (p=0.034) with nitrite. No adverse effects of nitrite were detected.

Conclusions

In this phase II study intra-coronary nitrite infusion did not alter infarct size although a trend to improved myocardial salvage index and a significant reduction in MACE was evident. In a sub-group of patients with TIMI flow≤1 nitrite reduced infarct size and MACE and improved myocardial salvage index indicating that a phase III clinical trial assessing intra-coronary nitrite administration as an adjunct to PCI in STEMI patients is warranted.

Clinical Trial Registration

URL: http://clinicaltrials.gov NCT01584453.

Introduction

ST-segment elevation myocardial infarction (STEMI) is thought to account for ~25-47% of all acute myocardial infarctions (AMI)^{1, 2}. Presently, timely and effective reperfusion with primary percutaneous coronary intervention (PCI) is the treatment of choice for reducing infarct size, preserving left ventricular ejection fraction, and preventing the onset of heart failure^{3, 4}. Myocardial reperfusion injury may account for up to 50% of final myocardial infarct size and is a major determinant of prognosis⁵, and this underlies interest in targeting reperfusing injury using adjunctive pharmacotherapy.

Whilst several therapeutic interventions have been tested in this regard and failed, in more recent years an improved understanding of the pathophysiological mechanisms underlying ischaemia/reperfusion injury has resulted in identification of some promising mechanical (ischaemic post-conditioning⁶, remote ischaemic pre-conditioning⁷), and pharmacological (cyclosporine⁸, exenatide⁹) strategies¹⁰. More recently an additional possibility has emerged in the form of inorganic nitrite (NO₂⁻). The activity of nitrite resides in its conversion to nitric oxide (NO) under the optimal conditions of low pO₂ and pH¹¹, conditions that prevail during ischaemic episodes. NO exerts a number of beneficial effects including anti-inflammatory and anti-platelet actions and prevents the opening of the mitochondrial permeability transition pore which is a critical and final common step in reperfusion^12,13.

We first demonstrated the cardioprotective effects of intra-coronary nitrite in isolated rat hearts¹⁴, an observation likewise demonstrated by others with intra-ventricular and intra-coronary nitrite administration both in vitro and in vivo^15-17,18. In all of these studies the beneficial effects were attributed to NO and were more often associated with local rather than systemic application of high local concentrations of nitrite (3-12 μmol/L), prior to reperfusion. Together, these observations provide the rationale for the investigation of the therapeutic potential of nitrite in the treatment of acute STEMI, where nitrite is delivered locally before balloon inflation at the time of primary PCI.

Methods

Study design and participants

This study was a double-blind, randomised, single-centre, placebo-controlled trial to determine whether intra-coronary injection of sodium nitrite reduces infarct size in patients with acute STEMI undergoing primary PCI. The trial was approved by an independent ethics committee, the Medicines and Healthcare Products Regulatory Agency, registered in approved registries (NCT01584453, EudraCT nr. 2011-000721-77) and performed in accordance with the Declaration of Helsinki (1996) and the principles of the International Conference on Harmonization–Good Clinical Practice (ICH-GCP) guidelines. Full details of the trial protocol have been published¹⁹. All appropriate subjects gave written informed consent before being included in the study (see supplement for further details). Consent in the emergency situation is challenging and thus patients who were unconscious, critically unstable (cardiogenic shock) or deemed unable to consent (pain, distress, language) were excluded. Consent was a 2 stage process where initially a study summary sheet consisting of a one-page sheet with diagrams, explaining the procedure and events,was given to the patient prior to randomization and full written consent taken at this time. A more detailed patient information sheet (PIS) was given following the procedure for reading. A second stage of consent was required for agreement to subsequent cardiac magnetic resonance imaging (CMR) analyses (see below).

All consecutive patients presenting to Barts Health Heart Attack Centre, based at The London Chest Hospital, suspected of an acute STEMI and candidates for primary PCI were considered eligible for participation. Inclusion criteria were symptoms of chest pain suggestive of myocardial ischaemia, time from onset of symptoms of ≤ 6 h, aged between 18 and 80 years of age and an ECG showing ST-segment elevation of 0.1 mV in two or more limb leads or 0.2 mV in two or more contiguous precordial leads, or presumed new left bundle branch block.

Patients with cardiac arrest, cardiogenic shock, previous AMI or CABG were not included in the study. Patients with known congenital methaemoglobinaemia, left ventricular systolic dysfunction due to pre-existing heart failure, chronic renal failure (i.e. with an estimated glomerular filtration rate<30mls/min) and women who were pregnant were not included. Finally, patients on pre-existing treatment with organic nitrate therapy (Nicorandil, isosorbide mononitrate), or had active malignancy, a life-threatening condition or had participated in any investigational drug or device study within the past 30 days were excluded.

Randomisation and Intervention

After coronary angiography, patients were randomised (1:1) to a high-dose bolus injection of intra-coronary sodium nitrite (1.8 μmol in 10 mL of 0.9% NaCl) or placebo (10 mL of 0.9% NaCl) administered prior to balloon inflation. After crossing the obstruction of the infarct-related coronary artery with a guide wire, an over-the-wire balloon (Emerge, Boston Scientific, Natick, MA, USA) was positioned beyond the obstruction. The guide wire was removed and the study drug solution injected by hand through the central lumen of the balloon catheter into the distal vascular bed over 30 seconds, irrespective of TIMI flow beyond the occlusion point. The guide wire was then reinserted through the balloon catheter and advanced to a distal position. The procedure was then continued as per operator preference with no restriction placed on vascular access route, type of stent or method of stenting (pre-dilatation or direct).

