WO2025096554A1

WO2025096554A1 - Intracoronary acetylcholine formulation for testing cardiac conditions

Info

Publication number: WO2025096554A1
Application number: PCT/US2024/053578
Authority: WO
Inventors: Samit Shah
Original assignee: Yale University
Current assignee: Yale University
Priority date: 2023-10-30
Filing date: 2024-10-30
Publication date: 2025-05-08
Anticipated expiration: 2026-04-30

Abstract

Dosage formulations of acetylcholine in sterile packaged units for injection into a catheter for delivery in a coronary artery are provided. These are selected from ACH doses of 2µg, 20µg, 50µg, 100µg, and 200µg compounded and diluted in sterile saline or phosphate buffered saline for direct bolus injection into the human coronary artery. These are injected into the coronary artery to diagnose whether or not a patient has coronary vasospasm.

Description

INTRACORONARY ACETYLCHOLINE FORMULATION FOR TESTING CARDIAC CONDITIONS CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No.63/594,167 filed October 30, 2023, which is hereby incorporated by reference in its entirety. FIELD OF THE INVENTION The invention is generally in the field of intracoronary acetylcholine dosage formulations for testing for ischemic heart disease. BACKGROUND OF THE INVENTION Chest pain is one of the most common presenting symptoms for patients who seek medical care. Nearly half of people with cardiac chest pain (related to a lack of blood flow to the heart), will have a coronary vasomotor disorder, or “ischemia and no obstructive coronary artery disease (INOCA).” Current professional society guidelines (2021 ACC/AHA Chest Pain; 2019 ESC Chronic Coronary Syndrome) recommend additional testing to characterize the underlying diagnosis (including coronary microvascular dysfunction or coronary vasospasm). However, comprehensive assessments of coronary physiology (including the epicardial vessels and microvascular bed) are currently not performed routinely in most medical centers due to existing protocols that are overly time consuming and require specialized equipment. As a result, the majority of patients with INOCA do not undergo guideline- recommended testing. This can lead to misdiagnosis and incorrect treatment. Technological advances allow diagnosis of abnormalities in coronary physiology that drive symptoms in many patients using validated invasive assessments of coronary artery pressure and flow. Unfortunately, adoption of invasive physiological testing remains low due to the increased effort and expertise required and a perceived limited clinical value in the absence of targeted therapies. It is therefore an object of the present invention to provide formulations and a protocol for diagnosis and treatment of patients with chest pain not resulting from blocked blood vessels. It is a further object of the present invention to provide methods of use, and interpretation of data obtained thereby, for the diagnosis and treatment of individuals with coronary vasospasm, which may occur more frequently in women. 1 ^45691327.1 BRIEF SUMMARY OF THE INVENTION Dosage formulations of acetylcholine (ACH) in sterile packaged units for injection into a catheter for delivery in a coronary artery are provided. These are selected from ACH doses of 2µg, 20µg, 50µg, 100µg, and 200µg compounded and diluted in sterile saline or phosphate buffered saline for direct bolus injection into the human coronary artery. In the preferred embodiments, these are in a luer lock syringe having a capacity of between one and three milliliters. The essential dosages in the sterile packaged units are 20µg, 100µg, and 200µg ACH. In the preferred embodiment, the dosage is provided in a volume of 2 mls. Drug Dose Volume Administered Concentration

