CA2832851C - Assessment of coronary heart disease with carbon dioxide - Google Patents

Abstract

The invention provides methods for diagnosing coronary heart disease in a subject in need thereof comprising administering an admixture comprising CO2 to a subject to reach a predetetmined PaCO2 in the subject to induce hyperemia, monitoring vascular reactivity in the subject and diagnosing the presence or absence of coronary heart disease in the subject, wherein decreased vascular reactivity in the subject compared to a control subject is indicative of coronary heart disease. Thc invention also provides methods for increasing sensitivity and specificity of BOLD MRI.

Description

ASSESSMENT OF CORONARY HEART DISEASE wrrn CARBON DIOXIDE
GOVERNMENT RIGHTS
The invention was made with government support under Grant No. HL091989 awarded by the National Institutes of Health, The government has certain rights to the invention.
FIELD OF INVENTION
The invention is directed to methods for detecting coronary heart disease using carbon dioxide (CO2) to induce hyperemia and monitor vascular reactivity.
BACKGROUND
The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Coronary artery disease (CAD) leads to narrowing of the small blood vessels that supply blood and oxygen to the heart. Typically, atherosclerosis is the cause of CAD.
As the coronary arteries narrow, blood flow to the heart can slow down or stop, causing, amongst other symptoms, chest pain (stable angina), shortness of breath and/or myocardial infarction.
Numerous tests help diagnose CAD. Such tests include coronary angiography/arteriography, CT angiography, echocardiogram, electrocardiogram (ECG), electron-beam computed tomography (EBCT), magnetic resonance angiography, nuclear scan and exercise stress test.
Functional assessment of the myocardium (for example the assessment of myocardium's oxygen status) requires that a patient's heart is stressed either via controlled exercise or pharmacologically.

Date Recue/Date Received 2021-02-26 Assessment of vascular reactivity in the heart is the hallmark of stress testing in cardiac imaging aimed at understanding ischemic heart disease. This is routinely done in Nuclear Medicine with radionuclide injection (such as Thallium) in conjunction with exercise to identify territories of the heart muscle that are subtended by a suspected narrowed coronary artery. In patients who are contraindicated for exercise stress-testing, this approach is typically used in conjunction with hyperemia inducing drugs, for example via adenosine infusion. Reduced coronary narrowing is expected to reduce hyperemic response and the perfusion reserve. Since nuclear methods are hampered by the need for radioactive tracers combined with limited imaging resolution, other imaging methods, such as ultrasound (using adenosine along with microbubble contrast) and MRI (also using adenosine and various conjugates of gadolinium (Gd) (fast-pass perfusion) or alterations in oxygen saturation in response to hyperemia, also known as the Blood-Oxygen-Level-Dependent (BOLD) effect) are under clinical investigation. Nonetheless, in patients who are contraindicated for exercise stress-testing, currently all imaging approaches require adenosine to elicit hyperemia.
However, adenosine has undesirable side effects (such as the feeling of "impending doom", bradycardia, arrhythmia, transient or prolonged episode of asystole, ventricular fibrillation (rarely), chest pain, headache, dyspnea, and nausea), making it less than favorable for initial or follow-up studies and many patients request that they do not undergo repeated adenosine stress testing. Nonetheless repeated stress testing is indicated in a significant patient population to assess the effectiveness of interventional or medical therapeutic regimens. In view of the side effects of hyperemia inducing drugs, there is a need for alternatives, which induce hyperemia in patients who are contraindicated for exercise stress-testing but do not cause the side effects caused by the existing hyperemia inducing drugs.
SUMMARY OF THE INVENTION
Applicants' invention is directed to the use of carbon dioxide to replace adenosine to induce hyperemia in subjects contra-indicated for exercise stress testing so as to diagnose coronary heart diseases but without the side effects of adenosine. In an embodiment, the CO2 levels are altered while the 02 levels are held constant. In another embodiment, the CO2 levels are

2 139678543.2 controlled by administering a blend of air and a controlled amount of a gas mixture comprising 20% oxygen and 80% carbon dioxide.
The invention is directed to methods for diagnosing coronary heart disease in a subject in need thereof comprising administering an admixture comprising CO2 to a subject to produce a hyperemic response corresponding to at least one selected increase in a subject's coronary PaCO2, monitoring vascular reactivity in the subject and diagnosing the presence or absence of coronary heart disease in the subject. The presence of coronary disease can be detected by monitoring a parameter indicative of a disease-associated change in a vasoreactive response to the at least one increase in PaCO2 in at least one coronary blood vessel or region of the heart. The inventors have found that such a change can be captured by monitoring the quantum of change in a parameter affected by a change in PaCO2. from an first PaCO2 level to a PaCO2 second level, for example a parameter correlated with vasodilation such as increased blood flow.
An observation of a change in a vasodilatory response can be extended to comparing responses among different subjects, wherein a decreased vascular reactivity in a subject in need of a diagnosis compared to that of a control subject is indicative of coronary heart disease.
Thus, according to one embodiment, the invention also provides a method for assessing hyperemic response in a subject in need thereof comprising administering an admixture comprising CO2 to a subject to reach a predetermined PaCO2 in the subject to induce hyperemia, monitoring vascular reactivity in the subject and assessing hyperemic response in the subject, wherein decreased vascular reactivity in the subject compared to a control subject is indicative of poor hyperemic response, thereby assessing hyperemic response in the subject in need thereof.
The invention may be directed to assessing organ perfusion in a subject in need thereof.

3 13567858.2 The invention may be directed to assessing vascular reactivity of an organ in a subject in need thereof.
The invention further provides methods of producing coronary vasodilation in a subject in need thereof comprising administering an admixture comprising CO2 to a subject to reach a predetermined PaCO2 in the subject so as to produce coronary vasodilation, thereby producing coronary vasodilation in the subject.
The invention also provides methods from increasing sensitivity and specificity for BOLD
MRI. The method includes administering an admixture comprising CO2 to a subject to reach a predetermined PaCO2 in the subject to induce hyperemia and imaging the myocardium using MRI to assess a hypermic response in response to a predetermined modulation in PaCO2. Optionally, imaging the myocardium comprises (i) obtaining free-breathing cardiac phase resolved 3D myocardial BOLD images; (ii) registering and segmenting the images to obtain the myocardial dynamic volume; and (iii) identifying ischemic territory and quantify image volume.
The invention is also directed to the use a CO2 containing gas for inducing hyperemia in a subject in need of a diagnostic assessment of coronary heart disease, wherein the CO2 containing gas is used to attain at least one increase in a subject's coronary PaCO2 sufficient for diagnosing coronary heart disease from imaging data, wherein the imaging data is indicative of a cardiovascular-disease-associated vasoreactive response to the least one increase in PaCO2 in. at least one coronary blood vessel or region of the h.eart.
The invention also provides a method for inducing hyperemia in a subject in need of a diagnostic assessment of coronary heart disease comprising administering a CO2 containing gas, attaining at least one increase in a subject's coronary PaCO2 sufficient for diagnosing coronary heart disease from imaging data and imaging the heart during a period in which the increase in PaCO2 is measurable, wherein the imaging data is indicative of a cardiovascular disease-associated vasoreactive response in at least one coronary blood vessel or region of the heart,

4 13967uss Optionally, the at least one increase in the subject's PaCO2 is selected to produce a coronary vasoreactive response sufficient for replacing a hyperemia inducing drug in assessing coronary disease.
Optionally, the use/method comprises attaining a particular predetermined PaCO2.
Optionally, the pre-determined PaCO2 is patient specific, for example an 8 to 20 mm Hg increase relative a baseline steady level measured at the time of testing.
Optionally, the use/method comprises administering carbon dioxide in a stepwise manner.
Optionally, the use/method comprises administering carbon dioxide in a block manner.
Optionally, the CO2 is administered via inhalation.
Optionally, the disease-associated coronary vasoreactive response is assessed relative to a control subject.
Optionally, the PaCO2 is increased and decreased repeatedly.
Optionally, the at least one PaCO2 produces at least an 8%-12% increase in a BOLD signal intensity.
Optionally, the disease-associated vasoreactive response is a compromised increase in blood flow.
Optionally, the imaging data is indicative of the presence or absence of a two-fold increase in blood flow in a coronary artery.