The dose of nitrite administered was chosen since studies in the forearm of healthy volunteers demonstrate bioactivity of local concentrations of 2.5-10 μmol/L following bolus administration^20-22: a range broadly associated with cardioprotection in reperfusion injury achieved though bolus dose administration in pre-clinical studies^{15, 18}. Manufacture of the interventions, blinding, coding and randomisation were conducted by the Pharmacy Manufacturing Unit at Ipswich Hospital prior to transfer to the London Chest Hospital Pharmacy. The randomisation list was computer-generated based on blocks of ten and kept in a sealed opaque envelope in the hospital pharmacy. No stratification factors were used. A total of 80 indistinguishable vials of sodium nitrite and placebo were provided. All study personnel were blind to treatment allocation until the study and all analyses had been completed. All patients underwent standard UK and Barts Health Trust care protocols prior to and post-primary PCI (Table 1).

Table 1. Baseline characteristics of the study population.

	Nitrite (n=40)	Placebo (n=40)
Age (yr) (Mean±SD)	56.35±11.16	57.60±13.20
Sex (M/F)	36/4	31/9
Diabetes mellitus	3 (7.5%)	3 (7.5%)
Body-mass index (kg/m2) (Mean±SD)a	28.97±5.14	28.58±5.17
Hypertension	20 (50.0%)	14 (35.0%)
Hypercholesterolaemia	16 (40.0%)	12 (30.0%)
Heart rate (BPM) (Mean±SD)	72.68±18.62	77.35±21.31
Systolic BP (mmHg) (Mean±SD)	124.48±29.92	136.13±26.99
Ischaemia time (min) (Mean±SD)b	207.05±76.34	171.63±67.72
Door to Balloon time (min) (Mean±SD)	46.45±13.76	42.35±11.94
Culprit Vessel
Left anterior descending	9 (22.5%)	12(30%)
Circumflex	5 (12.5%)	5 (12.5%)
Right coronary	26 (65.0%)	23 (57.5%)
TIMI flow before PCI
0	30 (75.0%)	31 (77.5%)
1	6 (15.0%)	3 (7.5%)
2	2 (5.0%)	5 (12.5%)
3	2 (5.0%)	1 (2.5%)
0/1	36 (90.0%)	34 (85.0%)
Syntax score (Mean±SD)	13.41±5.50	13.58±6.20
DES use	33 (82.5%)	30 (78.9%)
Treatment before PCI
Morphine	26 (43.3%)	34 (56.7%)
Treatment at time of PCI
Heparin	40 (100%)	40 (100%)
Aspirin	40 (100%)	40 (100%)
Clopidogrel/Prasugrel (No/No)	35/5	37/3
Glycoprotein IIb/IIIa inhibitor	40 (100%	40 (100%)

Values shown as number (%) unless otherwise stated.

Abbreviations: PCI, percutaneous coronary intervention;

TIMI, Thrombolysis in myocardial infarction; DES drug-eluting stent.

^aThe body-mass index is the weight in kilograms divided by the square of the height in meters.

^bIschaemia time determined from symptom to balloon times for each patient.

Endpoints

Primary End-Point: The primary end point was infarct size assessed by measurement of area under the curve (AUC) for creatine kinase (CK) in line with previous studies⁸.

Secondary end-points: The principal secondary end point was infarct size assessed by troponin T AUC and the area of delayed hyperenhancement evident by CMR, assessed on day 2 and 6 months after infarction.

Assessment of infarct size

Blood samples were obtained at admission and repeatedly over the next two days following treatment as per Barts Health Trust protocols. The AUC (expressed in arbitrary units) for CK and troponin T was measured in each patient by computerised planimetry (GraphPad prism v5.0, California).

Infarct size was also assessed using CMR. A CMR scans was offered as a sub-study with separate consent to participants and waswere conducted according to standard protocols (see online supplement for detail). CMR related measures in addition to the above-mentioned secondary endpoints were area at risk (AAR), myocardial salvage index, microvascular obstruction (MVO), left ventricular volumes and ejection fraction. The latter two are conventional measures of cardiac function the rest of the measures provide some insight into the potential mechanisms involved in any beneficial effects that might be seen.

Coronary angiography and sub-group

Coronary angiograms obtained before and after primary PCI were used to make an assessment of Thrombolysis In Myocardial Infarction (TIMI) flow grade and AAR using standard (BARI and APPROACH) validated approaches (see online supplement for detail). The TIMI flow assessments were then utilized to make an assessment of inclusion for a single sub-study analysis of the effect of nitrite in only patients with TIMI flow ≤1 at revascularsiation. This sub-group was specifically assessed since evidence demonstrates that cardioprotective strategies are most effective in such cohorts of STEMI patients⁷ and since the biochemistry of nitrite reduction indicates that activity of the anion is greatest in hypoxic environments.