ese are njec ed n o e coronary ar ery o d agnose w e er or not a patient has coronary vasospasm or coronary endothelial dysfunction. Starting dose: The administration of low-dose ACH (2µg to 20µg) is important for two reasons. First, it allows for evaluation of endothelial function without the induction of spasm (typically seen at higher doses). In most patients the recommended starting dose of ACH is 20µg and the typical highest dose is 100 µg of ACH, although an evaluation of the graded effect of ACH administration in producing spasm demonstrated that there was a modest increase in the incidence of spasm in men at 200µg compared with 100µg. In the event that the RCA is chosen for provocative testing, it is generally recommended to not administer more than 50 µg of ACH due to a higher risk of arrhythmia. The fundamental purpose of ACH testing is to evaluate endothelial function and test for coronary vasospasm. Although the total number of doses administered varies among institutional protocols, two doses are generally sufficient to complete this assessment. 2 ^45691327.1 BRIEF DESCRIPTION OF THE DRAWINGS FIG.1 is a schematic of the method: patient with suspected ischemia, ACH testing, then guidewire testing: FFR/CFR/IMR. Fractional flow reserve (FFR) is a ratio of coronary and aortic pressures characterizing epicardial conductance. Coronary flow reserve (CFR) assesses both the epicardial and microvascular compartments and is defined as the ratio between hyperemic and resting flows. PressureWire X guidewire (Abbott Vascular) is a wireless physiology pressure wire that enables measurement of pressure and temperature to calculate: Resting Full-Cycle Ratio (RFR), FFR and Index of Microcirculatory Resistance (IMR). FIG.2 is a schematic of the decision tree based on response to ACH to diagnose the cause of angina or other defects characteristic of non-obstructive coronary arterial disease. FIG.3 is a schematic of coronary ACH coronary reactivity to the formulations described herein and the resulting diagnosis. FIG.4 is an image of the five dosage units. DETAILED DESCRIPTION OF THE INVENTION The ACH dosage formulations and methods of use thereof is based on two discoveries: that many patients presenting with symptoms of a heart attack do not have obstructive coronary artery disease, but distinct disorders requiring vastly different treatment; and ACH dosage formulations that can be administered via cardiac catheterization can rapidly, safely and effectively diagnose conditions such as endothelial dysfunction or coronary vasospasm requiring treatment other than stents or angioplasty. The coronary vasculature comprises of epicardial arteries (>400 µm), pre-arterioles (100– 400 µm), arterioles (<100 µm) and capillaries (<10 µm). The epicardial arteries function as capacitance vessels and respond to shear forces by endothelium-mediated dilatation. Epicardial arteries are visible on coronary angiography but represent only 5–10% of the coronary vasculature. The pre-arterioles, arterioles and capillaries form the coronary microvasculature. At each level, vessels are lined with endothelium, which plays an important role in the modulation of vascular tone by synthesizing and releasing several vasodilator substances, such as nitric oxide (NO). Increased endothelial wall shear stress and ACH are determinants of coronary blood flow (CBF) in health. Both lead to the biosynthesis of NO, which acts on the neighboring smooth muscle cells to induce vasodilation via the NO pathway. 3 ^45691327.1 There is extensive safety data regarding the safety of intracoronary ACH administration (Takahashi et al J Am Coll Cardiol.2022 Jun, 79 (24) 2367–2378. However, due to the complexity of compounding (Figure 2), ACH testing is not routinely performed in more than 95% of the cardiac catheterization laboratories in the United States. I. Definitions Coronary artery disease: Obstructive coronary artery disease (“CAD”). This type occurs when a fatty substance called plaque builds up the coronary arteries, leading to artery narrowing. It's the most common type of coronary heart disease. Ischemia and no obstructive coronary artery disease (“INOCA”). There is no or minimal plaque buildup in the coronary arteries. Instead, narrowing in the coronary artery is caused by other conditions, including inappropriate constrictions (coronary vasospasm), heart tissue that compresses the coronary arteries (myocardial bridging), damage to the artery lining (endothelial dysfunction), and impaired opening of the microvascular vessels. Coronary blood flow is referred to as “CBF”. Coronary microvascular disease (“CMD”) is generally used to indicate abnormal findings during adenosine testing, and indicates problems in the non-endothelium dependent function of the microvasculature of the coronary arteries. Coronary flow reserve (“CFR”). CFR represents the ratio of hyperemic to baseline coronary blood flow (CBF), i.e., the capacity of the coronary circulation to deal with increased demand for myocardial perfusion, similar to a “stress test”. There are two important limitations to measuring CFR. The first is that it incorporates the function of the epicardial vessel and hence is not specific to the microvasculature. Second, CFR is a ratio that is dependent on the baseline CBF, and there is significant variability in resting blood flow, which can make it challenging to interpret CFR as it pertains to microvascular function. II. Compositions A. Acetylcholine Acetylcholine (“ACH”) is an ester of acetic acid and choline, which acts as a neurotransmitter. 