5 1396785u Optionally the imaging data are obtained by MR1 and the imaging method obtains input of a change in signal intensity of a BOLD MRI signal.
Optionally, the imaging method is PET or SPECT and the measure of a disease-associated vasoreactive response is the presence or absence of a threshold increase in blood flow.
Optionally, the at least one increase in PaCO2produces at least a 10% increase in intensity of a BOLD IvIR1 signal.
Optionally, the at least one increase in PaCO2 produces a 10-20% increase in, intensity of a BOLD MRI signal.
Optionally, the use/method comprises: (i) imaging the myocardium to obtain free-breathing cardiac phase resolved 31) myocardial BOLD images. (ii) registering and segmenting the images to obtain the myocardial dynamic volume and (Hi) identifying isehemic territory and quantifmg image volume.
Optionally, the at least one PaCO2 is at least a 10 mm Hg increase from a first level which is determined to be between 30 and 55 ram Hg. Optionally, the first level is first determined to be between 35 and 45 mm Hg.
Optionally, the sufficiency of the increase in PaCO2 is determined by increasing PaCO2 in a stepwise manner.
Optionally, the yasoreactive response is sufficient for obtaining a disease-associated change in BOLD MR1 signal obtained by administering CO2 in a manner effective to alternate between two or more PaCO2 levels over a period of time and using repeated BOLD
MRI
measurements to statistically assess the hyperemic response.

6 Optionally, the coronary vasoreactive response corresponds to a vasodilatory response produced by administering a hyperemia inducing drug for a duration and in amount per unit of time effective to assess coronary disease.
Optionally, the hyperemia inducing drug is adenosine, wherein adenosine is administered in a regimen of 140 milligrams/litre per minute over 4 to 6 minutes.
Optionally, the use/method comprises admixing air with a selected amount of a containing gas controlled to obtain a predetermined size increase in PaCO2 from a previous value, for example a measured baseline value.
The CO2 containing gas may contain, for example, 75 to 100% CO2. Optionally the CO2 containing gas comprises a percentage composition of oxygen in the 18-23%
range, optionally about 20%.
In one embodiment the invention is directed to a method for diagnosing coronary heart disease in a subject in need thereof comprising:
(i) administering an admixture comprising CO2 to a subject in a stepwise or block manner to reach a predetermined PaCO2 in the subject to induce hyperemia;
(ii) monitoring vascular reactivity in the subject; and (Hi) diagnosing the presence or absence of coronary heart disease in the subject, wherein decreased vascular reactivity in the subject compared to a control subject is indicative of coronary heart disease, thereby diagnosing coronary heart disease in the subject in need thereof.
As elaborated below, administering carbon dioxide to alter PaCO2 in block manner, is optionally repeated over time. Optionally carbon dioxide is administered so as to alternate between two or more levels of PaCO2 over a period of time.
Vascular reactivity may be monitored using any one or more of a variety of advanced imaging methods including positron emission tomography (PET), single photon emission

7 computed tomography/computed tomography (SPECT), computed tomography (CT), and magnetic resonance imaging (MRI), to name a few. Optionally, vascular reactivity may be measured using FFR.
A particularly advantageous admixture of CO2 and 02 for inducing hyperemia, particularly for blending a CO2 containing gas with air for inhalation is an admixture in which 02 is present in the range of 19-22%, for example about 20%. In this embodiment, CO2 may make up the rest of the admixture (81-78% respectively) or there may be a third gas in the admixture.
BRIEF DESCRIPTION OF FIGURES
Figure 1 depicts, in accordance with an embodiment of the present invention, the vascular reactivity in dogs as measured by the BOLD-effect using medical-grade Carbogen (5% CO2 and 95% 02) with and without coronary artery stenosis-Figure 2 depicts myocardial BOLD MRI with CO2 in canines under normocarbic and hypercarbic conditions under free breathing conditions.
Figure 3 depicts myocardial BOLD response to step-wise PaCO2 ramp up in canines while holding basal Pa02 constant.
Figure 4 depicts myocardial BOLD response to repeated (block) administration response.
Figure 5 depicts the Doppler flow through the left anterior descending artery in response to PaCO2 modulation while Pa02 is held constant.
Figure 6 depicts the Doppler flow through the LAD, RCA and LCX arteries in, response to PaCO2 modulation while Pa02 is held constant.

8 139678S2.2 Figure 7 is a bar graph depicting the territorial myocardial BOLD response to PaCO2 modulations in canines while Pa02 is held constant.
Figure 8 is a bar graph depicting the BOLD effect associated with PaCO2 modulation in blood, muscle and air while Pa02 is held constant, Figure 9 is a table summarizing the statistical BOLD data associated with the PaCO2 modulation in myocardial territories, blood, muscle and air, while Pa02 is held constant.
Figure 10 is a comparison of BOLD response to adenosine and PaCO2 (while Pa02 is held constant).
Figure 11 depicts the early findings of BOLD response to PaCO2 in humans, while Pa02 is held constant.
Figure 12(a) depicts a simulated BOLD signal for a change in PaCO2 (red line) with definitions for noise variability ( =o=2- 0) and response. Figure 12(b) depicts a relation between BOLD response (y-axis) and the number of measurements (x-axis) required to establish statistical significance (color-coded p-values). For a given BOLD response, the number of repeated measurements (N) required for reliable assessment (p<0.05) of a change from baseline condition lies at the right of the white dotted line. For e.g., to reliably detect a BOLD response from a voxel with peak BOLD signal response of 10%, greater than measurements are needed. The bar on the right gives the scale for p values associated with the statistical significance.
DETAILED DESCRIPTION OF THE INVENTION
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton Cr at., Dictionary of Microbiology and Molecular Biology rd ed, J.