Safety and tolerability

Following 6 months and 1 year after AMI, major adverse cardiac events (MACE) (defined as death, myocardial infarction, recurrent revascularization, stroke and heart failure) were recorded. MACE was assessed at clinic follow-up at 6 months and by telephone follow-up by trained research co-ordinators at 1 year. All events were verified with source documentation. Further safety measures included assessment of the acute safety and tolerability of intra-coronary nitrite (haemodyamics and level of methaemoglobin), and the incidence of major adverse events occurring within the first 48 hours after reperfusion, including death, heart failure, AMI, stroke, recurrent ischaemia, need for repeat revascularization, renal/hepatic insufficiency, vascular complications, and bleeding (see on line supplement for details).

Measurement of platelet reactivity and assessment of nitrite

Since nitrite has been shown to have important anti-platelet effects additional hypothesis generating biochemical and functional assessments of platelet function were made^{23, 24}. These included assessments of including platelet aggregation and P-selectin expression at baseline, 30 minutes post delivery of nitrite/placebo, 4, 24 hours and 6 months after infarction (see online supplement for details). In addition, to confirm successful administration of nitrite circulating plasma nitrite/nitrate levels (collectively termed NOx) were measured at baseline and 30 minutes post delivery of the study drug (see online supplement for details). Analysis of local coronary concentration following intervention administration was not possible due to the nature of the PCI procedure.

Statistical Analysis

Primary endpoint infarct size CK analysis: We hypothesised that nitrite would reduce the CK AUC by 30%, as per previous cardioprotective strategies namely cyclosporine⁸ and postconditioning⁶. We chose to assess CK rather than CK-MB as this matches previous studies^{6, 8} and since CK measurement is part of the routine clinical assessments made in the UK following PCI. Moreover, whilst CK-MB might be considered more specific for cardiac injury recent evidence suggests that CK AUC is comparable to CK-MB, Trop T or Trop I for the assessment of infarct size²⁵. For a statistical power of 80% and a probability of a type I error of 0.05 using a two-sided test, we calculated that the sample size should be 70 subjects (35 per group). Since 4-8% of patients will die by the time of the endpoint at 6 months and 10% will either not tolerate or fail to attend the CMR at 6 months an additional 10 patients were recruited to account for these eventualities, giving a total of 80 patients.

Secondary endpoint CMR: Based upon the assumption of a predicted relative decrease of CMR-determined infarct size of 20% (as per previous studies^8,29) calculations determined that 31 patients were needed in each patient group (statistical power of 80% and a probability of a type I error of 0.05 using a two-sided test assuming mean infarct of 25G and a SD of 7G).

Analysis was based on the intention-to-treat principle. Baseline demographic and clinical variables were summarised for each arm of the study. Descriptive summaries of the distributions of continuous baseline variables are presented in terms of percentiles (e.g. median, 25th and 75th percentile), while discrete variables are summarised in terms of frequencies and percentages.

Comparisons are between the sodium nitrite-treated and placebo control-treated group for the primary and secondary outcomes. The statistical comparison between the treatment groups for the primary endpoint of CK AUC was performed using the Wilcoxon rank-sum test for non-parametric data since previous studies clearly demonstrate a non Normal distribution for this biomarker⁸.

For all other hypothesis-generating outcome measures statistical comparisons between the groups were performed using unpaired Student’s t test for data with a Normal distribution or Wilcoxon rank sum tests for data with a non Normal distribution. For comparisons between treatment groups assessing platelet reactivity data are expressed as mean ± standard error and analysis performed using two-way repeated measures ANOVA.

To explore mechanisms associations between indices were measured using Pearson’s correlation coefficient with 95% confidence intervals to ascertain whether the CMR measures of infarct size were associated with biochemical measures of infarct size, to determine whether angiographic measures of AAR were associated with the CMR assessment of AAR and whether infarct size was associated with platelet reactivity. Statistical significance was established at p<0.05 (2-tailed) for all tests and performed using SPSS version 19, (SPSS Inc, Chicago, Ill).

Results

Characteristics of study population

Between April 2012 and December 2012, 430 patients were hospitalised for management of AMI at The Barts Health Heart Attack Centre. Of these patients 353 underwent PCI. Among these 353 patients,whilst 13 were not evaluated for enrollment because study personnel were not available. Another 251 were evaluated and excluded as depicted in Figure 1. This left 89 suitable patients, of which 9 declined. Data are thus presented for 80 patients (40 in the control group and 40 in the nitrite group, Figure 1).

Figure 1. Trial profile.

All baseline and procedural characteristics were similar between the treatment groups except ischaemia time (Table 1 and 2). The mean age of the trial participants was 57 years, with 84% male. 25% of the cohort had anterior infarcts with similar numbers in both treatment groups. Stenting of the culprit lesion was performed in 97.5% of all patients. In five patients, TIMI 3 flow was not achieved after PCI (3 in the nitrite group and 2 in placebo).

Table 2. Delivery of IMP, procedural and clinical outcomes.