4 ^45691327.1 ACH is the chief neurotransmitter of the parasympathetic nervous system, the part of the autonomic nervous system. a branch of the peripheral nervous system that contracts smooth muscles, dilates blood vessels, increases bodily secretions, and slows heart rate. ACH can stimulate a response or block a response and thus can have excitatory or inhibitory effects. ACH has dual effects on coronary arteries. It binds to the muscarinic 3 (M3) receptor on endothelial cells and leads to an influx of intracellular calcium (Ca2+) via the L-type calcium channels. Intracellular Ca2+ binds to the protein calmodulin, and the calcium-calmodulin complex activates the endothelial nitric oxide synthase (eNOS) enzyme, which catalyses the conversion of L-arginine into nitric oxide (NO). NO then diffuses into the neighbouring vascular smooth muscle cell (VSMC) and activates guanylate cyclase (GC) enzyme to catalyse the conversion of guanosine triphosphate (GTP) into cyclic GMP (cGMP). cGMP activates the protein kinase G (PKG), which, via a series of intracellular events, inactivates the calcium channels on the VSMC. This reduces the intracellular influx of Ca2+ into the VSMC, therefore leading to vasodilation. ACH also binds to the M3 receptor on the surface of VSMCs and, in the presence of endothelial dysfunction, leads to unopposed vasoconstriction. Endothelin-1 (ET-1) binds to its receptor (ETA) and activates Rho-kinase, which inhibits MLCP and leads to vasoconstriction. VSM relaxation, and therefore vasodilation, occurs when there is reduced phosphorylation of MLC. This can result from reduced intracellular Ca2+ concentration, inhibition of MLCK by increased intracellular concentration of cAMP and MLCP-activated MLC dephosphorylation. ACH chloride currently is only available as an intraocular solution, methacholine chloride is available as a powder, bethanechol chloride is available as tablets, and carbachol is available as an ophthalmic solution. MIOCHOL^®-E (acetylcholine chloride intraocular solution) is a parasympathomimetic preparation for intraocular use. It is packaged in a blister pack containing one vial and one ampoule. The vial contains 20 mg acetylcholine chloride and 56 mg mannitol. The accompanying ampoule contains 2 mL of a modified diluent of sodium acetate trihydrate, potassium chloride, magnesium chloride hexahydrate, calcium chloride dihydrate and sterile water for injection. 5 ^45691327.1 The reconstituted liquid is a sterile isotonic solution (275–330 milliosmoles/Kg) containing 20 mg acetylcholine chloride (1:100 solution) and 2.8% mannitol. The pH range is 5.0–8.2. https://www.bausch.com/globalassets/pdf/packageinserts/pharma/miochol-e-acetylcholine- chloride-intraocular-solution.pdf B. Reformulation into dosage units. ACH can be reformulated into dosage units using sterile ACH. However, since the only Food and Drug Administration approved ACH is the ophthalmic ACH, in the preferred embodiment the ophthalmic ACH (Bausch and Lomb MIOCHOL®) is compounded into sterile pre-filled syringes which can be stored and delivered to the cardiac catheterization laboratory for each procedure. Currently, coronary artery administration of ACH is an off-label use of an ophthalmic medication, and after reconstitution of a powder, each facility must make serial dilutions so that it can be safely injected into the coronary arteries. This must be performed for each individual patient that undergoes testing, placing a strain on hospital resources and staff. Stability testing data from Cedars-Sinai Medical Center (Los Angeles, CA) and facilities outside of the United States has shown that after compounding, ACH remains effective for a minimum of 7 days or