9 Date Recue/Date Received 2021-02-26 Wiley & Sons (New York, NY 2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5th ed, J. Wiley & Sons (New York, NY 2001); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2001), provide one skilled in the art with a general guide to many of the terms used in the present application.
One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.
"Beneficial results" may include, but are in no way limited to, lessening or alleviating the severity of the disease condition, preventing the disease condition from worsening, curing the disease condition, preventing the disease condition from developing, lowering the chances of a patient developing the disease condition and prolonging a patient's life or life expectancy.
"Mammal" as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs,. and the like. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.
"Treatment" and "treating," as used herein refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition, prevent the pathologic condition, pursue or obtain beneficial results, or lower the chances of the individual developing the condition even if the treatment is ultimately unsuccessful. Those in need of treatment include those already with the condition as well as those prone to have the condition or those in whom the condition is to be prevented.
13467858.2 "Carbogen" as used herein is an admixture of carbon dioxide and oxygen. The amounts of carbon dioxide and oxygen in the admixture may be determined by one skilled in the art.
Medical grade carbogen is typically 5% CO2 and 95% 02. In various other embodiments, carbon dioxide is used to induce hyperemia may be an admixture of ranges including but not limited to 94% 02 and 6% CO2, 93% 02 and 7% CO2, 92% 02 and 8% CO2, 91% 02 and 9%
CO2, 90% 02 and 10% CO2, 85% 02 and 15% CO2, 80% 02 and 20% CO2, 75% 02 and 25%
CO2 and/or 70% 02 and 30% CO2. Optionally, for blending with air, the CO2 containing gas comprises 20% oxygen.
"BOLD" as used herein refers to blood-oxygen-level dependence.
A "vascular-disease-associated" coronary vasoreactive response means a type and/or quantum of vasoreactive response elicited by cardiac stress testing (e.g.
exercise or administration of a hyperemic drug or a CO2 containing gas) as demonstrable in an imaging study using one or more diagnostic imaging parameters of the type suitable to diagnose coronary vascular disease. For example, with respect to PET and SPECT, a normal response would be considered a four to five fold increase in blood flow. With respect to BOLD MRI
imaging, a 10-12+% increase in BOLD signal would be considered normal. Disease associated responses are those which are not normal in varying significant degrees among which, as evidence of disease, benchmarks may be adopted to categorize differences with represent a clearer-cut diagnosis or a progression of disease that warrants greater follow-up or more proactive treatment, for example a less than two-fold increase in blood flow as measured by PET or SPECT (typically measured in ml. of blood/min/gm of tissue).
Accordingly, a benchmark which represent a change from a value that clinicians described as "normal" which is at least statistically significant and optionally is also comparable to a standard for cardiac stress testing adopted by clinicians with respect to inducing stress represents a clear-cut benchmark for using CO2 as a vasoactive stress stimulus.
A targeted increase in PaCO2 will be selected to cause a similar vasoreactive response in normal and diseased tissue. From the standpoint of statistical significance, it will be appreciated that selection of a discriminatory increase in PaCO2 may depend on whether or not repeat measurements are made, for example, the number of repeat measurements of a BOLD signal intensity that are made at while at lower and increased PaCO2 levels, Current methods for inducing hyperemia in subjects include the use of compounds such as adenosine, analogs thereof and/or functional equivalents thereof. However, such compounds (for example, adenosine) have adverse side effects including bradycardia, arrhythmia, transient or prolonged episode of asystole, ventricular fibrillation (rarely), chest pain, headache, dyspnea, and nausea, making it less than favorable for initial or follow-up studies.
The invention described herein is directed to the use of CO2 instead of hyperemia-inducing drugs, in view of their side effects, to assess myocardial response and risk of coronary artery diseases. To date, however, it has not been possible to independently control arterial CO2 and 02, hence direct association of the influence of partial pressure of CO2 (PaCO2) on coronary vasodilation has been difficult to determine. With the development of gas flow controller devices designed to control gas concentrations in the lungs and blood (for example, RespirACT1m, Thornhill Research,W0/2013/0082703), it is now possible to precisely control the arterial CO2, while, in some embodiments, holding 01 constant.
With such devices, the desired PaCO2 changes are rapid (1-2 breaths) and are independent of minute ventilation. The inventors are the first adopters of such devices for the assessment of myocardial response to CO2.
The claimed invention is believed to be the first to use modulation of CO2 levels to show that the carbon dioxide has the same effect as the clinical dose of other hyperemia-inducing drugs such as adenosine but without the side effects. The inventors induce hyperemia by administering an admixture comprising a predetermined amount of CO2 to a subject in need thereof to assess myocardial response, evaluate coronary artery disease and identify ischernic heart disease. In an embodiment, hyperemia is induced by independently altering the administered CO2 level while holding oxygen (02) constant to assess myocardial response, evaluate coronary artery disease and identify ischemic heart disease. A
subject's myocardial response after administration of CO2 may be monitored using various imaging techniques such as MRI.
Cardiac Stress Testing When exercise stress testing is contra-indicated (in nearly 50% of patients), every existing imaging modality uses adenosine (or its analogues such as dipyridamole or regadenoson) to induce hyperemia. However, as described above, adenosine or analogs thereof or functional equivalents thereof, are well known for their adverse side effects such as bradycazdia, arrhythmia, transient or prolonged episode of asystole, ventricular fibrillation (rarely), chest pain, headache, dyspnea, and nausea, malting it less than favorable for initial or follow-up studies. Direct measures of ischemic burden may be determined on the basis of single-photon emission computed tomography (SPECT/SPET), positron emission tomography (PET), myocardial contrast echocardiography (MCE), and first-pass perfusion magnetic resonance imaging (FPP-MRI). SPECT and PET use radiotracers as contrast agents. While SPECT and PET studies account for approximately 90% myocardial ischemia-testing studies, the sensitivity and specificity for both methods combined for the determination of severe ischemia is below 70%. Both MCE and FPP-MRI are relatively newer approaches that require the use of exogenous contrast media and intravenous pharmacological stress agent (adenosine), both carrying significant risks and side effects in certain patient populations.
BOLD-MRI
An alternate method, BOLD (Blood-Oxygen-Level-Dependent) MRI, relies on endogenous contrast mechanisms (changes in blood oxygen saturation, %02) to identify ischernic territories. The potential benefits of BOLD MRI for detecting global or regional myocardial ischemia due to coronary artery disease (CAD) were demonstrated by the inventors and others at least a decade ago. Although a number of pilot clinical studies have demonstrated the feasibility of using BOLD MRI for identifying clinically significant myocardial ischemia 13967858.2 due to CAD, the method is inherently limited by sensitivity and specificity due to low BOLD
contrast-to-noise ratio (CNR). The repeatability of BOLD MRI using CO2 provides the means to improve sensitivity and specificity, which is not possible using adenosine or analogs thereof The invention provides a method for increasing the sensitivity and specificity of BOLD MR'.
The method includes administering an admixture comprising of CO2 to the subject in need thereof to induce hyperemia and imaging the myocardium using MRI to assess a hypermic response in response to a predetermined modulation in PaCO2.
The proposed method utilizes (i) an individualized targeted change in arterial partial pressure of CO2 (PaCO2) as the non-invasive vasoactive stimulus, (ii) fast, high-resolution, 4D BOLD
MRI at 3T and (iii) statistical models (for example, the generalized linear model (GLM) theory) to derive statistical parametric maps (SPM) to reliably detect and quantify the prognostic ally significant ischemic burden through repeated measurements (i.e. in a data-driven fashion).
The method for increasing the sensitivity and specificity of BOLD MRI
comprises (i) obtaining free-breathing cardiac phase-resolved 3D myocardial BOLD images (under different PaCO2 states established via inhalation of an admixture of gases comprising of CO2) ; (ii) registering and segmenting the images to obtain the myocardial dynamic volume and (iii) identifying ischemic territory and quantify image volume_ Obtaining the images The first step in increasing the sensitivity and specificity of BOLD MRI is to obtain free-breathing cardiac phase resolved 31) myocardial BOLD images. Subjects are placed on the MRI scanner table, ECG leads are placed, and necessary surface coils are positioned.
Subsequently their hearts are localized and the cardiac shim protocol is prescribed over the whole heart. K-space lines, time stamped for trigger time are collected using Gine SSFP
acquisition with image acceleration along the long axis. Central k-space lines corresponding to each cardiac phase will be used to derive the center of mass (COM) curves along the z-axis via 1-D fast Fourier transform (FFT). Based on the COM curves, the k-space lines from each cardiac phase will be sorted into 1-30 bins, each corresponding to a respiratory state with the first bin being the reference bin (end-expiration) and the last bin corresponding to end inspiration.
To minimize the artifacts from under sampling, the data will be processed with a 3D filter, followed by re-gridding the k-space lines, application of a spatial mask (to restrict the registration to region of the heart) and performing FFT to obtain the under sampled 3D image for each respiratory bin. Using the end-expiration image as the reference image, images from all bins (except bin 1) are registered using kits such as Insight Tool Kit (freely available from www.itk.org), or an equivalent software platform, in an iterative fashion and the transform parameters will be estimated for rotation, scaling, shearing, and translation of heart between the different respiratory bins. The k-space data will again be divided into 1 to 30 respiratory bins, re-gridded, transformed to the reference image (3D affme transform), summed together, and the final 3D image will be reconstructed. Imaging parameters may be TR =
3.0 to 10 ms and flip angle = 10 to 900. In this fashion, 31) eine data under controlled PaCO2 values (hypo-and hyper-carbic states) are collected.
Registration and Segmentation of Images The next step in increasing the sensitivity and specificity of BOLD MRI is registeration and segmentation of the images to obtain the myocardial dynamic volume. The pipeline utilizes MATLAB and C4+ using the ITK framework or an equivalent software platform. The myocardial MR images obtained with repeat CO2 stimulation blocks will be loaded in MATLAB (or an equivalent image processing platform) and arranged in a four-dimensional (4D) matrix, where the first 3 dimensions represent volume (voxels) and the fourth dimension is time (cardiac phase). Subsequently, each volume is resampled to achieve isotropic voxel size. End-systole (ES) are identified for each stack based on our minimum cross-correlation approach. A 4D non-linear registration algorithm is used to find voxel-to-voxel correspondences (deformation fields) across all cardiac phases. Using the recovered 139785.2 deformation, all cardiac phases are wrapped to the space of ES, such that all phases are aligned to ES. Recover the transformations across all ES images from repeat CO2 blocks and bring them to the same space using a diffeomorphic volume registration tool, such as ANTS.
Upon completion, all cardiac phases from all acquisitions will be spatially aligned to the space of ES of the first acquisition (used as reference) and all phase-to-phase deformations and acquisition-to-acquisition transformations will be known. An expert delineation of the myocardium in the ES of the first (reference) acquisition will then be performed. Based on the estimated deformation fields and tranaforinations, this segmentation is propagated to all phases and acquisitions, resulting in fully registered and segmented myocardial dynamic volumes.
Image analysis to identity and Quanti,b, Ischemic Territories The final step needed for increasing the sensitivity and specificity of BOLD
MR1 is identifying ischernic territory and quantify image volume. Since BOLD
responses are optimally observed in systolic frames, only L systolic cardiac volumes (centered at ES) are retained from each fully registered and segmented 4D BOLD MR image set obtained above.
Only those voxels contained in the myocardium are retained and the corresponding R.PP
(rate-pressure-product) and PaCO2 are noted. Assuming N acquisitions per CO2 state (hypocarbic or hypercarbic) and K. CO2 stimulation blocks, and each cardiac volume consists of nxmxp (x--. multiplication) isotropic voxels, build a concatenated fully registered 4D
dataset consisting of mcmxpxt pixels, where xnultiplication and t = LxKxN, and export this dataset in NIFTI (or an equivalent) format using standard tools. The 41) dataset is loaded into a voxel-based statistical model fitting (such as FSL-FEAT developed for fMRI), to fit the model for each voxel. The statistical analysis outputs a P-statistic volume, i.e., the SPM, where for each voxel in the myocardium the p-value of the significance of the correlation to the model is reported. The statistical parametric maps (SPM) are thresholded by identifying the voxels that have p<0.05. Those voxels are identified as hyperemic for responding to the CO2 stimulation. The total number of hyperemic voxels (VII) are counted and their relative volume (Yu/ := \TR / total voxels in myocarditun) is determined. The voxels that do not respond to CO2 stimulation (on SPM) are identified as ischemic and used to generate a binary 3D map of ischemic voxels (3D-ISCH.p). In addition, total ischemic voxels (VI) and the relative ischemic volume (V51 --VI /total myocardial voxels) are determined.
The above methods provide ischemic volumes that can be reliably identified on the basis of statistical analysis applied to repeatedly acquire 4D BOLD images under precisely targeted changes in, PaCO2. These volumes are closely related to the clinical index of fractional flow reserve FFR.
FFR
An additional method, fractional flow reserve (FFR) is used in coronary catheterization to measure pressure differences across a coronary artery stenosis to determine the likelihood that the stenosis impedes oxygen delivery to the heart muscle (myocardial ischemia).
Fractional flow reserve measures the pressure behind (distal to) a stenosis relative to the pressure before the stenosis, using adenosine or papaverine to induce hyperemia. A cut-off point of 0.75 to 0.80 has been used wherein higher values indicate a non-significant stenosis and lower values indicate a significant lesion. FFR, determined as the relative pressure differences across the stenotic coronary artery has emerged as the new standard for determining clinically significant ischenlia (FFR. -E0.75). However, it is invasive, expensive, and exposes the patient to ionizing radiation and the side-effects of the use of adenosine. In view of the side-effects of adenosine discussed above, Applicants propose using carbon dioxide instead of adenosine to induce hyperemia, by administering to a subject an admixture .. comprising CO2 to reach a predetermined PaCO2 in the subject to induce hyperemia. In some embodiments, the admixture comprises any one or more of carbon dioxide, oxygen and nitrogen; carbon dioxide and oxygen; carbon dioxide and nitrogen; or carbon dioxide alone.
In one embodiment, the amounts of CO2 and 02 administered are both altered. In another embodiment, the amount of CO2 administered is altered to a predetermined level while the amount of 02 administered is held constant. In various embodiments, the amounts of any 13967858.2 one or more of CO2, 02 or N2 in an admixture are changed or held constant as would be readily apparent to a person having ordinary skill in the art.
Methods of the invention The invention is directed to methods for diagnosing coronary heart disease in a subject in need thereof comprising administering an admixture comprising CO2 to a subject to reach a predetermined PaCO2 in the subject to induce hyperemia, monitoring vascular reactivity in the subject and diagnosing the presence or absence of coronary heart disease in the subject, wherein decreased vascular reactivity in the subject compared to a control subject is indicative of coronary heart disease. In an embodiment, CO2 is administered via inhalation.
In another embodiment, CO2 levels are altered while the 02 levels remain unchanged so that the PaCO2 is changed independently of the 02 level. In a further embodiment, vascular reactivity is monitored using imagining techniques deemed appropriate by one skilled in the art, including but not limited to any one or more of positron emission tomography (PET), single photon emission computed tomography/computed tomography (SPECT), computed tomography (CT), magnetic resonance imaging (MR1), functional magnetic resonance imaging (fMRI), single photon emission computed tomography/computed tomography (SPEC/CT), positron emission tomography/computed tomography (PET/CT), ultrasound, electrocardiogram (ECG), Electron-beam computed tomography (EBCT), echocardiogram (ECHO), electron spin resonance (ESR) and/or any combination of the imaging modalities such as (PET/MR), PET/CT, and/or SPECT/MR. In an embodiment, vascular reactivity is monitored using free-breathing BOLD MR1. In some embodiments, the admixture comprises any one or more of carbon dioxide, oxygen and nitrogen; carbon dioxide and oxygen; carbon dioxide and nitrogen; or carbon dioxide alone. In one embodiment, the amounts of CO2 and 02 administered are both altered. In another embodiment, the amount of CO2 administered is altered to a predetermined level while the amount of 02 administered is held constant. In various embodiments, the amounts of any one or more of CO2, 02 or N2 in an admixture are changed or held constant as would be readily apparent to a person having ordinary skill in the art.