	Nitrite (n=40)	Placebo (n = 40)	P value
IMP Delivery
Systolic BP drop (Median (IQR)	8.8 (0.5-30.1)	11.0 (2.1-21.4)	0.90
Systolic BP drop >10%	18 (45%)	23 (57.5%)	0.37
MetHb (Median (IQR)	0.1 (0-0.13)	0 (0-0.2)	0.66
Angiographic AAR
APPROACH (Mean (95% CI))	30.89 (28.30-33.47)	26.63 (23.28-29.99)	0.05
BARI (Mean (95% CI))	27.41 (24.02-30.80)	25.17 (22.20-28.13)	0.32
Contrast	261.20±15.61	236.30±12.05	0.21
ST segment resolution (>70%)	5 (88.5%)	5 (88.5%)	0.99
Manual Thrombectomy	33 (82.5%)	31 (77.5%)	0.78
Procedural Success	37 (92.5%)	38 (95.0%)	0.62
Clinical events
48 hour	3 (7.5)	7 (17.5)	0.31
Death	0 (0)	0 (0)
Recurrent Ischaemia	0 (0)	1 (2.5)
Heart failure	2 (5)	3 (7.5)
CIN	1 (2.5)	3 (7.5)
6 month	N=40	n=40
MACE	0 (0)	4 (10.0)	0.04
Death	0 (0)	0 (0)
Repeat revascularisation	0 (0)	2 (5.0)
Recurrent myocardial infarction	0 (0)	1 (2.5)
Hospitilisation for heart failure	0 (0)	1 (2.5)
1 year	N=38	N=38
MACE	1 (2.6%)	6 (15.8%)	0.04
Death	0 (0)	0 (0)
Repeat revascularisation	1 (2.6%)	3 (7.9)
Recurrent myocardial infarction	0 (0)	1 (2.6)
Hospitilisation for heart failure	0 (0)	2 (5.3)
Medication at 1 year
Beta-blocker	33 (91.7%)	32 (88.9%)	0.69
ACE-i	33 (91.7%)	31 (86.1%)	0.45
ARB	3 (8.3%)	5 (13.9%)	0.45
Statin	36 (100%)	36 (100%)	1.00
Aspirin	36 (100%)	35 (97.2%)	0.31
ADP antagonist	30 (83.3%)	29 (80.6%)	0.76

Values shown as number (%) unless otherwise stated.

Abbreviations: AAR, area at risk; MACE, major adverse cardiac events; CIN, contrast-induced nephropathy; IMP, investigational medicinal product

Infarct size

There was no evidence of a difference in the CK AUC between the nitrite and control groups, with a median of 56,398 arbitrary units (IQR: 31185 to 83,531) in the nitrite group versus 48,195 (IQR: 27,726 to 82,841) in the control group (p=0.92). The median AUC for troponin T release was 140,782 arbitrary units (IQR, 84,949 to 218,133) in the nitrite group and 136,412 arbitrary units (IQR: 70,045 to 239,483) in the control group. This difference was not statistically different (p=0.85).

Of the 80 patients recruited 12 declined consent for the CMR protocols. In the remaining 68 patients no evidence of a difference in left ventricular volumes, mass or ejection fraction between the nitrite and placebo-treated groups were evident (Table 3). However, myocardial salvage index was improved in the nitrite group compared to placebo, although this difference fell on the borders of conventional statistical significance (p=0.05; Figure 2A). There was also a trend to smaller infarct size, incidence and MVO in the nitrite compared to placebo-treated group. CMR-assessed infarct size was positively associated with cardiac biomarkers (CK, r=0.770, p<0.01: troponin T, r=0.787, p<0.01, Online Figure I). The CMR-assessed AAR was associated with both angiographic risk scores (APPROACH: r=0.678, p<0.01, BARI: r=0.541, p<0.01, Online Figure II).

Table 3. CMR data for study population split by TIMI flow at presentation.