up to 28 days. Access to commercially available pre-filled ACH syringes drastically reduces the time and complexity required to perform invasive coronary function testing (CFT). As a result, this testing can be scaled to other hospitals. These dosage formulation units preferably are provided as a disposable 5-chamber storage and delivery system for the diagnostic drug. These units are characterized by being a single use, sterile system, which can be refrigerated for at least one month, with five separate dosage chambers (2 to 3mL per chamber, maximum of 5mL), with appropriate labeling, where volume is readily accessible, and which is compatible with standard male luer lock syringe (e.g., 10mL male luer lock syringe). The disclosed dosage forms provide an additional advantage in that the acetylcholine therein is stable for at least and up to 28 days reducing the burden on hospital resources and staff. For example, the dosage forms are stable for at least 5, 10, 15, 20, 25, and up to about 28 days. Stability can be determined in some forms, where the solution is clear and colorless. Methods for determining acetylcholine stability over time are known in the art (Sletten, et al., Journal of the Neurological Sciences, Volume 234, Issue 1, 1 – 3 (2005). In sum, the stability of acetylcholine in solution can be determined by reverse-phase HPLC with 6 ^45691327.1 electrochemical detection using an Acetylcholine/Choline Assay Kit. The inherent levels of Ch were used as the internal standard. The reconstituted ophthalmic ACH is a sterile isotonic solution (275–330 milliosmoles/kg) containing 20 mg acetylcholine chloride (1:100 solution) and 2.8% mannitol. The pH range is 5.0–8.2. The dosage unit formulations which have been developed are provided in five ACH doses (2µg, 20µg, 50µg, 100µg, and 200µg) compounded and diluted in sterile saline for direct bolus injection into the human coronary artery. Other ACH formulations that have been described for coronary use are neither these dosages, nor in a form that can be used for injection into the coronary artery. For example, Scuri et al.. J Appl Physiol (1985).1996 Jan;80(1):341-44. doi: 10.1152/jappl.1996.80.1.341. PMID: 8847325 describes a nebulized aerosolized formulation; Crippa et al. Cereb Cortex.1999 Jun;9(4):362-5. doi: 10.1093/cercor/9.4.362. PMID: 10426415 describes nanomolar dosing in saline with direct injection into the cerebral cortex; Phillips, et al. The British Medical Journal 1, no.4877 (1954): 1468–70. http://www.jstor.org/stable/20329271 describes milligram dosing, up to 100mg, not microgram dosing; and Francis et al. Amer, Heart J.196060(3):440-443, also describes milligram dosing in an aqueous mannitol solution. There is extensive safety data regarding the safety of intracoronary ACH administration (summarized here: https://www.jacc.org/doi/abs/10.1016/j.jacc.2022.03.385). However, due to the complexity of compounding, ACH testing is not routinely performed in > 95% of the cardiac catheterization laboratories in the United States. Chest pain is one of the most common presenting symptoms for patients who seek medical care. Nearly half of people with cardiac chest pain (related to a lack of blood flow to the heart), will have a coronary vasomotor disorder, or “ischemia and no obstructive coronary artery disease (INOCA).” Current professional society guidelines (2021 ACC/AHA Chest Pain; 2019 ESC Chronic Coronary Syndrome) recommend additional testing to characterize the underlying diagnosis (including coronary microvascular dysfunction or coronary vasospasm). C. Acetylcholine Dosage Units Dosage units consist of five 5 pre-diluted ACH doses: 2µg, 20µg, 50µg, 100µg, and 200µg, compounded and diluted in sterile saline for direct bolus injection into the human coronary artery. As described herein, the package for use in testing patients will include at least three different dosages, optionally five dosages. In most clinical practice only two or three of the 7 ^45691327.1 dosages are used. The five doses are: 2µg, 20µg, 50µg, 100µg, and 200µg. The essential doses are 20µg, 100µg, and 200µg. Preparation by Dilution into Single Syring Dosage Unites Ophthalmological ACH vial contains 20 mg un-reconstituted acetylcholine chloride powder. Reconstitute powder with 2 ml sterile diluent for 20 mg/2 ml concentrated solution. Desired