13967858.2 The invention also provides a method for assessing hyperemic response in a subject in need thereof comprising administering an admixture comprising CO2 to a subject to reach a predetermined PaCO2 in the subject to induce hyperemia, monitoring vascular reactivity in the subject and assessing hyperemic response in the subject, wherein decreased vascular reactivity in the subject compared to a control subject is indicative of poor hyperemic response, thereby assessing hyperemic response in the subject in need thereof.
This method may also be used to assess organ perfusion and assess vascular reactivity. In an embodiment, CO2 is administered via inhalation. In another embodiment, CO2 levels are altered while the 02 levels remain unchanged so that the PaCO2 is changed independently of the 02 level. In a further embodiment, vascular reactivity is monitored using imagining techniques deemed appropriate by one skilled in the art, including but not limited to any one or more of positron emission tomography (PET), single photon emission computed tomography/computed tomography (SPECT), computed tomography (CT), magnetic resonance imaging (1VIRI), functional magnetic resonance imaging (NRI), single photon emission computed tomography/computed tomography (SPECT/CT), positron emission tomography/computed tomography (PET/CT), ultrasound, electrocardiogram (ECG), Electron-beam computed tomography (EBCT), echocardiogram (ECHO), electron spin resonance (ESR) arid/or any combination of the imaging modalities such as (PET/MR), PET/CT, and/or SPECT/MR. In an embodiment, vascular reactivity is monitored using free-breathing BOLD MM.
In some embodiments, the admixture comprises any one or more of carbon dioxide, oxygen and nitrogen; carbon dioxide and oxygen; carbon dioxide and nitrogen; or carbon dioxide alone.
In one embodiment, the amounts of CO2 and 02 administered are both altered. In another embodiment, the amount of CO2 administered is altered to a predetermined level while the amount of 02 administered is held constant. In various embodiments, the amounts of any one or more of CO2, 02 or N2 in an admixture are changed or held constant as would be readily apparent to a person having ordinary skill in the art.
The invention is further directed to methods for producing coronary vasodilation in a subject in need thereof comprising providing a composition comprising CO2 and administering the composition comprising CO2 to a subject to reach a predetermined PaCO2 in the subject so as to produce coronary vasodilation in the subject, thereby producing coronary vasodilation in 13967858.2 the subject. In an embodiment, CO2 is administered via inhalation. In another embodiment, CO2 levels are altered while the 02 levels remain unchanged so that the PaCO2 is changed independently of the 02 level, In a further embodiment, vascular reactivity is monitored using imagining techniques deemed appropriate by one skilled in the art, including but not limited to any one or more of positron emission tomography (PET), single photon emission computed tomography/computed tomography (SPECT), computed tomography (CT), magnetic resonance imaging (MRI), functional magnetic resonance imaging (fMRI), single photon emission computed tomography/computed tomography (SPECT/CT), positron emission tomography/computed tomography (PET/CT), ultrasound, electrocardiogram (ECG), Electron-beam computed tomography (EBCT), echocardiogram (ECHO), electron spin resonance (ESR) andlor any combination of the imaging modalities such as (PET/MR), PET/CT, and/or SPECT/MR. In an embodiment, vascular reactivity is monitored using free-breathing BOLD MRI. In some embodiments, the admixture comprises any one or more of carbon dioxide, oxygen and nitrogen; carbon dioxide and oxygen; carbon dioxide and nitrogen; or carbon dioxide alone. In one embodiment, the amounts of CO2 and administered are both altered, In another embodiment, the amount of CO2 administered is altered to a predetermined level while the amount of 02 administered is held constant. In various embodiments, the amounts of any one or more of CO2, 02 or N2 in an admixture are changed or held constant as would be readily apparent to a person having ordinary skill in the .. art.
The invention also provides a method for assessing tissue and/or organ perfusion in a subject in need thereof comprising administering an admixture comprising CO2 to a subject to reach a predetermined PaCO2 in the subject to induce hyperemia, monitoring vascular reactivity in the tissue and/or organ and assessing tissue and/or organ perfusion by assessing the hyperemic response in the subject, wherein decreased vascular reactivity in the subject compared to a control subject is indicative of poor hyperemic response and therefore poor tissue and/or organ perfusion. In an embodiment, CO2 is administered via inhalation. In another embodiment, CO2 levels are altered while the 02 levels remain unchanged so that the PaCO2 is changed independently of the 02 level. In a further embodiment, vascular reactivity is monitored using imagining techniques deemed appropriate by one skilled in the 13967103a art, including but not limited to any one or more of positron emission tomography (PET), single photon emission computed tomography/computed tomography (SPECT), computed tomography (CT), magnetic resonance imaging (MRI), functional magnetic resonance imaging (fMRI), single photon emission computed tomography/computed tomography (SPECT/CT), positron emission tomography/computed tomography (PET/CT), ultrasound, electrocardiogram (ECG), Electron-beam computed tomography (EBCT), echocardiogram (ECHO), electron spin resonance (ESR) and/or any combination of the imaging modalities such as (PET/MR), PET/CT, and/or SPECT/MR. In an embodiment, vascular reactivity is monitored using free-breathing BOLD IvERI. In some embodiments, the admixture comprises .. any one or more of carbon dioxide, oxygen and nitrogen; carbon dioxide and oxygen; carbon dioxide and nitrogen; or carbon dioxide alone. In one embodiment, the amounts of CO2 and 02 administered are both altered. In another embodiment, the amount of CO2 administered is altered to a predetermined level while the amount of 02 administered is held constant. In various embodiments, the amounts of any one or more of CO2, 02 or N2 in an admixture are IS changed or held constant as would be readily apparent to a person having ordinary skill in the art.
In some embodiments, the admixture comprising CO2 is administered at high doses for short duration or at low doses for longer durations. Por example, administering the admixture comprising CO2 at high doses of CO2 for a short duration comprises administering any one or more of 40mmllg to 45mmHg, 45mmHg to 50mmHg, 50mmlig to 55mmHg, 55mmHg CO2 to 601orn. Hg CO2, 60mml-Ig CO2 to 65mm Hg CO2, 65mmlig CO2 to 70mm Hg CO2, 70mmHg CO2 to 75mm Hg CO2, 75mml-Ig CO2 to 80mm Hg CO2, 80mmHg CO2 to 85min Hg CO2 or a combination thereof, for about 20 minutes, 15 minutes, 10 minutes, 9 minutes, 8 .. minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute or a combination thereof. In various embodiments, the predetermined levels of CO2 are administered so that the arterial level of CO2 reaches the PaCO2 of any one or more of the above ranges.
For example, administering low doses of predetermined amounts of CO2 for a longer duration comprises administering the predetermined amount of CO2 at any one or more of 13967358.2 about 30nuntig CO2 to about 35rtunlig CO2, about 35mmlig CO2 to about 40mmHg CO2, about 40mmHg CO2 to about 45nunfig CO2or a combination thereof for any one or more of about 20 to 24 hours, about 15 to20 hours, about 10 to 15 hours, about 5 to 10 hours, about 4 to 5 hours, about 3 to 4 hours, about 2 to 3 hours, about 1 to 2 hours, or a combination thereof, before inducing hyperemia. In various embodiments, the predetermined levels of CO2 are administered so that the arterial level of CO2 reaches the PaCO2 of any one or more of the above ranges.
In one embodiment, CO2 is administered in a stepwise manner. In another embodiment, administering carbon dioxide in a stepwise manner includes administering carbon dioxide in 5nmilig increments in the range of any one or more of lOmmHg to 100roralig CO2, 20mmHg to 100mning CO2, 30mmHg to 100minHg CO2, 40mmHg to 100mmHg CO2, 50mnalig to 100mmHg CO2, 60mmHg to 100mmilg CO2, 10minHg to 90mrnHg CO2, 20mmlig to 90mmHg CO2, 30minHg to 90rmaillg CO2, 40mmHg to 90mrallg CO2, 50mmlIg to 90mmilg CO2, 60mrriHg to 90minHg CO2, 10mrnHg to 80mmilg CO2, 20mmHg to 80mmHg CO2, 30trunHg to 80rnialHg CO2, 40rnmElg to 80mmrig CO2, 50mml-Ig to 80mmI4g CO2, 60mmHg to 80mmllg CO2, 10nunHg to 70mmHg CO2, 20mrxitIg to 70=rdig CO2, 30inmHg to 70mmlig CO2, 40mmHg to 70mmHg CO2, 50mmllg to 70mml-Ig CO2, 60rainHg to 70rnmIlg CO2, lOnanalg to 60nunHg CO2, 20rnm.Hg to 70nainHg CO2, 30mnallg to 70inmflg CO2, 40minHg to 70mmlIg CO2, 50mnillg to 70mm11g CO2, 60mmlig to 70mm11g CO2, 10trimilg to 60ramHg CO2, 20mmHg to 60nunHg CO2, 30rrunflg to 60rattillg CO2, 40mmHg to 60mmlig CO2 or 50mmilg to 60mmilg CO2. In various embodiments, the predetermined levels of CO2 are administered so that the arterial level of CO2 reaches the PaCO2 of any one or more of the above ranges.
In another embodiment, administering carbon dioxide in a stepwise manner includes administering carbon dioxide in lOmmlig increments in the range of any one or more of 1 mmHg to 100mm1-ig CO2, 20mmHg to 100nunHg CO2, 30mmHg to 100nunHg CO2, 40namHg to 100nunHg CO2, 50innalg to 100mmlig CO2, 60mmlig to 100mmHg CO2, lOmmlig to 90mmHg CO2, 20mtnElg to 90mmHg CO2, 30mm11g to 90mm11g CO2, 40mmHg to 90mmHg CO2, 50mm1EEg to 90=i-1g CO2, 6OrnmHg to 90mmIt CO2, 1 OmmIlg to 80minlig CO2, 20trunlig to 80mmHg CO2, 30rainHg to 80mmHg CO2, 40mm1Hg to 80mm11g CO2, 50mmHg to 80mmilg CO2, 60mmtig to 80mmlig CO2,