	Whole Cohort			TIMI ≤1			TIMI >1
	Nitrite (n=33)	Placebo (n=35)	P value	Nitrite (n=27)	Placebo (n = 30)	P value	Nitrite (n=4)	Placebo (n = 5)	P value
Baseline CMR
LVEDVi (ml/m2)	76.13 (71.11-81.14)	70.58 (65.22-75.94)	0.13	75.69 (69.93-81.44)	70.33 (64.31-76.34)	0.19	78.01 (51.65-104.40)	71.99 (54.30-89.68)	0.58
LVESVi (ml/m2)	36.16 (32.56-39.75)	35.71 (30.94-40.48)	0.88	35.16 (31.20-39.12)	35.41 (30.62-40.20)	0.94	39.90 (18.85-60.94)	37.37 (12.14-62.80)	0.84
LVMi (g/m2)	63.02 (58.65-67.40)	58.23 (54.67-61.79)	0.09	61.07 (56.27-65.88)	58.07 (54.44-61.71)	0.31	74.11 (58.19-90.03)	59.12 (40.75-77.49)	0.13
LVEF (%)	52.87 (49.88-55.86)	50.07 (46.46-53.67)	0.23	53.86 (50.40-57.32)	50.00 (46.43-53.57)	0.12	49.55 (40.32-58.78)	50.50 (29.96-71.04)	0.92
IS (% LV)	17.10 (14.12-20.08)	19.55 (16.40-22.70)	0.25	15.31 (12.36-18.27)	20.08 (16.72-23.43)	0.03	21.43 (9.62-33.23)	15.86 (0.42-31.30)	0.40
AAR (% LV)	34.58 (31.62-37.55)	33.05 (29.40-36.72)	0.52	33.89 (30.54-37.24)	33.27 (29.12-37.42)	0.82	35.71 (21.15-50.28)	31.74 (21.62-41.87)	0.51
MSI	0.52 (0.46-0.58)	0.44 (0.39-0.49)	0.05	0.56 (0.50-0.62)	0.43 (0.37-0.49)	0.002	0.41 (0.31-0.51)	0.54 (0.30-0.78)	0.17
MVO
No. (%)	16 (48.5%)	23 (69.7%)	0.13	10 (37.0%)	21 (72.4%)	0.02	3 (75.0%)	1 (25.0%)	0.14
Amount (g) (median IQR)	2.98 (0-6.25)	3.47 (0-4.75)	0.34	1.00 (0.80-5.87)	4.50 (1-7.50)	0.003	1 (0-8.25)	1 (0-1)	0.09
6 month CMR	(n=29)	(n=33)		(n=25)	(n=29)		(n=2)	(n=4)
LVEDVi (ml/m2)	82.13 (74.80-89.46)	75.62 (69.90-81.34)	0.15	79.27 (72.01-86.54)	75.37 (69.20-81.53)	0.40	109.94 (−184.6-404.5)	80.57 (60.05-101.1)	0.16
LVESVi (ml/m2)	36.50 (31.48-41.52)	34.85 (30.53-39.17)	0.61	33.80 (30.08-37.54)	34.94 (30.16-39.73)	0.71	64.97 (−216.3-346.2)	37.42 (25.62-49.21)	0.13
LVMi (g/m2)	55.67 (51.66-59.68)	51.20 (48.17-54.24)	0.07	54.21 (50.35-58.07)	51.09 (47.75-54.44)	0.21	73.36 (−80.60-227.3)	52.14 (46.98-57.30)	0.05
LVEF (%)	55.93 (52.71-59.15)	54.75 (51.62-57.87)	0.59	57.19 (54.12-60.27)	54.51 (50.96-58.05)	0.25	42.43 (−61.45-146.30)	53.51 (43.38-63.65)	0.18
IS (%)	11.88 (9.52-14.24)	13.15 (10.75-15.56)	0.45	10.69 (8.38-13.02)	13.70 (11.16-16.24)	0.08	16.33 (−9.40-42.06)	10.59 (0.24-20.93)	0.32

Values shown as mean (95% CI). Abbreviations: LVEDVi, Indexed left ventricle end-diastolic volume; LVESVi, Indexed left ventricle end-systolic volume; LVMi, Indexed left ventricle mass; LVEF, left ventricle ejection fraction; AAR, area at risk; MSI, myocardial salvage index; MVO, microvascular obstruction

Figure 2. Effect of intracoronary nitrite on cardiac magnetic resonance imaging (CMR)-determined myocardial salvage index.

The myocardial salvage index on CMR is presented for (A) 35 TIMI flow=0-3 patients in the control and 33 patients in the nitrite group. (B) Myocardial salvage index is reduced in the nitrite-treated group of 27 TIMI≤1 patients vs 28 in the control. Significance evaluated using unpaired t test and data shown as mean±SEM.

Coronary angiography and sub-group analysis

Since nitrite bioactivity is thought to occur to a greater extent under ischaemic conditions we assessed the effect of nitrite on infarct size according to whether the culprit vessel was occluded or not at the time of drug administration. Angiographic analysis indicated that 66 of the 80 patients had TIMI flow≤1 pre-procedure and successful drug delivery (i.e. unsuccessful procedures excluded). In this sub-group ischaemia time was the same between the two groups as were all other baseline characteristics (Online Table I). Importantly, in this sub-group there was a significant reduction in myocardial infarct size assessed by CK AUC between the nitrite and control groups, with a median of 44,608 arbitrary units (IQR: 27,535 to 64,848) in the nitrite group versus 55,666 (IQR: 41,591 to 93,659) in the control group (p=0.030). This represents a 19% reduction in infarct size (Figure 3A). The median AUC for troponin T release was 131,410 (IQR: 71,337 to 183,452) in the nitrite group and 176,492 (IQR: 89,831 to 245,094) in the control group (p=0.16) (Figure 3B).

Figure 3. Intracoronary nitrite lowers infarct size by biomarker assessment in the TIMI flow ≤1 subgroup.

Serum CK was measured at baseline and between 4-48 hours after coronary reperfusion. Curves for the nitrite and control group are shown in Panel A. Serum troponin T was measured at the same time points as CK and curves shown in Panel B. T bars denote standard errors of the mean (SEM).

In the nine patients with TIMI >1 flow at time of infusion, baseline characteristics were similar between the groups aside from a significantly longer ischaemia time in the nitrite group (supplement Table S2). No evidence of a difference in infarct size assessed by CK or troponin T AUC was seen in the patients with TIMI flow >1 treated with nitrite compared to placebo, although there was a trend to increased values in the nitrite group (Online Table II).