concentrations for intracoronary administration are: Syringe 1: 2 µg/2 ml (1 µg/ml) Dose 2 µg Syringe 2: 20 µg/2 ml (10 µg/ml) Dose 20 µg Syringe 3: 50 µg/2 ml (25 µg/mL) Dose 50 µg Syringe 4: 100 µg/2 ml (50 µg/ml) Dose 100 µg Syringe 5: 200 µg/2 ml (100 µg/ml) Dose 200 µg All dilutions are in sterile saline or sterile phosphate buffered saline. syringe 1 (2 µg) (optional): a. withdraw 0.1 ml of 20 mg/2ml reconstituted ACH solution using a 1 cc syringe b. inject 0.1 ml of concentrated ACH into a 1 L bag of normal saline c. withdraw 2 ml from 1 l bag into a 3 cc syringe for a 2 µg/2 ml final concentration (10 mg/ml x 0.1 ml = 1 µg/ml x 1000 ml syringe 2 (20 µg): a. withdraw 0.1 ml of 20 mg/2ml reconstituted ACH solution using a 1 cc syringe b. inject 0.1 ml of concentrated ACH into a 100 mL bag of normal saline c. withdraw 2 ml from 100 ml bag into a 3 cc syringe for a 20 µg/2 ml final concentration (10 mg/ml x 0.1 ml = 10 µg/ml x 1000 ml syringe 3 (50 µg): a. withdraw 0.5 ml of 20 mg/2ml reconstituted ACH solution using a 1 cc syringe b. inject 0.5 ml of concentrated ACH into a 200 mL bag of normal saline c. withdraw 2 ml from 200 ml bag into a 3 cc syringe for a 50 µg/2 ml final concentration (10 mg/ml x 0.5 ml = 25 µg/ml x 1000 ml syringe 4 (100 µg): a. withdraw 0.5 ml of 20 mg/2ml reconstituted ACH solution using a 1 cc syringe b. inject 0.5 ml of concentrated ACH into a 100 mL bag of normal saline c. withdraw 2 ml from 100 ml bag into a 3 cc syringe for a 100 µg/2 ml final concentration (10 mg/ml x 0.5 ml = 50 µg/ml x 1000 ml syringe 5 (200 µg): 8 ^45691327.1 a. withdraw 1 ml of 20 mg/2ml reconstituted ACH solution using a 1 cc syringe b. inject 1 ml of concentrated ACH into a 100 mL bag of normal saline c. withdraw 2 ml from 100 ml bag into a 3 cc syringe for a 200 µg/2 ml final concentration (10 mg/ml x 1 ml = 50 µg/ml x 1000 ml 9 ^45691327.1 Alternatively, one can draw up from the 20 µg/ml solution: 1 ml: 20 µg 2.5 ml: 50 µg 5 ml: 100 µg 10 ml: 200 µg In another embodiment, the ACH can be prepared by dissolution of the ACH into sterile saline or phosphate buffered saline, if pharmaceutically acceptable ACH which has been produced under an appropriate GMP protocol is available. III. Methods of Diagnosis and Treatment Ischemic heart disease (IHD) is a leading cause of morbidity and mortality in women and men. The diagnostic pathway for suspected IHD involves stress testing and coronary angiography. Coronary angiography (or cardiac catheterization) is the gold-standard for diagnosing stable IHD and evaluating patients with suspected myocardial infarction (MI) because the etiology is often obstructive coronary artery disease (CAD). However, half of patients with stable symptoms have ischemia with no evidence of obstructive CAD (INOCA) and 25% with an acute MI have no evidence of obstructive plaque or thrombosis during coronary angiography (MINOCA). These syndromes encompass a heterogeneous pathophysiology, including coronary vasospasm and microvascular dysfunction, which are not identified by routine coronary angiography. As a result, many patients who undergo cardiac catheterization for suspected IHD are not given a specific diagnosis or a targeted therapeutic plan, contributing to ongoing symptoms, recidivism, and poor outcomes. Coronary microvascular dysfunction (CMD) is a type of non-obstructive coronary artery disease that causes the small blood vessels feeding the heart muscle to not work as they should. This can result in discomfort in the chest that can feel like a heaviness, tightness, pressure or squeezing, sweating, nausea and dizziness, stomach pain, difficulty breathing, being very tired (fatigue) and having low energy. Despite major advances in diagnosing INOCA, therapeutic strategies for coronary vasospasm remain limited and poorly defined. Treatments may include calcium channel blockers (“CCBs”) and nitrates constitute the treatment of choice. Lifestyle changes such as improving diet, doing regular exercise, not smoking, reducing obesity and controlling diabetes can often improve symptoms. A comprehensive physiology-guided diagnostic approach can improve the precision of patients with chest pain who present to the catheterization laboratory with suspected IHD. 10 ^45691327.1 However, comprehensive assessments of coronary physiology (including the epicardial