10=i-1g to 70mmHg CO2, 20mmElg to 70mmlig CO2, 30mmHg to 70rnmilg CO2, 40mmHg to 70mmHg CO2, 50mmHg to 70minfig CO2, 60nunlig to 70mmHg CO2, 1 OmmHg to 60nunlig CO2, 20mmlIg to 70nunHg CO2, 30mmilg to 70mmHg CO2, 40mmHg to 70mmilg CO2, 50nunHg to 70mmHg CO2, 60mraHg to 70mmHg CO2, 1 Omml-Ig to 60tninHg CO2, 20mralig to 60mmHg CO2, 30ramlig to 60mmHg CO2, =40mmHg to 60mmHg CO2 or 50mmHg to 6OrnmHg CO2. In various embodiments, the predetermined levels of CO2 are administered so that the arterial level of CO2 reaches the PaCO2 of any one or more of the above ranges.
In a further embodiment, administering carbon dioxide in a stepwise manner includes administering carbon dioxide in 20mmilg increments in the range of any one or more of 1 OmmHg to 100mmHg CO2, 20mmHg to 100rnmHg CO2, 30mrailg to 100nunHg CO2, 40mmHg to 100mmHg CO2, 50mmHg to 100mmllg CO2, 60mmHg to 100nunlig CO2.
lOmmlig to 9Ortunlig CO2, 20mmHg to 90nunkrg CO2, 30mtnlig to 90mmilg CO2, 40mmHg to 90mmHg CO2, 50nunHg to 90mmHg CO2, 60minHg to 90mmHg CO2, 1 mmHg to 80ml-11i-1g CO2, 20mmlig to 80nunHg CO2, 30tninHg to 80mmlig CO2, 40mmHg to 80mmHg CO2, 50mnilig to 80mmHg CO2,, 60mmHg to 80mmHg CO2, lOmmHg to 70mmHg CO2, 20mra11g to 70mratig CO2, 30mmHg to 70mmilg CO2, 40mmHg to 70mmHg CO2, 50mmlig to 70mm11g CO2, 60mmHg to 70mmIig CO2, lOmmHg to 60mmHg CO2, 20mmtig to 70mnifig CO2, 30mniHg to 70mmHg CO2, 40mmHg to 70mmHg CO2, 50mmHg to 70nunlIg CO2, 60trunEg to 70mmHg CO2, lOmmlig to 60mmHg CO2, 20mmilg to 60mrolig CO2, 30mnaig to 60mmHg CO2, 40mmHg to 60mmHg CO2 or 50mnalig to 60mmHg CO2. In various embodiments, the predetermined levels of CO2 are adrninistered so that the arterial level of CO2 reaches the PaCO2 of any one or more of the above ranges.
In a further embodiment, administering carbon dioxide in a stepwise manner includes administering carbon dioxide in 301runHg increments in the range of any one or more of 1 01/1121Hg to 100minHg CO2, 20mmllg to 100nunHg CO2, 30rnm1lg to 100ramllg CO2, 40mmHg to 100mmng CO2, 5Ornmilg to 100mmHg CO2, 60mmlig to 100mnal-Ig CO2, lOrrunHg to 90mmHg CO2, 20=111g to 90mmllg CO2, 30minHg to 90mm1-Ig CO2, 40minHg to 90mmlig CO2, 50mm11g to 90mraHg CO2, 60mm11g to 90nunHg CO2, 10nunHg to 80nurdig CO2, 2OrnmHg to 80mmilg CO2, 30mmHg to 80mtntig CO2, 40mmHg to 80mml1g CO2, 50namHg to 80mm1Hg CO2, 60minlig to 80mmlig CO2, lOmmHg to 70mmHg CO2, 20mm11g to 70mmHg CO2, 30mmlig to 70mmHg CO2, 40mmHg to 70mmHg CO2, 50mrnHg to 70mmlig CO2, 60mmHg to 70mmHg CO2.
10mmHg to 60mmHg CO2, 20mmHg to 70mmHg CO2, 30mmilg to 70mmHg CO2, 40mmHg to 7OrnmHg CO2, 50trimHg to 70.tumfig CO2, 60mm.Hg to 70mmHg CO2, lOmmlig to 60mmHg CO2, 20mmHg to 60rrunHg CO2, 30mmHg to 60mmHg CO2, 40ranHg to 60mmHg CO2 or 50mrn.lig to 60mmHg CO2. In various embodiments, the predetermined levels of CO2 are administered so that the arterial level of CO2 reaches the PaCO2 of any one or more of the above ranges.
In a further embodiment, administering carbon dioxide in a stepwise manner includes administering carbon dioxide in 40mmHg increments in the range of any one or more of lOmmHg to 100mmHg CO2, 20mm1-Ig to 100mmHg CO2, 30mtnifg to 100mmHg CO2.
40mmHg to 100mmHg CO2, 50ramHg to 100mmHg CO2, 60mmHg to 100mmlig CO2, lOrrunHg to 90mmlig CO2, 20mmilg to 90mmHg CO2, 30mmHg to 90mmHg CO2, 40mmHg to 90-minHg CO2, 50mtnHg to 90mm11g CO2, 60mmHg to 90nun11g CO2, lOmmHg to 80mmHg CO2, 20mm14g to 80rrunHg CO2, 30mmllg to 80nunHg CO2, 40rtunllg to 80mmHg CO2, SOrrinaHg to 80mmiig CO2, 60mmHg to 80mmHg CO2, lOmmHg to 70innfflg CO2, 20mmHg to 70mmHg CO2, 30mmlig to 70mmHg CO2, 40mmHg to 70mmlig CO2, 50nunHg to 70mmHg CO2, 60mmHg to 70mmHg CO2, lOmmHg to 60mmHg CO2, 20mnillg to 70mmHg CO2, 30mmHg to 70mmHg c02, 40mmHg to 70mmHg CO2, 50mtnHg to 70mmHg CO2, 60mmHg to 70mmllg CO2, lOmmilg to 60nunHg CO2, 20mml1g to 60mmllg CO2, 30mml1g to 60mmHg CO2, 4Ornmitg to 60nutHg CO2 or 50mmHg to 60mmHg CO2, In various embodiments, the predetermined levels of CO2 are administered so that the arterial level of CO2 reaches the PaCO2 of any one or more of the above ranges.