In the TIMI flow≤1 group there was a significant decrease in CMR-determined myocardial infarct size (15.31 (12.36-18.27) vs 20.08 (16.72-23.43), p=0.03) associated with an increased myocardial salvage index (0.56 (0.50-0.62) vs 0.43 (0.37-0.49), p=0.002) (Figure 2B) associated with a reduction in MVO (37% vs 72.4%) in the nitrite-treated patients (Table 3). No evidence of difference in infarct size or AAR was seen in patients with TIMI flow>1 treated with nitrite compared to placebo (Table 3).

Safety and tolerability of nitrite

Following administration of nitrite 45% of patients developed a >10% decrease in systolic blood pressure (within 10 minutes) however the magnitude and incidence was similar to the control group and did not alter clinical management. There was a small (but clinically insignificant) rise in met-Hb in the patients receiving nitrite however, the levels were not different to the control group (Table 2).

During the first 48 hours after reperfusion, 7 adverse clinical events were recorded in the control group compared to 3 in the nitrite group (Table 2). 1 year after infarction, 6 MACE events were recorded in the control group compared to 1 in the nitrite group (p=0.04, see Table 2). There were no differences in the prescription of prognostic medication between the treatment groups at discharge or at 1 year of follow-up (Table 2).

Plasma NOx levels

Similar nitrate and nitrate levels between the groups were evident at baseline, but an increase in circulating nitrite, but not nitrate, levels was evident at 30 minutes following sodium nitrite administration indicating successful administration (Figure 4).

Figure 4. Plasma NO₂⁻ and NO₃⁻ levels pre and post intervention.

Plasma NO₂⁻ and NO₃⁻ levels measured at baseline and 30 minutes after delivery of either intra-coronary nitrite or placebo in all patients. Each line representing the difference between baseline and 30 minute plasma NO₂⁻ and NO₃⁻ levels shown for each patient in the nitrite group in panel A and placebo in panel B. Error bars represent mean ± SD for each group. ***P<0.0001 using paired t-test. (NO₂⁻ = Nitrite, NO₃⁻ = Nitrate)

Platelet reactivity

Platelet aggregation and P-selectin expression changed substantially over time (Figure 5 and Online Figure III, IV), in both groups. In all conditions, platelet reactivity was greatest at baseline with no differences between the treatment groups in the whole cohort (e.g. unstimulated mean ± SD P-selectin expression in the whole cohort was 8.9±4.6% and 7.9±4.8% and ADP-induced P-selectin expression was 47.2±18.9 and 46.9±18.2 in the placebo and nitrite-treated groups respectively). In both groups there was a decrease at 4 hours, followed by a slight elevation at 24 hours and a further decrease by 6 months. However, these changes post-baseline were all suppressed in the nitrite group versus placebo in both the whole cohort (p<0.05 or 0.01, Figure 5, Online Figure III, IV) and in the TIMI ≤1 sub-group (p<0.01, Figure 5, Online Figure III, IV). Post-hoc analyses demonstrated that the reactivity of platelets to activating stimuli (only ADP shown for clarity) appears directly associated to CMR-determined infarct size (Figure 5) with 6 month CMR infarct size positively associated with both P-selectin expression (r=0.401, p=0.002, Figure 5E) and platelet aggregation (r=0.344, p=0.007, Figure 5F) at 6 months in response to ADP.

Figure 5. Platelet reactivity post-intervention.

Platelet reactivity measured at baseline, 30 minutes, 4 hours, 24 hours and 6 months after coronary reperfusion. Platelet P-Selectin expression assessed in whole blood in response to ADP (10 μmol/L) is shown for nitrite versus placebo for all patients in panel A. Panel B shows whole blood impedancce aggregometry in response to the same ADP stimulus in all patients. Panel C shows P-selectin expression in response to ADP in patients with TIMI flow <1. Panel D shows aggregation in response to ADP in the TIMI <1 subgroup. All panels show nitrite treated versus placebo. Data expressed as mean ± SEM. # =P<0.05, ##=P<0.01, for two-way repeated measures ANOVA (ADP: adenosine diphosphate). (E) There was a positive association between platelet P-selectin expression in response to ADP and LGE (late gadolinium enhancement) assessed infarct size on CMR at 6 months. Panel F depicts a similar positive association between platelet aggregation in response to ADP and LGE CMR infarct size at 6 months. Correlations determined using Pearson’s correlation coefficient.

Discussion

In this proof-of-concept phase 2 study intra-coronary administration of nitrite at the time of reperfusion in patients with AMI was not associated with a reduction compared to placebo in the primary outcome measure of infarct size as assessed by cardiac biomarkers. Although there was greater myocardial salvage index with an 18% increase in the nitrite group compared to placebo (this was on the boundaries of conventional statistical significance (p=0.05)), and significant reductions in MACE at 6 months and 1 year. In a single retrospective sub-group analysis of patients with TIMI flow ≤1, at the time of primary PCI, treatment with nitrite was associated with a 20% reduction in infarct size compared to placebo as assessed by cardiac biomarkers (AUC for CK). This effect was replicated by the CMR analyses demonstrating a reduction of infarct size of 25% associated with a greater myocardial salvage index and reduced platelet reactivity.