vessels and microvascular bed) are currently not performed routinely in most medical centers due to existing protocols that are overly time consuming and require specialized equipment. As a result, the majority of patients with INOCA do not undergo guideline- recommended testing and appropriate treatment. See Sinha A, et al. Coronary microvascular disease: current concepts of pathophysiology, diagnosis and management. Cardiovasc Endocrinol Metab.2020 Jul 16;10(1):22-30. doi: 10.1097/XCE.0000000000000223. PMID: 33634252; PMCID: PMC7901821; and Smilowitz, et al. The following Comprehensive Coronary Physiology Protocol can be performed in less than one hour using equipment that is commercially available, using the dosage formulations described herein. The protocol and specific dosage unit ACH formulations are scalable and performed routinely within the standard of care in this field. Testing of the Microcirculation and Epicardium with Acetylcholine. Because the endothelium is present in all-sized coronary vessels, the use of ACH during invasive coronary function testing (CFT) allows for evaluation of both the microcirculatory and epicardial circulation. In the regulation of coronary vasomotor tone, ACH acts on the muscarinic receptors (AChM3R) in both the endothelium and vascular smooth muscle. In coronary arteries with healthy endothelium, ACH induces endothelium- dependent dilation by release of nitric oxide, which predominates over ACH-induced smooth muscle– mediated constriction. In the presence of endothelial dysfunction and impaired release of nitric oxide, smooth muscle– mediated constriction predominates in response to ACH. Higher doses of intracoronary ACH may increase the likelihood of inducing vasoconstriction through direct stimulation of the smooth muscle muscarinic receptors, particularly in the setting of endothelial dysfunction or smooth muscle hyperreactivity. Although endothelial function can be assessed by other means, such as dobutamine, exercise, cold pressor testing, and mental stress, testing with ACH remains the gold standard for invasive testing of endothelial function. The overall prognosis of patients with coronary spasm is favorable; however, patients with epicardial spasm are at increased risk for myocardial infarction and repeat angiography, whereas microvascular spasm is associated with recurrent angina. Equipment and protocol. Doses of ACH vary widely in the literature and clinical practice, typically ranging from an ultralow dose of 0.36 mg to a high dose of 200 mg. All protocols recommend the administration of graded doses of ACH, monitoring of ECG and clinical symptoms, and performance of coronary angiography in a consistent view between doses 11 ^45691327.1 to allow for assessment of epicardial spasm before proceeding to the next dose. In addition, use of metallic letters or numbers on the cine pictures to differentiate doses can be helpful for later or outside review. Dosing of ACH. Although all centers recommend a graded administration of ACH, there are significant differences in the starting, the highest, and the total number of doses administered. Starting dose: The administration of low-dose ACH is important for two reasons. First, it allows for evaluation of endothelial function without the induction of spasm (typically seen at higher doses). Second, administration of higher doses can be avoided in the event that spasm does occur at a lower dose, although experience suggests that low- dose ACH administration rarely produces epicardial spasm. Starting dose of ACH at 20 µg is recommended. Highest dose: Most investigators have used protocols that stopped at the administration of 100 µg of ACH, although some centers have continued to 200 µg in the absence of demonstrated spasm with the 100µg dose. An evaluation of the graded effect of ACH administration in producing spasm demonstrated that there was a modest increase in the incidence of spasm in men at 200 µg compared with 100 µg, although there was no demonstrable increase in spasm among women above the 50 µg dose. In the event that the RCA is chosen for provocative testing, it is generally recommended to not administer more than 50 µg of ACH due to a higher risk of arrhythmia. Total number of doses administered: The fundamental purpose of ACH testing is to evaluate endothelial function and