13967855.2 In a further embodiment, administering carbon dioxide in a stepwise manner includes administering carbon dioxide in 50nmi11g increments in the range of any one or more of lOmmHg to 100romHg CO2, 20mmilg to 100mmlig CO2, 30mmHg to 100mmHg CO2, 40mmHg to 100mmilg CO2, 50mmHg to 100mrnHg CO2, 60mmHg to 100mmlig CO2, lOmmHg to 90mm11g CO2, 20mmHg to 90mmHe CO2, 30mmHg to 90mmlig CO2, 40minfig to 90mmHg CO2, 50mmHg to 90mmHg 002, 60mmHg to 90mmit CO2, lOmmHg to 80mmHg CO2, 20mm11g to 80mmHg CO2, 30mmHg to 80mmHg CO2, 40mmHg to 80mmHg CO2, 50mmHg to 80inmHg CO2, 60minHg to 80nuntig CO2, 10mralig to 70namHg CO2, 20nunHg to 70mmHg CO2, 30mmHg to 70mmllg CO2, 40mmlig to 70mmHg CO2, 50mmHg to 70mmHg CO2, 60mmHg to 70mmHg CO2, 1 ()mmHg to 60mmlig CO2, 20mmHg to 70rnmHg 002, 30mmHg to 70mmHg CO2, 40mmlig to 70mmHg CO2, 50mmHg to 70mmHg 002, 60mmHg to 70mmHg 002, 1 OmmHg to 60mm11g CO2, 20nunHg to 60mmHg CO2, 30mmHg to 60nuntig CO2, 40mnallg to 60mmHg CO2 or 50irimHg to 60mmHg CO2. In various embodiments, the predetermined levels of CO2 are administered so that the arterial level of CO2 reaches the PaCO2 of any one or more of the above ranges.
Other increments of carbon dioxide to be administered in a stepwise manner will a readily apparent to a person having ordinary skill in the art.
In a further embodiment, predetermined amount of CO2 is administered in a block manner.
Block administration of carbon dioxide comprises administering carbon dioxide in alternating amounts over a period of time. In alternating amounts of CO2 comprises alternating between any of 20trunlig and 40mmHg, 30mmHg and 40mtnHg, 20rtutHg and 50mmHg, 30mrnHg and 50mmHg, 40mmlig and 50imnHg, 20mmHg and 60mmHg, 30narnfig and 60mmHg, 40minHg and 60mmHg, or 50mmHg and 60mmHg. In various embodiments, the predetermined levels of CO2 are administered so that the arterial level of CO2 reaches the PaCO2 of any one or more of the above ranges. Other amounts of carbon dioxide to be used in alternating amounts over a period of time will be readily apparent to a person having ordinary skill in the art.
1390858,2 In one embodiment, vascular reactivity may be measured by characterization of Myocardial Perfusion Reserve, which is defined as a ratio of Myocardial Perfusion at Stress to Myocardial Perfusion at Rest. In healthy subjects the ratio may vary from 5:1 to 6:1. The ratio diminishes with disease. A decrease in this ratio to 2:1 from the healthy level is considered the clinically significant and indicative of poor vascular reactivity.
In another embodiment, vascular reactivity may be measured via differential absolute perfusion, which may be obtained using imaging methods such as first pass perfusion, SPECT/PET, CT perfusion or echocardiography in units of ml/sec/g of tissue.
In an embodiment the CO2 gas is administered via inhalation. CO2 may be administered using, for example, RespirACTrm technology from Thornhill Research. In various embodiments, CO2 is administered for 1-2 minutes, 24 minutes, 4-6 minutes, 6-8 minutes, 8-10 minutes, 10-12 minutes, 12-14 minutes, 14-16 minutes, 16-18 minutes and/or minutes. In a preferred embodiment, CO2 is silministered for 4-6 minutes. In an additional embodiment CO2 is administered for an amount of time it takes for the arterial PaCO2 (partial pressure of carbon dioxide) to reach 50-60 mmHg from the standard levels of 30 mmHg during CO2-enhanced imaging.
In one embodiment, carbon dioxide used to induce hyperemia is medical-grade carbogen which is an admixture of 95% 02 and 5% CO2. In various other embodiments, carbon dioxide is used to induce hyperemia may be an admixture of ranges including but not limited to 94% 02 and 6% CO2, 93% 02 and 7% CO2, 92% 02 and 8% CO2, 91% 02 and 9% CO2, 90% 02 and 10% CO2, 85% 02 and 15% CO2, 80% 02 and 20% CO2, 75% 02 and 25% CO2 and/or 70% 02 and 30% CO2.
In another embodiment, vascular reactivity and/or vasodilation are monitored using any one or more of positron emission tomography (PET), single photon emission computed tomography/computed tomography (SPECT), computed tomography (CT), magnetic resonance imagine (MRI), functional magnetic resonance imaging (fMRI), single photon 13967853.2 emission computed tomography/computed tomography (SPECT/CT), positron emission tomography/computed tomography (PET/CT), ultrasound, electrocardiogram (ECG), Electron-beam computed tomography (EBCT), echocardiogram (ECHO), electron spin resonance (SR) and/or any combination of the imaging modalities such as (PET/MR), PET/CT, and/or SPECT/MR In an embodiment, vascular reactivity is monitored using free-breathing BOLD MRI.
Imaging techniques using carbon dioxide involve a free-breathing approach so as to permit entry of CO2 into the subject's system. In an embodiment, the subjects include mammalian subjects, including, human, monkey, ape, dog, cat, cow, horse, goat, pig, rabbit, mouse and rat. In a preferred embodiment, the subject is human.
ADVANTAGES OF THE INVENTION
.. The methods described herein to functionally assess the oxygen status of the myocardium include administering an effective amount of CO2 to the subject in need thereof. In an embodiment, the 02 level is held constant while the CO2 level is altered so as to induce hyperemia. Applicants herein show the vascular reactivity in subjects in response to changes in PaCO2. The existing methods use adenosine, dipyridamole, or regadenoson which have significant side-effects described above. As described in the Examples below, CO2 is at least just as effective as the existing methods (which use, for example, adenosine) but without the side effects.
The use of CO2 provides distinct advantages over the use of, for example, adenosine, Administering CO2 is truly non-invasive because it merely involves inhaling physiologically sound levels of CO2. The instant methods are simple, repeatable and fast and most likely have the best chance for reproducibility. Not even breath-holding is necessary during acquisition of images using the methods described herein. The instant methods are also highly cost-effective as no pharmacological stress agents are required, cardiologists may not need to be present during imaging and rapid imaging reduces scan times and costs.