In this study we show statistically significant reductions of infarct size in the sub-group of patients with TIMI flow ≤1 but not in the whole cohort. Important determinants of infarct size after primary PCI include AAR and the duration of ischaemia²⁶, both of which may be confounding variables in the present study. Despite the use of best practice through randomisation and double-blinding nitrite-treated patients had a longer mean ischaemia time compared to the placebo treated group, which is known to adversely affect myocardial salvage and infarct size^{27, 28}. Importantly, in the sub-group analysis in those with TIMI flow ≤1 there were no differences in ischaemia time or any other baseline values between the groups. This result suggests that for nitrite to be most effective in reducing infarct size in STEMI patients it needs to be administered while the culprit artery is still occluded. The mean ischaemia time in the whole cohort (~189 minutes) reflects well when compared to other studies assessing potential cardioprotective strategies e.g. cyclosporine⁸ and postconditioning^{6, 29} with values ranging from 331-252 minutes. The comparatively reduced ischaemia time in the present study likely underlies the smaller infarct sizes seen herein in comparison to other published studies (e.g.^{6, 8}), a fact corroborated by the CMR analyses. It is noteworthy that, although not statistically different, the AAR assessed by angiographic scores displayed a trend to be larger in the nitrite-treated group in the whole cohort with increases of 5-14% depending on the method used: this could confound the results with theoretically larger infarcts in the nitrite group.

A recent study using intravenous nitrite in STEMI patients, with a pre-specified recruitment criteria of TIMI flow≤1 prior to reperfusion, showed no reduction in infarct size³⁰. These findings contrast directly with our sub-group assessment of TIMI flow≤1 patients where a substantial cardioprotective effect, in almost all measures of cardioprotection, was evident. This difference may relate to differences in the route of administration and dose. In the Frenneaux study nitrite was administered intravenously using a dose shown previously to achieve circulating levels of 6 μmol/L, in dogs, that was associated with profound cardioprotection¹⁸. This concentration sits within the previously demonstrated efficacious levels of 3-12 μmol/L in numerous pre-clinical studies in vivo in various species^15-17. Unfortunately in the Frenneaux study³⁰ this dose increased circulating levels of nitrite from 0.76 to 1.4 μmol/L only, suggesting that the pharmacokinetics of intravenously administered nitrite in humans is different from dogs. In our study we gave a bolus dose of nitrite directly into the coronary artery providing a local estimated concentration of ~10 μmol/L and at the very least 3 μmol/L. We believe that achieving this high local concentration prior to reperfusion is the key factor underlying the efficacy in the TIMI flow≤1 patients in our cohort.

In patients with significant coronary flow (TIMI flow>1) prior to infusion a lack of benefit is not unexpected, although the numbers are small. Extensive pre-clinical evidence demonstrates that the cytoprotective properties of nitrite in models of myocardial infarction^14-18 is most evident with application into or on the ischaemic organ with the culprit vessel occluded at time of drug delivery i.e. zero flow. Bioactivation of nitrite to nitric oxide within the circulation does occur under physiological conditions in humans^{20, 22}, however, this phenomenon is enhanced with decreasing oxygen tension and this underlies its improved bioactivity under hypoxic/ischaemic conditions^{11, 22}. Standard care pathways for STEMI patients presenting to hospitals for primary PCI include early and efficient administration of anti-platelet and anti-thrombotic therapies. Indeed, studies suggest that >40% of patients will have spontaneously reperfused in the infarct-related territory resulting in significant coronary flow (TIMI flow>1) within the culprit coronary artery prior to revascularisation³¹. Assessing whether nitrite would benefit, or indeed cause no harm, to a representative group of patients including those with TIMI flow>1 is important.

The mechanisms underlying the beneficial effects of nitrite have been attributed to its conversion to nitric oxide which improves mitochondrial function but also exerts anti-inflammatory and anti-platelet effects^{12, 13}. Interestingly, MVO was reduced in patients treated with nitrite. MVO has been implicated in worse clinical outcomes due to poor myocardial perfusion despite epicardial coronary artery revascularization³². In our study, the incidence of MVO at the initial CMR scan was reduced by ~20% and 35% in the nitrite-treated group in the whole cohort and the TIMI flow<1 subgroup respectively. It is worth noting that factors that impact on MVO, such as comorbid conditions and the use of antiplatelet and anticoagulant therapy, were similar between the two treatment groups. In addition, it is important to appreciate that whilst statistical differences were shown with two group statistical comparisons that we did not perform statistical correction for multiple comparisons in this study. Thus, further prospective studies powered for statistical significance across all of the CMR-related measures would be required to confirm the validity of our observations.

Our exploratory mechanistic analyses assessing platelet function suggest that the underlying reason for the difference in MVO may relate to reductions in platelet reactivity. Post-hoc analyses show direct correlation between platelet reactivity and infarct size, and we suspect that the reduced infarct size and as a consequence a reduced systemic inflammation might underlie this improvement with nitrite. Further analyses assessing levels of systemic inflammation and whether they correlate with infarct size/platelet reactivity are additional secondary outcome measures that should inform on this possibility. However, further prospective studies powered for assessment of platelet reactivity are warranted.