test for coronary vasospasm. Although the total number of doses administered varies among institutional protocols, two doses are sufficient to complete this assessment. Routine administration of 100 µg has a very low incidence of adverse events. Therefore, after the initial dose, proceeding directly to the 100 µg dose for spasm assessment will expedite the procedure without sacrificing safety. One should stop before the higher dose if spasm has been demonstrated at the lower dose, and one may consider giving an additional higher dose (200 mg) if spasm has not been demonstrated but is highly suspected. Recommendation: Two doses (20 µg and 100 µg). One dose is sufficient if spasm is demonstrated at the lower dose, and a third dose (200 µg) is optional if spasm is not demonstrated at the higher dose but is strongly suspected. Rate of ACH administration. Many protocols use a slow, gradual intracoronary infusion, generally over 2 to 3 minutes per dose, whereas others use a more rapid infusion (generally 30- 60 seconds). Recommendation: A low dose (20 µg) to assess endothelial function is given as a slow 12 ^45691327.1 infusion/injection over 2 to 3 minutes. A high dose (100 µg) to assess coronary spasm is given as a quicker bolus over 30 to 60 seconds, as tolerated (see comments on the safety of bolus administration in the Avoidance of Complications section later). Method of ACH administration. Some operators prefer infusing ACH directly through the guide catheter, whereas others prefer administration of ACH selectively into the coronary artery of interest (typically the LAD artery) by using a microcatheter. Theoretically, the infusion selectively into the LAD artery avoids the potential for concomitant left circumflex or left main artery spasm. Conversely, infusion directly through the guide catheter has the advantage of simplicity and avoidance of small potential for unwanted spasm, dissection, or other complication of placement of a microcatheter into a selected vessel. Infusion directly through the guide catheter allows for assessment of the left circumflex artery as well. Endothelial dysfunction is a failure to observe the expected epicardial vasodilation and increase in coronary flow in response to the administration of ACH. The definition of an abnormal angiographic response varies somewhat in the literature, but a practical definition is any epicardial constriction of >0% but <90% after the administration of ACH (ideally determined by quantitative coronary angiography [QCA]). Although the failure to vasodilate and the presence of mild vasoconstriction can often be identified visually, QCA measurements should be encouraged in borderline cases. Endothelial function can also be more precisely evaluated by measuring the changes in overall CBF after ACH administration (normal: >50% increase from baseline), although this requires Doppler velocity and QCA measurements (CBF ¼ p (average peak velocity / 2) (vessel diameter / 2)2). One study found evidence of endothelial dysfunction in over two-thirds of ANOCA patients without evidence of CMD to adenosine or spasm from ACH, thus highlighting the need for careful endothelial assessment in these patients. Microvascular spasm is more challenging to diagnose. Ideally, one demonstrates a decrease in CBF and an increase in coronary sinus lactate production during ACH administration without epicardial constriction. However, for practical purposes, microvascular spasm is currently defined as ischemic ECG changes and angina in the absence of any visible epicardial constriction. Nonetheless, some combination of chest pain and ECG changes may not always represent diffuse microvascular spasm, and other mechanisms, such as significant endothelial dysfunction, may be involved. Epicardial coronary spasm is generally considered to be present if there is visible severe constriction of the coronary artery, accompanied by ECG changes indicative of ischemia and clinical onset of angina. The definition of severe constriction has varied in the literature from 13 ^45691327.1 75% to 90% stenosis when compared with a normal reference segment, with most publications settling on a >90% narrowing definition. Prinzmetal (variant) angina is a clinical term referring to chest pain associated with transient ST-segment elevation on the ECG. This typically occurs at rest or with mild exercise and represents recurrent episodes of myocardial ischemia secondary to epicardial or microvascular spasm. Summary of Administration of Acetylcholine Dosages to the Coronary Artery Bolus Injection Initial Dose Second Dose Maximum Dose* Left coronary artery 20µg 100µg 200µg