13967856,2 Further, the improved BOLD MRI technique described above provides a non-invasive and reliable determination of ischemic volume (no radiation, contrast-media, or adenosine) and other value-added imaging biomarkers from the same acquisition (Ejection Fraction, Wall Thickening). Additionally, the subject does not experience adenosine-related adverse side effects and thus greater patient tolerance for repeat ischemia testing. There is a significant cost-savings from abandoning exogenous contrast media and adenosine/regadenoson.
Moreover, the proposed BOLD MRI paradigm will be accompanied by significant technical advances as well: (i) a fast, high-resolution, free-breathing 4D SSFP MRI at 31, that can impact cardiac MRI in general; (ii) Repeated stimulations of the heart via precisely targeted changes in PaCO2; and (iii) adoption of sophisticated analytical methods employed in the brain to the heart.
EXAMPLES
All imaging studies were performed in instrumented animals with a Doppler flow probe attached to the LAD coronary arteries for measurement of flow changes in response to CO2 and clinical dose of adenosine. In these studies, CO2 and 02 delivery were tightly controlled using Respiract. CO2 values were incremented in steps of 10 mmHg starting from 30 mmHg to 60 mmHg and were ramped down in decrements of 10 mmHg. At each CO2 level, free-breathing and cardiac gated blood-oxygen-level-dependent (BOLD) acquisitions were prescribed at mid diastole and Doppler flow velocities were measured. Similar acquisitions were also performed with block sequences of CO2 levels; that is, CO2 levels were alternated between 40 and 50 mmHg and BOLD images (and corresponding Doppler flow velocities) were acquired at each CO2 level to assess the reproducibility of the signal changes associated with different CO2 levels. Each delivery of CO2 using Respirgtct were made in conjunction with arterial blood draw to determine the arterial blood CO2 levels. Imaging-based demonstration of myocardial hyperemic response to changes in PaCO2 was shown in in health human volunteers with informed consent.
Example 1 13957858.2 The inventor has shown that CO2 can increase myocardial perfusion by a similar amount, as does adenosine in canine models. The inventor has also shown that in the setting of coronary artery narrowing, it is possible to detect regional variations in hyperemic response with the use of MRI by detecting signal changes in the myocardium due to chpnges in oxygen saturation (also known as the BOLD effect) using a free-breathing BOLD MRI
approach.
As show in Figure 1, the inventor found a 20% BOLD signal increase (hyperemic response) with medical-grade carbogen breathing in the absence of stenosis in dogs. With a severe stenosis, the hyperemic response was significantly reduced in the LAD (left anterior descending) territory but the other regions showed an increase in signal intensity (as observed with adenosine). First-pass perfusion images acquired with adenosine under severe stenosis (in. the same slice position and trigger time) is also shown for comparison. Heart rate increase of around 5-10% and a drop in blood pressure (measured invasively) by about 5% was also observed in this animal under carbogen. All acquisitions were navigator gated T2-prep 2D SSFP (steady-state free precession) and triggered at mid/end diastole (acquisition window of 50 ins). To date 10 dogs have been studied with medical-grade carbogen and have yielded highly reproducible results.
In detail, the color images (lower panel of figure 1) are color-coded to the signal intensities of grayscale images (above). The darker colors (blue/black) represent territories of baseline myocardial oxygenation and the brighter regions represent those regions that are hyperemic.
On average the signal difference between a dark blue (low signal) and orange color (high signal) is about 20%. Note that in the absence of stenosis, as one goes from 100% 02 to Carbogen, the BOLD signal intensity is elevated (second image from left) suggesting CO2 based vasoreactivity of the myocardium. The dogs were instrumented with an occluder over the left-anterior descending (LAD) coronary artery. As the LAD is occluded, note that the region indicated by an arrow (i.e. approximately between 11 o'clock and 1-2 o'clock (region supplied by the LAD)) becomes darker (3rd image from left), suggesting that vasodilation is no longer possible or is reduced. The first pass image (obtained with adenosine stress following BOLD images) at the same stenosis level also shows this territory clearly. The inventor has also been comparing the epicardial flow enhancements in response to Carbogen (with ETCO2 reaching 48-50 mm Hg) against clinical dose of adenosine and the responses have been quite similar (-20% response).
Example 2 Co-relation between inhaled CO2 and oxygen saturation Applicants assessed the difference between myocardial blood-oxygen-level dependent (BOLD) response under hypercarbia and nobnocarbia conditions in canines. The BOLD
signal intensity is proportional to oxygen saturation.
Top panels of Figure 2 depict the myocardial response under hypercarbia (60mm Hg) and normocarbia (30minHg) conditions and show an increase in BOLD signal intensity under hypercarbia condition. The lower panel depicts the difference as obtained by subtracting the signal under rest from that under stress. The myocardial BOLD signal difference between the two is depicted in grey and shows the responsiveness of canines to hypercarbia conditions.
Applicants further assessed the myocardial BOLD response to stepwise CO2 increase (ramp-up) in canines. As shown in Figure 3, as the amount of CO2 administered increases, the BOLD signal intensity increases which is indicative of an increase in hyperemic response to increased uptake of CO2 and oxygen saturation.
To further evaluate vascular reactivity and coronary response to CO2, Applicants measured the myocardial BOLD signal in response to block increases of CO2 in canines.
Specifically, the myocardial BOLL) signal was measured as the amount of CO2 administered to the canine subjects alternated between 40mm1Hg CO2 and 50mmHg CO2. As shown in Figure 4, an increase in CO2 level from 40mmHg CO2 to 50nnnHg CO2 resulted in an increase in BOLD
signal intensity and the subsequent decrease in CO2 level to 40mmilg resulted in a decreased BOLD signal. These results show a tight co-relation between administration of CO2 and vascular reactivity and coronary response.
13967858.2 Example 3 Co-relation between the amount of CO2 inhaled and Doppler flow Doppler flow, an ultrasound-based approach which uses sound waves to measure blood flow, was used to show that administration of CO2 leads to vasodilation which results in greater blood flow, while Pa02 is held constant. The Doppler flow was measured in the left anterior descending (LAD) artery. As shown in Figure 5, as the amount of administered increases the Doppler flow increases. The relative change in coronary flow velocity is directly proportional to the amount of CO2 administered.
Example 4 Each of the arteries which supply blood to the myocardium responds to the CO2 levels The myocardium is supplied with blood by the left anterior descending (LAD) artery, the right coronary artery (RCA) and the left circumflex (LCX) artery. Applicants measured the blood flow through each of these arteries in response to increasing CO2 supply. As shown in Figure 6 and summarized in Figure 7, in each of the three LAD, RCA and LCX
arteries, there is a direct correlation between the amount of CO2 administered and the signal intensity; as the amount of administered CO2 increases, the signal intensity, signaling the blood flow, in each of the three arteries increases. Further, as shown in Figure 6 and summarized in Figure 8, there is no response to CO2 modulation in control territories such as blood, skeletal muscle or air. As shown in Figure 9, the mean hyperemic response is approximately 16%.
Example 5 Vascular reactivity to CO2 comparable to adenosine Vascular reactivity of three canines that were administered with adenosine was compared 1.39678!2,2 with the vascular reactivity of canines that were administered with CO2. As shown in Figure 10, the hyperemic adenosine stress BOLD response is approximately 12% compared with 16% in response to CO2.
Further, as shown in Figure 11, early human data shows a hyperemic response of approximately 11% for a partial pressure CO2 (pCO2) change of lOnm-.Mg, from 35mmHg to 45mmHg.
Example 6 To derive a theoretical understanding of how repeated measurements may affect the BOLD
signal response, for a given BOLD response to PaCO2. Applicants performed numerical simulations of statistical fits assuming various peak hyperemic BOLD responses to two different PaCO2 levels (as in Fig.12a) along with known variability in BOLD
signals. The results (Fig. 12b) showed that as the BOLD response decreases, the number of measurements required to establish statistical significance (p<0.05) associated with the BOLD response increases exponentially. This model provides the basis for developing a statistical framework for identifying ischemie volume on the basis of repeated measures.
Various embodiments of the invention are described above in the Detailed Description.
While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and/or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well.
Unless specifically noted, it is the intention of the inventors that the waft and phrases in the specification be given the ordinary and accustomed meanings to those of ordinary skill in the applicable= art(s).
The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit Date Recue/Date Received 2021-02-26 the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.
While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects. It will be understood by those within the art that, in general, terms used herein are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least,"
the term "includes" should be interpreted as "includes but is not limited to," etc.).