Although small this study also shows no indication that intra-coronary nitrite administration has adverse effects in the cohort as a whole or within the sub-group. Specifically, we assessed blood pressure due to the known vasodilator and blood pressure lowering effects²¹ of raised circulating nitrite levels. The data demonstrate that blood pressure did drop in some patients although this was equally evident in both arms and likely due to bradycardia and hypotension which are a common feature of reperfusing occluded coronary arteries (Bezold-Jarisch reflex), particularly the right coronary artery. We also assessed methaemoglobinemia, due to the known interaction of nitrite with oxyhaemoglobin to generate methaemoglobin particularly occurring with systemic nitrite infusions³³, and here also no adverse effect was noted. Pre-clinical evidence indicates that nitrite is cytoprotective against ischaemia-reperfusion injury only when given at concentrations (3-12 μmol/L) that far exceed physiological (0.1-0.4 μmol/L) levels. We suggest that the lack of adverse effect despite the use of high (supra-physiological) levels of nitrite in this study reflects the advantage of intra-coronary nitrite administration i.e. achieving high local concentrations within the myocardium only. An additional advantage of the intra-coronary route is that it provides an option causing no delay in reperfusion. This is compared to other therapies including cyclosporine and exenatide where intravenous administration may result in both greater side-effects and a delay in reperfusion whilst administered.

Study Limitations

Despite the same method of drug delivery used in both randomised patient groups, there was a longer ischaemia time in the nitrite group, which will have limited the potential effect of the therapy seen in the study cohort. The study was powered based on the enrolment of all-comers to prevent any treatment delay and to test the therapy in as broad a group as possible. Despite this sufficient numbers of patients with TIMI flow≤1 were available to conduct powered statistical analyses.

Further studies powered for assessing safety in TIMI flow>1 patients are essential to determine the generalised safety of intra-coronary nitrite administration in patients presenting with an AMI, and could be incorporated into a larger phase 3 study assessing the therapeutic utility of nitrite in AMI.

For the CMR measures, our study was powered for single statistical comparison for the secondary outcome measure of infarct size, we did not conduct multiple testing. However, CMR provides information regarding several other features of cardiac structure and function as detailed in the tables. A further study powered sufficiently for statistical comparison of multiple CMR-derived measures of infarct size, MVO and other indices providing valuable information regarding cardiac function such as ejection fraction may be of value to confirm the observations in this study.

Infarct size is an intermediate outcome measure that is commonly used to assess cardioprotective strategies in STEMI patients. However, as an intermediate outcome measure this does not provide clear understanding on hard outcomes such as MACE. Our study was not powered to detect changes in MACE and although we saw evidence of benefit the low number of events prevent drawing of any reliable conclusion. We suggest that our data provide strong support for conducting a phase 3 study in patients with TIMI≤1 flow at point of revascularisation assessing the therapeutic potential of intra-coronary nitrite administration with MACE as the primary outcome measure.

Conclusion

This study demonstrates that intra-coronary nitrite infusion should be added to the list of ‘promising’ cardioprotective agents for potential use in AMI when administered intracoronary at the point of revascularisation. Further investigation of this potential in a larger Phase 3 clinical trial is warranted.

Novelty and Significance.

What is known

• Despite the introduction of primary percutaneous coronary intervention (PCI) for treatment of acute myocardial infarction (AMI) significant morbidity and mortality rates remain, in part due to the damaging effects of reperfusion following revascularization.

• Thus, reducing reperfusion injury is thought to offer a potential therapeutic avenue to improve outcome.

• There is extensive pre-clinical data demonstrating efficacy of sodium nitrite (delivered locally) in reducing reperfusion injury and subsequent infarct size.

What new information does this article contribute

• This phase 2 double-blind randomized placebo controlled clinical trial assessed the efficacy of intra-coronary nitrite infusion during primary PCI for AMI

• In a subgroup of patients with occluded culprit arteries at the time of PCI there was a significant reduction in infarct size.

• This effect was associated with apparent reductions in platelet reactivity over the 6 months following PCI and reductions in major adverse cardiac events at 6 months and 1 year.

The results of this clinical trial demonstrate that local intra-coronary administration of sodium nitrite reduces infarct size as assessed by measurement of cardiac enzyme release and scar size determined using cardiac magnetic resonance imagining methods, in patients with an occluded artery (TIMI flow≤1) at the time of PCI. The intra-coronary route for nitrite administration prior to balloon inflation offers a potential cardioprotective strategy that causes no significant delay in delivery of the primary angioplasty procedure. In addition this route enables high dose delivery of nitrite that is not associated with significant methaemoglobinemia or blood pressure decrease, the two potential concerns with nitrite delivery, indicating no adverse safety profile. These results suggest that intra-coronary nitrite administration to the culprit vessel of select patients presenting with AMI may provide a new therapeutic option as an adjunct to primary angioplasty and warrants further investigation in larger outcome studies.

Abbreviations

AAR

Area at risk

AUC

Area under the curve

CMR

Cardiac magnetic resonance imaging

MACE

Major Adverse Cardiac Event

MVO

Microvascular obstruction

NO

Nitric oxide

NO₂⁻

Nitrite

PCI

Percutaneous coronary intervention

STEMI

ST-segment elevation myocardial infarction

TIMI

Thrombolysis In Myocardial Infarction