coronary artery guide catheters. The site of placement is the right or left coronary artery. Provocative testing should preferentially be performed on the left coronary artery due to an increased risk of bradycardia and tachyarrhythmias with provocative testing of the right coronary artery. The right coronary artery may be assessed in select cases. In the majority of cases, provocative testing with ACH should be performed after diagnostic angiography and before guidewire-based physiology or intracoronary imaging. However, the sequence of testing are based on operator preference and should be documented according to the angiographic acquisition sequence labeling guidelines. Patients should be monitored with electrocardiographic monitoring according to the catheterization laboratory routine. Additional monitoring with a cardiac monitor with pacing / defibrillator functionality is recommended. The ACH dosage should be administered by a gradual bolus hand injection via a 6Fr guide catheter and patients should be monitored for bradycardia, atrioventricular block, tachyarrhythmias, or symptoms such as chest pain. See FIG.1. Patients should be monitored with electrocardiographic monitoring according to the catheterization laboratory routine. Additional monitoring with a cardiac monitor with pacing / defibrillator functionality is recommended. After a minimum of 30 seconds the patient should be assessed for any new symptoms and an electrocardiogram should be obtained from the telemetry monitor (ideally 12-lead, if possible). 14 ^45691327.1 After each dose a cine angiogram should be performed in the exact same projection as the baseline angiogram. The angiographic view is per the discretion of the operator. After waiting for at least one minute, the dose should be escalated to Treatment of each patient. If the patient experiences chest discomfort, there is > 1mm ST-elevation or > 1mm ST- depression on the EKG, and there is 90% vasospasm of the epicardial vessel, then the test is diagnostic for coronary vasospasm and no further doses should be performed. This procedure can be used to diagnose the following conditions based on the response to injection: Coronary artery vasospasm Epicardial coronary artery endothelial dysfunction Microvascular coronary artery endothelial dysfunction Coronary artery microvascular spasm Absence of coronary artery vasospasm This is illustrated in FIG.2 and FIG.3. The five dosage units ready for use during the procedure are shown in FIG.4. Treatment of Patients Based on Diagnosis Appropriate treatments of the patients are reported in the following table from Comprehensive Management of ANOCA, Part 2—Program Development, Treatment, and 15 ^45691327.1 Research Initiatives. J Am Coll Cardiol.2023 Sep, 82 (12) 1264–1279. https://doi.org/10.1016/j.jacc.2023.06.044, as shown in the following table.

16 ^45691327.1 Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims. 17 ^45691327.1

Claims

CLAIMS I claim: 1. A sterile packaging comprising dosage formulations of acetylcholine in sterile packaged units for injection into a catheter for delivery in a coronary artery, wherein the acetylcholine dosage are 20µg, 100µg, and 200µg. 2. The sterile packaging of claim 1 further comprising acetylcholine dosages of 2µg and 50µg acetylcholine. 3. The sterile packaging of claim 1 or 2 wherein the dosages are formulated with sterile saline or sterile phosphate buffered saline, optionally comprising mannitol, in a volume between one and three milliliters, preferably 2 milliters. 4, The sterile packaging of any of claims 1-3 wherein the dosage formulation is configured to connect to and inject the formulation into a catheter suitable for placement in a coronary artery. 5. A method to diagnoses if a patient coronary vasospasm comprising placing a catheter into a coronary artery, preferably the left coronary artery, injecting the formulation of any of claims 1-4, and assessing the response. 6. The method of claim 5 comprising administering a starting dose of ACH is 20 µg.

18 ^45691327.1