13967858.2

Claims (27)

What is claimed is:

1. A non-invasive method of diagnosing coronary heart disease in a subject, the non-invasive method comprising:
administering to the subject via inhalation a controlled amount of a carbon dioxide (CO2)-containing gas via a gas flow controller device in a non-therapeutic diagnostic setting;
attaining at least one non-therapeutic increase in the subject's coronary arterial partial pressure of CO2 (PaCO2) monitored via the gas flow controller device and corresponding to the administered CO2-containing gas for diagnosing coronary heart disease; and imaging the heart during a period in which the at least one increase in PaCO2 is measurable to obtain imaging data useable to determine cardiovascular disease-associated vasoreactive response in at least one coronary blood vessel or region of the heart, wherein the at least one non-therapeutic increase in the subject's coronary PaCO2 is in the range of 8 to 40 mmHg relative to a baseline steady level measured initially.

2. The non-invasive method of claim 1, comprising attaining the at least one increase in PaCO2 in a stepwise manner.

3. The non-invasive method of claim 1, comprising attaining the at least one increase in PaCO2 in a block manner.

4. The non-invasive method of any one of claims 1 to 3, wherein the non-therapeutic increase in PaCO2 is subject specific.

5. The non-invasive method of any one of claims 1 to 4, wherein the cardiovascular disease-associated vasoreactive response is a compromised increase in blood flow.

6. The non-invasive method of any one of claims 1 to 5, wherein the imaging method is positron emission tomography (PET) or single photon emission computed tomography Date Recue/Date Received 2022-11-22 (SPECT), and a presence or absence of a threshold increase in blood flow is detected by the measure of the cardiovascular disease-associated vasoreactive response.

7. The non-invasive method of any one of claims 1 to 6, wherein the imaging data is indicative of the presence or absence of a two-fold increase in blood flow.

8. The non-invasive method of claim 1, wherein the PaCO2 is increased or decreased in a block manner repeatedly.

9. The non-invasive method of claim 1, wherein the imaging data is obtained by magnetic resonance imaging (MRI).

10. The non-invasive method of claim 1, wherein the imaging data comprise a change in signal intensity of a blood oxygenation level dependent (BOLD) MRI signal.

11. The non-invasive method of claim 10, wherein the presence of coronary heart disease is assessed on the basis of the at least one increase in PaCO2 producing at least an 8%-20% increase in BOLD signal intensity.

12. The non-invasive method of claim 10, wherein the presence of coronary heart disease is assessed on the basis of the at least one increase in PaCO2 producing at least a 9%-12% increase in BOLD signal intensity.

13. The non-invasive method of claim 9, further comprising: (i) registering and segmenting MRI images to obtain the myocardial dynamic volume; and (ii) identifying ischemic territory and quantifying image volume.

14. The non-invasive method of claim 10, further comprising: (i) imaging the myocardium to obtain free-breathing cardiac phase resolved 3D myocardial BOLD
images; (ii) registering and segmenting the images to obtain the myocardial dynamic volume; and (iii) identifying ischemic territory and quantifying image volume.

15. The non-invasive method of claim 1, wherein the administration of the containing gas is by increments of a uniform size.

16. The non-invasive method of claim 1, furthering comprising: diagnosing coronary Date Recue/Date Received 2022-11-22 heart disease from the imaging data.

17. A non-invasive method for imaging vascular reactivity under hyperemia in a subject for diagnostic assessment of cardiovascular disease, the non-invasive method comprising:
administering to the subject a CO2-containing gas in a non-therapeutic diagnostic setting via a gas flow controller device to attain at least one selected increase in the subject's coronary PaCO2, thereby inducing hyperemia in the subject; and imaging the subject's heart during a period in which the selected increase in PaCO2 is measurable, wherein the imaging data indicates the vascular reactivity under the hyperemia, wherein the at least one selected increase in PaCO2 is in the range of 8 to 20 mm Hg relative to a baseline steady level measured initially.

18. The non-invasive method of claim 17, wherein the imaging data is comparable to reference data indicative of a cardiovascular disease-associated vasoreactive response.

19. The non-invasive method of claim 18, wherein the reference data is imaging data.

20. The non-invasive method of claim 18, wherein the cardiovascular disease-associated vasoreactive response is comparable to a vasodilatory response produced by a hyperemia-inducing drug.

21. The non-invasive method of claim 19, wherein the reference data is data obtained using adenosine-induced hyperemia.

22. The non-invasive method of claim 21, wherein the hyperemia-inducing drug is adenosine.

23. The non-invasive method of claim 1, wherein cardiovascular disease-associated vasoreactive response is obtained by controlling the administration of the CO2-containing gas to repeatedly alternate between at least two PaCO2 levels and obtaining repeat BOLD MRI measurements at each level to statistically assess a hyperemic response.

Date Recue/Date Received 2022-11-22

24. The non-invasive method of claim 1, wherein the cardiovascular disease-associated vasoreactive response is monitored using fractional flow reserve (FFR).

25. The non-invasive method of claim 1, wherein the subject is any one of human, monkey, ape, dog, cat, cow, horse, goat, pig, rabbit, mouse and rat.

26. Use of a CO2-containing gas for imaging hyperemia in a subject in need of a diagnostic assessment of coronary heart disease, wherein the CO2-containing gas is used to attain at least one increase in the subject's coronary PaCO2 for diagnosing coronary heart disease from imaging data, wherein the imaging data is useable to determine a cardiovascular disease-associated vasoreactive response to the least one increase in PaCO2 in at least one coronary blood vessel or region of the heart.

27. The non-invasive method of claim 1, wherein the at least one non-therapeutic increase in the subject's coronary PaCO2 is an increase of 10 mmHg, 15 mmHg, mmHg, 30 mmHg, or 40 mmHg.

Date Recue/Date Received 2022-11-22